Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in tropical and subtropical regions of the world and results in economic losses in the cattle industry. Major surface proteins (MSPs) have been used as markers for the genetic characterization of A. marginale strains and demonstrate that many isolates may occur in a given geographic area. However, in Brazil, little is known about the genetic diversity of A. marginale isolates within individual herds. This study was designed to examine the genetic variation among A. marginale infecting calves in a farm in the south of Minas Gerais State, Brazil. Blood samples collected from 100 calves were used to prepare Giemsastained smears that were microscopically examined for the presence of A. marginale. From each blood sample, DNA was extracted and analyzed by a polymerase chain reaction (PCR), followed by sequencing to determine diversity among the isolates. Examination of blood smears showed that 48% of the calves were infected with A. marginale, while the real-time PCR detected 70.2% positivity. Congenital infections were found in four calves. The microsatellite and tandem repeat analyses showed high genetic diversity among the isolates.Keywords: Anaplasma marginale, MSP1a, DNA sequencing, microsatellites, tandem repeats, Brazil. ResumoA anaplasmose bovina, causada pela rickettsia Anaplasma marginale e transmitida por carrapatos, é endêmica em regiões tropicais e subtropicais no mundo e causa grandes perdas econômicas na industria de bovinos. Proteínas principais de superfície (MSPs) foram usados como marcadores para a caracterização genética de amostras de A. marginale, demonstrando que diferentes isolados podem ocorrer numa certa região geográfica. Porém, no Brasil pouco se sabe sobre a variedade genética de isolados de A. marginale em rebanhos individuais. Este estudo teve como objetivo investigar a ocorrência de variação genética entre bezerros infectados com A. marginale numa fazenda do sul de Minas Gerais, Brasil. Amostras de sangue coletadas de 100 bezerros foram utilizadas para o preparo de esfregaços sanguíneos, corados pelo Giemsa, para detecção da infecção por A. marginale. Amostras de DNA extraídas de cada amostra foram analisadas através de PCR seguido de sequenciamento. O exame dos esfregaços demonstrou que 48% dos bezerros estavam infectados com A. marginale, enquanto que o PCR detectou 70,2% de positividade. Infecção congênita foi detectada em quatro bezerros. As análises de microsatélites e 'tandem repeats' comprovaram uma grande diversidade genética entre os isolados.Palavras-chave: Anaplasma marginale, MSP1a, sequenciamento de DNA, microsatélites, tandem repeats, Brasil.