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Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections

Published online by Cambridge University Press:  14 August 2017

ANDREA PAPARINI Open the ORCID record for ANDREA PAPARINI [Opens in a new window] ,
RONGCHANG YANG ,
LINDA CHEN ,
KAISING TONG ,
SUSAN GIBSON-KUEH ,
ALAN LYMBERY  and
UNA M. RYAN
ANDREA PAPARINI*
Affiliation:
Vector and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, WA, Australia
RONGCHANG YANG
Affiliation:
Vector and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, WA, Australia
LINDA CHEN
Affiliation:
Vector and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, WA, Australia
KAISING TONG
Affiliation:
Vector and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, WA, Australia
SUSAN GIBSON-KUEH
Affiliation:
Freshwater Fish Group and Fish Health Unit, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western 6150, Australia
ALAN LYMBERY
Affiliation:
Freshwater Fish Group and Fish Health Unit, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western 6150, Australia
UNA M. RYAN
Affiliation:
Vector and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, WA, Australia
*
*Corresponding author: Vector- and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Molecular and Biomedical Sciences, Murdoch University, 90 South Street, Murdoch WA, 6150, Australia. E-mail: A.Paparini@murdoch.edu.au
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Summary

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Keywords

Cryptosporidiumfishnext-generation sequencing (NGS)18S rRNA geneactin geneSanger sequencingmolecular phylogenymolecular systematicsparasitologymixed infections
Type
Research Article
Information
Parasitology , Volume 144 , Issue 13 , November 2017 , pp. 1811 - 1820
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Copyright
Copyright © Cambridge University Press 2017 

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