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.

Genomic analysis on Brazilian strains of Anaplasma marginale / Análise genômica de cepas Brasileiras de Anaplasma marginale

Abstract Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.

Resumo Anaplasma marginale é um patógeno transmitido por vetores que causam uma doença conhecida como anaplasmose. Até a presente data, não há genomas sequenciados de cepas brasileiras. O objetivo deste estudo foi comparar o genoma completo das cepas brasileiras de A. marginale (Palmeira e Jaboticabal) com os genomas de cepas de outras regiões (cepas dos EUA e Austrália). As sequências dos genomas das cepas brasileiras foram obtidas mediante sequenciamento de nova geração. As "reads" foram mapeadas usando-se como referência o genoma de A. marginale da cepa Florida. Foram identificados polimorfismos de nucleotídeo único (SNPs) e analisadas inserções/deleções (INDELs). As duas linhagens brasileiras se agruparam em um clado particular que, por sua vez, agrupou-se em um grupo maior junto com as linhagens norte-americanas. Além disso, foram identificadas diferenças significativas nas proteínas de superfície entre os dois isolados brasileiros. Esses resultados lançam luz sobre a história evolutiva de A. marginale e fornecem as primeiras informações de genomas de isolados sul-americanos. Avaliar as sequências de genomas de cepas de diferentes regiões é essencial para aumentar o conhecimento do pan-genoma dessa bactéria.

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.

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

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 msp1 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.(AU)

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 msp1 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.(AU)

Immune response of calves inoculated with proteins of Anaplasma marginale bound to an immunostimulant complex / Resposta imune de bezerros inoculados com proteínas de Anaplasma marginale ligadas a um complexo imunoestimulante

Despite our current knowledge of the immunology, pathology, and genetics of Anaplasma marginale, prevention in cattle is currently based on old standbys, including live attenuated vaccines, antibiotic treatment, and maintaining enzootic stability in cattle herds. In the present study, we evaluated the use of an immunostimulant complex (ISCOMATRIX) adjuvant, associated with a pool of recombinant major surface proteins (rMSP1a, rMSP1b, rMSP4 and rMSP5) to improve the humoral immune response triggered in calves mainly by IgG2. Ten calves were divided in three groups: 4 calves were inoculated with the ISCOMATRIX/rMSPs (G1); 2 calves were inoculated with ISCOMATRIX adjuvant (G2); and 4 calves received saline (G3). Three inoculations were administered at 21-day intervals. In G1, the calves showed significant increases in total IgG, IgG1 and IgG2 levels 21 days after the second inoculation, compared to the control group (p < 0.05), and G1 calves remained above the cut-off value 28 days after the third inoculation (p < 0.05). The post-immunized sera from calves in G1 reacted specifically for each of the rMSPs used. In conclusion, the ISCOMATRIX/rMSPs induced antigen-specific seroconversion in calves. Therefore, additional testing to explore the protection induced by rMSPs, both alone and in conjunction with proteins previously identified as subdominant epitopes, is warranted.

Apesar dos avanços da imunologia, patologia e genética de Anaplasma marginale, a prevenção em bovinos ainda é baseada nas vacinas vivas atenuadas, na terapia com antibiótico e estabilidade enzoótica dos rebanhos bovinos. No presente estudo, avaliou-se o uso de um complexo imunoestimulante (ISCOMATRIX), associado às proteínas recombinantes de superfície (rMSP1a, rMSP1b, rMSP4 e rMSP5) para melhorar a resposta imune humoral desencadeada em bezerros, principalmente por IgG2. Dez animais foram divididos em três grupos: 4 bezerros foram inoculados com o ISCOMATRIX/rMSPs (G1), 2 bezerros foram inoculados com ISCOMATRIX adjuvante (G2) e 4 bezerros receberam salina (G3). Três doses vacinais foram administradas em intervalos de 21 dias. No G1, os bezerros apresentaram aumentos significativos nos níveis de IgG total, IgG1 e IgG2 21 dias após a segunda inoculação, em comparação com o grupo de controle (p <0,05). Nos bezerros do G1 esses níveis de anticorpos permaneceram acima do ponto de corte 28 dias após a terceira inoculação (p < 0,05). Os soros pós-imunização de bezerros do G1 reagiram especificamente com cada uma das rMSPs utilizadas. Em conclusão, o ISCOMATRIX/rMSPs induziu soroconversão antígeno-específica em bezerros. Portanto, se justifica a realização de ensaios adicionais para explorar a proteção induzida pela rMSPs, tanto sozinhas como em conjunto com novas proteínas identificadas com epítopos subdominantes.

Immune response of calves inoculated with proteins ofAnaplasma marginale bound to an immunostimulant complex

Despite our current knowledge of the immunology, pathology, and genetics of Anaplasma marginale, prevention in cattle is currently based on old standbys, including live attenuated vaccines, antibiotic treatment, and maintaining enzootic stability in cattle herds. In the present study, we evaluated the use of an immunostimulant complex (ISCOMATRIX) adjuvant, associated with a pool of recombinant major surface proteins (rMSP1a, rMSP1b, rMSP4 and rMSP5) to improve the humoral immune response triggered in calves mainly by IgG2. Ten calves were divided in three groups: 4 calves were inoculated with the ISCOMATRIX/rMSPs (G1); 2 calves were inoculated with ISCOMATRIX adjuvant (G2); and 4 calves received saline (G3). Three inoculations were administered at 21-day intervals. In G1, the calves showed significant increases in total IgG, IgG1 and IgG2 levels 21 days after the second inoculation, compared to the control group (p 0.05), and G1 calves remained above the cut-off value 28 days after the third inoculation (p 0.05). The post-immunized sera from calves in G1 reacted specifically for each of the rMSPs used. In conclusion, the ISCOMATRIX/rMSPs induced antigen-specific seroconversion in calves. Therefore, additional testing to explore the protection induced by rMSPs, both alone and in conjunction with proteins previously identified as subdominant epitopes, is warranted.

Apesar dos avanços da imunologia, patologia e genética deAnaplasma marginale, a prevenção em bovinos ainda é baseada nas vacinas vivas atenuadas, na terapia com antibiótico e estabilidade enzoótica dos rebanhos bovinos. No presente estudo, avaliou-se o uso de um complexo imunoestimulante (ISCOMATRIX), associado ás proteínas recombinantes de superfície (rMSP1a, rMSP1b, rMSP4 e rMSP5) para melhorar a resposta imune humoral desencadeada em bezerros, principalmente por IgG2. Dez animais foram divididos em três grupos: 4 bezerros foram inoculados com o ISCOMATRIX/rMSPs (G1), 2 bezerros foram inoculados com ISCOMATRIX adjuvante (G2) e 4 bezerros receberam salina (G3). Três doses vacinais foram administradas em intervalos de 21 dias. No G1, os bezerros apresentaram aumentos significativos nos níveis de IgG total, IgG1 e IgG2 21 dias após a segunda inoculação, em comparação com o grupo de controle (p 0,05). Nos bezerros do G1 esses níveis de anticoprpos permaneceram acima do ponto de corte 28 dias após a terceira inoculação (p 0,05). Os soros pós-imunização de bezerros do G1 reagiram especificamente com cada uma das rMSPs utilizadas. Em conclusão, o ISCOMATRIX/rMSPs induziu soroconversão antígeno-específica em bezerros. Portanto, se justifica a realização de ensaios adicionais para explorar a proteção induzida pela rMSPs, tanto sozinhas como em conjunto com novas proteínas identificadas com epitopos subdominantes.

Antimicrobial peptides in Rhipicephalus (Boophilus) microplus / Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o

Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus / Antimicrobial peptides in Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by flies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale. Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the first line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire of antimicrobial peptides (AMPs). However, the Anaplasma sp. have developed evolutionary mechanisms to be able to adapt and survive in this arthropod which is the main biological vector this pathogen. The AMPs can be expressed constitutively by the immune system, induced by infection, or by the recognition of surface components of microorganisms such as lipopolysaccharides (LPS) and peptidoglycan (PNG). However, through evolutionary events, the Anaplasma marginale lost genes encoding these components characteristic of the cell wall of Gram-negative bacteria, and thus, is likely that the major surface proteins (MSPs) are involved in its strengthening as the resistance to AMPs. Although the mechanisms of action of AMPs have not been fully elucidated, models are proposed to demonstrate how the interactions between lipid bilayer and AMP happen. More than 1,000 AMPs have been described in several groups of eukaryotes. In particular, amphibian peptides account for 592 of total AMPs representing a rich source of these molecules. Additionally, another 166 AMPs were isolated from insects. However, in R. microplus, few studies have described the existence of AMPs. The known R. microplus antimicrobial peptides are defensin and ixodidin (both isolated from hemocytes), the microplusin (isolated from female hemolymph and eggs), VTDCE ( isolated gut and ovary), and other two peptides characterized as fragments of bovine hemoglobin, Hb 33-91 and Hb 98-114, (isolated from engorged female gut). Conclusion: Since the silencing of genes encoding AMPs expressed in R. microplus decreases the number of A. marginale, it is suggested that this bacteria could adapt to support the tick immune defense mechanisms generating a symbiotic relationship, a evidence that the expression of AMPs can be manipulated by the pathogen to assist in its multiplication by a mechanism not yet defined, thus the Anaplasma sp. and the tick vector can live together allowing the bacteria transmission by the host. More studies about antimicrobial peptides expressed in R. microplus against invading microorganisms are necessary in order to improve the comprehension of its immune system and its competence for bovine anaplasmosis as a vector.

Antimicrobial peptides in Rhipicephalus (Boophilus) microplus / Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o

Seroprevalence of IgG antibodies against Anaplasma marginale in cattle from south Mozambique / Soroprevalência de anticorpos de classe IgG contra Anaplasma marginale em bovinos da região Sul de Moçambique

The current study aimed to investigate the seroprevalence of IgG antibodies to Anaplasma marginale in cattle from Maputo, Gaza and Inhambane provinces, south Mozambique. A total of 809 serum samples from cattle were obtained and tested by indirect enzyme-linked immunosorbent assay (i-ELISA). The chi-square test at 5 percent significance was used to assess the association between seroprevalence and the variables gender, age and geographic origin of animals. The overall seropositivity was 76.5 percent (n = 619) and anti-A. marginale antibodies were detected in 89.1 percent (n = 156), 68.4 percent (n = 308) and 84.2 percent (n = 155) of the animals in the provinces of Maputo, Gaza and Inhambane, respectively. A significant association (p < 0.05) was found with the geographic origin of the animals, while sex had no significant relationship. The frequencies of seropositive in the age groups were 63.2 percent (n = 72), 80.0 percent (n = 92), 83.1 percent (n = 98) and 77.3 percent (n = 357) for animals <12; >12 and <24; >24 and <36; >36 months, respectively. These results indicate that in southern Mozambique there are areas of enzootic stability to A. marginale. Thus, epidemiological monitoring is required to monitor the immune status of animals in the region.

O objetivo do presente estudo foi investigar a soroprevalência de anticorpos da classe IgG contra Anaplasma marginale em bovinos de corte da região Sul de Moçambique. Para esse efeito, 809 amostras de soro foram coletadas e avaliadas pelo ensaio imunoadsorção enzimático indireto (ELISA-i). O teste Qui-Quadrado, a 5 por cento de significância, foi utilizado para avaliar a associação entre a soroprevalência e as variáveis sexo, idade e origem geográfica dos animais. A soropositividade geral foi de 76,5 por cento (n = 619), e anticorpos anti-A. marginale foram detectados em 89,1 por cento (n = 156), 68,4 por cento (n = 308) e 84,2 por cento (n = 155) dos animais nas províncias de Maputo, Gaza e Inhambane, respectivamente. Uma associação significativa (p < 0,05) foi observada entre a origem geográfica dos animais, enquanto o sexo não demonstrou uma relação significativa. A frequência de soropositivos com relação à faixa etária foi de 63,2 por cento (n = 72), 80,0 por cento (n = 92), 83,1 por cento (n = 98) e 77,3 por cento (n = 357) para animais de <12; >12 a <24; >24 <36; >36 meses, respectivamente. Os resultados demonstram que, no Sul de Moçambique, existem áreas de estabilidade enzoótica para A. marginale, em animais maiores de 12 meses. Assim, monitoramento epidemiológico deve ser realizado para o acompanhamento do status imunológico dos animais na região.

Seroprevalence of IgG antibodies against Anaplasma marginale in cattle from south Mozambique / Soroprevalência de anticorpos de classe IgG contra Anaplasma marginale em bovinos da região Sul de Moçambique

The current study aimed to investigate the seroprevalence of IgG antibodies to Anaplasma marginale in cattle from Maputo, Gaza and Inhambane provinces, south Mozambique. A total of 809 serum samples from cattle were obtained and tested by indirect enzyme-linked immunosorbent assay (i-ELISA). The chi-square test at 5 percent significance was used to assess the association between seroprevalence and the variables gender, age and geographic origin of animals. The overall seropositivity was 76.5 percent (n = 619) and anti-A. marginale antibodies were detected in 89.1 percent (n = 156), 68.4 percent (n = 308) and 84.2 percent (n = 155) of the animals in the provinces of Maputo, Gaza and Inhambane, respectively. A significant association (p < 0.05) was found with the geographic origin of the animals, while sex had no significant relationship. The frequencies of seropositive in the age groups were 63.2 percent (n = 72), 80.0 percent (n = 92), 83.1 percent (n = 98) and 77.3 percent (n = 357) for animals <12; >12 and <24; >24 and <36; >36 months, respectively. These results indicate that in southern Mozambique there are areas of enzootic stability to A. marginale. Thus, epidemiological monitoring is required to monitor the immune status of animals in the region.(AU)

O objetivo do presente estudo foi investigar a soroprevalência de anticorpos da classe IgG contra Anaplasma marginale em bovinos de corte da região Sul de Moçambique. Para esse efeito, 809 amostras de soro foram coletadas e avaliadas pelo ensaio imunoadsorção enzimático indireto (ELISA-i). O teste Qui-Quadrado, a 5 por cento de significância, foi utilizado para avaliar a associação entre a soroprevalência e as variáveis sexo, idade e origem geográfica dos animais. A soropositividade geral foi de 76,5 por cento (n = 619), e anticorpos anti-A. marginale foram detectados em 89,1 por cento (n = 156), 68,4 por cento (n = 308) e 84,2 por cento (n = 155) dos animais nas províncias de Maputo, Gaza e Inhambane, respectivamente. Uma associação significativa (p < 0,05) foi observada entre a origem geográfica dos animais, enquanto o sexo não demonstrou uma relação significativa. A frequência de soropositivos com relação à faixa etária foi de 63,2 por cento (n = 72), 80,0 por cento (n = 92), 83,1 por cento (n = 98) e 77,3 por cento (n = 357) para animais de <12; >12 a <24; >24 <36; >36 meses, respectivamente. Os resultados demonstram que, no Sul de Moçambique, existem áreas de estabilidade enzoótica para A. marginale, em animais maiores de 12 meses. Assim, monitoramento epidemiológico deve ser realizado para o acompanhamento do status imunológico dos animais na região.(AU)

PREVALÊNCIA DE ANTICORPOS ANTI Anaplasma marginale (RICKETTSIALES: ANAPLASMATACEAE) EM BOVINOS NA MESORREGIÃO DO MÉDIO PARAÍBA

Serumprevalence of antibodies against Anaplasma marginale were studied by the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Sera samples from 223 bovines of eight municipalities in the "Médio Paraíba" mesorregion, in the state of Rio de Janeiro state, were analysed. The prevalence showed 98.21% of positives, of which were: 36.32% with a titre of 1:500, 43.50% of 1:1000, 12.56% of 1:2000, 3.59% of 1:4000, 1.79% of 1:8000, 0.45% of 1:16000, and 1.79% were negative. The evaluation was done within three age groups: 1 to 3 years (n = 53), 3 to 6 years (n = 71) and > 6 years (n = 99), of which 96.23%, 100% and 97.98% were positive, respectively. Regarding to the breed, 100% of beef cattle (n = 38) and 97.84% of dairy cattle (n = 185), were positive. According to the sex, 98.91% of the females (n = 183) and 95.00% of the males (n = 40), were positive. There were no significant differences (P > 0.05) between the prevalence of age groups, breeding types and sexes. There were significant differences (P 0.0001) among the serumprevalence in the municipalities. The region was characterized as an enzootically stable area to A. marginale.

Realizou-se a pesquisa de anticorpos da classe IgG anti Anaplasma marginale através do ensaio de imunoadsorção enzimática (ELISA) indireto em 223 amostras de soros de bovinos provenientes de oito municípios da mesorregião do Médio Paraíba, estado do Rio de Janeiro. A prevalência observada foi de 98,21%. Das amostras positivas, 36,32% possuíam o título de 1:500, 43,50% com título de 1:1000, 12,56% com título de 1:2000, 3,59% com título de 1:4000, 1,79% com título de 1:8000, 0,45% com título de 1:16000 e 1,79% foram soronegativos. Procedeu-se a avaliação da prevalência por grupos etários de 1 a 3 anos (n = 53), 3 a 6 anos (n = 71) e > 6 anos (n = 99), na qual 96,23%, 100% e 97,98% dos animais foram soropositivos, respectivamente. A análise em relação à aptidão zootécnica revelou que 100% dos animais com aptidão para corte (n = 38) e 97,84% com aptidão para leite (n = 185) reagiram ao ensaio. Dos animais estudados, 183 eram fêmeas e 40 machos e, destes, 98,91% e 95,00% foram positivos, respectivamente. A análise estatística demonstrou não haver diferença significativa (P > 0,05), de prevalência, entre idades, aptidões zootécnicas e entre sexos. Houve diferença significativa (P 0,0001) de soropositivos entre os municípios. A região foi caracterizada como uma área de estabilidade enzoótica para A. marginale.

PREVALÊNCIA DE ANTICORPOS ANTI Anaplasma marginale (RICKETTSIALES: ANAPLASMATACEAE) EM BOVINOS NA MESORREGIÃO DO MÉDIO PARAÍBA

Serumprevalence of antibodies against Anaplasma marginale were studied by the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Sera samples from 223 bovines of eight municipalities in the "Médio Paraíba" mesorregion, in the state of Rio de Janeiro state, were analysed. The prevalence showed 98.21% of positives, of which were: 36.32% with a titre of 1:500, 43.50% of 1:1000, 12.56% of 1:2000, 3.59% of 1:4000, 1.79% of 1:8000, 0.45% of 1:16000, and 1.79% were negative. The evaluation was done within three age groups: 1 to 3 years (n = 53), 3 to 6 years (n = 71) and > 6 years (n = 99), of which 96.23%, 100% and 97.98% were positive, respectively. Regarding to the breed, 100% of beef cattle (n = 38) and 97.84% of dairy cattle (n = 185), were positive. According to the sex, 98.91% of the females (n = 183) and 95.00% of the males (n = 40), were positive. There were no significant differences (P > 0.05) between the prevalence of age groups, breeding types and sexes. There were significant differences (P 0.0001) among the serumprevalence in the municipalities. The region was characterized as an enzootically stable area to A. marginale.

Realizou-se a pesquisa de anticorpos da classe IgG anti Anaplasma marginale através do ensaio de imunoadsorção enzimática (ELISA) indireto em 223 amostras de soros de bovinos provenientes de oito municípios da mesorregião do Médio Paraíba, estado do Rio de Janeiro. A prevalência observada foi de 98,21%. Das amostras positivas, 36,32% possuíam o título de 1:500, 43,50% com título de 1:1000, 12,56% com título de 1:2000, 3,59% com título de 1:4000, 1,79% com título de 1:8000, 0,45% com título de 1:16000 e 1,79% foram soronegativos. Procedeu-se a avaliação da prevalência por grupos etários de 1 a 3 anos (n = 53), 3 a 6 anos (n = 71) e > 6 anos (n = 99), na qual 96,23%, 100% e 97,98% dos animais foram soropositivos, respectivamente. A análise em relação à aptidão zootécnica revelou que 100% dos animais com aptidão para corte (n = 38) e 97,84% com aptidão para leite (n = 185) reagiram ao ensaio. Dos animais estudados, 183 eram fêmeas e 40 machos e, destes, 98,91% e 95,00% foram positivos, respectivamente. A análise estatística demonstrou não haver diferença significativa (P > 0,05), de prevalência, entre idades, aptidões zootécnicas e entre sexos. Houve diferença significativa (P 0,0001) de soropositivos entre os municípios. A região foi caracterizada como uma área de estabilidade enzoótica para A. marginale.