Dataset Information

Multilocus typing of Cryptosporidium spp. in young calves with diarrhea in Korea.

ABSTRACT: We assessed the prevalence and performed molecular analysis of Cryptosporidium spp. in diarrheal feces of calves in Korea. Diarrheal fecal samples were collected from 951 young calves (<3months) on 425 farms. Cryptosporidium prevalence was assessed by PCR and ELISA, and molecular characterization was performed by targeting the 18S rRNA, heat-shock protein 70 (hsp70), and glycoprotein 60 (gp60) genes. Data were analyzed according to the sex, type of cattle, region, season, and type of diarrhea. PCR analysis revealed Cryptosporidium spp. in 9.9% (94/951) of diarrheal fecal samples. C. parvum and C. bovis/ryanae were present in 6.1% (58/951) and 4.1% (39/951) of diarrheal fecal samples, respectively. In addition, ELISA showed positive results for C. parvum in 9.7% (92/951) samples. Statistical analysis of the PCR and ELISA results revealed a lower prevalence of C. parvum in the hemorrhagic diarrheal samples (P<0.05). For C. bovis/ryanae, seasonality and high prevalence in hemorrhagic diarrhea were observed (P<0.05). Of the 951 samples tested for C. parvum, 903 samples showed agreement with a ? value of 0.65, indicating good agreement between the two tests. Although C. bovis and C. ryanae share highly similar 18S rRNA sequences, PCR based on hsp70 successfully distinguished C. bovis from C. ryanae. Sequence analysis of gp60 revealed that C. parvum belonged to the IIa families and was further subtyped as IIaA18G3R1 and IIaA16G3R1, which have not been previously reported in Asia. These findings indicate that Cryptosporidium spp. play an important role in diarrhea in young calves in Korea. Considering the zoonotic significance of C. parvum IIa subtype and dense rearing system of cattle in Korea, prevention and continuous monitoring of Cryptosporidium are required.

SUBMITTER: Lee SH

PROVIDER: S-EPMC7185498 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Multilocus typing of Cryptosporidium spp. in young calves with diarrhea in Korea.

Lee Seung-Hun SH VanBik Dorene D Kim Ha-Young HY Lee Yu-Ran YR Kim Jong Wan JW Chae Myeongju M Oh Sang-Ik SI Goo Youn-Kyoung YK Kwon Oh-Deog OD Kwak Dongmi D

Veterinary parasitology 20160928

We assessed the prevalence and performed molecular analysis of Cryptosporidium spp. in diarrheal feces of calves in Korea. Diarrheal fecal samples were collected from 951 young calves (<3months) on 425 farms. Cryptosporidium prevalence was assessed by PCR and ELISA, and molecular characterization was performed by targeting the 18S rRNA, heat-shock protein 70 (hsp70), and glycoprotein 60 (gp60) genes. Data were analyzed according to the sex, type of cattle, region, season, and type of diarrhea. P ...[more]

PMID: 27809984

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Project description:Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1−60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11−20 days (26.1%, 95% CI 17.1−35.1), whereas the lowest rate was observed in calves aged 21−30 days (7.7%, 95% CI 0.0−16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.

Project description:There is a need for an integrated genotyping approach for C. parvum; no sufficiently discriminatory scheme to date has been fully validated or widely adopted by veterinary or public health researchers. Multilocus fragment typing (MLFT) can provide good differentiation and is relatively quick and cheap to perform. A MLFT tool was assessed in terms of its typeability, specificity, precision (repeatability and reproducibility), accuracy and ability to genotypically discriminate bovine-derived Cryptosporidium parvum.With the aim of working towards a consensus, six markers were selected for inclusion based on their successful application in previous studies: MM5, MM18, MM19, TP14, MS1 and MS9. Alleles were assigned according to the fragment sizes of repeat regions amplified, as determined by capillary electrophoresis. In addition, a region of the GP60 gene was amplified and sequenced to determine gp60 subtype and this was added to the allelic profiles of the 6 markers to determine the multilocus genotype (MLG). The MLFT tool was applied to 140 C. parvum samples collected in two cross-sectional studies of UK calves, conducted in Cheshire in 2004 (principally dairy animals) and Aberdeenshire/Caithness in 2011 (beef animals).Typeability was 84 %. The primers did not amplify tested non-parvum species frequently detected in cattle. In terms of repeatability, within- and between-run fragment sizes showed little variability. Between laboratories, fragment sizes differed but allele calling was reproducible. The MLFT had good discriminatory ability (Simpson's Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44). Some markers were more informative than others, with MS1 and MS9 proving monoallelic in tested samples.Further inter-laboratory trials are now warranted with the inclusion of human-derived C. parvum samples, allowing progress towards an integrated, standardised typing scheme to enable source attribution and to determine the role of livestock in future outbreaks of human C. parvum.

Dataset Information

Multilocus typing of Cryptosporidium spp. in young calves with diarrhea in Korea.

Publications

Multilocus typing of Cryptosporidium spp. in young calves with diarrhea in Korea.

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