Investigation of Parasitic Nematodes Detected in the Feces of Wild Carnivores in the Eastern Qinghai-Tibet Plateau, China

Abstract

Wildlife shares grazing areas with herders in the eastern Qinghai-Tibet Plateau, and humans can be infected by zoonotic nematodes through direct contact with animals or contaminated water. In this study, fecal samples (n = 296) from wild carnivores were collected to explore the infection rate and molecular genetic characteristics of nematodes by stratified random sampling in the survey areas. Host species and the nematodes they carried were then identified using 16S rRNA and 18S rRNA gene sequencing, respectively. Statistical analysis, neutrality tests, genetic diversity analysis and Bayesian inferred trees were performed to complete the study. In total, 10 species of nematodes were detected in 240 feces from six species of carnivores identified (including dominant Vulpes ferrilata and Vulpes vulpes), namely Uncinaria stenocephala, Toxascaris sp., Crenosoma vulpis, Parapharyngodon bainae, Oesophagostomum muntiacum, Aspiculuris tetraptera, Mastophorus muris, Nematodirus spathiger, Muellerius capillaris, and Molineus patens. Among these nematodes, U. stenocephala (35.83%, 86/240) and Toxascaris sp. (14.58%, 35/240) were detected at higher rates than the other nematodes (χ2 = 516.909, p < 0.05). Of 17 and 18 haplotypes were found based on the ITS1 gene for U. stenocephala and nad1 gene for Toxascaris sp., respectively. For the first time, using molecular methods, we report the infection of V. ferrilata by U. stenocephala, a potential zoonotic parasite, and suggest Toxascaris sp. may be a newly discovered nematode that lives within the fox intestine.

Keywords: Qinghai-Tibet Plateau; Toxascaris sp.; Uncinaria stenocephala; Vulpes ferrilata; Vulpes vulpes; nematode.

Figure 1

Results of 16S rRNA sequencing of fecal samples for host identification. Note: Vulpes sp. indicates that 16S rRNA gene fragments of both Vulpes ferrilata and Vulpes vulpes were detected in one sample.

Figure 2

The haplotype network of Uncinaria stenocephala based on 122 ITS1 gene sequences and Toxascaris sp. based on 45 nad1 gene sequences. (a) Colors represent different hosts, the size of the circle is proportional to the number of sequences with that haplotype, the distances between circles are proportional to the base differences between haplotypes, and median vectors were inferred using NETWORK software. Hap-Us-1 differs from Hap-Us-16 by six bases, Hap-Us-3 differs from Hap-Us-11 by two bases, and Hap-Us-7 differs from Hap-Us-10 by two bases. (b) Median vectors3 differs from Hap-Ts-2 by three bases, Hap-Ts-2 differs from Hap-Ts-3 by three bases, Median vectors4 differs from Hap-Ts-11 by two bases, Median vectors4 differs from Hap-Ts-7 by two bases, Median vectors1 differs from Hap-Ts-16 by seven bases, and Hap-Ts-16 differs from Hap-Ts-12 by eight bases.

Figure 3

Bayesian phylogenetic trees for three nematodes detected in fox feces from Shiqu County and constructed using MrBayes 3.2.4. (a) Phylogenetic tree of 18 Toxascaris sp. haplotypes of the nad1 gene, with the nucleotide substitution model “HKY + I + G”. (b) Phylogenetic tree based on the Crenosoma vulpis 18S rRNA sequence, with the nucleotide substitution model “HKY + I”. (c) Phylogenetic tree of 17 Uncinaria stenocephala haplotypes of the ITS1 gene with the nucleotide substitution model “K80”. Bootstrap values are shown, and the sequences retrieved from the NCBI are shown with accession numbers.

Figure 4

Overlap of nematode species detected using the three nematode universal primers (Nem A/B/C).