Caracterização molecular de msp1 alfa da cepa AmRio1 de Anaplasma marginale em bezerro e carrapatos experimentalmente infectados

Abstract

Bovine anaplasmosis, caused by intraerythrocytic rickettsia Anaplasma marginale (Rickettsiales: Anaplasmataceae), is an economically important disease of cattle which is endemic in tropical and subtropical regions of the world. Major surface proteins (MSP) have been widely used in studies involving A. marginale, MSP1a being one of the most studied because it is an adhesin for bovine erythrocytes and tick cells, thus having an important role in the agent's transmission phenotype. At the same time, it is regarded as a stable marker for strains of A. marginale, since it has a highly conserved C-terminal region and a highly variable N-terminal region, but constant for strains. It is believed that this protein can undergo selective pressure because it has a neutralization-sensitive epitope in its variable region. Therefore, in random changes in the structure of tandem repeats due to mutation, well-adapted strains could prevail in the environment. Thus, the aim of this study was to evaluate the diversity of msp1 α gene of A. marginale in acute and chronic infection in one experimentally infected immunocompetent calf and in ticks. In the study, a calf, which was experimentally infected intravenously with AmRio 1 strain of A. marginale, was used. This calf was monitored by observing clinical signs, rectal temperature, blood smears and hematocrit of day-2 after experimental infection until the day 28 after infection. The bovine was subsequently infested with 1g of Rhipicephalus (Boophilus) microplus larvae. The calf's blood was collected from day 2 to day 119 after infection. Ticks were recovered in partenogenetic phase (n=61) and 43 underwent dissection to remove the gut and salivary gland, and 18 for saliva collection using pilocarpine 2%. The samples were submitted to Polymerase Chain Reaction (PCR), having msp1 α as target gene. The product of this reaction was submitted for sequencing in both directions. After inoculation of the strain AmRio 1 of A. marginale, the bovine showed clinical signs and changes in hematocrit and temperature, with supportive treatment necessary for the animal's recovery. This demonstrates the pathogenicity of the strain. All samples of the calf and the ticks were positive in PCR. The sequences revealed a stability in repeated sequences of MSP1a in both acute and chronic infection in the calf and all tick organs and saliva. Positivity of tick saliva suggests that the strain AmRio 1 of A. marginale is transmitted biologically by R. (Boophilus) microplus. In addition, tick organs had large amounts of DNA of A. marginale, indicating the agent's replication.