D. Verloo

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.

General expression of RoTat 1.2 variable antigen type in Trypanosoma evansi isolates from different origin

The variable surface glycoprotein of Trypanosoma evansi RoTat 1.2 variable antigen type (VAT) is used as an antigen in different antibody detection assays for T. evansi. To obtain more information on the predominant character of RoTat 1.2 and its diagnostic potential in antibody detection tests, we checked its expression in 10 different T. evansi stocks and clones from different parts of the world. Cryostabilates were injected into mice and the trypanosomes of the first peak parasitaemia were screened for the presence of RoTat 1.2 by VAT specific immunofluorescence. To monitor the appearance of RoTat 1.2 specific antibodies during infection, rabbits were infected and serologically tested at different time intervals with VAT specific immune trypanolysis, CATT/T. evansi, LATEX/T. evansi and ELISA/T. evansi. Test results confirm the predominant character of RoTat 1.2.

Towards a new reference test for surra in camels.

ABSTRACT Current serological diagnosis of Trypanosoma evansi infection in camels is based on the native variable antigen type RoTat 1.2. The goal of this study was to develop a novel serological diagnostic test based on a nonvariable protein and freed from the use of rats or mice for its production. An enzyme-linked immunosorbent assay using a recombinant extracellular domain of invariant surface glycoprotein 75 (ELISA/rISG75) was developed and tested on a collection of 184 camel sera. The results were compared to those obtained from three established antibody detection tests based on variable surface glycoprotein RoTat 1.2: an ELISA for T. evansi (ELISA/T. evansi), a card agglutination test for trypanosomiasis (CATT/T. evansi), and an immune trypanolysis (TL) assay. The ELISA/rISG75 and the ELISA/T. evansi showed a sensitivity of 94.6% (95% confidence interval [CI], 87.8 to 98.2%, at 19% positivity cutoff value) and 98.9% (95% CI, 94.1 to 99.8, at 12% positivity cutoff value), respectively. The ELISA/rISG75 had 100% specificity (CI, 95.9 to 100%), while the ELISA/T. evansi showed 98.9% specificity (CI, 95.9 to 100%). The ELISA/rISG75 demonstrated an almost perfect agreement with the TL assay, the CATT/T. evansi, and the ELISA/T. evansi, with kappa scores of at least 0.94. The ELISA/rISG75, having a performance comparable to that of the gold standard (the TL assay) and being independent of antigenic variation, may become a new reference test for surra in camels. It opens avenues for the diagnosis of T. evansi infections in other hosts as well as for the development of a pan-Trypanozoon test for detection of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum.

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.

Comparative in vitro isolation of Trypanosoma theileri from cattle in Belgium

Ten blood samples randomly collected from cows on a farm nearby Antwerp, Belgium, were inoculated into KIVI culture medium (Kit for In Vitro Isolation of trypanosomes) and RPMI 10%+feeder medium. Within 3 weeks of incubation all KIVI cultures and four RPMI 10%+feeder revealed presence of Trypanosoma theileri. Some practical implications regarding the use of KIVI for isolation of pathogenic African trypanosomes from cattle and other Bovidae are discussed.

Evaluation of an EDTA version of CATT/Trypanosoma bruceigambiense for serological screening of human blood samples

CATT/Trypanosoma brucei gambiense, a direct card agglutination test designed for field surveys on human African trypanosomosis, is currently used with freshly collected heparinized blood samples. When testing serum samples, it has been observed earlier that, at lower sample dilutions, a complement-mediated inhibition phenomenon may cause false negative test results. This can be avoided by adding an anticomplementary agent such as di-sodium ethylenediaminetetraacetate dihydrate (EDTA) to the reaction. As the sensitivity of the blood assay might be improved in the same way, this possibility has been examined under both laboratory and field conditions, by adding EDTA to the test buffer or, as an anticoagulant, to the blood samples. The CATT-EDTA versions proved up to 7% more sensitive but also 1-2% less specific than the current test. CATT buffer supplemented with EDTA remained stable for at least 2 years at +45 degrees C.

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.

Validation of a PCR-oligochromatography test for detection of Trypanozoon parasites in a multicenter collaborative trial

ABSTRACT A PCR-oligochromatography test for diagnosis of human and animal trypanosomiasis was evaluated through a multicenter ring trial with six laboratories testing a set of 21 blinded samples, resulting in qualitative data (positive or negative). Results showed an intralaboratory repeatability (accordance) of 88.7% (credible interval [CI], 84.4 to 92.5%) and an interlaboratory repeatability (concordance) of 88.1% (CI, 84.3 to 92.3%).

Expression of RoTat 1.2 cross-reactive variable antigen type in Trypanosoma evansi and T. equiperdum.

Abstract: The variable antigen type (VAT) RoTat 1.2 has been cloned from a T. evansi strain, isolated in 1982 from a water buffalo in Indonesia. All T. evansi isolates hitherto tested express this VAT. In a study on the differential diagnosis of T. equiperdum and T. evansi in horses, we investigated serological evidence for the expression of RoTat 1.2 in 11 T. evansi and six T. equiperdum populations originating from Asia, Europe, Africa, and the Americas. Preinfection sera and sera of days 7, 14, 25, and 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 experiment, all rabbits infected with T. evansi became positive in the three serological tests. Five out of six rabbits infected with T. equiperdum also became positive in the three tests. Only one T. equiperdum strain (the OVI strain from South Africa) did not induce the production of antibodies reactive with RoTat 1.2 and thus might not contain or express a VSG that shares epitopes similar to those on the RoTat 1.2 VSG. The data lead to the conclusion that T. equiperdum can express VSGs containing epitopes serologically similar to those in the T. evansi RoTat 1.2 VAT. This explains, in part, why the antibody detection tests based on Ro Tat 1.2 VSG cannot reliably distinguish between the infections caused by T. evansi and those caused by T. equiperdum. There are no data that contradict the possibility that the putative T. equiperdum strains, which express VSGs with epitopes similar to those on RoTat 1.2, are actually T. evansi.