Evaluation of different nested PCRs for detection of Anaplasma phagocytophilum in ruminants and ticks.

BACKGROUND Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in mammals, which has a broad geographical distribution and a high degree of clinical diversity. Currently, numerous PCR assays have been developed and used for the detection of A. phagocytophilum in various specimens. However, their performance varies. The aim of this study was to evaluate the performance of five nested PCR assays by detection of 363 ruminant and tick samples, and to select the most appropriate methods for the sensitive detection of A. phagocytophilum in environmental or clinical samples. RESULTS Positive PCR results for A. phagocytophilum were obtained in 75 (20.7%), 42 (11.6%) and 19 (5.2%) specimens with primer sets EC (EC9/EC12a and SSAP2f/SSAP2r), EE (EE1/EE2 and EE3/EE4) and ge (ge3a/ge10r, ge9f/ge2), respectively. The amplification of template DNA with the primer set MSP (MAP4AP5/MSP4AP3, msp4f/msp4r) could not be obtained in both ruminants and ticks, and a low specificity of the EL primers [EL(569)F/EL(1193)R, EL(569)F/EL(1142)R] in tick samples was observed. Our results revealed that the nested PCR with primer set EC complementary to the 16S rRNA gene was the most sensitive assay for detection of A. phagocytophilum in ruminant and tick specimens. A. phagocytophilum was detected in 47 (35.1%) sheep, 12 (10.4%) cattle, and 17 (14.9%) ticks. Two A. phagocytophilum genotypes were identified, that varied between sheep and cattle in sample collection sites. CONCLUSIONS This report provides more valuable information for the diagnosis and management of granulocytic anaplasmosis in China.