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Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India.

ABSTRACT: The present study reports the characteristics of the 5S rRNA spacer sequences of Dirofilaria repens microfilariae isolated from dogs. The 22 nucleotide long spliced leader 1 sequences located in the 5S rRNA spacer region are completely conserved in all nematodes. There is variation in the spliced leader 1 sequences and associated sites in the 5S rRNA spacer region of D. repens. Absence of canonical SL 1 sequences distinguishes D. repens from other filarial species.

SUBMITTER: Chirayath D

PROVIDER: S-EPMC7596136 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India.

Chirayath Deepa D Alex P C PC Pillai Usha Narayana UN

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20200806 4

The present study reports the characteristics of the 5S rRNA spacer sequences of Dirofilaria repens microfilariae isolated from dogs. The 22 nucleotide long spliced leader 1 sequences located in the 5S rRNA spacer region are completely conserved in all nematodes. There is variation in the spliced leader 1 sequences and associated sites in the 5S rRNA spacer region of D. repens. Absence of canonical SL 1 sequences distinguishes D. repens from other filarial species. ...[more]

PMID: 33184552

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Project description:BackgroundIn Lithuania, the first case of canine subcutaneous dirofilariosis was recorded in 2010. Since then, an increasing number of cases of canine dirofilariosis have been documented in different veterinary clinics throughout the country. Human dirofilariosis was diagnosed in Lithuania for the first time in September 2011. However, to the authors' knowledge, there are no published data on the presence and prevalence of autochthonous dirofilariosis in dogs and humans in the country. The present study provides information about the predominant species and prevalence of Dirofilaria in dogs and describes the cases of human dirofilariosis in Lithuania. It also outlines PCR detection of the bacterial endosymbiont Wolbachia that contributes to the inflammatory features of filarioid infection.ResultsA total of 2280 blood samples and six adult worms from pet and shelter dogs were collected in the central and eastern regions of Lithuania in 2013-2015. Based on their morphological appearance, morphometric measurements and molecular analysis, all the adult nematodes were identified as Dirofilaria repens. The diagnosis of microfilariae in blood samples was based on blood smear analysis and Knott's test. The PCR and sequence analysis of the ribosomal DNA ITS2 region and cox1 gene confirmed the presence of D. repens. Overall, 61 (2.7%) of the 2280 blood samples were found to be positive for the presence of D. repens. The infection rate of D. repens was significantly higher in shelter dogs (19.0%; 19/100) than in pet dogs (1.9%; 42/2180) (χ2 = 100.039, df = 1, P < 0.0001). Forty-nine DNA samples of D. repens-infected dogs were tested for the presence of the bacterial endosymbiont Wolbachia and, of these, 40 samples (81.6%) were found to be positive. Three ocular and six subcutaneous cases of human dirofilariosis were diagnosed in Lithuania in the period 2011-2018.ConclusionsTo the authors' knowledge, this is the first report of autochthonous D. repens infection in dogs and humans in Lithuania. The present data demonstrate that D. repens is the main etiological agent of dirofilariosis in Lithuania. The DNA of the filarioid endosymbiotic bacterium Wolbachia was detected in the vast majority of dogs infected with D. repens.

Project description:Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors' opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.

Project description:BackgroundHeartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA.MethodsTo provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results.ResultsDirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001).ConclusionsTo our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA.

Dataset Information

Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India.

Publications

Molecular characterization of 5S ribosomal spacer sequences of <i>Dirofilaria repens</i> microfilariae in dogs in Kerala, India.

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