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Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool

Hotchkiss, Emily J.; Gilray, Janice A.; Brennan, Marnie L.; Christley, Robert M.; Morrison, Liam J.; Jonsson, Nicholas N.; Innes, Elizabeth A.; Katzer, Frank

Authors

Emily J. Hotchkiss

Janice A. Gilray

Marnie L. Brennan

Robert M. Christley

Liam J. Morrison

Nicholas N. Jonsson

Elizabeth A. Innes

Frank Katzer



Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Citation

Hotchkiss, E. J., Gilray, J. A., Brennan, M. L., Christley, R. M., Morrison, L. J., Jonsson, N. N., …Katzer, F. (2015). Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool. Parasites and Vectors, 8(1), https://doi.org/10.1186/s13071-015-1107-8

Journal Article Type Article
Acceptance Date Sep 22, 2015
Publication Date Oct 1, 2015
Deposit Date Dec 12, 2016
Publicly Available Date Dec 12, 2016
Journal Parasites and Vectors
Electronic ISSN 1756-3305
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 8
Issue 1
Article Number 500
DOI https://doi.org/10.1186/s13071-015-1107-8
Keywords Cryptosporidium parvum, Multilocus, Genotyping, Bovine
Public URL http://eprints.nottingham.ac.uk/id/eprint/39322
Publisher URL http://dx.doi.org/10.1186/s13071-015-1107-8
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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