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Acta Parasitologica

, Volume 62, Issue 3, pp 549–556 | Cite as

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions

  • Fattaneh Mikaeili
  • Alexander Mathis
  • Peter Deplazes
  • Hossein Mirhendi
  • Afshin Barazesh
  • Sepideh Ebrahimi
  • Eshrat Beigom KiaEmail author
Article

Abstract

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.

Keywords

PCR-RFLP Toxocara canis Toxocara cati internal transcribed spacers (ITS) identification 

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References

  1. 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
  2. Borecka A. 2004. Differentiation of Toxocara spp. eggs isolated from the soil by the PCR-linked RFLP method. Helminthologia, 41, 185–187Google Scholar
  3. Borecka A., Gawor J. 2008. Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs. Journal of Helminthology, 82, 119–122. DOI:10.1017/S0022149X07877522CrossRefGoogle Scholar
  4. Chen J., Zhou D.H., Nisbet A.J., Xu M.J., Huang S.Y., Li M.W., Wang C.R., Zhu X.Q. 2012. Advances in molecular identification, taxonomy, genetic variation and diagnosis of Toxocara spp. Infection, Genetics and Evolution, 12, 1344–1348. DOI:10.1016/j.meegid.2012.04.019CrossRefGoogle Scholar
  5. Deplazes P., van Knapen F., Schweiger A., Overgaauw P.A. 2011. Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis. Veterinary Parasitology, 182, 41–53. DOI: 10.1016/j.vetpar.2011.07.014CrossRefGoogle Scholar
  6. Despommier D. 2003. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clinical Microbiology Reviews, 16, 265–272. DOI: 10.1128/CMR.16.2.265-272.2003CrossRefGoogle Scholar
  7. El-Shazly A.M., Mohammed R. M., El-Beshbishi S. N., Azab M. S., El-Ghareeb A. S., Abdeltawab A. H., Zalook T.K. 2009. Soilparasites particularly Toxocara eggs in Egypt. Journal of the Egyptian Society of Parasitology, 39, 151–162PubMedGoogle Scholar
  8. Epe C., Meuwissen M., Stoye M., Schnieder T. 1999. Transmission trials, ITS2-PCR and RAPD-PCR show identity of Toxocara canis isolates from red fox and dog. Veterinary Parasitology, 84, 101–112CrossRefGoogle Scholar
  9. 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
  10. Fogt R. 2006. Molecular techniques applied in species identification of Toxocara. Wiadomości Parazytologiczne, 52, 31–35PubMedGoogle Scholar
  11. Fogt-Wyrwas R., Jarosz W., Mizgajska-Wiktor H. 2007. Utilizing a polymerase chain reaction method for the detection of Toxocara canis and T. cati eggs in soil. Journal of Helminthology, 81, 75–78. DOI:10.1017/S0022149X07241872CrossRefGoogle Scholar
  12. 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
  13. Gawor J., Borecka A., Marczynska M., Dobosz S., Zarnowska-Prymek H. 2014. Risk of human toxocarosis in Poland due to Toxocara infection of dogs and cats. Acta Parasitologica, 60, 99–104. DOI: 10.1515/ap-2015-0012CrossRefGoogle Scholar
  14. Jacobs D.E., Zhu X., Gasser R.B., Chilton N.B. 1997. PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog fox and cat. Acta Tropica, 68, 191–200CrossRefGoogle Scholar
  15. Khademvatan S., Abdizadeh R., Tavalla M. 2014. Molecular characterization of Toxocara spp. From soil of public areas in Ahvaz southwestern Iran. Acta Tropica, 135, 50–54. DOI: 10.1016/j.actatropica.2014.03.016CrossRefGoogle Scholar
  16. Khazan H., Khazaei M., Tabaee S.S., Mehrabi A. 2012. Prevalence of Toxocara spp. eggs in Public Parks in Tehran City, Iran. Iranian Journal of Parasitology, 7, 38–42.PubMedPubMedCentralGoogle Scholar
  17. 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
  18. Li M.W., Zhu X.Q., Gasser R.B., Lin R.Q., Sani R.A., Lun Z.R., Jacobs D.E. 2006. The occurrence of Toxocara malaysiensis in cats in China, confirmed by sequence based analyses of ribosomal DNA. Parasitology Research, 99, 554–557. DOI: 10.1007/s00436-006-0194-zCrossRefGoogle Scholar
  19. Mahami-Oskouei M., Dalimi A., Forouzandeh-Moghadam M., Rokni M.B. 2011. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2). Iranian Journal of Parasitology, 6, 35–42PubMedPubMedCentralGoogle Scholar
  20. Mikaeili F., Mirhendi H., Hosseini M., Asgari Q., Kia E.B. 2013. Toxocara nematodes in stray cats from Shiraz, southern Iran: intensity of infection and molecular identification of the isolates. Iranian Journal of Parasitology, 8, 593–600PubMedPubMedCentralGoogle Scholar
  21. Mikaeili F., Mirhendi H., Mohebali M., Hosseini M., Sharbatkhori M., Zarei Z., Kia E.B. 2015. Sequence variation in mitochondrial cox1 and nad1 genes of ascaridoid nematodes in cats and dogs from Iran. Journal of Helminthology, 89, 496–501. DOI:10.1017/S0022149X14000133CrossRefGoogle Scholar
  22. Nichols R.L. 1956. The etiology of visceral larva migrans. I. Diagnostic morphology of infective second-stage Toxocara larvae. Journal of Parasitology, 42, 349–362CrossRefGoogle Scholar
  23. Nuchprayoon S., Junpee A., Nithiuthai S., Chungpivat S., Suvannadabba S., Poovorawan Y. 2006. Detection of filarial parasites in domestic cats by PCR-RFLP of ITS1. Veterinary Parasitology, 140, 366–372. DOI:10.1016/j.vetpar.2006.04.003CrossRefGoogle Scholar
  24. 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
  25. Shafiei R., Sarkari B., Moshfe A. 2013. A Consistent PCR-RFLP Assay Based on ITS-2 Ribosomal DNA for Differentiation of Fasciola Species. Iranian Journal of Basic Medical Sciences, 16, 1266–1269PubMedPubMedCentralGoogle Scholar
  26. 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
  27. Soulsby E.J.L. (Ed.) 1982. Helminths, Arthropods and Protozoa of Domesticated Animals. Bailliere Tindall London, UKGoogle Scholar
  28. 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
  29. Tavares R.G., Staggemeier R., Borges A.L.P., Rodrigues M.T., Castelan L.A., Vasconcelos J., et al. 2011. Molecular techniques for the study and diagnosis of parasite infection. The Journal of Venomous Animals and Toxins including Tropical Diseases, 17, 239–248CrossRefGoogle Scholar
  30. Yamaguti S. (Ed.) 1961. Systema Helminthum: Nematodes of vertebrates. Inter Science publisher Inc., New York, USAGoogle Scholar
  31. Zhu X.Q., Gasser R.B. 1998. Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes. Electrophoresis, 19, 1366–1373. DOI: 10.1002/elps.1150190828CrossRefGoogle Scholar
  32. Zhu X.Q., Jacobs D.E., Chilton N.B., Sani R.A., Cheng N.A., Gasser R.B. 1998. Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia. Parasitology, 117, 155–164CrossRefGoogle Scholar
  33. Zhu X.Q., Gasser R.B., Chilton N.B., Jacobs D.E. 2001. Molecular approaches for studying ascaridoid nematodes with zoonotic potential, with an emphasis on Toxocara species. Journal of Helminthology, 75, 101–108PubMedGoogle Scholar
  34. Zibaei M., Abdollahpour F., Birjandi M., Firoozeh F. 2010. Soil contamination with Toxocara spp. eggs in the public parks from three areas of Khorram Abad, Iran. Nepal Medical College journal, 12, 63–65PubMedGoogle Scholar

Copyright information

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2017

Authors and Affiliations

  • Fattaneh Mikaeili
    • 1
  • Alexander Mathis
    • 2
  • Peter Deplazes
    • 2
  • Hossein Mirhendi
    • 3
  • Afshin Barazesh
    • 1
  • Sepideh Ebrahimi
    • 1
  • Eshrat Beigom Kia
    • 4
    Email author
  1. 1.Department of Medical Parasitology and Mycology, School of MedicineShiraz University of Medical SciencesShirazIran
  2. 2.Institute of Parasitology, Vetsuisse FacultyUniversity of ZurichZurichSwitzerland
  3. 3.Department of Medical Parasitology and Mycology, School of MedicineIsfahan University of Medical SciencesIsfahanIran
  4. 4.Department of Medical Parasitology and Mycology, School of Public HealthTehran University of Medical SciencesTehranIran

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