Isolamento e caracterização molecular de duas novas cepas de Anaplasma marginale e estabelecimento de linhagens de células embrionárias do carrapato Rhipicephalus (Boophilus) microplus

Abstract

In the first chapter two new strains of Anaplasma marginale derived from the Seropédica, RJ were isolated and characterized in tick cells line of Ixodes scapularis (IDE8), enabling new studies in the diagnosis with the use of local strains. In the second chapter the first lines of embryonic cells of Rhipicephalus (Boophilus) microplus from Brazilian strains were developed and characterized. The strains of A. marginale were from blood of two naturally infected calves belonging to the Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica. After isolation, the strains were characterized morphologically by light microscopy and molecularly by nested PCR of msp1α gene. Two strains of A. marginale were isolated and named as AmRio1 and AmRio2. Small colonies were observed in cytospin smears stained with Giemsa’s 1:10, of each of the isolates, 60 days after culture initiation. Analysis of msp1α microsatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of one MSP1α tandem repeat from the strain AmRio1 resulted in a new sequence (named 162) with one amino acid change. The results of our phylogenetic analysis demonstrated that A. marginale strains from Brazil and Argentina formed two large clusters. In establishing of the strains of R. (B.) microplus, the ticks were from strain kept calves in Parasitic Diseases Laboratory, UFRRJ, Seropédica. After 13 days of laying, the pool of eggs was sterilized, macerated, filtered and centrifuged at 200 xg for 10 minutes. The pellet was suspended in Leibotiz’s L15 medium and divided into 6 flasks of 25 cm2, of which 3 were contaminated, one was deposited in the cell bank of the Pirbright Institute and others two were characterized in the present study (BMEBR1 and BMEBR2 lines) through molecular analysis of the 16S rDNA gene (PCR), growth curve and epifluorescence and light microscopy. Sixty days in culture, the cells began to show confluent aspect, reaching the formation of the monolayer. There was a complete similarity (100%) of new lines BMEBR1 and BMEBR2 with R. (B.) microplus in Brazil (EU918178.1), Costa Rica (EU918179.1), Paraguay (EU918180.1), among others. Both strains showed lower growth than the R. (B.) microplus line from Costa Rica, BME/CTVM2. The BMEBR1 line has increased phagocytic ability that the BMEBR2 and BME/CTVM2 lines. In addition, these strains have acidification characteristics, lipid corpuscle production and oxidative stress different from each other. The new strains were frozen and defrosted successfully, and were stable with respect to morphology and growth rate. Thus, these lines can be used in future studies to propagation of new strains of A. marginale isolated.