Molecular detection and genotyping of Toxoplasma gondii in stray cat feces from Khorramabad, West Iran

Abstract

Cats, being the definitive host of Toxoplasma gondii, have a significant impact on the spread and outbreaks of the parasite. An essential factor in comprehending the transmission pattern of this parasite is an analysis of the genetic diversity distribution in cats infected with T. gondii. This study was aimed at determining the prevalence rate and genotyping of T. gondii in stray cat feces from Khorramabad, West Iran. In the years 2016-2017, 200 cats were sampled to get fresh feces specimens. Parasitological methods were utilized for the identification of oocysts. The DNA was isolated from the feces using a commercially available Genomic Mini Kit. In order to identify the genetic composition of T. gondii, we employed PCR-RFLP, sequencing, and phylogenetic analysis of the GRA6 target gene. No one of the samples tested positive for parasitology techniques. A total of 6.5 % (13/200) samples were positive when using the GRA6-PCR method. Based on PCR-RFLP results, all 13 samples were of T. gondii type III genotype. The nucleotide sequences of two samples from this study were found to be 5 % different from those of 12 references of T. gondii and one strain of Hammondia hamondi that was used as an external control. Based on the findings, molecular tests are more sensitive than parasitological methods. The RFLP approach revealed that type III of T. gondii is the prevailing and important genotype in Khorramabad, West Iran.

Keywords: Genotyping; PCR-RFLP; Prevalence; Stray cats; Toxoplasma gondii.

Fig. 1

Agarose (1.5 %) gel electrophoresis showing the PCR products (791 bp) amplified from positive samples. Lane M: DNA size marker (50 bp), lanes 1–3: positive samples, P: positive control, N: negative control.

Fig. 2

Agarose (1.5 %) gel electrophoresis showing the PCR-RFLP pattern of the GRA6 gene cut with MseI endonuclease (544 bp and 97 bp bands). Lane M: DNA size marker (100 bp), lanes 1, 3, 5, and 7: uncut PCR products before using enzyme, lanes 2, 4, 6, and 8: RFLP pattern of the type III genotype of Toxoplasma. gondii.

Fig. 3

Multiple sequence alignments of the T. gondii GRA6 gene in stray cats based on reference sequences (MG692613, MG692612): accession numbers of sequences registered in GenBank from the present study.

Fig. 4

The phylogenic tree was constructed by the maximum likelihood method using the nucleotide sequence of reference strains and our isolates. The scale bar indicates a 5 % nucleotide difference. Hammondia hamondi was considered an outgroup branch. The isolates of the present study are separated by a red dot.