José Antonio Castro-Hermida

Prevalence of and risk factors involved in the spread of neonatal bovine cryptosporidiosis in Galicia (NW Spain).

n Abstractn n An epidemiological study was carried out on farms in Galicia (NW Spain) to investigate the prevalence of and the risk factors associated with the spread of infection by Cryptosporidium parvum in calves of less than 3 weeks of age. A total of 22 cattle farms (10 beef herds and 12 dairy herds) were visited once every 21 days between January and December 2000. A faecal sample was collected directly from the rectum of each of the 844 calves born during the study period. Each sample was studied macroscopically to establish its consistency as liquid, soft or solid, and the presence of mucus or blood noted. C. parvum oocysts were identified by direct microscopic examination and the intensity of infection established semiquantitatively as slight, moderate or severe. Of the 844 calves, 404 were found to have the parasite in their faeces, i.e. the prevalence was 47.9%. Statistical analysis of the risk factors such as general characteristics of the farm and the housing conditions of the calves, revealed three variables that significantly effect the risk of cryptosporidial infection in suckling calves: the method of cleaning, the type of flooring and the frequency of cleaning.n n

A study of cryptosporidiosis in a cohort of neonatal calves

During calving time on an experimental farm, 32 newborn calves were selected at random and monitored for infection with Cryptosporidium parvum for the first 30 days of their lives. The animals were fed pooled colostrum for 2-3 days after birth and housed in individual pens, which were washed daily using a pressure hose. Fecal smears were examined by microscopy after staining with carbol fuschin for visualization of oocysts. Oocyst shedding was scored semiquantitatively according to the average number of oocysts in 20 randomly selected fields at 1000x magnification. All the animals acquired the infection before 18 days of age. The period of maximum risk was between 9 and 12 days; 50% of the animals were infected by 9.4 days of age. It was found that the earlier the animals acquired the infection, the longer the patent period. Oocyst shedding, which did not always begin with the onset of diarrhea, lasted between 8 and 23 days (mean 12.4+/-3.3 days). Furthermore, fecal samples from 32 periparturient cows (within +/-7 days of giving birth) were filtered, concentrated and examined for oocysts using a fluorescent monoclonal antibody test, which revealed that six of the cows, although asymptomatic, were excreting C. parvum oocysts.

Herd-level risk factors for Cryptosporidium infection in dairy-goat kids in western France.

n Abstractn n We conducted a cross-sectional study of risk factors for herd-level kid positivity for Cryptosporidium parvum oocysts in dairy-goat farms (Deux-Sèvres, western France). From January to March 2003, faeces from a convenient sample of 879 5- to 30-day-old goat kids from 60 herds were examined microscopically after staining with carbol fuschin. Oocyst shedding was scored semi-quantitatively (0 to 4+) allowing us to obtain a cumulative score per herd. Standardized questionnaires with information about management practices were collected in each farm. We found positive kids in 32 of 60 herds (53.3%) and in 142 animals out of 879 (16.2%). We used logistic regression for two risk-factor model: (1) simple positive (case: herd score ≥1+, at least one positive kid in the herd, versus control: herd score=0), (2) strongly positive (case: overall herd score ≥3+ versus control: herd score <3+). Risk factors associated with simple positive herds were period of sampling compared to the peak of births (After versus Before, OR=4.2, 95% CI 1.2, 15.3) and practice of kid grouping by age or weight (Yes versus No, OR=4.4, 95% CI 1.0, 19.1). Risk factors associated with strongly positive herds were period of investigation (February/March versus January, OR=12.7, 95% CI 2.1, 76.6), exposure to graminaceous plants in forage (OR=11.6, 95% CI 1.7, 81.0) and type of ventilation in the goat premises (Vertical versus Wind effect, OR=14.7, 95% CI 2.1, 106.1). No important association was found between kid-management practices and herd positivity. These results suggest a major role of the environment of kids during their first hours of life in the adult-goat premises regarding the transmission of C. parvum infection.n n

Cryptosporidium Contamination in Harvesting Areas of Bivalve Molluscs

Cryptosporidium contamination was evaluated in areas in Galicia (northwestern Spain) where bivalve molluscs are harvested. Galicia is the main mussel-producing region in Europe. Data were collected on water contamination of effluents that are discharged into these areas. Cryptosporidium spp. were detected by immunofluorescence microscopy and molecular methods in 71% of the river water samples (n = 7), 64% of raw sewage samples (n = 11), 50% of effluents from wastewater treatment plants (n = 16), and 29.3% of the mussel samples (Mytilus galloprovincialis, n = 184). Cryptosporidium parvum was identified in all samples of contaminated mussels, Cryptosporidium muris was found in three samples of effluent from wastewater treatment plants, and Cryptosporidium baileyi was found in a sample of raw sewage. Further studies are needed to determine the parasitological quality of water in these shellfish harvesting and recreational areas. Cryptosporidium could be a public health risk from consumption of raw or undercooked contaminated molluscs and use of contaminated waters for recreational purposes.

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.

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

Abstract. 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 4xa0days, with 15–25% viable oocysts remaining thereafter. In addition the infectivity of these oocysts at 10 and 31xa0days post-contamination was demonstrated using a suckling murine model.

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.

Cooking mussels (Mytilus galloprovincialis) by steam does not destroy the infectivity of Cryptosporidium parvum.

The consumption of shellfish has increased considerably worldwide, with an associated increase in foodborne illnesses. Among the bivalves, the mussels are usually cooked by steam, which constitutes a typical dish in several regions. In this article, we demonstrate that this preparation is not sufficient to destroy completely the infectivity of Cryptosporidium parvum. Oocysts recovered from experimentally contaminated mussels (Mytilus galloprovincialis) were infectious to neonatal mice after cooking. Although, to date, no official cases of cryptosporidiosis linked to shellfish consumption have been reported, we recommend that people with reduced immunity avoid this type of food because they are at high risk of being infected with Cryptosporidium spp. after eating raw or undercooked contaminated bivalves.

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.