F. Freire-Santos

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.

Treatment with β-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions

Following the unexpected activity of the excipient β-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of β-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with β-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. β-cyclodextrin was well tolerated by all of the treated 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.

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).

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

Abstract. Two commercially valuable mollusc species (Ostrea edulis and 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.

UNEXPECTED ACTIVITY OF β-CYCLODEXTRIN AGAINST EXPERIMENTAL INFECTION BY CRYPTOSPORIDIUM PARVUM

An unexpected activity of β-cyclodextrin, an excipient used in pharmaceutical technology, was observed against Cryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to β-cyclodextrin (2.5% suspension), were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of the viability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7 days postinoculation by examination of intestinal homogenates, was significantly lower (P < 0.05) than in the control litters. The preventive and curative efficacies of β-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice. Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation.

Cryptosporidium parvum : An attempt at experimental infection in rainbow trout Oncorhynchus mykiss

A study was carried out on rainbow trout Oncorhynchus mykiss fed with a commercial feed contaminated with bovine-isolated Cryptosporidium parvum oocysts whose viability and infectivity were previously tested by inoculation of oocysts to neonatal Swiss CD-1 mice. Histological examination of hematoxylin-eosin-stained gastrointestinal sections from control and C. parvum-exposed fish revealed no life-cycle stages of Cryptosporidium in any part of the apical border of the digestive tract. However, sections of the stomach and pyloric region from exposed fish displayed large numbers of 5-7-microm spherical structures located deep within the epithelial tissue. Under conditions of host stress, the number of these structures increased remarkably. An immunofluorescence antibody test with IgG and IgM anti-cryptosporidial antibodies revealed fluorescence reactivity in these structures. Simultaneously, wild trout were analyzed in order to detect natural cryptosporidial infections; Cryptosporidium oocyst-like bodies were found in the intestinal content of 10% of the specimens.

Evaluation of β-cyclodextrin against natural infections of cryptosporidiosis in calves

The effectiveness of beta-cyclodextrin, excipient used in pharmaceutical industry, in the treatment of natural infection by Cryptosporidium parvum in suckling calves, was evaluated. Administration of the drug at a dose of 500 mg/kg body weight for 3 consecutive days from birth (prophylactically) or following confirmation of the infection (therapeutically) decreased the severity of diarrhoea and shortened the duration of oocyst shedding.

Efficacy of β-Cyclodextrin Against Experimental Cryptosporidiosis in Neonatal Lambs

The efficacy of β-cyclodextrin against experimental Cryptosporidium parvum infection was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 1 g/kg of body weight during 3 consecutive days. Preventive treatment was started within 1 day of birth, and therapeutic treatment was initiated at the onset of diarrhea following confirmation of infection. Disease development and drug efficacy were evaluated by monitoring the presence or absence of diarrhea and oocyst shedding from birth until 30 days of age. Weight gains at 15 and 30 days of age were also recorded. β-Cyclodextrin was highly effective as a prophylactic treatment; 1 animal did not acquire the infection, diarrhea was prevented in infected animals, and there was a considerable decrease in oocyst shedding. The therapeutic treatment was effective in decreasing the severity of diarrhea and the duration of oocyst shedding. The animals tolerated the drug well, and there was a significant increase in their body weights.

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.