Dataset Information

Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction.

ABSTRACT:

Background

Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR).

Methods

The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing.

Results

Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously.

Conclusion

This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

SUBMITTER: Oh IY

PROVIDER: S-EPMC5623924 | biostudies-literature | 2017 Jul-Sep

REPOSITORIES: biostudies-literature

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Publications

Molecular Detection of <i>Dirofilaria immitis</i> Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction.

Oh In Young IY Kim Kyung Tae KT Sung Ho Joong HJ

Iranian journal of parasitology 20170701 3

<h4>Background</h4><i>Dirofilaria immitis</i>, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting <i>D. immitis</i> include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of <i>D. immitis</i> in blood using polymerase chain reaction (PCR).<h4>Methods</h4>The study was conducted at Eulji University, Republic of Korea in 2016. To detect <i>D ...[more]

PMID: 28979354

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Project description:BackgroundAs a result of globalization and climate change, Dirofilaria immitis and Dirofilaria repens, the causative agents of dirofilariosis in Europe, continue to spread from endemic areas in the Mediterranean to northern and northeastern regions of Europe where autochthonous cases of dirofilarial infections have increasingly been observed in dogs and humans. Whilst D. repens was recently reported from mosquitoes in putatively non-endemic areas, D. immitis has never been demonstrated in mosquitoes from Europe outside the Mediterranean.MethodsFrom 2011 to 2013, mosquitoes collected within the framework of a German national mosquito monitoring programme were screened for filarial nematodes using a newly designed filarioid-specific real-time PCR assay. Positive samples were further processed by conventional PCR amplification of the cytochrome c oxidase subunit I (COI) gene, amplicons were sequenced and sequences blasted against GenBank.ResultsApproximately 17,000 female mosquitoes were subjected to filarial screening. Out of 955 pools examined, nine tested positive for filariae. Two of the COI sequences indicated D. immitis, one D. repens and four Setaria tundra. Two sequences could not be assigned to a known species due to a lack of similar GenBank entries. Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium, D. repens was found in a single Anopheles daciae and all S. tundra were demonstrated in Aedes vexans. All positive mosquitoes were collected between mid-June and early September.ConclusionThe finding of dirofilariae in German mosquitoes implies the possibility of a local natural transmission cycle. While the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, potential culicid vectors and reservoir hosts must prospectively be identified and awareness among physicians, veterinarians and public health personnel be created. The health impact of S. tundra on the indigenous cervid fauna needs further investigation.