title: Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool.
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.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. emily.hotchkiss@moredun.ac.uk.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Hotchkiss
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. janice.gilray@moredun.ac.uk.
authors.forename: Janice A
authors.initials: JA
authors.lastname: Gilray
authors.affiliation: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. marnie.brennan@nottingham.ac.uk.
authors.forename: Marnie L
authors.initials: ML
authors.lastname: Brennan
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Leahurst Campus CH64 7TE, Liverpool, L69 7BE, UK. robc@liverpool.ac.uk.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Christley
authors.affiliation: Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK. liam.morrison@roslin.ed.ac.uk.
authors.forename: Liam J
authors.initials: LJ
authors.lastname: Morrison
authors.affiliation: Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK. nicholas.jonsson@glasgow.ac.uk.
authors.forename: Nicholas N
authors.initials: NN
authors.lastname: Jonsson
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. lee.innes@moredun.ac.uk.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Innes
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. frank.katzer@moredun.ac.uk.
authors.forename: Frank
authors.initials: F
authors.lastname: Katzer
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000453:epidemiology / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000235:genetics*; D005243:Feces; D005838:Genotype; D058885:Multilocus Sequence Typing / Q000379:methods*; D015203:Reproducibility of Results; D006113:United Kingdom / Q000453:epidemiology