Prevalence and genetic characterization of Enterocytozoon bieneusi and Giardia duodenalis in Tibetan pigs in Tibet, China

Enterocytozoon bieneusi and Giardia duodenalis are important opportunistic enteric zoonotic pathogens that cause diarrhoea and intestinal diseases in animals and humans. China is the largest producer of pigs, but whether Tibetan pigs, a unique pig breed in Tibet, are infected with E. bieneusi and G. duodenalis is unknown. Therefore, we conducted a molecular epidemiological survey to determine the prevalence of E. bieneusi and G. duodenalis in Tibetan pigs in Tibet, China, and identified the genotypes of these causative agents. A total of 345 faecal specimens were collected from Tibetan pigs from three Tibet counties (Milin, Cuona and Gongbujiangda), examined by nested PCR and sequenced utilizing genetic markers in the ribosomal internal transcribed spacer (ITS) region of the rRNA and glutamate dehydrogenase (gdh) gene for E. bieneusi and G. duodenalis, respectively. Moreover, using multilocus sequence typing, the subtypes of E. bieneusi were identified based on four loci (MS1, MS3, MS4 and MS7). A total of 41 (11.88%) faecal samples from Tibetan pigs were E. bieneusi-positive, and 2 (0.58%) were G. duodenalis-positive. The multivariate logistic regression analysis showed that age was considered a risk factor for Tibetan pig infection of E. bieneusi. Two novel (GB11, GB31) and four known E. bieneusi genotypes (EbpC, EbpD, PigEBITS5 and CHS12) were identified and were all classified as zoonotic group 1 according to the phylogenetic analysis. Two MLGs (MLGI and MLGII) were further identified in the E. bieneusi EbpC genotype by multilocus sequence typing analysis. In addition, two G. duodenalis assemblages (D and E) were found in the present study. To our knowledge, the current study is the first to detect the prevalence and perform genetic characterization of G. duodenalis in Tibetan pigs in Tibet, China. The results could provide essential data for controlling E. bieneusi and G. duodenalis infections in Tibetan pigs that are in contact with other animals and humans, as Tibetan pigs could be a potential source for human infection by these pathogens.