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

Hotchkiss EJ, Gilray JA, Brennan ML, Christley RM, Morrison LJ, Jonsson NN, Innes EA, Katzer F - Parasit Vectors (2015)

Bottom Line: 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.The primers did not amplify tested non-parvum species frequently detected in cattle.The MLFT had good discriminatory ability (Simpson's Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44).

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. emily.hotchkiss@moredun.ac.uk.

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.

No MeSH data available.


Frequency distribution of multilocus genotypes (MLGs) of Cryptosporidium parvum identified in 118 samples from 19 farms in Scotland (light grey) and 20 farms in Cheshire (dark grey)
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Fig2: Frequency distribution of multilocus genotypes (MLGs) of Cryptosporidium parvum identified in 118 samples from 19 farms in Scotland (light grey) and 20 farms in Cheshire (dark grey)

Mentions: There was limited evidence of mixed genotype infections within individual calves, with 109/118 calves shedding single alleles at each locus. In addition single MLGs were detected on most farms. Overall 23 MLGs were identified in 118 samples, from 39 different farms in two regions of the UK (Fig. 2). The majority of MLGs (14/23) were only identified on one sampled farm, however some MLGs were more widely distributed with 4 MLGs common to both the Cheshire (2004) and NE Scotland (2011) studies. One MLG was particularly prevalent, having been identified on 8 farms representing both studies (MLG 12).Fig. 2


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

Hotchkiss EJ, Gilray JA, Brennan ML, Christley RM, Morrison LJ, Jonsson NN, Innes EA, Katzer F - Parasit Vectors (2015)

Frequency distribution of multilocus genotypes (MLGs) of Cryptosporidium parvum identified in 118 samples from 19 farms in Scotland (light grey) and 20 farms in Cheshire (dark grey)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4591062&req=5

Fig2: Frequency distribution of multilocus genotypes (MLGs) of Cryptosporidium parvum identified in 118 samples from 19 farms in Scotland (light grey) and 20 farms in Cheshire (dark grey)
Mentions: There was limited evidence of mixed genotype infections within individual calves, with 109/118 calves shedding single alleles at each locus. In addition single MLGs were detected on most farms. Overall 23 MLGs were identified in 118 samples, from 39 different farms in two regions of the UK (Fig. 2). The majority of MLGs (14/23) were only identified on one sampled farm, however some MLGs were more widely distributed with 4 MLGs common to both the Cheshire (2004) and NE Scotland (2011) studies. One MLG was particularly prevalent, having been identified on 8 farms representing both studies (MLG 12).Fig. 2

Bottom Line: 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.The primers did not amplify tested non-parvum species frequently detected in cattle.The MLFT had good discriminatory ability (Simpson's Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44).

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK. emily.hotchkiss@moredun.ac.uk.

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.

No MeSH data available.