Erratum to: Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

Unfortunately, the original version of this article [1] contained an error. Figure 1 in the original article, corresponded to the first coinertia analysis that was carried out with no data on the procyclin PE repeats for the T. brucei brucei strains. After including these data, the coinertia analysis was modified both in the directionality of the arrows in the Y Hyperspace and in the biplot generated by the interaction of the two coinertia axes. The modified coinertia analysis is included in Fig. 1.

Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

BACKGROUND: Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. METHODS: Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. RESULTS: We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. CONCLUSION: Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.

Molecular profiles of Venezuelan isolates of Trypanosoma sp. by random amplified polymorphic DNA method.

Nine Trypanosoma sp. Venezuelan isolates, initially presumed to be T. evansi, were collected from three different hosts, capybara (Apure state), horse (Apure state) and donkey (Guarico state) and compared by the random amplification polymorphic DNA technique (RAPD). Thirty-one to 46 reproducible fragments were obtained with 12 of the 40 primers that were used. Most of the primers detected molecular profiles with few polymorphisms between the seven horse, capybara and donkey isolates. Quantitative analyses of the RAPD profiles of these isolates revealed a high degree of genetic conservation with similarity coefficients between 85.7% and 98.5%. Ten of the primers generated polymorphic RAPD profiles with two of the three Trypanosoma sp. horse isolates, namely TeAp-N/D1 and TeGu-N/D1. The similarity coefficient between these two isolates and the rest, ranged from 57.9% to 68.4% and the corresponding dendrogram clustered TeAp-N/D1 and Te Gu-N/D1 in a genetically distinct group.

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.

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.

Ultrastructural alterations in the adrenal gland cortex of mice experimentally infected with a Venezuelan isolate of Trypanosoma evansi.

The ultrastructural study of adrenal gland from mice experimentally infected with Trypanosoma evansi, in addition to intravascular and intracellular trypanosomes, showed different degrees of cortical cell alterations and capillary wall modifications. Beside its biological scope, these results suggest a role for the adrenal cortex to partake in Surra's etiopathogenesis and describe for the very first time a T. evansi intracellular stage.

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.

Evaluation of an enzyme-linked immunosorbent assay using recombinant major surface protein 5 for serological diagnosis of bovine anaplasmosis in Venezuela.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of bovine anaplasmosis with purified recombinant major surface protein 5 (MSP5) of Anaplasma marginale produced in Escherichia coli. Serum antibody responses against MSP5 were detected in calves experimentally infected with A. marginale as early as 21 days postinfection and reached maximum titers at 28 days postinfection. The MSP5 ELISA performed with serum samples taken from field cattle from different regions of Venezuela showed a seroprevalence of 47%, which seems to be in accordance with the reported epidemiological status of bovine anaplasmosis in Venezuela. Positive results obtained in the MSP5 ELISA were further confirmed by immunoblotting, with the recombinant MSP5 as the antigen. Thus, these results confirmed the importance of MSP5 as a suitable antigen for the serological diagnosis of bovine anaplasmosis.

Antigen recognition on Anaplasma marginale and bovine erythrocytes: an electron microscopy study.

The reactivity of sera from Anaplasma marginale-infected bovine with red blood cells and with purified anaplasma bodies was analyzed by electron immunomicroscopy. Red blood cells from non-infected and from anaplasma-infected cows and A. marginale bodies separated from parasitized erythrocytes, were incubated with control, pre-immune and immune sera followed by anti-bovine IgG-Peroxidase. Immune sera from cows infected with the venezuelan and Florida isolate reacted with red blood cell membranes from normal and infected bovines, while sera from non-infected cows did not. The immune sera also recognized epitopes localized on the cell wall, membrane and on unidentified intracellular structures of the purified anaplasma bodies. Thus, we propose that A. marginale infection may cause structural and biochemical modifications of the plasma membrane of the bovine red blood cells during its intraerythrocytic cycle. This in turn could elicit an autoimmune type of response against its own cells that would stimulate erythrophagocitosis. The strong reactivity of the immune sera with the Anaplasma bodies suggests that the bovine immune system also recognizes epitopes located on the parasite.