Use of semi-nested PCR for the diagnosis and molecular identification of Babesiagibsoni infection in dogs

Diagnosis of canine babesiosis, caused by Babesiagibsoni is difficult, especially in dogs with chronic infection or carrier. A Semi-nested Polymerase Chain Reaction assay was developed and standardized by using three oligonucleotide primers targeting the hypervariable region of18SrRNA gene. The specific primers amplified theB. gibsoniDNA, while no nonspecific amplification was detected with DNA from non-infected dogs as well as from dogs infected with other species. All the samples were tested by blood smear examination as well as semi-nested PCR assay. Semi-nested PCR assay was found to be more sensitive than blood smear examination in diagnosis and molecular confirmation of Babesiagibsoni. Out of 273 suspected blood samples, collected from dogs presented to the Teaching Veterinary Clinical complex, Mannuthy, Kerala, India and 34 were confirmed for B.gibsoniby semi-nested PCR.Sequencing of 183bp PCR product revealed that the amplified product was from a region of 18SrRNA gene. The sequence obtained when analysed using BLAST revealed 100 per cent homology with query coverage of 100 per cent with the published B. gibsoni (Asia genotype) gene sequence.