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Prevalence of Cryptosporidium parvum in dairy calves and GP60 subtyping of diarrheic calves in central Argentina.

ABSTRACT: Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ² = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocyst-positive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.

SUBMITTER: Lombardelli JA

PROVIDER: S-EPMC7087732 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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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Prevalence of Cryptosporidium parvum in dairy calves and GP60 subtyping of diarrheic calves in central Argentina.

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