Development of real-time PCR assay for detection of Anaplasma marginale

Anaplasma marginale is a rickettsial pathogen responsible for bovine anaplosmosis, the acute disease in cattle herds which is associated with anemia, fever, rapid loss of milk production and weight, abortion, and, in some cases, death of the infected cattle. Anaplasma marginale is transmitted by ticks and biting insects. Diagnosis of bovine anaplasmosis is made by microscopic examination of blood smears stained with Giemsa stain, but this method is not useful to detect presymptomatic animals. Several serological tests have used extensively for epidemiological studies, but they do not discriminate between different Anaplasma species. A real-time polymerase chain reaction (PCR) combines high specificity with accurate measurement of DNA copy number and allows quantification of the targeted pathogen DNA. The goal of this study was to develop a real-time PCR assay for differential detection of A. marginale in the blood of cattle. The single-copy gene msp4 was chosen as a target DNA for PCR. Msp4 is a dominant immune protein of outer membrane of all Anaplasma knowen to date. The primers for phylogenetic analysis in А. marginale based on msp4 were reported earlier by J. de la Fuente et al. (2001), but they were not species-specific. The analysis of msp4 gene sequence of different A. marginale isolates and closely related species, including A. ovis, revealed species-specific areas, which were used for design of primers and TaqMan probe (MSP4-F 5′-CA-TGAGTCACGAAGTGGCT-3′ and MSP4-R 5′-GGCACACT-CACATCAATC-3′, MSP4-probe 5′-(Cy5)-AAGGGGGAGTAATGGGAGGTAGCT-3′) for amplification and detection of 177 bp fragment of msp4 gene by a real time PCR. In the amplified nucleotide sequences a 99 to 100 % homology to msp4 fragments was found in different isolates of A. marginale. To assess analytical sensitivity of our PCR test, we used pGEM -msp4, a constructed recombinant plasmid with 177 bp fragment of msp4 gene, diluted to obtain samples with 10 0-10 7 msp4 copies. It was shown that the assay was able to detect as few as 10 2 of A. marginale msp4 gene in the analyzed DNA sample. Analytical specificity of the developed primers and the MSP4-probe was proved in tests with DNA of sheep naturally infected by A. ovis, and also DNA isolated from cows with Sanguibacter keddieii, Propionibacterium acnes and Pseudomonas aeruginosa infection pre-detected by sequencing. In these samples no increased fluorescence characteristic of probes from animals infected by A. marginale was observed with no PCR products identified. Thus, the method specificity allowed to differ A. marginale and A. ovis. The developed method of A. marginale identification on the basis on amplification and detection of the msp4 gene fragment using a real time PCR differed from known analogues with high sensitivity, rapidity and opportunity of quantitative evaluation of the bacterial load. The developed method could be used for rapid differential detection and quantification of А. marginale in blood samples from infected cattle for confirmation of anaplasmosis and epidemiological studies