Absolute Quantification of Babesia canis in Stray Dogs by qPCR in Al Diwaniyah, Iraq

Canine babesiosis is a serious tick-borne disease that threatens dog health worldwide. It is caused by protozoan parasites of the genus Babesia, transmitted mainly by the brown dog tick. The disease leads to anemia, fever, and sometimes death. This study aimed to establish a quantitative PCR (qPCR) assay for accurate detection and measurement of Babesia canis in stray dogs in Al Diwaniyah Province, Iraq. A total of 45 blood samples were collected from stray dogs brought to private clinics. DNA was extracted and analyzed using PCR targeting the HSP70 gene of B. canis. Results showed that 34 out of 45 samples (75.5%) were positive for Babesia spp. The amplicons were sequenced, and their accession numbers were deposited in GenBank. Phylogenetic analysis confirmed high similarity between local strains and global isolates. A gradient protocol was applied to optimize qPCR conditions. Annealing at 60 °C produced the best amplification for both B. canis and the housekeeping gene GAPDH. Plasmid standards were synthesized and used as calibrators to calculate absolute copy numbers. The qPCR results showed a wide distribution of parasite loads among positive dogs. The normalized copy number of B. canis relative to GAPDH provided accurate quantification. This study confirms a high prevalence of B. canis in stray dogs and demonstrates the value of qPCR for sensitive diagnosis. The method can be applied in epidemiological studies and in monitoring infection levels to support better control of canine babesiosis in Iraq.