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ISSN No. 2320 – 8694 Peer Reviewed - open access journal Common Creative Licence - NC 4.0 Volume No – 4 Issue No(special issue) – 3(S) May, 2016 Journal of Experimental Biology and Agricultural Sciences Journal of Experimental Biology and Agricultural Sciences (JEBAS) is an online platform for the advancement and rapid dissemination of scientific knowledge generated by the highly motivated researchers in the field of biological sciences. JEBAS publishes high-quality original research and critical up-to-date review articles covering all the aspects of biological sciences. Every year, it publishes six issues. The JEBAS is an open access journal. Anyone interested can download full text PDF without any registration. JEBAS has been accepted by EMERGING SOURCES CITATION INDEX (Thomson Reuters – Web of Science database), DOAJ, CABI, INDEX COPERNICUS INTERNATIONAL (Poland), AGRICOLA (USA), CAS (ACS, USA), CABI – Full Text (UK), AGORA (FAO-UN), OARE (UNEP), HINARI (WHO), J gate, EIJASR, DRIJ and Indian Science Abstracts (ISA, NISCAIR) like well reputed indexing database. [HORIZON PUBLISHER INDIA [HPI] http://www.horizonpublisherindia.in/] Editorial Board Journal of Experimental Biology and Agricultural Sciences _______________________________________________________________________________ Editor-in-Chief Sebua Silas Semenya Department of Biodiversity University of Limpopo South Africa Email: [email protected] Co-Editor in Chief Kuldeep Dhama (M.V.Sc., Ph.D) NAAS Associate, Principal Scientist Division of Pathology, IVRI, Izatnagar, India- 243122 Email: [email protected] Managing Editor Kamal Kishore Chaudhary (M.Sc, Ph.D) INDIA Email: [email protected], [email protected] Associate Managing Editor Anusheel Varshney School of Environment & Life Sciences, University of Salford, England, United Kingdom [email protected] Technical Editors M K Meghvansi Scientist D Biotechnology Division Defence Research Laboratory, Tezpur, India E mail: [email protected] K L Meena Lecturer – Botany MLV Govt. College, Bhilwara, India E mail: [email protected] B L Yadav Head – Botany MLV Govt. College, Bhilwara, India E mail: [email protected] Gautam Kumar Room No – 4302 Computer Center – II IIIT-A E mail: [email protected] Yashpal S. Malik ICAR-National Fellow Indian Veterinary Research Institute (IVRI) Izatnagar 243 122, Bareilly, Uttar Pradesh, India E mail: [email protected]; [email protected] A. K. Srivastava Principal Scientist (Soil Science) National Research Center For Citrus A Nagpur, Maharashtra, India Email: [email protected] Neeraj Associate Professor and Head Department of Botany Feroze Gandhi College, RaeBareli, UP, India Md.Moin Ansari Associate Professor -Senior Scientist Faculty of Veterinary Sciences and Animal Husbandry Srinagar-190006, J&K, India Associate Editors Biswanath Maity Carver College of Medicine Department of Pharmacology University of Iowa – Iowa City, USA Email: [email protected] Wu Yao Senior Manager China Development Bank ChaoYang District Beijing, China Email: [email protected] Auguste Emmanuel ISSALI Forestry Engineer Head - Coconut Breeding Department at Marc Delorme Coconut Research Station, Port Bouet, Côte d’Ivoire Regional Coordinator -COGENT Email: [email protected] Omoanghe S. Isikhuemhen Department of Natural Resources & Environmental Design North Carolina Agricultural & Technical State University Greensboro, NC 27411, USA Email: [email protected] Vincenzo Tufarelli Department of Emergency and Organ Transplantation (DETO) Section of Veterinary Science and Animal Production University of Bari ‘Aldo Moro’ s.p. Casamassima km 3, 70010 Valenzano, Italy Email: [email protected] Sunil K. Joshi Laboratory Head, Cellular Immunology Investigator, Frank Reidy Research Center of Bioelectrics College of Health Sciences, Old Dominion University 4211 Monarch Way, IRP-2, Suite # 300, Norfolk, VA 23508 USA Email: [email protected] Assistant Editors A K Trivedi Senior Scientist (Plant Physiology) - NBPGR Nainital (Uttarakhand) INDIA – 263 132 E mail: [email protected] Rajnish Kumar Room No – 4302 (Biomedical Informatics Lab) Computer center – II, IIIT-A, Allahabad E mail: [email protected] Bilal Ahmad Mir Department of Genetics University of Pretoria South Africa-0002 E mail: [email protected]; [email protected] Amit Kumar Jaiswal School of Food Science and Environmental Health College of Sciences and Health Dublin Institute of Technology, Dublin 1, Ireland E mail: [email protected] Gurudayal Ram Assistant Professor Jacob School of Biotechnology and Bioengineering (JSBB) Sam Higginbottom Institute of Agriculture, Technology and Sciences(SHIATS) Allahabad, Uttar Pradesh – 211007 Rajveer Singh Chauhan Division of Phycology Department of Botany University of Lucknow, Lucknow, INDIA E-mail: [email protected] Y. Norma-Rashid (Norma Yusoff) Professor Institute of Biological Sciences – Faculty of Science University of Malaya, 50603 Kuala Lumpur MALAYSIA E-mail: [email protected] Oadi Najim Ismail Matny Assistant Professor – Plant pathology Department of Plant Protection College Of Agriculture Science University Of Baghdad, Iraq E-mail: [email protected], [email protected] Girijesh K. Patel Post Doc Fellow 1660 Springhill Avenue Mitchell Cancer Institute University of South Alabama, USA E-mail: [email protected] Anurag Aggarwal MD, DA, PDCC (Neuroanesthesia and Intensive Care), India E-mail: [email protected] Peiman Zandi Department of Agronomy I.A.University Takestan branch,Takestan,Iran E-mail: [email protected] ISSN No. 2320 – 8694 Volume No – 4 Issue No(special issue) – 3(S) May, 2016 Journal of Experimental Biology and Agricultural Sciences Publisher: HORIZON PUBLISHER INDIA [HPI] Message - Lead Guest Editor (Dr Kuldeep Dhama, MVSc, Ph.D) _______________________________________________________________________________ Dear Authors, As Lead Guest Editor of the special issue - Biomedical perspectives of advances in disease diagnosis and therapeutics (BPADDT) of JEBAS journal, I sincere convey thanks to my able team members - Dr Yashpal S. Malik, Dr Vincenzo Tufarelli and Dr Minakshi Prasad for their valuable contributions as its Guest Editors which made reach this issue a very successful event. With their high support and personal dedicated contributions, ten (10) quality papers got attracted in this special issue including few very comprehensive reviews on interesting and demanding topics focusing modern approaches and recent advances in molecular diagnosis, potent immunomodulatory agents and valuable alternate / complementary and novel therapeutics for safeguarding health of animals, boosting their growth and production, as well as being useful in protecting human health issues. My special thanks also goes with contributing authors of diverse field and expertise for making this issue meet its vision of biomedicinal value be achieved. Along with this, I also acknowledge help of all peerreviewers by whose valuable evaluations all the papers of this issue reached up to the mark of high quality scientific presentations having updated knowledge to be shared at global level. In the end I feel happy to extend a vote of thanks to Managing Editor and Management committee of jebas journal in providing us opportunity as well as a very cooperative and cordial environment at various time points to make publish this special issue timely. Very updated information provided in articles of this issue on various areas, will definitely help to disseminate useful scientific knowledge and attract good attention of scientific community including of veterinary and biomedicine professionals, animal producers and researchers / scholars. Thank You Dr Kuldeep Dhama (MVSc, Ph.D) Principal Scientist & NAAS Associate Division of Pathology ICAR-Indian Veterinary Research Institute (IVRI) Izatnagar-243 122, Bareilly, Uttar Pradesh, India Email: [email protected], [email protected] Guest Editors [BPADDT] Lead Guest Editor Dr. Kuldeep Dhama (MVSc, Ph.D) Principal Scientist & NAAS Associate Division of Pathology ICAR-Indian Veterinary Research Institute (IVRI) Izatnagar-243 122, Bareilly, Uttar Pradesh, India Email: [email protected], [email protected] Guest Editors Dr. Yashpal S. Malik (MVSc., Ph D, Post-Doc USA) National Fellow, ICAR-IVRI Principal Scientist, Division of Biological Standardization -IVRI Izatnagar 243122, Bareilly Uttar Pradesh, India Dr. Vincenzo Tufarelli (DVM, PhD) Researcher, Department of Emergency and Organ Transplantation (DETO) Section of Veterinary Science and Animal Production University of Study of Bari ‘Aldo Moro’ Valenzano - 70010, Bari, Italy Dr. Minakshi Prasad (PhD), Fellow NAAS Professor and Head Department of Animal Biotechnology College of Veterinary Sciences LALRUVAS, Hisar 125001, Haryana - India ISSN No. 2320 – 8694 Volume No – 4 Issue No(special issue) – 3(S) May, 2016 Journal of Experimental Biology and Agricultural Sciences Publisher: HORIZON PUBLISHER INDIA [HPI] Welcome Message - Managing Editor (Dr Kamal Kishore Chaudhary, M.Sc, Ph.D) _______________________________________________________________________________ Dear Readers, It is with much joy and anticipation that we celebrate the launch of Special Issue on Biomedical Perspectives of Advances in Disease Diagnosis & Therapeutics (BPADDT) of Journal of Experimental Biology and Agricultural Sciences (JEBAS). On behalf of the JEBAS Editorial Team, I would like to extend a very warm welcome to the readership of JEBAS. I take this opportunity to thank our authors, editors and anonymous reviewers, all of whom have volunteered to contribute to the success of the journal. I am also grateful to the staff at Horizon Publisher India [HPI] for making JEBAS a reality. JEBAS is dedicated to the rapid dissemination of high quality research papers on how advances in Biotechnology, Agricultural sciences along with computational algorithm can help us meet the challenges of the 21st century, and to capitalize on the promises ahead. We welcome contributions that can demonstrate near-term practical usefulness, particularly contributions that take a multidisciplinary / convergent approach because many real world problems are complex in nature. JEBAS provides an ideal forum for exchange of information on all of the above topics and more, in various formats: full length and letter length research papers, survey papers, work-in-progress reports on promising developments, case studies and best practice articles written by industry experts. Finally, we wish to encourage more contributions from the scientific community and industry practitioners to ensure a continued success of the journal. Authors, reviewers and guest editors are always welcome. We also welcome comments and suggestions that could improve the quality of the journal. Thank you. We hope you will find JEBAS informative. Dr. Kamal K Chaudhary Managing Editor - JEBAS May 2016 Editorial Biomedical Perspectives of Advances in Disease Diagnosis & Therapeutics (BPADDT) Kuldeep Dhama, Yashpal Singh Malik, Minakshi Prasad and Vincenzo Tufarelli The effective management of diseases, including elimination or eradication, largely depends upon adoption of suitable diagnostic procedures and preventive or therapeutic regime. With the advent of molecular tools in the field of laboratory disease diagnosis, their easygoingness and end-user friendliness, the diseases of utmost importance are now timely identified with implementation of efficient disease control measures. This special issue has focused mainly on modern approaches and advances in molecular diagnosis, and developing effective and potent immunomodulatory and therapeutic modules for control of infectious diseases, posing challenge to animals and having public health concerns. Upcoming alternate / complementary and novel therapeutic regimens like probiotics, phytonutrients, herbs, vitamins as growth promoters and safeguarding health have been given due importance, especially in the era of rising drug resistant microbial pathogens. Nutritional and immunomodulatory applications effectively would be helpful in safeguarding animal health and boosting growth and production as well as protecting health of human beings and general health problems will be of high interest. This special issue is published with 10 articles. The review on “Prospective and Applied Researches in Probiotics, Prebiotics and Synbiotics: An Overview on the Functional Food Concept” provides potential benefits of representative bioactive compounds (Probiotics, prebiotics and synbiotics) on human and animal health and an overview of meat and plant-based functional products. Another review on “Lantana camara: An alien weed, its impact on animal health and strategies to control” enriches the knowledge on the toxic and beneficial effects of this weed. This article discusses the information regarding its progression, mechanism by which it affect animals, pathological alterations, treatment and what strategies can be opted to get rid of this weed. The review on “Effect of Morinda citrifolia in Growth, Production and Immunomodulatory Properties in Livestock and Poultry” elaborates the wide range of medicinal properties it possesses. Around 200 neutraceutical compounds have been identified from the plant and all of its components have high demand in case of alternative medicines and herbal medicines. Poultry industry has undergone rapid growth mainly during last three decades with the use of antibiotics, either as growth promoters or therapeutic agents. The review “Exploring Alternatives to Antibiotics as Health Promoting Agents in Poultry” describes advantages of alternative approaches to antibiotics in poultry including the use of organic acids, probiotic microorganisms, and prebiotic substrates. The existing evidences reveal that dietary vitamin E supplementation may be useful in controlling the production of reactive oxygen species and continue to be explored as a potential feeding strategy to support avian reproduction. A review on “Antioxidant Activity of Vitamin E And its Role in Avian Reproduction” provide the insight over the usefulness of the vitamin E in normal reproduction in animals and humans. A large number of infectious diseases infects masses of population and may lead to loss of lives and also incur huge economic losses. The best way to control these diseases is by diagnosing them at a very primary level and taking necessary precautionary measures so as to avoid the spread. Since last few years, the diagnostic approach has changed from tedious molecular biological techniques, to easy and rapid diagnostic techniques. Molecular diagnostics incorporated with nanobiotechnology has improved clinical diagnosis and opened a new area for development of personalized medicine. Nanotechnology has also played a crucial role in designing of diagnostic assays for medical and veterinary use. The review on “Nanodiagnostics: A New Frontier for Veterinary and Medical Sciences” provides the useful information on applications of nanodiagnostics in identification of infectious agents at an early stage of infection. A review on “Canine Parvovirus- An Insight into Diagnostic Aspect” focuses on various biotechnological approaches used for diagnosis of the virus which affects the dogs. These approaches provide rapid, sensitive, optimal detection and effective control of infection. Another review on “Prevalence, Diagnosis, Management and Control of Important Diseases of Ruminants with Special Reference to Indian Scenario” highlights the adoption of improved monitoring and/or surveillance, rapid and confirmatory diagnosis, and networking of diseases, to go forward in the path of eradication of important diseases of ruminants. Bats have been identified as the reservoir host for several pathogens, which subsequently may cause significant illness in human and animals. Of the note, zoonotic viral diseases such as Ebola, Hendra, Nipah and rabies are the diseases of importance associated with causalities. Though bats are important reservoir hosts for several zoonotic viruses, very little information is available regarding host/virus relationships. The review on “Bats: Carriers of Zoonotic Viral and Emerging Infectious Diseases” addresses some of the issues and furthermore provide the insight into interactions of bats and zoonotic viruses. The special issue also includes a research paper on “Resistotypes of Rhodococcus equi Isolated from Foals with Respiratory Problems” keeping in view that R. equi is ranked among the most important disease problems in equines, which is zoonotic and has no effective vaccine. The study analyzed the distribution pattern of the resistotypes (R-types) of various isolates of R. equi in the Haryana and Rajasthan states of India. We are sure that the information compiled will be useful for veterinary and biomedicine professionals, livestock and poultry producers, researchers, students/scholars, public health experts, and would help in targeting development of valuable and effective disease diagnostics, medicines, nutraceuticals, pharmaceuticals and therapeutics for safeguarding various health issues and production performances in a better way. This special issue can serve as a basis to formulate a significant number of recommendations. These articles show that human and animal welfare and biotechnology is a very broad subject and that there are a number of subtopics that need further investigation. Potentially, a considerable amount of biomedical improvement can be achieved at little expense. Examples in this special issue show that there is a great deal of progress to be made, simply by increasing awareness and gaining a little knowledge. ISSN No. 2320 – 8694 Peer Reviewed - open access journal Common Creative Licence - NC 4.0 http://www.jebas.org/ Volume No – 4 Issue No(special issue) – 3(S) May, 2016 Journal of Experimental Biology and Agricultural Sciences Publisher: HORIZON PUBLISHER INDIA [HPI] - http://horizonpublisherindia.in/ INDEX ____________________________________________________________________________ Research Article Page No Resistotypes of Rhodococcus equi isolated from foals with respiratory problems Sourabh Chhabra, Khurana S K*, Kapoor P K and Richa Khirbat [doi: http://dx.doi.org/10.18006/2016.4(3S).242.248] 242.248 Review Articles Effect of Morinda citrifolia in growth, production and immunomodulatory properties in livestock and poultry: a review Jai Sunder*, Tamilvannan Sujatha and Anandamoy Kundu [doi: http://dx.doi.org/10.18006/2016.4(3S).249.265] 249.265 Antioxidant activity of vitamin e and its role reproduction Vincenzo Tufarelli* and Vito Laudadio [doi: http://dx.doi.org/10.18006/2016.4(3S).266.272] 266.272 in avian An overview on the functional food concept: prospectives and applied researches in probiotics, prebiotics and synbiotics Vincenzo Tufarelli* and Vito Laudadio [doi: http://dx.doi.org/10.18006/2016.4(3S).273.278] 273.278 Canine parvovirus- an insight into diagnostic aspect Minakshi P*, Basanti Brar, Sunderisen K, Jiju V Thomas, Savi J, Ikbal, Koushlesh Ranjan, Upendera Lambe, Madhusudan Guray, Nitish Bansal, Pawan Kumar, Vinay G Joshi, Rahul Khatri, Hari Mohan, C S Pundir, Sandip Kumar Khurana and Gaya Prasad [doi: http://dx.doi.org/10.18006/2016.4(3S).279.290] 279.290 Bats: carriers of zoonotic viral and emerging diseases Koushlesh Ranjan*, Minakshi Prasad and Gaya Prasad [doi: http://dx.doi.org/10.18006/2016.4(3S).291.306] 291.306 infectious Nanodiagnostics: a new frontier for veterinary and medical sciences Upendra Lambe, Minakshi P*, Basanti Brar, Madhusudan Guray, Ikbal, Koushlesh Ranjan, Nitish Bansal, Sandip Kumar Khurana and Manimegalai J [doi: http://dx.doi.org/10.18006/2016.4(3S).307.320] 307.320 Lantana camara: An alien weed, its impact on animal health and strategies to control Rakesh Kumar*, Rahul Katiyar, Surender Kumar, Tarun Kumar and Vijay Singh [doi: http://dx.doi.org/10.18006/2016.4(3S).321.337] 321.337 Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to indian scenario Mani Saminathan, Rajneesh Rana*, Muthannan Andavar Ramakrishnan, Kumaragurubaran Karthik, Yashpal Singh Malik and Kuldeep Dhama [doi: http://dx.doi.org/10.18006/2016.4(3S).338.367] 338.367 Exploring alternatives to antibiotics as health promoting agents in poultry- a review Ajit Singh Yadav*, Gautham Kolluri, Marappan Gopi, Kumaragurubaran Karthik, Yashpal Singh Malik and Kuldeep Dhama [doi: http://dx.doi.org/10.18006/2016.4(3S).368.383] 368.383 Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 RESISTOTYPES OF Rhodococcus equi ISOLATED FROM FOALS WITH RESPIRATORY PROBLEMS Sourabh Chhabra1, Khurana S K2,*, Kapoor P K1and Richa Khirbat3 1 Department of Veterinary Public Health and Epidemiology, LUVAS, Hisar, Haryana, India 125 004 National Research Centre on Equines, Hisar, Haryana, India 125 001 Department of Animal Biotechnology, LUVAS, Hisar, Haryana, India 125 004 2 3 Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).242.248 KEYWORDS Rhodococcus equi Foal Resistotype Marker ABSTRACT Rhodococcus equi has been recognized primarily as a respiratory pathogen of equines particularly of foals between one and four months of age. R. equi is ranked among the most important disease problems in equines especially because of its high prevalence and mortality rate. R. equi being an intracellular pathogen is very fastidious and requires prolonged specific antibiotic combination therapy lasting up to three months for successful treatment. This assumes further importance as no effective vaccination is available for prevention. It has zoonotic potential and may be responsible for infection in immunocompromised humans. This study is aimed at analyzing the distribution pattern of the resistotypes (R-types) of various isolates of R. equi in different areas of Haryana and Rajasthan, India. Antimicrobial susceptibility pattern of R. equi isolates was determined by Kirby Bauer disc diffusion method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. A total of twenty eight clinical isolates of R. equi from foals from different parts of Haryana and Rajasthan were used in this study. Antibiogram of R. equi isolates with 33 antibiotics revealed ten distinct R-types: R-type 1 to R-type 10, on the basis of variable results of four antibiotics i.e. amoxycillin, gentamycin, colistin and streptomycin. Out of ten resistotypes obtained the relative frequencies of R-1 resistotype and R-4 resistotype were found to be high i.e. 28.57% and 25%, respectively. Differentiation of R. equi strains into R-types is an important tool for therapeutics. In addition to direct foal management, it may have implication in identifying the source and spread of infection, and as epidemiological marker to correlate various isolates from various places, ultimately helping in therapeutics for timely control. * Corresponding author E-mail: [email protected] (Khurana S K) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 243 Chhabra et al 1 Introduction Rhodococcus equi is aerobic, non-sporulating, non-motile, gram-positive bacteria, found in soil, herbivore dung and the intestinal tract of cattle, horse, sheep, pig and some other animals. It is primarily a respiratory pathogen mainly affecting the foals between 1-4 months of age (Khurana et al., 2015). The disease is direct anthropozoonosis since the animals are primary reservoirs of the etiological agent (Khurana, 2014). It can be responsible for infection in humans compromised by immunosuppressive drug therapy, lymphoma or AIDS (Takai et al., 1995; Mizuno et al., 2005; Napoleao et al., 2005). R. equi has been ranked one of the most important disease problems in the horse industry especially because of its high prevalence and mortality rate (Blood-Horse, 2009; Muscatello, 2009; Elissalde et al., 1980; Mir et al., 2015). The symptoms in foals include pyrexia, respiratory distress, pulmonary lesions and chronic lung abscesses, which when left untreated lead to death due to asphyxiation (Wichtel et al., 1991; Lavoie et al., 1994). The infection can spread from the lungs to other organs and joints when granulomatous foci in the lung open up (Prescott, 1991; Giguere, et al., 2011). It has been reported that only a small proportion of all R. equi in soil are able to cause the infection and only R. equi carrying virulence plasmids can cause disease in foals (Muscatello et al., 2006). Foal survival requires successful antimicrobial therapy (Sweeny et al., 1987). Most of the conventional antibiotics are not effective against R. equi because it is an intracellular pathogen. R. equi is usually resistant to beta-lactam antibiotics such as penicillin G, oxacillin, ampicillin, carbenicillin and cefazolin (Kedlaya et al., 2001). Current treatment comprises the use of a combination of erythromycin and rifampin (Jacks, 2003). This combination facilitates the drugs to penetrate the lung abscesses, macrophages or neutrophils where the bacteria survive and multiply (Prescott, 1991). During the last decade the minimum inhibitory concentrations (MIC) of erythromycin and rifampin for R. equi are rising and there are reports of resistance to these antibiotics (von Bargen & Haas, 2009). Therapy with orally administered macrolides has greatly improved survival rates for foals with R. equi pneumonia (Sweeney et al., 1987). R. equi isolates resistant to the commonly used macrolides (azithromycin, clarithrimycin and erythromycin) as well as rifampin are emerging these days. The overall prevalence of R. equi isolates resistant to macrolides or rifampin has been reported as 4% (Giguere et al., 2010). Keeping in view, the emerging variable resistance to various important antibiotics and difficulty in identification of R. equi isolates due to inconsistent biochemical tests, the present study was undertaken to study the resistance pattern of various isolates of R. equi and to find out various resistotypes. 2 Materials and Methods A total of twenty eight R. equi isolates from foals with respiratory problems from various parts of Haryana and Rajasthan were used. Nasal swabs from each case were collected in Cary-Blair transport medium and processed in the laboratory for isolation. All the isolates of R. equi were maintained on nutrient agar slants. All the twenty eight isolates of R. equi were identified by cultural characteristics, gram staining and biochemical tests. After 48 hrs incubation of nutrient agar plates characteristic 1-2 mm irregularly round smooth, mucoid, glistening, semi transparent, salmon pink to yellow coloured and coalescing colonies on nutrient agar were observed. The antibiotic sensitivity test was conducted by Kirby Bauer disc diffusion method following the Clinical and Laboratory Standards Institute (CLSI) guidelines (Bauer et al., 1966). Table 1 Antibiotics used and their concentration. S. No Antibiotics 1 Azithromycin 2 Ciprofloxacin 3 Chloramphenicol 4 Ceftriaxone 5 Cefaparazone 6 Enrofloxacin 7 Erythromycin 8 Methicillin 9 Norfloxacin 10 Neomycin 11 Ofloxacin 12 Oxytetracycline 13 Rifampicin 14 Roxithromycin 15 Tobramycin 16 Vancomycin 17 Amikacin mcg = Micrograms Concentration 30 mcg 10 mcg 25 mcg 30 mcg 75 mcg 10 mcg 10 mcg 10 mcg 10 mcg 03 mcg 02 mcg 30 mcg 30 mcg 30 mcg 30 mcg 05 mcg 30 mcg _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org S. No 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Antibiotics Ampicillin Cloxacillin Kanamycin Nalidixic acid Oxacillin Penicillin G Sulphadiazine Trimethoprim Amoxycillin Colistin Gentamycin Streptomycin Tetracycline Lincomycin Pefloxacin Clindamycin Concentration 25 mcg 30 mcg 05 mcg 30 mcg 05 mcg 10 mcg 100 mcg 25 mcg 10 mcg 25 mcg 30 mcg 25 mcg 10 mcg 15 mcg 05 mcg 25 mcg Resistotypes of Rhodococcus equi isolated from foals with respiratory problems A few colonies of the R. equi were picked with a wire loop from the nutrient agar slant and inoculated into a test tube containing brain heart infusion broth (BHI). These tubes were incubated at 370C for 16-18 hours to produce a bacterial suspension of moderate cloudiness. The bacterial broth suspension was spread evenly onto the surface of Muellar Hinton Agar (MHA) plates covering whole of the plate with a sterile cotton swab. After the inoculums had dried, the disks were placed on the agar. The discs of thirty three antibiotics were used (Table 1). Plates were then incubated immediately at 370C for 24 hrs. After incubation, the diameter of zone of inhibition was measured with antibiotic zone reader and interpreted as per manufacturer interpretation values. The results of antibiotic sensitivity were analyzed to find out antibiotics which are sensitive or resistant to all isolates. Antibiotics which were showing variable results i.e. some of isolates were sensitive and some were resistant to antibiotics were identified. On the basis of antibiotic sensitivity/ resistance twenty eight R. equi isolates were classified into various resistotypes. 3 Results All the isolates were sensitive to 20 antibiotics (zone range with mean in mm): azithromycin (30-34, 32), ciprofloxacin(31-38, 34.5), chloramphenicol (20-30, 25), ceftriaxone (28-34, 31), clindamycin (20-28, 24), cefaparazone 244 (22-30, 26), enrofloxacin (28-34, 31), erythromycin (32-38, 35), lincomycin (30-34, 32), methicilin (20-28, 24), norfloxacin (21-30, 25.5), neomycin (20-32, 26), ofloxacin (24-32, 28), oxytetracycline (10-19,14.5), pefloxacin (25-31, 28), rifampicin (31-36, 33.5), roxithromycin(31-37, 34), tetracycline (25-33, 29), tobramycin (20-34, 27), vancomycin (22-28, 24). All the isolates were resistant showing no inhibition zone or zone of inhibition within resistant range to 9 antibiotics viz. amikacin, ampicillin, cloxacillin, kanamycin, nalidixic acid, oxacillin, penicillin-G, sulphadiazine, trimethoprim. However, 4 antibiotics viz., amoxycillin, colistin, gentamycin, streptomycin showed variable results (Table 2) which formed the basis to formulate resistotypes of R. equi. Ten resistotypes could be detected amongst 28 isolates of R. equi (Table 3). The resistotype R-1 represented eight isolates (NS-1, NS-44, NS-98, NS-117, NS-120, NS-151, NS244, NS-290) which showed 28.50 % relative frequency and found to be resistant to amoxycillin, gentamycin, streptomycin and sensitive to colistin. Resistotype R-2 represented only one isolate (NS-6) which showed 03.57% relative frequency and found to be sensitive with all the four antibiotics, Resistotype R-3 represented two isolates (NS-25, NS-276) which showed 07.14 % relative frequency and found to be resistant to all four anitibiotics, Resistotype R-4 represented seven isolates (NS36, NS-79, NS-113, NS-150, NS-231, FNS-4, FNS-5) which showed 25 % relative frequency and found to be resistant to gentamycin, streptomycin and sensitive to amoxycillin, colistin. Table 2 Antibiotic sensitivity of twenty eight isolates of Rhodococcus equi. Antibiotics to which all the isolates were Sensitive Resistant Azithromycin Amikacin Ciprofloxacin Ampicillin Chloramphenicol Cloxacillin Ceftriaxone Kanamycin Clindamycin Nalidixic acid Cefaparazone Oxacillin Enrofloxacin Penicillin-G Erythromycin Sulphadiazine Lincomycin Trimethoprim Methicilin Norfloxacin Neomycin Ofloxacin Oxytetracycline Pefloxacin Rifampicin Roxithromycin Tobramycin Tetracycline Vancomycin *Number in parentheses represent sensitive (S) and resistant (R) isolates _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Antibiotics to which isolates showed variable resistance pattern Amoxycillin (12S, 16 R) Colistin (21S, 7R) Gentamycin (4S, 24R) Streptomycin (5S, 23 R) 245 Chhabra et al Table 3 Resisotypes recognized amongst twenty eight Rhodococcus equi isolates. Resitotype R-1 Sensitive or Resistant to antibiotic A C G Str R S R R Sample No. (No. of Isolates) NS-1, NS-44, NS-98, NS-117, NS-120, NS-151, NS-244 NS-290 (8) R-2 S S S S NS-6 (1) R-3 R R R R NS-25, NS-276 (2) R-4 S S R R NS-36, NS-79, NS-113, NS-150, NS-231, FNS4, FNS-5 (7) R-5 R S S R NS-48, NS-216 (2) R-6 S S R S NS-62 (1) R-7 R R R S NS-77 (1) R-8 S R R R NS-121, NS-161, NS-288 (3) R-9 R R S R NS-170 (1) R-10 R S R S NS-202, NS-188 (2) A = Amoxycillin, C = Colistin, Str = Streptomycin, G = Gentamycin, R = Resistant, S = Sensitive Resistotype R-5 represented two isolates (NS-48, NS-216) which showed 03.57% relative frequency and found to be resistant to amoxycillin, streptomycin and sensitive to gentamycin, colistin., Resistotype R-6 represented one isolate NS-62) which showed 03.57% relative frequency and found to be resistant to gentamycin and sensitive to amoxycillin, colistin, streptomycin. Resistotype R-7 represented one isolate (NS-77) which showed 03.57% relative frequency and found to be resistant to amoxycillin, gentamycin, colistin and sensitive to streptomycin. Resistotype R-8 represented three isolates (NS-121, NS-161, NS-288) which showed 10.71 % relative frequency and found to be resistant to gentamycin, streptomycin, colistin and sensitive to amoxycillin. Resistotype R-9 represented one isolate (NS-170) which showed 03.57% relative frequency and Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 28.57% 03.57% 07.14% 25.00% 07.14% 03.57% 03.57% 10.71% 03.57% 07.14% found to be resistant to amoxycillin, colistin, streptomycin and sensitive to gentamycin , Resistotype R-10 represented two isolates (NS-202, NS-188) which showed 07.14% relative frequency and found to be resistant to amoxycillin, gentamycin and colistin, streptomycin ( Figure 1). Out of ten resistotypes obtained the relative frequencies of R-1 resistotype and R-4 resistotype were higher i.e. 28.57% and 25%, respectively. Resistotype R-1 included eight isolates of R. equi which were isolated from nasal swabs of foals belonging to various places i.e. Tohana (NS-1), Sorkhi (NS-44), Gangua (NS-98), Hanumangarh (Rajasthan) (NS-177), Ismaila (NS120), Muklan (NS-151), Barwala (NS-244) and Kaimri (NS290). Whereas resistotypes R-4 included isolates of R. equi from various places viz. Sorkhi (NS-36), Sachakhera (NS-79), Muklan (NS-113 and 150), Jakhal (NS-231) and Tohana (FNS4 and FNS 5). Four isolates of R. equi of Tohana were characterized into three resistotypes viz. R-1 (NS-1), R-2 (NS6) and R-4 (FNS-4 and FNS-5). Figure 1 Proportion of resistotypes of R. equi isolates. _________________________________________________________ Relative frequency Resistotypes of Rhodococcus equi isolated from foals with respiratory problems Discussion and conclusions For selection of appropriate agents to treat R. equi infections, both the in vivo and in vitro properties of the antimicrobial agents should be considered. Previous reports have shown that R. equi is susceptible to ampicillin/sulbactam, amoxycillin/clavulanic acid, gentamycin, erythromycin, tetracycline, rifampin, TMP-SMZ, imipenem, and vancomycin . The organism is usually less susceptible to penicillin, ampicillin, cephalosporins, or quinolones (Prescott, 1991; McNeil & Brown 1992; Nordmann & Ronco 1992; Giguere et al., 2011). All the twenty eight isolates of R. equi of this study were grouped into ten resistotypes on the basis of four antibiotics which showed variable results described vide supra. A total of sixteen resistotypes (2 n = 2 4 = 2x2x2x2 = 16) could be possible on the basis of four variable antibiotics if larger number of isolates could be studied for resistotyping. These results could not be compared to previous reports because of non availability of such type of study in earlier literature which shows that the present study is probably the first study of resistotypes of R. equi. However, in recent review Khurana (2015) reported emergence of antibiotic resistance to various antibiotics. Resistance to rifampicin in R. equi attributatable to monooxygenase like sequence has been reported (Anderson et al., 1997). Mutations in rpoB gene leading to rifampicin resistance have been reported (Asoh et al., 2013; Liu et al., 2014). These resistotypes could be used as epidemiological marker during outbreak studies due to R. equi in equines. In this study Resistotype R-1 represented eight isolates (NS-1, NS-44, NS98, NS-117, NS-120, NS-151, NS-244, NS-290) which showed highest relative frequency (28.50 %) and found to be resistant to amoxycillin, gentamycin, streptomycin and sensitive to colistin. These isolates were from various places i.e. Tohana (NS-1), Sorkhi (NS-44), Gangwa (NS-98), Hanumangarh, Rajasthan (NS-177), Ismaila (NS-120), Muklan (NS-151), Barwala (NS-244) and Kaimri (NS-290). All these isolates were from the areas near Hisar city within a radius of 70 km except isolate NS-177 from Hanumangarh, Rajasthan. Resistotype R-4 had second highest relative frequency (25%) represented by seven isolates (NS-36, NS-79, NS-113, NS-150, NS-231, FNS-4, FNS-5) resistant to gentamycin, streptomycin and sensitive to amoxycillin, colistin from various places viz. Sorkhi (NS-36), Sacchakhera (NS-79), Muklan (NS-113 and 150), Jakhal (NS-231) and Tohana (FNS4 and FNS 5). Four isolates of R. equi of Tohana were characterized into three resistotypes viz. R-1 (NS-1), R-2 (NS6) and R-4 (FNS-4 and FNS-5). This finding is very interesting and showing the use of resistotypes as epidemiological marker and could be useful for therapy in a particular endemic area/ farm after identifying the resistotype. In the present study all isolates of R. equi were found sensitive to rifampicin and macrolides antibiotics viz. azithromycin, _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 246 erythromycin and roxithromycin. Macrolide resistance in R. equi has also been reported (Burton et al., 2013; Liu et al., 2014). A glycopeptides resistance operon vanO having potential implications in R. equi therapy has been described (Gudeta et al., 2014). Cohen (2014) has warned about the challenges of emergence of resistance to macrolide due to nonavailability of effective alternative for R. equi therapeutics. Rifampicin along with macrolide is drug of choice for effective treatment of R. equi infections. However the emergence of resistance against these antibiotics poses a serious challenge in therapeutic management. But in this study such problem has not been observed, therefore the cases of foals respiratory diseases can be treated successfully in this area with standard antibiotic combination therapy used to treat R.equi infections. It is therefore, recommended that veterinarians must use antibiotics for treatment judiciously. Acknowledgements The authors are thankful to National Research Centre on Equines, Hisar and Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar for providing necessary facilities to carry out the research work. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References Anderson SJ, Quan S, Gowan B, Dabbs ER (1997) Monooxygenase like sequence of Rhodococcus equi gene conferring increased resistance to rifampin by inactivating this antibiotic. Antimicrobial Agents and Chemotherapy 41 : 218221. Asoh N, Watanabe H, Fines-Guyon M, Watanabe K., Oishi K, Kositsakulchai W, Sanchai T, Kunsuikmengrai K, Kahintapong S, Khanawa B, Tharavichitkul P, Sirisanthana T, Nagatake T (2013) Emergence of rifampin-resistant Rhodococcus equi with several types of mutations in rpo B gene among AIDS patients in northern Thailand. Journal of Clinical Microbiology 41: 2337-2340. doi: 10.1128/JCM.41.6.2337-2340.2003. Bauer AW, Kirby WMM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathoogy 45:493-6. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 EFFECT OF Morinda citrifolia IN GROWTH, PRODUCTION AND IMMUNOMODULATORY PROPERTIES IN LIVESTOCK AND POULTRY: A REVIEW Jai Sunder*, Tamilvannan Sujatha and Anandamoy Kundu Division of Animal Science, ICAR-Central Island Agricultural Research Institute, Port Blair, A and N Islands 744105 Received – April 18, 2016; Revision – May 02, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).249.265 KEYWORDS ABSTRACT Morinda citrifolia Livestock Poultry Growth Production Immunomodulatory Properties Morinda citrifolia L. is commonly known as Noni and has been found to have wide range of medicinal properties. It is usually found in the coastal region in many countries including Andaman and Nicobar Islands and belongs to the family Rubiaceae. This small evergreen tree is widely grown and well adapted to the tropics and can grow in fertile, acidic, alkaline and saline affected soils. It tolerates high soil salinity and brackish water stagnation. All the components of this plant have high demand in case of alternative medicines and herbal medicines. Due to its high demand and as a source of revenue generation the detail study on its nutritional benefits and therapeutic values are essential for its commercial exploitation. More than 200 nutraceutical compounds have been identified from the plant. Morinda citrifolia is reported to have broad spectrum biological activities such as antimicrobial, immunomodualtory, antioxidant wound healing etc. Apart from the in-vitro scientific validation of the activities and in-vivo trial in some lab animal model, the plant has been used for livestock and poultry health and production. A lot of reviews have been written on the different uses of Noni, however, scientific review on the use of this plant on the growth, production, immunomodulator and other pharmacological activities of M. citrifolia in livestock and poultry has not been compiled. Therefore this review discusses the compilation of the work done on the use of M. citrifolia in livestock and poultry. * Corresponding author E-mail: [email protected] (Jai Sunder) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 250 1 Introduction Livestock farming is considered to be a profitable complementary enterprise in agriculture and constitutes an important activity for accelerating the rural economy. They are being reared under small open ranging system to large scale intensive system in our country. This sector has been growing at steady pace; however, during the recent years due to indiscriminate usages of antibiotics in livestock and poultry the problem of multidrug resistance has evolved (Woolhouse et al., 2015). The high dosage of antibiotics, hormones and chemical derivatives for increase and sustain livestock production has increased the problem of its residual effects in their products thus causing a serious health concern (Landers et al., 2012). The organic farming has changed the scenario of the present agricultural production system; similarly the concept of organic livestock production system has been evolved. The use of traditional medicine and alternative medicine has been practiced by the farmers for sustainable production (Galav et al., 2013; Luseba & Tshisikhawe, 2013). A large number of medicinal plants with known medicinal properties are available and is being used by the farmers (Chinsembu et al., 2014; Verma, 2014; Luseba & Tshisikhawe, 2015). Morinda citrifolia is also known as Noni, belongs to the family Rubiaceae and is mostly available in the coastal region (Nelson, 2006). It is commonly known as Indian mulberry, Ba Ji Tian, Nono or Nonu, Cheese fruit and Nhau in various countries throughout the world (Bruggnecate, 1992; Whistler, 1992; Solomon, 1999; Chan-Blanco et al., 2006). In these islands it is commonly known as Lorang, Burmaphal, Pongee phal and Surangi by the tribal. M. citrifolia has a rich history in India, where it has been used for tens of centuries in the system of medicine known as Ayurveda. This small evergreen tree or sprint (10-20 ft) is native to India and also distributed to south-eastern Asia to Australia and now has tropical distribution widely adapted to the tropics (Dixon et al., 1999; Ross, 2001). It can grow in challenged environments viz, saline, acidic and alkaline soils. M. citrifoia has been known for its wide range of medicinal properties (Younos et al., 1990; Bruggnecate, 1992; Hiramatsu et al., 1993; Hirazumi et al., 1996; Solomon, 1999; Brown, 2012; Assi et al., 2015). Reports are available on the scientific studies of this plant and wide health benefits. The plant is also suitable for saline affected lands, owing to its saline resistant properties the plant can be gown as an alternative crop for the saline affected areas and its high demand both at national and international market, the studies on its phytochemicals and its effect on production and immune response is utmost importance. Polynesians used the whole plant for treatment of various kinds of illness as herbal remedies (Earle, 2001). Various reports are available for use of this plant for treatment of illness such as diabetes, blood pressure, cancers, arthritis, poor digestion etc. (Singh et al., 1984; Bruggnecate, 1992; Solomon, 1999; Whistler, 1992; Wang et al., 2002; Brown, 2012; Lee et al., 2012; Fletcher et al., 2013; Saminathan et al., 2013a; _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Sunder et al Saminathan et al., 2014b; Sharma et al., 2016). However, there is less information on the scientific validation of the Morinda fruits viz. treatment of colds and influenza reported by Solomon, 1999. Allen (1873) reported some information on the ethno botanical properties, which is considered to be one of the earliest report on the medicinal use of morinda. The history of use of M. citrifolia for livestock and poultry is very limited. Due to its strong smell and taste many animals do not consume the product and avoid contact with the fruit and seeds. The residents of South Pacific islands have noted health benefits for themselves and their animals by ingesting the M. citrifolia fruit (Whistler, 1985). Some animals such as pigs consume the fruit in its natural state (Fugh-Berman, 2003). Most animals have difficulty in consuming and digesting whole fruit. The available literatures revealed that the leaves are used for livestock fodder (eg. Niue, India) and to feed silk worms (India). Over the year’s research have been mainly carried out on use of fruit and leaf extract. Studies have been carried out on anthelmintic property (Raj, 1975; Morton,1992), anti-inflammatory (Basar et al., 2010; Fletcher et al., 2013), antitumor potential (Hirazumi & Furusawa, 1999; Furusawa, 2002; Brown, 2012; Jinhua et al., 2013; Saminathan et al., 2014a), utilization of its juice extract waste in diets of goats (Aregheore, 2005), enhanced mononuclear phagocytic activity of gram-negative bacteria in calves fed with the fruit puree (Schafer et al., 2008). Researchers have been carried out on the volatile components of ripe fruits and their effects on drosophila (Farine et al., 1996). Studies on the effect of its fruit and leaf extract in the total serum protein, creatinine and albumen level of poultry have been carried out (Sunder et al., 2011a). However, the scientific evidence of its use in animal model and its effect on production system is very rare (FughBerman, 2003; Sunder et al., 2007; Sunder et al., 2011b; Sunder et al., 2011b c). In the present review, information on the available literature on the use of M. citrifolia in livestock and poultry has been documented. 2 Growth and production promoting properties in poultry The growth performance ability of the M. citrifolia fruit was tested in Nicobari fowl; an indigenous poultry bird of Andaman & Nicobar Islands, India and broiler (Sunder et al., 2011b). Broilers were given fresh noni fruit juice (1.5 ml/bird/day). The performance of the morinda fed group was found more than the control birds in terms of body weight gain, feed conversion ratio and feed efficiency. The overall performance index of Morinda fed group was found to be superior (93.6±16.15) than other groups. Egg production was also recorded to be high in the morinda fed group (95.24%) than in control (83.11 %). Similarly, highest dressing % was obtained in the Morinda fed group (69.05%) than in control (68.38%). The highest body weight gain during the 3rd and 4th month of age was observed due to the growing phase of the bird during which the bird attained more body weight gain than compared to the other phase of the growth. The reports of Morinda on the growth was reported by Sunder et al. (2007) in Effect of Morinda citrifolia in growth, production and immunomodulatory properties in livestock and poultry: a review broiler birds, however, no reports are available on the effect on the egg production in the Nicoabri fowl. Studies on feeding of M. citrifolia fruit extract to Japanese quail showed better body weight gain, feed conversion ratio and performance index in Morinda fed group than in control group. The overall results revealed that higher body weight gain in Morinda fed group (162.11±0.06 g) compared to control group (153.005±1.05 g) for 0 to 7 weeks of age. FCR of the Morinda fed group (5.99 ± 0.17) was recorded better than control group (6.18 ± 0.16). The feed efficiency of Morinda fed group (0.22 ± 0.05) was also found to be higher (p<0.05) than control group (0.20±0.08). The hen day egg production was recorded better in the Morinda fed group when compared to the control group. The overall analysis of the growth and production performance of both the groups revealed that the M. citrifolia crude fruit extract fed @ 5% daily enhanced the body weight gain and the egg production performance of the Japanese quail (Sunder et al., 2013d). 3 Growth, production and imunomodulatory properties in livestock Ethno veterinary application of noni fruit puree has been studied in the livestock. Brooks et al. (2009) demonstrated that feeding of noni puree enhanced the immunity of neonatal calves and potentially long term health especially in the preweaning stage. In earlier findings, Brooks et al. (2009) reported that supplementation of juice from M. citrifolia enhanced the activation of CD4+ and CD8+ T cells in neonatal calves. Bactericidal effect of noni was evaluated via an ex vivo whole blood bactericidal assay by Schäfer et al. (2008). Result showed that noni fed group showed significantly more killing power at day 14 when compared to control calves. Advantages of supplementing Morinda Max have been demonstrated in newly received cattle. Yancey et al. (2013) studied the growth performance effect of feeding of noni in cattle. They have reported that feeding of pulp of M. citrifolia to cow at the level of up to 2% in the diet increased body weight gain with better feed conversion ratio. The gain was increased with the increase in concentration of Noni pulp in the diet. Ponce et al. (2011) found that feeding of Noni extract to calf increased the growth performance as well as the immunomodulatory properties. The immune enhancing effect including the antibacterial, antiinflammatory, anti cancer and anti oxidant effect of noni has been validated (Pawlus et al., 2005; Kusirisin et al., 2009; Nitteranon et al., 2011). Presence of Iridoid and polysaccharide fractions of noni has been shown to induce the release of several immune mediators, many of which have beneficial stimulatory effects and may help in the maturation of the neonatal immune system (Bui et al., 2006; Hirazumi & Furusawa, 1999; Deng et al., 2010). The growth promotion effect of M. citrifolia juice may be due to its rich nutrients value which contain all the essential amino acids, minerals, vitamins and other nutrients which are required for the growing cells (Chunhieng, 2003) It is very rich in proxeronine which is believed to be a precursor to _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 251 xeronine which helps in activation of xeroninase ( Heinicke, 2001). As the Morinda fruit contain several amino acid, vitamins, minerals, co-enzymes, carbohydrates and alkaloids which directly or indirectly help in metabolism of the nutrients and help in overall growth of the cell and tissues (Chan Blanco et al., 2006; Takashima et al., 2007; Assi et al., 2015). More than 200 nutraceutical compounds have been identified from the plant (Solomon, 1999; Singh, 2012). Leaf and fruit of this plant are reported to be used as feed for livestock and poultry (Fugh-Berman, 2003). However, further investigation on several bioactive compounds present in the M. ciirifolia will help in understanding the actual mechanism in detail and its use in livestock and poultry as a source of vitamins and minerals supplement for higher growth, production and immunity. 3.1 Anticholesterol properties Cholesterol is very important compounds used for many physiological functions; however, the unwanted increase in the level of bad cholesterol is the predisposing factors for many of the diseases. Lot of works have been carried out to lower the level of cholesterol in chicken meat by using addtivies, feed supplents, etc. (Chowdhury et al., 2002; Yalcin et al., 2006; Aydin et al., 2008; Azeke & Ekpo, 2009; Saki et al., 2014). Considering the importance of the cholesterol content the study was conducted to see the effect of M. citrifolia fruit and leaf extract supplementation on the blood cholesterol level. A group of 100 poultry birds were fed 5% crude fruit extract and leaf extract of M. citrifolia daily along with the normal basal ration. Studies on borilers and Nicobari fowl revealed that feeding of fruit extract (5%) and leaf extract (5%) daily along with the normal basal ration lowered the blood cholesterol level. The cholesterol level in the Morinda fruit extract fed group at 4th week (179+12.3) and at 6th week (201+9.4) was found to be lower than control group (185+11.4) and (233+10.5), respectively. The cholesterol level in the birds fed with leaves extract was found to be slightly higher than fruit extract. The cholesterol level in the Morinda leaf extract fed group at 4th week were (190±11.1) and at 6th week were (210±5.4) was again found to be lower than the control group (222±12.3) and (248±8.1) respectively. The result revealed that the feeding of M. citrifoiia extract daily in the poultry ration has lowered the level of cholesterol in the blood serum (Sunder et al., 2011a). Reports suggest that the Noni has phytochemicals and beta-sitosterol, a plant sterol with potential for anti-cholesterol activity (Palu et al., 2006; Wang et al., 2006). Research on lowering the cholesterol level in chicken meat and egg is going on worldwide. Research showed that cholesterol content may vary in eggs and blood and lot of work is being carried out on chicken eggs and meat either through the use of additives, dietary fibre, polyunsaturated fatty acids supplementation etc (Barroeta, 2007; Dalkilic et al., 2009). Recent study showed that feeding of morinda fruit extracts to calves reduced the level of total cholesterol, triglycerides, LDL-cholesterol in the morinda fed group than in the control group (Anantharaj et al., 2016). 252 3.2 Effect on blood biochemcial profile of poultry Concentration of serum protein is useful in monitoring various disease status. It increases during dehydration, multiple myeloma and chronic liver diseases, and decreases in renal diseases and liver failure. Creatinine is a waste product formed in the muscle from the high energy storage compound creatinine phosphate. It is useful indicator of renal function and increases in various renal diseases. Albumin is a plasma protein synthesized in liver from amino acids absorbed from ileum. It increases during dehydration and stasis during venipuncture. It decreases during excess protein loss and decreases synthesis due to dietary, hepatic disease or malabsorbtion (Babatunde et al., 1992). Lot of reports are available on the effect of feeding of herbal plants and its extracts on the blood biochemical profile of poultry, (Langhout, 2000; Kamel, 2001; Elagib et al., 2012; Hosseinzadeh et al., 2014; Kant et al., 2014). However, very few researches have been carried out to see the effect of feeding of noni in the blood biochemical profile of poultry. Sunder et al., 2011a found that feeding of noni decreases the level of total protein, serum creatinine and albumin in the poultry. There is no report available on the effect of M. citrifolia in blood biochemical parameters of poultry, however, similar to this study, Polat et al., 2011 found that feeding of rosemary reduces the blood cholesterol level and increases the creatinine level. Creatinine is a chemical waste molecule that is generated from muscle metabolism. The lower values indicates that no muscular wastage which might have been possibly cause by inadequacy of protein in animals. In the present study also the level of blood protein was found to be low. However, Ghazalah & Ali (2008) found that creatinine levels were all reduced by dietary rosemary leaves compared to controls. It is useful indicator of renal function and increase in various renal diseases. Albumin is synthesized in the liver from amino acids absorbed from the ileum. It increases during dehydration and decreases during excess protein loss. The level of total serum protein, albumin and creatinine was found to be better in the Morinda fed group. The effect of M. citrifolia on various biochemical parameters may be useful in monitoring physiological status and disease status as well as therapeutic evaluation of the products. Research showed that the feeding of M. citrifolia juice enhanced the immune response in poultry (Sunder et al., 2007). 3.3 Antimastitis properties in cattle Mastitis is a serious problem of the dairy cows and estimated loss due to direct and indirect looses has been estimated to the tune of $35 billion annually (Mubarack et al., 2011). Infection of the cow’s udder and the mastitis is considered as one of the major constraints for growth of dairy industry worldwide (Sasidhar et al., 2002; Osteras & Solverod, 2009). Due to development of multidrug resistance bacteria the use of herbal based preparation for treatment of mastitis and other diseases have been reported in livestock (Virmani et al., 2010; Kalayou et al., 2012; Mir et al., 2014; Zeedan et al., 2014). Several plants have been reported to be used for treatment of mastitis _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Sunder et al viz. Capsicum annuum, Lepidium sativum, Allium sativum, Sesamum indicum, Citrus limon, Zingiber officinale, Citrullus colocynthis, Curcuma longa, Amomum subulatum, Sesamum indicum, Cuminum cyminum, Rosa indica, Centratherum anthelmisticum, Triticum aestivum, Nigella sativa and Peganum harmala (Dilshad et al., 2009). In many of the reports, the antibacterial activity of the plant extracts were described against the organisms responsible for mastitis, however, Sunder et al. (2013a) reported the treatment of mastitis by feeding of M. citrifolia in dairy cows. Sunder et al. (2013a) observed that oral feeding of noni fruit juice (100 ml) to dairy cows is effective for treatment of mastitis. In the mastitis milk, level of electrical conductivity increases due to changes in ionic concentration which resulted in the decrease in the level of lactose and K+ and increase in the level of Na+ and Cl (Kitchen, 1981; Kaşikçi et al., 2012). Sunder et al. (2013a) found that feeding of M. citrifolia decreased the electrical conductivity from 5.5 mho to less than 5.0 mho, which resulted in decrease in the milk pH (7.0 to 6.6) thus reducing the total microbial load in the milk. Level of microbial load was also decreased from 5.13 x 108 to 3.54 x108. Among the normal cow no change was observed in the pH of cow’s milk with respect to different teat of the cow (Batavani et al., 2007). In case of mastitis Electrical conductivity will vary in the range of 5.58 mS. It is concluded that the feeding of M. citrifolia reduced the milk pH, electrical conducti8vity, microbial load in the mastitis affected milk. 4 M. citrifolia as feed supplement Plants are being used as a source of feed and feed additives in animal feeding since time immemorial. During the recent years due to ban of most of the antibiotic growth promoters use of medicinal plants as a feed additives, supplements for the livestock and poultry have been increased (Charis, 2000; Tipu et al., 2006 ; Mirzaei-Aghsaghali, 2012; Eevuri & Putturu, 2013; Mirzaei & Venkatesh, 2012). Use of M. citrifolia fruit as a feed ingredient was studied in Japanese quail (Sunder et al., 2013b,d). M. citrifolia fruits were cut into small pieces and sun dried. The dried fruits were grounded to make it in the form of small granules. Fruit granules were added in the quail ration up to 15% by replacing maize, rice bran and wheat by 5% each. The body weight at 2nd, 3rd and 5th week of age was higher significantly (p<0.05) in the Morinda fed group compared to the control group. Feed conversion ratio and egg production was found to be better in morinda fed group. The overall production performance revealed that at the end of 100 days of egg production the Morinda fed group showed 24% more egg production than the control group. The study showed that M. citrifolia can be used as feed ingredient in Japanese quail ration to the tune of 15% on dry matter basis. In an another study, Japanese quail were fed with fruit granules of M. citrifolia, 20% (w/w) as replacement of the normal concentrate mixture in the ration (Sunder et al., 2013 b). Results revealed the higher body weight gain in morinda group (109.4±7.22) than in control group (106.8±6.65) at 5th week of age. Egg production was also found better in morinda group (59.34±12.31) than in control group (56.80±10.71). It is Effect of Morinda citrifolia in growth, production and immunomodulatory properties in livestock and poultry: a review concluded that part-replacement of quail ration with dried ripe fruit granules of M. citrifolia (20%, w/w) would be costefficient in quails with no apparent side effects. This is the first report of use of M. citrifolia fruit granules as feed supplement in the poultry. There is no report available on the use of M. citrifolia fruit pulp as a feed for poultry. However, some reports are available on the use of leaf and fruit of this plant as feed for livestock and poultry (Fugh-Berman, 2003). Use of other medicinal plants viz. Curcuma longa, turmeric, Ocimum sanctum, tulsi, Aloe vera, were also reported to enahcned the body weight gain, better FCR, feed efficiency in poultry (Kumar et al., 2005; Durrani et al., 2006; Lanjewar, 2008; Al-Kassie et al., 2011; Eevuri & Putturu, 2013). The proximate analysis of the noni fruit pulp revealed that the crude protein content is only 5.8%, however, it is very rich in all amino acids, micro and macro minerals and vitamins which are essential for the vital functioning of the cells/tissues for growth and production. The M. citrifolia fruit is very rich in the nutraceutical compounds and contains rich amounts of minerals like K, Ca, Mg, Fe, Cu and Mn (Singh et al., 2008). High egg production and growth promoting effect may be due to the nutraceutical compounds and minerals present in the fruit which might have played important role in enhancing the growth and production. The earlier reports with the use of M. citrifolia fruit juice revealed that the supplementation of M. citrifolia juice @ 5% enhanced body weight gain in broiler and Japanese quail (Sunder et al., 2007, Sunder et al., 2011a). Morinda fruit contain several amino acids, vitamins, minerals, co-enzymes, carbohydrates and alkaloids which directly or indirectly help in metabolism of the nutrients and help in overall growth of the cell and issues hence in the Morinda fed group the overall performance was better than in control group. The studies on evaluation and utilization of Noni juice extract waste (NJEW) in goat’s diet revealed that the unusual taste and low degradability of essential nutrients may be the factors limiting the use of NJEW in ruminant diets. Therefore, research on its palatability quality is required so that the extracts and the fibres may be effectively utilized as a source of livestock food (Aregheore, 2005) 5 M. citrifolia as Immunomodulator Immune system of poultry generally benefits from the medicinal plants and herbs which are rich in flavonoids, vitamin C and carotenoids. During the last decade there has been substantial increase in the use of medicinal herbs as feed supplement, immunostimulant and growth promoters. Many plants are available which are very rich in these compounds including the M. citrifolia. There are several reports available with the use of medicinal herbs for immunostimulants in the poultry (Kumari et al., 1994; Emadi & Kermanshahi, 2007; Durrani et al., 2008; Lee et al., 2010; Mahima et al., 2012; Mirzaei-Aghsaghali , 2012; Dhama et al., 2015). Phytochemical analysis of the M. citrifolia revealed the presence of several compounds viz. carbohydrates, gums and mucilages, proteins, amino acids, fats and oils, anthraquinone glycosides, coumarin glycosides, flavonoids, alkaloids, tannins, phenolic compounds and citric acid which are _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 253 responsible for several activities including the immunostimulation (Nayak et al., 2012). Studies showed that M. citrifolia stimulated the immune system for anti tumor, anticancer, antioxidant activities both in-vivo and in-vitro (Hirazumi & Furusawa, 1999; Furusawa & Hirazumi, 2003; Palu et al., 2006; Nayak & Mengi, 2010; Mufidah et al., 2013; Assi et al., 2015; Krishnaiah et al., 2015). However, very few reports are available on the use of M. citrifolia as imnomostimulant in livestock and poultry (Sunder et al., 2007; Lohani, 2010; Sunder et al., 2011a; Benjamin et al., 2014). Studied showed that feeding 5% crude leaf extract of M. citrifolia to Nicobari fowl enhanced B cell mediated immune response. Furusawa et al. (2003) studied the antitumor activity in allogeneic mice and found that immunomodualtory property is due to a polysaccharide rich substance present in the fruit. Sunder et al. (2007) also studied the immunomodulatory properties of M. citrifolia in poultry. This was the first report of immunomodulaotry properties of M. citrifolia in poultry. They found that feeding of M. citrifolia fruit juice @ 5% in water enhanced both humoral (B cell mediated) and cellular (T cell mediated) immunity in broilers. The humoral immune response of the Morinda group was significantly better than control group (P<0.05). The peak response was observed at first week post inoculation (PI) in Morinda group (1.48±0.18) compared to control group (0.82±0.1). The direct challenge test of IBDV in the Morinda fed birds showed protection against the infection as only 6.6% mortality was recorded in this group compared to 25% in control. Humoral immunity and cell mediated immunity of M. citrifolia was studied by Sunder et al. (2011b) and found that supplementation of 1.5 ml of fruit juice enhanced B cell and T cell mediated immunity in Nicobari fowl. Literatures also indicated that Noni increases the defences and reinforces the immune system of the body, neutralize its function in all the cells and regenerates the affected cells (Heinicke, 1985). Thus it helps in preventing the diseases such as IBDV infection in the present study as it increases the immune response as observed. Reports also suggests the effect of M. citrifolia in inducing the release of interferons, interleukins and nitric oxide (Hirazamu & Furusawa,1999). 6 Synergistic effect of Morinda with other medicinal plant and probiotic During the last decade lot of works have been carried out in search of alternative to antibiotics which could be safely used as growth promoter, antimicrobial without any side effect or residual compounds in the end products. Probiotics are single or mixed group of bacteria when administered to the host showed many beneficial effects viz. growth promotion, enhancing nutrient uptake from the intestine, reducing the harmful microorganism, increases the immunity (Kabir, 2009; Brisbin et al., 2010). Reports are available on the use of lactobacillus as growth promoter and probiotics in livestock and poultry (Ibrahim et al., 2005; Salarmoini & Fooladi, 2011; Zamanzad-Ghavidel et al., 2011). Similarly, beneficial proprieties of medicinal plants have also been reported in the 254 the livestock and poultry by many workers (Mishra et al., 2008; Javed et al., 2009; Narimani-Rad et al., 2011). Use of probiotics and prebiotics together showed beneficial effect showed that However, Sunder et al. (2012) and Sunder et al. (2015) studied the use of combination of Morinda and lactobacillus in poultry. They reported that combination of M. citrifolia and lactobacillus showed synergistic effect in terms of body weight gain, immunomodulatory properties, and reduction in gut microbial count and feed efficiency. This is the only report available on the combined use of M. citrifolia and lactobacillus in poultry. Since the ban of antibiotics as growth promoters in poultry, the use of lactobacillus and herbal based nutraceuticals compounds have been increased. Role of nutraceuticals in improving the gut health and growth performance of poultry have been described by many workers (Muir et al., 2000; Yang et al., 2009; Zamanzad-Ghavidel et al., 2011; Adil & Magray, 2012; Das et al., 2012; Fallah et al., 2013; Sugiharto, 2015). However, Sunder et al. (2015) studied the feeding of noni and lactobacillus sin broiler which was not studied earlier. They found that feeding of lactobacillus and noni juice showed synergistic effect and enhanced the reduction in gut coliform load. Antimicrobial activity of M. citrifolia was also reported by some workers and they found that the activity is mainly due to presence of terpenoid compounds, phenolic compounds such as acubin, alizarin, acopoletin, anthraquinones in the noni fruit (Jin et al., 1998; Lavanya & Brahmaprakash, 2011; NarimaniRad et al., 2011; Salarmoini & Fooladi, 2011). Histology study of the chicken gut after feeding with noni fruit and lactobacillus showed significant changes in crypt depth and villi height, which is considered to be the main site for development of immune response and nutrient uptake (Sunder et al., 2014a). Similar to the finding of use of noni and lactobacillus, use of herbal based feed supplement as a growth promoter and effect on the gut function has also been reported by some workers (Hashemi et al., 2009; Lavinia et al, 2009; Abdulkarim et al., 2013; Kanduri et al., 2013). 7 Morinda citrifolia and Andrographis paniculata on expression of toll-like receptors Kalmegh (Andrographis paniculata) a promising medicinal plant has been scientifically validated to exhibit functions such as antiinflammaotry, immunomodulator (Sheeja & Kuttan, 2007; Abu-Ghefreh et al., 2009; Wang et al., 2010; Shen et al., 2013; Gao & Wang, 2016). Toll like receptors (TLRs) are innate immune receptors and induce fast and appropriate host defence reaction against pathogens. TLRs recognise the conserved microbial patterns such as flagellin, LPS, peptidoglycan in an efficient and non self reactive manner to initiate pro inflammatory cytokines. Role of TLR in the immunomodulation has been demonstrated and at least 10 TLRs have been identified in chickens, including TLR1A, 1B, 2A, 2B, 3, 4, 5, 7, 15 and 21 (Barjesteh et al., 2013; St Paul et al., 2013). Sunder et al. (2014b) studied that supplementation of Noni and Kalmegh influenced the expression levels of TLR_________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Sunder et al 2, TLR-3, TLR-4, TLR-5, TLR-15 and TLR-21 significantly (P<0.05). The gene expression level of TLR-3, TLR-4 and TLR-5 was found more which showed the immunomodulatory properties of herbal extract. The increased expression of TLR3, TLR-4 and TLR-5 may be due to the effects of phytochemicals on these TLR signal transduction pathway. Andrographolide has been found to induce the APK and PI3K signaling pathway which thereby involved in macrophage activation (Rao, 2001; Fukao & Koyasu, 2003; Wang et al., 2010). Park et al. (2009) also found that production of interferon is mainly due to the presence of quercetin in the noni fruit. This was also supported with the finding of Tanabe et al., 2003 and Tohyama et al., 2005. They also found that IFN-γ plays important role in expression of TLR-3, TLR-4 and TLR5. The high level of TLR-3, TLR-4 and TLR-5 and decreased TLR-7 gene indicated the antiviral and antibacterial activities. In another study the effect of feeding of M. citrifolia fruit to broiler was done and expression of TLR-4, TLR-5, IL-8, and IL-12 was found to be more while the TLR-7 and IL-6 level was lowered. The high level of interleukins and TLR may be responsible for antiviral and antibacterial activities in the noni fruits. 8 Grommune tonic for poultry Feeding of Grommune, a noni based herbal tonic showed more body weight gain, better FCR and enhanced B cell mediated immunity in broilers. The dose was standardized and found that feeding of Grommune @ 15 ml per bird up to 4 week and 30 ml per bird up to 8th week of age improved the body weight, feed conversion ratio and immune competency status of broilers (Sunder et al., 2014c). 9 Morical supplement for Japanese quail Sunder et al. (2013b,c) studied the feeding of different concentration of morical, a herbal based M. citrifolia feed supplement in the Japanese quail. They found that supplementation of 4% (w/w) of morical in feed showed more annual egg production (238.5) compared to control (215.4). They also observed that egg shell thickness, high Ca content in egg shell, egg yolk content, total weight of the egg increased with the increasing concentration of morical supplementation up to 8% in the feed. 10 Anticancer Activity Antitumor activity of the noni fruit was reported due to the presence of a polysaccharide rich substance (Hirazumi et al., 1994). Reports also suggests the role of M. citrifolia as anticancer and antitumor properties (Liu et al., 2001; Wang et al., 2001; Wang & Su, 2001; Wong, 2004; Issell et al., 2009; Brown, 2012; Saminathan et al., 2013a; Wu et al., 2015). Later, a lot of studies were carried out to find out the compounds responsible for the antitumor and anticancer activities directly or indirectly (Hiramatsu et al., 1993; Hisawa et al., 1999; Hirazumi & Furusawa, 1999; Mathivanan et al., Effect of Morinda citrifolia in growth, production and immunomodulatory properties in livestock and poultry: a review 2005). Studies on effect of Noni-ppt showed impvoemnt in the survival time and curative effect in treatment of cancer. Nonippt administration significantly prolonged the survival duration of inbred Lewis lung tumor-bearing mice (Hirazumi et al., 1996). Further studies on Noni-ppt suggested that it suppress the tumor growth through release of TNF- , IL-1ß, IL-10, IL12 p70, IFN- , and nitric oxide (Hirazumi & Furusawa, 1999). Some individual compounds from Noni juice were reported to function as ras inhibitors and thus suppressed the rasexpressing tumors (Wang et al., 1999). Liu et al. (2001) found that the antitumor potential of noni fruit due to the presence of glycosides and asperulosidic acid extracted from the noni fruit on the AP-1 transactivation and cell transformation in mouse epidermal JB6 cells. They found that both the compound suppressed the cell transformation and AP-1 activity. Wang & Su (2001) found that supplementation of noni juice to rats helped in prevention of DMBA-DNA adduct formation by 30% in heart, 41% in lung, 42% in liver, and 80% in kidney. Arpornsuwan & Punjanon (2006) tested the methanol extract from M. citrifolia fruits for cytotoxicity activity on the MTT assay. The appearance of cytotoxic changes after exposure to the extract was in a concentration dependent manner. The most sensitive cell line was baby hamster which showed median lethal concentration of 2.5 mg/ml followed by Vero cell (3.0 mg/ml) and human laryngeal carcinoma cell (5.0 mg/ml) respectively. In another study, Thani et al. (2010) found the cytotoxic activity of noni leaves against the KB cell line while Lv et al. (2011) found the anticancer potential in the root extracts. Gupta & Patel (2013) also demonstrated that combination of Noni and cisplatin were able to induced the anticancerous activity through the p53 and Bax proteins (pro-apoptotic) up regulating pathways and Bcl-2 (anti-apoptotic gene), survivin and Bcl-XL proteins down regulating mechanisms. Saminathan et al. (2013b) showed the antitumor activity of noni fruit against mammary tumours in rats. The frequency of the tumor incidence was found to be significantly decreased in the noni fruit treated group and only benign tumour was observed while in the control group malignant tumour was observed. 11 Hypotensive activity The first report of the hypotensive potential of the noni fruit was available in 1927, when Davison (1927) found that hot water extract of noni roots lowered the blood pressure of dog, the study was supported by the finding of Youngken et al., 1960; Youngken, 1958; Gilani et al., 2010. Later, Moorthy & Reddy (1970) and Yamaguchi et al. (2002) also reported the hypotensive effect of ethanol extract of noni dogs and rats respectively. Asahina et al., 1994 showed the diueretic effect of noni fruit juice. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 255 12 Anti-inflammatory activity The first scientific validation of the anti-inflammatory activity of noni juice was reported by Zhang et al. (1994). They studied the inhibitory activity of noni juice on COX-2 and COX-1 activities and compared with traditional anti-inflammatory drugs such as aspirin, indomethacin and celebrex. They found that activity of noni in COX-2 inhibition was on par with the celebrex. Later, Wang et al. (2002) also studied the antiinflammatory activity of noni juice and found that feeding of 10% noni juice in drinking water for 12 days decreases the inflammatory foci in and acute liver injury model in female rats induced by the CCl4. McKoy et al. (2002) showed that feeding of 200 mg of M. citrifolia prevented the formation of paw edema in the rats. This anti-inflammatory effect may be due to the inhibition of B2 receptor mediated mechanism of bradykinin Study showed that damnacanthal isolated from the M. citrifolia roots mediates anti-inflammatory activity through H1 receptor (Okusada et al., 2011). Dussossoy et al. (2011) reported that the anti-inflammatory activity of the noni is due to the presence of several polyphenols, flavonoids, phenolics compounds, irridoids and ascorbic acid. These compounds probably acts through the NO and PGE2 pathways by directly inhinbiting the cycloxygenase COX-1 and COX-2 activities. Palu et al. (2006) also described the use of noni seed oil as anti-inflammatory activity in skin. Noni seed oil inhibited the COX-2 and 5-LOX enzymes in a concentration dependent manner and found that it is safe for topical use for skin care applications and is non-comedogenic. 13 Analgesic effect The analgesic potential of M. ctirifolia was reported by Younos et al. (1990). They evaluated the root extracts of M. citrifolia in mice and found that it showed a dose-related analgesic activity in the writhing and hot plate tests in mice. The extract did not show any toxic effect and further, on administration of higher dose, it decreased all the behavioural parameters and induces sleeping which is suggestive of sedative properties of the M. citrifolia. The analgesic and tranquilizing properties of the noni fruit was also reported by Joseph Betz (1997) and Wang et al. (2002). They also observed the dose dependant analgesic properties of the M.citrifolia in mice. Punjanon & Nandhasri (2005) evaluated the different dose concentration of noni fruit extract viz. 1, 2, 3 and 4 g in mice and compared with the morphine sulphate. They found that 4 g/kg-1 concentration of noni fruit extract showed similar effect as produced by the morphine sulphate in inhibition of abdominal constriction induced by acetic acid. 256 This proves that M. citrifolia is having analgesic effect; however, detail studies are necessary for the identification of the chemical compounds and to study the mechanism of action. The analgesic efficacy of alcoholic extracts of Noni fruits was also demonstrated in the acetic acid induced writhing test (Okusada et al., 2011). In another study, Basar et al. (2010) demonstrated the analgesic activity of alcohol extract of noni fruit in reducing the pain and arthritis. 14 Antimicrobial Activity Many reports are available on the antimicrobial activity of M. citrifolia. Atkinson, 1956 reported that the antibacterial effect of noni is due to the presence of acubin, L-asperuloside, alizarin and anthraquinone. Reports also suggested that these compounds are responsible for antibacterial activity against Ps. Aeruginosa, Pr. Morgaii, B. subtilis, S. aureus, E. coli, Shigella and Salmonella as well as treatment of skin infection, cold fever and other bacterial infection (Bushnell et al., 1950; Tabrah & Eveleth, 1966; Leach et al., 1988; Locher et al., 1995). Duncan et al. (1998) showed that scopoletin, a compound available in the noni is responsible for antibacterial activity against E. coli and control of serious illness and even death. Umezawa (1992) demonstrated that anti HIV activity in the noni is due to the presence of a compound i.e 1-methoxy2-formyl-3-hydroxyanthraquinone which suppressed the cytopatheic effect of HIV infected cells. Broad spectrum antibacterial activity of various solvent extracts of M. ctirifolia have been reported against Gram positive and Gram negative microorganisms (Wei et al., 2008; Jayaraman et al., 2008; Selvam et al., 2009; Kumar et al., 2010; Usha et al., 2010; Sunder et al., 2012; West et al., 2012). The first report of the use of noni against tuberculosis was demonstrated by Saludes et al. (2002). They demonstrated that bactericidal activity of the noni leaf extracts was 89% compared to 97 % with rifampicin. The antifungal activity of M. citrifolia was demonstrated by Banerjee et al. (2006). They studied the antifungal activity in-vitro and found that M. citrifolia inhibited the growth of C. albicans in-vitro. They also found that the same extract also showed inhibitory activity against Apergillus nidulans spores. Sunder et al. (2012) have studied the wide spectrum antibacterial and antifungal activity of various parts of the M. citrifolia extracts. They have found that the methanol, ethanol, ethyl acetate, chloroform, acetone extracts of leaf, stem bark, fruit and seed showed broad spectrum antibacterial and antifungal activity in – vitro. Conclusion and future perspectives Farmers in several countries use medicinal plants in the maintenance and conservation of the healthcare of livestock. The last two decades have seen tremendous interest in the area of medicinal and aromatic plants. The role of plant derived drugs have been emphasized both national and international level. Based on the findings of the several researches on the _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Sunder et al use of Morinda citrifolia for livestock health and production, it is concluded that the M. citrifolia may be used as alone or in combination with probiotics and other medicinal plants for growth, production, immunomodualtor, antioxidant and many other properties in livestock and poultry. A lot of compounds with neturaceutical values have been identified from the Morinda citrifolia. Although lot of products are available in the market for human use, however, there are are plenty of scope for development of noni based herbal products for livestock health and production. The antibacterial potential of the plant should be explored and studied in detail to develop the drug against multidrug resistance bacteria mainly for the tuberculosis, malaria, HIV and other diseses. The feeding of noni fruit has been found to exhibit very good antioxidant, anti-cholesterol and growth and immunomodulatory property in livestock and poultry. Study on the palatability fo the fruit should be carried out to develop the cheap and best technology which is available at the famer doorstep for the livestock and poultry. Reports suggested that chicken, duck and pig consumes the raw fruit of noni. However, the palatability of the fruit should be improved for feeding as such to the other livestock which could save the production losses and post harvest losses. The high mineral richness of the fruit and leaf should be studied in detail to study the efficacy of mineral supplement in poultry and large animals. Since the ban of antibiotics as growth promoters by the European Union in 2006, lot of compounds, products etc have been studied as alternative to the antibiotics for probiotic, prebiotic, growth promotion effects in livestock and poultry. The presence of rich nutracrutical compounds in the noni might be useful for exploring this plant as an alternative to the antibiotic in poultry without giving any side effect. Morinda citrifolia trees are widely grown in coastal forest areas of A&N Islands. Owing to its high nutritive value for medicinal importance and having national and international market, increasing demand, there is a possibility for emerging as one of the most remunerative fruit crops to the island farmers. Recently, it has undergone a revival in Andaman and Nicolas Island as interest in plant with nutracentical properties has increased. Noni plant is distributed in almost all parts of the Island. It can be found near the coast, in open lands and Grassland, in gulches and in distributed forest of the dryer areas. It tolerates high soil salinity and brackish water stagnation. All the components of this plant have high demand in case of alternative medicines and herbal medicines. Due to its high demand and as a source of revenue generation the detail study on its effect on the livestock health and production should be carried out for its commercial exploitation. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. Effect of Morinda citrifolia in growth, production and immunomodulatory properties in livestock and poultry: a review References Abdulkarim A, Amad, Wendler KR, Zentek J (2013) Effects of a phytogenic feed additive on growth performance, selected blood criteria and jejunal morphology in broiler chickens. Emirates Journal of Food and Agriculture 25: 549–554. Abu-Ghefreh AA, Canatan H, Ezeamuzie CI (2009) In vitro and in vivo anti-inflammatory effects of andrographolide. International Journal of Immunopharmacology 9: 313–318. Adil S, Magray SN (2012) Impact and manipulation of gut microflora in poultry: a review. Journal of Animal and Veterinary Advances 11: 873–877. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 ANTIOXIDANT ACTIVITY OF VITAMIN E AND ITS ROLE IN AVIAN REPRODUCTION Vincenzo Tufarelli* and Vito Laudadio Department of Emergency and Organ Transplantation (DETO), Section of Veterinary Science and Animal Production, University of Study of Bari ‘Aldo Moro’, Valenzano 70010 Bari, Italy. Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).266.272 KEYWORDS ABSTRACT Vitamin E Antioxidant Reproduction Avian Species Oxidative stress, a state characterized by imbalance between pro-oxidant molecules comprising reactive oxygen and nitrogen species, and antioxidant defences, has been found to play an important in poultry reproduction in both male and female Increasing evidence suggests that vitamin E plays an important role in normal reproduction in animals and humans, and vitamin E supplementation is now recommended. Vitamin E comprises eight molecules composed by a chromanol ring and a phytol side chain having same functions: four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ). This article reports an overview on the currently available literature on the role of reactive species and oxidative stress in avian reproductive processes. Current evidences demonstrate that dietary vitamin E supplementation may be effective in controlling the production of reactive oxygen species and continue to be explored as a potential feeding strategy to support avian reproduction. * Corresponding author E-mail: [email protected] (Vincenzo Tufarelli) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 267 1 Introduction In overall, poor fertility is mostly related to male bird, even though females may be rather responsible for the flock fertility decline (Hocking & Bernard, 2000; Habibian et al., 2014). In addition, the strong genetic selection for large breast progeny, low frame size, reduction of libido, age, environment, feed and stress are some factors influencing fertility in poultry (RomeroSanchez et al., 2008; Khan et al., 2012). Therefore, the oxidative stress has been identified as one of the major factors affecting reproductive parameters and thus has been widely investigated in recent years. Carotenoids and vitamins A, D, E and K are liposoluble compounds naturally present in food or feed used as excipients in industrial fields such as pharmaceutics. Nevertheless, carotenoids do not belong to the common vitamins classification and they are commonly studied with liposoluble vitamins as fifty carotenoids among the over 600 carotenoids identified to this day are pro-vitamin A elements (Gonnet et al., 2010; Jones et al., 2013). Vitamins are receptive compounds, they must be preserved from pro-oxidant elements which could influence their chemical integrity and decrease their physiological benefits. Vitamin E includes eight molecules composed by a chromanol ring and a phytol side chain having same functions: four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ) (Górnaś, 2015). Tocopherols contain saturated side chain, while tocotrienols possess 3 conjugated double bonds. The α, β, γ and δ prefixes represent the methyl groups position on chromanol ring (Hincha, 2008). The α-Tocopherol is the richest in nature and one α-tocopherol molecule can catch two peroxyl radicals responsible of lipid oxidation start (Niki et al., 1984; Brigelius-Flohe & Traber, 1999). Thus, α-tocopherol molecule protects membrane lipids against oxidation (Niki et al., 1991), and it stabilizes mechanically the membranes (Srivastava et al., 1983). Vitamin E digestion is similar to vitamin A and carotenoids digestion. Vitamin E deficiency might occur in case of fat malabsorption being usually characterized by neurological problems due to poor nerve conduction, which are reversible by supplementation (Brigelius-Flohe & Traber, 1999). The α-Tocopherol is a main constituent of vitamin E in the leaves of plants. It is a capable antioxidant and through numerous studies it has been shown to play a key-role in protecting the photosynthetic apparatus of plants against oxidative damage especially under stress conditions (MunneBosch, 2005). Vitamin E plays an important role in the transport of amino acids and probably lipids in the intestine (Wang & Quinn, 2000). Vitamin E is also involved in iron metabolism and steroidogenesis (MacDonald et al., 1991), and it stimulates humoral and cellular immune responses against infectious diseases (Oliver et al., 1998). The symptoms and disorders of vitamin E deficiency vary, depending on the species affected (Baldi et al., 2013; Habibian et al., 2014). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Tufarelli et al Vitamin E deficiency may manifest itself in a number of disorders in organs and in adipose tissue (Matringe et al., 2008). Vitamin E deficiency may increase the risk of ischemic heart disease, breast cancer, and the incidence of infections (Hincha, 2003), and it promotes susceptibility to dietary and environmental stress in humans and animals (Oliver et al., 1998; Wang & Quinn, 2000; Khan et al., 2011). Compared to findings in humans, available data from animal trials show that vitamin E toxicity is low and that the vitamin E results not mutagenic, carcinogenic, or teratogenic (Munne-Bosch, 2005). Vitamin E has also a positive effect on fertility (Khan et al., 2012). In particular, the activity of vitamin E was first identified as an essential dietary factor for male and female reproduction in rats (Della Penna, 2005; Khan et al., 2011). Although vitamin E has a wide range of functions in the body, it is primarily crucial for fertility in humans and livestock species. In the poultry farming, males are added into flock of hens to produce fertile egg which dictate the final flock profitability (Ordas et al., 2015). For that reason, male fertility is essential in the net income of avian industry. 2 Free radicals and oxygen and lipid peroxidation In biological systems, the reactive oxygen species (ROS) or also free radicals are produced by the prooxidative enzyme systems, irradiation lipid oxidation, air pollutants and glycoxidation (Halliwel, 1997; Sabry, 2013; Kostadinović et al., 2015). The generation of free radicals induced oxidative stress which associated with many degenerative diseases, including atherosclerosis, vasospasms, cancers, stroke, hyperoxia, arthritis, heart attack, age pigments, dermatitis, liver injury and induction of apoptosis (Simon et al., 2000; Niki, 2014; Rahal et al., 2014). In animals, free radicals are also associated with metabolic disorders, diabetes and infectious diseases. In the contrary there are some benefits of free radicals have been reported. These benefits are the activation of nuclear transcription factors, gene expression and destructive effect to tumor cells and microorganisms (Packer & Weber, 2001). Superoxide radicals (O•-2) act as controller for the growth of cells (Bhattacharyya et al., 2014); in addition, it can assault a lot of pathogens stimulating inflammatory responses (Stief, 2003). Nitric oxide (NO•) is signaling molecules participating in cellular and organ function as a neurotransmitter and a mediator of the immune responses (Fang et al., 2002). In living organisms under aerobic conditions more than 90% of oxygen consumed is reduced directly to water by cytochrome oxidase in electron-transport chain via four-electron mechanisms without ROS release (Lushchak, 2014). Oxidative stress is caused by the imbalance between prooxidants and antioxidants at either cellular or individual level (Panda & Cherian, 2014; Rahal et al., 2014). The production of ROS, also defined as oxidants, has become a concern because of their potential toxic consequence, at higher levels, on semen quality and functions (Agarwal et al., 2003; Khan et al., 2012). Antioxidant activity of vitamin e and its role in avian reproduction Also, ROS are highly reactive agents belonging to the class of free radicals. All living cells including spermatozoa regularly face the oxygen paradox. The oxygen is necessary for maintaining life; nevertheless, its metabolites, such as ROS, must be neutralized constantly to support the small amount essential for physiological cell function (Niki, 2014; Surai, 2016). The spermatozoa PUFAs are extremely susceptible to lipid peroxidation, as a result, ROS are produced in high quantity, which are harmful to the fertilizing capability of semen (Agarwal et al., 2003). Due to an increased production of ROS, oxidative stress, which is the imbalance between prooxidants and antioxidants happens (Agarwal et al., 2003; Urso & Clarkson, 2003; Surai, 2016). Under normal conditions, the body generally has enough reserves of antioxidants to manage with the ROS production (Castillo et al., 2001). Nevertheless, in particular conditions of stress when ROS production exceeds the body's antioxidant capacity, oxidative stress occurs. The oxidative stress determines a reduction of sperm quantity, decreasing also spermatozoa motility and increasing the dead sperm (Sikka, 2001; Agarwal et al., 2003; Khan et al., 2012), determining reproductive problems. The lipid peroxidation can be described in overall as a process where the oxidant compounds (i.e. free radicals or non-radical species) assault lipids with carbon-carbon double bond, in particular PUFAs involving hydrogen abstraction from a carbon (Yin et al., 2011; Ayala et al., 2014). In reply to peroxidation of the lipid membrane, and along with specific cellular metabolic circumstances and restore capacity, the cell can support cell survival or stimulate cell death (Ayala et al., 2014). The impact of lipid oxidation in cells and how damages are implicated in physiological processes and pathological conditions have been investigated in previous studies (Yin et al., 2011; Volinsky & Kinnunen, 2013; Ayala et al., 2014). In modern poultry production is associated with various stress conditions that are responsible of a decrease in productive and reproductive traits of young poultry, breeders and layers (Celi et al., 2014; Surai, 2016). One of the very significant sources of lipid peroxidation is mitochondria, which having a key-role in ROS production through the NADH-dependent oxido-reductase system (Hallak et al., 2001). Mitochondria are present in adequate levels in gametes providing mechanical energy for motility. The inner mitochondrial membrane potential is very important in regulating sperm functions. Wang et al. (2003) reported that mitochondrial membrane potential decreased in spermatozoa of infertile subjects with high levels of ROS production and have a significant correlation with concentration of sperm. High ROS level disrupts the outer and inner mitochondrial membrane determining the release of cytochrome C protein and activate caspases to stimulate apoptosis (Khan et al., 2012). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 268 The damage of DNA and cross linking proteins can in addition decline the quality of semen (Sharma & Agarwal, 1996). The exposure of sperm to artificially produced ROS determines the damage in form of modification of all bases, frame shifts and DNA cross-links (Duru et al., 2000). Spermatozoa with dysfunctional DNA are unable to fertilize the oocyte, and thus, fertilisation rate decreases with increasing DNA damage (Sun et al., 1997; Khan et al., 2012). 3 Dietary vitamin E on reproduction activity in avian species Available published review papers report that dietary vitamin E supplementation of a balanced poultry ration significantly supports reproductive functions, including semen volume, sperm concentration, sperm viability, sperm motility, and sperm capacity, in avian species (Khan et al., 2012; Rakha et al., 2015; Rengaraj & Hong, 2015). The vitamin E is getting substantial interest in poultry feeding due to its key- role as a dietary antioxidant to prevent oxidative stress (Dhama et al., 2014). Vitamin E is a well-documented fat-soluble antioxidant and has been shown to inhibit free radical-induced damage to sensitive cell membranes (Panda & Cherian, 2014; Rengaraj & Hong, 2015). Vitamin E is supplemented to the diet to maintain and enhance performance in layers, broilers, broiler breeders, and turkey (Sunder et al., 1997; Panda et al., 2009; Khan et al., 2012). The results obtained varied depending upon the level and duration of feeding diets supplemented with vitamin E, genetic stocks, age, assessment criteria and stress conditions (Panda et al., 2009; Panda & Cherian, 2014). Vitamin E is found in turkey semen, with a higher concentrations within sperm cells than seminal plasma (Surai, 1981). Moreover, it is a natural stabilizer of sperm plasma and membranes of mitochondrion (Surai & Ionov, 1992) and it has been demonstrate to boost sperm mobility and viability during storage (Donoghue & Donoghue, 1997). Comparatively, lower vitamin E levels are present in both chicken and drake semen than in turkey, with significant low vitamin E found in gander (Surai & Ionov, 1992). Furthermore, this vitamin is splitted between seminal plasma and spermatozoa, with higher amount in sperm cells of turkey than in seminal plasma as reported by Surai (1981). Even though observed in semen, supplementation of this vitamin in semen extenders produced conflicting findings in reducing peroxidation. It was assessed that neither 10 nor 40 µg of vitamin E was adequate to limit peroxidation under aerobic storage condition (Long & Kramer, 2003). Additionally, the effect of vitamin E on the mobility and viability of sperm is somewhat contradictory. In fact, in a study on semen from turkeys, the supplementation of vitamin E increased sperm mobility as well as the membrane integrity (Donoghue & Donoghue, 1997), whereas it was demonstrated no influence on mobility or viability when supplementing vitamin E (Long & Kramer, 2003). Studies on mammalian species demonstrated an opposite relationship between lipid peroxidation degree and sperm mobility with vitamin E. 269 To improve poultry male reproductive traits, supplementing vitamin E in diet is used regularly in avian production. The dietary increase in the vitamin E inside semen was demonstrated to determine a significant decrease in the lipid peroxidation susceptibility (Niki, 1991; Lin et al., 2005). Moreover, Biswas et al. (2007) also reported that fertility was low in absence of vitamin E in a basal diet and when vitamin E was supplied, fertility was restored in birds. Cerolini et al. (2006) reported that sperm levels were enhanced in male breeders by adding vitamin E at 200 mg/kg of diet. In addition, Biswas et al. (2009) found that including vitamin E in diet of cockerels significantly decreased the abnormal and dead spermatozoa proportion improving the birds fertility. The age decreases the fertility in cockerels and it was also linked with low levels of vitamin E in testes and this can be reinstated supplementing vitamin E (Surai et al., 2000). Lately, Ebeid (2014) demonstrated in male chicken that vitamin E in diet in combination with organic selenium has a synergistic influence in reducing lipid peroxidation and enhancing the antioxidative status in plasma of domestic fowl, which almost certainly translated into improved spermatozoa count, motility and decreased dead spermatozoa percentage under heat stress conditions. In addition, Ipek & Dikmen (2014) found that a dietary combination of vitamin E may affect significantly sexual maturity, egg mass and hatching traits of quails reared under heat stress. Increased dietary vitamin E supplementation of the maternal diet was associated with increased vitamin E concentrations in the egg yolk, embryonic tissues and their increased resistance to oxidative stress (Surai et al., 2016). In addition, Urso et al. (2015) reported that hatchability of the eggs of breeders fed 120 mg vitamin E/kg feed was higher than those fed diet containing 30 mg vitamin E/kg of feed. Conclusion The present literature review shows that vitamin E is required for the development and function of the reproductive tissues in both sexes, possibly due to its key role in the modulation of antioxidant balance. Biological systems are under a continuous influence of oxidative stress because of excessive generation of ROS. Although biological systems are affected in different ways by oxidative stress, there are sufficient antioxidant protections that can decrease the progression of the damage. Excessive ROS production and resulting OS may contribute to aging and several diseased states affecting reproduction. However, when an imbalance exists between levels of ROS and the natural antioxidant defenses, various measures can be used to protect humans against the oxidative stress -induced injury. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 AN OVERVIEW ON THE FUNCTIONAL FOOD CONCEPT: PROSPECTIVES AND APPLIED RESEARCHES IN PROBIOTICS, PREBIOTICS AND SYNBIOTICS Vincenzo Tufarelli* and Vito Laudadio Department of Emergency and Organ Transplantation (DETO), Section of Veterinary Science and Animal Production, University of Study of Bari ‗Aldo Moro‘, Valenzano 70010 Bari, Italy. Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).273.278 KEYWORDS ABSTRACT Probiotics Prebiotics Synbiotics Functional food The principal role of diet is to supply adequate nutrients providing energy to sustain physiologic functions and well-being. Every foods and feeds are functional and consumption of bioactive molecules is facilitated by diet. All probiotics, prebiotics and synbiotics are functional components able to exercise significant influences on human and animal wellbeing. Emphasizing these positive activities is one possible approach for improving the health image of meat and plants and developing functional products. Discovering of new prebiotic/probiotic/synbiotic functional foods is linked to the interest of the food industry to renew constantly through introduction of products with enhanced nutritional value, but also with health advantage for consumers. This review provides potential benefits of representative bioactive compounds on human and animal health and an overview of meat and plant-based functional products. Besides the increase of scientific reports, there is a necessary need to update consumers of the feeding value of novel functional foods. * Corresponding author E-mail: [email protected] (Vincenzo Tufarelli) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 274 Tufarelli et al 1 Introduction Within the last decade, knowledge of the significance of diet in human and animal health and well-being has considerably increased and nutritionists have identified specific foods playing a key-role in supporting the consumers‘ health status. Beyond meeting nutritional requirements, it is extensively recognized that dietary factors are able to change the detrimental development of different chronic diseases (Alkerwi, 2014; Peiretti et al., 2015). In the developed world, there has been an explosion of consumer attention in the active role of foods in the well-being and life prolongation as well as in the prevention of initiation, promotion and development of cancer, cardiovascular diseases and osteoporosis (Pandey & Rizvi, 2009). As a result, a new term ―functional food‖ was proposed (Dimer & Gibson, 1998; Sanders, 1998; Pisulewski & Kostogrys, 2003; Grajek et al., 2005). Functional foods may improve the overall conditions of body, reduce the risk of some diseases and could even be used for curing some illnesses (Laudadio et al., 2015). It was demonstrated that there is a high demand for functional food as many studies reported that the medical service of the aging population is quite costly (Menrad, 2003; Mark-Herbert, 2004; Side, 2006). functional food. The European Commission‘s Concerted Action on Functional Food Science in Europe (FuFoSE), directed by the International Life Science Institute (ILSI) Europe described functional food as a product that can be only considered functional if belong the basic nutritional effect it has positive influences on one or more functions to human organism, diminishing the risk of the development of diseases. However, the European Legislation, does not take into account functional foods as specific categories, but only as a concept (Stanton et al., 2005; Coppens et al., 2006). In agreement to the European Union rule on health claims made on foods (EC No. 1924/2006), a record of official claims has to be published for all member countries, and nutrient specifications also has to be defined for food having health claims. Health claims can be ―function claims‖ and ―reduction of disease risk claims‖. Thus, to better understand functional food it is first essential to comprehend how the science of nutrition itself has changed. Nutrition has progressed from the prevention of nutritional deficit and the institution of dietary standards, guidelines and food/feed guides, to the support of a state of health and the reduction of the risk of disease (Siro et al., 2008; Bigliardi & Galati, 2013; Vella et al., 2013; Vella et al., 2014; Asher & Sassone-Corsi, 2015). 2 Probiotics The concept of functional food was first defined by researchers in Japan in 1984 who investigated the correlations between nutrition, sensory quality, and physiological systems modulation (Siro et al., 2008). Later, the Ministry of Health introduced in 1991 the regulations for approval of a detailed health-related food class named Food for Specified Health Uses (FOSHU) including the institution of definite health claims for this food (Kwak & Jukes, 2001; Menrad, 2003; Siro et al., 2008). According to Gibson & Williams, (2005) the unique features of functional foods are being a conventional or everyday food; to be consumed as part of the normal/usual diet; composed of naturally occurring (as opposed to synthetic) components perhaps in unnatural concentration or present in foods that would not normally supply them; having a positive effect on target function(s) beyond nutritive value/basic nutrition; may enhance well-being and health and/or reduce the risk of disease or provide health benefits so as to improve the quality of life including physical, psychological and behavioral performances; have authorized and scientifically based claims There is no precise legislative definition in many countries related to the term and drawing a border line between conventional food and functional food is demanding even for scientists of nutrition and food (Mark-Herbert, 2004; Niva, 2007). Up to now, a number of national authorities and academic institutions have proposed the description for _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Based on the currently established definition by FAO/WHO in 2001, probiotics are defined as ―live microorganisms which when administered in adequate amount confer a health benefits on the host‖. Using fermented food leads to helpful bacteria and bacterium dietary supplements, consumers are supplied with live bacteria that pass the gastric to replicate themselves in the large intestine (Michalak & Chojnacka, 2016). The researches sustaining the efficacy of live bacteria are copious, and there are a small amount of contradictory results on the effects of the same strain supplied in either viable or nonviable form. As a result, due to well-established definitions and for the sake of better information to consumers, the term probiotic is to be reserved for a product including vital and living cells (Aureli et al., 2011). Probiotics that are commonly used are Lactobacilli, and Bifidobacteria as well as nonpathogenic yeast. The Bifidobacteria have been used also in microbial food supplements destined to infants (Milner & Roberfroid, 1999), individually (Langhendries et al., 1995) or along with Lactobacilli (Marteau et al., 1997; Jahromi et al., 2015). Other microorganisms have probiotic properties such as: Escherichia coli Nissle, Streptococcus thermophilus, Enterococcus francium, Saccharomyces boulardii, Propionibacterium, Leuconostoc, and Pediococcus, however some of these strains can be pathogenic (Seno et al., 2005). Earlier available reviestudiesws have reported that probiotics can excite the immune system (Tasvac, 1964; Rezaei et al., An overview on the functional food concept: prospectives and applied researches in probiotics, prebiotics and synbiotics 2015), decrease the intolerance to lactose (Conway, 1996), diminish incidence of diarrhea, reduce blood cholesterol (Fernandes & Shahani, 1990) operate as a antibiotic, repress tumors and defend against cancer by sustaining the adequate balance of the intestinal microflora (Lee & Salminen, 1995). To achieve a probiotic status, microorganisms must fulfill a number of criteria related to safety, functional effects and technological properties (FAO/WHO, 2001). From the safety point of view, the probiotic microorganisms should not be pathogenic, have no connection with diarrhoeagenic bacteria and no ability to transfer antibiotic resistance genes, as well as be able to maintain genetic stability (Saarela et al., 2002). In the literature, the use of different solid surface models, such as mucosa, alginate, carrageenan, gelatin, collagen, glass, polystyrene and carboxymethylcellulose are also described (An & Friedman, 1997). However, numerous investigations have shown that none of the simple models exhibit comparable adhesion properties to those presented by epithelial cell cultures. It should be stressed that the results obtained with the in vitro models are not sufficient and require confirmation in double blind, randomized, placebo-controlled human trials. From the practical point of view, the technological aspects of probiotic production also play a very important role. During the technological processing bacteria cells are exposed to different stresses (Knorr 1998; Mattila-Sandholm et al., 2002). 3 Prebiotics According to the most recent definition ―A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health‖ (Gibson et al., 2004; Macfarlane et al., 2006). Many criteria have to be rewarded when a molecule is to be defined as a prebiotic: stability, safety, resistance to digestion in the upper bowel and fermentability in the colon, organoleptic property, and ability to improve the growth of useful bacteria in gut (Gibson et al., 2004; Chen et al., 2014). Carbohydrates as oligofructose, inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), soybeanoligosaccharides, transgalacto-oligosaccharides, glucooligosaccharides, gentio-oligosaccharides, xylooligosaccharides, lactulose, isomalto-oligosaccharides, and polysaccharides as pectins and starch are considered to be efficient prebiotic substances (de Vrese & Schrezenmeir, 2008). Nevertheless, most of the studies on prebiotics investigated the inulin-type fructans (inulin, FOS) and GOS which selectively stimulate the Bifidobacteria growth and have been related to long-lasting safe commercial utilize (Macfarlane et al., 2006; Brown et al., 2015; Rezaei et al., 2015). A prebiotic is a non-viable food constituent moving the colon and having a selective fermentation. The advantage to host is _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 275 mediated throughout a selective stimulus of growth or activity of one or a restricted number of colonic bacteria. Food ingredients classified as prebiotics must not be hydrolyzed or absorbed in the upper gastro-intestinal tract, need to be a selective substrate for one or a limited number of colonic bacteria, must alter the microbiota in the colon to a healthier composition and should induce luminal or systematic effects that are beneficial to host health (Gibson & Roberfroid, 1995). Bifidobacteria and/or lactobacilli are good target organisms. Thus, the plan of future investigations to study the outcome of prebiotics in both humans and animals should consider the length of supplementation period, the choice of populations, and the type of vehicle utilized to augment the prebiotics consumption in diet, as these variables may have effect on the outcome of the studies. 4 Synbiotics A synbiotic is defined as: ―A mixture of a prebiotic and a probiotic that beneficially affects the host by enhancing the survival and the implantation of live microbial dietary supplements in the gut, by selectively stimulating growth and/or activating the metabolism of a specific or few number of health-promoting bacteria‖ (Gibson & Roberfroid, 1995; Roberfroid, 2002). Consequently, a synbiotic is a combination of the concept of probiotics and prebiotics (Mousavi et al., 2015). This mix would benefit the host by improving survival and implantation of the selected microbial supplements. Because of the nutritional benefits associated with microflora management approaches, foods are the main vehicle for pro-, pre- and synbiotics. However, there may also be potential pharmaceutical applications, but to date most evidence for this is still hypothetical. The synbiotics offer a additional option; in fact, the employ of synbiotics as functional food components is a novel and increasing area and few animal and human studies have been conducted to investigate their outcome on risk factors for coronary heart disease (Roberfroid, 2002). The synbiotics development is a worthwhile area of enhanced functional food compounds. Scientists are intensely interested in synbiotic theory as it leads to the combination of probiotics and prebiotics. The influence of synbiotic is directed towards two different target traits of the gut, both the small and large intestinal tracts. Prebiotic oligosaccharides stimulate probiotic bacteria in the colon, moreover prebiotic carbohydrate is used by a probiotic strain for its growth and replication in gut will be selectively supported (Deng et al., 2015). This mixture could enhance the survival of probiotic organisms, due to its specific substrate is promptly available for fermentations, determining healthier host composition. There are many evidences related to animal investigations on the potential positive effects of synbiotics: in one of the comparative in vitro studies of a number of strains of Bifidobacterium longum, Bifidobacterium animalis and 276 Tufarelli et al Bifidobacterium catenulatum grew best on FOS with much more lower growth rate found on inulin. Specific synbiotics were supplied to rat or chicken and their faeces were characterized for coliforms, bifidobacteria, and total cell counts (Mousavi et al., 2015; González-Herrera et al., 2015; Paturi et al., 2015). Higher levels of Coliforms and Bifidobacteria were found in animals fed both FOS and synbiotics (Bielecka et al., 2002). Synbiotics are believed to amplify the persistence of probiotics in gut was supported by studies including the preparation of Lactobacillus acidophilus and FOS has been investigated as in vitro model of human gut (Gmeiner et al., 2000). References Moreover, it has been believed that synbiotics consumption decrease cancer risk factors in patients with colon cancer (Rafter et al., 2007). Studies on animal reported that combining probiotic and prebiotic apply defensive effect against the development of tumor in colon, however, human data sustaining this suggestion are few (Liong, 2008). Moreover, the intervention of synbiotics resulted in significant modification of fecal flora: Lactobacillus and Bifidobacterium were augmented and Clostridium perfringens reduced. In addition, it is imperative to select a mixture of a definite substrate and a microorganism for a synbiotic product that can enhance the advantageous effect when compared to a product including a probiotic or a prebiotic only (Capela et al., 2006; Huebner et al., 2007). Aureli P, Capurso L, Castellazzi AM, Clerici M, Giovannini M, Morelli L, Poli A, Pregliasco F, Salvini F, Zuccotti GV (2011) Probiotics and health: an evidence-based review. Pharmacological Research 63: 366-376. Conclusions and future perspectives Substantial improvement has been made in the knowledge to identify and characterize the functional effects of foods and feeds. High-quality health is strongly linked to a good lifestyle, particularly to good quality dietary behavior conforming diet guidelines, the established suggestions and the most recent science on nutrition. Certainly the improvement of the body functions and the progress of well-being and health through a specific diet and the reduction of the risk to develop dietrelated diseases by means of appropriate food choices are priorities for involved parties. These comprise researchers, food/feed industries, consumers and governments. To date, the notion of gut flora modulation is enjoying novel attractiveness and it is essential that human studies are utilized to assess and confirm probiotics, prebiotics and synbiotics. The progress of functional food proposes huge potential for enhancing health and value of life for people. So, it is crucial that the scientific evidence for functional foods be properly substantiated before the possible health benefits are extensively communicated to the consumers. This will make sure the credibility of the claimed benefits of the functional foods. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 CANINE PARVOVIRUS- AN INSIGHT INTO DIAGNOSTIC ASPECT Minakshi P1,*, Basanti Brar1, Sunderisen K1, Jiju V Thomas2 , Savi J1, Ikbal1, Koushlesh Ranjan3, Upendera Lambe1, Madhusudan Guray1, Nitish Bansal1, Pawan Kumar1, Vinay G Joshi1, Rahul Khatri4, Hari Mohan4, C S Pundir5, Sandip Kumar Khurana6 and Gaya Prasad3 1 Department of Animal Biotechnology LUVAS, Hisar, Haryana- 125004, India University of Minnesota, USA SVPUAT, Meerut, U.P. India 4 Centre for Medical Biotechnology, Maharshi Dayanand University, Rohtak, Haryana-124001, India 5 Department of Biochemistry, MDU, Rohtak, Haryana-124001, India 6 NRCE, Hisar, Haryana, India 2 3 Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).279.290 KEYWORDS ABSTRACT CPV Antigenic variation Diagnosis PCR VP2 gene Canine parvovirus (CPV) leads to an acute disease, characterized by hemorrhagic gastroenteritis, vomiting and myocarditis in dogs. The disease can affect dogs of any age but is fatal in pups. CPV has undergone genetic variations that have led to emergence of various CPV-2 antigenic variants such as 2a, 2b and 2c with replacement of the original CPV-2 circulating in the dog population. CPV genome is made up of 5.2 Kb nucleotides. Viral protein VP2 plays a very important role in determining antigenicity and host range specificity of CPV. The antigenicity as well as host range of CPV is determined by virus specific VP2 protein. That’s why the mutations that affect the VP2 gene are the main source of different antigenic variants. It spreads rapidly in the wild population of canines as well as domestic animals, infected feces serve as a main source of infection because the virus is shed in large quantity in the feces particularly 4 - 7 days post infection. The present review is focused on various * Corresponding author E-mail: [email protected](Minakshi P) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 280 Minakshi et al biotechnological approaches used for diagnosis of CPV along with some conventional techniques including gold standard virus isolation in animal cell culture, hemagglutination test, electron microscopy, enzyme linked immunosorbent assay (ELISA). The biotechnological approaches such as polymerase chain reaction (PCR), Real-time-PCR, Loop-mediated isothermal amplification (LAMP), Bead based multiplexing, Microarray chip and DNA probe etc. have also assured their application. These approaches provide rapid, sensitive, optimal detection and effective control of CPV infection. 1 Introduction Canine Parvoviruses (CPVs) are small, non-enveloped viruses belonging to the Genus Parvovirus in the family Parvoviridae. Its genome is comprised of linear, negative-sense, single standard DNA of about 5.2 kb size, and encodes two structural (VP1 and VP2) and two non-structural (NS1 and NS2) proteins (Mochizuki et al., 1993). The first evidence of CPV infection in dogs dates back to 1970s and was identified as canine parvovirus type 2 (CPV 2) (Burtonboy et al., 1979). This CPV2 isolate was likely to be a variant of Feline panleukopenia virus (FPV) because of detection of active circulation of intermediate viruses between FPV and CPV-2 in wild carnivores (Truyen, 2006). These two differed in at least six amino acids which are mostly located on VP2 protein (Truyen et al., 1995). Since then, CPV-2 has been identified globally and now, it is endemic in most populations of wild canines (Driciru et al., 2006, Ramsauer et al., 2007). Two new antigenic types of CPV-2 i.e. CPV-2a and CPV-2b differed at two amino acid positions, N426D and I555V (Truyen, 2006) and have became wide spread (Hoelzer et al., 2008). The Asp-426 Glu substitution in capsid protein of CPV-2 generate a new variant known as CPV-2c which may infects several canine breeds (Buonavoglia et al., 2001, Decaro et al., 2006; Castro et al., 2007; Gombac et al., 2008). During acute phase of infection dogs may excrete virion particles up to 109/gram of faeces (Carmichael & Binn, 1981). Moreover, CPV-2 virion particles are very stable in environment which facilitates its transmission through faecal-oral route. Canine parvovirus (CPV) infection is a highly infectious viral disease of dogs of great concern for pet owners, veterinarians and scientists due to its high morbidity and mortality rates associated. Parvovirus infects dogs of all age groups, but puppies are more affected than adults. The initial clinical signs of CPV infection are nonspecific and include anorexia, depression, lethargy, and fever. Within 24 to 48 hours, most affected dog starts vomiting and hemorrhagic small-bowel diarrhea results severe dehydration. With severe dehydration, protein loss, concomitant infection, and inability to produce a rapid immune response, further weakening the dog. In last, all of these factors can lead to shock and death (Bargujar et al., 2011). The current knowledge of epidemiology, pathogenesis, clinical findings and diagnosis of canine parvoviral enteritis was briefly highlighted (Geetha, 2015; Shim et al., 2015). CPV in faecal samples has been detected by several methods based _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org on virus isolation in cell culture, haemagglutination (HA), electron microscopy, enzyme linked immunosorbent assay (ELISA) and DNA hybridization. Several molecular diagnostics assays such as polymerase chain reaction (PCR), multiplex PCR, Reverse Transcriptase PCR (RT-PCR), nucleic acid sequencing, Real Time-PCR, DNA probe etc have provided the unparalleled identification and discrimination ability for several viral pathogens (Minakshi et al., 2014; Kaur et al., 2015). Such diagnostic techniques should be transferred to end users for proper applications. 2 Conventional Methods 2.1 Viral Isolation Various cell cultures viz. Crandell Feline Kidney cell line (CRFK), Madin Darby canine Kidney cell line (MDCK), Walter Reed Canine Cell line (WRCC) have been used for the isolation of CPV from the clinical samples for diagnosis of canine parvovirus. CPV is primarily isolated in laboratory in canine lung and kidney cell line. It produces characteristics cytopathic effects (CPE) such as presence of intranuclear inclusion bodies in host cell, detachment and rounding of cells (Figure 1A, B). However, cell culture is not used as a routine diagnostic test because it is a time consuming process, require permissive cell lines, low sensitivity and requires skilled personnel. Moreover, the sensitivity of different cell lines for CPV multiplication may also vary. CPV-2 can be isolated from cell culture only after few days of inoculation (Desario et al., 2005). 2.2 Electron Microscopy Electron microscope can also be used for morphological identification of CPV2. Under electron microscope CPV may be seen as either single virion particle or in group of few viruses (Amo et al., 1999). On 3rd to 9th day of infection viruses are excreted in large quantity in faeces, thus, this period is best for electron microscopic study of CPV-2 from fecal samples. However, electron microscopy needs large quantity of virus to confirm a sample as positive, because electron microscopy is less sensitive in comparison to other molecular tests (Esfandiari & Klingeborn, 2000). Electron microscopy was successfully used for diagnosis of canine parvoviral enteritis in fecal sample (Klingeborn & MorenoLópez, 1980). Canine parvovirus- an insight into diagnostic aspect 281 Figure 1. A) Micro-photograph of CRFK Cell line after 48 hours of growth; B) Micro-photograph of CRFK Cell line showing CPE after 48 hours of infection. 2.3 Haemagglutination (HA) Assay 2.5 Counter immuno electrophoresis Haemagglutination is specific, rapid and inexpensive test for CPV diagnosis. Haemagglutination is one of the important properties of Canine parvovirus. CPV has binding ability for sialic acid receptors on cell surface and agglutinates the RBCs of several animal species such as rhesus monkey, African green monkey, and Porcine etc (Burtonboy et al., 1979; Carmichael et al., 1980; Parrish et al., 1988). Seroprevalance studies among CPV2a infected dogs was reported using haemagglutination inhibition assay in North Korea (Klingeborn & Moreno-López, 1980; Deepa & Saseendranath, 2012). Two diagnostic assays was compared for their sensitivity and specificity and found that diagnostic accuracy of the ELISA was significantly greater than the IFA (Luren et al., 2012). However, the serology based diagnostic assays must be used with caution because in most of the cases dogs may show positive serological tests for CPV-2. This may happen due to administration of CPV-2 vaccine in large dog population and also the endemic nature of virus in several areas which may lead to inapparent infection and generation of antibody titer. Monoclonal antibodies based antigenic typing of canine parvovirus (CPV2a and CPV2b) was reported (Shankar et al., 2014). A laboratory technique used to evaluate the binding of an antibody to its antigen. Counter immuno electrophoresis uses electric field in diffusion medium which is made up of polyacrylamide gel or agarose. The electric field facilitates the rapid migration of antibody and antigen towards each other so that line of precipitation will form at earlier than simple diffusion reaction. The line of precipitation indicates the binding of antibody with antigen hence positive result. Mixed infections for coronavirus antigen with canine parvovirus was detected by counter immuno electrophoresis in fecal samples (Ganesan et al., 1990). The prevalence of canine parvovirus infection was reported in clinically suspected dogs AGID and CIEP (Deepa & Saseendranath, 2012). 2.4 Latex agglutination test In latex agglutination tests the latex micro beads are coated with microbes specific either antigen or antibody, which can be used for detection of either microbe specific antibody or antigen in agglutination reaction. Positive result is detected by visualization of agglutination reaction which is characterized by clumping of micro beads with microbial antigen or antibody. Bodeus (1988) detected the CPV specific antibodies from field sample through latex agglutination test. Moreover, a new technology called SAT-SIT technology can be used for rapid detection of several other emerging hemagglutinating viruses from animals and humans (Marulappa & Kapil, 2009). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 2.6 Fluorescent antibody test In fluorescent antibody test an antibody tagged with fluorescent dye is used for detection of specific antigen. Based on tagging of fluorescent dye either on primary antibody or secondary antibody, the test may be either direct or indirect fluorescent tests. In direct fluorescent antibody test, the antibody binds directly with specific antigen and gives specific fluorescence signals for antigen detection. Fresh frozen tissues and formalin fixed are used to detect CPV using immunofluroscence (IFA) and immunoperoxidase (PAP). PAP gives more permanent, high resolution and clear intracellular localization of antigen than IFA (MaCartney et al., 1986). A semiquantitative ELISA and an immunofluorescence assay (IFA) were conducted for senstivity and specificity of canine parvovirus (Gray et al., 2012). An indirect fluoroimmunoassay using magnetic protein micro bead was validated for identification of antibodies against canine viruses such as CPV, CDV and rabies (Wang et al., 2011). 282 Minakshi et al Table 1 PCR assays developed for detection of canine parvovirus. S. No 1. Technique VP2 gene amplified by PCR and cloned in pTargeT mammalian expression vector. VP2 gene was selected on the basis of restriction enzyme analysis and further confirmed by sequencing. The present work has shown that the recombinant plasmid could be used as DNA vaccine against canine parvovirus infection. Epidemiological study of canine parvovirus infection and analyzed by PCR assay. CPV-2a strains detected by PCR-RFLP Gene/ target VP2 gene Reference Gupta et al., 2005 sequences specific for CPV variant strains VP2 gene Behera et al., 2015 VP2 gene 5. PCR was carried for VP2 capsid gene to detect all types of CPV, (CPV-2a/2b/2c) including the new CPV-2c Genomic typing of canine parvovirus using PCR 6. CPV and its variants typing using PCR 7. Characterization of canine parvovirus by PCR 9. Analysis of VP2 gene sequences of canine parvovirus isolates 10. molecular characterization and phylogenetic analysis of canine parvovirus by PCR Typing of canine parvovirus using mini-sequencing based SNP analysis Detection of canine parvovirus by PCR assay 2. 3. 4. 11. 12. sequences specific for CPV variant strains sequences specific for CPV variant strains sequences specific for CPV variant strains VP2 gene sequences specific for CPV variant strains sequences specific for CPV variant strains VP1 and VP2 gene Demeter et al., 2010 Silva et al., 2013 Costa et al., 2005 Shankar et al., 2014 Pereira et al., 2000 Chinchkar et al., 2006 Mohan Raj et al., 2010 Naidu et al., 2012 Singh et. al., 2013 2.7 Enzyme Linked Immunosorbent Assay 3.1 Polymerase Chain Reaction (PCR) The IgM antibodies indicate the recent infection of pathogen. These antibodies were derived in a number of laboratories; all appear to bind to the amino-terminal region of the major core protein. The sensitivity of ELISA tests is found to be much higher that other serological assays such as immunodiffusion test, HA or HI test (Banja et al., 2002). The sensitivity and specificity of sandwich ELISA for detection of CPV in dog fecal sample was found much higher than HA test (Rimmelzwaan et al., 1991; Drane et al., 1994). A point-ofcare ELISA test kit yielded accurate results and highly sensitive and specific for detection of both CPV as well as CDV infection under field conditions. The Point-of-care ELISA system was used for identification of antibodies against CPV and CDV. This assay can be useful in animal vaccination programme and their care and management for outbreak of such disease (Litster et al., 2012). CPV antigens can also be identified in fecal samples by Sandwich ELISA (Deka et al., 2015) PCR is a modern diagnostic assay which utilizes the specific amplification of desirable DNA sequence using template specific primer and DNA polymerase enzyme. It can also be used for diagnosis of those pathogens which may not be grown in laboratory condition. PCR assay has been used for diagnosis of several animal and human viruses. It can also detect viruses at early stage of infection before eliciting immune response and onset of clinical symptoms. Thus PCR may help in formulating policies for control and prevention of disease at early (Sharma et al., 2012). It can detect CPV from a samples having very minute quantity of virus. This assay is also much rapid and specific that gel filtration test. The samples having fecal inhibitory substances can be passing through spin column to remove inhibitory substances (Uwatoko et al., 1995). Molecular typing of CPV was done by using PCR based assays and CPV-2a and CPV-2b types were detected (Gauri et al., 2012). The PCR is a rapid, sensitive and specific method for detecting canine parvovirus (Savi et al., 2010, Figure. 2A, B). There are different researcher were used the PCR techniques for the diagnosis and detection of canine parvovirus as Table 1. Now a says several modifications of PCR such as multiplex PCR, Real-time PCR, nested PCR etc are used for molecular detection of viruses. Moreover, PCR amplicons can be used for nucleic acid sequencing and phylogenetic study for confirmatory diagnosis and tracking evolutionary history of virus. 3 Nucleic Acid Based Methods Various nucleic acid based detection technique has been developed for the confirmation of CPV in the clinical samples. These techniques are fast, sensitive and specific and are discussed below: _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Canine parvovirus- an insight into diagnostic aspect 283 Figure 2 A) Genotyping of CPV isolate 2a; Lanes: M-100bp Marker, lane 3: control, lane 2: vaccine lane 1: field samples; B) genotyping of CPV isolate 2b, lanes: M-100bp Marker, lane 1: control, lane 2: vaccine lane 3 to 5: field samples 3.2 Multiplex PCR Multiplex PCR utilizes the power of PCR using several primer sets of different amplicons size for different pathogens in a single reaction. Multiplex PCR enables the presence of nucleic acids from several pathogens to be checked for in one test, but care must be taken to avoid interference between primer pairs or templates. It is a time as well as cost effective methods because it can detect several pathogens simultaneously. Multiplex PCR assay can also be used for simultaneously identification of canine Leptospira sp and CPV (Ramadass & Latha, 2005). The CPV-2a and CPV-2b strains were also differentiated using multiplex PCR assay (Parthiban et al., 2010). 3.3 Real-Time PCR The real-time PCR is used for quantification of PCR product in reaction which can be used for estimation of viral load in sample. TaqMan assay based Real time PCR (RT-PCR) has been used for the detection of CPV-2 DNA in sample and an attractive tool for revealing single nucleotide polymorphisms in the capsid protein gene between CPV types 2a and 2b and CPV types 2b and 2c (Decaro et al., 2006). The advantage of the real time PCR is that there is no need to analyse the PCR product by agarose gel electrophoresis. Everything will be graphically shown on the monitor of the computer. Another advantage is that amount of the DNA present in the sample can be quantitated. Recently, SYBR Green based real time PCR has been developed for quantitation of CPV-2 variants in faecal samples of dogs (Kumar et al., 2010). Canine parvovius infection was detected in feces of free-ranging wolves using real time PCR and the assay was 100% sensitive and specific with a minimum detection threshold level (David et al., 2012). RT-PCR method was used for the amplification of rotavirus RNA, BTV as well as for CPV viruses using Taqman probe _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org and SYBR Green chemistry (Decaro et al., 2005; Anamul et al., 2015; Feng et al., 2015). The SYBR Green-based real-time PCR assay was used for the amplification of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/μL of target DNA per reaction and this study have been used successfully in veterinary diagnostic laboratory and have been helpful tools for the diagnosis and quantification of parvovirus infection in canines, felines and swine (Lin et al., 2014). 3.4 Multiplex Real-Time PCR The term multiplex real-time PCR is used to describe the use of two to four fluorogenic oligoprobes for the discrimination of multiple amplicon. To date, there have been only a few truly multiplexed realtime PCR assays described in the literature. The use of non-fluorescent quenchers and the continuous development of better light sources in the machines are now in use and first applications for virus detection are becoming available. The vp2 gene based Multiplex Real-Time PCR was validated for simultaneously identification of CPV, FPV and PPV. Multiplex real time PCR have been used to detect and quantify CPV (Decaro et al., 2007; Wei et al., 2009; Zhao et al., 2013). Further, Kaur et al., 2016 reported that multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types. 3.5 PCR-Restriction Fragment Length Polymorphism (PCRRFLP) RFLP uses specific restriction enzymes for study of restriction pattern of viral nucleic acid. However, this is a time consuming technique. However, through PCR small quantity of viral nucleic acid can be amplified and used for RFLP analysis. The RFLP technique was successfully used for differentiation of CPV-2 antigenic variants (Savi et al., 2009; Zhang et al., 2010). 284 Minakshi et al Figure 3 A) Insilco RE of 747bp amplified product, M-100bp Marker, lane 1: undigested 747bp product, lane 2: vaccine digested to give548, 149 and 50bp products: lane 3& 4: field isolates digested to give 402bp, 148 and 145bp 50 bp products. B) Restriction enzyme digestion of 747 bp PCR product Lane M: 100bp ladder (MBI Fermentas) 1: Uncleaved PCR product of Canine parvovirus field strains (747bp) 2: PCR product of vaccine strain digested by Rsa I to give 548,149 & 50 bp products 3-5: PCR product of samples digested by Rsa I to give 402,149,146; & 50 bp . 6: Water control. RFLP technique was also engaged in differentiation of CPV-2b and CPV-2c strains (Gauri et al., 2012). The partial VP2 gene specific PCR assay was standardized with corresponding consensus primers to amplify the desired length (747bp) of product. The PCR assay was carried out using the published partial VP2 gene specific primers (Sakulwira et al., 2001). Amplified 747 bp product was used for in silico restriction enzyme profiling. Reference sequences for vaccine and field strains were retrieved from the NCBI and loaded into the Insilco restriction enzyme profiling software (SNAP GENE SOFTWARE) & the resultant profiles were observed and images were obtained (Figure 3A). In wet-lab restriction enzyme profiling restriction enzyme RsaI (New England Biolabs) was used for template digestion. The different Restriction enzyme gave different RE digested products from both vaccines as well as field strain. The resultant digested products were resolved in 4% agarose gel electrophoresis (Figure. 3 B). 3.6 Peptide nucleic acid-based (PNA) array Peptide nucleic acid (PNA), are considered as a stable nucleic acid analogue. It contains pseudo-peptide skeleton in place of sugar phosphate backbone which is chemically and biologically highly stabile. PNAs hybridize to cRNAs or cDNAs more efficiently than DNA. It possibly happens due to electrically neutral nature of PNA backbone. Peptide nucleic acid-based (PNA) array was used to discriminate between the four CPV-2 antigenic types (CPV-2, -2a, -2b, and -2c) during ante-mortem diagnosis of dogs, using newly developed PNADNA hybridization assay. The PNA array has high sensitivity and specificity compared with a real time PCR using the TaqMan assay, a gold standard method (An et al., 2012). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 3.7 Nucleic acid hybridization assay The VP1 and VP2 protein specific digoxigenin labeled probe have been developed for detection of CPV. This probe may also be used for in situ hybridization and detection of CPV from immobilized tissue samples in formalin and paraffins (Nho et al., 1997). Further, Decaro et al. (2005) develop two minor groove binders (MGB) fluorophores (FAM and VIC) labeled probe for rapid quantification of CPV-2 variants in dog fecal samples. The MGB probe was able to detect SNPs in CPV 2a/2b and 2b/2c. Both the MGB probe assays were found to be highly specific, sensitive and reproducible as compared to other methods used to detect the virus. 3.8 Loop-mediated isothermal amplification (LAMP) assay LAMP assay is considered as alternative to conventional nested PCR. LAMP assay can be used for detection of DNA of several viruses of animal and human origin. In comparison to nested-PCR, LAMP assay are proved to be more rapid, sensitive and fairly reproducible method. It did not amplify other canine pathogens (Parthiban et al., 2012). Detection of canine parvovirus in fecal samples was reported using loopmediated isothermal amplification (Cho et al., 2006). A detection system based on the application of LAMP in conjunction with ELISA and LFD for convenient visual detection of CPV with high sensitivity and specificity was developed (Sun et al., 2014). Mukhopadhyay et al. (2012) standardized a highly sensitive and specific LAMP assay for detection of CPV DNA from fecal samples. The assay showed result within one hour. Recently, VP2 gene based LAMP PCR assay has been developed (Figure 4). The assay is 30 times more sensitive than conventional PCR (unpublished data). Canine parvovirus- an insight into diagnostic aspect 285 Figure 4 A) Hydroxy Napthol Blue based visual LAMP assay for colorimetric discrimination of positive and negative samples for parvovirus B) PCR amplification of LAMP assay; 2-10 Tenfold serial dilutions of template 109 to 101, 11: Clinical CPV sample 12: Negative control, 1&13: molecular Marker. 3.9 Whole genome amplification and sequencing Complete coding sequence of canine parvovirus genome could be amplified by sequence specific primer. Pick-primer software is available in the NCBI BLAST to design these primers. To attain the maximal coding sequences overlapping of primers with their forward and reverse sequence is preferred. Optimization of PCR reaction mixture and thermocyclic conditions is very important for amplification. The annealing temperature may vary with each primer and it should be standardized for the amplification. The products resolved in 1% agarose gel to observe the amplification (Figure 5). 3.10 Phylogenetic analysis The nucleotide sequences of pathogens are used for phylogenetic study which shows its evolutionary relationship and closeness with other strains of same or different virus species. The phylogenetic studies of CPV vaccine strains in India have been done (Nandi et al., 2010). Phylogenetic studies also revealed the fact that Indian CPV variants are closely related among themselves. The CPV variants also showed little divergence from their ancestor MEVs (Singh et al., 2014). The sequences were aligned against the other published CPV VP1/VP2 gene sequences using software from DNASTAR. The amino acid sequence, phylogenetic maps and percentage homology were deduced and analyzed from the sequences using the same software. The phylogenetic tree revealed that CPV2 and both CPV vaccine strains were in separate monophyletic group. The VP gene sequences of the Haryana isolate and gene sequences of various global isolates were used for phylogenetic analysis. The phylogenetic tree developed on the basis of VP gene indicated that the Hisar isolate is clustering with the Chinese isolate (Acc. No. JQ686671.1) as a separate group than rest of the Chinese isolates, Russian and USA isolates which indicates that the Hisar field isolate and the Chinese isolate originated from a common ancestor CPV (Figure 6). Figure 5 whole genome amplification of CPV field isolate (sample no; 915/H) by primer walk technique. M-100bp Marker, lane 1to 9: Amplified primer products by primers 2 to 10, lane 10: primer 12 th amplified product. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 286 Minakshi et al Figure 6: Phylogenetic tree based on VP gene obtained from Hisar isolate to that of global isolate. 3.11 Biosensor Biosensor is an analytical device which converts biological responses to electrical signals. It is used for rapid diagnostic method which helps to detect disease in low sample with high selectivity and specificity in seconds. Disadvantage is that heat sterilization is not possible. A biosensor is also developed to detect CPV infection using Quartz Crystal Microbalance (QCM) biosensor and prolinker B to rapidly diagnose CPV infection. ProLinker™ B enables antibodies to be attached to a gold-coated quartz surface in a regular pattern and in the correct orientation for antigen binding. QCM biosensor is 95.4 % sensitive and 98% specific compared to PCR. It is rapid and accurate clinical diagnostic tool for CPV infection (Kim et al, 2015). 4 Prevention and control The prevention and control of CPV infection depends primarily on an effective immunization program; but disinfection, animal movement control and husbandry practices also must be considered especially in shelters. In most of the cases of CPV treatment supportive therapy is used which is based on suppression of symptoms and prevention of further complications. Since disease is very acute the supportive intravenous fluid therapy should be started as soon as possible. The dog may recover within 2-3 days. However, the treatment may not always be successful. Care should be taken that infected dogs should not be allow to come in contact with other healthy dog, because CPV may infect other healthy dog easily. The disease can be prevented through proper vaccination. However, vaccination cannot be always successful because of prevalence of a large number of distinct antigenic variants of CPVs. For CPV control live attenuated as well as inactivated vaccines are used. There has been extensive research on these vaccines and their use in protecting dogs (Appel et al., 1979; Carmichael et al., 1983). First vaccination to puppies should be given at 8 weeks of age. Later on, vaccination is done at every 3-4 weeks for up to 4 months. The disease caused by CPV-2c can be prevented by vaccination of puppies at 6 weeks of age (Glover et al., 2012). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org The hygiene in kennel should be appropriate for disease prevention, because CPV can be live on some surfaces years’ together. The bleaching solution in water in a ratio of 1:30 can be used to kill the CPV. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 BATS: CARRIERS OF ZOONOTIC VIRAL AND EMERGING INFECTIOUS DISEASES Koushlesh Ranjan1,*, Minakshi Prasad2 and Gaya Prasad3 1 2 3 Department of Veterinary Physiology and Biochemistry, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India, 250110 Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India, 125004 Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India, 250110 Received – April 18, 2016; Revision – May 05, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).291.306 KEYWORDS ABSTRACT Bat Reservoir host Vector Zoonosis Emerging infectious disease Bats are reported as reservoir host for several viruses, which cause significant illness in human and animals. Some of the bat transmitted zoonotic viral diseases such as Ebola, Hendra, Nipah and rabies may cause severe human casualties. They also harbor several other viruses such as MERS and SARS corona viruses, which may cause disease in human through direct spillover to human or through an intermediate host or vectors. Being reservoir hosts bats do not get affected by these viruses. This probably may happen due to the specificity of bat immune system, which reacts differently with viral pathogens in comparison to their other mammalian counterparts. Although bats are important reservoir hosts for several zoonotic viruses, very little information is available regarding host/virus relationships as only few experimental studies have been done on bat colonies, lack of expertise for study of bat immunology and antiviral responses and difficulty in conducting field work. However, with the advancement in epidemiology and molecular biology, these problems can be addressed, which will provide the insight into interactions of bats and zoonotic viruses. It may also clarify regarding virus persistence in nature and various associated risk factors which might facilitate viral transmission to animals and humans. * Corresponding author E-mail: [email protected] (Koushlesh Ranjan) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 292 Ranjan et al 1 Introduction 2 Bat Immunology Bats are the most diverse, abundant and geographically dispersed member of vertebrate family. Despite enough information since ancient age, still reliable information is required to explain the diversity in their lifestyle, anatomy, role in ecosystems ecology and as reservoir hosts for viruses of medical and veterinary importance. Bats can survive in diverse climate. They are found in all continents except Antarctica. Different bat species may feed on several food materials such as mammals, blood, insects, fish, pollen and fruit. Bats are also recognized as reservoir hosts for several zoonotic viruses which can infect humans and animals (Hayman et al., 2013). Although they can transmit several zoonotic viruses, they are also valuable elements of terrestrial biotic communities. They play a significant role in insect control, pollination of plants and reseed the cut forests which are essential for survival of human and animal life (Hill & Smith, 1984; Kunz & Fenton, 2003). It is observed that several viruses which are highly pathogenic for human and animals can infect and persist in healthy bats without causing significant harm to them. Possibly it may be due to the fact that bats were evolved earlier among mammalian species and their acquired and innate immune responses have significant differences from other animal species such as rodents and primates. It is also assumed that bat’s immune system react differently with pathogens which lead to control virus replication with persistence of infectious virus in bat tissues (Schountz, 2014). This results in prevention of immunopathological responses in infected bat tissues. However, within several bat species significant differences in immune responses against viral infection may be found. Bats harbor a range of emerging infectious viral pathogens. Many of such emerging infectious diseases (EIDs) are zoonotic in nature (Woolhouse & Gowtage-Sequeria, 2005; Jones et al., 2008; Dhama et al., 2013). In developing countries, the zoonotic viral infections especially caused by RNA viruses such as rabies, Ebola etc. have been recognized as significant threats for human health (Maudlin et al., 2009; Dhama et al., 2015). In addition to rabies and other lyssaviruses (Streicker et al., 2010), bats have also been reported as reservoir hosts for several other viral pathogens such as Hendra virus (HeV) (Halpin et al., 2000; Edson et al., 2015), severe acute respiratory syndrome-coronavirus (SARS- CoV) (Li et al., 2005; Vijaykrishna et al., 2007), Ebola virus (EBOV) (Leroy et al., 2005), Nipah virus (NiV) (Chua et al., 2002a; Chua et al., 2002b; Reynes et al., 2005) and Marburg virus (MARV) (Peterson et al., 2004; Towner et al., 2007). In USA, a new lineage of influenza A virus has also been reported from little yellow shouldered bats (Sturnira lilium) (Tong et al., 2012). Several other Paramyxoviruses have also been reported from bats from various regions of the globe (Drexler et al., 2012). Since, bats are reported as reservoirs for several viral EIDs (Table 1), it is crucial to understand that how bat ecology may influence zoonotic disease outbreaks and their role as reservoirs for emerging viral pathogens (Messenger et al., 2003; Calisher et al., 2006; Wong et al., 2007; Hayman et al., 2013). Several new viral pathogens are identified in bats every year which need to be characterized for their zoonotic potential to human population. Most of such studies are mainly focused on zoonotic infectious diseases of medical and veterinary importance. This review paper is focused on bat associated zoonotic viruses causing diseases to animals and human. Several bat species play important role in maintenance and transmission of zoonotic viruses which explains the requirement of special consideration for characterization of bats from other mammals. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Although very little is known about bat immunology, several studies have shown that bat immune responses also have some similarities with mammals that evolved after bats. Several immunoglobulin classes such as IgG, IgA, and IgM found in mammals have also been purified from great fruit-eating bat (Artibeus lituratus) sera (McMurray et al., 1982). The lymphoid development of bats and other mammals are also very similar which is evidenced by identification of B and T lymphocytes, Macrophages and cells expressing surface Ig in bone marrow of Indian bats (Pteropus giganteus) (Schountz et al., 2004). The serological assays against several viruses such as severe acute respiratory syndrome-coronavirus (SARSCoV) like viruses, Hendra virus and Ebola virus in bats (Lau et al., 2005; Leroy et al., 2005) indicate that virus specific adaptive B and T cell responses might occur despite persistent virus infection. However, further study is required to understand the mechanism of antibody synthesis, cytokine synthesis, lymphocyte proliferation etc. in bats. 3 Zoonotic viruses in bats Bats harbor several viruses as reservoir host. Many of these viruses have not been reported to transmit from bats to human or other mammals. However, several viruses of bats such as Nipah and Hendra virus, rabies virus and related lyssaviruses, SARS-CoV-like virus etc may be transmitted to human and animals and lead to highly pathogenic disease (Table 1). Some other viruses such as certain flaviviruses, alphaviruses and bunyaviruses may also infect bats via vectors. However, it is not established that whether bats act as important reservoir hosts for such viruses. 3.1 Rabies Virus A lot of scientific information is available regarding rabies virus, its transmission and pathogenesis in human and animals. Rabies was described in ancient literature in around 4000 years ago. However, its scientific study started in late 19th century. Louis Pasteur amplified the rabies virus in spinal cord of rabbit and prepared vaccine for post exposure prophylaxis. Bats: Carriers of zoonotic viral and emerging infectious diseases 293 Table 1 Zoonotic viruses causing disease in human and their bat reservoir hosts. S. No. 1 Disease Rabies Virus Rabies virus and other lyssaviruses Reservoir Host Several bat species distributed world wide 2 Ebola virus disease Ebolaviruses 3 Marburg virus 7 Marburg virus disease Middle east respiratory syndrome Severe acute respiratory syndrome Severe acute febrile disease Encephalitis Franquet’s epauletted fruit bat (Epomops franqueti), Hammer headed bat (Hypsignathus monstrosus), little collared fruit bat (Myonycteris torquata) Egyptian fruit bat (Rousettus aegyptiacus) Egyptian tomb bat (Taphozous perforatus) Chinese horseshoe bat (Rhinolophus spp.) Rousettus spp. 8 Encephalitis Tioman virus Pteropus hypomelanus 9 Menangle disease Menangle virus Little red flying foxes and gray headed flying foxes 4 5 6 virus MERS-CoV SARS-CoV Sosuga virus Nipah viruses and Hendra Rabies virus belongs to the family Rhabdoviridae, genus Lyssavirus and transmitted between several mammals, including bats. Rabies transmission is primarily mediated by bite inoculation of virus available in saliva of rabies infected individuals. Three species of bats viz. Diaemus youngi (whitewinged vampire bat), Diphylla ecaudata (hairy-legged vampire bat) and Desmodus rotundus (vampire bat) have been reported to be involved in rabies transmission. However, further studies have shown that mainly Desmodus rotundus (vampire bat) is important in rabies transmission (Turner, 1975; Anderson et al., 2014). In USA, bats have been reported as reservoir vector in over 90% of human rabies cases. Among bats tricolored bat (Perimyotis subflavus) are reported as major reservoir host (Gilbert et al., 2015). The bat rabies virus variants isolated from Latin America in free tailed bats (genus Tadarida) and vampire bats (Desmodus rotundus) have been found to be close to earliest rabies virus. The study also suggest that adaptation of rabies virus in bats occurred earlier in colonial genera (Myotis and Eptesicus) than in bats of solitary genera (Pipistrellus, Lasionycteris, and Lasiuris) (Hughes et al., 2005). Globally, approximately 55,000 annual human deaths are caused by rabies virus which can be associated with bats (Knobel et al., 2005). Rabies viruses of bat origin may sporadically spill over to infect human and other mammals. It has been reported in USA that most rabies victims do not recall the incidence of bitten by bat. This may be due to unusual circumstances during bat bite or being small size of the biting animal (Rupprecht et al., 2004). Recent studies also suggest that all rabies virus variants affecting terrestrial carnivores _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Some flying foxes (Pteropus spp.) References Rupprecht et al., 1995; Calisher et al., 2006; López-Roig et al., 2014 Leroy et al., 2005 Towner et al., 2007 Memish et al., 2013 Lau et al., 2005 Albarino et al., 2014; Amman et al., 2015b Chua et al., 2002; Halpin et al., 2000 Chua et al., 2001; Yaiw et al., 2008 Philbey et al., 1998; Barr et al., 2012 might be originated from cross-species transmission and genetic exchange from bat associated rabies virus. 3.2 Other lyssaviruses Bats are also reservoir for several other lyssaviruses including Duvenhage virus (DUVV), Shimoni bat virus (SHIBV), Irkut virus (IRKV), West Caucasian bat virus (WCBV), Australian bat lyssavirus (ABLV), European bat lyssavirus 1 (EBLV-1) and European bat lyssavirus 1 (EBLV-2). EBLV-1 and EBLV2 are reported in Europe from Eptesicus fuscus and Myotis spp of bat respectively. Some of the sporadic cases of human rabies have been reported from EBLV-1 and EBLV-2 (Kuzmin & Rupprecht, 2007; Kuzmin et al., 2011). However, in terrestrial mammals some of the sporadic cases of EBLV-1 infection were also reported which might be a potential source for human exposure (Dacheux et al., 2009). In France, neutralizing antibodies against EBLV-1 were detected in six species (Pipistrellus pipistrellus, P. kuhlii, Hypsugo savii, Plecotus austriacus, Eptesicus serotinus and Tadarida teniotis) of bats (López-Roig et al., 2014). Recently, in Germany, EBLV-1 and EBLV-2 were detected from Eptesicus serotinus and Myotis daubentonii bat species (Schatz et al., 2014). The complete genome sequences of EBLV-1 have been extracted from Eptesiscus isabellinus bat in Spain (Marston et al., 2015). Some of the insectivorous bat species such as Murina leucogaster harbor IRKV (Botvinkin et al., 2003). IRKV may also cause human rabies. IRKV was reported from a human rabies case in Russia. The human patient was a victim of an 294 insectivorous bat bite (Leonova et al., 2010). Some of the suspected human rabies cases caused by IRKV were also detected in Ukraine and China (Botvinkin et al., 2006). IRKV was also first time isolated in China from brain of northeastern bat (Murina leucogaster) which showed maximum nucleotide and amino acid identity with IRKV isolated from Russia. Virus produced rabies like symptoms in adult mice (Liu et al., 2013a). On experimental pre-exposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) analysis with rabies vaccine against IRKV in hamster model showed that routine PrEP with three doses of vaccine may generate complete protection. However, for complete protection from IRKV higher doses of PEP agent such as anti-rabies immunoglobulins are required (Liu et al., 2013b). The WCBV was isolated in south-eastern Europe from insectivorous bat Miniopterus schreibersii. Since, WCBV are most divergent Lyssavirus, all the anti-rabies biological are inefficient in providing protection against this virus (Hanlon et al., 2005). Although, the public health significance and ecology of WCBV is still unknown, the experimental infection in bats and laboratory animals, developed typical rabies symptoms which led to death (Kuzmin et al., 2008). Other member of Lyssavirus, SHIBV was also isolated from a bat (Hipposideros commersoni) (Kuzmin et al., 2011). The biological significance of SHIBV for public health is unknown. However, they may cause pathogenesis in experimentally infected laboratory animals, which develop rabies and finally die (Markotter et al., 2009; Kuzmin et al., 2010). Due to their antigenic differences, current rabies biologicals cannot protect from SHIBV (Hanlon et al., 2005). DUVV also causes dreadful human rabies in Africa. Despite availability of anti-rabies biological, it still causes human casualties because of inadequate knowledge of disease. Some of insectivorous bat species such, Miniopterus sp may transmit DUVV to human (Markotter et al., 2008). In 2007, a Dutch tourist was bitten by a bat in Kenya. The patient was allowed for medical help. However, due to lack of adequate anti-rabies PEP administration, rabies symptom was developed and patient died from DUVV infection (van Thiel et al., 2009; Koraka et al., 2012). The ABLV was discovered in ‘rabies-free’ Australia in 1996. The ABLV was identified first in black flying fox (Pteropus alecto) (Fraser et al., 1996). Now, it is assumed that all bats in Australia may potentially carry ABLV (http://www.health.nsw.gov.au/). Later on, it was reported that some of the insectivorous bat species such as Saccolaimus flaviventris may also harbour ABLV (Gould et al., 2002). Two fatal human cases of ABLV infection with clinical symptoms compatible with rabies have been detected (Gould et al., 2002; Warrilow et al., 2002). 3.3 Henipavirus (Hendra and Nipah virus) _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ranjan et al An outbreak of acute respiratory illness was reported in human and horses during 1994 to 2004 in Hendra, Australia (Field et al., 2011). The etiological agent reported was from genus Henipavirus and family Paramyxoviridae. Later on it was named as Hendra and Nipah virus (Murray et al., 1995). Several bat species such as fruit bats (flying foxes) of the genus Pteropus, including gray headed flying fox (Pteropus poliocephalus), black flying fox (P. alecto), spectacled flying fox (P. conspicillatus) and little red flying fox (P. scapulatus) were reported as probable reservoir hosts of Hendra virus (Field et al., 2011; Wang et al., 2013). The qRT-PCR assay showed that P. alecto is potent reservoir host than P. poliocephalus and P. scapulatus for Hendra virus in Australia (Edson et al., 2015). However, a little knowledge is available about the dynamics of Hendra virus infection and maintenance in bat. The horses probably get Hendra virus infection from flying foxes by spillover (Field et al., 2011). The periodic outbreaks of Hendra virus in local flying fox population lead to an increased incidences of spillover infection to horses. The Hendra virus infection in flying foxes increases when threshold number of susceptible flying foxes is reached and virus enters the flying fox population from a nomadic individual or group. This concept was well studied for related morbilliviruses (Bolker & Grenfell et al., 1996; Swinton et al., 1998). Nipah virus was isolated form adult male human and pigs showing symptoms of respiratory illness, fever and encephalitis in Malaysia in 1999 (Chua et al., 1999). The disease was found highly fatal for human patients. Further, investigation showed that most of the human patients were having history of direct pig contact (Chua et al., 2000). Later on, variable flying fox (Pteropus hypomelanus) and large flying fox (P. vampyrus) were found as natural reservoir hosts for Nipah virus (Chua et al., 2002a; Chua et al., 2002b). Nipah virus associated disease was also reported from human in Bangladesh (Sazzad et al., 2013; Chakraborty et al., 2016). Nipah virus outbreak in Bangladesh was very similar to Malaysian outbreak in several aspects such as fever, central nervous system signs, delayed recognition and a high case fatality rate. However, in Bangladesh human cases were not directly associated with disease in pigs, and some evidence of human to human transmission was also reported (Hsu et al., 2004). The serological surveys in Bangladesh suggested that Nipah virus is transmitted by only the Indian flying foxes (Hsu et al., 2004). Nipah virus infections were also reported from human in India (Chadha et al., 2006). Later on, neutralizing antibodies against Nipah virus was also reported from large flying foxes in Cambodia (Olson et al., 2002) and Indonesia (Sendow et al., 2006). Thus, henipaviruses are reported from human and bats in several countries across the globe (Halpin et al., 2000). The detail molecular genetics study also evidenced that Nipah and Hendra viruses are circulating in their natural hosts, flying foxes since ancient days (Gould, 1996). However, the recent outbreak of Nipah and Hendra virus in human population suggests some major changes in behavior and habitat change in bats. The emergence of flying fox populations under stress Bats: Carriers of zoonotic viral and emerging infectious diseases conditions due to habitat loss altered the foraging and behavioral patterns which results in virus niche expansion and closer proximity to livestock and human population. This may be the pathway of Nipah virus outbreak in human (Chua et al., 2002a). 295 transcription-PCR (RT-PCR) from some of the seronegative animals suggesting acute infection. However, the continuous virus shedding from seropositive animals also suggested the presence of persistent infections in some animals (Guan et al., 2003). Further study also proved the palm civets act as an incidental host for SARS-CoV rather than principal host. 3.4 Menangle and Tioman Viruses Menangle virus of genus Rubulavirus and family Paramyxoviridae was originally isolated from stillborn piglets near Menangle in Australia in 1997 (Philbey et al., 1998). The affected litters were characterized by mummification, autolyzing, stillborn and live piglets. Several teratogenic defects such as brachygnathia, arthrogryposis and kyphosis were also reported (Barr et al., 2012). It has been proved that Menangle virus has significant tissue tropism for secondary lymphoid organs in pigs and humans and for intestinal epithelium in weaned piglets (Bowden et al., 2012). Serological analysis of persons in contact with the infected pigs also showed the high titers of antibodies against Menangle virus along with clinical signs of febrile illness with measles like rash. However, none of the persons were in direct exposure to flying foxes (Chant et al., 1998). Further study showed that bats living in mixed colonies of little red flying foxes and gray headed flying foxes near the pig farm had neutralizing antibodies against the virus (Philbey et al., 1998). Although the virus isolation from flying foxes were unsuccessful, the paramyxovirus like virion particles labeled with antibody against Menangle virus was reported from flying fox feces collected near the pig farm and a convalescent sow by electron microscopy. The Tioman virus is a rubulavirus and is distinct from Menangle virus. It is antigenically related to Menangle virus and harboured by Pteropid fruit bats (Yaiw et al., 2008). It was isolated from variable flying foxes in Malaysia. It was discovered accidently during identification of natural host of Nipah virus which caused large scale outbreaks of encephalitis in pigs and humans in Singapore and Malaysia in 1998-1999. It is a newly recognized paramyxovirus and little is known about its pathogenesis and host range (Chua et al., 2001). 3.5 SARS-CoV like viruses An unrecognized corona virus from family Coronaviridae was reported as causative agent of severe acute respiratory syndrome in humans in 2002 (Rota et al., 2003). The virus was later named as severe acute respiratory syndrome-corona virus (SARS-CoV), which is a distant relative of group 2 coronaviruses of rodents, dogs, cattle, pigs, and humans (Gorbalenya et al., 2004). The epidemiologic studies suggested that SARS outbreaks were directly associated with wildlife meat industry. The SARS-CoV like viruses were also isolated from some of the wildlife species such as raccoon dogs (Nyctereutes procyonoides) and masked palm civets (Paguma larvata). The SARS-CoV specific antibodies were also detected in hog badger (Arctonyx collaris) in China (Guan et al., 2003). The viral RNA was detected by reverse _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Later on, it was reported that some of the bats (Chinese horseshoe bats; family Rhinolophidae and genus Rhinolophus) possessed either antibody against SARS-CoV or infected with SARS-CoV like viruses (Li et al., 2005). The genome sequences of SARS-CoV from humans and civets were also found phylogenetically close to bat SARS-CoV like viruses (Li et al., 2005). It suggests the origin of humans and civets SARS-CoV is associated with bat viruses in China. Further study also suggest origin of human SARS-CoV might be from unrecognized SARS-CoV like virus of bat origin which was transmitted to amplifying hosts viz. raccoon dogs, masked palm civets and hog badger and spilling over to human population through close contact with these animals or their tissues. Later on adaptive mutations in virus genome lead to human to human transmission of virus (Song et al., 2005). The disease potential of a SARS like virus, SHC014-CoV from Chinese horse shoe bat (Rhinolophidae) was studied using reverse genetics system where a chimeric virus was prepared which expressed spikes of bat coronavirus SHC014 in a mouse adapted wild type SARS-CoV backbone (Menachery et al., 2015). In mouse, chimeric virus developed severe pathogenesis which was found untreatable with anti-SARS immunotherapeutics. Moreover, chimeric virus replicated in primary human airway cell line and produced an equivalent titer of SARS-CoV outbreak from human (Ge et al., 2013; Menachery et al., 2015) which indicates a vital threat of reemergence of human SARS-CoV from wild bat population. 3.6 Middle East respiratory syndrome (MERS) MERS causes severe respiratory illness in human. MERS was first time reported from Saudi Arabia in 2012 (Bermingham et al., 2012). Later on, it has been spread to several other countries. Most people suffered with this disease develop symptoms of severe acute respiratory illness such as cough, fever and shortness of breath. MERS is caused by a corona virus called MERS-CoV. For MERS the case-fatality rate is reported as about 45%.It may cause infection to pregnant woman and develop severe respiratory signs (Alserehi et al., 2016). MERS-CoV and SARS-CoV are very similar, which suggests that bats may also play a role in transmission of MERS CoV to human population. The partial RNA sequence of betacoronavirus from faecal pellet of an Egyptian tomb bat Taphozous perforates showed 100% nucleotide identity with virus isolated from human index case patient (Memish et al., 2013). One of the camel species (Camelus dromedarius) may harbor this virus in nature, because MERS-CoV can be experimentally established in camel (Adney et al., 2014; Raj et al., 2014; Omrani et al., 2015). 296 3.7 Ebola Virus The Filoviridae family of virus consists of genus Ebolavirus (Ebola Sudan virus, Ebola Zaire virus, Ebola Reston virus and Ebola Ivory Coast virus) and Marburgvirus (Marburg virus). The natural reservoirs of these viruses are not yet confirmed. However, the RNA genome of Ebola virus has been identified in terrestrial mammals in Central African Republic (Morvan et al., 1999). Ebola virus may cause highly fatal haemorrhagic disease in human, which may also infect other mammals (Dhama et al., 2015). The high viral loads in body fluids allow virus transmission from human to human (To et al., 2015). A serious Ebola virus outbreak was started in December 2013 in West Africa which also reached to other continents (Gumusova et al., 2015). Experimentally, Ebola Zaire virus was also replicated in little free-tailed bat (Chaerephon pumilus), Angola free tailed bat (Mopscondylurus) and Wahlberg’s epauletted fruit bat species (Epomophorus wahlbergi) (Swanepoel et al., 1996). The serological surveillance also showed presence of IgG immunoglobulin in 4% of bat population of six species viz. Hypsignathus monstrosus, Epomops franqueti, Myonycteris torquata, Mops condylurus, Micropteropus pusillus and Rousettus aegyptiacus (Pourrut et al., 2009). Later on Ebola virus RNA was also detected in spleen and liver tissues of some fruit bats species viz. Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata (Leroy et al., 2005). The qPCR assay have successfully detected the Reston ebolavirus (RESTV) specific RNA segments from oropharyngeal swabs of several bat species (Miniopterus schreibersii, M. australis, C. brachyotis and Ch. plicata) from Philippines (Jayme et al., 2015). The detection of Ebola virus RNA from bats is a fascinating finding, but only based on nucleic acid detection it is difficult to establish the bat as reservoir host. It is also suggested that there might be a nonpathogenic undetected Ebola virus spreading in bat population which may give rise to pathogenic strain by mutations in other mammals (Monath, 1999). However, until and unless virus is isolated from bat species, the experimental infections unambiguously demonstrate that virus is persisting as well as transmitting from bat species to other mammals. 3.8 Marburg virus Marburg virus was first reported from an epidemic in Frankfurt and Marburg in Germany and Belgrade in the former Yugoslavia. Marburg virus belongs to Filoviridae family. It causes highly fatal disease in human called Marburg virus disease (MVD). Although it is a rare disease, it may cause high fatality in human during outbreak. The case fatality rate of MDV was reported from 25% in the initial laboratory based study in 1967, to more than 80% during outbreaks in Democratic Republic of Congo in 1998-2000 and in Angola in 2005 (http://www.who.int/csr/disease/marburg/en/). This virus is transmitted either by direct contact with the tissues, blood and other body fluids of infected persons or handling dead or ill infected animals such as fruit bats and monkeys. Some of the study in Uganda showed that fruit bat of Rousettus _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ranjan et al aegyptiacus species might be a natural reservoir for Marburg virus (Amman et al., 2012). The Marburg virus specific IgG and nucleic acid (RNA) was detected in naturally infected individual fruit bat (Rousettus aegyptiacus) in Gabon indicating the Rousettus aegyptiacus as natural reservoir for Marburg virus (Towner et al., 2007). Later on, serological surveillance also revealed the presence of antibody against Marburg virus in 1% of bat population of Hypsignathus monstrosus and Rousettus aegyptiacus species (Pourrut et al., 2009). The experimental infection of Marburg virus to Rousettus aegyptiacus species of bats also showed the wide distribution of virus in bat tissues followed by recovery of large quantity of viral RNA which suggested the natural reservoir potential of Rousettus aegyptiacus species of bat (Jones et al., 2015; Amman et al., 2015a). 3.9 Sosuga virus Sosuga virus is a novel paramyxovirus which may cause severe acute febrile condition in human. In 2012, a female wildlife biologist reported the malaise, fever, generalized myalgia, headache, arthralgia, neck stiffness and sore throat after a short field expedition for collection of bats and rodents in South Sudan and Uganda (Albarino et al., 2014). However, the patient recovered successfully with adequate medical support. The metagenomics studies of pathogen nucleic acid suggest that the etiological agent might be a novel paramyxovirus related to rubula like viruses of fruit bats origin (Albarino et al., 2014). The new virus was named as Sosuga virus (on name of South Sudan and Uganda). It was also established that virus is most likely originated in bats. However, the efforts to virus detection in African bats are still under way. To establish the fact regarding bat as potential reservoir, the bat tissues collected during the last three week period prior to onset of clinical symptoms were tested for presence of Sosuga virus (Amman et al., 2015b). It was reported that several Egyptian rousette bats (Rousettus aegyptiacus) were found positive for Sosuga virus. Further analysis of Egyptian rousette bat tissues collected from other locations in Uganda were also found positive for Sosuga virus (Amman et al., 2015b). This suggests that Egyptian rousette bats could be a potential natural reservoir for Sosuga virus. 4 Routes for transmission of bat-borne viruses to human Many of the bat associated viruses are restricted to specific geographical regions with availability of bat reservoir host, such as Egyptian fruit bats associated Ebola virus in Africa and flying foxes associated Hendra and Nipah virus in Australia and Southeast Asia. However, how bat transmit diseases to human is a mystery because most of the bat species remain away from human dwellings in tropical rain forests and in caves. The studies of bat transmitted zoonotic diseases revealed that most probably these diseases are transmitted to humans either via intermediate host or direct contact with bats (Figure 1). Therefore some of the hypotheses for transmission of bat borne disease to human have been proposed. Bats: Carriers of zoonotic viral and emerging infectious diseases 297 Figure 1 Common routes of transmission of bat associated EIDs between bats, animals and human. Thick arrows represent the most significant pathways whereas thin arrows represent less common or less known pathways for bat-associated EIDs transmission. 4.1 Transmission through direct contact Bats usually reside in dark caves and deep forests. Therefore the direct contact of bat with human is a rare incidence. However, people may get infection of bat associated viruses by bat bite and handling of live bats during capture and consumption of bat meat (Marí Saéz et al., 2015). The capture and selling of wild animals including bats increases the risk of zoonotic virus outbreak in human population (Figure 1). In 2007, Ebola hemorrhagic fever virus outbreak costs life of 186 human in Democratic Republic of Congo (DRC). The epidemiological investigation reported that infection reach to human population by consumption of infected fruit bats meat (Leroy et al., 2009). The transmission by direct contact or ingestion of food infected with bat droppings, is an important source because several viral nucleic acid have been extracted from bat droppings (Halpin et al., 2000; Marí Saéz et al., 2015). Sometimes, accidental bat bite may also result in human rabies. In South Africa human death was reported by Duvenhage virus (DUVV) infection by bat scratch (Adjemian et al., 2011). 4.2 Transmission through intermediate host It is proposed that bats may transmit disease to human through an intermediate host which is close to human and may amplify the virus. The remaining contaminated fruits eaten by fruit bats _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org may be consumed by intermediate hosts such as horses, pigs and non-human primates. Human may get infection from these intermediate hosts by direct contact or consuming their products. In tropical Australia and Southeast Asia, Hendra and Nipah viruses are transmitted by flying foxes. During Nipah virus outbreak in 1998 in Malaysia, it was hypothesized that pigs get infection of Nipah virus by consuming the half consumed mangoes by flying foxes. Mangoes were a major food for flying foxes, and half consumed mangoes contaminated by urine and saliva of bats was accidently consumed by pigs (Figure 1). This results in cross-species infection of pigs followed by subsequent infection to human (Chua et al., 2002a). Horses may get Hendra virus infection by consuming contaminated fruit, grass, feed or water by bat’s saliva, urine and feces and subsequently infection may reach to human (Plowright et al., 2015). Camels play major role in human life in Middle East countries for transportation as well as entertainment. It was hypothesized that dromedary camels act as intermediate host for MERS-CoV infection from bats to humans (Memish et al., 2014). MERS-CoV was also detected in camel milk (Reusken et al., 2014). Thus, virus may be excreted in milk and poses a high risk of infection for people either during milking process or consumption of unpasteurized milk. In 2003, severe SARS outbreak was reported in China. The SARS-CoV was transmitted from bat to palm civet and 298 subsequently to human (Liu, 2003). In Central Africa, Ebola virus was transmitted to apes by consumption of fruit contaminated by bats (Leroy et al., 2005). 4.3 Transmission through aerosol Bat may spread large number of viruses in air. Thus, air may get contamination by bat borne viruses especially in caves. People may get infection by bat borne viruses by inhalation of contaminated air (Figure 1). The lethal viral hemorrhagic fever outbreak in Cynomolgus macaques was reported by inhalation of aerosols containing Marburg virus (MARV-Angola) (Alves et al., 2010). Some reports suggest that human may get infection of Marburg virus by visiting in caves in Africa. The most probable route of transmission in this condition might be by aerosol transmission (Timen et al., 2009). 5 Isolation and characterization of virus Viruses from several tissues samples can be grown in a variety of cell culture system in laboratory. For molecular diagnostic study nucleic acid isolation from cell culture material is a good choice. Nucleic acid isolation followed by PCR assays is extremely rapid and sensitive technique. Several other sensitive diagnostic assays such as multiplex PCR, RT-PCR, Real-time PCR etc. are also used for viral emerging infectious diseases (EIDs) diagnosis (Rihtaric et al., 2010; Huang et al., 2012; Freuling et al., 2013; Suin et al., 2014). For identification of a newly recognized virus, PCR amplification of viral nucleic acid followed by nucleic acid sequence data analysis is used. The nucleic acid sequence data of viral pathogens are compared with available sequences in GenBank database (http://www.ncbi.nih.gov/GenBank/) to search for sequence similarities with nucleic acid sequences of known viruses. Moreover, recombinant viral proteins expressed in other expression system can also be used for serodiagnostic tests. During diagnosis of EIDs extra precaution should be taken to avoid misdiagnosis. For example, the first report of Nipah virus infection in Malaysia in 1999 was misdiagnosed as Japanese encephalitis virus (JEV) infection (Calisher et al., 2006). Although, all the human patients were of adult age male already vaccinated against JEV and pigs also suffered a fatal disease, the disease was misdiagnosed as JEV. Later on the failure of intensive vaccination in clinical disease control forced the medical and scientific community to think about new emerging disease. But, by the time there is a huge economic and human life loss was reported. Such incidences force us for certain degree of intellectual preparedness in terms of reagents, equipments and scientific knowledge that could be used for development of rapid diagnostic assays during outbreak of newly emerged viruses. 6 Diagnostic limitations Several diagnostic assays based on serological techniques such as ELISA, immunofluorescence assay etc and molecular techniques viz. PCR, Real-Time PCR, multiplex PCR, nucleic acid sequencing etc are available for sensitive detection of _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ranjan et al viruses of bat origin. However, for diagnosis of previously unrecognized viruses, new assays and reagents are required. For identification of new viruses PCR will be useful which needs knowledge of nucleic acid sequences of recognized bat associated viruses such as viruses of Mononegavirales order (family Bornaviridae, Filoviridae, Rhabdoviridae, and Paramyxoviridae) for suitable primer designing (Pringle, 1991). In addition, specific antibody conjugates may also be required for enzyme-linked immunosorbent assays or immunofluorescence assays to identify either virus specific antibodies in sera samples or antigens in tissue samples. Some of the classical methods such as hemagglutination or hemagglutination inhibition tests were also used for viral diagnosis. However, these assays are broadly cross-reactive. Several cell cultures and animal inoculations can also be used for virus isolation. However, for bat associated zoonotic viruses this technique is potentially hazardous, and it should not be used without appropriate biocontainment. With the advancement in molecular biology techniques for viral nucleic identification, virus isolation technique is not much appreciated. However, virus isolation technique will provide us virus in bulk quantity which may be used in many areas of research and development such as development of vaccines, suitable diagnostics and animal disease model to study the pathogenesis of virus. 7 Bats and emerging viruses More than 200 different viruses under 27 families are detected in some species of bats (Moratelli & Calisher, 2015). However, only few viral diseases such as SARS, MERS, Ebola virus disease etc are transmitted from bats to human (Moratelli & Calisher, 2015). Because a large proportions of bats under mammalian species (about 20%), their diverse habitats, biology and natural history, it assumed that bats may harbor several other viruses of human and animal importance (O'Shea et al., 2014; Brook & Dobson, 2015). However, the transmission of zoonotic viruses through bat is mostly based up on assumptions. Proper investigation is still required for establishment of role of bat in zoonotic virus transmission (Fenton et al., 2006). In most of the study same viruses are detected both in bats and humans, but this does not prove the bats as reservoir host. Many of the viral nucleic acid sequences have been isolated from bat tissues or excreta. The virus might be entered to bat body through food chain. It only indicates that bats may act as temporary host for those viruses (Calisher et al. 2006; Melaun et al., 2014). Bats share several immunophysiological parameters to human. This probably occurred due to the fact that bats are in close contact with human population since several years in many parts of the world for habitat and food requirements. Such interaction of bat with human and other animals favors the chance of potential spillover of diseases. Some of the phylogenetically related species of bats may act as intermediate host for bat transmitted viruses. It explains the Bats: Carriers of zoonotic viral and emerging infectious diseases transmission of Hendra and Nipah diseases to human. However, in some of the cases spill over infection is also caused by other animal species such as palm civets, pigs, raccoon dogs and horses. In Malaysia, it was established that Nipah virus was spilled over to human population through pigs from fruit eating bats (Chua et al., 2002b; Dobson, 2006). Some of the insects such as Haematophagous sp. may also transmit virus from bat to human (Melaun et al., 2014). It is also reported that mechanical transmission of bat associated zoonotic viruses to human population is also possible. 8 Control and prevention of bat-associated emerging infectious diseases (EIDs) Several factors progressing from primary to more proximate drive disease emergence from bats. For bat originated viral disease control such factors should be taken in consideration. Several steps should be taken for control of bat transmitted zoonotic viruses. Such steps should be initiated at individual level, population level and at societal level. 8.1 Individual level control In most of the cases no specific medical therapy has been found beneficial in bat associated viral EID. In human rabies therapeutic measures are very challenging and in most of the cases they fail to save the patient life. The early diagnosis i.e. before onset of fulminant stage in animal may allow effective prophylaxis in human. The prognoses of fulminant rabies carry a very poor and unfavorable result. In medical history the first case of successful experimental rabies treatment (Milwaukee Protocol) was reported in a 15 year old girl bitten by a bat in 2004 (Willoughby et al., 2005). Later on, extension of Milwaukee Protocol (consisting of antiviral drugs therapy, therapeutic coma and intensive medical care) did not show much successful in many other patients (Rupprecht, 2009; Rubin et al., 2009). The suitable prophylaxis measures before the onset of illness, has proved a much higher success rate in several other bat associated EIDs. For treatment of viral diseases modern molecular biology approach may also be used. The currently untreatable infection of henipaviruses may be treated with small interfering RNA (siRNA) molecules homologous to viral RNA (Mungall et al., 2008). Although, siRNA has capability to treat several viral infections, it is still under developmental phase. Several issues related to siRNA such as its delivery, efficacy in humans and cost effectiveness has yet to address. Moreover, the possibility of potential use of Ebola virus as bioweapons has forced scientific community for development of an effective vaccine product for any emergency outbreak. In mouse model of hemorrhagic Ebola virus infection, the vesicular stomatitis virus based recombinant vaccine has proved its safety and efficacy in preventing clinical signs of disease (Jones et al., 2007). Ebola virus vaccines can also be delivered through mucosal surface route. This vaccine delivery approach is very rapid and may prove advantageous during sudden disease outbreak. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 299 8.2 Population level control The bat associated viral EIDs should be addressed intensively at population level. The population level study of several bats associated EIDs have been carried out. Rabies is studied in depth and public health guidelines including vaccination of pets and other animals on public display, vaccination of humans in high risk groups, separation of domestic and pet animals from the wildlife reservoirs of rabies, public awareness regarding rabies etc was issued for rabies control. The current recommendation also advocates about pre and post exposure prophylaxis for high risk group individuals such as animal handlers, veterinarians, rabies researchers and laboratory workers and long term travelers to rabies endemic areas (NASPHV, 2009). Despite advances in epidemiology, molecular biology and vaccination science the proper control of bat associated viral EIDs remains challenging in many parts of the world. To reduce the bat associated viral EIDs outbreaks in human population, measure should be taken to control either the bat population or viral infection to bat population. In one of such measure anticoagulant on vampire bats can be applied and subsequently bats should release in wild condition (Kuzmin & Rupprecht, 2007). This will lead to consumption of anticoagulant by other vampire bats during grooming. It is well established that vampire bats can digest only coagulated blood. Thus, they may die by blood feeding which will remain uncoagulated in their digestive system. The anticoagulant can also be applied on animal skin to control bat population (Kuzmin & Rupprecht, 2007). 8.3 Societal level control The recent global emergence of Henipavirus and SARS coronavirus of Bat origin has started a new discussion on how to control disease emergence. The possible reasons of emergence of bat associated viral EIDs are environmental changes, increased human mobility and overpopulation. Therefore, to control viral EIDs monitoring of increased global mobility with other practical measures such as surveillance of transportation can be initiated. The intensive monitoring of borders and ports can be initiated for ill passengers and animals. Proper care and management facility should be provided which will benefit the ill animal and human as well as population moving from there. Moreover, for international travelers specific health measures such as pre-travel vaccination as well as post-travel health checkup should be initiated. Environmental conservation is also essential for sustenance of biodiversity and natural habitat. It is reported that many of the wild animals including bats are now reaching to human dwellings for food and shelter which also carry the EIDs to human population. The evidence show that environmental degradation play a major role in increased rates of disease emergence especially EIDs. However, the exact role of loss of environmental conservation in EIDs is still not understood, therefore further study is needed to establish the facts. 300 Ranjan et al Conclusion and future perspective References Bats cover the diverse group of mammalian species. They harbor several zoonotic viruses in their body than any other animal species (Hayman et al., 2013; Luis et al., 2013). It explains the necessity of knowledge of immune resistance mechanisms of bat that allow bats to harbor viral pathogens, mechanisms underlying disease emergence and pathogenic basis of viral diseases in bats (Dobson, 2006; Daszak et al. 2013; Mandl et al. 2015). To address such issues field epidemiological studies along with intensive laboratory experiments on bat associated virus using live bats and bat cell cultures are required. The bat cell lines from bats of different species need to establish to facilitate the in vivo and in vitro experiments. Adjemian J, Farnon EC, Tschioko F, Wamala JF, Byaruhanga E, Bwire GS, Kansiime E, Kagirita A, Ahimbisibwe S, Katunguka F, Jeffs B, Lutwama JJ, Downing R, Tappero JW, Formenty P, Amman B, Manning C, Towner J, Nichol ST, Rollin PE (2011) Outbreak of Marburg hemorrhagic fever among miners in Kamwenge and Ibanda Districts, Uganda, 2007. The Journal of Infectious Diseases 204 (Suppl. 3): S796–S799. doi: 10.1093/infdis/jir312. There is always a threat of experimental introduction and release of virus through laboratory animals, especially laboratory animals of foreign origin in new geographical area. Therefore, to reduce such risks only native laboratory animals should be used for animal experimentations for viruses. These animals should be kept for captive breeding. After few generations of captive breeding physiologically uniform animal strains could be obtained, that can be used in study of zoonotic diseases (Eger & Gardner, 2008). It is also necessary to design field studies for continuous search regarding new viral pathogen circulation in bats, their zoonotic potential, role of various abiotic and biotic factors affecting bat populations and their role in disease spillovers to humans (Parrish et al., 2008, Daszak et al., 2013, Marí Saéz et al., 2015). For adequate control of bat associated EIDs epidemiologists as well as wildlife experts should work together to minimize the risk of viral outbreak in human population. The joint expertise of bat biologists, veterinary and medical professionals and molecular biologists can be utilized for control and prevention of bat associated zoonotic viruses. After all, it is difficult to say that bats are responsible for emergence of zoonotic viruses. Only the intensive laboratory research using epidemiological and ecological approaches conducted by molecular biologists, bat biologists, veterinary and medical researchers may provide useful and satisfactory evidence regarding zoonotic virus transmission from bat. The accurate statement will be based on concrete laboratory evidence only. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 NANODIAGNOSTICS: A NEW FRONTIER FOR VETERINARY AND MEDICAL SCIENCES Upendra Lambe1, Minakshi P1,*, Basanti Brar1, Madhusudan Guray1, Ikbal1, Koushlesh Ranjan2, Nitish Bansal1, Sandip Kumar Khurana3 and Manimegalai J1 1 Department of Animal Biotechnology, LUVAS, Hisar, Haryana, India Department of Veterinary Physiology and Biochemistry, SVPUAT, Meerut, U.P. India 3 NRCE, Hisar, Haryana, India 2 Received – April 28, 2016; Revision – April 26, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).307.320 KEYWORDS ABSTRACT Nanotechnology Biosensors Diagnostics Veterinary Medical Infectious diseases are one of the greatest threats to animal and human population living in the developing world. These diseases have capacity to instigate in a small area and then open out very fast to the rest of the world and causing a heavy pandemic situation, for example; avian influenza pandemic. Such diseases infect large masses of population and may lead to loss of lives and also incur huge economic losses. Therefore, the best way to control these diseases is by diagnosing it at a very primary level and taking necessary precautionary measures so as to avoid the spread. Since last few years, the diagnostic approach has changed from tedious molecular biological techniques, to easy and rapid diagnostic techniques. Nanotechnology has extended the molecular diagnostics limit to nanoscale. These developed techniques do not require sophisticated laboratories and expert personnel, and hence are a cheap diagnostic approach. These assays can also be performed at the field level where the patient is present and get the results there itself. Hence, they are also called as pen side test or lab on chip diagnostic assays. The biological tests using nanotechnology become quicker, more flexible and more sensitive. These techniques have greatly influenced the diagnostic approach in the veterinary as well as medical field. Especially in the developing countries such as India, where the laboratory services are not * Corresponding author E-mail: [email protected] (Minakshi P) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 308 Minakshi et al available at the village level, these techniques have facilitated the disease diagnosis the most. Nanotechnology also applies the tools and processes for nanofabrication which is used to build devices for studying biosystems. Molecular diagnostics incorporated with nanobiotechnology has improved clinical diagnosis and opened a new area for development of personalized medicine. Nanotechnology has also played a crucial role in designing of diagnostic assays for medical and veterinary use. The nano materials have many versatile optical properties, piezo-electric properties, thermal properties, electro-chemical properties, enzyme mimicking properties etc. By exploiting these properties, the workers have designed different approaches for diagnosis. In this review, different nano-diagnostic approaches for detection of pathogen have been stated. 1 Introduction Bacteria, viruses and other microorganisms are omnipresent creatures which are responsible for causing disease in the humans and livestock. These organisms may affect multiple host species including humans. Therefore, they are of zoonotic importance and important in the public health concern. Some infectious agents can also be used as a part of biological warfare agent (MacKenzie, 2015). Hence, the correct diagnosis of the infectious agent gets primary importance, especially in case of livestock, because they are directly or indirectly linked to the humans through food webs. Several reasons can be attributed towards the diagnosis such as sub-clinical infections, persistently infected animals (PI), carrier or reservoir hosts, organisms transmitted through insect vectors or intermediate hosts (Rivera-Benitez et al., 2016; Navarro et al., 2016; Weber et al., 2016). Therefore, if the infection can be detected at the very primary level before maximum population is affected, proper control measures can be planned and huge economic losses can be prevented (Cascio et al., 2011; Stephen et al., 2015). Biosensors are commonly used in medical and veterinary diagnostics because of their higher sensitivity, simplicity in operation, ability to perform multiplex analysis, etc. (Patel et al., 2016). Since last two decades tremendous research in the field of diagnostic science has resulted in the development of numerous tools for detection of pathological agents and various diseases they cause in the humans and the animals. These new techniques have so many advantages over the previous techniques (Wei & Erkang, 2013). They are very handy, can be performed and interpreted by a layman, do not require sophisticated laboratories, very quick results with good specificity and sensitivity at a very cheap and affordable rate. Besides, there is no need of transportation of samples to the labs, as the test can be performed at the point where the animal is standing, thus reducing sample upset (Baptista, 2014; Alharbi & Al-Sheikh, 2014). Meanwhile, there is risk of spread on infectious disease, severe diseases conditions and even death due to absence of appropriate control measures (Dahlhausen, 2010). Apart from delayed diagnosis, other disadvantages such as possibilities of variations induced by transportation of samples, processing and testing conditions and even lack of uniform diagnostic platforms may further _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org complicate the result and results generated may be doubtful. Now there are different strategies designed for the diagnosis of disease either by detection of Ag or Ab, for which different types of biosensors are designed. In a Biosensor the physiological interaction between the ligand and the biorecognition element is converted by transducer, into measurable electric signal which can be further enhanced by a computer aided readout system for the user or sometimes can be read by naked eye only (Arora et al., 2010). Generally for the diagnosis of the disease, Ab based biosensors are preferred (Conroy et al., 2009). Mostly, the sensors are designed to diagnose the disease of veterinary importance as well as having zoonotic importance and vice versa (Stringer et al., 2008; Tran et al., 2012). Some have developed the sensors for surrogate human viruses so as to avoid the direct contact with the human viruses (Connelly et al., 2012). Therefore we need other techniques which can diagnose the disease at the point where the patient is present. Such techniques are the requirement for the developing countries like India. Nanotechnology is an emerging field which has contributed the most for the development of the biosensor technological approach (Syed, 2014). A biosensor is a compact analytical device which employs a ligand-specific bio-recognition element, such as an antibody, enzyme, receptor, nucleic acid, aptamers, peptide/protein, cells, tissue or whole organisms. These elements are immobilized on a sensor surface which is integrated with a signal conversion unit or transducer (Ayyar & Arora, 2013). Nanotechnology employs use of nanomaterials which exhibit physiochemical properties such as electrochemical (Rathee et al., 2016), chemical luminescence (Roda et al., 2016), optical (Tereshchenko et al., 2016), which are completely different than the actual material (Krejcova et al., 2015). These properties are generally exploited in designing of biosensors. These days even smartphone integrated biosensors have developed (Diming & Qingjun, 2016; Cevenini et al., 2016; Roda et al., 2016). There are many reports on nanoparticles having properties mimicking the properties of certain enzymes, thus these particles can be used in designing immunoassays. In this review, the Nano-diagnostic biosensors for the detection of pathogens which are human and veterinary importance are discussed. 17 Nanodiagnostics: a new frontier for veterinary and medical sciences 309 2 Immuno assays These are the label free assays which can detect the substrate without labeling the biomolecules with any enzyme. The AgAb reaction is detected by exploiting diverse properties of nanoparticles. Previously, immuno sensors exploited the very specific binding affinity of antibodies for a specific compound or antigen. The binding of antigen to antibody follows the lock and key hypothesis of interaction. The antigen-antibody binding usually result in generation of a detectable signals from secondary molecules such as enzymes, fluorescent molecules or radioisotopes tagged with either antigen or antibody (Marazuela & Moreno, 2002). Figure 1 Types of Nano-diagnostic Biosensors There are various approaches being used for the development of nano-diagnostic assays. The nano diagnostic can be classified into two categories, in-vitro and in-vivo. In-vivo is the diagnostic imaging techniques in case of live animals. On the other hand, the in-vitro techniques include, different antibody based immune assays and different nucleic acid based hybridization assays coupled to the nanoparticles (Figure 1). Several types of biosensor technologies have been used for detection of biomolecules. But due to advancements in nanotechnology, the need of labelling the biomolecule with enzyme or radioisotope is not required when Nano-particles are used (Tianshu et al., 2015). Several types of antibody/antigen interaction detection systems are available which are currently used for detecting diseases, (Table 1, Figure 2). IgG antibody based detection systems have been developed for diagnosis of autism (Gogolinska & Nowak, 2013). For antigen/antibody based detection several types of silver and gold nanoparticles are used. Similarly, silver nanoparticles have been used for diagnosis of H1N1 virus (Yanxia et al., 2014) and gold nanoparticles have been used for diagnosis of Salmonella (Giyoung et al., 2015), Human T lymphotrophic virus and Hepatitis B Virus (Randolph et al., 2016) etc (Table 2). Figure 2 Different approaches for designing antigen/antibody based nano-diagnostic tools. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 310 Minakshi et al Table 1 Lateral Flow assay for detection of various biological agents. Agent Nanoparticle Detection Limit Reference HIV-1 GNP 0.24pg/ml Xiuli et al., 2016 HIV MYO GNP 1.56ng/ml Ruihua et al., 2016 Mycoplasma pneumonea AF-647 0.3830 Liming et al., 2016 TB GNP 100pg/ml Corstjens et al., 2016 Prostate specific Ag Photon up-converting NPs 41ng/liter Juntunen et al., 2016 Hepatitis C GNPs - Hwan et al., 2015 Enterobacteriaceae GNP - Jyoti et al., 2015 Mycotoxin MNP Xie et al., 2015 Table 2 Antigen/antibody interaction based system for detection of different pathogens. Organism Adeno virus H1N1 Encephalomyocarditis virus Salmonella Duck Hepatitis virus HIV Salmonella pullorum Salmenella Human T lymphotrophic virus Hepatitis B Virus Orchid Virus General Virus H1N1, H5N1, H7N9 H1N1 Nano Particle Triangular AuNPs Silver NPs Triangular AuNPs AuNPs Silicon wafers Fe-Au shell Blue Silica & MNPs Quantum dots GNPs GNPs Gold Nano rods GNP Chip ZnO Nano rods GNPs Type of detection Raman Scattering Fluorescence OPDA Raman Scattering Microfluidic Ellipsometry Imaging Amperometric Sandwich assay Magnetic sensor Immunoaffinity assay Immunoaffinity assay SPR Fluorescence PDMS Micro fluidic system 2.1 Optical Biosensor The optical properties of nano-particles are exploited in an optical biosensor (Radhika et al., 2012). The Optical biosensors utilize several sensor techniques such as resonant mirrors, surface plasmon resonance and waveguides can be widely used for analysis of biomolecular interactions without using any molecular tag. Advances in instrumentation and experimental design have led to the increasing application of optical biosensors in many areas of diagnosis (Matthew, 2002). This means that when the conjugated nanoparticles bind to the Ag/Antibody Polyclonal Monoclonal Polyclonal Polyclonal Polyclonal Glycoprotein 160 Polyclonal Polyclonal Monoclonal Monoclonal Label free Fluorescence Microscopy Electrochemical Aptamers specific molecules, they change their refractive index (Xudong et al., 2008) and therefore, change their color which is directly proportional to the number of interacting molecules or mass of the interacting molecules at that given instant. The techniques such as immune dot-blot assay, lateral flow assay work on the same principle. Several types of biosensors have been designed on optical detection principles (Figure 3), such as Surface plasmon resonance based biosensors; interferometer-based biosensors and optical waveguide based biosensors etc (Jeremy, 1997; Baird & Myszka DG, 2001). Figure 3 Basic principle of biosensors _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Reference Chia et al., 2011 Yanxia et al., 2014 Chia et al., 2011 Giyoung et al., 2015 Cheng et al., 2011 Ning et al., 2009 Qian et al., 2016 Giyoung et al., 2015 Randolph et al., 2016 Randolph et al., 2016 Lin et al., 2014 Yen et al., 2016 Ji-Hoon et al.,2016 Tseng et al., 2016 17 Nanodiagnostics: a new frontier for veterinary and medical sciences 311 Figure 4 Surface plasmon resonance based principle. 2.1.1: Surface plasmon resonance (SPR) biosensor It was first demonstrated for biosensing in 1983 by (Liedberg et al., 1983). Nanoparticles display unique physical properties due to their nano-size. Metallic nanoparticles have intense absorbance and scattering properties due to Surface Plasmon Resonance (SPR). When an oscillating electric field interacts with the free conductive band of electrons at the surface of the AuNP, collective dipolar oscillation of the electrons occurs. This is called Surface Plasmon (Radwan & Azzazy, 2009). SPR has been extensively explored and has gradually become a very powerful label-free tool to detect the pathogens (Pattnaik, 2005; Homola, 2003). In SPR, a surface plasmon wave (SPW) which is a charge density oscillation occurs at the interface of two media with dielectric constants of opposite signs, such as a metal (gold or silver) and a dielectric (Figure 4). This technique has been successfully used for the detection of viruses and bacteria (Boltovets et al., 2004). Gold nanoparticles embedded PVA matrix is used as sensing material (Rithesh et al., 2016). Detection can be performed by visual colour change observations, photometry or resonance light scattering by interacting molecules on surface of nanoparticles deciphered by changing refractive index. This has a very wide range of applications in the areas of environmental, pharmaceutical and biological analysis and clinical diagnosis (Yanlin et al., 2016). Gurpreet et al. (2016) has reported the use of this type of biosensors in the detection of Niesseria meningitides. SPR sensors can visualize living cell interactions which can be used for malignant cell detection in cellular diagnostic systems (Yanase et al., 2014). SPR based rapid immunoglobulin M (IgM) diagnostic test has been successfully used for detection of dengue from human serum in only 10 minutes with 100% specificity and 83-93% sensitivity (Jahanshahi et al., 2014). The SPR biosensor based assay was also used for simultaneous _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org detection of multiple TB antibodies in patient serum with high sensitivity and specificity in real-time (Hsieh et al., 2012). 2.1.2 Interferometer-based biosensors Optical interferometers have already used in detection of surface bound bio-reactants such as bacteria, spores, toxins, viruses, and proteins (Schneider et al., 2000; Schmitt et al., 2007). These devices are based on evanescent field sensing. Light is confined within the core of the waveguide, and extends into the surrounding media so that its field can interact with the environment. Therefore, a biomolecular interaction takes place between a receptor molecule, previously deposited on the waveguide surface, and its complementary analyte produces a change in the refractive index at the sensor surface that induces a variation in the optical properties of the guided light via the evanescent field. Interferometric assays have an advantage in detection of intact bacterial or viral particles. Influenza virus has been detected in oral-nasal secretion of patients at concentrations of a few ng/mL through this technique. Recent study shows that microorganism growth can also be detected using hollow-core photonic fiber based FabryPerot interferometer (Xiaohui et al., 2016). A label-free DNA biosensor based on microfiber-assisted Mach-Zehnder interferometer for in-situ real-time DNA hybridization kinetics detection has been experimentally demonstrated by (Binbin et al., 2016). While Mach–Zehnder interferometer point-of-care system for rapid multiplexed detection of microRNAs in human urine specimens is done by (Qing et al., 2015). Sandwich assay for detection of Streptavidin was demonstrated by (Wenjie et al., 2016) with detection limit of 0.02 nM. The Interferometric biosensor was used for detection of Aflatoxin M1. The test result was highly reproducible and reusable (Chalyan et al., 2016). A fiber-optic interferometer based optic biosensor operating at 1550 nm was evaluated for quantification of gelatin (protein) in water (Yadav et al., 2014). 312 Minakshi et al Table 3 Enzymatic interactions based detection of different agents associated with health concern. Compound Norepinephrine IFN Gamma Protein estimation IL-3 Stem cell factor SCF Nano Mass Nanoparticle FeMoO4 rods AuNP MNPs AuNP GNP Graphene films Type of sensor Cyclic voltammetry ITO Colorimetric iPCR iPCR Ultrasound frequency shift 2.1.3 Optical waveguide based biosensors Optical waveguides based biosensor utilize fluorescence resonance energy transfer (FRET) triggered by the binding event between multivalent protein and dye-tagged receptors (Song et al., 2000). It is successfully adapted to the detection of biomarkers for complex biological material. The spatial filtering of wave-based detection is a distinct advantage as it ensures that the bulk biological material is not irradiated. This arrangement effectively minimizes background fluorescence and eliminates the need for extensive sample preparation when analyzing complex samples. Mukundan et al. (2009) have successfully used this approach to detect extremely low concentrations of disease biomarkers in patient samples. Optical wave guide biosensors are used for the detection of RNA in the samples (Carrascosa et al., 2016). 3 Enzymatic interactions based nanodiagnostics Enzymes are very popular bioreceptors due to their specific binding capabilities and catalytic activity. Enzymatic interaction is used for specific analyte recognition (Pohanka, 2013). The enzyme based biosensors provide specific advantages such as ability to catalyze several reactions, can detect many analytes such as substrates, products, modulators Detection molecule Without modification HPR-Ab Punctates Polyclonal Ab Polyclonal Ab Piezoelectric crystal and inhibitors. Moreover, enzymes are not consumed in reactions. Therefore, biosensor can be used continuously without loss of activity. Enzymatic interactions methods can detect much lower limit of analytes (Patel et al., 2016). However, the sensor lifetime depends on enzymatic stability (Lucie et al., 2011).There are several types of enzymatic interactions detection systems are available which are currently used for detection of agent associated with health concern (Table 3). Several biological molecules such as IL-3 (Lucie et al., 2011), IFN Gamma (Yaru et al., 2016), total protein (Gero et al., 2016) etc., in disease conditions have been estimated using enzymatic interaction based biosensor. Recently, there has been little advancement in these types of biosensors like, the accumulation of insulin causes type 2 diabetes. To detect this condition a biosensor called Nano-cage-mediated refolding of insulin by PEG-PE micelle has been developed (Xiaocui et al., 2016). Cholin a breast cancer marker, detected form serum by nano interface technology (Thiagarajan et al., 2016). Similarly, blood glucose level is monitored by a noninvasive saliva biosensor (Wenjun et al., 2015). Aptamer based GnRH biosensor in equine urine has been demonstrated by (Richards et al., 2016). Figure 4 Approaches for making Nucleic acid based diagnostics _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org References Kunda et al., 2016 Yaru et al., 2016 Gero et al., 2016 Lucie et al., 2011 Lucie et al., 2011 Li & Wang, 2016 17 Nanodiagnostics: a new frontier for veterinary and medical sciences 313 Table 4 Nucleic acid interactions based nano-diagnosis detection of different agents associated with disease. Organism Arabis Mosaic Virus Lily Symptomless Virus HSV GYSVD HBV HBV Dengu Canine adeno Salmonella HBV Influenza virus White spot syndrome virus Porcine epidemic diarrhea Influenza HCV Nanoparticle SMP SMP SMP SMP AuNP MNPs 3D Graphene GNPs GNPs Cu Nano cluster CdZnTeS Quantum dots GNPs GNPs Sugar chain GNP GNPs Sensor type Magnetic Magnetic Magnetic Magnetic Barcode amplification Hybridization Impedimetric sensor Microarray chip LFICA Colorimetry Fluorescence LAMP Nano RT-PCR RT qPCR Hybridization 4 Nucleic acid interactions based nanodiagnostics The nucleic acid based Biosensors are known as genosensors. The analyte recognition is based on principle of nucleotide base pair complementarity, such as A: T and C: G in DNA. Complementary (probe) sequences are synthesized from target nucleic acid sequence, labeled with suitable dye and immobilized on bio sensor chip. Thus, probe will hybridize with target gene followed by generation of optical signals (Marazuela and Moreno, 2002). There are several types of Nucleic acid (DNA/RNA) interaction detection systems available which are used for detection of several viruses or other disease associated agents (Table 4; Figure 4). The DNA genosensors can be combined with PCR amplification for detection of several microorganisms. The DNA genosensors based assays lead to direct detection of hybridization process using electrochemical redox mediators, enzyme amplification or nanoparticles labeled ingredients (Pedrero et al., 2011). Nucleic acid based biosensors have also used for screening of allergens in food materials because of high stability of DNA in comparison to proteins even after processing of food (Mafra et al., 2008). The assay is based on selection of DNA target sequences coding allergenic proteins. Such techniques are also used for animal meat identification. Bovine and sheep meat samples were detected by targeting highly repetitive satellites DNA (∼250 bp and 430 bp, respectively) (Mascini et al., 2005). A more reliable and faster genosensors based technique has been developed for chicken, bovine and swine meat identification. This method uses a combination of isothermal amplification of DNA along with electrochemical detection of DNA on disposable carbon based electrochemical printed chips (Ahmed et al., 2010). Genosensors are also used for monitoring of genetically modified organisms (GMO) having specific genes (transgene) introduced into their DNA using genetic engineering to _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Nucleic acid RNA RNA RNA RNA DNA oligos DNA oligo RNA DNA 16s rRAN DNA Molecular beacons DNA oligo RNA SYBRgreen 5‟UTR DNA Reference Ning et al., 2014 Ning et al., 2014 Ning et al., 2014 Ning et al., 2014 Wang et al., 2010 Wang et al., 2010 Seon et al., 2016 Yadav et al., 2015 Cheng et al., 2013 Xiaoxia et al., 2016 Oluwasesan et al., 2016 Yortyot et al., 2013 Wanzhe et al., 2015 Yasuo et al., 2015 Sherif et al., 2010 improve crop production (by insect or herbicide resistance) or to enhance nutritional properties. Target gene selections for such genosensors are relatively easy because the transgenic inserts sequences are completely known and available in open databases. Several genosensors have been developed for detection of transgene from GMOs (Yang et al., 2007a; Yang et al., 2007b; Yang et al., 2008; Feng et al., 2008; Jiang et al., 2008; Ma et al., 2008; Zhang et al., 2008; Yang et al., 2009; Zhou et al., 2009; Bonanni et al., 2009; Jiang et al., 2011; Yang et al., 2012; Arugula et al., 2014; Manzanares-Palenzuela et al. 2015). 5 DNA based nanotechnology DNA nanotechnology utilizes newly designed artificial nucleic acid structures for analytical purposes. In such assays, nucleic acids are used as non-biological engineering materials rather than as carrier of genetic information. Some researchers have designed static structures with DNA, such as DNA computers and molecular machines (Seeman & Nadrian, 2004). There are different DNA based technology such as Microarray, Rolling circle amplification, Threshold mediated strand displacement (TMSD) and L shaped DNA probes in which nanoparticle were used to facilitate the process (Shi et al., 2014; Ravan, 2016; Elham et al., 2016) (Table 5). The nano-biotechnology system may be used for creation of a DNA robot which can recognize infected cells and induce apoptosis to kill such cells (Douglas et al., 2012). The DNA robot was an elegant model system which has shown great potential for uses as a smart drug. The DNA nanotechnology science has also been used as carriers for Doxorubicin (anticancer drug) (Jiang et al., 2012; Zhao et al., 2012). This showed increased potency of Doxorubicin as compared to normal medication. Thus, DNA nanotechnology has shown breathtaking pace in recent years. It leads to control of structure and function at molecular level with unparallel efficiency (Tørring & Gothelf, 2013). 314 Minakshi et al Table 5 Nanoparticles facilitated nucleic acid based technologies. Technique Micro Array Rolling circle amplification Threshold mediated strand displacement L shaped DNA probes RNA quantification Nanoparticle GNPs GNPs GNPs GNPs GNPs Nano-Immuno-PCR Nano-Immuno-PCR has additional sensitivity than other conventional methods because it utilizes combined effect of nucleic acid amplification along with an antibody-based assay (Guangxin et al., 2015). It uses a DNA-antibody conjugate as a bridge which links the immunoreaction with PCR reaction. This method has better specificity and 109 fold more sensitivity than conventional ELISA assay (Ruiyan & Huisheng, 2015; Chang et al., 2016). The latest advancements in this technique include better production of DNA-antibody conjugate and better readout methods. It also has broad range of applications in clinical diagnostics because it is an ultrasensitive protein detection assay (Chang et al., 2016). Several developed NanoImmuno-PCR assays for disease diagnosis have been listed in the Table 6. Conclusion Nanomaterials offer a vast number of breakthroughs such as cost effective, lower risk to consumers and faster approach that will further enhance the clinical aspect of veterinary sciences in future and conceived that bacterial infections can be eliminated in the patient within minutes, instead of using treatment with antibiotics over a period of weeks. Nanotechnology has found its way into the food industry to improve food shelf life, safety and quality control. In coming years it can be expected that nanotechnology may practically apply in artificial creation of cells, tissues and organs. The artificial cells can be used in replacement of defective cells and organs, especially in metabolic disorders. Nanotechnologies have power to extent the modern molecular diagnostics to personalized medicine and therapeutics. Such techniques have Sensor type Pixel sensors SPR TMSD Hybridization Colorimetry Nucleic acid DNA DNA probes RNA DNA PNA peptide nucleic acid Reference Wang et al., 2010 Shi et al., 2014 Ravan, 2016 Elham et al., 2016 Joshi et al., 2013 been used in the field of pathogen detection, DNA detection assay, biomarker discovery and cancer diagnosis. Nano medicine also has important role in future therapeutics as well as diagnostic assays. Although nanotechnologies have several applications and benefits, it is still in the early stages of its development and yet to apply throughout the world for routine diagnostics and therapeutics approaches. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 Lantana camara: AN ALIEN WEED, ITS IMPACT ON ANIMAL HEALTH AND STRATEGIES TO CONTROL Rakesh Kumar*, Rahul Katiyar, Surender Kumar, Tarun Kumar and Vijay Singh ICAR-IVRI, Izatnagar, Bareilly, U.P, India - 243122 Received – April 28, 2016; Revision – April 09, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).321.337 KEYWORDS Lantana camara Lantadenes Allelopathy Hepatotoxic Poisonous ABSTRACT Lantana camara is one of the most commonly known noxious weed distributed worldwide. The red flower variety (L. camara var. aculeata) of this weed is mainly toxic and usually prevalent in tropical and sub-tropical countries. Lantana leads to hepatotoxicity, photosensitization and intrahepatic cholestasis almost in all the animals. LA is the main toxic pentacyclic triterpenoid present in this weed. Lantadene toxicity leads to fatty degeneration, bile duct hyperplasia, gall bladder edema, degeneration of parenchymal cells and portal fibrosis observed on histopathological examination. L. camara toxicity causes fluctuation in hematological as well as in biochemical parameters. The management of toxic effects can be achieved by activated charcoal, vaccination and supportive therapy but are not much effective. Besides the harmful effects of this plant, there are some beneficial effects also including antiinflammatory, hepatoprotective action, antitumor action etc. The control of this weed is difficult because of its allelopathic action. Nowadays this plant is used in many recent advanced techniques like phytoremediation of particulate pollution, phytoextraction of heavy metals and many others. Thereby the use of this plant in the field of research can be an effective way to manage this alien weed. As far as the toxicity is concerned it can be prevented by the using conventional therapeutic methods along with immunological, nanotechnological and biotechnological approaches. The aim of this article is to discuss the information regarding its progression, mechanism by which it affect animals, pathological alterations, treatment and what strategies we can opt to get rid of this weed. * Corresponding author E-mail: [email protected] (Rakesh Kumar) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 322 1 Introduction Toxic plants are of major concern to veterinarians because of their harmful effects to livestock in terms of causing mortality and reduction in productivity (Sharma et al., 2007; Diaz, 2011). The severity of toxic effects caused by poisonous plants varies among species and depends upon the nature, part and amount of toxic component taken, environmental conditions, species, age, size and body condition of the animals (Sharma et al., 2007). Along with the toxic effects to livestock, these invasive species are supposed to be the one of the major threat to biodiversity and ecosystem after habitat destruction (Drake et al., 1989; Holmes, 1990; Buckley & Roughgarden, 2004; De Milliano et al., 2010; Osunkoya & Perrett, 2011; Zhang & Chen, 2011). These invasive plants have turned to predators and are responsible for causing diseases in animals as well as in plants (Ehrenfeld, 2006; Chambers et al., 2007; Drenovsky et al., 2012). Among poisonous plants L. camara is one of the most commonly known noxious (Pereira et al., 2003; Mello et al., 2005) and invasive weed worldwide (Palmer et al., 2000; Baars et al., 2003; Totland et al., 2005; Moura et al., 2009; Van Driesche et al., 2010). This weed is responsible to cause heavy mortality of livestock as well as responsible to cause loss of agro and forest ecosystem (Day et al., 2003; Mello et al., 2005; Sharma et al., 2007). L. camara Linn. was introduced as an ornamental shrub by a British in Calcutta Botanical Garden in year 1809, belongs to family Verbenaceae (Bouda et al., 2001; Kumar, 2001; Yadav & Tripathi, 2003; Munsif et al., 2007). The word Lantana is derived from a Latin word lento, which means ―to bend‖ (Ghisalberti, 2000). This weed is locally known as bunch berry, baraphulnoo, red or wild sage (Sharma et al., 2007). This plant shows change in inflorescence with age and season that’s why very difficult to classify taxonomically (Munir, 1996). The binomial name of this plant was given by Linnaeus in year 1753 (Kumarasamyraja et al., 2012). The main varieties of Lantana on the basis of flower colour includes Pink L. camara, White L. camara, Red L. camara, Pink edged red L. camara and Orange L. camara. Other important species of the genus lantana includes L. indica, L. crenulata, L. trifolia, L. lilacina, L. involuerata and L. Sellowiance but red flower variety (L. camara var. aculeate) is most toxic (Sharma et al., 2007). A pink variety of Lantana camara called as Taxon is usually grazed by animals in New Zealand and it is nontoxic (Black & Carter, 1985). This plant attains a height of 2-3 m and the branches carry curved prickles. The leaves are oval, cuneate, rounded at the base and rugose and crenate at the upper portion, which are rough at maturity and give an offensive odor (Sharma et al., 2007). The fruits are greenish in early stages and become dark blue later on. The green immature fruits are poisonous, while the ripened dark blue fruits are tasty so often taken by birds as well as human beings (Sharma et al., 2007). Many species of lantana are native to Africa and America and has covered many of the neighboring countries (Day et al., 2003). But later _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Rakesh et al on this species has displaced the invertebrate population and other native populations in Africa (Samways et al., 1996). Lantana camara is among 100 most notorious weeds in the world and got entry approximately in 60 countries (GISD, 2010; Lüi, 2011). This weed has been found as a major weed in 12 countries and listed among the 5 most noxious weeds prevalent in Australia and has covered 60% pastures in Queensland (Holm et al., 1979; Anderson et al., 1983; Ghisalberti, 2000). This weed has replaced Quercus leucotrichphora and Pinus roxburghii forests in Kumaun hills (U.P.) (Bhatt et al., 1994); invaded the teak plantations in Tamil Nadu (Clarson & Sudha, 1997); covered Western Ghats (South India) (Muniappan & Viraktamath, 1993) and heart water region of Garhwal (U.P.) (Rajwar, 1998). In Himachal Pradesh, heavy outbreaks of lantana toxicity have been reported from Rampur Bushair and sporadic cases of toxicity have also been reported from cattle, buffaloes and small ruminants (Sharma, 1984). In general for the success and impact of any weed many biotic and abiotic environmental factors are responsible (Sheppard et al., 2012). One of the most important factor for the huge prevalence of this weed throughout world is its phytotoxic or allelopathic action which is due to the presence of phenolic compounds (umbelliferone, methylcoumarin, salicylic acid etc.) and lantadenes i.e. LA (lantadene A) and LB (lantadene B) (Achhireddy et al., 1984; Jain et al., 1989; Singh et al., 1989; Ferguson & Rathinasabapathi, 2003). The suppressive allelopathic action of this plant has been seen on certain plant species like Glycine max (Linn), Cyclosorus dentatus Forsk, Triticum aestivum L., Zea mays L. and Lolium multiflorum Lam (Achhireddy et al., 1985; Sharma et al., 2007). This weed is mainly disseminated by droppings of moving animal flocks/ birds, cutting and pollination (Ghazoul 2002; Sharma et al., 2007). 2 Toxic components of Lantana camara The most important toxic components present in this weed are lantadenes. Lantadenes are pentacyclic triterpenes (Table. 1) and often led to hepatotoxicity, photosensitization and jaundice (Sharma et al., 1979; Sharma & Makkar, 1981; Sharma et al., 2007). There are 2 forms isolated from lantana toxin i.e. crystalline and amorphous. The amorphous form is found to be icterogenic to guinea pigs (Sharma et al., 1988a). Among the known compounds present in lantana, LA is the most hepatotoxic component while certain other compounds like naphthoquinones, oil constituents (citral), iridoid glycosides (Theveside) and some of the oligosaccharides are of lesser importance as far as toxicity is concerned (Ajugose) (Dominguez et al., 1983; Abeygunawardena et al., 1991). The lantadenes are mainly present in the leaves of this plant (Sharma et al., 2007) having varying toxic effects among different species and strains of mammals/livestock. The toxic effects of this plant are evident both in ruminants as well as in non-ruminants (Sharma et al., 2007). Lantana camara: An alien weed, its impact on animal health and strategies to control 323 Table 1 Chemical compounds obtained from Lantana camara and their mechanism of actions. S.No. Triterpenoids References 1. Hepatotoxic Action LA, LB, LC, RLA and icterogenin 2. Antimicrobial and antibacterial activity LA, LB, oleanolic acid, ursolic acid, 4Epihederagonic acid and 24-Hydroxy-3-oxours12-en-28-oic acid 3. Protein kinase C inhibitor 4. Anti-inflammatory Verbascoside Brown et al., 1963; Johns et al., 1983a; Sharma et al 1991; Verma et al., 1997; Wachter et al., 2001; Khan et al., 2003; Srivastava et al., 2005; Kong et al., 2006; Parimoo et al., 2015 Brown et al., 1963; Sharma et al 1991; Inada et al., 1995, 1997; Verma et al., 1997; Wachter et al., 2001; Kong et al., 2006; Kumar et al., 2006; Barreto et al., 2010; Hussain et al., 2011; Sousa & Costa, 2012 Herbert et al., 1991 5. Antitumor LA, oleanolic acid, ursolic acid, Camaraside and Lantalucratins A-F 6. Anxiolytic action (Psychiatric disorder) 7. Antitubercular UASG 8. Allelopathy LA, Umbelliferone, Hydroxycoumarin, 6methylcoumarin, Salicylic acid, gentisic acid, Vanillic acid and Quercetin 9. Antiviral 10. Hepatoprotective LA, LB, LC, RLA, RLB and 22beta-Hydroxy-3oxolean-12-en-28-oic acid Oleanolic acid and ursolic acid 11. Leukotriene inhibitor Oleanonic acid 12. Anti-hyperlipidemic Oleanolic acid and ursolic acid 13. Antimutagenic 22beta-Dimethylacryloyloxylantanolic acid Hart et al., 1976b; Johns et al., 1983b; Singh et al., 1990, 1991; Liu, 1995; Siddiqui et al., 1995 Hart et al., 1976b; Johns et al., 1983b; Giner-Larza et al., 2001 Hart et al., 1976b; Liu, 1995, Liu, 2005; Mishra et al., 1997; Verma et al., 1997; Chen et al., 2005, Chen et al., 2006 Barre et al., 1997; Mello et al., 2005 14. Nematicidal Camarinic acid, Linaroside and Lantanoside Siddiqui et al., 1995; Begum et al., 2000 15. Antiprotozoal Triterpnes from Lantana montevidensis Mohameda et al., 2016 16. Antithrombin O’Neill et al., 1998; Weir et al., 1998 18. Cardio active 5,5-Trans-fused cyclic lactone containing euphane triterpenoids Apigenin, Cirsilineol, Eupafolin, Eupatorin and Hispidulin Martynoside 19. Insecticidal action Bioactive molecules without any cross resistance 20. Anti-diabetic UASG Seyoum et al., 2002; Dua et al., 2010; Rajashekar et al., 2012 a; Rajashekar et al., 2012 b; Rajashekar et al., 2012 c Venkatachalam et al., 2011; Kazmi et al., 2013 21. Inhibitor of larval hatch and exsheathing Lantana decoction in combination with A. zerumbet, M. villosa and T. minuta 17. Antiproliferative Oleanolic acid, ursolic acid and Oleanonic acid LA Hart et al., 1976b; Johns et al., 1983b; Liu, 1995; Verma et al., 1997; Giner-Larza et al., 2001; Benites et al., 2009; Ghosh et al., 2010; Hussain et al., 2011; Sousa & Costa, 2012 Brown & Rimington, 1964; Seawright & Hardlicka, 1977; Mahato et al., 1994; Deena & Thoppil, 2000; Ghisalberti, 2000; Hayashi et al., 2004; Gomes de Melo et al., 2010; Bisi-Johnson et al., 2011 Kessler et al., 1994; Awad et al., 2009; Kazmi et al., 2013 Seawright & Hardlicka, 1977; Verma et al., 1997; Wachter et al., 2001; Kong et al., 2006 Brown et al., 1963; Johns et al., 1983a; Singh et al., 1989; Sharma et al 1991; Verma et al., 1997; Wachter et al., 2001; Kong et al., 2006; Verdeguer et al., 2009 Johns et al., 1983a; Inada et al., 1995 Nagao et al., 2002 Syah et al., 1998 Macedo et al., 2012 Abbreviations: Lantadene A (LA), Lantadene B (LB), Lantadene C (LC), Reduced Lantadene A (RLA), Reduced Lantadene B (RLB), Ursolic acid stearoyl glucoside (UASG) _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 324 Rakesh et al Among ruminants cattle, buffalo and sheep are highly susceptible, while goats are little resistant to lantadene toxicity (Lal & Kalra, 1960; Sharma et al., 1988b; Sharma et al., 2007). Guinea pigs show most typical signs of lantana toxicity (Sharma et al., 1988b), while male rats are often resistant to lantana toxicity because of the action of testosterones (Pass et al., 1979a; Pass et al., 1985; Sharma et al., 1992; Sharma et al., 2007). The toxic effects of lantana have been seen in Kangaroos and Ostriches also (Johnson & Jensen, 1998; Cooper, 2007). Green fodder scarcity is the major causes of lantana toxicity in animals, mainly in those who are often send to pastures without feeding any prior feed (Sharma & Makkar, 1981). In spite of having many toxic effects this weed is also having anticancer (Gomes et al., 2010; Sathish et al., 2011), antibacterial (Rwangabo et al., 1988; Barreto et al., 2010), antifungal (Sharma et al., 2007), anti-diabetic (Garg et al., 1997), anti-inflammatory, analgesic, antimotility (Ghosh et al., 2010), anti-feedant, larvae repellent (Moffitt et al., 2010), anticonvulsant (Bisi-Johnson et al., 2011), antiulcer and antioxidant actions (Sathish et al., 2011). Oleanolic acid and ursolic acid are the major components, while LA and LB are the minor constituents obtained from Townsville prickly orange variety of lantana (Hart et al., 1976a). Figure 1 Flow diagram showing different chemical compounds present in Lantana camara. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Lantana camara: An alien weed, its impact on animal health and strategies to control 325 Figure 2 Flow chart of absorption and mechanism of action of lantadenes. 3 Absorption and mechanism of action of lantadenes This toxin has been found to be absorbed through entire GIT (gastrointestinal tract), mainly small intestine (Sharma et al., 2007). The retention time of lantadenes in GIT plays a significant role in progression of effect (Pass et al., 1981a). Bile has not been found to be having any role in toxin absorption. L. camara mainly attacks liver and kidneys of ruminants and leads to photosensitization. The animals are died within 2-4 days in acute cases. In sub acute lantadene toxicity study a dose dependent mortality was reported (Parimoo et al., 2015). Sluggishness, weakness, bloody diarrhea, edematous ears and eyelids, cracks and fissurs on muzzle and other non-hairy parts, conjunctivitis, ulceration of the tip and under surface of the tongue (if un-pigmented), pale conjunctival, vulvar or vaginal mucous membranes and sclera of eye are some of the clinical signs observed in lantana toxicity. The acute lantana toxicity can be induced either by the leaf powder or by partially purified lantadene powder (Sharma & Makkar, 1981). In sheep, the oral administration of lantadene leaf powder (at the dose of 4 and 8 g/kg body weight) leads to photosensitization, conjunctivitis and bile stained liver while administration of lantadene leaf powder in goats diarrhoea, anorexia and jaundice is evident, but no photosensitization has been seen (Obwolo et al., 1990). The LD50 value of lantadene in sheep is 1-3 mg/kg body weight, when administered by intravenous route, while the LD50 value is 60 mg/kg body weight when administered by oral route, because of show absorption (Nellis, 1997). The oral administration of lantadenes at the dose rate of _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 25 mg/kg body weight did not lead to mortality in guinea pigs, but produced hepatotoxic and nephrotoxic effects which were evident on histopathology and on biochemical estimation and were indicative of sub-acute toxicity (Parimoo et al., 2015). Transfer of lantana toxins to milk, placenta, or to the offspring has not been reported, but some teratological effects has been seen in rats (Mello et al., 2005; Sharma et al., 2007). Lantadenes are also having effect on reproductive system, as found to interfere with the sperm count, daily sperm production, and sperm morphology (Sharma et al., 2007). 4 Hepatotoxic action of lantadenes Lantana toxins cause intrahepatic cholestasis along with the inhibition of bile secretions without widespread hepatic necrosis (Pass et al., 1979b). Hepatocellular damage precedes the intense and prolonged jaundice observed during lantana poisoning (Sharma et al., 2007). Significantly, in lantana toxicity, the cells located around the central vein remain normal, while parenchymal cells lying to the periphery of the liver are damaged. Generally, changes associated with intrahepatic cholestasis include dilation of bile canaliculi, loss of microvilli, alterations in enzyme activities and composition of the canalicular membrane (Trauner et al., 1998). Phylloerythrin, a degradation product of chlorophyll formed by the action of microorganisms in the GIT gets accumulated in the liver and leads to photosensitization (Rimington & Quin, 1934). This type of photosensitization is also called as hepatogenous photosensitization, which occurs due to the impaired hepatobiliary excretion (Kellerman & Coetzer, 1985). This impaired hepatobiliary excretion of phylloerythrin leads 326 Rakesh et al to its accumulation in plasma. The inhibition of bile secretion leads to accumulation of bilirubin and ultimately leads to jaundice (Trauner et al., 1998). L. camara toxicity leads to collagen fibres formation in advanced stages, which extends into periportal areas of the liver and can be seen when stained with Foot’s reticulin and Van Gieson stain (Gopinath & Ford, 1969). 5 Clinical signs (de Mello et al., 2003; Sharma et al., 2007) A. I. II. III. IV. The dose of lantadenes determines the severity of ictericity (Gopinath & Ford, 1969). The clinical signs follow a definite pattern as given below: V. B. I. II. III. IV. V. VI. Loss of appetite and decrease in ruminal motility (within 24 h) Photosensitization in un-pigmented areas leads to necrosis later on (within 24-48h) Icterus (yellowish sclera and other mucus membranes, within 48-72h) In acute/ more severe cases (death within 2 to 4 days) In less severe cases (death within 1-3 weeks) In female rats, fetal abnormalities, embryo toxicity and implantation losses have been reported 6 Pathology Seawright (1965) was the first to study the effects of oral administration of lantana leaf extracts on guinea pigs and observed pathological lesions in heart, lungs, liver, gall bladder and kidneys. C. Gross pathology: Liver: Swollen, fragile, pale yellow, mottled with rounded edges (Sharma et al., 1991, 1992). Gall bladder: 3–4 times distended with dark opaque and viscous contents (Sharma et al., 2007). Kidneys: Swollen, pale and yellowish brown (Seawright & Allen, 1972). Stomach: Gas accumulation (Sharma et al., 1991; Sharma et al., 1992). Mucus membranes: Pale (Sharma et al., 1991, 1992). On histopathological examination lantadenes showed degeneration of the periportal parenchymal cells, distended bile canaliculi, fatty degeneration, portal fibrosis, hyperplasia of bile ducts, and edema of gall bladder walls in cattle (Dwivedi et al., 1971; Uppal & Paul, 1978). Hematological examination in cattle reveals, increase in blood clotting time and hematocrit values but decrease in erythrocyte sedimentation rate has been reported (Hussain & Roychoudhury, 1992). There was an increase in direct and total bilirubin, increase in the phylloerythrin levels, increase in serum AST, ALP, GLDH, serum total protein, serum albumin, and serum globulin and decrease in albumin/globulin ratio in cattle (Dwivedi et al., 1971; Seawright & Hrdlicka, 1977). The fibrous tissue formation is seen in chronic liver conditions irrespective of etiology, as in chronic diseases the myofibroblasts produce type 1 collagen which leads to fibrosis. Table. 2 Histopathological alterations in different animal species. S. No 1 Species Cattle 2 Goats 3 Sheep 4 Guinea Pigs and Rats 5 Rabbits Histopathological alterations Degeneration of the periportal parenchymal cells, distended bile canaliculi, fatty degeneration, portal fibrosis, hyperplasia of bile ducts, edema of gall bladder in cattle. Hemorrhages of inter-sinusoidal spaces, coagulative necrosis, cirrhosis and proliferation of bile ductules, fatty degeneration of proximal convoluted tubules of kidneys, proliferation of bile ductules in the liver occurs. Centrilobular cells vacuolation with bile mainly in chronic cases. Periportal vacoular degeneration, fatty degeneration, haemorrhages, bile duct proliferation with yellow-brown bile plugs, portal fibrosis in liver. Fatty degeneration of PCT, vacuolar degeneration of tubular epithelium of cortex, hyaline cast in kidneys. Oedema and haemorrhagic ulcer in gall bladder. Subepicardial petechial haemorrhages in heart along with pulmonary oedema and haemorrhages in lung. Portal fibrosis, bile canaliculi dilatation, degeneration and swelling of hepatic cells, biliary hyperplasia, biliary cirrhosis in the liver. Tubular nephrosis, inflammatory interstitial reaction, degeneration of tubules in the kidneys. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org References Dwivedi et al., 1971; Uppal & Paul, 1978 Sharma et al., 2007 Sharma et al., 2007 Sharma et al., 1992; Parimoo et al., 2015 Sharma 2007 et al., Lantana camara: An alien weed, its impact on animal health and strategies to control 327 Table. 3 Hematological examination in different animal species. S. No. 1. Species Cattle 2. Sheep 3. Goat 4. Guinea pigs Hematological parameters Increase in blood clotting time and hematocrit values but decrease in erythrocyte sedimentation rate. Transient increase in the hematocrit value and neutrophils number but a decline in number of thrombocytes seen. Progressive decrease in packed cell volume, hemoglobin, and total erythrocyte count while increase in leukocyte count and blood clotting time observed. Increase in hematocrit, erythrocyte and leukocyte number, hemoglobin and urea levels in acute lantana toxicity. Significant increase in PCV and TLC, but not in TEC observed in sub-acute lantadene toxicity study. References Hussain & Roychoudhury, 1992 Seawright, 1963 Ali et al., 1995 Sharma et al., 2007; Parimoo & Sharma, 2014 Table. 4 Biochemical Alterations in different animal species. S. No. 1. Species Cattle 2. Sheep 3. 4. Goats Guinea pigs Biochemical Alteration Increase in direct and total bilirubin, increase in the phylloerythrin levels, increase in serum AST, ALP, GLDH, serum total protein, serum albumin, and serum globulin and decrease in albumin/globulin ratio. No change in the serum ALP, AST and ALT levels. Rise of serum bilirubin, AST, creatinine, GGT and BUN levels. Marked increase in conjugated form of bilirubin, AST, LDH, GLDH, BUN, ALT and SDH. No significant increase in total proteins, ACP and creatinine levels were observed in sub-acute toxicity of lantadenes while ALT, AST and ALP were significantly elevated. 7 Treatment Specific treatment for lantana toxicity is still lacking, the preventive measures are more effective than curative measures to decline the harmful effects of this notorious weed (Oyourou et al., 2013), but there are some conventional treatment methods which can be applied (McSweeney & Pass, 1982; Sharma et al., 2007): I. Keep the intoxicated animals away from light; provide fluid therapy and adequate feed. II. Administration of activated charcoal 5g/kg body weight with electrolyte in stomach tube within 24h, which reduces the absorption of lantadenes. III. Administration of bentonite 5g/kg body weight. It is much cheaper than charcoal but takes longer time to show desired effect. IV. Administration of Tefroli powder obtained from Tephrosia purpurea plant. V. Oral administration of liver tonics like Liv-52. VI. Vitamin B-complex administration. VII. Enzymatic removal of bilirubin by bilirubin-oxidase, which is effective in jaundice. VIII. Herbal tea i.e. Yin Zhi Huang (YZH) from Artemisia capillaries, effective in neonatal jaundice. IX. Herbal plants like Tinospora cordifolia, Gingko biloba, Berberis lycium and Hippophae salicifolia also show ameliorative effect on L. camara-induced toxicity in guinea pigs. Gingko biloba has also shown the protective effect against CCl4 (Shenoy et _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org X. XI. XII. References Dwivedi et al., 1971; Seawright & Hrdlicka, 1977 Seawright, 1963; Dwivedi et al., 1971 Obwolo et al., 1991 Sharma et al., 1992; Sharma et al., 2007; Parimoo et al., 2015 al., 2001; Chavez- Morales et al., 2011) and rifampicin (Naik & Panda, 2008) leads to decrease ALT and AST levels when fed to rats. Ginko biloba also shows hepatoprotective action against glyphosate, uranium and CCl4 toxicity, which are potent hepatotoxicant (Yapar et al., 2010; Cavusoglu et al., 2011; Guo et al., 2011). Vaccination can also be done but it is not an effective measure. Bacterial strains like Pseudomonas picketii, Alcaligenes faecalis and Alcaligenes odorans can be used which degrades the LA. Rumenotomy can be done to evacuate the entire GI tract. 8 Prevention It is the cost effective way of controlling the accidental introduction of lantana into the ecosystem. The different ways by which lantana infestation can be prevented includes (Priyanka et al., 2013): i. The international standards for trading partner countries in a well targeted form must be implemented. ii. The adequate surveillance and monitoring system for early detection of lantana infestation must be implemented. iii. Implementation of strict border controls, transport controls and quarantine methods should be followed. 328 Rakesh et al iv. v. The biosecurity and quarantine system should be strengthened in an organized form. Collaboration with government agencies, so that outline can be made to prevent the spread of lantana. Involvement of all the agencies concerned with invasive species management is must. Educate and communicate people regarding the harmful effects of this alien weed which can be done by organizing campaigns and training programs. 8 Control and Management Against this alien weed 41 biological agents are introduced worldwide since 1902 which covers the largest and longest running control program for weed control, but no satisfactory success has been achieved till date (Baars & Neser, 1999; Sheppard, 2003; Zalucki et al., 2007). In past years a huge man power and different ways were used to eradicate lantana. Many mechanical, biological techniques, use of fire etc. were used in India but no success was achieved. In Australia (Haseler, 1979) and South- Africa (Marsh, 1978) efforts were made to eradicate this weed but everything was vain. iv. v. 9 Strategies which can be opted for controlling L. camara includes 1. Monitoring of lantana population by mapping, remote sensing, GPS/GNSS techniques and satellite; assessment and implementation of control measures like crop rotation, sowing the pastures, plantation etc. are the key steps to be taken for successful control of this alien weed (Priyanka et al., 2013). 2. The maximum use of this weed in our routine life can decrease the incidences of its prevalence. So, the small scale research projects can be supported to utilize this plant in many different ways like: i. ii. iii. Train the people for making furniture, baskets, mosquito repellent cakes, incense sticks etc. from lantana. This method is followed in few states of India like Tamil Naidu. This plant is a part of folk medicines for many ailments like cancers, asthma, respiratory infections etc. (Deena & Thoppil, 2000; Ghisalberti, 2000; Bevilacqua et al., 2011). In many parts of the world, this weed is used in the treatment of many ailments like wound healing, scratches, rheumatism, fever, toothache, rashes and malaria (Chharba et al., 1993; Ghisalberti, 2000; Silva et al., 2005). Because of its multifarious applications in health, this weed is also called as traditional and tropical folk medicinal plant (Taviano et al., 2007; Awad et al., 2009; Moffitt et al., 2010; Pour & Sasidhara, 2011). In India because of human health concerns and environmental hazards the insecticides are never mixed with grains, and biofumigants are often proven as very good model against the insects and _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org vi. vii. viii. ix. x. have no risk of cross resistance as well (Rajashekar et al., 2012a; Rajashekar et al., 2012b). The extracts obtained from different parts of lantana have many beneficial properties like anthelminthic, antibacterial, anti-ulcerogenic, anti-inflammatory, termiticidal, antifungal, antiprotozonal, antipyretic and many more (Siddiqui et al., 1995; Barre et al., 1997; Kumar et al., 2006; Rajesh & Suman, 2006; Hussain et al., 2011; Sousa & Costa, 2012). The leaves of this weed contain many bioactive compounds and also have insecticidal activities (Khan et al., 2002; Dua et al., 2010; Rajashekar et al., 2012c). Essential oils obtained from L. camara leaves have adulticidal activity against mosquitoes (Dua et al., 2010). The essential oils obtained from the leaves and flowers of this weed, also shows fumigant action (Alitonou et al., 2004; Zoubiri & Baaliouamer, 2012). The leaf extracts of this weed are having inhibitory effect on aquatic weeds like Microcystis aeruginosa and Eichhornia crassipes (Sharma et al., 2007; Rai, 2013) and are often used for controlling pests and almond moths in an environment friendly way (Gotyal et al., 2010; Rajashekar at al., 2012c; Rajashekar et al, 2013). It also improves the hydraulic properties which is often beneficial to certain crops like wheat and rice (Bhushan & Sharma, 2005; Rai, 2013). The fruit eating populations consume dark blue ripened fruits of this plant as a food (Gosper & Vivian-Smith, 2006; Sharma et al., 2007; Rai, 2013). So it can be used as a source of food. The methanolic extract of L. camara can reduce lipid peroxidation and can elevate the level of glutathione, thereby can prevent free radicals induced damage (Loganayaki & Manian, 2010; Sathish et al., 2011). L. camara along with L. montevidensis shows antioxidant activity (Sousa et al., 2015). This weed can be used as a bio-fuel and in Kraft pulping (Naithani & Pande, 2009; Bhatt et al., 2011). Lantana camara nowadays is being utilized for vermicomposting (Hussain et al., 2015). 3. Chemical control includes the use of chemical weapons like Brush killer 64, Gramoxone, Bladex-H etc. which can reduce the spread of lantana. 4. The biological control is supposed to be the cost effective and long term solution to get rid of this alien weed (Hunt et al., 2008). Risk assessment is most effective tool to check the stability of biological control agents used against lantana (Arnett & Louda, 2002; Baars, 2003; Berner & Bruckart, 2005; Briese, 2005; Sheppard et al., 2005; Wright et al., 2005; Ding et al., 2006; Hunt et al., 2008). Biological control includes: Lantana camara: An alien weed, its impact on animal health and strategies to control 329 Table. 5. List of some useful products obtained from different parts L. Camara. S. No. 1. 2. 3. 4. Part Leaves, stem Leaves, stem, roots Aerial parts Leaves Compounds Oleanonic acid Oleanolic acid Camarinic acid, Lantanoside Lactones containing euphanes Action Anti-inflammatory Antimicrobial, antitumor, anti-inflammatory Nematicidal Anti-thrombin 5. Leaves Apigenin Anti-proliferative 6. 7. Leaves Leaves and branches Camaraside Martynoside Antitumor Cardioactive (Sources: Sharma et al., 2007; Hussain et al., 2011; Sousa & Costa, 2012) i. Use of certain biological agents like plume moth (Lantanophaga spp.), seed fly (Ophiomyia spp.), fungus (Corynespora cassiicola) (Pereira et al., 2003) and Tingid bug (Leptobyrsadecora). ii. Some of the plants like Aconophora compressa and Citharexylum spinosum can be introduced for the biological control of this weed as in Australia (Palmer et al., 1996; Dhileepan et al., 2006; Manners & Walter, 2009; Manners et al., 2010). 5. In some of the states like Himachal Pradesh the state forest department has introduced a ―Cut Root Stock (CRS) ―method for the eradication of this weed. 6. Use of lantana in research can be done e.g. the ripened berries of lantana are often used for preparing silver nanoparticles nowadays (Kumar et al., 2015). 7. In many metal polluted tropical and sub-tropical countries this weed is used in phytoextraction of heavy metals especially lead (Jusselme et al., 2012; Jusselme et al., 2013; Jusselme et al., 2015) and phytoremediation of particulate pollution (Rai, 2012; Rai, 2015a; Rai, 2015b). 10 Differential diagnoses It is little bit difficult to differentially diagnose lantana toxicity from other plant toxicities, because almost similar kind of lesions and symptoms are produced by these plants e.g. Senecia, Crotolaria, Helenium spp (Sneezeweed) produce hepatotoxicity like lantana poisoning. The oak poisoning also produces similar signs. Therefore clinical history, clinical signs, presence of plant in feed and ruminal contents are quite informative to assess the lantana toxicity. plantations. The allelopathic effect is the major contributor for hampering the growth of surrounding vegetation and flare up wherever it finds place. The lantadenes are the major toxic components present in this plant which are responsible to cause toxicity in almost all the animals thereby leads to economic losses to the farmers by causing diseases and mortality. Specific treatment for lantana toxicity is not available and only preventive measures are supposed to be more effective. Certain methods for the management of toxicity are often used but are not much effective. Besides many harmful effects this weed is having many advantages. But the harmful effects often supervenes the utility of this weed. So, it is very important to develop the measures to control this weed in a desirable and cost effective way. Many approaches are applied to destroy this weed but most of them are not effective. Only the utilization of this plant is supposed to be an effective method for managing this weed. This utilization approach can only be capable to get rid of the negative impact of this weed on environment and can help to promote economic upliftment of rural economy. It is also very important to develop rational therapies against lantana toxicity by using immunological and biotechnological approaches, so that along with utilization the therapeutic measures can be evolved for livestock treatment. Already many pharmacological effects of this weed have been known, but still there is a scope to use this plant in the field of nanotechnology and therapeutics which can provide long term solutions to avoid the cruelty of this weed to the livestock, mankind, vegetation and our ecosystem. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 PREVALENCE, DIAGNOSIS, MANAGEMENT AND CONTROL OF IMPORTANT DISEASES OF RUMINANTS WITH SPECIAL REFERENCE TO INDIAN SCENARIO Mani Saminathan1, Rajneesh Rana2,*, Muthannan Andavar Ramakrishnan3, Kumaragurubaran Karthik2, Yashpal Singh Malik4 and Kuldeep Dhama1 1 Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India 3 Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar Campus, Uttarakhand - 263 138, India 4 Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India 2 Received – May 05, 2016; Revision – May 09, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).338.367 KEYWORDS ABSTRACT Ruminant Diseases Prevalence Diagnosis Prevention Management Control India India possess huge livestock population, which is endangered by different endemic infectious diseases (bacterial, viral, protozoan and parasitic), which collectively causes significant economic losses to the landless poor farming community. Infectious diseases impose economic losses by causing morbidity, mortality, decreased production (milk, meat, wool etc.), decreased feed conversion ratio which results in reduced weight gain, decreased draught power and fertility. Furthermore, economic burden is also due to the cost of treatment, abortion, consequences on internal livestock movement, germplasm and international trade. In addition, some of the diseases are zoonotic and inflicts considerable impact on public health. Uncertain agrarian climate, unpredictable weather, drought, floods, migration of livestock, scarcity of fodders, and unhygienic zoo-sanitary and healthcare practices together resulted in endemicity of diseases ultimately leads to more incidence and prevalence of livestock and poultry diseases throughout the year. Synchronized monitoring and surveillance of disease throughout the country is a fundamental requirement for sustainable livestock production. With fairly developed telecommunication in India, following technologies like interactive voice response system, SMS through mobile/cell phones and toll-free landline phones (voice mail) are required for enhancing the effectiveness and efficiency of * Corresponding author E-mail: [email protected] (Rajneesh Rana) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 339 Saminathan et al disease monitoring and surveillance. Multidisciplinary approaches at international, national and local levels are required for management, control and eradication of endemic and transboundary diseases of livestock. Improved monitoring and/or surveillance, rapid and confirmatory diagnosis, and networking of diseases are required to go forward in the path of eradication. Vaccination is the main strategy for control and eradication of many diseases. Good management practices consisting of stringent biosecurity measures, strick sanitation and hygiene practices in the farm, isolation and quarantine of diseased animals, and trade restrictions are necessary for successful operation of control programmes. This review highlights prevalence, pathogenesis, diagnosis, and prevention and control measures for important diseases of ruminants with special reference to India. 1 Introduction India possesses rapid growing animal husbandry sector and moving towards to attain self-sufficiency in the production of livestock products (Dhama et al., 2014a). The animal husbandry department is a major contributor to the Indian economy and overall contribution is 28-32% in agricultural GDP and 4 to 6% of the national GDP. It also contributes 810% of the country‟s labour power (Hemadri & Hiremath, 2011). India possess largest livestock population in the world with 528 million of domesticated animals; first place in the world in buffalo population (105.3 million), second in cattle (199 million) and goat (140.5 million) population, and third in sheep (71.5 million) population (Hemadri & Hiremath, 2011; Biswal et al., 2012; Dhama et al., 2014a; Chand et al., 2015). Livestock population in India is threatened by disease outbreaks, droughts, floods and other climatic anomalies. There are several diseases affecting livestock that causes serious effect on the production of animals, human health, trade of livestock and animal products, as a result the overall economic development will be affected (Gibbs, 1981; Depa et al., 2012; Dhama et al., 2014a). Improved quality and quantity of livestock products is necessary, in order to compete in the international market, which intern needs disease free animal health status (Bhanuprakash et al., 2011; Awase et al., 2013; Bayry, 2013; Chand et al., 2015). In recent times, emerging and re-emerging diseases of livestock, poultry and humans have tremendously increased. Many of the diseases like brucellosis, tuberculosis, glanders, corona, influenza, Nipah and Hendra viral diseases are of zoonotic significance (Chakraborty et al., 2014; Dhama et al., 2014a; Kumar et al., 2015a). and human population. Higher occurrences of emerging and reemerging diseases might be due to various factors like crowded livestock and human population, deforestation, lack of public awareness and increased contact between livestock and humans with wild animals and birds (Depa et al., 2012; Chakraborty et al., 2014). The global expansion of cultivating land, population growth, intensive industrialization, climate changes, movement of vectors, illegal and unregulated trade, hiding/reduced reporting of the disease outbreaks are other reasons for the emergence and spreading of the disease (Gibbs, 1981; Dhama et al., 2014a,). Two-thirds of the global population including human and livestock are living in the developing countries and majority of the diseases were emerged from developing countries. International and national collaboration along with sincere scientific implementations and political decisions are necessary to tackle such emerging infectious diseases (Bhanuprakash et al., 2011; Hemadri & Hiremath, 2011; Biswal et al., 2012). An effective management for emerging and re-emerging diseases needs multidisciplinary activities like surveillance, rapid reporting, collection and transport of clinical materials for diagnosis of the etiological agents, strengthening of basic research, epidemiological modelling and prediction, forecasting model development, development of novel vaccine candidates and suitable adjuvants etc. (Biswal et al., 2012; Depa et al., 2012; Chand et al., 2015). The present review discusses the prevalence, pathogenesis, diagnosis and prevention and control measures for important diseases of ruminants with special reference to India. 2 Viral diseases of ruminants 2.1 Foot and Mouth Disease (FMD) Beside that several other viral diseases of animals in India such as foot and mouth disease (FMD), bluetongue (BT), peste des petits ruminants (PPR), sheeppox, goatpox, camelpox, infectious bovine rhinotracheitis (IBR), malignant catarrhal fever (MCF) and bacterial disease like haemorrhagic septicaemia (HS), black quarter (BQ), anthrax and brucellosis were endemic and has potential of crossing continental boundaries (Arya & Bhatia, 1992; Benkirane & De Alwis, 2002; Bhanuprakash et al., 2011; Biswal et al., 2012; Saminathan et al., 2013; Bayry, 2013; Chand et al., 2015; Kumar et al., 2015a). Emergence of new serotypes in various pathogens creates additional risk and warning to the livestock _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Foot and mouth disease (FMD) is endemic in India from 1864 onwards (Subramaniam et al., 2013). Animal population in India is threatened by FMD owing to unrestricted movements of animals, incomplete vaccinations, and inapparent infection in small ruminants which act as reservoirs. Direct loss of 20,000 crore/annum has been estimated due to the disease (Venkataramanan et al., 2006). FMD causes huge economic losses and decrease in milk yield causes 8% of total direct loss (Mathew & Menon, 2008). The other economic losses were huge expenditure spent for FMD control programmes throughout the country by the government, increased cost for Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario treatment, decreased productivity (meat, wool etc.) and draught power. The disease causes severe damage to the production and international trade. Because of its severity, the OIE and FAO declared it as “High Priority Disease”. The body also appealed to different countries to make effective groups for its strategic control (FAO & OIE, 2012). FMD affects wide variety of host including 529 million of livestock as well as captive and free-living wild even toed animals (Verma et al., 2008; Verma et al., 2012). Among 7 serotypes (O, A, C, Asia1, SAT-1, SAT-2 and SAT-3), only O, A, C, and Asia-1 were recorded in India. Since 1995, C serotype has not been reported from India and the globally the last outbreak was reported in Ethiopia during 2005 (Rweyemamu et al., 2008). About 70-80% of outbreaks are due to „O‟ followed by Asia 1 (3-10%) and A (3-6.5%) (Hemadri & Hiremath, 2011; Biswal et al., 2012; Pattnaik et al., 2012). The FMD incidence is increased in India due to the execution of schemes for indigenous cattle genome improvement by cross-breeding of local cattle breeds with exotic cattle. FMD incidences were more during pre-monsoon and winter season; however incidence of FMD were reported regularly all the months of year. The probable reason for its continuous persistence is due to uncontrolled animal movements throughout the country. Besides that because of the weak financial status of some of the livestock owners; their herd remains unvaccinated which posses‟ additional threat to the surrounding vaccinated herds. It has been reported that among 10 genotypes of serotype A, only 2 called as VI and VII were circulating in India from past 20 years. In Asia 1 serotype, VIA and VIB genotypes were circulating in India (Verma et al., 2010; Hemadri & Hiremath, 2011; Biswal et al., 2012; Pattnaik et al., 2012; Subramaniam et al., 2013). The control and eradication of FMD in India, progressive control pathway (PCP) was implemented (Rweyemamu et al., 2008). The FMD control programme (FMDCP) was executed in 54 districts from 8 states of India during 10th five year plan (based on epidemiological data obtained from more than 35 years) covering the population of 30 million cattle and buffalos. In the control program implemented areas, there has been gradual build up of herd immunity and substantial fall in the disease incidence (Biswal et al., 2012; Depa et al., 2012; Pattnaik et al., 2012; Verma et al., 2012; Subramaniam et al., 2013). 2.2 Peste des Petits Ruminants (PPR) Peste-des-petits-ruminants (PPR) is a highly contagious, acute and transboundary viral disease of goats and sheep caused by the genus Morbillivirus belonging to the family Paramyxoviridae. Clinically, the disease is manifested as conjunctivitis, high fever, oculonasal discharge, necrotizing and erosive stomatitis, enteritis and bronchopneumonia followed by either mortality or recovery from the disease (Taylor, 1984). Currently, as South Asia is more focused for PPR, it percolates serious losses to the countries like Afghanistan, Pakistan, Nepal, Bangladesh and India. The disease was first observed in Tamil Nadu in 1987 (Shaila et al., 1989). On the genomic basis the virus is grouped into four _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 340 lineages i.e. I, II, III, and IV out of which lineage IV is common in Asia (Balamurugan et al., 2012). In India, till date, only lineage IV viruses have been reported. In North-eastern regions the seroprevalence rate of PPR in goats was 13.8% in Assam (Begum et al., 2016) and 2.11% in Tripura. The PPRV goat strain isolated during 2013 outbreak in Tripura region of India shared 99.2 to 99.6% nucleotide identities with the Bangladeshi strains (Muthuchelvan et al., 2014). The disease outbreak is most commonly observed in the months of April to October followed by winters. Goats were more susceptible to PPR and manifest severe clinical form of disease than sheep. A report from central India showed occurrence of dual infection of PPR and Goatpox in indigenous goats (Malik et al., 2011a). Several PPR outbreaks were encountered and the disease is enzootic in most of the southern states of India like Karnataka, Andhra Pradesh and Tamil Nadu; western states of India like Maharashtra; eastern states of India like West Bengal and Orissa; northern states of India like Rajasthan and Himachal Pradesh; central states of India like Madhya Pradesh (Dhar et al., 2002; Balamurugan et al., 2012; Singh et al., 2013; Muthuchelvan et al., 2015). The expected yearly losses due to this disease may reach up to 1800 million rupees. In northern region of India, outbreaks were most frequent in goats whereas in southern regions of India, the outbreaks were most frequent in sheep (Balamurugan et al., 2012; Balamurugan et al., 2014). The economic losses per animal in sheep and goats ranges between Rs. 523 to 945 (Thombare & Sinha, 2009; Awase et al., 2013). The growth of goat industry is hampered by PPR owing to high morbidity (50-90%) and mortality (50-85%) rates. Kids more than 4 months and less than one year of age are more susceptible to PPR. The occurrence of disease outbreaks was higher during March to June (51.7%) when compared to remaining months (Thombare & Sinha, 2009; Awase et al., 2013). The economic impact of PPR includes trade limitations; a hindrance to intensive livestock production development due to the impedance to the import of new breeds, which in turn cause scarcity of animal protein to humans. The control of PPR can be achieved by effective vaccination measures. Infected animals should be kept in quarantine for the period of one month. In the infected area, the movement of the animal should be restricted strictly. Practically, sanitary and control measures are difficult to follow in India due to vast nature and PPR endemicity. However, the effective measure for PPR control is mass vaccination using the effective vaccine, along with quarantine measures (Sen et al., 2010; Balamurugan et al., 2014; Muthuchelvan et al., 2015). The control measures should contain the “bottom-up” approach such as from livestock owners to field veterinarians to policy makers (Singh, 2011). Further, increasing the production of PPR vaccine, enhancing the disease diagnostic facilities, strengthening the quality control units and improved infrastructure facilities for field workers is necessary for management and control of diseases. The NCP-PPR control 341 scheme was implemented in 2010 and presently extended to all the states of the country (Balamurugan et al., 2014; Muthuchelvan et al., 2015). 2.3 Bluetongue (BT) Bluetongue (BT) disease is now endemic in India and the first case of BT was reported from Maharashtra during 1964 (Sapre, 1964). Later on, the disease has been reported from several parts of the country based on virus isolation and/or detection of BTV-specific antibodies. BTV belongs to the genus Orbivirus of the family Reoviridae (Pringle, 1999). In serological survey, the presence of antibodies against BTV in Indian cattle, buffalos, goats, camels as well as in some wild ruminants has been observed. However, in cattle and buffalos, clinical form of BT has not been reported. The clinical signs vary from asymptomatic to fatal form which is determined by the BTV serotype, animal species, breed and age (Elbers et al., 2008). Recently, 27 BTV serotypes have been identified worldwide with the addition of 2 more new serotypes (Maan et al., 2011; Zientara et al., 2014). In India, 22 serotypes have been recognised on the basis of virus isolation and/or serology (Rao et al., 2016). Presently, 13 serotypes namely, BTV-1, 2, 3, 4, 6, 9, 10, 12, 15, 16, 17, 18, 21 and 23 were isolated from India especially from southern states (Maan et al., 2012; Minakshi et al., 2012; Rao et al., 2012; Chauhan et al., 2014). Genomic studies of these serotypes exhibit their variation with the standard reference strain (Rao et al., 2016). Culicoides spp. is the major vector for BTV. Among 1400 species were identified worldwide, minimum 39 species have been identified as vector for BT in India (Maheswari, 2012). The analysis of outbreak data reveals that the intensity of disease outbreak was severe in Karnataka followed by Andhra Pradesh and Tamil Nadu (Hemadri & Hiremath, 2011). During 2007-08, a severe outbreak of the disease was happened in India, afterwards only a mild clinical form of disease was observed (Hemadri & Hiremath, 2011). Diagnosis of BT can be done by epidemiology, vector species, clinical signs, postmortem findings and molecular tests (Afshar, 1994). Confirmatory diagnosis can be done by virus isolation, detection of anti-BTV antibodies by serological methods and nucleic acid detection by RT-PCR. BTV can be isolated from chicken embryonated eggs and competitive ELISA (c-ELISA), virus neutralization test (VNT) and agar gel immunodiffusion (AGID) assay (Afshar et al., 1989; Pathak et al., 2008). By using RNA-PAGE, cell culture adapted Indian BTV with more than 10 segments have been reported (Ramakrishnan et al., 2005b). For successful control of BT in India, it is required to initiate vector and sentinel control measures and rapid disease diagnosis. The control measure for BT includes vaccination of animals with an inactivated BTV vaccine. Inactivated monovalent BT vaccines were developed using BEI (Ramakrishnan et al., 2006) and hydroxylamine inactivants (Ramakrishnan et al., 2005a). Recently, for control of BT in India, inactivated pentavalent vaccine consisting of BTV-1, 2, 10, 16 and 23 was developed and commercialised (Reddy et al., 2010). Control of BT in India is a difficult task due to more _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Saminathan et al susceptible host species and more BTV serotypes. However, control of BT can be achieved by protecting the susceptible animals from vector Culicoides but always this is also not possible. Control of vector can be done by using insecticides, but it is expensive and does not provide complete relief from the vector. In India, modified live vaccines (MLVs) are not preferred due to nomadic nature of animals‟ results in spreading of the disease. Therefore, for management and control of BT, inactivated multivalent vaccines are preferred (Chand et al., 2015). 2.4 Sheeppox and goatpox Sheeppox virus (SPPV) and goatpox virus (GTPV) belongs to a member of the genus Capripoxvirus of the family Poxviridae. The sheeppox and goatpox diseases are notifiable to Office Internationale des Epizooties (OIE) due to its economic impact. In India, sheeppox and goatpox diseases are endemic, which are host specific and pose a serious economic loss to small ruminant husbandry (Bhanuprakash et al., 2006; Bhanuprakash et al., 2010; Bhanuprakash et al., 2011). Although, SPPV and GTPV are considered as host-specific they can cross the species barrier i.e. these viruses can infect both species (Santhamani et al., 2015). The outbreak of capripox was first reported in 1936 in India, and since then, frequent outbreaks have been reported throughout the country in almost all the states where sheep and goats are reared. The occurrence of the disease is usually observed throughout the year, however, most frequently seen during the rainy season (Garner et al., 2000). Mortality in young animals can exceed 50%. A recent report revealed that the morbidity and mortality rates in the flock were 18.4% and 6.3%, respectively (Hemadri & Hiremath, 2011). Exotic and young animals are highly susceptible (Bhanuprakash et al., 2006). Certain predisposing factor for spreading out the disease is the presence of a virus on the wool of the recovered animal. The virus can be transmitted by aerosols from diseased animals, through direct contact with abraded skin and mucosa or indirectly by vectors through mechanical transmission (Kitching & Mellor, 1986). Direct losses due to mortality are low, however, morbidity and post-disease impacts on leather quality, wool, and meat productivity are more (Bhanuprakash et al., 2011). In the Maharashtra state of India, the economic losses due to capripox diseases are calculated as Rs. 105 million (US$2.3 million) with an average morbidity and mortality of 63.5% and 49.5%, respectively, and after 6 years the flock recovered from outbreak (Garner et al., 2000). By the estimation of these losses, the predicted total annual loss is Rs. 1250 million (US$ 27.47 million) at the national level (Bhanot et al., 2009). The recovered animals from infection shall acquire lifelong immunity. In India, the slaughter policy and movement restrictions for disease control are difficult to follow due to various socio-economic factors. Therefore, an economical and sustainable approach for disease control is mass vaccination. Recently, P32-gene based PCR-RFLP and RPO30 and GPCR genes based sequencing analyses have been applied for the differentiation of Indian strains of SPPV and GTPV from field Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario samples (Santhamani et al., 2013; Santhamani et al., 2014; Santhamani et al., 2015). Vaccination is practiced for many years mainly for sheep pox to decrease the endemic nature of the disease. Recently, live goat pox vaccine has been developed to reduce the disease incidence in goats (Bhanuprakash et al., 2011). 2.5 Infectious Bovine Rhinotracheitis (IBR) Bovine herpesvirus 1 (BoHV-1) causes IBR, which consist of various clinical manifestations such as pustular vulvovaginitis, rhinotracheitis, balanoposthitis, infertility, abortion, mastitis, conjunctivitis and encephalitis in cattle. Mehrotra et al. (1976) first reported the disease in Uttar Pradesh, India and isolated the virus from calves affected with keratoconjunctivitis. Afterwards, the disease was reported from various parts of the country in India. The disease prevalence was more in crossbred and exotic breeds of cattle when compared to local breeds of cattle (Majumder et al., 2015a). IBR is usually transmitted by semen posing a serious risk to productivity and reproductive health (Huck et al., 1971). BoHV-1 is considered as most commonly found viral agent in the semen of bovines. Akin to other herpes viruses, BoHV-1 can cause latent infection and animals turn into reservoirs of the virus in the herd. Reactivation of virus from latent infection leads to shedding of the virus in the bull semen. BoHV-1 is maintained in the environment due its short cycle of infection, latent infection, resistance to environmental factors and reactivation during stress conditions (Muylkens et al., 2007; Nandi et al., 2009). BoHV-1 can cause considerable economic losses due to loss of body condition, abortion, milk yield, temporary failure of conception, loss of newborn calves, insufficient feed conversion, secondary bacterial pneumonia, cost of treatment and impact on national and international trade on germplasm and livestock (Gibbs, 1981; Majumder et al., 2015a). The mortality and morbidity rates differs among the breeds of cattle, which was lesser in milch breeds of cattle viz., 3% mortality and 8% morbidity when compared to beef breeds and feed lot cattle, which shows higher mortality rates of 2030% (Barenfus et al., 1963). The disease is not sex dependent as male and females are equally susceptible. Crossbred cattle are more susceptible than indigenous breed (Krishnamoorthy et al., 2015). Field virus infection or immunization using an avirulent strain of BoHV-1 virus resulted in development of latent infection. Sero-epidemiological data of BoHV-1 during 2009-10 in cattle and buffalo showed that the prevalence of disease was more in Tamilnadu (67% prevalence rate) and Meghalaya state is being lowest in prevalence (Selvaraj et al., 2008). The significant levels of antibodies were detected in bovines having a history of reproductive problems and abortions. A study revealed high antibody prevalence of BoHV-1 in cattle (50.9%) and buffaloes (52.5%) from India (Renukaradhya et al., 1996). The disease was reported in the yak with the overall prevalence of 40.8% (Rahman et al., 2007) and Mithun with overall prevalence of 19% (Rajkhowa et al., 2004). In general in India, the prevalence rate of disease was 34% with differing _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 342 prevalence rates in various parts of the nation includes 39% north-eastern, 37% northern, 25% central, 24% western and 17% eastern (Selvaraj et al., 2008; Bandyopadhyay et al., 2009a; Sarmah et al., 2015). In a recent report, 18% seropositivity was reported in bovine serum samples in Tripura (Majumder et al., 2013a). The BoHV-1 can be detected by virus isolation, SNT, FAT in infected tissues, ELISA, and IPT. Virus isolation using cell culture has been commonly practised (Majumder et al., 2015a). There are very limited information is available on molecular characterization of Indian BoHV1. Very recently, gB, gC, and gD genes of Indian BoHV-1 isolate were characterized (Majumder et al., 2013b; Majumder et al., 2014, Majumder et al., 2015b). The increased percent prevalence of the disease in recent past indicates that the problem should be tackled by adopting strict sanitary measures, maintaining 2-3 weeks of quarantine period before introducing new stock to the herd and segregation of virus positive animals, vaccination and use of semen from disease free animals. In order to get rid of the virus from a herd, identification and slaughtering of infected animals are required due to the possibility of reactivation of virus from latent infection (Majumder et al., 2015a). 2.6 Bovine viral diarrhoea (BVD) BVD is an endemic disease worldwide and causes considerable economic losses in cattle farming community. It is caused by bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae. Based on the pathogenic behaviour in cultured cells, cytopathic and non-cytopathic biotypes of BVDV are identified. BVDV consists of two genotypes (type 1 and 2) and further subdivided into several genetic and antigenic variants. Both the genotypes cause acute and persistent infection. Although the main focus of the research in cattle, the virus can infect wider range of species including domestic and mountain goats (Nelson et al., 2015). The economic losses are due to prenatal infections, infertility, abortions, congenital anomalies in calves, more neonatal deaths and persistently infected (PI) calves, which die due to mucosal disease. The incidence of BVD is usually unnoticed due to 70 to 90% of infections were in subclinical form (Neibergs et al., 2011). The BVDV was isolated from clinical samples for the first time in India by Mishra et al. (2004). The incidence of BVD in cattle (Sood et al., 2007), sheep and goats (Mishra et al., 2009) and buffaloes (Mishra et al., 2008) in India has been reported using molecular confirmatory tools. Commonly circulating genotype in BVDV in Indian cattle is BVDV-1b (Mishra et al., 2004). BVDV-2 was identified from sheep and goats (Mishra et al., 2007; Mishra et al., 2008) and cattle (Behera et al., 2011) might be due to unrestricted migration and trading in ruminants. Various serological studies have indicated the prevalence of BVDV antibodies in Indian cattle population (Nayak et al., 1982; Mukherjee et al., 1989; Sudharsana et al., 1999; Mishra et al., 2011). Post infected (PI) animals maintain the virus and play a major role in the spread of the virus among cattle population (Broderson, 2014). PI animals acquire the infection 343 by in utero from non-cytopathic biotype of BVD virus during 45 to 125 gestation period results in immunotolerance (Hessman et al., 2012). PI animals excrete more viruses throughout their lifespan and act as a major source of virus spread between and within the farms. Because PI animals give negative results for a serological test for antibody detection, however, positive for BVD antigen, hence assays targeting viral antigens detection are ideal for their diagnosis in a herd. 2.7 Picobirnaviruses The picobirnaviruses (PBV) have been identified as the emerging pathogens associated with enteric and respiratory infections in a number of mammalian and avian species. These are small structured viruses of nearly 35 nm with doublestranded bisegmented RNA genome. PBVs have been designated as genogroup I (GGI), genogroup II (GGII) and genogroup III (GGIII) based on sequence analysis of genome segment 2. In India, PBV was for the first time detected in bovine in West Bengal in 2009 (Ghosh et al., 2009) and subsequently from central India (Malik et al., 2011b). A PBV strain isolated from western Maharashtra from a buffalo calf showed huge genetic divergence (Malik et al., 2013a). Recently, we have identified and characterized a novel genogroup II picobirnavirus from a cattle calf (Malik et al., 2014), which is the first report of genogroup II detection from bovines. PBVs are detected together with other major enteric viruses such as rotavirus, astrovirus, coronavirus etc and are gaining importance these days. It is presumed that PBVs could be a potential threat for growing livestock industry due to leading gastroenteritis and associated economic issues. 2.8 Bovine rotaviruses Rotavirus (RV) leads to severe gastroenteritis and has become a major health problem throughout the world. The RV infections enforce colossal economic losses mainly due to increased morbidity and mortality, treatment and poor growth performance of enteritis-affected animals. Though considerable research has been carried out on RV disease in humans in India, information is scanty on animal rotaviruses epidemiology. The RV associated clinical signs may vary from asymptomatic/subclinical to severe enteritis. The RV prevalence has been reported between 3.25% to 42% using serological and molecular methods during 1990-2001 (Mittal et al., 1991; Shah & Jhala, 1992; Agarwal & Singh, 1993; Gulati et al., 1995; Jindal et al., 2000; Khurana & Pandey, 2001). The information generated through VP7 gene (G) and VP4 gene (P) genotyping of bovine RVs in various epidemiologic surveys confirms that (i) G3, G6, G8 and G10 constitute more commonly circulating G genotypes and P[11] as the P genotype in the country; (ii) other G types, such as G15 with P[15] and P[21] types have been detected but are localized in some parts of the country; (iii) multiple G and P types can cocirculate within the same region (Malik et al., 2012) and can cross the inter-geographical boundaries of the states (Malik et al., 2013b). Concurrent infection with two or more pathogens is a common phenomenon and interactions among multiple _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Saminathan et al pathogens often appear to generate a more severe or chronic outcome than is observed with the individual pathogen (Minakshi et al., 2009). Still very little is known about the specific interactions which occur in concurrent infections. Minakshi et al. (2009) reported the occurrence of dual infection of bovine group A rotavirus in a diarrhoeic calf in one of the northern state, Haryana, India. Malik et al. (2012) described the detection of G3 genotype in combination with G8 or G10 genotype. The control of the virus infection in both humans and animals is dependent on regular monitoring of newly emerging RVs and production of an effective vaccine to control rotavirus associated enteritis in young calves remains a challenge throughout the world. 3 Bacterial diseases of ruminants 3.1 Haemorrhagic septicaemia (HS) Haemorrhagic septicaemia (HS) is the most significant bacterial contagious infection in cattle and buffaloes with proven endemicity in India (Shivachandra et al., 2011). The disease usually causes devastating and alarming problem in buffaloes and cattle. The course of disease becomes fatal when the aetiological agent enters in non-endemic areas of reared bovines. The disease was more severe in buffaloes when compared to cattle and young and young adult animals manifest more severe form of disease than older animals (Singh et al., 2014a). Moreover, the disease is widely reported from bison, African buffalo, camels, elephants, horses, donkeys as well as from yak. HS is an acute, fatal and septicemic disease of cattle and buffaloes caused by Pasteurella multocida (Rajeev et al., 2011; Shivachandra et al., 2011). P. multocida causes HS in cattle and buffaloes, and pneumonic pasteurellosis in sheep and goats. The most prevalent serotypes in India are B:2, A:1, A:1,3, A:3, A:4, A:3,4,12, F:3, D:1, D:3, F:1, F:4 and F:4, 12. In an Indian molecular study of MLST conducted by Sarangi et al. (2016), 10 different sequence type were identified as ST 122, ST 50, ST 9, ST 229, ST 71, ST 277, ST 129, ST 280, ST 281 and ST 282. The B:2 serotype in India in pigs causes sporadic septicaemic disease. Apart from buffaloes, sporadic occurrence of the disease with 62% outbreak rates in goats, 102% in sheep and 5% in pigs has been reported during 2007-2010. An estimated economic loss due to haemorrhagic septicaemia in India is Rs. 225 million per year (Singh et al., 2008a). The economic loss due to HS has been calculated as Rs. 6816 per infected cattle and Rs. 10901 per infected buffalo. The total economic loss was Rs. 5255 crore estimated from throughout India. Among that, about 80.3% economic losses are due to direct effects of HS and 19.7% economic losses are due to indirect effects of HS. In HS, about 74.8% of the economic losses are due to affections in calves and 25.2% are due to loosses in adults (Singh et al., 2014a). In India, HS is the leading cause of mortality and second most commonly encountered disease during 1991 to 2010 reported by National Animal Diseases Referral Expert System (NADRES). According to NADRES report, approximately 97% of the HS Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario outbreaks were occured in cattle and buffaloes (Gajendragad & Uma, 2012). Epidemiological data of HS obtained during 1974 to 1986 revealed that it was the leading cause of mortality and second leading cause of morbidity than other enzootic diseases like RP, FMD, BQ and anthrax (Dutta et al., 1990). During the last 4 decades, it has been found that HS caused 46 to 55% of mortality in bovines in India (Benkirane & De Alwis, 2002). Diagnosis can be made on the basis of clinical signs and post-mortem findings and confirmatory diagnosis can be done by isolation and identification of etiological agent with appropriate staining (Rajeev et al., 2011). Vaccination is a major tool for the control of disease especially 2 to 3 months before the high-risk monsoon season. Although various HS vaccine types like oil adjuvant, alum-precipitated and various emulsion vaccines are commercially available, the search for suitable vaccines with long-lasting immunity and the good protective response is required. Good sanitary measures, early diagnosis, quarantine, isolation of infected animals, immediate antibiotics treatment, deep burial or incineration of carcasses and restriction of animal movements to disease-free areas are essential. Awareness of the disease among farmers is required for effective disease reporting system (Benkirane & De Alwis, 2002; Shivachandra et al., 2011). P. multocida has a zoonotic potential and infection to human spread through bites and scratches of the animals (especially dogs and cats) (Aski & Tabatabaei, 2016). The infection may lead to ocular infection to fatality in humans (Corchia et al., 2015; Talley et al., 2016). Shivachandra et al. (2013) studied the genetic relatedness of ptfA gene among P. mutocida isolates of different species and observed that avian isolates are divergent from mammalian isolates (Shivachandra et al., 2013). Recombinant Omp87 protein of P. multocida serogroup B:2 strain P52 elicited increase in IgG response and provided a different level of protection against homologous and heterologous challenge (Kumar et al., 2013). Similarly, rVacJ protein elicited antigenspecific IgG response in immunized mice (Shivachandra et al., 2014b) and comparative amino acid sequence of different P. Mutocida isolates showed absolute homogeneity (Shivachandra et al., 2014a). In another study, it was demonstrated that recombinant transferrin binding protein A (rTbpA) elicited I antigen specific IgG response and provided a different level of protection in mice challenge (Shivachandra et al., 2015). 3.2 Black quarter (BQ) BQ is a highly fatal and acute bacterial infection of cattle caused by Clostridium chauvoei affecting buffaloes, sheep, and goats. Young cattle and buffaloes with 6 to 24 months of age and good body condition are highly susceptible. C. chauvoei is normally present in the intestine of animals. In the soil, spores remain viable for many years and can act as a source of infection to animals. BQ is a soil-borne infection and outbreaks occur most commonly during the rainy season, in areas with moderate rainfall, where dry-crop cultivation is commonly _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 344 practised. This disease is widely prevalent in cattle belonging to Karnataka, Andhra Pradesh, Tamil Nadu and Maharashtra states of India. One sporadic outbreak of the disease from eight HF crossbred cows has also been reported by Zahid et al. (2012) from Ludhiana, Punjab. During 2009-10, the highest outbreak of disease were reported from West Bengal with 95 outbreaks, in which 293 animals were affected and 122 animals were died and then in Maharashtra with 37 outbreaks, in which 128 animals were affected and 96 animals were died (Hemadri & Hiremath, 2011). Combined prophylactic vaccine consists of ium aluminium hydroxide adsorbed and formalin inactivated cultures of C. chauvoei and P. multocida gives protective immunity against BQ and HS. Prior to operation in ruminants, proper disinfection of surgical instruments is necessary to avoid infection. 3.3 Anthrax Anthrax is a highly fatal, acute and febrile zoonotic disease. It is enlisted in top five diseases of zoonotic importance in India where attention has to be prioritizing (Sekar et al., 2011). Cattle and sheep are highly susceptible to anthrax followed by horse, mules and pig. It is a soil-borne infection, caused by Bacillus anthracis and outbreaks generally occur after the climatic change. The disease is endemic in south Asia and Bangladesh (Thapa et al., 2014), especially enzootic in India and endemic in Karnataka, Tamil Nadu, Andhra Pradesh, West Bengal, Orissa, Maharashtra and Jammu and Kashmir (Gunaseelan et al., 2011). The "incubator zones" for the presence of anthrax is an alkaline soil pH and dry periods which provides the microenvironment for spore survival and increased exposure to susceptible hosts. Majority of the disease incidence were encountered in cattle followed by sheep or goat, buffalo, pig and elephant in India. Although the disease is restricted to herbivores, but few sporadic cases from wildlife captive animals like hyena of Nandanvan zoo, Chattisgarh have also been reported in recent past (Patil, 2010). The possible role of the insects like house fly (Musca domestica) in spreading the disease among animals also cannot be ruled out (Fasanella et al., 2010). Beside that another important source of spread of infection is a bone meal which is an essential additive of animal feed as well as in fertilizers; being frequently imported or exported among developing countries (Davies & Harvey, 1972). The epidemiological data collected during 2002-2010 from bovine outbreaks of anthrax, showed maximum outbreaks of disease in West Bengal, in which 631 animals were affected with disease and 564 animals were died. In small ruminants especially sheep, the highest outbreaks of disease were encountered from Andra Pradesh and Karnataka which might be due to more sheep population in these states and also unresetricted migratory patterns. The important tools for the prevention of anthrax are vaccination, avoiding opening of the carcass, proper carcass disposal, burning of the bush, appropriate treatment, and in order to avert a future outbreak, annual revaccination is necessary for the outbreak area for at least three years (Chelsea et al., 2008). 345 Saminathan et al 3.4 Enterotoxemia (ET) 3.5 Brucellosis Enterotoxemia (ET) is a severe disease of ruminants caused by C. perfringens types B, C and D with more case fatality rates results in considerable economic losses to the farmers (Rood & Cole, 1991). ET is otherwise called as pulpy kidney or overeating disease. The disease affects cattle, sheep, and goats. Sheep and goats of all the age group are affected however younger animals are more prone to infection. The bacteria are usually present in fewer numbers in the intestine of sheep and goats. Where there is sudden change either in food or in the environment, the disease precipitates. Overgrowth of the C. perfringens occurs due to excessive consumption of milk or large amounts of grain, immunosuppression, heavy gastrointestinal parasitism, ration rich in carbohydrates and low in roughage, and reduced gastrointestinal motility. In India, during the commencement of monsoon season, frequent disease outbreaks of enterotoxaemia in sheep were encountered every year, in spite of frequent vaccinations against C. perfringens Type D (Kumar et al., 2014). Severe enteritis and sudden death in lambs are caused by type B and C infections. Kumar et al. (2014) reported the prevalence of C. perfringens type C for the first time in India. Major causative agent for enterotoxemia is C. perfringens type D and C. C. perfringens type B and D produces epsilon toxin, which is responsible for lethal ET (Arya & Bhatia, 1992). Epsilon toxin is initially secreted as inactive prototoxin, which undergoes trypsin digestion leads to conversion of active toxin by losing N-terminal peptide (Bhown & Habeeb, 1977). Brucellosis is one of the five main notifiable bacterial diseases of zoonotic importance in the world. Brucellosis is a disease of animals with humans as an accidental host (Joshi & Parkash, 1971). Brucella is a Gram-negative facultative intracellular bacteria and bovine brucellosis is caused by B. abortus, less frequently by B. melitensis and rarely by B. suis. Cattle and buffaloes harbor predominantly B. abortus biotype-1; followed by biotype-3; rarely biotypes-2, 4, 5, 6 and 9 (Renukardhya et al., 2002). Out of 6 Brucella spp., B. melitensis, B. abortus, B. suis and B. canis can cause infection and clinical symptoms in man in the descending order of pathogenicity (Smits & Kadri, 2005). The disease has been eradicated from the livestock populations of most European countries, Japan, Canada and the USA. The disease is highly endemic in different states of the country and reported in different animal species like cattle, buffalo, sheep, goats, camel, yak and pig (Smits & Kadri, 2005). But, the highest prevalence is seen in dairy cattle. There are various reasons of its endemicity viz. ignorance of carrier animals, ineffective test and slaughter policy in most of the Indian states, improper and unplanned vaccination, no effective quarantine and uncontrolled trans-state migration of animals (Renukaradhya et al., 2002). By seeing the intensification of disease in recent past, it seeks to review the ongoing policies implemented for its eradication and/ or control. Bovine brucellosis is now have been eradicated from many countries but as it is still prevailing in many states of our country; we are lagging behind in various ruminants‟ production like milk and meat etc. (Singh et al., 2015a). The mature epsilon toxin is responsible for highly lethal, dermo necrotic and edematous activities. The clinical signs in sheep are colic, diarrhoea and neurological symptoms. Postmortem lesions are widespread vascular congestion, with cerebral, cardiac, pulmonary, and renal oedema (Uzal et al., 2004). A sporadic case of the disease outbreak has also been reported in camel calf with the association of C. perfringens type A. During 2006, highest number of outbreak was encountered in India. Majority of the disease outbreaks were recorded from AP, Tamil Nadu, Karnataka, Maharashtra and Jammu and Kashmir. An estimated 12,929 sheep and 619 goats have died due to ET since 2002 (http://www.icar.org.in/files/Vision%202030_PDADMAS-1101-2012.pdf). In the recent past, the number of disease outbtreaks became reduced significantly due to the efficient vaccination strategies. The animals affected with disease should not be vaccinated, because it will flare up the disease outbreak. The vaccination strategy for young animals includes primary dose at 4 weeks of age and booster revaccination at 1 month later of primary dose and all the adult animals should be vaccinated yearly once. Recently, an inactivated whole-cell vaccine was developed and commercialised for the control and eradication of ET, however major disadvantage of this vaccine is local reactions at the site of inoculation. Recently, for sheep combined aluminum hydroxide adjuvanted epsilon toxoid (recombinant) and live attenuated freeze-dried sheep pox vaccine is developed (Chandran et al., 2010). In India, brucellosis was first recognized in 1887 and since then the cases are being observed in almost all the states of the country. In India, on an average, the disease causes revenue losses of INR 420 per cattle, INR 1100 per buffalo, INR 42 per sheep, INR 30 per goat and INR 36 per pig with the total economic loss of Rs. 350 million. It is also reported from the Yak (Renukaradhya et al., 2002; Bandyopadhyay et al., 2009b; Singh et al., 2015a). Higher prevalence of disease was reported from goats in Bihar and Madhya Pradesh states and in sheep Rajasthan and Karnataka states of India. The overall prevalence of disease was 8.85% in goats and 6.23% in sheep in India (Joshi et al., 1975; Chatterjee et al., 1986; Suresh et al., 1993; Gill et al., 2000; Renukardhya et al., 2002; Hemadri & Hiremath, 2011). In spite of significant improvements made in diagnosis and therapeutic advances, brucellosis emerged as a widespread and highly prevalent disease in many developing countries. The sero-epidemiologial data collected during 1994 to 2001 from various states of India revealed that the disease in cattle and buffalo was more prevalent in Union Territory of Delhi followed by Andaman and Nicobar islands, West Bengal, Tamilnadu, Kerala, Gujarat, Maharashtra and Punjab. The annual sero-prevalence data reveals increasing trend of the disease in India. The representative samples collected from different states of the country showed that the prevalence rate become increased from 34.15% during 2006-07 to 67.28% during 2010-11 (Gill et al., 2000; Renukaradhya et al., 2002; Hemadri & Hiremath, 2011). Control and management of _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario animal brucellosis include careful herd management, hygiene, and vaccination. Many countries attained brucella free status by employing test and slaughter policy. However, in India, only “test and segregation” policy is practically adaptable to control the disease in conjunction with efficient preventive measures and control of animal movements. Vaccination is the practical, feasible and effective approach for the control of brucellosis in our country. Hygienic disposal of uterine discharges, foetus, foetal membranes is required. Increased public awareness through health education programmes is necessary. Vaccination with B. abortus strain 19 vaccine (Bruvax; Indian Immunologicals Limited, Hyderabad) is in use for female cattle and buffalo calves between 4 to 6 months of age followed by annual revaccination and all adult females just after parturition. The advantages of calf hood vaccination are the prevention of abortions in the herd produces short-lived antibody response up to 6 to 8 months after vaccination, no booster vaccination required and builds herd immunity in 3-5 years period. The vaccine should not be administered to pregnant animals, bulls and male calves (Kulshreshtha et al., 1978; Renukardhya et al., 2002; Hemadri & Hiremath, 2011; Shome et al., 2012). 3.6 Leptospirosis Leptospirosis is a re-emerging zoonotic disease in Asia especially in India, Africa and Latin America and widespread in those countries having a hot and humid temperature. Till date, 210 pathogenic Leptospira serovars have been identified which have been placed into 24 serogroups based on antigenic similarity. The genus Leptospira consists of 2 species known as L. interrogans and L. biflexa. The pathogenic species are L. interrogans, L. borgpetersenii, L. inadai, L. meyeri, L. noguchii, L. santlilrosai, L. weilli, L. wolbachii, L. fainei, L. alexanderi, L. parva and L. kirschneri. L. biflexa is the saprophytic species. Most commonly reported serovars responsible for the infection in man and animals are Autumnalis, Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Hebdomadis, Australis, and Hardjo. These serovars have been also commonly reported from wild animals and natural carriers such as rodents (Srivastava, 2008; Himani et al., 2013). The disease leptospirosis was first reported in 1988 as Andaman haemorrhagic fever. It has attained significant consideration in recent past as the incidences are being increased among various livestock species in India. Leptospirosis is endemic in India, from 20th century onwards and many outbreaks of disease have been encountered in coastal regions of West Bengal, Maharashtra, Gujarat, Tamil Nadu, Kerala, Orissa, Karnataka and Andaman and Nicobar Islands (Varma et al., 2001). Majority of the disease incidences are happening between October to November which correlates with the monsoon season. The disease was initially reported from Indian cattle by Adinarayanan et al. (1960). Subsequently, several other workers also reported its incidence and prevalence (Srivastava et al., 1991; Varma et al., 2001; Sivaseelan et al., 2003). _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 346 The disease is of significant economic importance in terms of losing livestock through abortions and increasing treatment cost for repeat breeding cases. The disease mostly precipitates with the monsoon season. The reservoir for the etiological agent is rodents in principal along with cattle, pig, and dog. Sero-epidemiological data of leptospirosis conducted during 1995 from different states of India revealed that highest prevalence in dogs (15.9%) followed by horses (14.6%), sheep (12.5%), cattle (7.5%) and buffaloes (5.4%) (Srivastava et al., 1991; Sivaseelan et al., 2003; Srivastava, 2008). During 2007 outbreak in Karnataka, a total of 1,516 cases and southern parts of Gujarat in 2011, 130 deaths within a span of two months were reported (http://www.gideononline.com; Promed, 2011). Recently, 209 cases of disease with 12 cases of deaths in Kochi and Kerala, and 16 deaths from districts of Surat and Valsad of Gujarat were recorded in October 2012 (http://www.healthmap.org). A seroprevalence rate of 71.12% was reported from Kerala where highly prevalent serovar is Leptospira interrogans serovar Autumnalis followed by Australis, Pomona, Canicola, Pyrogenes, Icterohaemorrhagiae, Javanica and Patoc (Ambily et al., 2013). These alarming reports highlight the constant risk of leptospirosis and emergence of new serovars other than the vaccine serovars demands the inclusion of these serovars in the vaccines due to serovar-specific immunity in leptospirosis (Sambasiva et al., 2003). Since leptospires are fastidious and slow growing organisms, isolation of organism is difficult due to 6 weeks required for organisms to grow. Most widely used test for diagnosis of leptospirosis is microscopic agglutination test (MAT) and a titre of 1:100 or more indicates infection in seroprevalence studies (Srivastava, 2008; Himani et al., 2013). The prevention and control of leptospirosis in domestic animals and man are difficult to achieve due to the widespread distribution of leptospires in wildlife and long-term carriers. The diseased animals should be immediately isolated for at least 2 weeks and the premises should be thoroughly disinfected. The carrier animals shedding the organisms in the urine should preferably be slaughtered and buried or burnt. Since leptospirosis is an occupational hazard all the persons directly involved with animals or its environment should use gumboots, gloves, aprons etc. Rodent control using rodenticides, better hygienic practices and environmental hygienic measures to avoid the risk of water, soil and food contamination are necessary to check the transmission of leptospirosis (Sambasiva et al., 2003; Srivastava, 2008; Himani et al., 2013; Patil et al., 2014). Although in wild animals vaccination is not possible, and in domestic animals vaccination strategy can be applied for control and prevention of leptospirosis using outer membranes of bacteria and/or whole cell inactivated with chemical agents (Palaniappan et al., 2002). 347 3.7 Listeriosis Listeriosis also known as circling disease, silage disease and meningoencephalitis, and a fatal disease of ruminants like sheep, goat, cattle, buffalo, camel, and non-ruminants like horse, pig, canine, rodent, wild animals, birds and also humans (Malik et al., 2002; Barbuddhe et al., 2012; Dhama et al., 2013a, 2015a). Among small ruminants sheep are mostly susceptible. Recently, listeriosis caused 23,150 illnesses, 5463 deaths and 172,823 disability-adjusted-life-years worldwide (De Noordhout et al., 2014). It is an important foodborne zoonotic disease caused by intracellular pathogen Listeria monocytogenes, which does cell to cell entry results in the crossing of blood-brain, intestinal and placental barrier (Hernandez-Milian & Payeras-Cifre, 2014; Dhama et al., 2015a). The organism can survive at various temperatures ranges from 4 to 37 °C (Janakiraman, 2008). The organism has several virulence factors like internalins, hemolysin, metalloprotease, listeriolysin-O (LLO), fibronectin-binding protein-A (FbpA), phospholipases and bile exclusion system which are necessary for intracellular multiplication, adhesion and pathogenisis (Vera et al., 2013). The disease occurs as sporadic or epidemic form throughout the world; however during an outbreak, it causes severe damage (Dhama et al., 2015a). Mostly the disease in animals occurs as subclinical but severe forms can also occur. The clinical manifestations of listeriosis include septicaemia, meningoencephalitis, abortion with placentitis in last trimester, stillbirth, perinatal infections and gastroenteritis (Janakiraman, 2008; Barbuddhe et al., 2012; Limmahakhun & Chayakulkeeree, 2013; Dhama et al., 2015a). Listeriosis has unique seasonal occurrence during December to May in northern hemispheres due to the seasonal feeding of silage. More cases of abortions in sheep occurs during February and March due to late pregnancies. In humans, mortality due to listeriosis varies from 20- 30% (Barbuddhe et al., 2012; Dhama et al., 2013a, 2015a). Listeriosis can be transmitted by ingestion of feed and water contaminated with saliva, nasal secretions, faeces and aborted materials from infected animals and also inhalation of dust and soil contaminated with bacteria (Brugere-Picoux, 2008). Ready-toeat foods and animal origin foods like milk, meat, and their products play a crucial role in the transmission of listeriosis to humans (Rebagliati et al., 2009). Young lambs (under 5 weeks of age) will suffer from septicemic form while the encephalitic form is noticed in older lambs (4-8 months). It causes abortion and stillbirth in pregnant women and in foetuses it causes abscesses and granulomas in various organs like lungs, liver, and spleen (Drevets & Bronze, 2008). In India, genital listeriosis is very common and correct epidemiological data are not available due to under-reporting and poor diagnostic facilities (Malik et al., 2002). Studies regarding the prevalence of Listeriosis in various developing countries are necessary to identify the accurate status of disease throughout the world (De Noordhout et al., 2014). Treatment of listeriosis is a difficult task due to the invasion of all cell types. Drugs of choice for listeriosis are erythromycin and ampicillin. Control of listeriosis is difficult due to ubiquitous nature of bacteria in the _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Saminathan et al environment, lack of a simple method for identification of Listeria and poor understanding of risk factors except silage (Malik et al., 2002). Good management practices like proper disposal of contaminated materials, beddings and litters, and infected carcasses should be done carefully by incineration or burning methods. Consumption of unpasteurized milk or uncooked meat should be avoided (Rebagliati et al., 2009). 3.8 Tuberculosis Bovine tuberculosis (bTB) is a chronic bacterial zoonotic disease and easily spreads to humans through inhalation of aerosols or ingestion of unpasteurized infected milk (Prasad et al., 2005). Tuberculosis is caused Mycobacterium tuberculosis complex (MTC) belongs to the genus Mycobacterium. MTC has four species namely, M. tuberculosis, M. bovis, M. africanum and M. microti. M. tuberculosis mainly affects humans, whereas M. bovis causes bovine tuberculosis and affects wide host range including domestic as well as wild animals (Grange, 2001; Verma et al., 2014a). The bTB is widely prevalent and causes 10-25% loss in productivity (Verma et al., 2014b). In developing countries, there is increased the incidence of M. bovis infection in humans causing serious public health problem due to sharing of the same habitat in domesticated animals and humans (Grange, 2001). In Africa, bTB endemic zones nearly 85% of cattle and 82% of the human populations were living together (Michel, 2002). Organisms are excreted in exhaled air, sputum, faeces, urine, milk, vaginal and uterine discharges, and discharges from open peripheral lymph nodes (Verma et al., 2014a). There are limited reports available from India and many epidemiological and public health aspects of the infection remain largely unknown (Neeraja et al., 2014a; Neeraja et al., 2014b; Baqir et al., 2014; Verma et al., 2014b). An overall prevalence of bTB in India is 14.31 to 34.42% (Thakur et al., 2010). M. bovis mainly causes extra-pulmonary forms of tuberculosis and major route of transmission is the oral route. Bovine tuberculosis has been classified as list B disease by OIE due to various socio-economic and public health concerns at the national level as well as the international trade of livestock and their products. Various wild animals like badgers, brushtail possums, deer, bison and African buffalo play an important role in the maintenance of M. bovis infection in wildlife communities and the spread to domestic animals. The test and slaughter policy is effective for tuberculosis control, however in India difficult to follow due to various social and economic constraints and the existence of more wildlife reservoir. In the early stage of infection, test and segregation are recommended while in the terminal stage of infection, test and slaughter is followed. Hence, it is difficult to eradicate bTB infection from livestock until transmission between wildlife and domestic animals has prevented. Better diagnostic tests for rapid screening of disease at the field level should be developed (Sandhu, 2011). The main diagnostic test used for screening of bovine tuberculosis is the tuberculin test (Baqir et al., 2014; Neeraja et al., 2014a; Neeraja et al., 2014b). For control of bTB in India requires Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario 348 vaccination in livestock and captive wildlife species with collaborative efforts between agriculture, wildlife, environmental and political authorities. Pasteurization of milk before marketing and organized abattoirs with carcasses can be routinely tested for TB (Sandhu, 2011). Bacillus of Calmette and Guerin (BCG) vaccine is used for TB, which is a live, laboratory-attenuated strain of M. bovis (Waters et al., 2014). sensitive diagnostic test for diagnosis of infected animals before clinical signs develop. Due to chronic nature of the disease, it is necessary to diagnose the infection early to decrease the production losses and to avoid the infection spread to susceptible animals and humans (Tripathi, 2008). 3.9 Paratuberculosis (or) Johne's disease 4.1 Fascioliasis Paratuberculosis also known as Johne's disease (JD) is infectious, chronic granulomatous enteritis of cattle and buffaloes caused by Mycobacterium avium subspecies paratuberculosis (MAP) (Singh et al., 2014b). JD is a deadly, emaciating and chronic wasting disease characterized by weight loss and profuse diarrhoea. JD mainly affects domestic and wild ruminants and poses serious economic loss to the dairy industry. JD is endemic in farms throughout the country. Most of the time, the disease unnoticed due to chronic nature of the disease and unfamiliar symptoms to the clinicians and farmers (Tripathi, 2008). It is a zoonotic disease and causes Crohn's disease (CD) in humans (Greenstein, 2003). High prevalence of MAP antibodies (Indian bison type) was recorded from animal attendants having gastrointestinal problems, who were worked in endemic JD infected goat flocks in India (Singh et al., 2008b). CD is a non-specific chronic inflammatory condition of the gastrointestinal tract and clinical signs are reduced appetite, bloody diarrhoea, abdominal pain, vomiting, tiredness and weight loss. JD is a list B of OIE listed disease and animals require certification due to trade restrictions. It is calculated that almost 40% of US dairy herds are infected with JD and economic losses exceed 1.5 billion/year in dairy industry (Wells & Wagner, 2000). Animals are often infected during early life by faecal oral route. The Mycobacterium infect the M cells of the follicle in intestinal epithelium and then engulfed by intestinal macrophages leads to replication and viable for several months to years and development of disease (Momotani et al., 1988; Tripathi, 2008). Fascioliasis is caused by Fasciola gigantica and F. hepatica. In India, fascioliasis is more common caused by F. gigantica. Fascioliasis is more common in sheep and causes high economic loses to sheep rearing farmers. The incidence rate of fascioliasis in various climatic regions like tarai, hills and plains in northern region of India was recorded as 10.79% in cattle, 13.90% in buffaloes, 2.78% in sheep and 2.35% in goats (Sharma et al., 1989; Garg et al., 2009). One of the studies conducted in Gorakhpur district showed that about 94% of the buffaloes are infected with F. gigantica (Singh & Agarwal, 1981). Lymnaea acuminate has been identified to be the intermediate host (Agarwal & Singh, 1988). Livestock of Tarai region is having the maximum incidence of fasciolosis when compared to hills and plains (Malone et al., 1998). In cattle and buffaloes, high prevalence of disease was noticed during winter months (15.57% buffaloes, 11.84% cattle) followed by summer and rainy season. The disease was first reported in cattle in Lahore of undivided India, followed by another case in 1917 from a Military Dairy Farm, Hisar. Since then, many cases have been reported throughout the country with an incidence rate of 1.78 to 1.9%. MAP infecting the animals in North India has been genotyped as Bison type (Sevilla et al., 2004; Sevilla et al., 2005). High seroprevalence of JD average 29% (29.8% in cattle and 28.6% in buffalo) has been found in domestic animals by using indigenous, sensitive and MAP-specific ELISA kits in North India. The seroprevalence of JD in Uttar Pradesh (31.9%), Punjab (23.3%), Gujarat (13.39%) and Andhra Pradesh (16.26%) has been reported (Sivakumar et al., 2005; Tripathi, 2008; Singh et al., 2011; Mohan et al., 2013). In spite of very high morbidity rates and lower productivity, economic losses in production go unnoticed in India due to chronicity. The insertion element IS900 has been regularly used to identify the MAP in clinical samples (Garg et al., 2015). Management and control of JD are difficult due to the absence of rapid and _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 4 Parasitic diseases of ruminants However, in sheep and goats, high prevalence of disease (2.71% goats and 4.60% sheep) was noticed during rainy season. An Abattoir studies exhibits its more prevalence in buffaloes (31.14%) when compared to sheep and goats (Garg et al., 2009; Mir et al., 2013; Swarnakar & Sanger, 2014). Further, 5.48% of Lymnaea auricularia snails were carrying the immature stages of F. gigantica. The snails present in the Tarai region causes higher incidence (7.28%) of disease when compared to snails present in plains (1.57%). During 2002, higher incidence of Fasciola was recorded from Orissa with a total of 545 outbreaks, in which 4993 animals were affected and 34 animals were died, followed by Haryana, Manipur, Mizoram, and Bihar. Nationwide fascioliasis collective data shows outbreaks were maximum in Orissa followed by Bihar, Mizoram, Rajasthan, Haryana, Kerala, Karnataka, Maharashtra, Tamil Nadu and Gujarat (Gupta et al., 1986; Sheikh et al., 2007; Hemadri & Hiremath, 2011). One of the methods to control the disease is to effectively control the snail population (Kumar et al., 2009). In a present era which is more concern with the environment pollution, the researcher should exercise in the direction to identify more such products which are of plant origin as well as to ensure about its biodegradability. The experiments conducted by some workers under this direction had shown proven activity of some plants like Alstonia scholaris, Thevetia peruviana, Euphorbia pulcherima and Euphorbia hirta act as snailicidle (Singh et al., 2010). 349 4.2 Trypanosomiasis Trypanosoma evansi causes Trypanosomiasis, which is a significant haemoprotozoan infection of wild animals, dogs, horses, camels, donkeys, cattle and buffaloes. The outbreaks are reported in water buffaloes in India (Tewari et al., 2013; Pandey et al., 2015). The disease was originated from North Africa and through the Middle East it reaches into India (Desquesnes et al., 2013). All domestic animals except camels, the disease is commonly known as Surra and in camels the disease is known as Tibersa. T. evansi is the most commonly occurring trypanosome species in India and causes major economic losses in horses and camels (Tewari et al., 2013). Tabanidae flies (Horse flies and Deer flies) are the vectors responsible for the spreading of the disease. There are 244 species of this vector reported in India (Banerjee et al., 2015). In dogs, the prevalence of T. evansi was observed higher in Mongrel than that of other recognized breeds. The young ones below 2 years of age are more commonly affected (Prasad et al., 2015). Absence of the disease transmitting vector is the major reason for non-prevalence of other trypanosome species. Due to subclinical infection, the incidence of trypanosomiasis in cattle and buffaloes has been unnoticed in India and buffaloes may act as reservoirs (Jaiswal et al., 2015). When the animals become stress due to long transportation, hard work, overcrowding, malnutrition, inclement weather and other concurrent infections, the infection flare up and become prominent and visible infection (Rani et al., 2015). The acute form of the disease in bovine is manifested as emaciation, high fever, lachrymation, corneal opacity, reduced milk yield, nervous signs and mortality often happens within 24 hours of onset of clinical signs. Chronic Surra is characterized by weight loss with loss of reproductive performance (Radostits et al., 2007). In addition to economic losses, the disease also causes immune suppression along with huge mortality in precious animals. The disease occurs in all age groups of animals. The incidence of infection is more common during 1.5 to 2 months after rain, because more availability of rain water lodged breeding areas for disease spreading vectors (Rani et al., 2015). Highest incidence of disease was reported during 2001-02 whereas least incidence of disease was reported during 2009-10. The researcher should more focus on the development of novel chemotherapeutic agents having selective and promising therapeutic potential (Sivajothi et al., 2013). T. evansi can be controlled by using trypanocidal drugs, control of vectors and trypanotolarent cattle breed development (Tewari et al., 2013). Diminazene aceturate (7 mg/kg b. wt) is the prescribed drug for the treatment of trypanosomiasis in ruminants and should be administered deep intramuscularly. 4.3 Theileriosis Bovine tropical theileriosis is an inapparent disease in large ruminants, caused by Theileria annulata and highly fatal diseases of indigenous cattle breeds and cross breeds due to extensive cross-breeding programmes. T. annulata and T. parva are considered to be the most pathogenic species of Theileria. The incidence rate of T. annulata is 14.94% by _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Saminathan et al blood smear examination whereas, 30 to 60% of seropositivity was reported in cross-bred cattle in India, except Himalayan region due to unfavourable climate for tick survival (Singh et al., 1993; Naik et al., 2010; Kohli et al., 2014; Kumar et al., 2015b). Theileriosis causes a serious economic loss due to mortality and reduced milk yield. In India, the tropical theileriosis is one of the major hurdles in genetic improvement programme of Indian taurus cattle breed. The estimated economic loss due to theileriosis is approximately 800 million US dollars (Devendra, 1995). The incidence of disease is seasonal due to availability of ticks on the host, which carries the organism, was higher during summer months like May to October. Other predisposing factors favouring the occurrence of disease are stress, harse climate, concurrent disease, transportation and vaccination. Hyalomma anatolicum anatolicum trnamits T. annulata, which is normally present in the semi desert desert and steppe. Adult and its immature stages of three host tick were present in cattle. The adult stages of tick were more active during late spring season. The immature phase of tick usually feed on on the animals, and nymphs and larvae can obtain the disease by feeding on the infected animal and transmit the disease to other animals following moulting, adults or nymphs feed on other animals. The incidence of the disease is higher during March to November because this is adult feeding season; however, the nymphs have the feeding season of July to September. The occurrene of T. annulata is higher during late spring and early summer season (Roy et al., 2004; Magona et al., 2011; Vahora et al., 2012; Kohli et al., 2014). The epidemiological data reveals that the highest number of outbreaks was occurred from Orissa followed by Bihar, West Bengal, Jharkhand and Haryana. In India, on nucleotide heterogeneity study, it was observed that in T. annulata there is a strain variability as hetrogenicity varies between 0.1 to 8.6. Therefore, it is also required to go for an elaborative study on the genetic diversity of the parasite which is an overlooked area till now (George et al., 2015). The disease epidemiological data showed that the incidence of disease was low during 2008 whereas the incidence was more during 2006 (Hemadri & Hiremath, 2011). 4.4 Babesiosis In India, babesiosis in bovines is mainly caused by Babesia bigemina and Babesia bovis. The disease is prevalent in many states of the country like U.P., Orissa, Kerala, Punjab, Arunachal Pradesh, Mizoram, Meghalaya and Assam (Ravindran et al., 2002; Wadhwa et al., 2008; Singh et al., 2009a; Sharma et al., 2013; Saravanan et al., 2013). The Babesia is transmitted to the susceptible animal by Boophilus microplus, which is the one host tick. Transovarial route is the major mode of spread within the tick. The infected female adult tick can spread the disease up to 32 generations. There are some novel serological tests like slide enzyme-linked immunosorbent assay (S-ELISA), indirect fluorescent antibody test (IFAT) and molecular test like PCR available for the detection of the disease in bovines (Singh et al., 2009a; Harkirat et al., 2013). Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario The disease epidemiological data showed that incidence of disease was more during 2001, whereas incidence of disease was less during 2008-09. The analysis of the epidemiological data reveals that the incidence of the disease were maximum in Orissa followed by Assam, Haryana, Jharkhand, Bihar, Madhya Pradesh, Rajasthan and Himachal Pradesh (Ananda et al., 2009; Singh et al., 2012). Different diseases of ruminants and the species affected are presented in Fig. 1. 5 Emerging Diseases Recently, many outbreaks of emerging diseases like bovine spongiform encephalopathy (BSE), paramyxovirus infection in pigs (Nipah) and horses (Hendra), severe acute respiratory syndrome (SARS), rabies, tuberculosis, bovine corona virus, Lyme disease, Crimean-Congo hemorrhagic fever, West Nile virus and zoonotic H5Nl avian influenza were detected, which causes significant morbidity and mortality in the developing world (Hansa et al., 2013; Verma et al., 2014a; Goswami et al., 2014; Madhu et al., 2016). Many diseases re-emerge with new strains that facilitate them to escape from present control measures. Emerging and re-emerging diseases arise due to genetic changes because of immunological pressure and recombinantions with other viral or cellular genes. Currently, microbes and concerned vectors are changing themselves because of continuous change in agriculture versus animal husbandry practices. A faster mode of trades of animals and their products have facilitated the spread of microbes and the vectors involved; to newer zones of territories. The 350 possibilities of emergence of vector borne diseases can also be co-related with air travelling (Kumar et al., 2015a). Because of high urbanization, forest area is constantly shrinking which may result to lead more interaction between human, captive and wild lives resulting in emerging novel microbes. Sometimes, already existing organisms may transform into an actually novel, and until now unidentified organisms. Emerging diseases are more threatening to ruminants because of less information available about their origin, magnitude of economic losses as well as epidemiology. Recently, numerous resistant bacteria to many drugs are arising with unique pathogenic potential for human through food chain like Campylobacter, Salmonella enterica, Enterococcus spp. etc (Mishra et al., 2011; Kumar et al., 2015a). 6 Exotic Diseases There has been a constant risk of emergence of novel pathogens/ diseases into a disease free country results in a grave effect on animal health due to high morbidity and mortality. Exotic (non-native) diseases/organisms, once entered into a country, can become more intense into an epidemic as being overlooked by the veterinarians due to its non-recognition or being prejudice of its non-occurrence. The disease may also intensify due to non-availability of suitable drugs or vaccine for control, the absence of disease resistance in the host and inadequate facilities in diagnose and limiting the spread of the disease. Figure 1 Disease map and ruminant species affected. Different diseases and the species affected are shown in the figure. Map also represents the place and year of few diseases reported for the first time in India. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 351 Therefore, it is needed to put extra precaution while importing the animals from disease suspected countries, which may harbour the pathogens. The WTO has granted permission to take legal actions in countries to implement their sovereign rights to save their livestock from fatal diseases (Bayry, 2013; Kumar et al., 2015a). Saminathan et al serosurveillance or clinical prevalence, ELISA is a rapid, simple and sensitive assay. MAb-based competitive ELISA (cELISA), immunocapture ELISA, sandwich ELISA (s-ELISA), simple and aqueous phase ELISA (SNAPELISA) and blocking ELISA (B-ELISA) are often used for disease diagnosis. ELISA has a higher diagnostic sensitivity and specificity for detection of antigen/antibody in samples (Afshar et al., 1989; Afshar, 1994; Balamurugan et al., 2014; Chakraborty et al., 2014). 7 Diagnosis of animal diseases 7.2 Molecular diagnostic techniques A tentative diagnosis of the diseases can be done on the basis of clinical signs, species affected, epidemiological pattern, post-mortem lesions and laboratory confirmation by isolation of etiological agent, various serological and molecular methods. Serological tests such as counterimmunoelectrophoresis (CIE), agar gel immunodiffusion test (AGID) or agar gel precipitation test (AGPT), ELISA, complement fixation test (CFT), serum or virus neutralization test (VNT), hemagglutination (HA) and hemagglutination inhibition (HI) test are often used for diagnosis. Virus isolation, immunohistochemical detection, immunoperoxidase test (IPT) and indirect immunofluorescence test (IFAT), RT-PCR, real-time RT-PCR and nucleic acid hybridization are also commonly used for diagnosis of diseases (Rajeev et al., 2011). The novel proteomic techniques interventions like 2-D gel electrophoresis, HPLC and MALDITOF have also given better insight to the disease diagnosis by characterizing the microbes‟ aetiology (Balamurugan et al., 2014; Chakraborty et al., 2014; Dhama et al., 2014a). 7.1 Conventional tests/assays The major disadvantage of the conventional assays is more labour and time consuming, less sensitive hence not suitable for proper diagnosis, however, used for secondary confirmatory diagnosis and retrospective epidemiological studies. To overcome the disadvantages of conventional assays, novel molecular biological techniques like real-time RT-PCR, loop-mediated isothermal amplification (LAMP) assays, and real-time LAMP have been developed and used frequently for the rapid and sensitive diagnosis of infectious RNA and DNA from clinical materials at nano and picogram level (Rajeev et al., 2011; Sharma et al., 2015). Out of these, for the diagnosis of the diseases, isolation of the microbes stands the gold standard. Virus/ bacterial isolation always cannot be performed as routine diagnostic tests since they are cumbersome, time-consuming, and need cell culture or other selective growth media facilities. Beside that the chances of missing the organism are more due to unfair handling while processing the samples. The traditional culture techniques are also not as sensitive as molecular tools like RT-PCR (Balamurugan et al., 2014; Chakraborty et al., 2014; Dhama et al., 2012). Several researchers are undergoing towards the development of molecular diagnostics tools for the early, rapid and specific detection of diseases. For serological diagnosis of diseases and mass screening of samples for seromonitoring or _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Due to the advent of improved biotechnological tools results in better understanding of the genome of pathogens and nucleic acid-based specific and sensitive assays were developed. 7.2.1 Nucleic acid hybridization Nucleic acid hybridization has been widely used for the identification and differential diagnosis of diseases with the limitations of cumbersome and time-consuming, and feasible for regular diagnosis with number of samples. Despite high sensitivity, radio-labelled probes were not broadly used due to a short half life of 32P, the risk of biohazard needs isotopes handling facility and fresh samples. To overcome disadvantages of radio-labelled probes, non-radioactive probes were developed using digoxigenin (DIG) labelled oligonucleotides or biotinylated DNA. This test provides very specific and rapid results moreover its sensitivity is also not less than that of radioactive labelled probes (Rajeev et al., 2011; Sharma et al., 2015). Some more advance assays like Fluorescent in situ hybridisation (FISH) can also be used to localise the targeted nucleic acid sequences within the cellular material (Balamurugan et al., 2014; Chakraborty et al., 2014). 7.2.2 Polymerase chain reaction (Nucleic acid amplification) RT-PCR has been commonly used for detection and differential diagnosis of diseases. The two-step and one-step RT-PCR has been commonly used for the rapid diagnosis of RNA and DNA in the clinical samples in a single step, which has the capability to replace the existing PCR. A highly sensitive RT-PCR-ELISA for the diagnosis and differentiation of diseases has been developed using RT-PCR product labelled with DIG. The test can identify viral RNA with a titre minimum of 0.01 TCID50/100 µl in the infected tissue culture fluid. PCR-ELISA is 10,000 times more sensitive than RTPCR for identification of early and late stages of infection (Rajeev et al., 2011; Sharma et al., 2015). For rapid specific detection and quantification of antigen, real-time PCR techniques targeting genes of pathogens was developed using SYBR Green or TaqMan hydrolysis probe, which is highly sensitive. Multiplexing of Real Time PCR technique can curtail the time further as able to quantify more than single aetiology in one run (Afshar et al., 1989; Afshar, 1994; Balamurugan et al., 2014; Chakraborty et al., 2014; Dhama et al., 2012). Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario 7.2.3 Pen-side tests In the recent years, rapid, simple, specific and highly sensitive novel field diagnostic device known as latex beads based agglutination tests (Rana et al., 1999) and LAMP (LoopMediated Isothermal Amplification) were developed. This test provides a rapid and sensitive diagnosis in a rural environment, where no laboratory equipment was available. LAMP can be performed where thermocyclers cannot be easily maintained/ procured (Rekha et al., 2014). The advantage of these tests is no need of agarose gel electrophoresis. Lateral flow test (Arun et al., 2014), simple dot-ELISA, dipsticks, immunofiltration, and antigen-competition ELISA were developed for the diagnosis of antigen/antibody in samples. These tests can be used for screening of large number of clinical samples and suitable for animal disease diagnosis in the field and can be used as a pen-side test for identification of disease. A suitable LAMP test was developed for the diagnosis of Mycoplasma agalactiae the causative agent of contagious agalactia in goats with the detection level of 20 fg DNA. The test could be performed with 70 min. at 58°C constant temperature (Rekha et al., 2015). These assays had high diagnostic sensitivity and specificity and bear the advantages of rapid, user-friendly, more economic and do not need technical skill or expertise (Balamurugan et al., 2014; Chakraborty et al., 2014; Dhama et al., 2014b; Sharma et al., 2015). 7.2.4 Recombinant antigen-based assays Nowadays, production of recombinant proteins becomes simple and more efficient, due to the progression of recombinant DNA and gene expression technology. Due to the advantage of genetic modification, the quality and quantity of recombinant proteins became improved and the maintenance of post-translational modifications usually determines the choice of the host systems. Various efforts have been made to develop the different expression systems like bacterial, mammalian, yeast and insect cells for expression of different proteins of pathogens and to evaluate the possible use of recombinant proteins in different diagnostic tests. The limitations lay here with that of biosafety issues which need to be taken utmost care (Balamurugan et al., 2014; Chakraborty et al., 2014; Sharma et al., 2015). 7.2.5 Nanotechnologies The tests involved here are able to detect molecular interactions. The benefits of such tests are their smaller dimensions by using nanoarrays and nanochips as platforms. Another superiority of such arrays is their potential to analyse a sample for an array of all the probable infectious agents which are having common overlapping clinical signs in single DNA chip. The technique is of use in detecting Influenza strains and vesicular viral diseases. Beside that the nanoparticles like gold nanoparticles, nanobarcodes, quantum dots (cadmium selenide) labelled antibodies/ antigens are also used for identification of specific pathogens or molecules _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 352 (Rajeev et al., 2011; Balamurugan et al., 2014; Chakraborty et al., 2014; Sharma et al., 2015). 7.2.6 Biosensors Biosensors based assays are the tools used either to detect antigen or an antibody. In such assays, there is a use of receptor (antibody) as well as a transducer which converts a biological interaction reaction into a quantifiable signal. The common transducer technologies are electrochemistry, fluorimetry, reflectometry and/ or resonance. The Biosensors are attached to very high sensitive instruments able to capture readable signals. The limitations of such applications are as it needs high skilled persons for interpretations of the result, the high cost of equipment and high sample processing charges (Balamurugan et al., 2014; Chakraborty et al., 2014; Rajeev et al., 2011; Sharma et al., 2015). Diagnostic techniques used for the diagnosis of different diseases along with their merits and demerits are presented in Fig. 2. 8 Strategies for prevention, control and eradication of diseases Generally, for prevention, control and eradication of infectious diseases, strict biosecurity measures, quarantine, and isolation of infected and disease suspected animals should be appropriately implemented. Added these, effective disease surveillance, monitoring and networking programmes with suitable vaccination and treatment strategies are of utmost importance for the successful control of various diseases (Dhama et al., 2013b; Dhama et al., 2014b; Verma et al., 2014b). Supported with conventional diagnostic tools, recent advancements in diagnostics viz., multiplex PCR, LAMP, biochips, microarrays, nanotechnology, gene sequencing and phylogenetic analysis need to be strengthened for early and confirmatory diagnosis of ruminant diseases (Kawadkar et al., 2011; Dhama et al., 2012; Dhama et al., 2014a; Dhama et al., 2014b; Hurk & Evoy, 2015; Karthik et al., 2016). Together with conventional live and killed vaccines in practice, recent advances in the field of vaccines and vaccinology need to be exploited to their full capacity to counter important diseases of ruminants and help alleviate economic losses of animal producers. These include DNA vaccines, subunit vaccines, recombinant vaccines, plant/edible vaccines, virus‐like particles, nano vaccine etc. (Dhama et al., 2008, Dhama et al., 2013c; Delany et al., 2014; Finco & Rappuoli, 2014; Kim et al., 2014; Singh et al., 2014c; Pany et al., 2015; Singh et al., 2015b). Along with this, for designing, developing and producing effective vaccines the usage of modern adjuvants (TLRs), immunomodulators, and delivery systems is the need of the hour for providing better protection to animals after vaccination (Reed et al., 2013; Dhama et al., 2015b; Singh et al., 2015b). 353 Saminathan et al Flaring up of several emerging and re-emerging diseases due to several predisposing factors including immune pressures, evolution and mutations in microbes, global warming and rising drug resistance in microbes also demands research attention for exploiting alternative and promising therapeutic options of probiotics, cytokines, si-RNA, egg yolk antibodies (IgY), phages, toll-like receptors, nanomedicines, herbs and nutritional immunomodulators (Blecher et al., 2011; Dhama et al., 2013d; Dhama et al., 2013e; Dhama et al., 2014a; Dhama et al., 2015b; Mahima et al., 2012; Malik et al., 2013c; Tiwari et al., 2013; Tiwari et al., 2014). Inspite of huge coordinated efforts for the eradication and control of animal diseases like CBPP, RP, and FMD, until now in India rinderpest is the only disease successfully eradicated (Sekar et al., 2011; Bayry, 2013; Balamurugan et al., 2014). India was declared provisionally free from Contagious Bovine Pleuropneumonia (CBBP) from October 2003, however from May 2007, OIE declared India free from CBPP infection (Singh & Rana, 2014). Similar attempts are required to control and eradicate the enzootic diseases present in India, which causes more economic losses every year. The disease control programmes in India includes National Control Programme on Brucellosis (NCPB), Foot-and-Mouth Disease Control Programme (FMDCP), avian influenza, preparedness, control and containment, and National Control Programme of Peste des Petits Ruminants (NCPPPR) could not be progressed up to satisfactory level further. In the 12th five year plan, the focus is also given for monitoring and control of certain economically important animal diseases (Sekar et al., 2011). There should be strict implementation on controlled movements/ transport of animals from one state to another as well as strict quarantine measures should be adopted. As being a tropical status of the country, more emphasis should be given on the research and development work for Thermo-durable vaccines so that the dependency over cold chain may be avoided and effective vaccination programme can be implemented (Singh et al., 2009b; Verma et al., 2014b; Sharma et al., 2015). Figure 2 Diagnostic techniques used for diagnosis of different diseases, their merits and demerits. _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario 8.1 Mass vaccination is a key for disease control Mass vaccination has been the practical approach for control and eradication of several infectious diseases worldwide. In humans, for the control and eradication of polio, mass vaccination approach was followed by World Health Organization (WHO). Few successful examples are smallpox and polio in humans and rinderpest and CBPP in cattle. Effective control measures along with proper vaccination will get rid of the epidemics of livestock diseases (Sekar et al., 2011). Vaccines will safeguard the livestock by decreasing the spread of infectious agents. Recent molecular advancements like reverse vaccinology resulted in the development of subunit DNA, recombinant and non-pathogenic virus-vectored vaccines leads to production of economically feasible low cost vaccines that are used at field level. In rural areas, basic infrastructure facilities like cold storage needs to be established in veterinary dispensaries to offer improved livestock health services (Bhanuprakash et al., 2010; Bhanuprakash et al., 2011; Dhama et al., 2014a). More work needs to be done in the area of cheaper vaccine development, so as to attract more and more livestock owners to get their animals vaccinated. 354 disease and reporting actions at the district level. The clinicians and diagnostic laboratory personals have to collect the samples from various places frequently like animal fares, veterinary hospitals, slaughterhouses and livestock farmers who are the direct stakeholders (Singh et al., 2009b; Verma et al., 2014b; Sharma et al., 2015). 8.3 Cost analysis of control program India is still developing the country, analysis of cost for control program is essential. For the implementation of control and eradication program for any diseases needs a considerable amount of financial support from the Government of India. Any disease control programme needs fund for vaccines production and/or purchase, manpower (scientific, technical and supporting), diagnostics, equipment, contingent expenses and infrastructure. In some circumstances, manpower can be arranged by state veterinary departments, research institutes, and colleges. Further to reduce the cost infrastructure facilities developed during the execution of the National Project on Rinderpest Eradication (NPRE) could be used for any disease control and eradication programs after required up gradation (Singh et al., 2009b; Verma et al., 2014b; Sharma et al., 2015). 8.2 OIE pathway 8.4 Public-private partnership (PPP) Due to social, ethical and political reasons, restriction of animal movement and test and slaughter policy are difficult to follow in India. Mass vaccination strategy is the feasible, best viable and economical method for control and eradication of diseases (Verma et al., 2014b). India has successfully eradicated the rinderpest by adopting the OIE pathway and in future for successful eradication of any enzootic diseases and to announce the country free from diseases, it is necessary to follow the OIE pathway. OIE pathway includes initial mass immunization, followed by serological surveillance for two years and then no vaccination. These approaches definitely push the country towards free from enzootic diseases results in the declaration of provisional absence of disease. To attain total disease eradication status in the nation a request/report needs to be submitted to the OIE to officially announce as free from disease, after 3 years of the initial declaration. Two consequent yearly serological screening are necessary during this 2 year period. Therefore, a total of 8 to 10 years is needed for officially to declare any disease free from a particular country (Singh et al., 2009b; Verma et al., 2014b; Sharma et al., 2015). It is essential to keep the disease database and disease registry to confirm efficient reporting and monitoring of disease outbreaks. The trained technical, scientific, supporting manpower is required to run a successful disease control program. Surveillance is an effective tool to undertake the disease control program along with mass vaccination. Effective disease forecasting models and programmes need to be developed which have applied applications. These sound models may generate a high confidence level among stakeholders. Further, this may incline them more towards adopting animal husbandry practices to earn their livelihood (Sekar et al., 2011). Veterinarians and para-veterinarians are required to be trained and equipped for rapid diagnosis of _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org A lot of successful stories are available regarding publicprivate partnership in India like ongoing polio control and eradication program. Various non-governmental organizations (NGOs) are significantly contributing to animal husbandry development programmes all over the India. Veterinary vaccines and other biological products are produced by government organizations belonging to both state and central in India. In India, there are 29 biological (vaccines) production centres, among which 22 are under the control and aid of government and 7 are under the control and aid of private sector. Further, many cooperatives are actively contributing for vaccinations, deworming, housing and sheep/goat breeding in many states. The involvement of cooperatives and NGOs are necessary to run the control and eradication programs of diseases. The livestock population is more in India, a large volume of vaccines is needed to implement the programme and hence it is essential to have an association with private manufacturers. Moreover, various NGOs and top private vaccine manufacturers voluntarily undertake the low-cost vaccine production technologies, for launching the control and eradication program (Singh et al., 2009b; Verma et al., 2014b; Sharma et al., 2015). 8.5 Social and Political Concerns Another most important success-determining factor is the political support for disease eradication programmes. To involve the political support, the selected disease for eradication should be need based, economical, international relevance, zoonotic priority and technically feasible. Therefore, clear goal and commitment at various levels are necessary especially in the field staff should be created awareness 355 regarding goals and modalities and should be motivated with incentives. The important social requirement for disease eradication programmes from livestock farmers is perseverance. Presently, both social and political importance of many diseases have been understood and could more be taken advantage to support the disease eradication programme at an international level (Singh et al., 2009b; Sharma et al., 2015; Verma et al., 2014b). 8.6 Constraints in eradication of disease Initially, the incidence of the disease in animals is reported by the village livestock farmers or sarpanch of the village or local village assistant to field veterinarians. At that time, the disease is diagnosed on the basis of clinical signs described by the livestock farmers and majority of infected animal dies by the time veterinarian reaches to the village due to insufficient transport facilities. Due to this limitation, a collection of samples from infected animals and subsequent confirmation of the disease would not be possible. Further, many outbreaks of the disease are not reported frequently, hidden and affected animals were sold at low cost. The areas where a veterinarian can arrive the animal before removal, they are unable to collect the samples because of lack of facilities for collection, preservation and transportation to the adjacent laboratory for diagnosis (Chakraborty et al., 2014; Dhama et al., 2014a). A major problem in the control and eradication of diseases using test and slaughter policy is inadequate compensation to the owner for the culling of infected animals. This encourages the owners to hide the clinical sign of the disease in affected areas results in the existence of the disease and animals will act as carriers (Verma et al., 2014c). 8.7 Disinfection of the infected animal premises and their products Materials and by-products from infected animals like meat and meat products, offal, wool, skin, and hide should be thoroughly disinfected by chemical inactivation, heat treatment, and ionizing irradiation. Milk and dairy products from infected/suspected animals need to be disposed off. Bedding materials from infected animals, feed stuff, excretory and secretary products including dung and urine, and clothing of person working in infected animal houses should be destroyed properly (Chakraborty et al., 2014; Chand et al., 2015). 8.8 India‟s Potential to control and eradicate the diseases India is a developing country, with 29 states and 7 union territories. The veterinary infrastructure facilities for control and eradication of diseases were available at both central and state levels. A total of 8732 veterinary hospitals (polyclinics), 18,830 veterinary dispensaries and 25,195 veterinary aid centers/stockhome centers/mobile dispensaries were available in the country (Bhanuprakash et al., 2011; http://dahd.nic.in/.). In addition, India has 53 veterinary colleges, and 13 veterinary and animal science universities distributed throughout the _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Saminathan et al country. Further, more than 31 non-governmental organizations (NGOs), 642 Krishi Vigyan Kendras (KVKs) and 23 state biological units were available in the country. Veterinary and animal science has the supreme apex body called as Indian Council of Agricultural Research (ICAR) under which 9 national institutes in the country like, Indian Veterinary Research Institute (IVRI) and National Dairy Research Institute (NDRI), etc., 2 Project Directorates, 6 National Research Centres and one National Bureau of Animal Genetic Resources are there. ICAR is the central organization to plan, implement and execute the control programs with the help of department of animal husbandry under the ministry of agriculture and animal husbandry. The main referral laboratory for disease diagnosis is Centre for Animal Disease Research and Diagnostic (CADRAD), which connects the regional disease diagnostic laboratories (RDDLs) located throughout the country at four zones namely south, north, east and west (Bhanuprakash et al., 2011). With the support of these facilities, India has the potential to control and eradication of diseases in future like rinderpest. India has been declared provisionally free from CBBP from October 2003 and OIE have declared freedom status of India from CBPP infection from May 2007 onwards. Likewise, other animal diseases like foot-and-mouth disease, PPR, Bluetongue, HS, brucellosis, etc., are possible to control and eradication in the future (Bhanuprakash et al., 2011; Verma et al., 2014b). Conclusion and Future Perspectives The animal diseases present in India require a serious attention and needs improved research facilities especially in the field of epidemiology and huge funding. Valid, state-wise, comprehensive research data especially in the field of epidemiology are necessary for planning and control of diseases that are endemic in India, otherwise implementation of control measures will be difficult and eradication will be impossible. Animal diseases are not only danger to the Indian economy but also equally important in reaspect to the human health. During the recent years, majority of the infectious emerging diseases affecting the human are originated from animals. Thus, it is logical to say safeguarding the animal health is most important for maintenance of human health. For successful control of diseases, epidemiological forecasting, quick and correct diagnosis, safer and quality vaccines, sanitation measures along with adequate infrastructure facilities for cold storage and transport facilities to reach the vaccines for the remote areas, where end users are living; are necessary. Advanced diagnostic assays have reduced the puzzle in the diagnosis and differentiation of diseases from others. Door delivery of veterinary services and better extension services for greater awareness to farmers will significantly enhance the possibility of eradication of diseases thus helping in control programmes. The major constraints in the control of disease in the developing country like India are poor vaccination coverage, lack of financial support and insufficient infrastructure, which interferes the building of herd immunity. An interdisciplinary approach like veterinarians, scientist (animal health), medicos, para-veterinary officers, and Prevalence, diagnosis, management and control of important diseases of ruminants with special reference to Indian scenario NGOs need to take a leadership role while implementing the control programmes for controlling and eradication of important diseases of livestock. This will increase the livestock production and their sustainability, which ultimately results in alleviation of poverty in the rural areas of the country. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. 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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 EXPLORING ALTERNATIVES TO ANTIBIOTICS AS HEALTH PROMOTING AGENTS IN POULTRY- A REVIEW Ajit Singh Yadav1,*, Gautham Kolluri1, Marappan Gopi1, Kumaragurubaran Karthik2, Yashpal Singh Malik2 and Kuldeep Dhama2 1 ICAR-Central Avian Research Institute, Izatnagar-243122, UP, India ICAR-Indian Veterinary Research Institute, Izatnagar-243122, UP, India 2 Received – May 05, 2016; Revision – May 09, 2016; Accepted – May 21, 2016 Available Online – May 25, 2016 DOI: http://dx.doi.org/10.18006/2016.4(3S).368.383 KEYWORDS Poultry production Antibiotics Probiotics Prebiotics Synbiotics Organic acids Plant extracts Phytobiotics Herbs ABSTRACT Poultry industry has undergone rapid growth during last three decades. For which even higher usage of antibiotics, both as growth promoters as well as therapeutic agents, has been adopted. However, due to the fear of resistance development in bacterial populations to antibiotics, presence of antibiotic residues in poultry products and increasing consumer demand for products free from antibiotic residues, search for alternatives that could replace antibiotics without causing loss to productivity or product quality has accelerated. Such alternatives in poultry include the use of organic acids, probiotic microorganisms, prebiotic substrates that benefit proliferation of beneficial bacterial populations or synbiotic (combinations of prebiotics and probiotics) ensuring better production and maintaining health of the birds. Others include vitamins and minerals, herbal drugs, plant extracts, phytobiotics and antimicrobial peptides. Probiotic organisms provides competition to pathogenic organisms for intestinal colonizing sites, reduce the diversion of nutrients for harmful microbes and the toxins produced by them and stimulates the immune systems. Similarly, prebiotic offers an alternative, as it alters the intestinal microbes and immune system to reduce colonization by pathogens and allows proliferation of beneficial microflora in the gut. Even using synbiotic is a better strategy for enhancing poultry production, however, more research is needed for selection of probiotic, prebiotics or synbiotics either alone or in combination that can result in the selection of strains capable of performing effectively in the gastrointestinal tract. The contents of this review will be useful for researchers to enrich their knowledge on alternatives of antibiotics in poultry birds without compromising performance of birds and bird welfare. * Corresponding author E-mail: [email protected] (Ajit Singh Yadav) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. 369 1 Introduction Poultry industry has seen an unparalleled growth during last three decades and is now recognized as one of the fastest growing component of agriculture sector. This has happened due to increased consumption of eggs and meat with their easy availability, relatively inexpensive cost and rich in all essential nutrients which can meet the deficiencies of critical dietary minerals, vitamins and amino acids (Dhama et al., 2014a). High growth contributing factors have been exploited for optimal genetic potential of birds, availability of quality of the feed, providing optimal environmental condition and preventing disease outbreaks. In recent years, gut health of poultry birds has been the area of intense studies in poultry production for higher production (Rinttila & Apajalahti, 2013), as this is the main site where nutrient uptake takes place. The gastro-intestinal tract is the organ which exposed to majority of environmental pathogens next to skin (Yegani & Korver, 2008). Thus, the main thrust in poultry production is to maintain sound gut health and function ensuring proper health and production. Due to impaired gut function and health, both the digestion and absorption of nutrients are affected and thus the overall health and performance of birds will be compromised which ultimately affect economics of poultry production. Antibiotics have been widely used in poultry production worldwide due to their easy availability and low cost. It has revolutionized the intensive poultry to promote growth, production and feed conversion efficiency by improving gut health and reduction of sub-clinical infections. Antibiotics inclusion at low concentration augment gut health by reducing the pathogen load and helps in preventing sub-clinical infection normally present continuously in the birds even in the well-organized poultry units. The beneficial effects of using antibiotics include the thickening of intestine which leads to more nutrient absorption. Thus, it can spare the critical nutrients for the host by reducing the competition between host and pathogens and by preventing the microbial adherence and invasion to the gut wall lowers the production of toxic amines thus preventing stress to birds. The effect of antibiotics is more pronounced when the birds are kept under unhygienic conditions and are maintained on relatively vitamins and or amino acid imbalance /deficient diet that clearly indicate the nutrient sparing effect of antibiotics. Penicillin G was the first antibiotic introduced in veterinary medicine in 1947 for use as intramammary infusions. Since that time the use of antibiotics has become an integral part of managing animal health in agriculture. Antibiotics are administered to food animals including poultry by several different routes including injections, orally in feed and water. Commonly employed antibiotics for preventative and therapeutic purposes in poultry are chlortetracycline (Athar & Ahmad, 1996; Kodimalar et al., 2014), furazolidones (Oluwasile et al., 2014), fluroroquinolones (Anderson & Macgowan 2003; Luangtongkum et al., 2006; Martinez et al. 2006; Billah et al., 2015), oxytetracycline (Adel Feizi, 2013), sulphonamides _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ajit et al (Persoons et al., 2012), gentamicin (Alm El Dein & Elhearon, 2010), Quinolones (Naeem et al., 2006). Despite tremendous beneficial use, the practice of using antibiotics in poultry is being questioned, owing to increased resistance to antibiotics (Tiwari et al., 2014a). The mechanism of resistance development in antibacterial population happens when an antibiotic is applied in food animal at sub-therapeutic level, which results in eliminating the sensitive population of bacteria leaving the variants having unusual traits and resists the effect. These resistant bacteria then multiply becoming the predominant. The resistant population so produced transmits the resistance which is genetically defined to subsequent progeny and also to other bacterial strains via mutation or plasmid mediated (Catry et al., 2003). Human may get exposure to such resistant bacteria population through consumption and handling of meat contaminated with such pathogens (Van den Bogaard & Stobberingh, 2000). Once these are acquired, such resistant bacteria can colonize the intestinal tract of human and the genes coding resistance to antibiotics in these bacteria can be transferred to other bacteria belonging to the endogenous microflora of humans (Ratcliff, 2000; Stanton, 2013), thus causing impediments in effective treatment of bacterial infections. Because of drug resistance associated with use of antibiotics in poultry production, there has been a big push to find alternative treatment methods for common poultry ailments. The alternatives to antibiotics are needed to maintain the gut health and performance by controlling pathogens and increased nutrient digestion and absorption. Some of the ways to minimize antibiotics in poultry include use of whole grain cereals, live microbial cultures, use of fermentable sugars and processing/ sterilization of feeds. Prominent alternatives in poultry production include organic acids, probiotics, prebiotics, synbiotics, herbal drugs, vitamins, minerals and plant extracts (essential oils) etc. (Dhama et al., 2014a). The attributes of alternatives are as follows: 1. 2. It should improve performance effectively It should have little therapeutic use in human or veterinary medicine 3. It should not cause deleterious disturbances of the normal gut flora 4. It should not be involved with transferable drug resistance 5. It should not be absorbed from the gut into edible tissue 6. It should not cause cross-resistance to other antibiotics at actual use level 7. It should not promote Salmonella shedding 8. It should not be mutagenic or carcinogenic 9. It should not give rise to environmental pollution 10. It should be readily biodegradable 11. It should be non-toxic to the birds and its human handlers. Exploring alternatives to antibiotics as health promoting agents in poultry- a review Even combined supplementation of prebiotics and probiotics which is referred as symbiotic is a better strategy for enhancing production, however, more research is needed for selection of probiotic, prebiotics or synbiotics either alone or in combination that can result in the selection of strain/s capable of performing effectively in the gastrointestinal tract. The present review discusses the valuable alternatives to antibiotics as health promoting agents in poultry production system, including of organic acids, probiotics, prebiotics, synbiotics, vitamins and minerals herbal drugs, plant extracts, phytobiotics and antimicrobial peptides. The contents of the review will be useful for researchers to conduct more research on alternatives of antibiotics in poultry birds without compromising performance of birds and bird welfare. 2 Alternatives to antibiotics as health promoting agents in poultry 2.1 Organic acids Organic acids are being considered as one of the effective alternative of the antibiotics in recent years because of their antimicrobial activity against wide range of pathogenic bacteria because of their ability to induce a pH reduction in the gut and these can improve nutrient utilization in poultry diets (Eidelsburger et al., 1992; Boling et al., 2000; Kil et al., 2011). These have been used either as single acid or combination of several acids (Wang et al., 2009). Use of organic acids and their salts in poultry has been permitted as safe by the European Union (Adil et al., 2010). Basically, organic acid includes carboxylic acids and fatty acids having a chemical formula of R-COOH, where R represents chain length of the acids. In poultry feeding, organic acids of short chain length like formic (C1), acetic (C2), propionic (C3) and butyric acid (C4) had been tried more often. Other carboxylic acids used include citric, lactic, fumaric, malic and tartaric acids (Dibner & Buttin, 2002). Generally, organic acids are weak acids and these are dissociated only partly and most organic acids possessing antimicrobial activity have a pKa value (defined as the pH at which the acid is half dissociated) in the range of 3 to 5. Organic acids are also available as calcium, potassium or sodium salts. The salts are being preferred as these are odorless and easy to handle during feed processing owing to their less volatile property and solid in their state. Further, the organic acids are less corrosive in nature and more soluble in water (Huyghebaert et al., 2011). These can be used both in water and feed. The proposed sequential mechanisms of action as bactericidal exhibited by organic acid are as follows: Initially acid form of organic acids can penetrate across the bacteria cell wall and subsequently penetrated organic acids within bacterial cells dissociate into the conjugated base form (non-protonated form) leading to a reduction in cellular pH and the decreased intracellular pH creates a stressful environment for bacteria leading to cellular dysfunctions thereby preventing bacterial growth (Mani_________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 370 Lopez et al., 2012). On other hand, sorbic acid increases the permeability of the bacterial cell as well as causing interference with membrane proteins (Abdelrahman, 2016). Role of organic acids in poultry production include lowering down the pH of the poultry feed and gastrointestinal tract (GIT), improved nutrient utilization in diets by increasing nutrient retention, preventing the growth of pathogens (Afsharmanesh & Pourreza, 2005; Mroz, 2005). Capacity to decrease pH in the feed and GI tract by the organic acid likely dependent on the pH conditions of the GIT and pKa values of the organic acid used (Kim et al., 2005). The pH reduction in GI tract is more pronounced in the upper part. Application in drinking water ensures pathogen reduction and subsequently crop besides regulating normal gut flora (Açkgöz et al. 2011; Hamed & Hassan 2013). Organic acids are readily absorbed in the proximal part of gastro-intestinal tract. Their ability to acidify the gut environment results in increased intestinal protease enzyme activity which in-turn increases the nutrient digestibility and utilization. This may be due to fact that acidic digesta may be retained for longer time in GI tract, and therefore provide more time for nutrient digestion in the GIT (Kidder & Manners, 1978; Mayer, 1994). Inhibition of undesirable microbes not only prevents the accumulation of toxic metabolites, but also spares more nutrients available for the host, ensuring higher feed utilization efficiency. Moreover, stabilization of intestinal pH also increases the efficacy of all digestive enzymes. Organic acids are used in feed sanitation programme, acting as feed additives and preservative. By preventing the growth of pathogenic bacteria it prevents the feed deterioration and extends the shelf-life of perishable food ingredient. Organic acids commonly used to reduce the pathogenic microbial load (like Salmonella and Escherichia coli) include short chain fatty acids, volatile fatty acids and weak carboxylic acids. Organic acids also reduce the colonization of pathogens on intestinal wall, preventing damage to epithelial cells. Daily application of short chain fatty acids increases epithelial cell proliferation; quick repairing of intestine, increased villous height and in turn increased absorptive capacity. Medium chain fatty acids (MCFA) destroys the bacteria by penetrating its phospholipid layer and alters the cell membrane through the formation of pores resulting in leakage of contents (Hermans & De Laet, 2014). It provides an early pathogen barrier for the inhabiting pathogens. Propionic acid is an effective mold inhibitor (Zha & Cohen, 2014) and can completely inhibit feed mycotoxin. Continuous feeding of propionic acid to chicks reduced Salmonella Gallinarum count of crop and caecal contents. Addition of 0.36% Calcium formate also reduced Salmonella level both in carcass and caecal samples. 371 Akyurek et al. (2011) observed increase in Lactobacilli population and reduction in coliforms and Clostridia in ileum in broilers fed blends of organic acids than the antibiotic groups. Similarly, reduction is Salmonella in caecum through synthesis of antimicrobial peptides in chickens fed with acetate, propionate and butyrate salts (Sunkara et al., 2011; Sunkara et al., 2012). Organic acids cocktail (Hassan et al., 2010; Hamed & Hassan, 2013) is reported to have more synergistic effect with better efficiency compared to antibiotic growth promoters against intestinal colonized pathogens viz. E. coli, Salmonella. N-heterocyclic dicarboxylic acids and pyridyl-mercapto-thiadiazoles are the new generation organic acid types as a future broad-spectrum inhibitors of the metallob-lactamases (MbLs) which can be used in conjunction with beta lactam antibiotics for counteracting drug resistant serotypes (Abdelrahman, 2016). The availability of calcium especially in egg producing chickens is influenced by the presence of oxalic acid which is present in plant sources. This oxalic acid form insoluble calcium oxalate salts (Jadhav et al., 2015). An increase in calcium solubility and availability was observed in the studies of Tang et al. (2007) who fed the birds with Lactobacillus strains which is attributed to its ability to reduce the gut pH due the production of lactic and acetic acids. Organic acids also reduce contamination of litter with pathogens and diminish the risk of re-infection, thus reducing the bacterial challenge to poultry birds. Organic acids possess potent property to reduce pH and have been found to reduce pathogens in GI tract, however, more studies are needed to elucidate the mode of action of dietary organic acids and their effects on growth performance of broiler chickens by various combinations of acids and their concentration in feed or drinking water. 2.2 Probiotics Probiotics are either single and/or mixture of live microbial culture which promote health benefits to the host (Fuller, 1992). Mode of probiotic bacteria involves competition with receptor sites in the intestinal tract, production of specific metabolites (short organic fatty acids, hydrogen peroxide, other metabolites possessing antimicrobial activity) and immune stimulation effect (Madsen et al., 2001; Sherman et al., 2009). Microorganisms used as probiotics include Lactobacillus, Streptococcus, Enterococcus, Bacillus, Clostridium, Bifidobacterium species and E. coli while yeast and fungus used as probiotics include Saccharomyces cerevisiae and Aspergillus oryzae (Fuller, 1999). Bacteria and yeasts have been included as spores or as living microorganisms. Probiotics classified as non-colonizing species such as Saccharomyces cerevisiae and Bacillus spp. (spores) while colonizing species include Lactobacillus and Enterococcus spp. Saccharomyces known to offer a source of good quality protein and B complex vitamins. Due to immunomodulatory properties, yeast extract, the non-antibiotic functional product is suggested to be the potential non-antibiotic alternative for decreasing pathogenic bacteria in turkey production (Huff et _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ajit et al al., 2010). Currently, yeast cell derivatives are gaining importance as zootechnical feed additives (Świątkiewicz et al., 2014). Similarly, feeding of Aspergillus awamori (0.05%) improved growth performance through the release of growth promoters (Yamamoto et al., 2007) and meat quality by increasing unsaturated fatty acid content in breast meat (Saleh et al., 2012) of broiler chickens. The reason behind the use of probiotics has been primarily to establish normal intestinal flora with broad target of prevention or minimizing the disturbances caused by enteric pathogens (Dhama et al., 2008). Probiotics are not the substitute of antibiotics in birds with serious infections but these are useful in restoring the normal bacterial population. The effect of probiotic depend on physiological state of the bird, type and concentration of probiotic strain, persistence in intestine, ability to survive during feed processing and gastrointestinal track and compatibility with natural microbiota of the intestine. The strain of probiotic to be called as ideal should be resistant to acid, bile salts and digestive enzymes. It should also possess property of rapid multiplication so as to produce microbial population required for producing desirable effect. Further, the strain used should not impart antibiotic resistance into the intestinal microflora (Seema & Johri, 1992; Pal & Chander, 1999; Dhama et al., 2011; Mookiah et al., 2014). Benefits of probiotics: 1. Improves the health of gut health by upholding a desired equilibrium in its microbial population and reducing incidences of diarrhea. 2. Inhibits growth of pathogens and reduces the mortality 3. Results in better feed conversation efficiency 4. Improves growth rate and body weight gain 5. Improves the digestive enzymes and in turn nutrient absorption 6. Reduces circulating cholesterol level through regulation of lipid metabolism 7. Enhances efficacy of vaccines 8. Plays important role in fast detoxification of mycotoxins 9. Reduce stress associated with administration of antibiotics, temperature, vaccination, transportation etc. 10. Synthesis Vitamin B complex vitamins 11. Improves litter quality via. enteric and also litter ammonia production 12. Enhances the intestinal short chain fatty acids which could alter the microbial composition in gut 13. Leaves no residues effects in products 14. Decreases environmental pollution One mode of action associated with probiotics is the competitive exclusion as these produce some substances which inhibit growth of pathogens. Moreover, the pathogens also compete with them for a place in the intestinal epithelium. Exploring alternatives to antibiotics as health promoting agents in poultry- a review The substances produced by probiotic bacteria are short-chain organic acids (lactic, acetic, propionic), hydrogen peroxide, bacteriocins which includes nisin, acidolina, lacocydyna, lacatcyna, reutryna, entrocine, laktoline. Bacteriocins produced by probiotics possess a high antibacterial activity against Salmonella, Campylobacter, Escherichia coli and Clostridium perfringens. Probiotic (s) supplementation in feed is considered to be the potential candidate strategy for controlling necrotic enteritis (Mahmood et al., 2014) and Eimeria acervulina and E. tenella with effective reduction of oocysts (Lee et al., 2007). Another mode of action of probiotics is by stimulation of immune system due to their ability of adhesion to the intestinal mucosa which allows creating a natural barrier for entry of pathogens thereby enhancing immunity. Further, probiotic stimulation of the immune system exhibited higher production of immunoglobulins, stimulation macrophages and lymphocytes activity and also by augmentation of the production of γ-interferon (Yang & Choct, 2009). Ensuring antibiotic efficacy without therapeutic involvement, consumer’s demand for antibiotic free products and animal welfare promotion are considered to be the key drivers for increased use of probiotics in poultry production currently (Blanch, 2015). A latest approach in probiotics feeding especially in poultry is the in ovo injection of probiotic culture. As the newly hatched chick will have a sterile gastro-intestinal tract, so it harbors the microflora when they are exposed to various microbes in the environmental on its arrival to its rearing house system. Colonization in chicks takes place after hatching (AmitRomach et al., 2004) but presence of few numbers of microbes in their intestine during pre-natal stage itself was reported by Pedroso (2009) and Bohorquez (2010). Various available scientific reports showed that feeding of probiotics in birds reduced the impact of various stress conditions. Similarly, the newly hatched chicks are being exposed to different types of stresses like hatching, sexing vaccination, dehydration, starvation, transport, etc. Various in ovo injection studies have shown that embryonic administration of essential amino acids, minerals, carbohydrates, fatty acids reduced the impact of these stress and enhanced the growth performance in broilers. Hence, the administration of probiotic culture in in ovo condition could also be help in overcoming various stresses during early life. In an experiment in broilers, in ovo injection with combination of probiotic organisms at 17.5 days of incubation significantly reduced the Salmonella counts in intestine (de Oliveira, 2014). 372 include alteration of GI microflora, immune stimulation, preventing colon cancer and reducing pathogen invasion, reduction of cholesterol and odor compounds (Cummings & Macfarlane, 2002), improve gut health through intestinal microbial balance, promotion of enzyme reaction, reduction in ammonia and phenol products and ultimately reducing production cost (Ghiyasiet al., 2007; Khksar et al., 2008; Peric et al., 2009). The predominant prebiotics tried in chickens are gluco-oligosaccharides (GOS), fructo-oligo-saccharides (FOS), mannan-oligo-saccharides (MOS), stachyose and oligochitosan (Jiang et al., 2006). Some attributes for being a good prebiotic include (i) it should neither hydrolyzed nor absorbed in the upper part of the gastrointestinal tract, (ii) induce systemic effects to enhance health of the host and palatable as feed ingredient and (iii) easy to process in large scale. Addition of prebiotics to poultry diets can minimize the use of antibiotics ultimately reducing bacterial drug resistance (Patterson & Burkholder, 2003). Further, use of prebiotics in poultry diet can reduce colonization of pathogens such as Escherichia coli, Vibrio cholera, S. Typhimurium, S. Enteritidis etc. (Bailey et al., 1991). Supplementation of oligosaccharides reduced total viable counts in meat and caecum. Prebiotics also promotes the growth of Bifidobacteria and Lactobacillus and reduces the harmful intestinal pathogens (Dhama et al., 2007) Thus, prebiotics can be used as one of the alternative of antibiotics with an aim to improve poultry health and performance through alteration of intestinal microbial population and stimulating immune system by pathogen reduction, however, more studies are needed to elucidate exact role and mode of action as single component or in combination. The presence of microfloral population in gastro-intestinal tract influences the growth and immune system in chickens. Prebiotics are well known for its ability to enhance the establishment of good microbes (Gibson, 1999; Van Loo et al., 1999) but they also involved in altering the innate immune response through binding with receptors, promotes endocytosis, cytokines and chemokines (Di Barolomeo et al., 2013). Inulin, a polymer of fructose is widely used as prebiotic in both human as well as in animals. Even though, they are indigestible in the intestinal tract but serves as a substrate for the growth of Bifidobacteria (Niness, 1999; Kelly, 2008). Inulin also promotes the production of secretory immunoglobulin A (SIgA) at ileum (Nakamura et al., 2004) and increases the immunity against invading bacteria in the gut (Buddington et al., 2002). 2.4 Synbiotics 2.3 Prebiotics Prebiotics are certain non-digestive feed components that benefit the host by selectively accelerating growth rate and /or proliferation of one or more of a limited number of bacteria in the colon of host so that the health of the gut can be improved. These provide the substrate to the beneficial intestinal microorganisms. The main function associated with prebiotics _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org The mixture of probiotics and prebiotics (synbiotics) which provides the live culture and feeding them from better survival in the bird’s intestinal tract (Yang et al., 2009; Gaggia et al., 2010). Fructo-oligosaccharides and bifidobacteria, and lactitol and lactobacilli are the commonly known combinations of pro and prebiotics for use as synbiotics (Yang et al., 2009). 373 Ajit et al Microflora of intestine play important role in bird health and if this balance between useful microorganisms gets disturbed, then the health and overall performance of the bird is affected. This invites to explore role of dietary supplementation in the form of prebiotics which can be supplemented to support the growth of beneficial microflora so that production in poultry birds can be enhanced. The supplementation of prebiotics which ensure growth of probiotics is called synbiotics (Huyghebaert et al., 2011). The supplementation of both probiotics and prebiotics could improve the survival and persistence of the useful organism in the gut of birds as specific substrate is available for fermentation (Yang et al., 2009; Adil & Magray, 2012). Synbiotics were effective in improving the growth of broiler in the diet of chickens (AbdelRaheem et al., 2012; Mookiah et al., 2014). Feeding of synbiotics in broiler chicken was found to have beneficial effect on intestinal morphology and nutrient absorption leading to enhanced performance (Awad et al., 2008; Hassanpour et al., 2013). Very few studies have reported the optimal benefits of synbiotics in poultry (Li et al., 2008). Much attention has to be paid to find out the best combination of pro and prebiotic and its subsequent evaluation of their synergistic effects for use as potential synbiotics to ensure maintenance of proper health. An investigation by Madej et al. (2015) in broilers revealed that in ovo administration of inulin (prebiotic) along with Lactobacillus organism altered the development of various immune organs. increased poultry’s performance (NRC, 1994). Minerals like iron has growth promoter and inhibitor role while phosphorus has role in weight gain of broilers (Abudabos, 2012). Vitamin Q is most commonly known as ubiquinone due to its distribution among various systems. This is produced endogenously as lipid soluble compound which plays an important role in the energy transformation process inside the cellular mitochondria (Gopi et al., 2015). However, their synthesis will not be sufficient as the age advances. Similarly, in birds especially of fast growing varieties their endogenous production might not be sufficient along with various stress conditions. Gopi (2013) reported an improvement in feed efficiency in broilers fed high energy diet. Moreover, intake of the compound increases their anti-oxidant defence mechanism especially lipophilic systems. Intake of vitamin Q increases the host defence against various microbes bacteria, virus, protozoa (Bliznakov, 1978), activation of macrophages (Hogenauer, 1981) through increased energy availability. Folkers et al. (1982) and Gopi (2013) observed an increase in immunoglobulin G production and haemeagglutination titer (HI) against Newcastle disease virus in broilers, respectively. 2.5 Vitamins and minerals as growth promoters Phytochemicals (commonly known as phytobiotics) as the plant derived compounds have wide range of activities in plants, animals and humans. These compounds are the secondary metabolites produced by the plant which possesses characteristic flavor and taste, primarily for its self-protection from being grazed/ eaten by animals and from pest attack. Over the years, more than 80, 000 compounds have been identified so far like phenols, flavonoids, tannins, saponins, essential oils, etc. Initially, these compounds were considered as waste, anti-nutritional and health affecting ones. But, now-adays the approach towards them is changing globally as an antioxidants, digestive enhancer nutraceutical and health promoting substances (Narimani-Rad et al., 2011). Since, the identification of its anti-microbial activity across different groups of organisms (Brut, 2004; Murali et al., 2012) (both gram positive and gram negative organisms). In view of animal production especially in monogastrics (pigs and poultry production) they are mainly used as an alternative antibiotic growth promoter (Khaksar et al., 2012; Karangiya et al., 2016). Although, the exact mechanism of action is not yet known they have been found to favourably alters the gut micro-flora by reducing the number of pathogenic organisms (Salim, 2011). The probable mechanism of action is the through the alteration in membrane permeability to hydrogen ions (H+). In addition to its antibacterial activities, it also shows antiviral, antiprotozoan and anti-fungal actions. Their anti-fungal actions are getting more importance as these compounds are now being incorporated in to fungicide preparations which are cost effective as well as environmental friendly (Afzal et al., 2010) and also as fly repellent (Mansour et al., 2011). Use of minerals and different vitamins can improve the health status of the poultry which has been proved in the growth of broilers. Minerals and vitamin supplements has increased the poor health status of birds hence increasing the cost benefit ratio of the farm (Prescott & Baggot, 1993; Peric et al., 2009). Several beneficial effects like improved immune status of the bird increased feed conversion ratio, alteration of beneficial microflora in the gut and intestine. Vitamins like vitamin C has a major role to reduce stress mainly during summer months, increases feed intake thereby improving metabolism of the feed (Sahin et al., 2003). Other health promoting effects of vitamin C include reduction of weight loss in birds mainly due to summer stress. Antioxidant vitamin C is synthesized naturally in birds using an enzyme gulonolactone oxidase that is absent in guinea pigs and human (Lin et al., 2006; Khan, 2011). There is no recommended dose for vitamin C in birds but it may aid in suppressing stress by its antioxidant nature. Study reveals that broilers fed with vitamin C have shown good performance even under different environmental stress (McKee & Harrison, 1995). Vitamin C plays an important role in the metabolism of amino acids and promotes the absorption of minerals mainly iron by maintaining them in the reduced ferrous state (McDowell, 1989). Supply of L-arginine along with vitamin C has improved meat quality in broilers. Another vitamin namely vitamin E also showed improvement in feed conversion ratio and improved growth performance in poultry. Recommended dose of vitamin E is 5 to 25 IU/kg of feed for normal functioning of bird though higher doses has also _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Some of the beneficial applications of probiotics, prebiotics, vitamins, antimicrobial peptides and herbs as growth promoters in poultry are presented in figure. 1. 2.6 Antimicrobials of plant origin / phytobiotics Exploring alternatives to antibiotics as health promoting agents in poultry- a review 374 Figure 1 Beneficial effects of probiotics, prebiotics, vitamins, antimicrobial peptides and herbs as growth promoters in poultry. The anti-parasitic property is very well studied especially of tannins (condensed tannins) which are more potent against gastro-intestinal parasites of sheep and goats. They also show potent anti-coccidial activity against chicken coccidia. In case of large animals (cattle, buffaloes) the phyto-compounds especially essential oils, exhibits methanogenic suppression effect (reduces the methane enteric methane production). Phytochemicals possesses antioxidant (both hydrophilic and lipophilic activity). Due their antioxidant activity these compounds are being used during stress periods including the heat stress conditions (Wei & Shibamoto, 2007). Their antioxidant property could be helpful in improving the keeping quality of processed meats and also reduces the muscle drip loss during thawing of cold stored products (Windisch et al., 2008). These plant derived compounds shows typical flavors which could be exploited in human and pig foods. These compounds attract the consumers and increase their intake. Currently, essential oils are being used in preparation of icecream and others. However, their role as a flavoring agent in poultry production is still questionable. The dietary addition of active principles or its ingredient source increases the digestive process in the body. They were found to increase the secretion of digestive enzymes mainly trypsin, amylase and bile from the pancreas and liver respectively (Gopi et al., 2014a). This will _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org help to improve the overall digestibility of the feed and feed efficiency. However, the higher level of incorporation of certain compounds especially polyphenols which lead to negative effects on digestive efficiency due to their ability to bind with the digestive enzymes. Staying with digestion these substances also increases the nutrient absorption capacity through increase in the intestinal villi length and crypt depth. They also alter the lipid metabolism in the system by inhibiting the activity of hepatic 3– hydroxy–3–methylglutaryl coenzyme A (HMG–CoA) reductase which reduces the cholesterol synthesis in the liver (Lee et al., 2004). This effect could be utilized for production of low cholesterol meat and eggs (Mohamed et al., 2012). Although, these compounds are generally recognized as safe (GRAS) their level of use is still debatable due to their unknown mechanisms for various activities and their possibility of deposition in the body. Shift towards herbal medicine in the recent years is more due to advantages of these over chemical drugs which include reduced or zero toxicity, available naturally and possess ideal qualities as feed additive (Khan et al., 2010; Khan et al., 2012a). Plant parts such as herbs and spices are well known to have antimicrobial activities (Nychas & Skandamis, 2003). 375 The products derived from plant parts, specifically essential oils, are known to possess active ingredients that exhibit antimicrobial activity against bacteria, yeast and molds. Among the major groups of principle ingredients that impart antimicrobials property in their essential oils (EOs) include thymol, eugenol, saponins, flavonoids, carvacrol, terpenes and their precursors. Essential oils are volatile compounds due to which they possess characteristics fragrance of their origin and named after them (Oyen & Dung, 1999). The portion of plant from which essential oils can be derived include bulbs of onion and garlic, seeds of parsley, fruits, rhizomes, leaves of basil and tea plant, clove buds and other plant parts (Nychas & Skandamis, 2003). For example, cinnamon barks having high levels of cinnamamic aldehyde and spices with a high level of eugenol are reported to have potent antimicrobial activity (Davidson & Naidu, 2000). Essential oils from plants are reported to exhibit a broad antimicrobial spectrum against a wide range of bacterial and fungal agents (Tiwari et al, 2009). The antimicrobial property also depends on many biological factors (plant species, growing location and harvest stage), manufacturing processes (extraction/distillation) and conditions during storage (temperature, light, oxygen level and time). Thus, it remains a subject of investigation to identify and quantify the multitude of actions and claims improving feed efficiency and health status of poultry birds. The antimicrobial potential of essential oils also depends on the structural conformation of active ingredients and their concentration. Currently, herbs targeting bacterial quorum sensing disruption (Goossens, 2016) are gaining interest. Antimicrobial property of essential oils such as thymol and carvacrol has been widely studied against range of bacteria such as L. monocytogenes, S. Typhimurium, and Vibrio parahaemolyticus (Karapinar & Aktug, 1986; Tassou et al, 1995; Dhama et al., 2015a). Cinnamic aldehyde present in cinnamon oil have been found to exhibit antimicrobial action against a broad spectrum of bacteria such as L. monocytogenes, C. jejuni, and S. Enteritidis (Smith-Palmer et al, 1998). Eugenol present in clove essential oil has been widely studied for antimicrobials and antifungal activities (Deans et al., 1995; Smith-Palmer et al., 1998). Use of EOs in poultry ration has been found to exert beneficial effect on body weight again and feed efficiency in broilers (Cross et al., 2002; Bampidis et al., 2005; Cabuk et al., 2006). Similarly, feeding of turmeric powder enhances the circulatory anti-oxidant defence and in turn immune system (Madpouly et al., 2011). Similarly, incorporation of garlic at 3% level as feed additive has been found to enhance growth and performance of broiler chicks (Elagib et al., 2013). Incorporation of blends of different essential oils (lemon, basil, oregano, tea, etc.) in diet showed higher body weight gain (Khattak et al., 2014) in broilers, egg production with better feed conversion efficiency in laying quails (Cabuk et al., 2014). Recently, Salmonella Enteritidis and Salmonella Typhimurium has been found to be inactivated on skin of broiler birds using acidified sodium _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Ajit et al chlorite, trisodium phosphate or carvacrol (Karuppasamy et al., 2015, Yadav et al., 2016). Several plants and its derivates are extensively studied and used in poultry production including Aloe vera, Astragalus membranaceus, Ginger, Garlic, Noni, Onion, Turmeric and Thyme (Dhama et al., 2015b). These compounds have improved growth of broilers and also increased egg production of layers (Guo et al., 2004; Sunder et al., 2013; Sunder et al., 2014). Natural resin acid composition (RAC), resinol is shown to possess antibacterial, antifungal and antiparasitic properties and its inclusion in feed reduced the percentage of gram positive population in vitro and modulated the intestinal microbiota besides improving the growth performance (Vuorenmaa, 2015). Zhang et al. (2012) observed higher growth performance in broilers fed fermented leaves of Ginkgo biloba along with Aspergillus niger. Many active principles of the herbs have been identified but mechanism of action for all has not been elucidated though for some it has been reported. Reports reveal that active principles of these herbs improves the normal microbiota of the gut thereby increasing the nutritional metabolism, absorption leading to better growth and production (Hashemi & Davoodi, 2011). The increase in pancreatic enzymes (trypsin, chymotrypsin, amylase and lipase) activity due to feeding of turmeric, which is being attributed towards its active principle curcumin, has been seen (Khan et al., 2012b). Ginger increases secretion of enzymes like enterokinases and other enzymes important for digestion hence improving the digestion and metabolism of feed (Zhao et al., 2011). Similarly, addition of essential oils in feed has also improved secretion of digestive enzymes, increasing feed assimilation, overall activity of broilers were improved (AlKassie et al., 2011). These active principles also possess antioxidant properties thereby reducing the free radicals that are produced in the cells. Herbal products not only possess antioxidant and digestive properties but also possess antimicrobial, antiparasitic and immunomodulating properties. Though immunostimulants are available they possess side effects warranting for a replacement hence herbal drugs can be a better alternative as an immunostimulant. There are established reports regarding the potential of flavonoids, lectins, polysaccharides, peptides and tannins as immunomodulators. Plants like Neem, Ashwagandha, Guduchi, Noni etc., possess immunomodulatory properties and its effects are well documented (AbdElslam et al., 2013; Bhatt et al., 2013; Latheef et al., 2013a; Latheef et al., 2013b; Tiwari et al., 2014a; Tiwari et al., 2014b). Herbs like cinnamon, nishyinda and black pepper has been reported to have promising growth promoter effects without exhibiting side effects in broilers (Chowdhury et al., 2009; Mode et al., 2009; Molla et al., 2012; Saminathan et al., 2013). Several herbal extracts exert antibacterial action when fed to poultry thereby preventing infectious diseases and enhancing growth of the poultry (Dhama et al., 2014b; Dhama et al., 2015b). Active ingredients of thyme namely thymol and carvacrol shows antimicrobial action especially against gram negative bacterial pathogens by Exploring alternatives to antibiotics as health promoting agents in poultry- a review penetrating the cell wall and causing damage to the cells by binding to the amine and hydroxylamine groups (Juven et al., 1994; Helander et al., 1998; Abd El-Hack et al., 2016). Curcumin has better action against Eimeria spp. that causes coccidiosis in poultry (Khalafallah et al., 2011). Garlic increases phagocytic activity, production of interferon, interleukin and tumor necrosis factor α (Hanieh et al., 2010). Allicin, the bio-active component of garlic is reported to have the ability to infiltrate pathogen’s cellular membranes and subsequent binding to key enzymes that results in blockage of cellular activities. Cineol and eucalyptol of eucalyptus oil provides relaxing effect on air sacs with appropriate ventilation during respiratory tract infections of bird (Nakielski, 2015). Comprehensive knowledge about the single active compound or their possible synergistic or negative effects is required for the solution oriented developments in herbal treatment (Heinzl & Borchardt, 2015). Several benefits of phytobiotics have been elucidated in past (Lee et al., 2004; Windisch et al., 2008; Salim, 2011; Gopi et al., 2014b; Dhama et al., 2014b; Karangiya et al., 2016) and summarized as below: Salient benefits of phytobiotics are as follows: 1. 2. 3. 4. 5. 6. 7. 8. Favorably alters the microbial population for maintaining the gut health Reduces the insult of pathogenic bacteria, virus and parasites in the gut thereby reduces the need for anti-biotic therapy Improves the body weight gain and feed efficiency Increases the anti-oxidant defense against oxidative stress Decreases cholesterol content through inhibiting hepatic enzyme activity Stimulates the digestive enzyme secretions and nutrient absorption Ameliorate the negative effects of heat stress Environmental friendly insecticide and pesticide 2.7. Antimicrobial peptides Antimicrobial peptides are also termed as host defense peptides which are present in all living organisms with an amino acid length of about 30 to 60 numbers. These peptides possess immunomodulatory and antimicrobial activity that can damage bacteria (by targeting cell membrane), virus and also fungus (Li et al., 2012; Parachin et al., 2012). Several of these antimicrobial peptides are identified and many were tested for their beneficial effects like growth promoter activity in poultry. Antimicrobial peptides like colicin and cecropin, especially cecropin A (1-11)-D (12-37)-Asn (CADN) has been studied as growth promoter in poultry which indicated that this could be a possible alternative for antibiotics as growth promoters (Liu-Fa & Jian-Guo, 2012). In vitro studies indicated that, peptides isolated from chicken leukocytes have significantly inhibited L. monocytogenes and E. coli, Candida albicans (Harwig et al., _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 376 1994). Bacteriocins, the non-toxic ribosomal antimicrobial peptides secreted by bacteria on their cell surface are observed to effectively reduce the campylobacter colonization in poultry (Svetoch & Stern, 2010). These are new generation antimicrobials that may have potential to eradicate drug resistant bacteria. Nisin is the extensively studied bacteriocin for its use in food and therapeutic purpose in poultry (Joerger, 2003). Extraction of antimicrobial peptides from transgenic plants and application in poultry feed have been thought of (van t’ Hof et al., 2001). Conclusions Antibiotics have ruled the poultry industry since several decades as a growth promoter. However, due to their over usage bacteria has developed resistance against them thus threatening human community with the emergence of extremely drug resistant pathogens. Hence, it is must to eliminate the use of antibiotics as growth promoters and search for alternatives that can aid in beneficial activities. Recently much research has been diverted towards the search for antibiotic alternatives and which in turn has resulted in the enhanced use of probiotics, prebiotics herbal drugs, etc. The use of probiotics, prebiotics, synbiotics, plant extracts and organic acid has many potential benefits including improvement in digestion and absorption of nutrients, modification of birds’ metabolism, immunomodulation, and improvement in functioning and health of gut through exclusion and inhibition of pathogens in intestinal tract and improvement in safety of poultry products for human consumption. However, additional studies are still needed which would explore various combinations of these alternatives with specific target to enhance the production. Moreover, keeping in view the consumers demand for functional foods, efforts are being needed to explore further possibilities where alternatives of antibiotics in poultry production and poultry products with desirable attributes without affecting the welfare of the poultry birds, can be used. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. Acknowledgments All the authors acknowledge the support from their respective institutions and universities. References Abd El-Hack ME, Alagawany M, Farag MR, Tiwari R, Karthik K, Dhama K, Zorriehzahra J, Adel M (2016) Beneficial impacts of thymol essential oil on health and production of animals, fish and poultry: a review. 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