Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions
The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.
KeywordsPCR-RFLP Toxocara canis Toxocara cati internal transcribed spacers (ITS) identification
Unable to display preview. Download preview PDF.
- Aryaeipour M., Rouhani S., Bandehpour M., Mirahmadi H., Kazemi B., Rokni M.B. 2014. Genotyping and Phylogenetic Analysis of Fasciola spp. Isolated from Sheep and Cattle Using PCRRFLP in Ardabil Province, Northwestern Iran. Iranian Journal of Public Health, 43, 1364–1371PubMedPubMedCentralGoogle Scholar
- Borecka A. 2004. Differentiation of Toxocara spp. eggs isolated from the soil by the PCR-linked RFLP method. Helminthologia, 41, 185–187Google Scholar
- Fahrion A., Schnyder M., Wichert B., Deplazes P. 2011. Toxocara eggs shed by dogs and cats and their molecular and morphometric species-specific identification: is the finding of T. cati eggs shed by dogs of epidemiological relevance? Veterinary Parasitology, 177, 186–189. DOI: 10.1016/j.vetpar.2010.11.028CrossRefGoogle Scholar
- Gasser R.B., Zhu X.Q., Jacobs D.E., Hu M., Chilton N.B. 2006. Molecular genetic characterisation of members of the genus Toxocara–taxonomic, population genetic and epidemiological considerations. In: (Eds: C.V. Holland, H.V. Smith), Toxocara: The Enigmatic Parasite. Wallingford, United Kingdom, 18–31Google Scholar
- Lee C.C., Cheng N.A.B.Y., Bohari Y. 1993. Toxocara canis from domestic cats in Kuala Lumpur. Tropical Biomedicine, 10, 79–80Google Scholar
- Pawar R.M., Lakshmikantan U., Hasan S., Poornachandar A., Shivaji S. 2012. Detection and molecular characterization of ascarid nematode infection (Toxascaris leonina and Toxocara cati) in captive Asiatic lions (Panthera leo persica). Acta Parasitologica, 57, 67–73. DOI: 10.2478/s11686-012-0012-yCrossRefGoogle Scholar
- Sheng Z.H., Chang Q.C., Tian S.Q., Lou Y., Zheng X., Zhao Q., Wang C.R. 2012. Characterization of Toxascaris leonina and Tococara canis from cougar (Panthera leo) and common wolf (Canis lupus) by nuclear ribosomal DNA sequences of internal transcribed spacers. African Journal of Microbiology Research, 14, 3545–3549. DOI: 10.5897/AJMR12.414Google Scholar
- Soulsby E.J.L. (Ed.) 1982. Helminths, Arthropods and Protozoa of Domesticated Animals. Bailliere Tindall London, UKGoogle Scholar
- Spotin A., Gholami S., Nasab A.N., Fallah E., Oskouei M.M., Semnani V., et al. 2015. Designing and conducting in silico analysis for identifying of Echinococcus spp. with discrimination of novel haplotypes: an approach to better understanding of parasite taxonomic. Parasitology Research, 114, 1503–9. DOI: 10.1007/s00436-015-4334-1CrossRefGoogle Scholar
- Yamaguti S. (Ed.) 1961. Systema Helminthum: Nematodes of vertebrates. Inter Science publisher Inc., New York, USAGoogle Scholar