Genetic Characterization of Toxocara vitilorum in Turkey by Mitochondrial Gene Markers (cox1)

Abstract

Background: Toxocara vitulorum is a involved in the Ascaridoidea family and is a large roundworm with a semi translucent, soft body surface and pinkish color. Female worms measure 8-30cm in length, male worms 6-25cm. The major hosts of T.vitulorum are buffalo (Bubalus bubalis) and cattle (Bos species) in the humid tropics of Asia, Africa and South America. The diagnosis of T. vitulorum infections is usually made by observing characteristic eggs in routine fecal examination. Serological methods are also used to diagnose Toxocariasis. However, in recent years, PCR, a new generation molecular diagnostic method, has been used. The genetic structure of T. vitulorum is little known compared with data available from other parasites. The present sutudy was designed to determine the T. vitulorum isolates by the genetic characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) gene.

Materials, Methods & Results: Adult worms were collected from the feces of two calves (East Anatolian Red) during visits to the clinic at the Department of Internal Medicine of Van Yuzuncu Yil University, Faculty of Veterinary Medicine. Worms were washed thoroughly in 0.85 % saline to remove any debris and fixed into 70 % ethanol. After repeated and thoroughly washing the specimens, total genomic DNA of parasite extraction was performed be employing DNA extraction reagent kit (Thermo, GeneJET Genomic DNA Purification Kit) according to manufacturer’s recommendations. After DNA amplification, a 446 bp fragment of cox1 of T. vitulorum were obtained in all three isolates. All generated sequences were registered in GenBank database with accession numbers including MG905159, MG911729 and MG911730. The cox1 of T. vitulorum examined differed from another two isolates extracted from Germany beef cattle (KY313642.1) and Sri Lanka buffalo calf (FJ664617.1) at NCBI database. The MEGA 7 software was employed to calculate intra-species distance and similarity. The intra-species distance rate and similarity among the isolates were 0.005 and 99.995%, respectively. The cox 1 sequence of T. vitulorum did not differ from an isolate from Germany, but differed more from isolate from Sri Lanka. The phylogenetic tree that was constructed using the Neighbor-Joining (NJ) method. Bootstrap support (Bp) for ML trees was calculating 1000 bootstrap replicates. This results indicate that both the different species of Toxocara are host-specific and each member of the genus Toxocara spp. has a different about the molecular sequences. We used the phylogenies from the Maximum Parsimony (MP) method to construct another phylogenetic tree based on the cox1 (mtDNA) gene. The results again display that the cattle-calves (East Anatolian Red) isolates from Turkey homology with that obtained from the Germany beef cattle (accession no. KY313642.1).

Discussion: The genetic analysis of parasites is a crucial factor in terms of determining epidemiology and the control parasitic diseases of humans and animals. Toxocara vitulorum is the most common gastrointestinal helmints infecting ruminants particularly in tropical regions. Phylogenetic analysis revealed that T. vitulorum is 100% homology with related to sequence of T. vitulorum from Germany. The characterization of cox1 region can provides a foundation for accurate identification of some helminth species using PCR. Even though the small sample size,  the obtained results might provide useful information for further phylogenetic studies on the family Ascaridae.