title: Suitability of loci for multiple-locus variable-number of tandem-repeats analysis of Cryptosporidium parvum for inter-laboratory surveillance and outbreak investigations.
abstract: Cryptosporidium parvum is the major cause of livestock and zoonotically-acquired human cryptosporidiosis. The ability to track sources of contamination and routes of transmission by further differentiation of isolates would assist risk assessment and outbreak investigations. Multiple-locus variable-number of tandem-repeats (VNTR) analysis provides a means for rapid characterization by fragment sizing and estimation of copy numbers, but structured, harmonized development has been lacking for Cryptosporidium spp. To investigate potential for application in C. parvum surveillance and outbreak investigations, we studied nine commonly used VNTR loci (MSA, MSD, MSF, MM5, MM18, MM19, MS9-Mallon, GP60 and TP14) for chromosome distribution, repeat unit length and heterogeneity, and flanking region proximity and conservation. To investigate performance in vitro, we compared these loci in 14 C. parvum samples by capillary electrophoresis in three laboratories. We found that many loci did not contain simple repeat units but were more complex, hindering calculations of repeat unit copy number for standardized reporting nomenclature. However, sequenced reference DNA enabled reproducible fragment sizing and inter-laboratory allele assignation based on size normalized to that of the sequenced fragments by both single round and nested polymerase chain reactions. Additional Cryptosporidium loci need to be identified and validated for robust inter-laboratory surveillance and outbreak investigations.
authors.affiliation: Cryptosporidium Reference Unit,Public Health Wales Microbiology,Singleton Hospital,Swansea SA2 8QA,UK.
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Chalmers
authors.affiliation: Cryptosporidium Reference Unit,Public Health Wales Microbiology,Singleton Hospital,Swansea SA2 8QA,UK.
authors.forename: Guy
authors.initials: G
authors.lastname: Robinson
authors.affiliation: Moredun Research Institute,Pentlands Science Park,Bush Loan,Penicuik,Edinburgh EH26 0PZ,UK.
authors.forename: Emily
authors.initials: E
authors.lastname: Hotchkiss
authors.affiliation: Scottish Parasite Diagnostic and Reference Laboratory,Glasgow Royal Infirmary,10-16 Alexandra Parade,Glasgow G31 2ER,UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Alexander
authors.affiliation: Cryptosporidium Reference Unit,Public Health Wales Microbiology,Singleton Hospital,Swansea SA2 8QA,UK.
authors.forename: Sophie
authors.initials: S
authors.lastname: May
authors.affiliation: Moredun Research Institute,Pentlands Science Park,Bush Loan,Penicuik,Edinburgh EH26 0PZ,UK.
authors.forename: Janice
authors.initials: J
authors.lastname: Gilray
authors.affiliation: Scottish Parasite Diagnostic and Reference Laboratory,Glasgow Royal Infirmary,10-16 Alexandra Parade,Glasgow G31 2ER,UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Connelly
authors.affiliation: Cryptosporidium Reference Unit,Public Health Wales Microbiology,Singleton Hospital,Swansea SA2 8QA,UK.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hadfield
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology; D003457:Cryptosporidiosis / Q000175:diagnosis* / Q000453:epidemiology; D016785:Cryptosporidium parvum / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D004196:Disease Outbreaks; D062665:Epidemiological Monitoring / Q000662:veterinary*; D056426:Genetic Loci; D005838:Genotype; D060005:Genotyping Techniques; D018598:Minisatellite Repeats; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D016054:DNA, Protozoan
keywords: 
                 Cryptosporidium parvum
              ; genotyping; multi-locus; outbreak; reproducibility; surveillance; validation; variable-number tandem-repeat