CONICET | Buscador de Institutos y Recursos Humanos

A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of Anaplasma marginale. Only two molecular markers have been described for A. marginale so far. Major surface protein 4 (MSP4) nucleotide variations provides phylogeographic resolution for A. marginale isolates while MSP1a variable number of tandem repeat units in the 5-end region of the gene has been used to discriminate between genotypes. In addition, A. marginale has a reduced genome that render low frequency of variable number of tandem repeats after Tandem Repeat 82 Finder software analysis. This means that VNTR would not represent an accurate approach for A. marginale characterization. The aim of this study was to develop the MLST strategy to discriminate between A. marginale isolates. For this purpose, 14 housekeeping candidate genes were initially amplified and sequenced. These genes were selected mainly because of their use in previous bacterial MLST studies. After testing specific amplification against Babesia sp. and Bos taurus DNA and comparing the number of polymorphic sites in each gene, we selected 8 genes for further MLST studies. These genes were: dnaA, groEL, ftsz, lipA, sucB, sodB, recA, secY. Genetic variation among these 8 genes sequences was evaluated using the recently available genomes of A. marginale strains (str. Mississippi, str. Puerto Rico, str. Florida, and str. Virginia) and sequences from 4 other isolates (Mercedes, Salta, Quitilipi and South Idaho). Phylogenetic analysis was conducted using the program MEGA version 3.1. Neighbor-joining trees were generated selecting Kimura-2-parameter model; confidence values were determined by bootstrap analysis with 1000 replicates. Anaplasma phagocytophylum was considered as outgroup. Notably, two well supported clusters were found, where Argentinean isolates cluster split from North American one. Taking into account that more isolates must be included in the MLST analysis, these results show that this strategy, in accordance to the discriminatory power described for other bacteria, seems to be a promising tool for clearly distinguish A. marginale isolates.Anaplasma marginale. Only two molecular markers have been described for A. marginale so far. Major surface protein 4 (MSP4) nucleotide variations provides phylogeographic resolution for A. marginale isolates while MSP1a variable number of tandem repeat units in the 5-end region of the gene has been used to discriminate between genotypes. In addition, A. marginale has a reduced genome that render low frequency of variable number of tandem repeats after Tandem Repeat 82 Finder software analysis. This means that VNTR would not represent an accurate approach for A. marginale characterization. The aim of this study was to develop the MLST strategy to discriminate between A. marginale isolates. For this purpose, 14 housekeeping candidate genes were initially amplified and sequenced. These genes were selected mainly because of their use in previous bacterial MLST studies. After testing specific amplification against Babesia sp. and Bos taurus DNA and comparing the number of polymorphic sites in each gene, we selected 8 genes for further MLST studies. These genes were: dnaA, groEL, ftsz, lipA, sucB, sodB, recA, secY. Genetic variation among these 8 genes sequences was evaluated using the recently available genomes of A. marginale strains (str. Mississippi, str. Puerto Rico, str. Florida, and str. Virginia) and sequences from 4 other isolates (Mercedes, Salta, Quitilipi and South Idaho). Phylogenetic analysis was conducted using the program MEGA version 3.1. Neighbor-joining trees were generated selecting Kimura-2-parameter model; confidence values were determined by bootstrap analysis with 1000 replicates. Anaplasma phagocytophylum was considered as outgroup. Notably, two well supported clusters were found, where Argentinean isolates cluster split from North American one. Taking into account that more isolates must be included in the MLST analysis, these results show that this strategy, in accordance to the discriminatory power described for other bacteria, seems to be a promising tool for clearly distinguish A. marginale isolates.

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