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

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.

Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax.

Salivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48-67 kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended.

Modeling the pulsed light inactivation of microorganisms naturally occurring on vegetable substrates.

Pulsed light (PL) is a fast non-thermal method for microbial inactivation. This research studied the kinetics of PL inactivation of microorganisms naturally occurring in some vegetables. Iceberg lettuce, white cabbage and Julienne-style cut carrots were subjected to increasing PL fluences up to 12J/cm(2) in order to study its effect on aerobic mesophilic bacteria determined by plate count. Also, sample temperature increase was determined by infrared thermometry. Survivors' curves were adjusted to several models. No shoulder but tail was observed. The Weibull model showed good fitting performance of data. Results for lettuce were: goodness-of-fit parameter RMSE=0.2289, fluence for the first decimal reduction δ=0.98±0.80J/cm(2) and concavity parameter p=0.33±0.08. Results for cabbage were: RMSE=0.0725, δ=0.81±0.23J/cm(2) and p=0.30±0.02; and for carrot: RMSE=0.1235, δ=0.39±0.24J/cm(2) and p=0.23±0.03. For lettuce, a log-linear and tail model was also suitable. Validation of the Weibull model produced determination coefficients of 0.88-0.96 and slopes of 0.78-0.99. Heating was too low to contribute to inactivation. A single low-energy pulse was enough to achieve one log reduction, with an ultrafast treatment time of 0.5ms. While PL efficacy was found to be limited to high residual counts, the achievable inactivation level may be considered useful for shelf-life extension.