Detecting and Identifying Wolbachia pipientis in Occult Dirofilariasis Using a High-resolution Melting Real-time PCR in Stray Dogs

Background: Iran is one of the endemic areas of Dirofilaria immitis. The modified Knott test is the common diagnostic test based on detecting and identifying microfilaria in blood samples. Still, the results may be compromised by D. immitis occult infection (D. immitis infection without microfilariae). Wolbachia pipientis in a dog’s blood is an endosymbiont of D. immitis and an indicator of dirofilariasis. Objectives: This study aimed to investigate the occult infection of D. immitis in stray dogs in Guilan, Qazvin, and Mazandaran provinces, Iran, based on W. pipientis DNA tracking.Methods: Blood samples (n=۱۳۸) were collected, and the presence of W. pipientis was evaluated by amplifying a partial sequence of the FtsZ gene (۲۶۷ bp). Results: The results showed that ۱۷.۴% of the samples were positive by the modified Knott method, while ۴۷.۸% were positive by the molecular methods. The results of high-resolution melting (HRM) real-time PCR showed that the melting temperature of cox۱ gene amplicons of D. immitis and Acanthocheilonema reconditum were obtained at ۷۶.۶۷±۰.۰۳ °C and ۸۱.۳۸±۰.۰۵ °C, respectively as well as it was achieved ۸۰.۳۰±۰.۰۴ °C for FtsZ gene of W. pipientis. The results of the present study showed that it is necessary to use molecular methods to diagnose dirofilariasis occult infection accurately.Conclusion: The HRM real-time PCR analysis is a simple post-PCR step that exploits the thermal characteristics of the amplicons to detect the occult infection of dirofilariasis based on W. pipientis DNA tracking and identification.

کلیدواژه ها:

Acanthocheilonema reconditum ، Dirofilaria immitis ، High-resolution melting (HRM) real-time PCR ، nematodes ، Stray dogs