Armando Reyna-Bello

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DETECTION OF ANTI-TRYPANOSOMA EVANSI EQUINE ANTIBODIES

The standardization of ELISA for the detection of anti-Trypanosoma evansi antibodies in naturally and experimentally infected horses is described. Bayesian analysis was used to establish the cutoff between positive and negative sera. In order to determine the assessment of the ELISA test, the results obtained were compared with those from an IFA. A relative sensibility of 98.39%, a specificity of 95.12% and a predictive value of 96.83% were determined. The standardized technique was used to evaluate the antibody production against trypanosome in an experimentally infected equine, in which the sera converted 15 days after infection. The test was also used for a study of sera prevalence in a non-random sample from two different populations. A prevalence of 81.7% in workhorse and 57.14% in stable horses was found.

Transcriptome analysis of the bloodstream stage from the parasite Trypanosoma vivax

BackgroundTrypanosoma vivax is the earliest branching African trypanosome. This crucial phylogenetic position makes T. vivax a fascinating model to tackle fundamental questions concerning the origin and evolution of several features that characterize African trypanosomes, such as the Variant Surface Glycoproteins (VSGs) upon which antibody clearing and antigenic variation are based. Other features like gene content and trans-splicing patterns are worth analyzing in this species for comparative purposes.ResultsWe present a RNA-seq analysis of the bloodstream stage of T. vivax from data obtained using two complementary sequencing technologies (454 Titanium and Illumina).Assembly of 454 reads yielded 13385 contigs corresponding to proteins coding genes (7800 of which were identified). These sequences, their annotation and other features are available through an online database presented herein. Among these sequences, about 1000 were found to be species specific and 50 exclusive of the T. vivax strain analyzed here. Expression patterns and levels were determined for VSGs and the remaining genes. Interestingly, VSG expression level, although being high, is considerably lower than in Trypanosoma brucei. Indeed, the comparison of surface protein composition between both African trypanosomes (as inferred from RNA-seq data), shows that they are substantially different, being VSG absolutely predominant in T. brucei, while in T. vivax it represents only about 55%. This raises the question concerning the protective role of VSGs in T. vivax, hence their ancestral role in immune evasion.It was also found that around 600 genes have their unique (or main) trans-splice site very close (sometimes immediately before) the start codon. Gene Ontology analysis shows that this group is enriched in proteins related to the translation machinery (e.g. ribosomal proteins, elongation factors).ConclusionsThis is the first RNA-seq data study in trypanosomes outside the model species T. brucei, hence it provides the possibility to conduct comparisons that allow drawing evolutionary and functional inferences. This analysis also provides several insights on the expression patterns and levels of protein coding sequences (such as VSG gene expression), trans-splicing, codon patterns and regulatory mechanisms. An online T. vivax RNA-seq database described herein could be a useful tool for parasitologists working with trypanosomes.

Trypanosoma ( Duttonella ) vivax and Typanosomosis in Latin America: Secadera/Huequera/Cacho Hueco

The disease caused by T. vivax is commonly called Nagana in Africa and “secadera/cachera/cacho hueco/huequera” in parts of South America. This chapter will focus on the disease and its causative agent, reviewing new diagnostic methods, economic impact, chemotherapy, phylogenetic analysis of T. vivax isolates from Africa and South America, epidemiological studies in Latin America, and the analysis of recent genomic and transcriptomic data. T. vivax has a significant economic impact on livestock production in sub-Saharan Africa, where it is transmitted by the tsetse fly, and elsewhere in the African continent and in Central and South America, where it is transmitted mechanically. T. vivax is enzootic in most Latin American countries, and recurrent epizootic outbreaks causing significant morbidity and mortality have been reported over the past decades. Several significant landmarks in T. vivax research have been achieved in the last 2 years, including the publication of high-quality draft genome sequences and partial RNA-seq data for the Y486 strain, as well as the complete transcriptome of the LIEM-176 strain. Comparative analysis of the T. vivax, T. brucei, and T. congolense genomes revealed important differences in the surface proteins responsible for host immune response evasion in these species, and data from the T. vivax LIEM-76 transcriptome support the participation of other surface proteins, in addition to the VSG, in immune evasion. Proteins of the trans-sialidase family have been identified as important virulence factors that catalyze the desialylation of the host red blood cell, which in turn triggers the erythrophagocytosis that results in anemia. These findings will provide novel tools to tackle the challenge of controlling animal trypanosomosis caused by T. vivax in the developing world.

Kinetoplast adaptations in American strains from Trypanosoma vivax

The mitochondrion role changes during the digenetic life cycle of African trypanosomes. Owing to the low abundance of glucose in the insect vector (tsetse flies) the parasites are dependent upon a fully functional mitochondrion, capable of performing oxidative phosphorylation. Nevertheless, inside the mammalian host (bloodstream forms), which is rich in nutrients, parasite proliferation relies on glycolysis, and the mitochondrion is partially redundant. In this work we perform a comparative study of the mitochondrial genome (kinetoplast) in different strains of Trypanosoma vivax. The comparison was conducted between a West African strain that goes through a complete life cycle and two American strains that are mechanically transmitted (by different vectors) and remain as bloodstream forms only. It was found that while the African strain has a complete and apparently fully functional kinetoplast, the American T. vivax strains have undergone a drastic process of mitochondrial genome degradation, in spite of the recent introduction of these parasites in America. Many of their genes exhibit different types of mutations that are disruptive of function such as major deletions, frameshift causing indels and missense mutations. Moreover, all but three genes (A6-ATPase, RPS12 and MURF2) are not edited in the American strains, whereas editing takes place normally in all (editable) genes from the African strain. Two of these genes, A6-ATPase and RPS12, are known to play an essential function during bloodstream stage. Analysis of the minicircle population shows that its diversity has been greatly reduced, remaining mostly those minicircles that carry guide RNAs necessary for the editing of A6-ATPase and RPS12. The fact that these two genes remain functioning normally, as opposed to that reported in Trypanosoma brucei-like trypanosomes that restrict their life cycle to the bloodstream forms, along with other differences, is indicative that the American T. vivax strains are following a novel evolutionary pathway.

Identification and characterization of corpuscular, soluble and secreted antigens of a Venezuelan isolate of Anaplasma marginale.

Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.

Assessment of Chromogen Suitability in ELISA for the Detection of Anaplasmosis and Trypanosomosis

Abstract Two different ELISAs were routinely performed in our laboratory to detect bovine trypanosomosis and anaplasmosis. The ELISA test for trypanosomosis involved the adsorption of a soluble fraction of parasites as the antigen; and, the ELISA for anaplasmosis was performed with a purified recombinant protein MSP5r adsorbed to the plate. With the purpose of assessing the merit of ABTS and TMB, we compared the absorbance obtained from positive and negative control sera from both assays. The results obtained, suggest that TMB is more adequate for recombinant antigens and that ABTS is preferred when partially purified antigenic extracts are used in the ELISA test.

Trypanosoma vivax Adhesion to Red Blood Cells in Experimentally Infected Sheep

Trypanosomosis, a globally occurring parasitic disease, poses as a major obstacle to livestock production in tropical and subtropical regions resulting in tangible economic losses. In Latin America including Venezuela, trypanosomosis of ruminants is mainly caused by Trypanosoma vivax. Biologically active substances produced from trypanosomes, as well as host-trypanosome cellular interactions, contribute to the pathogenesis of anemia in an infection. The aim of this study was to examine with a scanning electron microscope the cellular interactions and alterations in ovine red blood cells (RBC) experimentally infected with T. vivax. Ovine infection resulted in changes of RBC shape as well as the formation of surface holes or vesicles. A frequent observation was the adhesion to the ovine RBC by the trypanosomes free flagellum, cell body, or attached flagellum in a process mediated by the filopodia emission from the trypanosome surface. The observed RBC alterations are caused by mechanical and biochemical damage from host-parasite interactions occurring in the bloodstream. The altered erythrocytes are prone to mononuclear phagocytic removal contributing to the hematocrit decrease during infection.