Molecular Characterization of Wolbachia Endosymbionts and Their Association With Canine Dirofilariasis in Colombo District, Sri Lanka.

Abstract

Canine dirofilariasis is caused by Dirofilaria, a type of filarial parasite that can also infect humans and is becoming a growing concern in Sri Lanka. Previous studies have noted that Sri Lanka has some of the highest numbers of dirofilariasis cases in Asia. This study was aimed at detecting Wolbachia bacteria-which are found inside Dirofilaria parasites-using molecular methods and also at identifying which Dirofilaria species are present in dogs in the Colombo District, Sri Lanka. Blood was collected from 368 dogs of various breeds between May and December 2025. Of these, only 35 samples (9.51%) tested positive for microfilaria using a microscope. These samples were then analyzed using DNA extraction and PCR with both general Dirofilaria and species-specific primers. At the same time, samples were also screened for Wolbachia, a bacterium that is important for Dirofilaria survival and reproduction, using Wolbachia-specific primers (wsp). Of the 35 microfilaria-positive samples, 18 (51.43%) were positive for Dirofilaria, and of those 18, only three (16.67%) were positive for Wolbachia. Among the Dirofilaria-positive samples, one (5.56%) was identified as Dirofilaria immitis, 11 (61.11%) as Dirofilaria repens, and three (16.67%) as Dirofilaria asiatica; a few samples did not match any of the species-specific primers. The PCR products for both Dirofilaria and Wolbachia were verified by gel electrophoresis and sequencing. The sequence results showed the presence of D. repens and D. asiatica in dogs. Importantly, this is the first molecular evidence of D. immitis in Sri Lanka and the first molecular identification of Wolbachia in Dirofilaria species in the country. The study also found that both D. repens and D. immitis can infect the same dog. These findings provide new information about canine dirofilariasis in Sri Lanka and highlight the need for Wolbachia-targeted parasite control and accurate molecular diagnosis to guide treatment and control efforts.