CRYPTOSPORIDIUM IN BIVALVE MOLLUSCSAuthor:
GÓMEZ COUSO HIPÓLITO.
Year: 2005.
University:
SANTIAGO DE COMPOSTELA.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.
Summary: POLLUTION STUDY OF NATURAL During the period between October 2000 and September 2001, investigated the presence and viability of the oocysts of Cryptosoporidium spp., In 241 samples of bivalve shellfish for human consumption provided by the Unit Control Shellfish Institute of Aquaculture at the University of Santiago de Compostela. Through a technique of PDI, which joined the coloring vital fluorogénico iodide propidio were observed Cryptosporidium oocysts spp. In 83 samples (34.4% of pollution). Of these, 44 (53.0%) contained potentially viable oocysts. Considering the origin of the samples, contamination was detected in 69 of the 203 samples collected on the coast galleo (34.0% and 49.3% of pollution feasibility ooquística) and in 14 of 38 samples from other countries in the European Union (36.8% and 71.4% of pollution feasibility ooquística). It found a lack of correlation (2, P = 0.20) between pollution Cryptosporidium spp., And the purification process to which they were subjected some of the samples (from one to more than 14 days). Nor there was a significant association (2, P = 0.85) between the levels of microbiological contamination and contamination by Cryptosporidium spp., Detected in bivalve molluscs analyzed. Prior to the molecular characterization of the isolated Cryptosoporidium spp. Was confirmed the absence of inhibitors of PCR technique in DNA extracted from shellfish and contaminated with DNA cryptosporidial. In addition, using 49 samples of bivalve molluscs randomly selected from the 241 analyzed earlier, a significant association was found (Fisher exact test, P = 0019) between the results obtained through the techniques of PCR and PDI. The combination of techniques RFLP, cloning and DNA sequencing to determine the existence of C.parvum and C.hominis, species currently considered responsible for the majority of human infections. The study conducted on samples of water (7 rivers, 16 treated wastewater and 11 raw sewage) and 184 biological samples (mussel M.galloprovincialis) collected in 4 estuaries (Rias Baixas), major production areas of bivalve molluscs and interest tourism, showed a broad environmental contamination by Cryptosporidium and Giardia. By PDI technique, it was found the presence of Cryptosporidium oocysts spp. In 5 samples of water river (71.4%), 8 treated wastewater (50.0%), 7 raw sewage (63, 6%) and 42 biological samples (22.87%). Likewise, were detected cysts of Giardia spp. In 6 samples river (85.7%), 14 treated wastewater (84.5%), 10 wastewater gross (90.9%) and 77 samples the mussels (41.8%). There was a large variability between quantitative values for both parasites and found a prevalence of Giardia spp. Vs. Cryptosporidium spp. Not only in the type of sample analyzed, but also in the concentrations detected, except in the biological samples that Cryptosporidium spp. introduced higher values of concentration. It showed an increase in pollution both enteroparásitos grows levels of bacterial contamination, both in the biological sample in the area under cultivation. However, only found a statistically significant association with the presence of cysts of Giardia spp. (Fisher's exact test, P = 0026; 2, P = 0008). The molecular characterization of the isolated Cryptosporidium spp. In environmental and biological samples, helped identify C.muris as the kind pollutant in three samples of purified wastewater: C.bailey in a sample of wastewater and crude C. delas parvum in all biological samples positive by PCR technique (n = 26). PILOT STUDY OF CONTAMINATION studies pollution pilot took place at the facilities of the Institute of Aquaculture at the University of Santiago de Compostela for the cultivation ym 8 antenimi 105b nt of bivalve molluscs. We used seeds of two species, O.edulis and T.decussatus, with a size of lava from 5-7 mm (approximately 8 months old) that were provided by the Center for Crops Mariños Ribadeo (Lugo) and the Experimentation Center in Aquaculture of Couso (A Coruna), belonging to the Network of Research Centers Mariña of the Ministry of Fisheries and Maritime Affairs of the Galician regional government. The seeds were maintained over the studies of 60 liter tank with natural seawater, at a constant temperature of 15 +-3Â ° C with aeration and controlled. The food consisted of a mixture composed equally by five species of microalgae and pollution pilot was carried out with different isolates cooquistes purified of C.parvum. We evaluated the survival of oocysts of C.parvum captured by the two species of bivalve molluscs above, comprobadose a sharp decline in the viability ooquística during the first four days of the study (97% vs. 30%). From the day four and until 10 post-contaminación, loss of viability ooquística was gradual, reaching the final day of the test values of 12% and 18% paralos oocysts retained by the oyster and clam seeds, respectively. Inoculation intragástrica of sediments derived from seed clam and oyster on the day 10 post-contaminación two litters of newborn mice Swiss CD-1, showed that all animals were inoculated infected at seven days post-inoculation. Likewise, it was found the maintenance of infectivity ooquística to 31 days post-contaminación. By histological examinations were studied transit of oocysts of C.parvum through seed clam T.decussatus. During the 10-day trial, was viewed oocysts in the gut: a 24-hour were measured over 10 oocysts Field microscopic x40 and on several occasions during the experiment were observed between 5 and 10 oocysts (days 2.4, 6.7 and 10 post-contaminación). The gills were also detected oocysts, from day 2 until 8 post-contaminación consecutively and always in a number not exceeding 5 oocysts Field microscopic x40. In this tissue, the oocysts are located in spaces interfilamentarse as in the mucus branquial. To a lesser average, the oocysts of C.parvum were found in the stomach, using siphons and in the hepatopancreas. At no point of the study was observed by the techniques used, that the parasite were internalized by cells in the mollusk, keeping the oocysts free or mixed with stomach contents and / or intestinal epithelia branquial without passing through and / or gastrointestinal. Moreover, assessed the release of oocysts of C.parvum from live and dead seeds and their subsequent capture by seeds of a different kind. When we studied the transmission of oocysts between coexisting species alive, it was observed in species receiving a decrease in the number of parasite and maintaining the viability oocysts in its viability in the kind of mollusk receiving until its demise before the end the study. Having found that bivalve molluscs represent a potential source of human infection by Cryptosporidium and mussels steamed being a typical dish in certain countries, finally studied, on a pilot basis, if this method of cooking inactivates the infective forms of the parasite. The results obtained in mice infants showed that the use of steam does not completely eliminate the ability of infective oocysts of C.parvum
