Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections

Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n = 14), Cryptosporidium huwi (n = 8), piscine genotype 2 (n = 4), piscine genotype 3-like (n = 1), piscine genotype 4 (n = 2), piscine genotype 5 (n = 13), piscine genotype 5-like (n = 1) and five novel genotypes (n = 5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.