Download Clinical Presentation and Pathology of Savannah isolate of

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Clinical Presentation and Pathology of Savannah isolate of Trypanosoma vivax in
experimentally infected Yankasa Sheep
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C.I. Ogbaje1*, I. A. Lawal2, J. O. Ajanusi2
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Department of Veterinary Parasitology and Entomology, College of Veterinary Medicine,
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University of Agriculture, Makurdi1.
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Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine,
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Ahmadu Bello University, Zaria2.
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*Corresponding author:
C. I. Ogbaje, Federal University of Agriculture, Makurdi, College of Veterinary Medicine,
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Department of Veterinary Parasitology and Entomology, PMB 2373, Makurdi, Benue State,
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Tel.: +234 803 529 5570; e-mail address: [email protected];
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Key words: Trypanosoma, Infection, Congestion, Parasite, Hyperemia, Necrosis, Pathology
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Abstract
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A study was conducted to assess the clinical presentation and pathology associated with
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experimental infection of Yankasa sheep with Trypanosoma vivax strain isolated from the field
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at Guinea Savanna region of Nigeria. Eighteen adult Yankasa sheep of both sexes were used.
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They were screened for haemo, endo, and ectoparasites, treated and conditioned for 2 weeks.
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They were divided into 3 groups (A, B & C) of 6 animals each. Two groups (A and B) were
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infected with 2 ml of infected blood containing approximately 2.0×106 of T. vivax. Group A were
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left untreated and monitored for 7 weeks while group B was treated at the peak of parasitaemia
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with a trypanocide. Group C served as uninfected control. The clinical signs observed were;
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anorexia, rough hair coat, ocular discharge, pale mucous membrane, diarrhoea, severe weakness,
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depression, oedema of the eye lids, teeth grinding, emaciation and torticollis-like central nervous
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system disorder. Pathological lesions seen were serous atrophy of pericardial fat, focal
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haemorrhages of diaphragmatic lobe of the right lung, hydropericardium (17 ml), diffuse pin
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point haemorrhages of the liver and kidney surfaces and empty gall bladder. Microscopically, the
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brain revealed areas of neuronal degeneration, spleen showed areas of haemosiderosis in the red
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and white pulps. The Kidney revealed necrotic renal tubules and glomerular degeneration, lung
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was congested with areas of hemorrhages and thickened alveoli wall. There was degeneration of
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the skeletal muscles with absence of sperm cells in the epididymis.
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Introduction
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Trypanosoma vivax accounts for up to half of total prevalence of African animal trypanosomosis
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in West Africa especially in Nigeria where it is considered to be the predominant pathogen for
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domestic animals (Osorio et al., 2008). This could be ascribed to the mechanical transmission or
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shorter development cycle in the anterior part of the tsetse fly (Sinshaw et al., 2006; Batista et
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al., 2012). The trypanosome readily persists in areas free of tsetse flies (for example, in Central
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and South America and in the Caribbean), where it is transmitted mechanically by biting flies or
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contaminated needles, syringes, and surgical instruments, (Batista et al., 2012). Trypanosoma
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vivax infects large variety of domestic and wild animals (Fikru et al., 2012).
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Anaemia which is considered to be the cardinal clinical sign observed in AAT (Cherenet et al.,
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2006; Biryomumaisho et al., 2007) is not specific for only trypanosomosis since anaemia is seen
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in all the diseases caused by blood parasites and some gastrointestinal helminthes. The individual
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host, breed susceptibility and the species of trypanosomes have utmost effect on the severity of
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the clinical response (Fikru et al., 2012). Other factors such as poor nutrition and concurrent
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infection, plays a role in the disease process. Trypanosoma vivax has been reported to have a
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variable incubation period and also considered to be less virulent for cattle than Trypanosoma
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congolense, but mortality rates of over 50 percent can occur (Njiru et al., 2011).
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Control of African animal trypanosomosis is dependent largely on vector control and use of
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drugs (chemotherapy and chemoprophylaxis), (Achenef and Birasa, 2013). In sub-Saharan
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Africa, sheep provide almost 30% of the meat consumed and around 16% of the milk produced
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(McClintock, 1983).
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In Nigeria, Sheep contributes about 50% of the total domestic meat production with a population
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of about 22.1 million (Opasina and David-West, 1984). They thrive in a wide variety of
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environments in the tropics and sub-tropics and they require little capital since they can be
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completely maintained on pastures and agricultural waste products (Francis, 1987).
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Trypanosomosis is becoming of increasing clinical importance in small ruminants in Nigeria
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because of the heavy economic loss from the effect of the disease (Omotainse and Anosa, 2009).
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This work was designed to carefully study the clinical manifestation and pathology produced by
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experimental infections of Trypanosoma vivax strain of Southern Guinea Savannah of Nigeria in
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Yankasa Sheep, since it has been reported that clinical response to the disease is dependent on
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the species and strain of the trypanosome and the breed of the affected animal. The outcome of
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this study will assist field Veterinarians to carry out effective treatment in areas where laboratory
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facilities are not available.
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Materials and Methods
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Ethical permission and Study Area
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Ethical approval was obtained from the Institutional Ethical Unit of the College of Veterinary
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Medicine of the Federal University of Agricultural, Makurdi, Nigeria. The Trypanosoma vivax
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used in the study was isolated from white Fulani cattle in Makurdi, Benue State, Nigeria.
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Makurdi, the administrative headquarter of Benue state, lies approximately between latitude
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7°441N and longitude 8°541E. The town is located along the coast of the River Benue (the
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second biggest River in Nigeria). The climatic condition in Makurdi is influenced by two air
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masses: the warm, moist south westerly air mass, and the warm, dry northeasterly air mass. The
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southwesterly air mass is a rain – bearing wind that brings about rainfall from the months of May
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to October. The dry northeasterly air mass blows over the region from November to April,
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thereby bringing about seasonal drought. The mean annual rainfall in Makurdi is about 1,290
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mm (Akintola, 1986). Temperature in Makurdi is however, generally high throughout the year,
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with February and March as the hottest months. Temperature in Makurdi varies from a daily
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maximum of 40°C and an annual mean minimum of 22.3°C (NBS, 2010).
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Parasite Identification
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The Trypanosoma vivax was confirmed in the laboratory by wet mount and thin blood smear
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examinations and molecular test.
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The molecular test was conducted through the used of multiplex and Trypanosoma vivax species-
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specific
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5’GCGTTCAAAGATTGGGCAATG-3’ R: 5’-CGCCCGAAAGTTCACC-3’) and Trypanosoma
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vivax species-specific (TVW A- d: 5’-GTG CTC CAT GTG CCA CGT TG-3’ TVW B- d: 5’-
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CAT ATG GTC TGG GAG CGG GT-3’) PCR were supplied by Inqaba Biotechnical Industries
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LTD, Hatfield- Pretoria, South Africa.
PCR.
The
primers
for
the
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multiplex
Trypanosoma
(F:
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The multiplex Trypanosoma PCR was performed as described by Desquesenes et al. (2001) and
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Trypanosoma vivax species-specific PCR was conducted as described by Masiga et al. (1996).
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The multiplex Trypanosoma PCR was conducted to rule out the possibility of mixed infections in
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the sample whereas the Trypanosoma vivax species-specific PCR was performed on the isolate
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for species identification (i.e. confirmatory test).
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Source and Management of Experimental sheep
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Eighteen Yankasa sheep, aged between 2-3 years and of both sexes were purchased from an
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open market at Karfur in Kastina State (Northern Nigeria). On arrival, the animals were
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screened for ecto, endo and haemo-parasites as described by Soulsby (1986) and Woo (1971)
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respectively.
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All the experimental sheep were dewormed using Albendazole® at the dose rate of 7.5 mg/kg.
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Ecto-parasites infestations were treated and controlled with cypermecthrin pour-on preparation
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and asuntol® spray (coumaphos). Those found with Anaplasma infections were treated with
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oxytetracycline long acting at the dose rate of 20 mg/kg body weight. Those infected with
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coccidia were treated with Amprolium for 5 days according to the manufacturer
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recommendation. All the sheep were also vaccinated each with 1 ml subcutaneous injection of
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monoclonal pestes de petit ruminantes (PPR) vaccine against PPR because of the endemicity of
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the disease in the experimental site. They were then introduced into arthropod-free pens of the
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Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine,
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Ahmadu Bello University, Zaria and pre-conditioned for two weeks before the commencement
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of the experiment.
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The animals were fed on cotton seed cake mixed with maize offal, ground nut husk, Digiteria
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hay, then salt lick and clean drinking water were provided ad libitum. The feed were sourced
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from National Agricultural Research Institute (NAPRI), Shika, Zaria.
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Animal Identification and Grouping
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The experimental animals were ear tagged and randomly divided into 3 groups (A, B and C) of 6
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animals per group. Base line data were obtained from each of the animals in all the groups for a
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period of 1 week prior to infection. Each of the sheep in groups A and B was infected through
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intravenous inoculation of 2 ml containing approximately 2.0×106 Trypanosoma vivax as
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quantified using the improved Naubauer haemocytometer (Petana, 1963). Group C served as
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uninfected control. Group A animals were treated with trypanocide (Diminazene aceturate at the
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dose rate of 3.5 mg/kg body weight) when the parasitaemia was massive (++++).
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Gross and Histopathological Examination
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At day 56 post- infection (pi), some of the sheep in the groups (A, B, and C) were picked at
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random, sacrificed and necropsied. Samples of the spleen, lungs, brain, liver, heart, kidney,
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testicle, ovary, uterus, adrenal gland and pancreas were taken immersed in phosphate buffered
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formol saline, dehydrated in graded concentration of absolute alcohol and xylene, and embedded
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in paraffin. Thin sections (5μ) mounted on a clean glass slides were stained routinely for
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histopathological examination by light microscopy using haematoxylin and eosin (H&E).
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Results
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The results of the multiplex PCR and Trypanosoma vivax species-specific PCR revealed that the
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sample was pure Trypanosoma vivax isolates since it was negative to multiplex primers PCR for
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other trypanosomes, but positive for Trypanosoma vivax which yielded an expected amplicon of
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175 bp. These two tests confirmed that the isolate used in the study was Trypanosoma vivax.
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Prepatent Period of six (6) days was recorded for all the infected sheep but with different levels
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of parasitaemia.
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The following clinical manifestations were observed following the experimental infection of the
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sheep with Trypanosoma vivax; anorexia, rough hair coat, ocular discharge, pale mucous
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membrane, diarrhoea, severe weakness, depression, oedema of the eye lids, teeth grinding,
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emaciation and torticollis- like central nervous system (CNS) disorder.
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The gross pathological lesions observed in the infected untreated animals were serous atrophy of
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pericardial fat, foci haemorrhages on the diaphragmatic lobe of the right lung, hydropericardium
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(17 ml), diffuse pin point haemorrages of the liver (Plate I), kidney and empty gall bladder.
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Those infected and treated 10 days post infection revealed serous atrophy of pericardial fat,
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congestion of the entire right apical lobe and parts of the diaphragmatic lobe and focal areas of
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haemorrhages. There were no pathological changes seen in the uninfected control sheep.
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Histopathology section of the brain of the infected untreated Yankasa sheep revealed areas of
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neuronal degeneration (Plate II), whereas the spleen showed areas of haemosiderosis in the red
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and white pulps. The Kidney of the same animal revealed necrotic renal tubules and glomerular
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degeneration (Plate III). The Lung was congested with areas of haemorrhages and thickened
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alveoli membranes (pneumonia), (Plate IV). There was atrophy and degeneration of the skeletal
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muscles with absence of sperm cells in the epididymis (Plate V).
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Photomicrograph of the lungs of the infected-treated sheep revealed thickened alveolar
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membranes (Pneumonia) and uterine necrosis. There were focal areas of tubular and glomerular
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necrosis in the kidneys. The spleen revealed haemosiderosis of the red and white pulps.
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Discussion
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In this study, gross pathological changes were observed in infected untreated sheep with severe
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clinical manifestations. This observation is a confirmation of previous report by other authors
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(Taylor and Authie, 2007; Osorio et al., 2008) that pathology in tissues is associated with the
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relative ability of Trypanosoma vivax to invade extravascular spaces and organs. The more
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pathogenic the strain of parasite used, the severe the clinical manifestations and the pathological
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lesions presented and verse versa. This explains why the observed lesions were more severe in
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organs of the infected untreated sheep.
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Viscera organs were variously affected because of the strategic physiological roles.
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Consequently, liver was more affected grossly because of its important function as the major site
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of erythrocytes and parasites clearance from the body system (Losos and Ikede, 1972). These
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were therefore common finding in the infected untreated animals in this study.
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Histologically also, the liver in the infected untreated animals revealed hepatic necrosis,
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degeneration and congested sinuses. The hepatic necrosis may have resulted from the insufficient
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blood supply to the liver due to anaemia and partial blockage of liver vessels by the parasites and
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their products at a certain time. The respiratory involvements were exhibited in all the humane
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sacrificed infected animals by the thickened alveolar membranes, congested and haemorrhagic
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lungs. The associated pneumonia may be due to secondary bacterial infections as a result of
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immunosuppression commonly seen in trypanosomosis (Taylor, 1998).
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The histological sections of infected female’s uterus revealed uterine necrosis. This may be one
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of the reasons for abortion that have been widely reported in the disease by other researchers
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(Maikaje et al., 1991; Bawa et al., 2000; Akinwale et al., 2006; Batista et al,. 2007). Section of
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the epididymis from infected males revealed normal epithelium that was devoid of spermatozoa.
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This finding is not in agreement with the earlier report that trypanosomosis causes epididymitis
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(Batista et al., 2007; Bezerra et al., 2008).
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In the kidney of the infected untreated animals, there were foci areas of necrosis in the renal
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tubules and glomeruli, these findings are in good agreement with the early report by Takeet and
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Fagbemi, (2009). Torticolis-like form of nervous signs were observed in two infected and
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untreated sheep which had areas of neuronal degeneration in the brain; nervous in coordination
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have been reported by Batista et al. (2007) from Brazil. This lesion may be due to the direct
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effect of the parasites on the brain or as a result of hypoxia from anaemia or partial occlusion of
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the brain vessels by the parasites/or their products.
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In the pancreas, there were areas of necrosis in the glands; similar inflammatory changes in the
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glands of the body have been reported by Losos and Ikede, (1972). There was also necrosis of
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the adrenal glands of the infected, untreated animals. Enlarged adrenal glands, characterized by
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inflammatory changes have been reported by Ogwu et al. (1992). Such inflammatory reaction if
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prolonged, can lead to necrosis, so this finding is in agreement with the former report.
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The haemosiderin and follicular depletion seen in the spleen of the infected animals may have
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been caused by hemolysis and anaemia which are associated with the disease. Similar
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observations have been reported by Taiwo et al. (2003), Fatihu et al. (2008), Takeet and
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Fagbemi, (2009). The mild atrophy of the skeletal and cardiac muscles associated with the
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infection may be due to anorexia and poor feed utilization by the infected animals. Similar
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muscle atrophy has been reported by Fatihu et al. (2008) in bovine trypanosomosis. This muscles
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atrophy may be one of the reasons why the disease is being referred to as wasting disease.
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In conclusion, the study shows that the Trypanosoma vivax strains used has high affinity for
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both vascular and extravascular organs and is highly pathogenic to the breed of sheep (Yankasa
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sheep) used.
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Acknowledgements: We sincerely appreciate TETFUND for the funding of this research and
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the contributions of Drs.M. Bisalla, J. Sambo; J. Natala, O.O.Okubanjo, R. Ofukwu, Mr. Bale,
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Professors R. Ezeokonkwo, N. U. Useh (for editing the manuscript) and staff of DNA laboratory
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of National Veterinary Research Institute, Vom and Biotechnology laboratory of Ahmadu Bello
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University, Zaria, Nigeria.
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diagnosis of