Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli in Dairy Goat Kids with Diarrhea in Partial Regions of Shaanxi Province, China

Abstract

Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are important diarrheal pathogens threatening the health of humans and various animals. Goats, especially pre-weaned goat kids, that carry these pathogens are important reservoirs related to human infection. In the present study, PCR-based sequencing techniques were applied to characterize Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in 202 fecal samples of diarrheal kids for Guanzhong dairy goats from five locations in Shaanxi Province. The positive rates of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli were 37.6% (76/202), 16.3% (33/202), 55.4% (112/202) and 78.7% (159/202) in these goat kids, respectively. Co-infection of two to four pathogens was found in 114 of 202 fecal samples. Significant differences (p < 0.001) in the positive rates of Cryptosporidium spp. and G. duodenalis were found among locations and age groups. Furthermore, two Cryptosporidium species (C. parvum and C. xiaoi), two G. duodenalis assemblages (E and A), nine E. bieneusi genotypes (CHG3, CHG1, BEB6, CHG5, CHG2, SX1, CHG28, COS-II and CD6) and two E. coli pathotypes (EPEC and EHEC) were identified. As for Cryptosporidium, two (IIdA19G1 and IIdA19G2) and two (XXIIIa and XXIIIg) subtypes were recognized in samples positive for C. parvum and C. xiaoi, respectively. A phylogenetic analysis based on the ITS locus of E. bieneusi indicated that all nine genotypes of E. bieneusi identified in this study belonged to the group 2. Four virulence factors (ehxA, eae, stx2 and stx1) of EPEC and EHEC were found in E. coli strains. Collectively, this study explored the colonization frequency of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats in Shaanxi Province and expanded our understanding of the genetic composition and zoonotic potential of these pathogens in goats.

Keywords: Cryptosporidium spp.; Enterocytozoon bieneusi; Escherichia coli; Giardia duodenalis; Shaanxi; goat.

Figure 1

Geographical distribution of sampling sites of dairy goat kids with diarrhea in partial regions of Shaanxi Province in the present study.

Figure 2

Phylogenetic relationships of Cryptosporidium spp. from sheep and goats based on the SSU rRNA gene by maximum likelihood analysis using general time-reversible model. Red-filled circles before the bold sample names represent species identified in the present study. Bootstrap values over 50 are presented at the nodes. Eimeria tenella (AF026388.1) is used as the outgroup. Representative sequences of each sequence type in this study are included in the phylogenetic analysis.

Figure 3

Phylogenetic relationships of Giardia duodenalis based on the tpi gene by maximum likelihood analysis using general time-reversible model. Red-filled circles before the bold sample names represent assemblages identified in the present study. Bootstrap values over 50 are presented at the nodes. Representative sequences of each sequence type in this study are included in the phylogenetic analysis.

Figure 4

Phylogenetic relationships of representative sequences for the ITS genotypes of E. bieneusi identified in this study with reference sequences by maximum likelihood analysis using general time-reversible model. Red-filled circles before the bold sample names represent genotypes identified in the present study. Bootstrap values over 50 are presented at the nodes. Genotype SW3 from stormwater (KF591679.1) is used as the outgroup.

Figure 5

Percentage (%) of infection types within co-infections of two, three and four pathogens. C+B/C+E/G+B/G+E/B+E/C+G+B/C+G+E/C+B+E/G+B+E/C+G+B+E represent co-infection of Cryptosporidium spp. and E. bieneusi/Cryptosporidium spp. and E. coli/G. duodenalis and E. bieneusi/G. duodenalis and E. coli/E. bieneusi and E. coli/Cryptosporidium spp., G. duodenalis and E. bieneusi/Cryptosporidium spp., G. duodenalis and E. coli/Cryptosporidium spp., E. bieneusi and E. coli/G. duodenalis, E. bieneusi and E. coli/Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli, respectively. Numbers above bars denote sample size (n).