Serodiagnosis of bovine trypanosomosis caused by non-tsetse transmitted Trypanosoma (Duttonella) vivax parasites using the soluble form of a Trypanozoon variant surface glycoprotein antigen

• p64 from T. equiperdum is a potent diagnostic tool for acute bovine trypanosomosis in Latin America.
• T. equiperdum p64 is a potent reagent to diagnose chronic bovine trypanosomosis in Latin America.
• A significant agreement was seen between assays using p64 and RoTat 1.2 VSG.
• A successful agglutination test was designed using p64 coupled to latex particles.

Previous studies have shown that a 64-kDa antigen (p64) that was purified from the Venezuelan TeAp-N/D1 isolate of Trypanosoma (Trypanozoon) equiperdum corresponds to the soluble form of its predominant variant surface glycoprotein (VSG), and exhibited cross-reactivity with Trypanosoma (Duttonella) vivax. The course of experimental acute infections of bovines with T. vivax were followed by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect enzyme-linked immunosorbent assay (ELISA). The value of p64 to diagnose bovine trypanosomosis was also examined using 350 sera from healthy and T. vivax-infected cows living in a trypanosomosis-endemic and enzootic stable area, and 48 sera obtained during a trypanosomosis outbreak. Serological assays showed that ∼70–80% of the infected sera contained anti-p64 antibodies, based on the comparative immunodetection of the T. equiperdum clarified antigenic fraction used as a reference test. In the absence of a gold standard, Bayesian analysis for multiple testing estimated a sensitivity and specificity of 71.6% and 98.8%, respectively, for the indirect ELISA using p64 as antigen. An apparent prevalence of 37.7% for bovine trypanosomosis infection was also estimated with a Bayesian approach when the p64 ELISA test was used. Employing blood from acute infected cows, the indirect ELISA response against p64 was contrasted with the microhematocrit centrifuge method and analyses by polymerase chain reaction (PCR) using specific primers targeting the inter-specific length variation of the internal transcribed spacer 1 region of the 18S ribosomal gene. The efficiency of p64 for the detection of anti-trypanosome antibodies in acute infected bovines was also corroborated serologically by comparing its response to that of the Indonesian Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2 VSG, which possesses high specificity and sensitivity. As expected, PCR was the best method to detect parasites and diagnose bovine trypanosomosis; however, a substantial level of concordance (Cohen’s κ = 0.667) was obtained when serological tests using p64 and RoTat 1.2 VSG were compared. Additionally, an agglutination assay was designed using p64 covalently coupled to carboxylate-modified latex microparticles, which was proven here to be suitable for a fast qualitative diagnosis of bovine trypanosomosis.