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Anaplasma marginale is an etiologic agent of bovine anaplasmosis. This study aimed to molecularly detect and characterize A. marginale that is prevalent in Mongolian cattle populations. A highly specific and sensitive nested PCR (nPCR) method based on the Msp5 gene was developed to detect A. marginale (Msp5 nPCR). The method detected A. marginale from the positive DNA samples obtained from different countries, while no amplicons were observed from DNA samples of several other bovine blood pathogens tested. The detection limit of Msp5 nPCR was determined to be 2 copies/μl. The method was tested against field blood DNA samples prepared from 300 Mongolian cattle in 2010. Results indicated a prevalence rate of 8.7% (26 of 300). On the other hand, partial DNA fragments of an Anaplasma sp. closely related to A. ovis (with 95.0% identity) were detected using a different nPCR method based on groEL gene. The phylogenetic analyses based on the Msp5, groEL and 16S rRNA genes demonstrated that A. marginale isolates in Mongolia were not divergent from the isolates distributed in other countries. The present study successfully established a new nPCR assay that can detect A. marginale, and reported the first molecular detection and characterization of A. marginale and an Anaplasma sp. closely related to A. ovis in Mongolian cattle populations.

Anaplasma marginale has been detected in the Philippines only by peripheral blood smear examination and serological methods. This study generally aimed to molecularly detect and characterize A. marginale in cattle and ticks in Cebu, Philippines. A total of 12 bovine blood samples and 60 Rhipicephalus (Boophilus) microplus ticks were collected on the Cebu Island in 2011. 16S rRNA-based screening-PCR and DNA sequencing revealed 8 cattle (66.7%) and 8 ticks (13.3%) to be positive for A. marginale, and 1 tick (1.7%) to be positive for A. centrale. Selected positive DNA samples were further characterized based on 16S rRNA (longer sequence), Msp5, Msp1α,gltA and groEL genes for phylogenetic analyses. Sequence identities of partial DNA fragments of A. marginale from the Philippines revealed 99.1-100% (16S rRNA, gltA, groEL and Msp5) and 94.3-97.6% (Msp1α) identities to the closest isolates from other countries. Moreover, sequence analysis of the Msp1α, gene showed 3 variants, including a case of co-infection with 2 variants. Phylogenetic analyses based on Msp1α and Msp5 genes revealed that Philippine A. marginale isolates formed a monophyletic lineage, and were phylogenetically related to Brazilian and Chinese isolates. On the other hand, a highly specific and sensitive nested PCR based on groEL, with a detection limit of 2 copies/PCR, was developed to detect A. marginale in the Philippines. This study reported the first molecular detection and characterization of A. marginale in cattle and R. microplus in Cebu, Philippines.