Hematological and biochemical changes in water buffalo calves (Bubalus bubalis) infected with Trypanosoma evansi

Four water buffalo calves (Bubalus bubalis) were each inoculated intravenously with 10(6)T. evansi (camel isolate) and the fifth calf kept as non-infected control. The blood and sera of all calves were examined every 4 days during the first month post-inoculation (pi) and then once weekly until the end of the experiment (88 days pi). They were examined for hematological and biochemical changes, liver and kidney function tests. Hemoglobin concentration (Hb%), packed cell volume (PCV) and red blood cell count were significantly decreased. Total leucocytic count, lymphocytes and monocytes showed significant increase. Liver function tests revealed significant elevation in the activity of lactate dehydrogenase enzyme (LDH), globulin, total biliruben and indirect biliruben while alkaline phosphatase enzyme showed significant decrease. Kidney function tests revealed significant decrease of both creatinine and urea.     

A comparative evaluation of parasitological tests and a PCR for Trypanosoma evansi diagnosis in experimentally infected water buffaloes

In this study five parasitological methods and a polymerase chain reaction (PCR) were compared for the diagnostic sensitivity for Trypanosoma evansi in experimentally infected water buffaloes over a period of 15 weeks. The combined estimates of sensitivity (CE(se)) of the PCR proved to be highest at 78.2%, closely followed by the mouse inoculation (MI), the micro-haematocrite centrifugation technique (MHCT) and the mini-anion-exchange centrifugation technique (MAECT) with CE(se) of, respectively, 74.0, 69.6 and 62.4%. The CE(se) of the buffy-coat technique (BCT) at 38.6% and the sodium dodecyl sulfate (SDS) clarification technique at 25.1% were considerably lower. PCR detected consistently all buffaloes infected from week 3 post-infection (PI) onwards. For MI this occurred after 5 weeks PI while for MHCT and MAECT these sustainable high levels were reached in the 7th week PI. BCT and SDS never detected all buffaloes infected. The influence of time and temperature on the viability of T. evansi in heparinized blood from water buffalo was also studied. In general we observed that the survival time tends to be longer when blood is kept at 4 degrees C. In samples kept in direct sunlight parasites became undetectable with the MHCT after 30min. After treatment of the water buffaloes with diminazene aceturate, the PCR signal disappeared within 24h.     

Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction.

The mechanically transmitted haemoflagellate, Trypanosoma evansi causes 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection of T. evansi is important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed from T. evansi repetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA of T. evansi derived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 microliters crude blood samples. Following experimental infection of calves with 5 x 10(5) T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle, Babesia bigemina and Theileria annulata were not amplified with the primers.     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

The influence of T. evansi infection on the immuno-responsiveness of experimentally infected water buffaloes.

In order to define the immuno-suppressive capacity of Trypanosoma evansi infections in buffaloes on the induction of immune responses against heterologous antigens, infected and non-infected buffaloes were vaccinated against Pasteurella multocida (haemorrhagic septicemia) and were simultaneously immunised with a control antigen, human serum albumin (HSA). Antibody responses against HSA were significantly reduced in T. evansi-infected animals, but no conclusive data were obtained on the antibody responses against P. multocida. Conversely, the local inflammatory response at the site of Pasteurella vaccination, as measured by increase in size, was significantly reduced in T. evansi-infected animals.These results indicate that the inductive capacity to mount humoral and cell-mediated immune responses against heterologous antigens is suppressed in T. evansi-infected animals. Consequently, T. evansi infection might interfere with the development of protective immunity upon heterologous vaccinations and could explain the poor protection of Pasteurella-vaccinated buffaloes in T. evansi-endemic areas of Vietnam.     

Trypanosoma evansi infections in Himalayan black bears (Selenarctos thibetanus).

The Asiatic or Himalayan black bear (Selenarctos thibetanus) is an endangered species. In South Asian countries, captive tamed Himalayan bears are commonly used by roving bear-charmers to entertain the people in rural and urban areas. In captivity, this species confronts several psychophysical traumas and communicable diseases, which are prevalent in other domestic species. The present report describes four cases of Trypanosoma evansi infection in live Himalayan charming bears, which originated from the Faisalabad and Jhang districts of Pakistan. The condition was characterized by pyrexia, accelerated pulse, tachypnea, depression, anemic mucous membranes, and ataxia (n = 3). Microscopic examination of peripheral blood films revealed moderate (n = 2) or high (n = 2) numbers of T. evansi. All four bears were treated twice at 3-day intervals with suramin sodium by using almost twice the dosage recommended for common domestic animals (10 mg/kg). The treated bears were found aparasitemic on repeat blood testing on days 5, 7, and 10 post-treatment. No adverse effects were noted and all four cases recovered in 3-7 days after completion of the second round of treatment. One bear died 8 days after the second treatment (day 11). This is the first report of T. evansi in bears.     

Trypanosoma evansi infection in cattle, buffaloes and horses in Indonesia

Cattle, buffaloes and horses in several areas of Indonesia were examined for evidence of infection with Trypanosoma evansi by the microhaematocrit centrifugation technique (MHCT) and an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to T. evansi. Evidence of infection was found in animals at each sampling site although differences were seen in prevalence rates between sites. Prevalence rates in buffalo were usually higher than in cattle in the same area while in horses they were much lower than in cattle or buffalo. An age-dependent prevalence rate was seen in buffalo and cattle with the highest rates seen in animals older than 2 years. These results concur with the view that T. evansi infection is widespread throughout most of the livestock-producing areas of Indonesia. The apparent lack of any obvious disease owing to T. evansi infection in the sampled animals suggests that a form of stability exists in most endemic areas which serves to ameliorate the effect of T. evansi infection and has an immunological basis linked to the parasite's limited antigenic diversity.     

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.     

Trypanosoma evansi experimental infection in the South American coati (Nasua nasua): clinical, parasitological and humoral immune response.

The course of Trypanosoma evansi infection in coatis (Carnivora, Procionidae) was followed for 262 days. Parasites were detected in all infected animals from day 2 post infection until the end of the study. No correlation between temperature and parasitemia was observed. Animals of the infected group demonstrated depression, weakness, lethargy and pale mucous membranes. Indirect fluorescent antibody tests detected anti-T. evansi antibodies within 7 to 14 days post infection and showed high levels until the end of the experimental period. The persistent parasitemia in coati and their relative tolerance to clinical signs suggested that this species develops a chronic disease and plays an important role in the epidemiology of trypanosomosis due to T. evansi in enzootic regions.     

Animal-level risk factors for Trypanosoma evansi infection in camels in eastern and central parts of Kenya

Point prevalences and animal-level risk factors for Trypanosoma evansi infection were investigated in a cross-sectional study that involved 2227 camels from eastern and central parts of Kenya. The screening tests used were haematocrit centrifugation technique (HCT), mouse inoculation and latex agglutination (Suratex). All camels were screened with HCT, while 396 and 961 of them were, in addition, screened with mouse inoculation and Suratex tests, respectively. Parasitological and Suratex test results were used in parallel to determine the number of camels exposed to T. evansi infections. Statistical analyses were conducted using Statistical Analysis Systems. Parasitological and Suratex test results in parallel were dependent variables in multivariable logistic regression models that determined risk factors for T. evansi infection. Herd-level clustering was corrected with general estimation equations. The prevalences were 2.3% and 19.6%, using parasitological and Suratex tests, respectively, and 21.7% when both tests were used in parallel. There was a positive association between the screening tests (McNemar's test = 104.8, P = 0.001) although the strength of association was low (Kappa = 0.2; 95% CI: 0.1-0.3). Before accounting for herd-level clustering, dry season (OR = 1.5; 95% CI: 1.0, 2.1) and nomadic pastoralism (OR = 1.8; 95% CI: 1.1, 3.2) were associated with increased odds of a camel being exposed to T. evansi infection compared to wet season and ranching, respectively. Following this correction, only nomadic pastoralism was significantly associated (OR = 3.1; 95% CI = 1.0, 14.4) with T. evansi infection compared to ranching. It is concluded that camels managed under nomadic pastoralism had higher risk of being exposed to T. evansi infections than camels from ranching systems of management.     

Serological responses to Neospora caninum in experimentally and naturally infected water buffaloes (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important natural host for Neospora caninum. Serologic responses to N. caninum were studied in experimentally and naturally infected water buffaloes in Brazil. Antibodies were assayed by the indirect fluorescent antibody test (IFAT) using a cut off value of 1:25. Six buffaloes were each inoculated subcutaneously with 5 x 10(6) live culture-derived tachyzoites of the cattle Illinois strain of N. caninum, and two calves were kept as uninoculated controls. Post-inoculation (p.i.) blood samples were collected weekly for 8 weeks and then monthly until 1 year p.i. All inoculated buffaloes developed IFAT titers of 1:100 or more between 7 and 11 days p.i. and the titers remained elevated until 7 weeks p.i. Antibody titers peaked to 1:1600 in 1, 1:800 in 3 and 1:400 in 2, usually by 3 weeks p.i. Antibody titers declined to 1:25 or 1:50 in all the six buffaloes by 12 months p.i. IFAT titers to N. caninum remained at an undetectable level (< 1:25) in both control uninoculated buffaloes. To follow the dynamics of N. caninum antibodies, sera from 29 buffaloes and their calves were collected for 1 year and assayed for N. caninum antibodies; 23 of 29 calves were seropositive (IFAT of 1:100 or more) at 1-2 day of age. Of these 23 calves, 17 remained seropositive during the study, while six became seronegative at four (two calves), six (one calf) seven (two calves) and eight (one calf) months of age. These findings suggest a high rate of neonatal transmission of N. caninum in buffaloes.     

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.     

Enzootiology of Trypanosoma evansi in Pantanal, Brazil

In order to better understand the enzootiology of trypanosomiasis caused by Trypanosoma evansi in the Brazilian Pantanal we examined domestic and wild mammals by microhematocrit centrifuge technique (MHCT), immunofluorescence antibody test (IFAT) and polymerase chain reaction (PCR). T. evansi infection was detected in all species sampled with exception of the sheep and the feral pig. High parasitemias were observed in capybaras (5/24), coatis (18/115), horses (31/321) and dogs (3/112). Among these species, only the capybaras did not develop anemia. Low parasitemias, only detected by PCR, were found in buffaloes (18/43), bovines (29/331), marsupials (1/4), small rodents (14/67), bats (7/18), and one armadillo (1/8). The highest prevalence of T. evansi infection was recorded in horses (73%), although no neurological signs in infected horses were observed. Diagnosis through standard parasitological tests and IFAT should be used with caution since they may overlook comprovedly infected horses. The relationship between ranch management and T. evansi infection in horse was investigated. The importance of other transmission mechanisms apart from the tabanids and reservoir hosts are discussed.     

Characterization of Trypanosoma (Trypanozoon) evansi from dromedary camels in Kuwait by isoenzyme electrophoresis

Blood from 115 camels in Kuwait was examined for blood parasites. Two camels of a local herd (1.7%) were found to be infected with Trypanosoma (Trypanozoon) evansi and three camels (2.6%) with microfilarial nematodes. The Trypanosoma stocks isolated from these two camels were screened for isoenzyme patterns of 10 enzymes using thin-layer starch-gel electrophoresis. The results revealed that these two stocks were identical to camel stocks of T. evansi from certain countries in Africa, as well as to two stocks isolated from dogs in Kuwait. This is the first record of Trypanosoma (Trypanozoon) evansi isolates and microfilariae from camels in Kuwait.     

伊氏锥虫在小鼠体内抗原变异规律及免疫保护试验

为了探索伊氏锥虫抗原的变异规律及其用于免疫预防的可能性，首先对伊氏锥虫安微株单虫克隆2个早期变异体ShTatl.1、ShTat1.2采用蛋白酶抑制剂TLCK处理，分离纯化这2种变异体的VSG，用ShTat1.1 VSG免疫KM小鼠，ShTat1.1锥虫攻击免疫鼠后6d分离锥虫，经间接免疫荧光试验（IFT）和班点酶标记试验（Dot-EIA)鉴定为ShTat1.2，说明同一克隆锥虫感染兔，小鼠第1次发生抗原变异后产生的变异体相同。依据这一规律，设计了免疫预防保护试验，试验小鼠分3组：一组为未免疫对照组，一组为ShTat1.1VSG单一抗原免疫组，另一组为ShTat1.1 VSG,ShTat1.2 VSG混合免疫抗原免疫组，各组均以ShTat1.1锥虫攻击，结果ShTat1.1 VSG ShTat1.2VSG免疫组小鼠全部获得保护，单用ShTat1.1 VSG免疫组小鼠和未免疫小鼠血中全部出虫、死亡，前者比后者出虫时间、存活时间延长。提示运用伊氏锥虫抗原变异这一规律设计的这种复合抗原，能激发宿主克服虫体抗原变异对免疫保护的干扰。     

水牛伊氏锥虫间接ELISA检测方法的建立

利用伊氏锥虫的优势变异型虫体作为可溶性抗原,通过试验研究和条件优化,建立了一种水牛伊氏锥虫间接ELISA方法.测试结果表明,该法灵敏度高,特异性强,对实验感染样本的检测获得了较好的效果,为一种有效的ELIAS方法,为广西水牛伊氏锥虫病的诊断奠定了技术基础.     

Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya

Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (kappa = 0.12), PCR (kappa = 0.11) and MHCT (kappa = 0.05). However, there was a higher agreement between farmers' classification of disease with the PCR test (kappa = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 +/- 0.5) and from infected calves (19.5 +/- 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable "pen-side" diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.     

Trypanosoma evansi infection in bovine and buffalo calves in Indonesia.

Fifteen bovine and 11 buffalo calves born on different farms in a Trypanosoma evansi-endemic area of West Java were monitored for the presence of T. evansi and T. evansi antibody at monthly intervals until they were 12 months of age. Fifty percent of the bovine and 83% of the buffalo calves sampled in the first month of life were antibody positive. This antibody was considered to be of colostral origin. Antibody developing later in life persisted for up to 12 months and was considered to have arisen in response to T. evansi infection. No protective function could be ascribed to the colostral antibody.     

Acetylcholinesterase activity and lipid peroxidation in the brain and spinal cord of rats infected with Trypanosoma evansi

Neurological and locomotor clinical signs are described in animals infected with Trypanosoma evansi. These disturbances may be related to changes in the amount of acetylcholine (neurotransmitter) in the synaptic cleft. Therefore, changes in acetylcholinesterase (AChE) activity and lipid peroxidation in brain and spinal cord of T. evansi-infected rats were investigated. Each rat was intraperitoneally infected with 10(6) trypomastigotes kept in fresh (group A; n=13) and cryopreserved blood (group B; n=13). Thirteen served as uninfected (not-infected; group C). In days 4 and 30 post-infection (PI) the rats were anesthetized and subsequently decapitated to obtain the brain and the spinal cord (between vertebrae L1 and S2). The brain was removed and dissected (cerebellum, cerebral cortex, striatum and hippocampus) to measure the activity of AChE and lipid peroxidation, determined by TBARS levels. To verify if T. evansi was present in the central nervous system (CNS), brain structures of three rats of each group were processed by PCR T. evansi-specific. AChE activity was significantly increased in all brain structures and decrease in spinal cord in infected rats in 4 PI (P<0.05). The levels of TBARS were decreased in the brain structures, differently from spinal cord, which showed increased lipid peroxidation in 4 PI. The AChE activity in striatum, cerebral cortex, hippocampus and spinal cord reduced concomitantly with the increase of the enzyme in cerebellum of the infected rats (P<0.05), and the TBARS levels increased in cerebellum, striatum and spinal cord of infected rats compared to non-infected animals in 30 PI. The PCR was positive for T. evansi in all structures of the brain, confirming the presence of the parasite in the CNS. Based on the results, we conclude that the changes in AChE activity and lipid peroxidation in the CNS are induced by infection with T. evansi, suggesting that the parasite interferes with the cholinergic neurotransmission in this experimental condition.     

First outbreak of Trypanosoma evansi in camels in metropolitan France

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.