Cryptosporidium spp. in Freshwater Bivalves in Portugal

Abstract

RYPTOSPORIDIUM spp. in water has been implicated inseveral human cryptosporidiosis outbreaks (Fayer 2004).Studies on Cryptosporidium oocysts in surface waters are madedifficult by the low concentrations of organisms present due todilution. Since bivalves are filter-feeders oocysts in suspended inwater can be removed by this filtration and the mollusc digestiveenzymes reduce oocyst viability (Freire-Santos et al. 2002). Ifoocysts survive the bivalve digestive tract and are excreted, thedense fecal material containing viable oocysts are expected tosedimenttothebottomoftheriverbed.However,bivalvefiltrationmechanisms can also concentrate Cryptosporidium oocysts pres-ent in the water. Hence it is possible to recover higher numbersof oocysts from the bivalves compared with that recoverable fromsurface waters (Graczyk et al. 2004).Human consumption of freshwater clams and mussels is com-mon in northeast Portugal and in the Tejo and Guadiana riverbasins. These bivalves might be a source of human Cryptospo-ridium infection therefore, it is important to assess the publichealth risks of eating contaminated bivalves. As bivalves can beuseful bioindicators of superficial water contamination (Graczyket al. 2004), we examined clams in Portugal for the presence ofCryptosporidum oocysts examined evaluated considering thatclams can be.MATERIALS AND METHODSThree freshwater bivalves, Anodonta anatina, Unio pictorum,and Corbicula fluminea, were analyzed. Sixteen sampling areas inthe Guadiana river hydrographic basin in southeast Portugal wereselected and the clams were randomly collected by searching andby kick sampling. A total of 55 samples were obtained betweenMay 2003 and June 2005: 69% (24/55) were ofU. pictorium,50%(19/55) were C. fluminea, and 31% (12/55) were A. anatine. Vari-ation in the water levels, turbidity, and unfavourable conditionsprecluded, being able to collect all three species at each samplingarea each time.Gills and the gastrointestinal tracts of each species were dis-sected from three to 15 bivalves (depending on their size) and thetissues were pooled to provide uniform sizes of preparations fromdifferent bivalves. The tissues were homogenized and oocystswere concentrated using a modification of the methods describedby Ritchie (Pereira da Fonseca 2000) and purified by discontinu-ous sucrose density centrifugation. The material collected at theinterface of the two layers with densities of 1,103 and 1,064 waswashed and concentrated into a pellet by centrifugation and thenresuspended in distilled water to a final volume of 1.5ml.Detection of oocysts was performed by direct immunofluores-cent assay(IFA)using themonoclonal antibody(Mab) kitCrypto/Giardia Cel (Cellabs, Brookvale, Australia). The isolated oocystpreparation was incubated with the Mab and observed by fluor-escence microscopy at 400 magnification. The size and shapeof Cryptosporidium oocysts enabled us to distinguish these fromGiardia cysts.Genotyping using 18S suRNA gene probes was performed on47 samples by nested PCR analysis (Xiao et al. 1999). Genom-ic DNA was extracted by the proteinase K method and the QiagenDNA extraction kit. Qiagen, Hilden, Germany. The primers usedwere 5