Identification of Cryptosporidium Species in Fish from Lake Geneva (Lac Léman) in France

Abstract

Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of vertebrates including humans, is increasingly recognized as a parasite of a diverse range of wildlife species. However, little data are available regarding the identification of Cryptosporidium species and genotypes in wild aquatic environments, and more particularly in edible freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were collected from fishery suppliers around the lake. Nested PCR using degenerate primers followed by sequence analysis was used. Five fish species were identified as potential hosts of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis, and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. parvum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100 analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. Histological examination confirmed the presence of potential developmental stages of C. parvum within digestive epithelial cells. These observations suggest that C. parvum is infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes found in terrestrial mammals would be an additional source of infection for humans and animals, and may also contribute to the contamination of the environment with this parasite. Moreover, the risk of human transmission is strengthened by the observation of edible fillet contamination.

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

“…Previous studies have also reported large variation in prevalences ranging from 0.8 to 100% (Sitjà-Bobadilla et al, 2005;Murphy et al, 2009;Reid et al, 2010;Zanguee et al, 2010;Morine et al, 2012;Koinari et al, 2013;Certad et al, 2015;Ryan et al, 2015). In the present study, the majority of fish screened were ornamental fish, for which the prevalence was 7.0% (38/545).…”

“…In that study, the prevalence of C. parvum was 86.7% (13/15), the prevalence of C. molnari was 6.7% (1/15) and 6.7% (1/15) were mixed C. parvum and C. molnari infections (Certad et al, 2015). C. molnari was identified in the stomach, while developmental stages of C. parvum were found in the stomach and intestine indicating that C. parvum is infecting fish, rather than being passively, carried which has important public health implications.…”

“…However, this sequence matched C. molnari-like isolates 100% over the 530 bp of sequence indicating that this too is a C. molnari-like isolate. Similarly, the C. molnari sequences from Northern pike (Esox lucius) (KP939352 and KP939354) (Certad et al, 2015) were not included as they only overlapped over 330 bp but over this length of sequence, they exhibited five single nucleotide polymorphisms (SNP's) with C. molnari-like isolates. The genetic distances between C. molnari and the C. molnari-like isolates at both the 18S and actin loci suggests that the C. molnari-like isolates are in fact a separate species as these differences are greater than many currently accepted species.…”

“…A recent study identified a high overall prevalence (36.6%-15/41) of Cryptosporidium in freshwater fish from Lake Geneva (Lac Léman) and identified C. parvum and C. molnari (Certad et al, 2015). In that study, the prevalence of C. parvum was 86.7% (13/15), the prevalence of C. molnari was 6.7% (1/15) and 6.7% (1/15) were mixed C. parvum and C. molnari infections (Certad et al, 2015).…”

Public health significance of zoonotic Cryptosporidium species in wildlife: Critical insights into better drinking water management

“…Of these, only C. parvum , C. hominis and C. scrofarum are of public health interest. Cryptosporidium scrofarum was identified in a whiting (Reid et al., 2010); C. parvum was found in School whiting, Nile tilapias, a Silver barb, Arctic char and European whitefish and C. hominis was reported in Mackerel scad (Reid et al., 2010, Gibson-Kueh et al., 2011, Koinari et al., 2013, Certad et al, 2015). In one of the most recent studies, C. parvum was identified in freshwater fish from Lake Geneva (Lac Léman) by both histology and molecular analysis (Certad et al., 2015).…”