Cleber Oliveira Soares

Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World - a review

The biology, epidemiology, pathogenesis, diagnostic techniques, and history of the introduction of Trypanosoma (Duttonella) vivax in the New World are reviewed. The two main immunological responses of trypanosome-infected animals - antibody production and immunodepression - are discussed in the context of how these responses play a role in disease tolerance or susceptibility. Isolation and purification of T. vivax are briefly discussed. The recent reports of bovine trypanosomiasis diagnosed in cattle on farms located in the Pantanal region of the states of Mato Grosso do Sul and Mato Grosso, Brazil, are also discussed.

Coexistence of antibodies to tick-borne agents of babesiosis and Lyme borreliosis in patients from Cotia county, State of São Paulo, Brazil

This paper reports a case of coinfection caused by pathogens of Lyme disease and babesiosis in brothers. This was the first case of borreliosis in Brazil, acquired in Cotia County, State of S o Paulo, Brazil. Both children had tick bite history, presented erythema migrans, fever, arthralgia, mialgia, and developed positive serology (ELISA and Western-blotting) directed to Borrelia burgdorferi G 39/40 and Babesia bovis antigens, mainly of IgM class antibodies, suggestive of acute disease. Also, high frequencies of antibodies to B. bovis was observed in a group of 59 Brazilian patients with Lyme borreliosis (25.4%), when compared with that obtained in a normal control group (10.2%) (chi-square = 5.6; p < 0.05). Interestingly, both children presented the highest titers for IgM antibodies directed to both infective diseases, among all patients with Lyme borreliosis.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.

Borrelioses, agentes e vetores

Borrelioses are infectous diseases caused by spirochaetes of the genus Borrelia. They are born mainly through ticks at animals and/or human beings. In this review are shown and discussed five groups of diseases determined by borrelia, general characteristics of the spirochaetes, aspects related to transmission by arthropods, biological and pathological aspects of the diseases in domestic and wild animals, Lyme disease as an important zoonosis, the association of borrelia with other hematozoa agents, the diagnostic methods and the comparative epidemiology with data obtained from Brazil and other countries. The borrelioses have pathological, clinical and epidemiological characteristics which vary according to physiographic regions due to the existence of different species, genospecies and strains of borrelia, of arthropod vectors, vector-agent relationship and of different ecocystems.

Ocorrência de Borrelia spp. em cultura de células embrionárias do carrapato Boophilus microplus (Acari: Ixodidae) no Estado do Mato Grosso do Sul, Brasil

The aim of the present work was to report the occurrence of Borrelia spp. in embryonic cell cultures from naturally infected Boophilus microplus. Seven days after the beginning of a primary culture of embryonic cells of B. microplus at 31oC was noted that the cells start suffering degeneration. Under examination at phase contrast microscope, the presence of prolongated microorganisms with great mobility was detected. Microscopic slides of the culture supernatant, hemolymph and egg mass, were stained by May Grunwald-Giemsa, allowing the visualization of the spirochetes. The morphologic examination of the microorganism and its visualization in. B. microplus, suggest to be Borrelia spp.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Genetic conservation of potentially immunogenic proteins among Brazilian isolates of Babesia bovis.

Bovine babesiosis caused by Babesia bovis remains an important constraint for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these vaccines have a number of drawbacks, which justifies the search for better, safer vaccines. In recent years, a number of parasite proteins with immunogenic potential have been discovered. However, there is little information on the genetic conservation of these proteins among different parasite isolates, which hinders their assessment as immunogens. The aim of the present study was to evaluate the conservation of the genes ama-1, acs-1, rap-1, trap, p0 and msa2c among five Brazilian isolates of B. bovis. Through polymerase chain reaction, genetic sequencing and bioinformatics analysis of the genes, a high degree of conservation (98-100%) was found among Brazilian isolates of B. bovis and the T2Bo isolate. Thus, these genes are worth considering as viable candidates to be included in a recombinant cocktail vaccine for cattle babesiosis caused by B. bovis.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.