Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India
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ORIGINAL ARTICLE
Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India Deepa Chirayath1 • P. C. Alex1 • Usha Narayana Pillai1
Received: 2 April 2020 / Accepted: 30 July 2020 Ó Indian Society for Parasitology 2020
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. Keywords Dirofilaria repens 5S rRNA spacer Spliced leader 1 (SL 1) sequence Kerala India
Introduction Dirofilaria repens is a zoonotic filarial nematode that affects dogs and other carnivore. Filarial parasites are highly evolved nematodes which complete their life cycle in two hosts. Definitive hosts are vertebrates which can be either human beings or animals. Intermediate hosts are mostly different species of mosquitoes. Blood sucking mosquitoes can feed on both human beings and animals during their life span and thus a wide variety of filariae of animals may infect humans. D. repens infection is common in dogs in Kerala, India (Radhika et al. 2001; Ambily et al. 2011; Chirayath et al. 2015) and it is now considered as an
& Deepa Chirayath [email protected] 1
Department of Veterinary Clinical Medicine, Ethics and Jurisprudence, College of Veterinary and Animal Sciences, Mannuthy, Kerala Veterinary and Animal Sciences University, Pookode, Wayanadu, India
emerging zoonosis causing subcutaneous dirofilariosis in humans (Sabu et al. 2005). The present paper reports the molecular peculiarities of canine D. repens.
Materials and methods A total of 1600 dogs above six months of age, presented to the Medicine Unit of Veterinary Hospital Mannuthy and Kokkalai with various complaints, check up and for vaccinations were screened by wet blood film examination during morning hours between 8.30 am to 12 noon. Blood smears of microfilaremic animals were subjected to Giemsa staining and acid phosphatase staining using the naphthol AS-TR- phosphate method of Barka (Chalifoux and Hunt 1971). Blood was collected from dogs positive for D. repens microfilariae for DNA isolation, after speciation of microfilariae by the staining patterns. DNA samples were subjected to Polymerase chain reaction using two sets of primers for 5S ribosomal RNA sequences of filariae (D. Rep F1 & D. Rep R1—Rishiniw et al. 2006 and S2 &S16—Xie et al. 1994). The primer sequences were D.rep—F1-50 TGT TTC GGC CTA GTG TTT CGA CCA 30 D.rep- R1-50 ACG AGA TGT CGT GCT TTC AAC GTG 30 and S2 50 GTT AAG CAA CGT TGG GCC TGG 30 S16 50 TTG ACA GAT CGG ACG AGA TG 30 respectively. Ten microlitre of PCR products were analysed by using 1.5% agarose gel electrophoresis, stained by ethidium bromide and visua
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