High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil.

Anaplasma marginale is an obligate intracellular Gram-negative bacterium found in ruminants' erythrocytes and is the etiological agent of bovine anaplasmosis. The bacterium's genetic diversity has been characterized based on sequences of major surface proteins (MSPs), such as MSP1α. The aim of the present study was to investigate the genetic diversity of A. marginale in cattle in the state of Maranhão, northeastern Brazil. To this end, 343 blood samples were harvested and subjected to iELISA assays using the recombinant surface protein MSP5. Out of 343 blood samples, 235 (68.5%) were randomly chosen and submitted to DNA extraction, qPCR and conventional PCR targeting the msp1α gene to determine amino acid sequences and classify the genotypes. The iELISA results showed 81.34% seropositivity (279/343), whereas qPCR revealed 224 positive samples (95.32%). Among these qPCR-positive samples, 67.4% (151/224) were also positive in the cPCR. Among the 50 obtained sequences, 21 strains had not been previously reported. Regarding the genotypes, H (26/50) and E (18/50) were identified most often, while genotypes F and C were only identified twice each and B and G once each. In conclusion, high prevalence and genetic diversity for A. marginale were observed in dairy cattle herds in the state of Maranhão.

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.

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.

Assessment of transplacental transmission of Neospora caninum in dairy cattle in the Agreste region of Pernambuco.

The transplacental transmission is the primary route of Neospora caninum infection in bovine herds around the world. This study aimed to determine the frequency of transplacental transmission of the parasite in dairy cattle of Agreste region of Pernambuco through serological tests (IFAT and ELISA). Three hundred sixteen serum samples from cows and heifers and their offspring were analyzed. The transplacental transmission rate was 72.22% (13/18) for cows and 69.23% (9/13) for heifers by IFAT. ELISA test showed transplacental transmission rate of 43.58% (17/39) for cows and 50% (9/18) for heifers. The transplacental transmission rates were similar, in both groups in test, but a higher seropositivity was found in cows by IFAT. Data were statistically analyzed using the chi-square and Fisher's exact test. A significant relationship of dependence between seropositivity of mothers and their offspring was found. The more frequent IFAT antibody titers and ELISA levels for N. caninum were, respectively, 200 and between four (cows) and five (heifers and offspring). In the Spearman correlation, no association was found between the magnitude of antibody titers for N. caninum between mothers and their offspring. The kappa test showed an index of 0.35, indicating a mild correlation between the serological tests used. The study suggests that cows and heifers are the main transmitters of N. caninum in the studied region and that vertical transmission is the major form of transmission in dairy herds of the Agreste region of Pernambuco.

High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil / Alta diversidade genética de Anaplasma marginale em bovinos de leite no nordeste do Brasil

Abstract Anaplasma marginale is an obligate intracellular Gram-negative bacterium found in ruminants' erythrocytes and is the etiological agent of bovine anaplasmosis. The bacterium's genetic diversity has been characterized based on sequences of major surface proteins (MSPs), such as MSP1α. The aim of the present study was to investigate the genetic diversity of A. marginale in cattle in the state of Maranhão, northeastern Brazil. To this end, 343 blood samples were harvested and subjected to iELISA assays using the recombinant surface protein MSP5. Out of 343 blood samples, 235 (68.5%) were randomly chosen and submitted to DNA extraction, qPCR and conventional PCR targeting the msp1α gene to determine amino acid sequences and classify the genotypes. The iELISA results showed 81.34% seropositivity (279/343), whereas qPCR revealed 224 positive samples (95.32%). Among these qPCR-positive samples, 67.4% (151/224) were also positive in the cPCR. Among the 50 obtained sequences, 21 strains had not been previously reported. Regarding the genotypes, H (26/50) and E (18/50) were identified most often, while genotypes F and C were only identified twice each and B and G once each. In conclusion, high prevalence and genetic diversity for A. marginale were observed in dairy cattle herds in the state of Maranhão.

Resumo Anaplasma marginale é uma bactéria Gram-negativa intracelular obrigatória de eritrócitos de ruminantes e responsável pela anaplasmose bovina. A diversidade genética de A. marginale tem sido caracterizada com base nas sequências das principais proteínas de superfície (MSPs), como a MSP1α. O objetivo deste estudo foi investigar a diversidade genética de A. marginale em bovinos no estado do Maranhão, Nordeste do Brasil. Dessa forma, 343 amostras de sangue foram submetidas ao ensaio iELISA, utilizando-se a proteína recombinante MSP5. Das 343 amostras de sangue, 235 (68,5%) foram escolhidas aleatoriamente e submetidas à extração de DNA, qPCR e PCR convencional para gene msp1α, para determinação das sequências de aminoácidos e classificação dos genótipos. Os resultados do iELISA mostraram 81,34% de soropositividade (279/343), enquanto qPCR revelou 224 amostras positivas (95,32%). Dentre estas na qPCR, 67,4% (151/224) mostraram-se positivas no PCR convencional. Das 50 sequências obtidas, 21 cepas não haviam sido relatadas anteriormente. Em relação aos genótipos, H (26/50) e E (18/50) foram os mais frequentes, enquanto os genótipos F e C foram identificados apenas duas vezes cada, e B e G uma vez cada. Em conclusão, alta prevalência e marcante diversidade genética de A. marginale foram observadas em rebanhos leiteiros no estado do Maranhão.

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 / 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.

Assessment of transplacental transmission of Neospora caninum in dairy cattle in the Agreste region of Pernambuco / Avaliação da transmissão transplacentária do Neospora caninum em bovinos de leite na região Agreste de Pernambuco

Abstract The transplacental transmission is the primary route of Neospora caninum infection in bovine herds around the world. This study aimed to determine the frequency of transplacental transmission of the parasite in dairy cattle of Agreste region of Pernambuco through serological tests (IFAT and ELISA). Three hundred sixteen serum samples from cows and heifers and their offspring were analyzed. The transplacental transmission rate was 72.22% (13/18) for cows and 69.23% (9/13) for heifers by IFAT. ELISA test showed transplacental transmission rate of 43.58% (17/39) for cows and 50% (9/18) for heifers. The transplacental transmission rates were similar, in both groups in test, but a higher seropositivity was found in cows by IFAT. Data were statistically analyzed using the chi-square and Fisher’s exact test. A significant relationship of dependence between seropositivity of mothers and their offspring was found. The more frequent IFAT antibody titers and ELISA levels for N. caninum were, respectively, 200 and between four (cows) and five (heifers and offspring). In the Spearman correlation, no association was found between the magnitude of antibody titers for N. caninum between mothers and their offspring. The kappa test showed an index of 0.35, indicating a mild correlation between the serological tests used. The study suggests that cows and heifers are the main transmitters of N. caninum in the studied region and that vertical transmission is the major form of transmission in dairy herds of the Agreste region of Pernambuco.

Resumo A transmissão transplacentária é a principal via de infecção do Neospora caninum nos rebanhos bovinos em todo o mundo. O presente estudo teve como objetivo determinar a frequência da transmissão transplacentária do parasita em bovinos leiteiros do Agreste Pernambucano, por meio de testes sorológicos (RIFI e ELISA). Foram analisadas 316 amostras de soro de fêmeas bovinas (vacas e novilhas) e de suas crias. A taxa de transmissão transplacentária pela RIFI foi de 72,22% (13/18) para vacas e 69,23% (9/13) para as novilhas. O ELISA teste mostrou taxa de transmissão transplacentária de 43,58% (17/39) para as vacas e 50% (9/18) para as novilhas. As taxas de transmissão transplacentária foram similares para os dois testes em geral, porém uma maior soropositividade foi encontrada nas vacas pela RIFI. Os dados foram estatisticamente analisados pelo teste de qui-quadrado e teste exato de Fischer. Foi encontrada uma relação significativa de dependência entre a soropositividade das mães e de suas crias. Os títulos de anticorpos anti- N. caninum foi de 200 na RIFI e posicionados entre o nível quatro (vacas) e cinco (novilhas e bezerros) pelo ELISA. Pela correlação de Spearman, não foi observada associação entre a magnitude de títulos de anticorpos anti- N. caninum de fêmeas com o de suas crias. O teste de concordância kappa revelou um índice de 0,35, indicando uma concordância leve entre os testes sorológicos utilizados. O estudo sugere que vacas e novilhas são as principais transmissoras do N. caninum na região estudada, sendo a transmissão vertical, a principal forma de transmissão do agente em rebanhos leiteiros do Agreste de Pernambuco.

Doença renal policística em bezerro / Polycystic kidney disease in a calf

Este relato descreve um caso de doença renal policística em um bovino, macho, mestiço, com um ano de idade. Ao exame clínicoobservou-se estado nutricional ruim, mucosas pálidas, desidratação moderada (8% a 10%), úlceras na região ventral da língua,áreas multifocais de hipotricose recobertas por crostas por todo o corpo e decúbito esterno-lateral permanente. Na avaliação dohemograma observou-se principalmente anemia arregenerativa e leucocitose devido à neutrofilia com desvio à [confirmar] esquerda.Na necropsia, os rins estavam aumentados de volume, pálidos e com a superfície natural finamente irregular. Histologicamente, haviasubstituição quase completa da cortical renal por múltiplos e pequenos cistos, distensão dos espaços de Bowman, regeneraçãotubular, fibrose, edema e leve infiltrado inflamatório linfoplasmocítico intersticial.

This report describes a case of polycystic kidney disease in a male, mixed breed bovine aged one year. On clinical examinationrevealed a poor nutritional status, pallid mucous membranes, moderated dehydration (8 to 10%), ulcers on the ventral portion ofthe tongue, multifocal areas of hypotrichosis covered with scabs throughout the entire body and permanent sternolateral decubitus.The blood exam revealed aregenerative anemia and leukocytosis (neutrophilia) with deviation to the left. The necropsy revealedenlarged, pallid kidneys with a finely irregular natural surface. The histological analysis revealed the nearly complete replacementof the renal cortex by numerous small cysts, distension of the Bowman space, tubular regeneration, fibrosis, edema and mildinterstitial lymphoplasmacytic inflammatory infiltrate.

Doença renal policística em bezerro / Polycystic kidney disease in a calf

This report describes a case of polycystic kidney disease in a male, mixed breed bovine aged one year. On clinical examination revealed a poor nutritional status, pallid mucous membranes, moderated dehydration (8 to 10%), ulcers on the ventral portion of the tongue, multifocal areas of hypotrichosis covered with scabs throughout the entire body and permanent sternolateral decubitus. The blood exam revealed aregenerative anemia and leukocytosis (neutrophilia) with deviation to the left. The necropsy revealed enlarged, pallid kidneys with a finely irregular natural surface. The histological analysis revealed the nearly complete replacement of the renal cortex by numerous small cysts, distension of the Bowman space, tubular regeneration, fibrosis, edema and mild interstitial lymphoplasmacytic inflammatory infiltrate. Keywords: diseases of cattle, kidney disease, lesions uremia, nephropathy