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Occurrence and genotyping of Giardia duodenalis and Cryptosporidium in pre-weaned dairy calves in central Sichuan province, China.

ABSTRACT: Giardia duodenalis and Cryptosporidium spp. are common human and animal pathogens. They have increasingly been reported in dairy calves in recent years; however, multilocus genotyping information for G. duodenalis and Cryptosporidium infecting pre-weaned dairy calves in southwestern China is limited. In the present study, the prevalence of G. duodenalis and Cryptosporidium spp. in pre-weaned dairy calves in central Sichuan province was determined and the pathogens were analyzed molecularly. Of 278 fecal samples from pre-weaned dairy calves, 26 (9.4%) were positive for G. duodenalis and 40 (14.4%) were positive for Cryptosporidium spp. Cryptosporidium bovis (n = 28), Cryptosporidium ryanae (n = 5) and Cryptosporidium parvum (n = 7) were detected. All seven C. parvum isolates were successfully subtyped based on the gp60 gene sequence, and only IIdA15G1 was detected. Multilocus sequence typing of G. duodenalis based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes revealed 19 different assemblage E multilocus genotypes (two known and 17 unpublished genotypes). Based on eBURST analysis, a high degree of genetic diversity within assemblage E was observed in pre-weaned dairy calves in Sichuan province. To the best of our knowledge, this is the first study using multilocus sequence typing and eBURST analysis to characterize G. duodenalis in pre-weaned dairy calves in southwestern China.

SUBMITTER: Zhong Z

PROVIDER: S-EPMC6121785 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are common enteric pathogens in humans and animals. Data on the transmission of these pathogens are scarce from Guangdong, China, which has a subtropical monsoon climate and is the epicenter for many emerging infectious diseases. This study was conducted to better understand the prevalence and identity of the three pathogens in pre-weaned dairy calves in Guangdong. METHODS:The occurrence and genetic identity of three pathogens were analyzed by polymerase chain reaction. PCR-positive products were sequenced to determine the species and genotypes. A Chi-square test was used to compare the prevalence of pathogens among sampling dates, age groups, or clinical signs. RESULTS:The detection rates of Cryptosporidium spp., G. duodenalis and E. bieneusi were 24.0% (93/388), 74.2% (288/388) and 15.7% (61/388), respectively. Three Cryptosporidium species were detected, including C. bovis (n = 73), C. parvum (n = 12) and C. ryanae (n = 7); one animal had concurrence of C. bovis and C. parvum. C. parvum was the dominant species during the first two weeks of life, whereas C. bovis and C. ryanae were mostly seen at 3-9 weeks of age. Sequence analysis identified the C. parvum as subtype IIdA19G1. Assemblage E (n = 282), assemblage A (n = 1), and concurrence of A and E (n = 5) were identified among G. duodenalis-positive animals using multilocus genotyping (MLG). Altogether, 15, 10 and 17 subtypes of assemblage E were observed at the bg, gdh and tpi loci, respectively, forming 49 assemblage E MLGs. The highest detection rate of G. duodenalis was found in winter. Sequence analysis identified genotypes J (n = 57), D (n = 3) and one concurrence of J and D among E. bieneusi-positive animals. The detection rate of E. bieneusi was significantly higher in spring (38.0%; 41/108) than in summer (7.2%; 8/111) and winter (7.1%; 12/169). CONCLUSIONS:These results indicate a common occurrence of C. parvum subtype IIdA19G1, G. duodenalis assemblage E, and E. bieneusi genotype J in pre-weaned dairy calves in Guangdong. More studies are needed to understand the unique genetic characteristics and zoonotic potential of the three enteric pathogens in the province.

Project description:BACKGROUND: Cryptosporidium and Giardia are important causes of diarrhea diseases in humans and animals worldwide, and both of them are transmitted by the fecal-oral route, either by direct contact or by the ingestion of contaminated food or water. The role of flies in the mechanical transmission of Cryptosporidium and Giardia has been receiving increasing attention. To date, no information is available in China about the occurrence of Cryptosporidium and Giardia in flies. We here investigated Cryptosporidium and Giardia in flies on dairy farms in Henan Province, China, at the genotype and subtype levels. METHODS: Eight hundred flies were randomly collected from two dairy farms from July 2010 to September 2010 and were divided evenly into 40 batches. The fly samples were screened for the presence of Cryptosporidium and Giardia with nested PCR. Cryptosporidium was genotyped and subtyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The identity of Giardia was determined by sequence analyzing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and ?-giardin (bg) genes. RESULTS: Forty batches of flies had 10% of contamination with Cryptosporidium or Giardia, with a mixed infection of the two parasites in one batch of flies. The Cryptosporidium isolates were identified as C. parvum at the SSU rRNA locus, and all belonged to subtype IIdA19G1 at the gp60 locus. The Giardia isolates were all identified as assemblage E of G. duodenalis at the tpi, gdh, and bg loci. One novel subtype of assemblage E was identified based on the gdh and bg loci. CONCLUSIONS: This is the first molecular study of Cryptosporidium and Giardia in flies identified at both genotype and subtype levels. SSU rRNA and gp60 sequences of C. parvum in flies was 100% homologous with those derived from humans, suggesting flies act as an epidemiological vector of zoonotic cryptosporidiosis. The variable PCR efficiencies observed in the analysis of Giardia at different loci suggest that we should use the multilocus genotyping tool in future studies to increase the detection rate, and importantly, to obtain more complete genetic information on Giardia isolates.

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Occurrence and genotyping of Giardia duodenalis and Cryptosporidium in pre-weaned dairy calves in central Sichuan province, China.

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