Serological occurrence for tick-borne agents in beef cattle in the Brazilian Pantanal.

This study investigated the seropositivity for five different tick-borne agents, namely Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, and Trypanosoma vivax in beef cattle in the Brazilian Pantanal. The serum samples collected from animals (200 cows; 200 calves) were used in indirect enzyme-linked immunosorbent assays (iELISA) to detect IgG antibodies against A. marginale, B. bovis, B. bigemina, and T. vivax, and Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum. No correlation was observed between seropositivity for C. burnetii and A. phagocytophilum with other agents whereas moderate correlation was observed for A. marginalexB. bigemina x B. bovis. Cows were more seropositive for T. vivax whereas calves were more seropositive for B. bovis and B. bigemina. The highest number of seropositive animals by a single agent was observed for T. vivax (15.2%). Co-seropositivity for T. vivax + A. marginale was higher in cows (25.5%) and for T. vivax + B. bovis + B. bigemina + A. marginale was higher in calves (57.5%). The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, Rhipicephalus microplus, in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, may explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.

Serological occurrence for tick-borne agents in beef cattle in the Brazilian Pantanal / Ocorrência sorológica para agentes transmitidos por carrapatos em bovinos de corte no Pantanal Brasileiro

Abstract This study investigated the seropositivity for five different tick-borne agents, namely Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, and Trypanosoma vivax in beef cattle in the Brazilian Pantanal. The serum samples collected from animals (200 cows; 200 calves) were used in indirect enzyme-linked immunosorbent assays (iELISA) to detect IgG antibodies against A. marginale, B. bovis, B. bigemina, and T. vivax, and Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum. No correlation was observed between seropositivity for C. burnetii and A. phagocytophilum with other agents whereas moderate correlation was observed for A. marginalexB. bigemina x B. bovis. Cows were more seropositive for T. vivax whereas calves were more seropositive for B. bovis and B. bigemina. The highest number of seropositive animals by a single agent was observed for T. vivax (15.2%). Co-seropositivity for T. vivax + A. marginale was higher in cows (25.5%) and for T. vivax + B. bovis + B. bigemina + A. marginale was higher in calves (57.5%). The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, Rhipicephalus microplus, in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, may explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.

Resumo Este estudo teve como objetivo determinar a co-soropositividade para agentes transmitidos por carrapatos, como Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, e Trypanosoma vivax em bovinos de corte do Pantanal Brasileiro. Amostras de soro foram colhidas de 400 animais (200 vacas; 200 bezerros) e submetidas a Ensaios Imunoenzimáticos Indiretos (iELISA) para detecção de anticorpos IgG anti- A. marginale, anti- B. bovis, anti- B. bigemina e anti- T. vivax, e à Reação de Imunofluorescência Indireta (RIFI) para detecção de anticorpos IgG anti -C. burnetii e anti- A. phagocytophilum. Ausência de correlação foi vista entre os animais soropositivos para C. burnetii e A. phagocytophilum com os outros agentes e correlação moderada ocorreu entre A. marginale x B. bigemina x B. bovis. Vacas foram mais soropositivas que bezerros para T. vivax e bezerros mais soropositivos que vacas para B. bovis e B. bigemina. Maior número de animais soropositivos para um único agente foi visto para T. vivax (15,2%). Vacas demonstraram maior co-soropositividade para T. vivax + A. marginale (25,5%) e bezerros para T. vivax + B. bovis + B. bigemina + A. marginale (57,5%). A alta correlação entre a soropositividade para A. marginale x B. bovis x B. bigemina é provavelmente devida à presença do vetor biológico, o carrapato Rhipicephalus microplus, nas fazendas estudadas. As vias de transmissão comuns, mediadas por dípteros hematófagos e fômites, podem explicar a alta co-soropositividade das vacas para A. marginale e T. vivax. A baixa soropositividade para C. burnetii é provavelmente devida ao tipo de sistema de criação empregado (extenso). A soropositividade para A. phagocytophilum em apenas um animal sugere a ocorrência de reação sorológica cruzada com outro agente do gênero Anaplasma.

Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal.

The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1ß gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America.

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.

Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal / Filogeografia de genótipos msp4 de Anaplasma marginale em gado de corte no Pantanal brasileiro

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

Resumo O gene msp4 de A. marginale é unicodon, estável e pouco heterogêneo, sendo considerado como um marcador útil para caracterização filogeográfica desta bactéria. Este trabalho teve como objetivo analisar a filogeografia de A. marginale com base no gene msp4 em bovinos de corte do Pantanal Brasileiro, comparativamente a outra regiões do mundo. Alíquotas de sangue foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Como resultado, 56,75% (227/400) mostraram-se positivas para A. marginale pela qPCR para o gene msp1β e destas, 8,37% (19/227) amostras foram positivas na PCR convencional para o gene msp4. Na análise de distância Network, 14 sequências do Pantanal brasileiro foram agrupadas em um único grupo com as da Thailândia, Índia, Espanha, Colômbia, Paraná (Brasil), México, Portugal, Argentina, China, Venezuela, Austrália, Italia e Minas Gerais (Brasil). Dentre 68 sequências do Brasil e do mundo, constatou-se a presença de 15 genótipos, sendo o genótipo número um (#1) o mais distribuído. As sequências msp4 de A. marginale detectadas em bovinos de corte no Pantanal brasileiro apresentaram baixo polimorfismo com formação de dois genogrupos filogeneticamente relacionados àqueles encontrados em ruminantes de países das América do Sul e Central, Europa e Ásia.

Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal.

There are few studies on the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially about beef cattle. The objective of this study was to evaluate the genetic diversity of A. marginale, based on the msp1α gene in Bos taurus indicus sampled from the Brazilian Pantanal. Aliquots of blood with and without EDTA were taken from 400 cattle (200 cows and 200 calves) across five extensive farms. The samples were submitted to the indirect immunoenzymatic assay (iELISA), quantitative real-time PCR (qPCR) for the msp1ß gene and to the semi-nested (sn) PCR for the msp1α gene. Positive samples were sequenced by the Sanger method and subjected to diversity analysis using the RepeatAnalyser software. The percentage of positive animals by iELISA, qPCR and (sn) PCR was 72.2% (289/400), 56.7% (227/400) and 23% (52/227), respectively. Cows (154/200) showed to be significantly more seropositive than calves (135/200). In qPCR, the number of calves and average quantification value (138/200; 1.3 × 106) A. marginale msp1α copies per µL proved to be higher when compared to that found for the cows (89/200; 3.9 × 104). The microsatellite analysis of the 26 sequences obtained from the msp1α gene revealed the presence of E (77%), C (15.4%) and B (7.7%) genotypes. Fourteen A. marginale strains were identified in the studied region, with eight that have never before been described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-ß-ß-ß-100; EV7-11-10-15; τ-11-11-27-18; τ-11-10-15; τ-27-13-18). Beef cattle are highly exposed to A. marginale in the Brazilian Pantanal. Moreover, a high genetic diversity of A. marginale, with eight new strains, was found in the studied region. While cows may act as chronic carriers, perpetuating the pathogen within the herd, male beef calves sold to other regions may disperse these strains.