18S rDNA next-generation sequencing uncovers the biodiversity of Gastrointestinal parasites in Tibetan grazing ruminants in China

Abstract

Background: Free-range yak, Tibetan sheep and Tibetan goat, predominantly distributed across the Qinghai-Tibetan Plateau (QTP) in China, are highly susceptible to a wide range of parasite infections, resulting in underestimated economic losses. We aimed to investigate the biodiversity of gastrointestinal parasites in local ruminants based on 18 S SSU ribosomal DNA gene (18 S rDNA) using next-generation sequencing.

Methods: Following DNA extraction from 79 fecal samples collected from yak, Tibetan sheep and goat in the southeast part of QTP, we proceeded to amplify the V3-V4 fragments of the18S rDNA gene. Subsequently, we assessed the diversity of parasitic protozoa and helminths. To identify parasitic infection patterns, correlation studies were conducted in different factors, including ages, health conditions and seasons.

Results: A total of 192 operational taxonomic units (OTU) were identified, including 10 phyla and 27 genera. High prevalence was observed in Entamoeba (93.67%), Blastocystis (75.95%) and Trichostrongylus (68.35%). By phylogenetic analysis, we identified a potential new Entmoeba species, along with zoonotic species/subtypes, such as Trichostrongylus colubriformis and Blastocystis ST10, ST12, and ST14. Two rarely reported zoonotic protozoa, Colpoda and Colpodella, were particularly noted for their high prevalence of infection and potential association with diarrhea. Juveniles and adults shared the similar species of parasites. A significant reduction in helminth diversity and infection prevalence was documented during autumn.

Conclusions: This study provides critical insights into the diversity of gastrointestinal parasites in QTP ruminants, thereby enhancing our understanding of the infection risk in grazing livestock.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12917-025-04887-6.

Keywords: 18S; Parasites diversity; Tibetan goat; Tibetan sheep; Yak.

Fig. 1

Geographical location of samples. Colors represent sampling counties, and stars represent the exact sampling location. Maps are downloaded at the https://datav.aliyun.com. (A) Sampling sites in northwest Sichuan, southeast of QTP, China. (B) Sampling sites in Aba Tibetan and Qiang autonomous prefecture and Ganzi Tibetan Autonomous Prefecture

Fig. 2

The Hierarchical clustering tree at the genus level. The length of branches represents the distance between samples. Red branches represent yak samples and green branches represent samples of Tibetan sheep/goats. The colored blocks represent mean abundance of different species at the genus level; Relative abundance < 0.01 categorized as others

Fig. 3

Comparation of gastrointestinal parasites biodiversity between yak and Tibetan sheep/goat. (A) Statistical differences of mean abundance between yak and Tibetan sheep/goat were evaluated using Wilcoxon rank-sum test, based on Sobs and Shannon index at the OTU level; ^NSP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) The principal component analysis (PCoA) based on genus level, with circles presented as confidence ellipses. (C) The Circos plots revealed the distribution proportion of high abundance genera. Relative abundance < 0.01 categorized as others

Fig. 4

Analysis of similarities and differences between healthy and diarrheic animals. (A) Statistical differences of mean abundance between healthy and diarrheic animals were evaluated using Wilcoxon rank-sum test, based on Sobs and Shannon index at the OTU level; ^NSP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) The principal component analysis (PCoA) based on genus level, with circles presented as confidence ellipses. (C) The Circos plots revealed the distribution proportion of high abundance genera. Relative abundance < 0.01 categorized as others. (D) Linear discriminant analysis effect size (LEfSe) identifies characteristic species with significantly different abundances within groups. Only species with LDA thresholds > 3 are shown; larger LDA scores indicate greater influence on differential effects

Fig. 5

Analysis of similarities and differences between juveniles and adults. (A) Statistical differences of mean abundance between juveniles and adults were evaluated using Wilcoxon rank-sum test, based on Sobs and Shannon index at the OTU level; ^NSP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) The principal component analysis (PCoA) based on genus level, with circles presented as confidence ellipses. (C) The Circos plots revealed the distribution proportion of high abundance genera. Relative abundance < 0.01 categorized as others

Fig. 6

Analysis of similarities and differences among seasons. (A) Statistical differences of mean abundance among seasons were evaluated using Wilcoxon rank-sum test, based on Sobs and Shannon index at the OTU level; ^NSP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***. P < 0.001. (B) The principal component analysis (PCoA) based on genus level, with circles presented as confidence ellipses. (C) The Circos plots revealed the distribution proportion of high abundance genera. Relative abundance < 0.01 categorized as others

Fig. 7

Phylogenetic tree of five OTU of Entamoeba spp. based on 18 S rDNA gene sequences. Nucleotide sequences were aligned with the Clustal W algorithm and phylogenetic analysis was performed using the Neighbor-Joining (NJ) method. The GenBank accession numbers follow the taxon names. Bootstrapping with 1,000 replicates was used to support the clades. Bar: 0.20 substitutions per site. The black filled circles represent 5 identified OTU