Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale

Anaplasma ovis and Anaplasma marginale are tick-transmitted bacteria that cause anaplasmosis in domestic and wild animals. Recent results show that some domestic and wild animals and ticks are susceptible to both A. ovis and A. marginale, thus supporting the need to differentiate between these species in hosts and ticks diagnosed with Anaplasma infection. However, although anaplasmosis is one of the most common diseases of grazing animals worldwide, rapid and effective tests are not available for the detection of and discrimination between these 2 Anaplasma species. The objective of this research was to develop an easy and reliable method to identify and discriminate between the closely related pathogens A. ovis and A. marginale. A. ovis and A. marginale major surface protein 4 (msp4) gene sequences were retrieved from different geographic strains and aligned to design 2 sets of primers in a region with significant differences between the 2 species, but completely conserved among strains. PCR reactions using these primers were 100% species-specific and detected all strains from each pathogen previously identified with other methods. The 2 sets of primers designed for the specific PCR amplification of A. ovis and A. marginale allow easy-to-detect and discriminate between the 2 pathogens, thus avoiding the time-consuming sequencing or multi-gene amplification procedures. This PCR provides a tool for the detection of A. ovis and A. marginale in ticks and in wildlife and domestic hosts.