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

Paparini, Andrea, Yang, Rongchang, Chen, Linda, Tong, Kaising, Gibson-Kueh, Susan, Lymbery, Alan, and Ryan, Una M. (2017) Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections. Parasitology, 144 (13). pp. 1811-1820.

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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.

Item ID: 59114
Item Type: Article (Research - C1)
ISSN: 1469-8161
Keywords: 18S rRNA gene, Actin gene, Cryptosporidium, Fish, Mixed infections, Molecular phylogeny, Molecular systematics, Next-generation sequencing (NGS), Parasitology, Sanger sequencing
Copyright Information: © Cambridge University Press 2017.
Funders: Murdoch University, Australian Research Council (ARC), Bayer Healthcare, Bayer Australia Ltd, Western Australia Water Corporation, WaterRA
Date Deposited: 13 Aug 2019 23:04
FoR Codes: 31 BIOLOGICAL SCIENCES > 3105 Genetics > 310509 Genomics @ 50%
30 AGRICULTURAL, VETERINARY AND FOOD SCIENCES > 3009 Veterinary sciences > 300904 Veterinary diagnosis and diagnostics @ 50%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50%
97 EXPANDING KNOWLEDGE > 970107 Expanding Knowledge in the Agricultural and Veterinary Sciences @ 50%
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