Evaluation of diagnostic tests for Trypanosoma evansi in experimentally infected pigs and subsequent use in field surveys in north Vietnam and Thailand.

This study is concerned with the evaluation of established diagnostic tests for diagnosis of Trypanosoma evansi in pigs. The immune trypanolysis test (TL), card agglutination test (CATT), latex agglutination test (LATEX), enzyme-linked immunosorbent assay (ELISA), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI) tests were initially evaluated in experimentally infected fattening pigs. All infected pigs were confirmed parasitologically positive with both MHCT and MI. Results of the serological assays indicated that the TL could be a reference test for the presence of RoTat 1.2 antibodies in pigs. The results of the CATT and LATEX were inconsistent with the TL while the ELISA results correlated with the TL results. The four serological assays were subsequently used in two field surveys in Vietnam and Thailand. Results of the two agglutination assays (CATT and LATEX) were not consistent and did not correlate with TL results. The ELISA at percentage positivity of 22 appeared to have good ability to discriminate between seropositive and seronegative animals. Of the 437 samples collected at smallholder pig premises in northern Vietnam, no positive pigs were detected with the TL test. In Thailand, 77 samples were collected from five farrowing farms with a history of surra. Two parasitologically positive sows were found and on each farm seropositive sows were detected.

The effect of Trypanosoma evansi infection on pig performance and vaccination against classical swine fever.

Although Trypanosoma evansi is not considered as an important pathogen in pigs, it may interfere with other pathogens or vaccinations by its immunosuppressive nature. In order to determine whether T. evansi alters pig performance and induces immunosuppression in pigs, induction of immune responses by vaccination against classical swine fever (CSF) and by immunization with a control antigen, human serum albumin (HSA), was assessed in T. evansi-infected and non-infected animals. Although T. evansi infection did not have a significant influence on growth performance, feed conversion or PCV, antibody responses against both the test antigen HSA and the CSF vaccine were significantly reduced in T. evansi-infected animals as compared to uninfected animals. Moreover, the reduced response against the CSF vaccine appears to be accompanied by a less well-developed protection against CSF with higher fever responses and leukopenia. This immunosuppression might explain the accounts of poor protection of CSF-vaccinated pigs reported in T. evansi-endemic areas of Vietnam, and suggests that prior treatments with trypanocidal drugs to improve the efficacy of CSF vaccination, may be justified.

Evaluation of whole fresh blood and dried blood on filter paper discs in serological tests for Trypanosoma evansi in experimentally infected water buffaloes.

In this study we investigated if whole blood could substitute for serum in the direct card agglutination test (CATT/Trypansosoma evansi) and the indirect card agglutination test (LATEX/T. evansi) for the sero-diagnosis of T. evansi in buffaloes. Likewise blood spots on filter paper were compared with sera for use in the indirect enzyme-linked immunosorbent assay/T. evansi (ELISA) and immunotrypanolysis test (T.L./T. evansi). Samples were collected weekly from experimentally T. evansi infected- and non-infected water buffaloes. To estimate test agreement between serum and respectively whole fresh blood and dried blood spots on filterpaper of the tests, kappa values with 95% confidence intervals were calculated, 0.75+/-0.11 for the CATT/T. evansi; 0.80+/-0.11 for the ELISA/T. evansi; 0.84+/-0.11 for the LATEX/T. evansi and 0.93+/-0.11 for the T.L./T. evansi. In addition kappa values with 95% confidence intervals were computed to assess agreement between results obtained in the reference T.L./T. evansi test and those obtained in the other assays; 0.70+/-0.10 for the CATT-Serum; 0.75+/-0.11 for the LATEX-Blood; 0.77+/-0.11 for the LATEX-Serum; 0.81+/-0.10 for the CATT-Blood; 0.81+/-0.11 for the ELISA-Serum and 0.84+/-0.11 for the ELISA-Confetti. Based on the high kappa values as calculated, we conclude that serum can be replaced by fresh whole blood for the agglutination assays or blood on filter paper for the ELISA/T. evansi and T.L./T. evansi.

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.

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.

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.