Application of msp1? PCR and 16S rRNA semi nested PCR-RFLP for detection of persistent anaplasmosis in tick infested cattle

Molecular diagnostic techniques viz; PCR targeting msp1? and semi nested PCR targeting 16SrRNA gene with subsequent restriction digestion of nested PCR products were employed for detection and identification of Anaplasma marginale DNA in tick infested cattle. A total of 120 blood samples along with ticks were collected from tick infested dairy cattle in and around Ludhiana district of Punjab, India. Prevalence of Rhiphicephalus (Boophilus) microplus infestation was highest (89.16%), followed by Hyalomma anatolicum anatolicum (7.50%) and mixed infestation (3.33%). The detection percentage of A. marginale by stained thin blood smear examination of blood samples was found to be 15.83%. The BAP-2 and AL34S set of oligonucleotide primers used for amplifying msp1? gene of A. marginale detected DNA in 39.16% (47/120) cattle blood and detection percentage was more in adult cattle (42.30%) as compare to calves (18.75%). The semi nested PCR specific for Anaplasma genus revealed amplicon of 577 bp in 40.83 % (49/120) samples. Further restriction digestion of 577 bp semi-nested PCR products by Bst 1107I at A. marginale specific GTATAC site confirmed 42 out of 49 specific for A. marginale while remaining 7 undigested products indicate the presence of other species of Anaplasma in the region.