FIRST REPORT OF CRYPTOSPORIDIUM MOLNARI-LIKE GENOTYPE AND CRYPTOSPORIDIUM PARVUM ZOONOTIC SUBTYPES (IIaA15G2R1 AND IIaA18G3R1) IN BROWN TROUT (SALMO TRUTTA)

This study reports for the first time the molecular characterization of Cryptosporidium spp. in Salmo trutta. A total number of 613 brown trout was captured by local anglers in 44 Galician rivers within 10 river basins (NW Spain) during the 2015 fishing season (March-August) and classified into groups according to their size. The gastrointestinal tracts were dissected and differentiated in pyloric ceca and intestine, which were homogenized and concentrated in phosphate-buffered saline 0.04 M pH 7.2/diethyl ether (2:1). Cryptosporidium oocysts were observed by immunofluorescence microscopy in 103 of 613 specimens (16.8%), with a mean intensity of 326.7 oocysts/trout. The highest prevalence rate was detected in specimens <2 yr (23.1%). Considering the anatomical location, Cryptosporidium oocysts were observed in pyloric ceca (72 trout, 69.9%), intestine (15 trout, 14.6%), or in both locations (16 trout, 15.5%), showing statistically significant differences between the 2 locations (P < 0.01). The prevalence rate in the pyloric ceca increased with the age/size the fish (62.2% vs. 70.8% vs. 83.3% for trout <2, 2-3, and >3 yr, respectively). By contrast, the prevalence rate in the intestinal location decreased with the age/size of specimens (21.6% vs. 12.5% vs. 7.7% for trout <2, 2-3, and >3 yr, respectively), but statistically significant differences were not determined. The microscopic observation of clusters of 4-20 oocysts in the pyloric ceca from 5 specimens of 20-28-cm body length is remarkable. By polymerase chain reaction amplification and sequencing of fragments of small-subunit ribosomal DNA (SSU-rDNA), GP60, hsp70, and actin loci, Cryptosporidium molnari-like genotype was identified in 1 trout and Cryptosporidium parvum (subtypes IIaA15G2R1 and IIaA18G3R1) in 47 fish, including those specimens in which oocyst clusters were observed. This finding may indicate a true infection by C. parvum, as the homogenization process would break the epithelial cells, releasing oocysts, free or in clusters. Cryptosporidium oocysts were detected in wild trout captured from 27 of 44 rivers sampled in Galicia (61.4%), belonging to 9 of the 10 river basins considered, confirming the presence of this protozoan parasite in Galician rivers and proving their wide dispersion in aquatic freshwater environments. The identification of the zoonotic species C. parvum in brown trout may indicate a risk to public health as trout may be a potential source of infection to humans. Thus, edible wild fish extend the range of foodstuffs involved in the transmission of cryptosporidiosis.