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The infectivity and virulence of seven Trypanosoma evansi and Trypanosoma equiperdum Venezuelan strains isolated from horses, donkeys and capybaras were compared in a mouse model up to 41 days, for parasitemia, animal weight, survival rates, packed cell volume, haemoglobin and erythrocyte count. Two T. equiperdum strains and three of the T. evansi strains resulted in 100% mice mortality, while the two T. evansi donkey strains exhibited lower infectivity and mortality. T. equiperdum strains had shorter pre-patent periods (4 days) than the T. evansi strains (4-12 days). In terms of pathogenicity, only the T. evansi horse strain and the two capybara strains produced a significant decrease of the packed cell volume, in haemoglobin concentration and in red blood cell count. In contrast, the T. evansi donkey strains did not show any changes in the hematological parameters. From the seven variables studied, only pre-patent period, day of maximum parasitemia, day of first parasitemia peak and number of parasitemia peaks were statistically significant. Weight decrease was only observed in mice infected with the T. evansi horse strain. T. equiperdum strains showed the highest mice lethality (7% survival by day 8 post-infection) with no change in the hematological parameters. The three T. evansi horse and capybara strains showed 80%, 87% and 97% survival rates, respectively by day 12 post-infection. However, by day 20 post-inoculation all the mice infected with the T. evansi horse strain died, while 53% and 27% capybara strains infected survived. Whereas by day 40 post-infection, 53 and 73% of the mice infected with the T. evansi donkey strains had survived. These results demonstrate striking infectivity and virulence differences between Venezuelan T. evansi and T. equiperdum strains in NMRI mice and open new possibilities to characterize inter and intra-species variations that may contribute to the pathogenicity of these two species.

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Regioselective copper-catalyzed boracarboxylation of vinyl arenes with bis(pinacolato)diboron and carbon dioxide has been achieved. New boron-functionalized α-aryl carboxylic acids, including nonsteroidal anti-inflammatory drugs (NSAIDs), are obtained in moderate to excellent yields. The synthetic utility of the transformation was shown through subsequent derivatization of the carbon-boron bond yielding formal hydroxy- and fluorocarboxylation products as well as anionic difluoroboralactones.

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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.

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The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guárico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela.

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The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were detected as seropositives to T. equi. In addition, 128 samples (35.56%) were seropositives to both hemoparasites. There were no significant differences between the seropositivity to B. caballi and T. equi with the factors sex and age of the horses. These results show that Lara State is an enzootic area for equine piroplasmosis, and are a contribution to a partial knowledge of the dynamic of this disease in Venezuela.

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Este estudio tuvo como objetivo efectuar una caracterización molecular de Trypanosoma vivax en ovinos de dos hatos en los cuales estos rumiantes, conjuntamente con vacunos y búfalos de agua, comparten la misma área agroecológica, estableciendo el potencial papel de los ovinos como fuente de infección de tripanosomosis por T. vivax para los grandes rumiantes. La técnica de microcentrifugación capilar (TMC) fue usada para establecer el porcentaje de infecciones activas por tripanosomas existente en los animales evaluados. Se empleó un ensayo de reacción en cadena de la polimerasa (PCR) para confirmar la identificación de especie, mientras que un ensayo de PCR-RFLP (polimorfismo en la longitud de los fragmentos de restricción) permitió evaluar la variabilidad intraespecífica entre los aislados de T. vivax detectados en ovinos vs. aquellos provenientes de bóvidos (vacunos y búfalos de agua), colectados en la misma área de producción. De las 320 muestras de sangre de ovinos colectadas, la TMC detectó positividad en 11 (4,35 por ciento), lo que es de gran relevancia epidemiológica debido a la baja sensibilidad de esta metodología. Los resultados de PCR permitieron caracterizar a T. vivax como la especie presente en todas las infecciones activas detectadas. Todos los animales infectados mostraron un valor de hematocrito inferior (P<0,05) al registrado en animales no infectados (22,435 vs. 31,450). El ensayo de PCR-RFLP permitió observar la existencia de perfiles de restricción similares entre los aislados de T. vivax evaluados, sugiriéndose la ausencia de variación intraespecífica para el marcador molecular en estudio, independientemente del origen de hospedador del que provino la muestra (ovinos, vacunos, búfalos de agua). Estos resultados permiten sugerir que los aislados de T. vivax que infectan ovinos, vacunos y búfalos de agua en el área de estudio pudiesen estar estrechamente relacionados desde un punto de vista genético y, consecuentemente...

This study was made to achieve a molecular characterization of Trypanosoma vivax in two Venezuelan farms where both small ruminants (mainly ovines) and bovines (cattle and water buffaloes) share the same agroecological area. In addition, it was made to assess the role of sheep as source of T. vivax infection for cattle and buffalo herds. The microhematocrit centrifugation technique (MHC) was used to establish the percentage of current trypanosome infection. A PCR-based assay was used to confirm the species identification while a PCR-RFLP assay was used for studying intra-specific variation among T. vivax from sheep vs. those from other livestock from the same area. From 320 sheep blood samples, MHC detected 11 (4,35 percent) which is of remarkable epidemiological significance due to the low sensitivity of this method. Based on PCR results, T. vivax was characterized as the only species responsible for all sheep infections. All infected animals showed a lower packed cell volume value (P<0,05) when compared with the non-infected (22,435 vs. 31,450). The PCR-RFLP technique revealed similar profiles among T. vivax isolates suggesting a non intra-specific variation within the molecular marker amplified regardless the host (sheep water buffaloes or cattle). Thus, it was suggested that T. vivax infecting sheep, cattle, and buffaloes in the study area could be genetically closely related. These findings show that sheep may play an important role in the epidemiology of livestock trypanosomiasis in this area and they might be incorporated into therapeutic and preventive programs against livestock trypanosomiasis.

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Animal trypanosomosis is one of the most severe constraints to agricultural development in sub-Saharan Africa and is also an important disease of livestock in Latin America and Asia. The causative agents are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extracellular position of trypanosomes obliges us to consider both the parasite and its excreted/secreted factors in the course of the physiopathologic process. The advent of proteomics led us to propose a comparative approach of the proteome (i.e., the whole parasite content) and the secretome (i.e., naturally excreted/secreted molecules) of T. congolense and T. evansi with particular attention to common and specific molecules between strains of differing virulence and pathogenicity. The molecular identification of differentially expressed trypanosome molecules correlated with either the virulence process or the pathogenicity will provide new potential molecular targets for improved field diagnosis and chemotherapy of animal trypanosomosis.

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Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

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