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.

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax.

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.

Purification of a 64 kDa antigen from Trypanosoma evansi that exhibits cross-reactivity with Trypanosoma vivax.

Trypanosoma evansi and Trypanosoma vivax are the most extensively distributed trypanosomes responsible for diseases in livestock. Western blot and indirect immunofluorescence assays revealed a high immunological cross-reaction between these two parasites. An antigen with an apparent molecular mass of 64 kDa (p64), which exhibited cross-reactivity with T. vivax, was purified to homogeneity from a Venezuelan isolate of T. evansi. This antigen is glycosylated, contains a glycosyl-phosphatidylinositol anchor and appeared to be localized through the cell except in the nucleus, indicating that it could primarily be confined to the parasite surface. These results, together with its relative abundance and apparent molecular weight, suggest that p64 probably corresponds to the soluble form of a variable surface glycoprotein from T. evansi. Anti-p64 polyclonal antibodies, raised on mice, recognized a 53 kDa polypeptide band from a Venezuelan isolate of T. vivax on Western blots. Additionally, sera obtained from naturally infected animals also recognized p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, demonstrating that another relevant cross-reacting epitope, different than the inositol-1,2-cyclic phosphate of the cross-reacting determinant, must exist in p64. To date, p64 represents the first antigen isolated and partially characterized from T. evansi.

Immune response of cattle infected with African trypanosomes.

Trypanosomosis is the most economically important disease constraint to livestock productivity in sub-Saharan Africa and has significant negative impact in other parts of the world. Livestock are an integral component of farming systems and thus contribute significantly to food and economic security in developing countries. Current methods of control for trypanosomosis are inadequate to prevent the enormous socioeconomic losses resulting from this disease. A vaccine has been viewed as the most desirable control option. However, the complexity of the parasite's antigenic repertoire made development of a vaccine based on the variable surface glycoprotein coat unlikely. As a result, research is now focused on identifying invariant trypanosome components as potential targets for interrupting infection or infection-mediated disease. Immunosuppression appears to be a nearly universal feature of infection with African trypanosomes and thus may represent an essential element of the host-parasite relationship, possibly by reducing the host's ability to mount a protective immune response. Antibody, T cell and macrophage/monocyte responses of infected cattle are depressed in both trypanosusceptible and trypanotolerant breeds of cattle. This review describes the specific T cell and monocyte/macrophage functions that are altered in trypanosome-infected cattle and compares these disorders with those that have been described in the murine model of trypanosomosis. The identification of parasite factors that induce immunosuppression and the mechanisms that mediate depressed immune responses might suggest novel disease intervention strategies.

[International and regional standardization of immunoenzyme tests: methods, concerns and limitations]. / Standardisations internationale et régionale des épreuves immuno-enzymatiques: méthodes, intérêts et limites.

Numerous attempts have been made to standardise immuno-enzyme techniques (enzyme-linked immunosorbent assay: ELISA) used for the diagnosis of infectious diseases, in order to improve the reproducibility of the tests, expression of results, choice of a positive threshold, and selection of reference samples. The international standardisation of reagents and test protocols appears essential for quality control and the comparison of results between laboratories, but the interpretation of results can encounter major differences depending on the geographical sector under study. Based on these studies, and in the light of a model indirect ELISA for detecting antibodies against Trypanosoma vivax in cattle, the author proposes the international standardisation of reagents, test protocol, and the expression of results of ELISA using international reference samples. For local standardisation, the following proposals are made: sampling of representative local populations. Establishment of the distribution patterns of infected and uninfected local populations. Selection of representative controls from local populations (secondary reference samples). Expression of test results in comparison with these controls. Establishment of internal quality control based on the response of controls. Determination of a positive threshold, in accordance with the requirements of the user. Adaptation of the positive threshold according to the prevalence observed in the geographical sector under study.

Potential of trypanotolerance as a contribution to sustainable livestock production in tsetse affected Africa.

Tsetse transmitted trypanosomiasis is possibly the major constraint on livestock and agriculture development in Subsaharan Africa. Control of the disease has been based on vector control as well as on the use of trypanocidal drugs to treat or prevent infection in animals. Both control methods are effective but have proven not to be sustainable. Moreover, the development of a vaccine against trypanosomiasis is unlikely to be successful in the near future. On the other hand, trypanotolerant cattle, like the N'Dama can survive and produce in tsetse affected areas without interventions. This taurine breed has been indigenous to Africa for approximately 7,000 years and forms presently about 6% of the bovine population of Africa. Generally the N'Dama are kept in the rural areas by the small-scale farmer as a multi-purpose animal. Recent studies have defined management characteristics and assessed the production potential at the village level and under ranching conditions of N'Dama cattle exposed to various levels of tsetse challenge. Furthermore, experimental infections showed conclusively the superior resistance to the effects of infection of the N'Dama cattle when compared to zebu cattle and have confirmed that trypanotolerance is innate in N'Dama cattle. Studies have been conducted on development of protective humoral and cellular responses, the regulation of parasite multiplication and control of anaemia. These studies provided tools for identifying components of trypanotolerance. The ability to resist the development of anaemia in the face of infection, has shown to be correlated with the capacity to be productive; moreover, PCV values can serve as selection criterium for trypanotolerance. Subsequently, repeatabilities and heritabilities of trypanotolerance and performance traits were estimated.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the indirect fluorescent antibody test for detecting Trypanosoma vivax in South American cattle.

The indirect fluorescent antibody test (IFAT) as used in Africa for detecting bovine trypanosomiasis was adapted for use in South America and evaluated. Antigen consisted of Trypanosoma vivax laden bovine blood fixed in a 60 : 40 : : acetone : methanol solution. The test detected initial titres of 1 : 50 and 1 : 100 at an average of 13.1 and 15.9 days post parasitaemia (PP). Maximum titres as high as 1 : 400 developed in eight calves at an average of 23.4 days PP. In another calf, 109 days PP were required. Efficacy in detecting sero-positive calves throughout the course of infection was 81.1 and 96.4 per cent at serum dilutions of 1 : 100 and 1 : 50 respectively. No false positive reactions occurred when sera from 36 haemoparasite-free calves were tested. Cross reactivity did not occur when sera from calves singularly infected with Trypanosoma theileri, T evansi, Anaplasma marginale, Babesia argentina, B bigemina and Eperythrozoon spp were similarly tested in the IFAT. No significant differences were found in IFAT results of surveys in which both conventional serum samples and sera eluted from dried blood samples on filter paper from the same calf were used.