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. METHODS AND RESULTS: Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. CONCLUSIONS: Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. SIGNIFICANCE AND IMPACT OF STUDY: This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.

A histological study of the transit of Cryptosporidium parvum oocysts through clams (Tapes decussatus).

A histological study was carried out to investigate the transit of Cryptosporidium parvum oocysts through the clam Tapes decussatus. Spat of approximately 5-7 mm shell length were maintained in a tank of natural sea water contaminated with purified C. parvum oocysts. The experiment lasted 240 h and, every 24 h, five specimens were killed, placed in Bouin's fixative, and processed routinely for histological examination. Sections (3 mum) cut from the all body tissues were stained with modified Gomori's trichrome for their accurate identification; the oocysts were detected by a direct immunofluorescence procedure. Oocysts were detected in siphons, gills, stomach, digestive diverticula, and intestine. The oocysts present in the intestine were free or mixed with the intestinal contents; therefore release of these oocysts with the feces should favour dissemination of contamination. Oocysts were found in branchial mucus and within the interfilamentary spaces, which suggests the occurrence of repeated filtrations and the possibility that the retained oocysts maintain their infective capacity.

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR.

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.

Anticryptosporidial activity of furan derivative G1 and its inclusion complex with beta-cyclodextrin.

The capacity of beta-cyclodextrin (betaCD) to form a complex with a new furanic derivative, G1, was investigated. Interactions of the drug and betaCD in solution and in the solid state were studied using phase solubility techniques, thermal methods, X-ray, and IR spectroscopy. Preparation of a kneaded mix of G1/betaCD increased both the aqueous solubility and the dissolution rate of the furan derivative. The anticryptosporidial efficacies of the drug and of the inclusion complex were evaluated using a suckling murine model. Oral administration of G1 considerably decreased the intensity of the infection, but betaCD showed similar anticryptosporidial activity to that of the betaCD-G1 complex and higher activity than G1 alone.

Contamination of bivalve molluscs by Cryptosporidium oocysts: 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.

In vitro and in vivo efficacy of alpha-cyclodextrin for treatment of experimental cryptosporidiosis.

The efficacy of alpha-cyclodextrin against infection by Cryptosporidium parvum was evaluated using in vitro and in vivo models. Cyclodextrins are water-soluble cyclic hexamers of glucose units with hydrophobic cavities capable of solubilizing lipophiles and are widely used as drug excipients in the pharmaceutical industry. The viability of purified C. parvum oocysts, exposed for 30, 60, 90, 120 min and 24h to different concentrations of alpha-cyclodextrin (2.5, 5, 7.5, 10, 12.5 and 15%), was evaluated by inclusion or exclusion of two fluorogenic vital dyes and by an excystation technique. Preventive and curative efficacies against cryptosporidial infections, at different doses (2.5 and 5%) and regimes of administration of alpha-cyclodextrin, were determined in an experimental neonatal mice model. Results of the viability assay showed a decrease in oocyst viability that was associated with an increase in exposure time, for each of the concentrations used. Moreover, a high proportion of nonviable oocysts (81%) was observed when C. parvum oocysts were exposed to alpha-cyclodextrin (2.5%) for 24h. The intensity of infection, determined 7 days post-inoculation by examination of intestinal homogenates, was significantly lower (P<0.05) than in the control litters, for all the assays carried out with alpha-cyclodextrin. Only 38.8% of the animals became infected when the alpha-cyclodextrin solution (5%) was administered 2h before inoculated oocysts, and every 24h at 1 and 2 days post-inoculation.

Environmental dispersal of Cryptosporidium parvum oocysts and cross transmission in cultured bivalve molluscs.

Two commercially valuable mollusc species ( Ostrea edulisand Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. A direct immunofluorescent antibody technique and inclusion/exclusion of the fluorogenic vital dye propidium iodide were used to test for the presence and viability of the oocysts, showing that transmission of contamination occurred between coexisting species. There was a decrease in the viability of oocysts in the initially uncontaminated molluscs as well as a large decrease in the number of oocysts retained when dead molluscs were used as the source of contamination. The results show the potentially important role that these molluscs play in spreading contamination in depuration plants and areas where aquatic organisms are cultivated.

Survival of Cryptosporidium parvum oocysts recovered from experimentally contaminated oysters (Ostrea edulis) and clams (Tapes decussatus).

Samples of two species of shellfish that form part of the human food chain (the oyster Ostrea edulis and the marine clam Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. Changes in the viability of oocysts subsequently recovered from the shellfish were evaluated by means of an immunofluorescent antibody technique (IFAT) and inclusion/exclusion of the fluorogenic vital dye propidium iodide. There was a sharp decrease in oocyst viability during the first 4 days, with 15-25% viable oocysts remaining thereafter. In addition the infectivity of these oocysts at 10 and 31 days post-contamination was demonstrated using a suckling murine model.

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.

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

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.

The efficacies of diloxanide furoate, beta-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 beta-cyclodextrin, an excipient used in pharmaceutical technology.

Serological response to Cryptosporidium parvum in adult cattle from the Andean region of Colombia.

Single faecal and serum samples were individually collected from 135 asymptomatic adult cows on seven farms in Cundinamarca (Colombian Andean region). Tests for the presence of oocysts of Cryptosporidium parvum (carbol fuchsin stain) and Eimeria spp (flotation in saturated saline solution) revealed that none of the animals had coccidia in their faeces. The IgG antibody levels to C. parvum were measured by an enzyme-linked immunosorbent assay (ELISA) technique and the reactivity to C. parvum antigens by a Western blotting procedure. Cryptosporidial antibodies were detected in cattle from all farms, with 53.3% (72 animals) being seropositive. Sera recognized 5-11 protein fractions with molecular masses ranging from 12 14 kDa to 97-100 kDa. Sera considered as positive by ELISA reacted intensely and more frequently with protein fractions of approximately 20-22, 42-48, 51-57 and 60-69 kDa, whereas only the 42-48 kDa antigen was strongly recognized by sera without IgG antibodies. The presence of IgG antibody against C. parvum in most animals, as well as the reactivity to major proteins of C. parvum, could be indicative of continuous exposure to this parasite.

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.

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.

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.

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%).

Effect of ultraviolet disinfection of drinking water on the viability of Cryptosporidium parvum oocysts.

Demineralized water was enriched with a known number of Cryptosporidium parvum oocysts obtained from fresh calf feces, which were purified and exposed to ultraviolet (UV) light (15,000 mW/sec) for different lengths of time. Coccidium-free litters of CD-1 neonatal mice then were inoculated intragastrically with the treated water. Seven days postinoculation mice were killed and C. parvum infection prevalence and intensity determined. In mice inoculated with C. parvum-enriched water that had been exposed to UV light for at least 150 min, no infection occurred.

Efficacy of activated sludge in removing Cryptosporidium parvum oocysts from sewage.

Primary clarifier effluent (procedure B) and final effluent (procedure A) from a wastewater treatment plant were enriched with Cryptosporidium parvum oocysts obtained from the feces of naturally infected calves. Procedure B samples alone were subjected to a laboratory simulation of activated-sludge treatment. Coccidium-free neonatal CD-1 mice were then inoculated intragastrically with procedure A or procedure B samples. Seven days after inoculation, the intensity of oocyst infection in procedure B mice was 91% less than in procedure A mice (controls).