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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
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ISSN No. 2320 – 8694
Volume No – 4
Issue No(special issue) – 3(S)
May, 2016
Journal of Experimental Biology and Agricultural Sciences
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
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Dr Kuldeep Dhama (MVSc, Ph.D)
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Division of Pathology
ICAR-Indian Veterinary Research Institute (IVRI)
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Guest Editors
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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
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
Experimental Biology and Agricultural Sciences (JEBAS). On behalf
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JEBAS is dedicated to the rapid dissemination of high quality
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Thank you. We hope you will find JEBAS informative.
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Managing Editor - JEBAS
May 2016
Biomedical Perspectives of Advances in Disease Diagnosis & Therapeutics (BPADDT)
Kuldeep Dhama, Yashpal Singh Malik, Minakshi Prasad and Vincenzo
The effective management of diseases, including elimination or
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”
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
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
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.
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
nanobiotechnology has improved clinical diagnosis and opened a
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
experts, and would help in targeting development of valuable and
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
Volume No – 4
Issue No(special issue) – 3(S)
May, 2016
Journal of Experimental Biology and Agricultural Sciences
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
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
Antioxidant activity of vitamin e and its role
Vincenzo Tufarelli* and Vito Laudadio
An overview on the functional food concept: prospectives and
applied researches in probiotics, prebiotics and synbiotics
Vincenzo Tufarelli* and Vito Laudadio
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
Bats: carriers of zoonotic viral and emerging
Koushlesh Ranjan*, Minakshi Prasad and Gaya Prasad
Nanodiagnostics: a new frontier for veterinary and medical
Upendra Lambe, Minakshi P*, Basanti Brar, Madhusudan Guray,
Ikbal, Koushlesh Ranjan, Nitish Bansal, Sandip Kumar Khurana
and Manimegalai J
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
Prevalence, diagnosis, management and control of important
diseases of ruminants with special reference to indian scenario
Ramakrishnan, Kumaragurubaran Karthik, Yashpal Singh Malik and
Kuldeep Dhama
Exploring alternatives to antibiotics as health promoting
agents in poultry- a review
Kumaragurubaran Karthik, Yashpal Singh Malik and Kuldeep Dhama
Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
Sourabh Chhabra1, Khurana S K2,*, Kapoor P K1and Richa Khirbat3
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
Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016
Available Online – May 25, 2016
Rhodococcus equi
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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.,
Table 1 Antibiotics used and their concentration.
S. No
mcg = Micrograms
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
S. No
Nalidixic acid
Penicillin G
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
(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,
Table 2 Antibiotic sensitivity of twenty eight isolates of Rhodococcus equi.
Antibiotics to which all the isolates were
Nalidixic acid
*Number in parentheses represent sensitive (S) and resistant (R) isolates
Journal of Experimental Biology and Agricultural Sciences
Antibiotics to which isolates showed variable resistance
Amoxycillin (12S, 16 R)
Colistin (21S, 7R)
Gentamycin (4S, 24R)
Streptomycin (5S, 23 R)
Chhabra et al
Table 3 Resisotypes recognized amongst twenty eight Rhodococcus equi isolates.
Sensitive or Resistant to antibiotic
Sample No. (No. of Isolates)
NS-1, NS-44, NS-98, NS-117, NS-120, NS-151,
NS-244 NS-290 (8)
NS-6 (1)
NS-25, NS-276 (2)
NS-36, NS-79, NS-113, NS-150, NS-231, FNS4, FNS-5 (7)
NS-48, NS-216 (2)
NS-62 (1)
NS-77 (1)
NS-121, NS-161, NS-288 (3)
NS-170 (1)
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
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
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
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.,
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
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.
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.
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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
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
Morinda citrifolia
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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
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
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
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
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).
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
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
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
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
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
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)
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
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
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.
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.,
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
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
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.
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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
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
Vitamin E
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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
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.,
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.,
Journal of Experimental Biology and Agricultural Sciences
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
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.
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
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.
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. Diet forms an important component of the antioxidant
protection system; it supplies the major antioxidants such as
vitamin E. Thus, vitamin E is a crucial element due to its
function in sustaining poultry well-being and reproductive
Journal of Experimental Biology and Agricultural Sciences
Tufarelli et al
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
ISSN No. 2320 – 8694
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
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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
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
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.,
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
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
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.,
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. The association
between the many disciplines involved in food and nutritional
science, consequently, is indispensable for credible and
successful innovation in functional food.
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
ISSN No. 2320 – 8694
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
Department of Animal Biotechnology LUVAS, Hisar, Haryana- 125004, India
University of Minnesota, USA
SVPUAT, Meerut, U.P. India
Centre for Medical Biotechnology, Maharshi Dayanand University, Rohtak, Haryana-124001, India
Department of Biochemistry, MDU, Rohtak, Haryana-124001, India
NRCE, Hisar, Haryana, India
Received – April 18, 2016; Revision – April 25, 2016; Accepted – May 21, 2016
Available Online – May 25, 2016
Antigenic variation
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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
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
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
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).
Minakshi et al
Table 1 PCR assays developed for detection of canine parvovirus.
S. No
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
Gupta et al., 2005
sequences specific for
CPV variant strains
VP2 gene
Behera et al., 2015
VP2 gene
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
CPV and its variants typing using PCR
Characterization of canine parvovirus by PCR
Analysis of VP2 gene sequences of canine parvovirus isolates
molecular characterization and phylogenetic analysis of canine
parvovirus by PCR
Typing of canine parvovirus using mini-sequencing based SNP
Detection of canine parvovirus by PCR assay
sequences specific for
CPV variant strains
sequences specific for
CPV variant strains
sequences specific for
CPV variant strains
sequences specific for
CPV variant strains
sequences specific for
CPV variant strains
VP1 and VP2
Demeter et al.,
Silva et al., 2013
Costa et al., 2005
Shankar et al., 2014
Pereira et al., 2000
Chinchkar et al.,
Mohan Raj et al.,
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.,
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
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
Canine parvovirus- an insight into diagnostic aspect
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.,
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
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.,
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.,
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
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
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
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
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
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,
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
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. Owner should not allow the dog to go
outside and mix with other stray or infected dogs. Proper care
should be taken in waste matter disposal especially faeces of
infected dogs.
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
ISSN No. 2320 – 8694
Koushlesh Ranjan1,*, Minakshi Prasad2 and Gaya Prasad3
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
Reservoir host
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
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International License Based on a work at
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,
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
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
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
Table 1 Zoonotic viruses causing disease in human and their bat reservoir hosts.
S. No.
Rabies virus and other
Reservoir Host
Several bat species distributed world
Ebola virus disease
Marburg virus
respiratory syndrome
respiratory syndrome
Severe acute febrile
Franquet’s epauletted fruit bat (Epomops
collared fruit bat (Myonycteris torquata)
Chinese horseshoe bat (Rhinolophus
Rousettus spp.
Tioman virus
Pteropus hypomelanus
Menangle virus
Little red flying foxes and gray headed
flying foxes
Sosuga virus
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
Some flying foxes (Pteropus spp.)
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;
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
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
( 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
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.,
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
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
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).
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.
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 ( 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
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
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
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
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 ( 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
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
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
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.
Ranjan et al
Conclusion and future perspective
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
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,
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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.
Authors are thankful to Sardar Vallabhbhai Patel, University of
Agriculture and Technology, Meerut, Uttar Pradesh, India for
providing facility to prepare the manuscript.
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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
Upendra Lambe1, Minakshi P1,*, Basanti Brar1, Madhusudan Guray1, Ikbal1, Koushlesh Ranjan2,
Nitish Bansal1, Sandip Kumar Khurana3 and Manimegalai J1
Department of Animal Biotechnology, LUVAS, Hisar, Haryana, India
Department of Veterinary Physiology and Biochemistry, SVPUAT, Meerut, U.P. India
NRCE, Hisar, Haryana, India
Received – April 28, 2016; Revision – April 26, 2016; Accepted – May 21, 2016
Available Online – May 25, 2016
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.
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Journal of Experimental Biology and Agricultural Sciences
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
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
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
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
Minakshi et al
Table 1 Lateral Flow assay for detection of various biological agents.
Detection Limit
Xiuli et al., 2016
Ruihua et al., 2016
Mycoplasma pneumonea
Liming et al., 2016
Corstjens et al., 2016
Prostate specific Ag
Photon up-converting NPs
Juntunen et al., 2016
Hepatitis C
Hwan et al., 2015
Jyoti et al., 2015
Xie et al., 2015
Table 2 Antigen/antibody interaction based system for detection of different pathogens.
Adeno virus
Encephalomyocarditis virus
Duck Hepatitis virus
Salmonella pullorum
Human T lymphotrophic virus
Hepatitis B Virus
Orchid Virus
General Virus
H1N1, H5N1, H7N9
Nano Particle
Triangular AuNPs
Silver NPs
Triangular AuNPs
Silicon wafers
Fe-Au shell
Blue Silica & MNPs
Quantum dots
Gold Nano rods
GNP Chip
ZnO Nano rods
Type of detection
Raman Scattering
Fluorescence OPDA
Raman Scattering
Ellipsometry Imaging
Sandwich assay
Magnetic sensor
Immunoaffinity assay
Immunoaffinity assay
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
Glycoprotein 160
Label free
Fluorescence Microscopy
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
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
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
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).
Minakshi et al
Table 3 Enzymatic interactions based detection of different agents associated with health concern.
IFN Gamma
Protein estimation
Stem cell factor SCF
Nano Mass
FeMoO4 rods
Graphene films
Type of sensor
Cyclic voltammetry
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
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
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
Table 4 Nucleic acid interactions based nano-diagnosis detection of different agents associated with disease.
Arabis Mosaic Virus
Lily Symptomless Virus
Canine adeno
Influenza virus
White spot syndrome virus
Porcine epidemic diarrhea
3D Graphene
Cu Nano cluster
CdZnTeS Quantum dots
Sugar chain GNP
Sensor type
Barcode amplification
Impedimetric sensor
Microarray chip
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
Nucleic acid
DNA oligos
DNA oligo
16s rRAN
Molecular beacons
DNA oligo
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).
Minakshi et al
Table 5 Nanoparticles facilitated nucleic acid based technologies.
Micro Array
Rolling circle amplification
Threshold mediated strand displacement
L shaped DNA probes
RNA quantification
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.
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
Nucleic acid
DNA probes
PNA peptide nucleic acid
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
ISSN No. 2320 – 8694
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
Lantana camara
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]
rights reserved.
Journal of Experimental Biology and Agricultural Sciences
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
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
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.,
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
Table 1 Chemical compounds obtained from Lantana camara and their mechanism of actions.
1. Hepatotoxic
LA, LB, LC, RLA and icterogenin
2. Antimicrobial
antibacterial activity
LA, LB, oleanolic acid, ursolic acid, 4Epihederagonic acid and 24-Hydroxy-3-oxours12-en-28-oic acid
3. Protein kinase C
4. Anti-inflammatory
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
(Psychiatric disorder)
7. Antitubercular
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
Apigenin, Cirsilineol, Eupafolin, Eupatorin and
19. Insecticidal action
Bioactive molecules without any cross resistance
20. Anti-diabetic
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
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.,
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
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
Lantana camara: An alien weed, its impact on animal health and strategies to control
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
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
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
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,
5 Clinical signs (de Mello et al., 2003; Sharma et al., 2007)
The dose of lantadenes determines the severity of ictericity
(Gopinath & Ford, 1969). The clinical signs follow a definite
pattern as given below:
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
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.
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,
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
Table. 2 Histopathological alterations in different animal species.
S. No
Guinea Pigs
and Rats
Histopathological alterations
Degeneration of the periportal parenchymal cells, distended bile canaliculi, fatty
degeneration, portal fibrosis, hyperplasia of bile ducts, edema of gall bladder in
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
Dwivedi et al.,
1971; Uppal &
Paul, 1978
Sharma et al.,
Sharma et al.,
Sharma et al.,
1992; Parimoo et
al., 2015
Lantana camara: An alien weed, its impact on animal health and strategies to control
Table. 3 Hematological examination in different animal species.
S. No.
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.
Roychoudhury, 1992
Seawright, 1963
Ali et al., 1995
Sharma et al., 2007;
Parimoo & Sharma,
Table. 4 Biochemical Alterations in different animal species.
S. No.
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):
Keep the intoxicated animals away from light;
provide fluid therapy and adequate feed.
Administration of activated charcoal 5g/kg body
weight with electrolyte in stomach tube within 24h,
which reduces the absorption of lantadenes.
Administration of bentonite 5g/kg body weight. It is
much cheaper than charcoal but takes longer time to
show desired effect.
Administration of Tefroli powder obtained from
Tephrosia purpurea plant.
Oral administration of liver tonics like Liv-52.
Vitamin B-complex administration.
Enzymatic removal of bilirubin by bilirubin-oxidase,
which is effective in jaundice.
Herbal tea i.e. Yin Zhi Huang (YZH) from
Artemisia capillaries, effective in neonatal jaundice.
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
Dwivedi et al., 1971;
Seawright & Hrdlicka,
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
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):
The international standards for trading partner
countries in a well targeted form must be
The adequate surveillance and monitoring system
for early detection of lantana infestation must be
Implementation of strict border controls, transport
controls and quarantine methods should be followed.
Rakesh et al
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.
9 Strategies which can be opted for controlling L. camara
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:
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
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
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.,
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
Table. 5. List of some useful products obtained from different parts L. Camara.
S. No.
Leaves, stem
Leaves, stem, roots
Aerial parts
Oleanonic acid
Oleanolic acid
Camarinic acid, Lantanoside
Lactones containing euphanes
Antimicrobial, antitumor, anti-inflammatory
Leaves and branches
(Sources: Sharma et al., 2007; Hussain et al., 2011; Sousa & Costa, 2012)
Use of certain biological agents like
plume moth (Lantanophaga spp.), seed
(Corynespora cassiicola) (Pereira et al.,
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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Wickramaratne DBM (1991) Furanonaphthoquinones from
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L. camara is an invasive toxic weed which is dominating
globally and is capable of over-run neighbouring young
Achhireddy NR, Suigh M, Achhhireddy LL, Nigg HN, Nagy S
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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
Mani Saminathan1, Rajneesh Rana2,*, Muthannan Andavar Ramakrishnan3, Kumaragurubaran
Karthik2, Yashpal Singh Malik4 and Kuldeep Dhama1
Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India
Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India
Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar Campus, Uttarakhand - 263 138, India
Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India
Received – May 05, 2016; Revision – May 09, 2016; Accepted – May 21, 2016
Available Online – May 25, 2016
Ruminant Diseases
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]
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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
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.,
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
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
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
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
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
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
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
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
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
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
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.
(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
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).
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
( 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
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
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
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 (; 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
( 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.,
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.,
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
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
vaccination in livestock and captive wildlife species with
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
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,
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).
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
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
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
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
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
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
(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
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).
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
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.
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
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
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
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.,
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;
In addition, India has 53 veterinary colleges, and 13 veterinary
and animal science universities distributed throughout the
Journal of Experimental Biology and Agricultural Sciences
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.
All the authors acknowledge the support from ICAR-Indian
Veterinary Research Institute, Izatnagar.
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Journal of Experimental Biology and Agricultural Sciences, June - 2016; Volume – 4(3S)
Journal of Experimental Biology and Agricultural Sciences
ISSN No. 2320 – 8694
Ajit Singh Yadav1,*, Gautham Kolluri1, Marappan Gopi1, Kumaragurubaran Karthik2, Yashpal Singh
Malik2 and Kuldeep Dhama2
ICAR-Central Avian Research Institute, Izatnagar-243122, UP, India
ICAR-Indian Veterinary Research Institute, Izatnagar-243122, UP, India
Received – May 05, 2016; Revision – May 09, 2016; Accepted – May 21, 2016
Available Online – May 25, 2016
Poultry production
Organic acids
Plant extracts
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
* Corresponding author
E-mail: [email protected] (Ajit Singh Yadav)
Peer review under responsibility of Journal of Experimental Biology and
Agricultural Sciences.
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International License Based on a work at
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
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.,
Luangtongkum et al., 2006; Martinez et al. 2006; Billah et al.,
2015), oxytetracycline (Adel Feizi, 2013), sulphonamides
Journal of Experimental Biology and Agricultural Sciences
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:
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
5. It should not be absorbed from the gut into edible
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
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
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
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.
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,
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
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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
3. Results in better feed conversation efficiency
4. Improves growth rate and body weight gain
5. Improves the digestive enzymes and in turn nutrient
6. Reduces circulating cholesterol level through
regulation of lipid metabolism
7. Enhances efficacy of vaccines
8. Plays important role in fast detoxification of
9. Reduce stress associated with administration of
antibiotics, temperature, vaccination, transportation
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
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).
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
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
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).
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
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
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
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).
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
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,
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:
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
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
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).
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.
All the authors acknowledge the support from their respective
institutions and universities.
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