Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats

Abstract

A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P ‫؍‬ 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.

A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine

“…Hence, in our study, a mimic EIC composed of an artificial sequence used in another study was utilized. 34 …”

Prevalence of Dirofilaria immitis Infection in Stray Cats by Nested PCR in Korea

“…Dirofilariosis has been reported in many warm-blooded animals, including snow leopards [ 1 ], coyotes [ 2 ], ferrets [ 3 ], cats [ 4 ], and humans [ 5 ]. The prevalence of feline heartworm infection is 10-20% of the prevalence in dogs within the same enzootic region [ 6 , 7 ]. Canines are a definitive host for Dirofilaria immitis , but its life cycle can also be completed in cats [ 8 ].…”

“…The natural resistance of cats to D. immitis and variations in mosquito feeding preferences partly explains the lower prevalence of infection in cats [ 9 ]. Additionally, the testing frequency is estimated to be 0.06% among cats, which is very low compared to 33% testing frequency in dogs [ 7 , 10 , 11 ]. Previous studies have reported that adult heartworms are present in 2.1-4.9% of shelter cats at necropsy [ 12 , 13 ].…”

“…Genomic DNA was extracted from whole blood isolates using a PrimePrep Genomic DNA isolation kit (GeNet Bio, Daejeon, Korea), and samples were stored at 4℃ until analysis by nested PCR. The following 2 PCR primer pairs were used to amplify the cytochrome c oxidase subunit I ( COI ) gene [ 7 ]: forward (5'-ATTGGTGGTTT TGGTAATTGGATGTTG-3') and reverse (5'-CAGAAGTCCCCAATACAGCAATCC-3'), which amplified a 634-bp fragment in the first round PCR and forward (5'-GGGTCCTGGGAGTAGTTGAAC-3') and reverse (5'-TTCACTAACAATCCCAAA CACCG-3'), which amplified a 406-bp fragment in the second round PCR. Nested PCR was performed using a method reported previously [ 7 ].…”

“…The following 2 PCR primer pairs were used to amplify the cytochrome c oxidase subunit I ( COI ) gene [ 7 ]: forward (5'-ATTGGTGGTTT TGGTAATTGGATGTTG-3') and reverse (5'-CAGAAGTCCCCAATACAGCAATCC-3'), which amplified a 634-bp fragment in the first round PCR and forward (5'-GGGTCCTGGGAGTAGTTGAAC-3') and reverse (5'-TTCACTAACAATCCCAAA CACCG-3'), which amplified a 406-bp fragment in the second round PCR. Nested PCR was performed using a method reported previously [ 7 ]. The PCR products were resolved on an ethidium bromide-stained 2% agarose gel in TAE buffer, and amplicons were visualized under UV light ( Fig.…”