M.E. Ares-Mazás

Contamination of bivalve molluscs by Cryptosporidiumoocysts:The need for new quality control standards

A yearlong study was carried out to investigate the presence and viability of Cryptosporidium oocysts in 203 samples of cultured shellfish from Galicia (NW Spain) and 38 samples imported from other European Union (EU) countries. Shellfish samples included mussels, oysters, clams and cockles. Cryptosporidium oocysts were detected, using a direct immunofluorescence antibody test (IFAT), in 34.4% of the samples analyzed; use of the fluorogenic dye propidium iodide (PI) revealed viable potentially infective oocysts in 53.0% of these samples. There was no relation between the presence of Cryptosporidium oocysts and the microbiological contamination detected in the samples expressed as Most-Probable-Number (MPN) of fecal coliforms, the different species of mollusc, or the month of sampling. One important finding was that the depuration process was ineffective in totally removing oocyst contamination. Furthermore, the existence of viable oocysts in samples with microbiological contamination levels lower than 300 fecal coliforms/100 g, which in accordance with current legislation are considered suitable for human consumption, suggests the need to include parasitological analyses in the quality control for these molluscs.

Effect of salinity, temperature and storage time on mouse experimental infection by Cryptosporidium parvum.

Cryptosporidium parvum oocysts collected from a naturally infected calf were exposed to different salinity and temperature for 2, 21 and 40 days, and then inoculated intragastrically into coccidium-free neonatal mice. The intensity of infection as determined seven days later by examination of intestinal homogenates were statistically analysed. Salinity, time and salinity-time interaction were the only factors with significant effect on the infection intensity.

Viability and infectivity of oocysts recovered from clams, Ruditapes philippinarum, experimentally contaminated with Cryptosporidium parvum

Abstract. This study confirms the important role of marine bivalve molluscs, destined for human consumption, as transmitters of cryptosporidiosis, zoonotic diarrhoeal disease caused by Cryptosporidium parvum. C. parvum oocysts recovered from seawater clams (Ruditapes philippinarum) were viable and infective in five of eight infected neonatal CD-1 Swiss mice. Oocysts were observed in clam gill and gastrointestinal tract tissue homogenates as well as in gill histological sections, by an immunofluorescent antibody technique. In vitro viability of recovered oocysts was also determined using fluorogenic vital dyes (75% viability).

Inhibition of Cryptosporidium infection in mice treated with a cyclodextrin inclusion complex with diloxanide furoate

Abstract. The efficacies of diloxanide furoate, β-cyclodextrin and a cyclodextrin inclusion complex against Cryptosporidium parvum were evaluated in a suckling murine model. Efficacy was established by numbers of oocysts recovered from the intestinal tract of mice on day 7 postinfection. The level of infection in treated mice was significantly lower than in control mice and, surprisingly, the most efficacious treatment was β-cyclodextrin, an excipient used in pharmaceutical technology.

Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

Aims:  To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish.

In vitro and in vivo efficacy of lasalocid for treatment of experimental cryptosporidiosis.

In vitro viability of purified Cryptosporidium parvum oocysts, exposed for 30, 60, 90 and 120min to 0.27mg/ml lasalocid suspension was evaluated by inclusion or exclusion of two fluorogenic vital dyes and an excystation technique. Continuously, preventive and curative efficacies at different doses (9, 6.75, 5.625 and 4.5mg/kg body weight) and regimens of lasalocid against cryptosporidial infection were evaluated on an experimental neonatal mice model. In vitro assays demonstrated a decrease in the oocyst viability related to an increase in exposure time for exposure to the lasalocid suspension. The infection was eradicated when the suspension was administered with a dose of > or = 6.75mg/kg body weight. No apparent toxic effects were observed.

Viability and infectivity of two cryptosporidium parvum bovine isolates from different geographical location

The viability of two Cryptosporidium parvum bovine isolates from Spain and Colombia was evaluated by in vitro excystation, inclusion/exclusion of two fluorogenic vital dyes (DAPI and PI) and infectivity assay in a suckling murine model. Excystation percentages were similar for both Spain and Colombia isolates (83% and 87%, respectively). The total viability of the Spain isolate, measured by inclusion/exclusion of two fluorogenic vital dyes, was 71% in comparison with that detected for oocysts of the Colombia isolate, 32.3%. The bovine C. parvum oocysts of both isolates were viable and infectious for suckling Swiss CD-1 mice. However, infectivity percentage and the mean intensity of infection were consistently higher in the Spain isolate than those from Colombia isolate. It was not possible to obtain a good correlation between in vitro excystation, inclusion/exclusion of vital dyes and in vivo infectivity for the Colombia isolate, while data obtained with the Spain isolate indicated that there was an apparent strong correlation between excystation efficiency, total viability and the infectivity. Although a comparative analysis of genetic variation among these isolates from different geographical location is necessary, variations observed between the both isolates seemed to be a result of parasite adaptation to environmental stresses such as temperature which appears to have a direct effect on the permeability of the oocysts.

Oocysts, IgG levels and immunoblot patterns determined for Cryptosporidium parvum in bovine examined during a visit to a farm (northeastern Spain).

Single fecal and serum samples were individually collected from 101 bovines selected at random during a visit to a farm in northeastern Spain (Group I, 26 animals aged 2-36 days; Group II, 34 animals aged 1.5-4.5 months; Group III, 41 animals aged 20-24 months). Testing for the presence of Cryptosporidium parvum oocysts in feces (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) indicated that 26% animals were infected (81% of Group I, 15% of Group II and 0% of Group III). Serological testing (ELISA for detection of specific anti-C. parvum IgG) indicated that 59% animals were seropositive (12% of Group I, 74% of Group II and 78% of Group III). Immunoblotting results indicate that cattle sera recognize C. parvum antigens of widely varying molecular weights and that the number of antigens recognized increases with age. Immunoblots revealed that some of the sera belonging to the Group I reacted with protein fractions between 15 and 20 kDa but none recognized the 21-23 kDa antigen. Only few sera in the Group II recognized the protein fraction between 15 and 20 kDa. The recognition of 21-23 kDa fraction was observed by four sera from uninfected and seropositive animals. Sera from all the seronegative Group II animals recognized few antigens and always with molecular weight greater than 50 kDa. Serum samples from both seropositive and seronegative animals belonging to the Group III recognized antigens with molecular weight ranging 15-20 kDa. Surprisingly, the protein fractions between 21 and 28 kDa reacted with approximately 30% of the sera from seropositive animals and only one of the nine sera from seronegative animals. The recognition of 42-46 kDa antigens increased with the age and only reacted with the sera from uninfected animals.

Cryptosporidium parvum oocyst antigens recognized by sera from infected asymptomatic adult cattle

Gel electrophoresis and Western blotting were used to investigate the recognition of Cryptosporidium parvum oocyst antigens by sera from asymptomatic C. parvum-seropositive cattle with or without coccidian oocysts in their faeces (C. parvum or/and Eimeria spp.). Most sera from coprologically C. parvum-positive animals (71%) recognized fractions in the 17-20 kDa range; these fractions were not recognized by sera from coprologically negative animals. In addition, most sera with antibodies to C. parvum (whether from coprologically positive or negative animals) showed moderate or strong reaction with antigenic fractions in the 47-49.5 kDa range (43%) and the 56.5-69 kDa range (80%).

Determination of immuno-cross-reactivity between Cryptosporidium parvum and Eimeria spp.

Immuno-cross-reactivity between Cryptosporidium parvum and Eimeria spp. was studied by the indirect fluorescent antibody test (IFAT) and Western blot procedure. Thirty-seven sera from asymptomatic (non-diarrheic) cattle, with known coprological (presence-absence of coccidia) and serological data respecting C. parvum, were tested by IFAT using Eimeria oocysts as antigen. Most sera (54%) displayed immunofluorescence around the surface of the Eimeria oocysts. Simultaneously, serum samples from rabbits naturally infected with Eimeria spp. (E. magna, E. intestinalis and E. residua), but free of C. parvum infection, were used to investigate the recognition of C. parvum oocyst antigens by the Western blot procedure. Fractions in the 11.5-94 kDa range, as well as others with molecular masses over 94 kDa, were recognized by sera from rabbits. Sera collected during patency period showed low or moderate reaction with antigenic fractions in the 11.5-25 kDa range. However, 29, 58 and 71 to 75 kDa proteic fractions were moderately or strongly recognized even after rabbits finished oocyst excretion.