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ThesisItem Open Access STUDIES ON Dirofilaria immitis IN DOGS AND ITS ASSOCIATION WITH Wolbachia SPECIES(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781022, 2013-06) Borthakur, Sonjoy Kumar; Deka, Dilip Kr.Dirofilaira immitis is an important canine filarial nematode. An epidemiological study was carried out to record the prevalence of D. immitis in dogs in two different geographical locations viz., Guwahati, Assam and Aizawl, Mizoram of North Eastern Region in India, from February, 2011 to July, 2012. The study also included to evaluate the persistence of Wolbachia endosymbiont with D. immitis. In the present study, dogs were grouped into three categories, i.e., stray, pet and working dogs, their respective numbers being 413, 266 and 103 irrespective of the study regions. Three different methods were used for the study, i.e., microscopy (wet film and KCT), immunological (Ag ELISA by SNAP®4Dx kit) and molecular techniques (PCR). The study revealed overall heartworm prevalence in Guwahati to be higher (18.23%) than in Aizawl (17.68%) irrespective of categories of dogs. Sex-wise, the infection was higher in male (18.12%) than in female (17.90%), though the difference was statistically non-significant. The overall efficacy percentage for detection of heartworm by wet film, KCT, Ag ELISA and PCR test revealed 6.26, 11.38, 18.03 and 13.93 percent, respectively. Ag ELISA test was found to be the best amongst the three types of tests compared. Using molecular tools, prevalence of D. immitis in dogs was 13.52 percent in Guwahati and in Aizawl was 14.62 percent. With PCR, 4 cases of D. repens could be diagnosed in stray dogs from Guwahati. The study revealed overall 22.69 percent occult infection, of which, highest cases were recorded in working dogs (60%). Occult infection was calculated by finding the difference between heartworm prevalence based on Ag ELISA and PCR test. Dot ELISA test using monoclonal antibody of D. immitis for detection of heartworm antigen in dog blood samples was standardized. The test revealed 72% specificity against known positive D. immitis blood samples at SNAP®4Dx commercial kit. Molecular technique using PCR was standardized to detect D. immitis using published primers with slight modification of thermal condition. Two different primers were used viz., specific primers for D. immitis only and another, pan filarial primers for detecting six different canine filariids. Both the primers resulted desired amplification product size against different filarial parasites. Molecular cloning and characterization of D. immitis for ITS-2 region of Guwahati isolates were conducted. The results showed the Guwahati isolates had a close relationship with that of South Asian isolates of D. immitis. Pair-wise homology analysis revealed 98.6 - 98.9% similarity with a few sequences available at NCBI GenBank. Similarly, phylogenetic analysis of D. repens encountered in Guwahati isolate was also done. Endosymbiont Wolbachia association with D. immitis worm as well as in heartworm infected blood was revalidated by PCR method. The findings were substantiated with the presence of the organisms in the worm’s lateral cord by fine structural studies conducted through transmission electron microscopy (TEM). Molecular evidence followed by sequence analysis of Wolbachia revealed 99.4 to 99.8% similarities with other sequences available in NCBI GenBank for Wolbachia endosymbiont of D. immitis. Finding of the present studies establish the endemicity of D. immitis in North East India and validates the association of Wolbachia endosymbiont in D. immitis. Record of D. repens warrants further detail studies owing to its zoonotic significance.