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Journal of Veterinary Advances Clinical Signs of Experimental Trypanosoma evansi Infection in Donkeys: Ameliorative Effects of Isometamidium chloride and Buparvaquone Treatments Garba U. M., Sackey A. K. B., Lawal I. A. and Esievo K. A. N. J Vet Adv 2015, 5(4): 891-901 DOI: 10.5455/jva.20150413121927 Online version is available on: www.grjournals.com GARBA ET AL. ISSN: 2251-7685 Short Communication Clinical Signs of Experimental Trypanosoma evansi Infection in Donkeys: Ameliorative Effects of Isometamidium chloride and Buparvaquone Treatments 1 Garba U. M., 2Sackey A. K. B., 3Lawal I. A. and 4Esievo K. A. N. 1 Veterinary Clinic, Equitation Dept, Nigerian Defence Academy, Kaduna, Nigeria. Department of Veterinary Medicine, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Nigeria. 3 Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Nigeria. 4 Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Nigeria. 2 Abstract Trypanosoma evansi (T. evansi) infection causes a progressively wasting and ultimately fatal animal trypanosomosis mainly in camels, horses, donkeys, zebra, mules and cattle in Africa, Asia, Middle East and South America resulting in reduced traction power and annual death of thousands of affected animals. The aim of this study was to evaluate the ameliorative effects of Isometamidium chloride (standard trypanocide) and Buparvaquone (anti-theilerial drug) treatments on the clinical signs of T. evansi infection in donkeys as part of study on the efficacies of the drugs against T. evansi infection. Twenty four apparently healthy donkeys were used for the experiment. The animals were housed, fed on hay and concentrate feed, and water was provided adlibitum. Animals were identified with neck-tags and grouped into 4 namely; groups A1, A2, A3 (T. evansiinfected groups) and B (control) of 6 animals each (3 males and 3 females) at random. Two milliliters of buffered, parasitaemic Wister rat blood containing 2.0x106 of T. evansi (Sokoto isolate) was used to infect each of all donkeys in the ‘A’ category through jugular vein. On day28 post-infection, groups A2 and A3 animals were treated with Isometamidium chloride and Buparvaquone respectively. Groups A1 and B remained as Infected-Untreated and Un-infected-Untreated respectively. Animals were monitored and evaluated postinfection and post-treatment for clinical signs including vital parameters and body weight changes. The effects of treatments on the observed-parameters were evaluated. Result showed that T. evansi infection in donkeys is predominantly a chronic disease, with an incubation period of 3-7 days. Isometamidium chloride treated group showed greater reduction in prevalence of signs than buparvaquone treated group which did not differ much from the Un-treated group. It was concluded that Trypanosoma evansi infection in donkeys is a chronic disease and treatment with Isometamidium chloride, ameliorates the clinical signs while buparvaquone does not. Keywords: Ameliorative effects, buparvaquone, clinical signs, donkeys, isometamidium chloride, trypanosoma evansi, infection. Corresponding author: Veterinary Clinic, Equitation Dept, Nigerian Defence Academy, Kaduna, Nigeria. Received on: 04 Mar 2015 Revised on: 14 Mar 2015 Accepted on: 13 Apr 2015 Online Published on: 30 Apr 2015 891 J. Vet. Adv., 2015, 5(4): 891-901 CLINICAL SIGNS OF EXPERIMENTAL TRYPANOSOMA EVANSI INFECTION IN … Introduction Trypanosoma evansi infection causes a progressively wasting and ultimately fatal animal trypanosomosis mainly in camels, horses, donkeys, zebra, mules and cattle in Africa, Asia, middle East and South America resulting in great economic losses due to reduced traction power (ILRI, 1997) and annual death of thousands of affected animals (Mihret and Mano, 2007). Other animals affected are dogs and pigs (OIE, 2004). Trypanosoma evansi infection is referred to with over 30 different ethnic terms which describe the chronic devastating outlook of infected camels and equids in different geographical regions of the world as reviewed by Hoare (Stephen, 1986) such as surra in Asia (OIE, 2004) which is an Indi word meaning ‘rotten’ (Vittoz, 1955), El-debab in Algeria (Atarhouch et al., 2003), Mbori in Sudan, Guifar in Chad and Pmenchaca amongst Tuaregs of Niger (Antoine-Moussiaux et al., 2007). Successful cure and consequently, amelioration of the accompanying signs of surra is affected by the problem of reported cases of resistance by T. evansi strains to the available range of trypanocides namely; Diminazene aceturate, quinapyramine, cymelarsan (De Koning, 2001; Suswam et al., 2001) and suramin (FAO, 2012) in different countries (El Rayah et al., 1999; Zhou et al., 2004). In this study, the clinical signs of Trypanosoma evansi infection in donkeys were collated as they emerged and the ameliorative effects of treatments using Isometamidium chloride; a standard trypanocides (Shapiro and Englund, 1990: Aliu, 2007) and Buparvaquone; an anti-theilerial drug (Dhar et al., 1987; Mutugi et al., 1988) were also evaluated as part of study conducted on the efficacies of Isometamidium chloride and buparvaquone in the search for drugs with efficacies against Trypanosoma evansi infection in donkeys. Materials and Methods Experimental Animals Twenty four apparently healthy donkeys comprising of 12 males and 12 females were used. The experimental animals were between 11 and 15 months old as estimated in the animal market prior 892 to purchase using eruption and wear features of the incisors teeth described by Wayne and Melvin (2000) and Joe (2012). Housing and Acclimatization of Animals Topical sweat-resistant fly-repellant spray containing pyrethroids (Endure®, Farnam co. inc., USA) was applied on the animals as recommended by the manufacturer, to keep flies off the animals prior to housing. The animals were housed in prepared and fly-proofed pen in the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, where the experiment was conducted. Zaria city is located in North Western, Nigeria (Geographical coordinates of Zaria; 11° 4' 0" North and 7° 42' 0" East). The experimental animals were acclimatized for 14 days (Wolfensohn and Lloyd (2013). Pre-Infection Evaluation of Experimental Animals During the acclimatization period, the animals were examined for external parasites, 5g faecal sample was collected per rectum into labeled polyethene bags and examined for gastrointestinal parasites using floatation and sedimentation methods (Charles, 2007). The animals were dewormed using Fenbendazole bolus (Fenacure®, Ashish Life Sciences PVT ltd, India) at the dose rate of 10mg/kg bd wt orally, once (Aliu, 2007). Four milliliters (4mL) of jugular vein blood was collected from each donkey using 10mL syringe and 19G X 11/2 inch needle (Weiser, 2012; Wolfensohn and Lloyd, 2013). Three milliliters (3ml) of the blood was anti-coagulated in EDTA+k3 (2mg/mL of blood) bottle which was used to prepare 2 Giemsa stained thin blood smear slides and 2 haematocrit centrifugation technique (HCT) tubes (Wosu, 2002) from each samples and were examined at x100 objective, across 20 light microscope fields. The balance of 1ml collected blood from each donkey was immediately used for mice inoculation test (MIT). Two (2) representative mice per donkey were each inoculated with 0.5ml of donkey’s blood per mouse using 1ml sterile disposable syringe with 29G X ½ inch needle intraJ. Vet. Adv., 2015, 5(4): 891-901 GARBA ET AL. peritoneally as support test for direct microscopy. Inoculated mice were monitored for parasitaemia at 48 hours intervals for 2 weeks using collected tail blood and examined on Wet Film and HCT (OIE, 2010) to ensure that the experimental donkeys were haemoparasites-free. Feeding of Animals The animals were provided with feed materials equivalent to 5% of their mean body weight in forms of Sorghum/Maize stovers, grass hay and legume hay in the ratio 4:1 while the concentrate feed consisted of a mixture of coarsely ground sorghum grain, bran, and dried groundnut cake which was served daily in two divided rations for morning and evening. Multi-minerals/vitamins lick blocks and clean drinking water were provided adlibitum (Aganga et al., 2000). Identification and Grouping of Animals Animals were identified with seriallynumbered neck-tags and assigned to experimental groups of 6 animals (3 males and 3 females) each at random, namely; groups A1, A2, A3 and B (Aviva and Poul, 2013). Baseline Data Collection Baseline data was collected on respiratory rate (costo-abdominal movement), pulse rate (pulsation of External Maxillary Artery), rectal temperature and body weight of the animals at 7 days intervals over 28 days period (Wosu, 2002). Body weights of the donkeys were determined using sling scale as reported by FAO (2014) and all parameters were evaluated between the hours of 06.00 and 08.00 local time, prior to provision of feed in order to minimize the influence of gut-fill on body weight values (De Aluja et al., 2005). Propagation of Trypanosoma Evansi Trypanosoma evansi originally isolated from camel in Sokoto, Nigeria (Sokoto isolate) and maintained in Wistar rats in the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria, was propagated in rats to obtain sufficient inoculum. To achieve this, 1mL of highly parasitaemic blood (determined on Wet Blood Film) was collected from an infected Wister rat via ocular vein into heparinised sample bottle 893 J. Vet. Adv., 2015, 5(4): 891-901 and diluted with 1mL phosphate buffered saline glucose solution to obtain 2mL inoculum. Immediately, 4 donor rats were inoculated each with 0.5mL of the inoculum, intra-peritoneally using sterile 1mL disposable syringe, 25GX 11/2 inch needle (Wolfensohn and Lloyd, 2013). The 4 rats were monitored for parasitaemia levels at 48 hours intervals using tail blood for Haematocrit Centrifugation Techniques (HCT) test. Twelve days post-inoculation, 3 of the 4 rats had attained +4 (>20 parasites per microscope field) while 1 rat had +2 (6-10/field) parasitemia levels. The donor rats were sacrificed and their blood pooled into a sterile conical flask containing heparin sodium 20 Units/mL of blood. A total of 15mL of pooled blood was obtained, the parasitemia was re-evaluated using haemocytometer (Elaine and Margi, 2007), then diluted with phosphate buffered saline glucose solution to obtain about 1.0 x106 T. evansi /mL of inoculum (Monzon et al., 1990). Experimental Infection of Animals The prepared inoculum was immediately injected at the rate of 2mL (containing 2.0x106 T. evansi) into each of the experimental donkeys in groups A1, A2 and A3 through jugular vein and the day of infection was considered as day0 of infection. Group B remained as Un-infected (control) group. Post-Infection Monitoring Post-treatment, the infected animals were monitored over 28 days period as follows; From day1 to day7; the animals were monitored daily while from day8 to day28; the animals were monitored twice per week for parasitemia levels using ear vein blood examined on HCT (OIE, 2010; FAO, 2014) and scored according to Woo (1970) technique to ensure establishment of infection in the infected groups of animals. Clinical signs, including respiratory rate, pulse rate and rectal temperature were also evaluated as described by Wosu (2002). Body weight was evaluated at 7 days intervals during the post-infection monitoring. Treatment of Specific Infected Animals On day 28 post-infection (PI), groups A2 animals were treated with Isometamidium chloride (Securidium®, Laprovet, France) 1% solution at the CLINICAL SIGNS OF EXPERIMENTAL TRYPANOSOMA EVANSI INFECTION IN … dose rate of 0.5mg/kg bd wt by deep intramuscular injection once while group A3 were treated with Buparvaquone (Butalex®, Coopers K-brand Ltd, Kenya) 5% w/v at dose rate of 2.5 mg/kg bd wt by deep intramuscular injection twice at 72 hours interval respectively as recommended by the manufacturers. Treatment day was considered as day0 of treatment. Post-Treatment Monitoring Post-treatment (PT) monitoring of experimental donkeys was conducted for 148 days during which clinical signs were further evaluated daily from day1-7, twice per week from day8-28 and at weekly intervals from day29 -148. Body weight was evaluated at 7 days intervals during the posttreatment monitoring. Ethical Considerations All procedures on the animals were performed as recommended by the European Union ‘Directive 2010/63’ contained in the report by Wolfensohn and Lloyd (2013) and approved by the Research and Ethics Committee of Ahmadu Bello University, Zaria, Nigeria. Statistical Analysis Group prevalence of clinical sign= Number of animals with the clinical sign in a group x100 Total number of animals in that group Means of parameters were determined for groups in all phases of experiments, tables and graphs were prepared using Microsoft Excel 2010. Analysis of Variance (ANOVA) was used to compare means of variables between experimental animals groups and within groups at 95% confidence level, where P< 0.05 was considered statistical significant (Mead et al., 2002). Clinical Signs Observed in Trypanosoma evansi Infection in Groups of Donkeys The incubation period observed was 3-7 days and pre-patent period of 3-4 days for experimental Trypanosoma evansi infection in donkeys. The clinical signs of infection were more pronounced in Infected-Untreated group of animals and started with mild pyrexia of 38.50C (control; 36.670C) (Table 1). Results Table 1: Prevalence of clinical signs observed in donkeys experimentally infected with Trypanosoma evansi based on infection and treatment status. Clinical Signs Post-infection (Day3 – Post-treatment (Day1 – 148 PT; 28 PI) equivalent to Day29-176 PI ) A1 (n=6) A2 (n=6) A3 ( n=6) A1(I-U, A2 A3 n=6) (I-Isomet, (I-Buparv, n=5) n=5) Mild pyrexia (38.50C, control; 36.670C) 6(100) 6(100) 6(100) NA NA NA Lethargy/Recumbency 6(100) 6(100) 6(100) 6(100) NA 5(100) Rough hair coat 3(50) 5(83.3) 5(83.3) 6(100) NA 5(100) Urticaria/alopecia on trunk 3(50) 4(66.7) 2(33.3) 2(33.3) NA 2(40) Prominence of ribs and hip bones 6(100) 6(100) 6(100) 6(100) NA 2(40) Pica appetite 6(100) 6(100) 6(100) NA NA NA Penile erection (in males) 3(100) 3(100) 3(100) NA NA NA Rectal prolapse 1(16.7) 1(16.7) 1(16.7) NA NA NA Pale ocular and oral mucosae (mild) 6(100) 6(100) 6(100) NA NA NA Intermittent diarrhoae 1(16.7) NA 2(33.3) NA NA NA Bilateral seromucous epiphora NA NA 2(33.3) NA NA 2(40) Isolation (seclusion) NA NA 2(33.3) NA NA 2(40) Ventral abdominal oedema NA NA NO 6(100) 1(20) 1(20) Lymphadenopathy 6(100) 6(100) 6(100) 6(100) NA 2(40) 894 J. Vet. Adv., 2015, 5(4): 891-901 GARBA ET AL. Morbidity Mortality 6(100) NA 6(100) 1(16.7) 6(100) 1(16.7) -------1(16.7) --------NA -------NA Post-infection (PI): Letter A1, A2 and A3 represented the T. evansi-infected groups of animal. Post-treatment (PT): A1 (I-U) = Infected-Untreated, A2 (I-Isomet) = Infected and Isometamidium chloride-treated, A3 (I-Buparv) = Infected and Buparvaquonetreated groups. n= sample size in animals group and NA = Not applicable (the referred clinical sign was not observed in the experimental group). Values in parentheses are percentages. Prevalence of ‘penile erection’ was based on the total number of male animals in the groups. Mean Respiratory Rate Changes The mean respiratory rate was significantly (P<0.05) elevated in all groups of animals infected with T. evansi from day28 to 35 post-infection (PI) and variously persisted to day56 PI. 895 J. Vet. Adv., 2015, 5(4): 891-901 The isometamidium-treated group showed decline in values not significantly different (P>0.05) from pre-inf. value as as early as day35 PI. After day56 rate returned to near pre-inf values (Fig 1). CLINICAL SIGNS OF EXPERIMENTAL TRYPANOSOMA EVANSI INFECTION IN … Mean Pulse Rate Changes All groups of animals, irrespective of infection status showed decline in mean pulse rate on day28 896 PI. The decline is apparently due to severe drop in ambient temperatue from 24±2 0C to 19±20C during the 1 week period (day 21-28 PI) (Fig 2). J. Vet. Adv., 2015, 5(4): 891-901 GARBA ET AL. Mean Rectal Temperature Changes All T. evansi-infect groups of animals showed increase in mean rectal temperature value from day7 to 21 PI(2 weeks period) with peaks on day14 PI. Rectal temperature undulated (Fig 3). Mean Body Weight Changes All T. evansi-infect groups of animals showed decline in mean body weight values from day7 to 21 PI(2 weeks period). Between day28 and 84 PI (8 weeks period), there was slight stability in mean body weight at the levels of pre-inf. value which were later, on day91 followed by a gradual decline trend to end of PT observation (Fig 4). 897 J. Vet. Adv., 2015, 5(4): 891-901 CLINICAL SIGNS OF EXPERIMENTAL TRYPANOSOMA EVANSI INFECTION IN … Discussion The Mild pyrexia and incubation period of 3-7 days may reflect the level of pathogenicity of the parasite isolate (Habila et al., 2012). The classical and non-specie specific signs (Gardiner and Mahmoud, 1990) observed were; lymphadenopathies due to immunologic response by lymphoid tissue to the infecting T. evansi (Klaus, 1996), pale mucous membranes suggesting reduced peripheral circulation as a result of anaemia in the infected animals (Rickman and Cox, 1983). The rough hair coat and urticaria might be due to immune complexes deposition, tissue inflammation and damages to organs (Enwezor and Sackey, 2005) while the bilateral seromucous epiphora may partly be due to entry of T. evansi into anterior chamber of eye as reported in horses (Stephen, 1986) resulting in inflammation (Enwezor and Sackey, 2005). The lethargy, prominent hipbone/ribs and the isolation behavior are the aftermaths of hypoglycaemia (Habila et al., 2012), hypocalcemia because of the role of calcium in muscle contraction (Bohn, 2012). Other infrequent signs observed in acute phase were persistent penile erection due to intravascular coagulopathy as reported earlier in horses by Lingard in 1893 (Stephen, 1986). The erection was not due to libido because at the age of 11 to 15 months when they were used for the experiment, the animals were not matured; until they attain 3-4 years of age (Starkey, 1995). The diarrhoae was precipitated by hypoalbuminaemia, and digestive disorders (Allison, 2012) resulting in an increased fluidity and volume of gut content and diarrhea. Also, immunosuppression of the host animal by T. evansi could allow flare of intestinal microflora such as Salmonella serovars and Clostridium perfringens in the colon and caecum and may precipitate hypermotility of intestines resulting in decreased transit time of intestinal content and diarrhea (Stewart, 2013). The rectal prolapse might be due to loss of rectal muscle tone during straining associated with intermittent constipation and diarrhoea (Stewart, 2013). Ventral abdominal oedema observed in the chronic phase of the infection in all groups of infected animals and more severe amongst Infected–Untreated animals while 898 the Infected and treated with Isometamidium or buparvaquone showed lower but the same severity possibly due to treatment in the latter 2 groups while the untreated group suffered full blown disease (OIE, 2010) precipitated by parasitesinduced metabolic enzymes failure (Enwezor and Sackey, 2005: Bal et al., 2012) and hypoalbuminaemia with loss of vascular fluid to the interstitial tissue especially in dependent areas (Allison, 2012). The observed 100% morbidity showed that T. evansi (Sokoto isolate) is pathogenic to donkeys and the low mortality rate in acute phase and the relapse observed in the chronic phase of the study suggests that the course of surra in donkeys is predominantly chronic if not treated or ineffectively treated (Faye et al., 2001; Desquesnes, 2013). The infected but Isometamidium chloride-treated animals showed decrease in the over-all signs of the disease in donkeys suggesting amelioration of signs following clearance of parasitaemia by the drug (Gutiérrez et al., 2013). The buparvaquone-treated group of animals showed persistence of the signs to chronic phase but with reduced prevalence suggesting that there was no amelioration of clinical signs by buparvaquone. The hyperpnoea observed in all infected groups of animals is apparently compensatory and accompanied T. evansi-induced anaemia which reduced available haemoglobin to transport oxygen to tissues resulting in tissue anoxia (Gardiner and Mahmoud, 1990). The isometamidium chloride treated animals showed return of respiratory rate to pre-infection level, a week post-treatment (PT) suggests amelioration by isometamidium as against persistence of hyperpnoea for longer period in infected-untreated and buparvaquone-tread groups. The genaral decline in mean pulse rate on day28 PI suggest severe drop in ambient temperatue (from 24±2 0C to 19±20C) during the 1 week period. Although, only the buparvaquone-treatment group showed significant tachycardia PI, there was tachycardia in other groups too post-infection (PI) is due to hypovolemia associted with anaemic situation induced by T. evansi infection (Gardiner and Mahmoud, 1990). The pyrexia observed in all T. evansi-infect groups of animals took a mild trend possibly due to relatively low susceptibility of donkeys (Silva et al., J. Vet. Adv., 2015, 5(4): 891-901 GARBA ET AL. 1995; Desquesnes et al., 2013) and immunotolerance to T. evansi (Herrera, et al., 2004). The absence of pyrexia in the chronic phase of infection may be as a result of low parasitaemia characteristic of chronic form of surra as in camel (Rami et al., 2003; Wolkmer et al., 2009). The decline in mean body weight values of all groups of T. evansi-infected animals occurred sideby-side high parasitaemia which results in build up of peroxides and free radicals in the body of infected animal and damage all components of the cell, including proteins and lipids (Murray et al., 2003; Saleh et al., 2009, uptake of host glucose by T. evansi for its matabolic activity (Maudlin et al., 2004; Hunt, 2010) and parasites-induced metabolic enzymes insufficiencies in the host (Enwezor and Sackey, 2005) despite good appetite resulting in progressive weight loss (emaciation) and stunted growth in chronic form of surra (Omer et al., 2007). Isometamidium treated group gained weight suggesting amelioration. The infected buparvaquone-treated and untreated groups experienced gentle but steady weight loss in chronic stage, perhaps due to persistence of pathogenic parasite in the animals without amelioration of signs resulting in un-abated metabolic failures and tissue damages (Enwezor and Sackey, 2005). Conclusion The clinical course of T. evansi infection in donkeys is partly acute but predominantly chronic. Some of the acute signs include transient mild pyrexia, hyperpnoea, tarchycadia, pale mucous membranes, lethargy, urticaria with alopecia, low mortality in acute stage, rarely observed signs are penile erection, intermittent diarrhea and rectal prolapse. The chronic signs include severe urticaria, progressive emaciation or stunted growth, anaemia, ventral abdominal oedema, recumbency and death. Early Isometamidium chloride treatment significantly ameliorates all clinical signs of surra in donkeys but buparvaquone does not significantly. Acknowledgement We are grateful to Prof. J.A. 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