Contribuições para o diagnóstico sorológico da tripanossomíase bovina no estado da Bahia

Abstract

Trypanosoma vivax is a hemoprotozoan responsible for trypanosomiasis in cattle, a disease that causes productive and reproductive losses, resulting in economic damage in Brazil. There are few reports of the disease in Bahia, despite frequent outbreaks, possibly due to diagnostic limitations, as clinical diagnosis is nonspecific, making it essential to associate it with laboratory testing. The Indirect Immunofluorescence Assay (IFA) requires the separation of parasites from blood cells in a laborious and costly purification process, which is the main limitation of the technique. Furthermore, the absence of T. vivax in vitro culture necessitates the use of animals for antigen production. This study aimed to standardize and validate IFA for the detection of anti-T. vivax antibodies using blood smears from sheep experimentally infected with T. vivax. Immunosuppression protocols and clinical and hematological alterations were described to support future research on antigen production. For this purpose, six adult sheep were intravenously infected with T. vivax trypomastigotes (Ipameri-GO strain, isolated from cattle) following different immunosuppression protocols. Once the animals reached peak parasitemia, blood samples were collected in tubes containing EDTA, heparin, and sodium citrate. Subsequently, thick blood smears were prepared and fixed with either acetone or methanol. For comparison, antigen purification using Percoll at different centrifugation speeds was performed, and recovered parasites were quantified using Brener’s method. To validate the technique, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated using bovine serum samples with known positive and negative status. Additionally, the serological status of 49 bovine samples from farms experiencing disease outbreaks was investigated. Cross-reactivity of IFA with other hemoparasites was also assessed. The best protocol involved blood smears from sodium citrate treated samples fixed with acetone. The technique demonstrated a sensitivity of 91.48%, specificity of 96.87%, and accuracy of 93.67%, with no cross-reactions observed with Babesia bovis, Babesia bigemina, Anaplasma marginale, Neospora caninum, Toxoplasma gondii, or Leishmania infantum. The variation in antigen yield with Percoll purification at different centrifugation speeds was only 15.25%, indicating that the use of standard centrifuges (6000×g) does not compromise results. Serological analysis revealed a 71.4% positivity rate among samples collected from farms in Bahia with reported trypanosomiasis outbreaks—16% higher than parasitological diagnoses. These findings are promising for conducting xiii seroepidemiological surveys in Bahia, allowing for more accurate mapping of parasite circulation. The immunosuppression protocol involving five doses of 2 mg/kg dexamethasone administered intravenously on alternate days yielded the best results for increasing parasitemia in a shorter period. It was concluded that using blood smears for the detection of anti-T. vivax IgG antibodies via IFA is effective, less costly, and less labor-intensive compared to antigen purification techniques described in the literature. Furthermore, to avoid sensitization of slides with antigens adsorbed by antibodies and nonspecific reactions, parasites should be collected during the first peak of parasitemia, within the first 10 days of infection, prior to the animal’s seroconversion period.