Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions
Authors: Mikaeili, Fattaneh; Mathis, Alexander; Deplazes, Peter; Mirhendi, Hossein; Barazesh, Afshin; Ebrahimi, Sepideh; Kia, Eshrat Beigom
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.
Comparison of in Vitro Efficacy of Six Disinfectants on the Hatching of Larval Eggs of Toxocara canis
IRANIAN JOURNAL OF PARASITOLOGY
Authors: Romero, Camilo; Heredia, Rafael; Bolio, Manuel; Miranda, Laura; Reyes, Laura; Arredondo, Mauricio; Flores, Ariadna
Background: The environmental contamination with Toxocara canis eggs increases the risk of dissemination and transmission of the parasite in dogs and paratenic hosts such as humans. We aimed to evaluate different disinfectants to compare their effect on T. canis eggs. Methods: For its realization, 850 embryonated eggs were obtained, which were suspended in a solution of 5% formaldehyde and distilled water in Eppendorf tubes. In the tubes containing the 850 embryonated eggs, researchers was added 0.5 mL of each solution (enzymatic solution, sodium hypochlorite, iodopovidone, quaternary of ammonium, benzalkonium chloride, and super oxidation solution). After mixing, an aliquot was taken, observed under the microscope, and the number of broken eggs counted at different times to find the most effective ovicidal moment. Results: The enzymatic disinfectant present a significant difference (P = 0.05) with 276.06 broken eggs followed by ammonium with 105.20 broken eggs. After 10 min, the ammonium solution was the one that showed a significant difference of 50.50 hatched eggs, followed by the enzymatic 26.80 and hypochlorite 25.00 treatments. After 20 min, the enzymatic solution treatment showed a significant difference with the other solutions showing an increase of 98.80 broken eggs. In the 30 and 40-min times, only the enzymatic treatment showed a significant difference of 334.10 and 381.70 of broken eggs respectively. Conclusion: The enzymatic solution has the greatest ovicidal effect against the eggs of T. canis to present a greater number of broken eggs in a given time between 20 and 40 minutes.