Studies of the dinamycs of Anaplasma marginale strains in the vector and the mammalian host through genotyping

PEREZ AGUSTINA; SARMIENTO, NESTOR; PERTILE CARLA; FARBER MARISA D; GUILLEMI, ELIANA

La Plata

Congreso; II Congreso de la Sociedad Latinoamericana de Ecología de Vectores (LA SOVE 2022); 2022

Sociedad Latinoamericana de Ecologia de Vectores

Anaplasma marginale is a gram-negative obligate intracellular tick-borne bacterium that infects erythrocytes of ruminants and other mammalian host species from tropical and subtropical world regions. In Argentina, the transmission of A. marginale is mainly associated to the one-host tick, Rhipicephalus microplus. Although the presence of A. marginale DNA was previously confirmed in R. microplus larvae by PCR, the transovarial transmission of the bacterium in the tick remains neglected. The aim of the present study was to detect and molecularly characterize A. marginale in the bovine and in organs from R. microplus engorged female removed from the host, in order to determine the A. marginale genotypes involved in the transmission process. For this purpose, blood and tick samples were obtained from a bovine with acute Anaplasmosis at an endemic region in Corrientes province, Argentina. In order to get rid of the host blood meal, we dissected a female engorged tick and extracted the ovaries and salivary glands. DNA was extracted from 400 μl of the bovine blood sample using the ADN PuriPrep-S kit (INBIO Highway) and from the tick tissues using the NucleoSpin Tissue kit (Macherey-Nagel). For A. marginale identification, we used a species-specific PCR protocol that amplifies two copies of the msp1β gene. We also amplified, cloned and then sequenced the 5’ end repetitive region of the msp1α gene, broadly used for genotyping A. marginale strains. We detected A. marginale in the bovine blood sample and in the tick organs. We analyzed 30 clones from the bovine blood sample and 8 clones for each tick organ. We then determined the number and type of tandem repeats for each MSP1α coding region. There was a great diversity of genotypes in the bovine (19) that were formed by different combinations of 20 repeats, with a minimum of one (AR12) and a maximum of 8 (AR9-62-61-AR10-62-62-61-AR14). Engorged females organs showed a fewer number of different genotypes (ovaries: 2; salivary glands: 3) and repeats involved (8). Two genotypes found in the engorged female (1 in salivary glands -AR15-62-62-62-61- and other in ovaries –AR9-62-66-62-61-) were not detected in the bovine host which could be the result of a previous transovarial transmission process of A. marginale. The high number of A. marginale genotypes detected in the infected bovine and the reduce number in the tick organs suggest a diverse genotype fitness at least for transovarial transmission. Moreover, these results help to explain how a monoxenic cycle tick as R. microplus, could be responsible for the large distribution of anaplasmosis in enzootic regions.