Genotyping Of Three Enteric Protozoans In Sheep From Heilongjiang Province And Assessment Of Their Zoonotic Potential

Parasitic diseases contribute significantly to the burden of infectious diseases worldwide. Many enteric protozoa can be the causative agents of diarrhea and even cause host deaths, especially the immune-compromised or-suppressed individuals. Enterocytozoon bieneusi, Giardia duodenalis and Cryptosporidium spp. are important zoonotic intestinal protozoa that infect humans, livestock, and wildlife. Children and AIDS patients who suffer from the three parasites typically experience severe malnutrition and slow growth behavior. At present, lots of studies focus on the investigation of the transmission pathways for the three important protozoa, while quite few data have been generated for sheep and the role of such host in zoonotic transmission of cryptosporidiosis, microsporidiosis, and giardiasis remains unknown. This study was performed to survey the prevalence and genetic traits of the three important protozoa in sheep from three cities of Heilongjiang and assess the zoonotic potential of the isolates identified.This study investigated fecal specimens from 489 sheep in multiple cities in northeast China for the prevalence and genetic characteristics of E. bieneusi by PCR and sequencing of the ribosomal internal transcribed spacer. Sixty-eight sheep specimens(13.9%) were positive for E. bieneusi. Sequence polymorphisms enabled the identification of 5 known genotypes(BEB6, CM7, CS-4, Ebp C and OEB1) and 6 new genotypes(NESH1 to NESH6). The genotypes formed two genetic clusters in a phylogenetic analysis, with CS-4, EbpC and NESH1 to NESH3 distributed in zoonotic group 1 and BEB6, CM7, OEB1 and NESH4 to NESH6 distributed in potentially host-adapted group 2. Nearly 70% of cases of E. bieneusi infections in sheep were contributed by human-pathogenic genotypes BEB6, CS-4 and Ebp C. The cooccurrence of genotypes BEB4, CS-4, EbpC, I, and J in domestic ruminants and children in northeast China and the identification of BEB6 and Ebp C in humans and water in central China imply the p ossibility of zoonotic transmission. This study also summarized E. bieneusi genotypes obtained from ruminants worldwide and displays their host ranges, geographical distributions, and phylogenetic relationships. The data suggest a host range expansion in s ome group 2 genotypes(notably BEB4, BEB6, I, and J) that were previously considered to be adapted to ruminants. We should be concerned about the increasing zoonotic importance of group 2 genotypes with low host specificity.We also performed a multilocus sequence typing(MLST) analysis on the 34 ITS locus-positive isolates from cities Harbin, Daqing, and Qiqihar. The isolates were previously genotyped as BEB6, CS-4, and NESH1 to NESH3. Phylogeny based on the ITS locus has shown the host-adapted features of BEB6 and zoonotic potential of CS-4 and NESH1 to NESH3. Six of the isolates were positive at the MS1 locus and belong to three genotypes. Likewise, three of the isolates were positive at the MS7 locus and belong to two genotypes. We obtained no positives at the loci MS3 and MS4. Phylogenetic analysis at the MS1 and MS7 loci revealed the close genetic relationships of the sheep E. bieneusi isoltes with the ones reported in humans. The result differed from that gained at the ITS locus, but supported the zoon otic importance of BEB6 isolates that were previously considered host-adapted.Giardia duodenalis was also identified in the speimens by PCR amplification of the genetic loci beta-giardin(bg), glutamate dehydrogenase(gdh), and triose phosphate isomerase(tpi). Among the 489 specimens examined, 11, 5, and 0 specimens were positive at the loci tpi, bg, and gdh, respectively. Sequence polymorphisms of tpi and bg facilitated the identification of zoonotic assemblage A and ruminant-adapted assemblage E in the isolates.We also found two of 489 sheep(0.4%) were infected with Cryptosporidium by PCR amplification of the small subunit ribosomal RNA(SSU rRNA) gene using the genus-specific primers. The two isolates belong to C. xiaoi via sequence analysis.As inferred from the above findings, sheep could serve as the potential reservoir and source of human microsporidiosis and giardiasis. The data should be useful in prevention and control of microsporidiosis and giardiasis in human and animals in the study area.