Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina.

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis.

Cyclosporiasis is an emerging worldwide infection caused by an obligate intracellular protozoan parasite, Cyclospora cayetanensis. In immunocompetent patients, it is mainly manifested by self-limited diarrhea, which is persistent and may be fatal in immunocompromised patients. The standard treatment for cyclosporiasis is a combination of two antibiotics, trimethoprim and sulfamethoxazole. Gastrointestinal, haematologic and renal side effects were reported with this combination. Moreover, sulfa allergy, foetal anomalies and recurrence were recorded with no alternative drug treatment option. In this study, silver nanoparticles were chemically synthesized to be evaluated for the first time for their anti-cyclospora effects in both immunocompetent and immunosuppressed experimental mice in comparison to the standard treatment. The effect of silver nanoparticles was assessed through studying stool oocyst load, oocyst viability, ultrastructural changes in oocysts, and estimation of serum gamma interferon. Toxic effect of the therapeutic agents was evaluated by measuring liver enzymes, urea and creatinine in mouse sera. Results showed that silver nanoparticles had promising anti-cyclospora potentials. The animals that received these nanoparticles showed a statistically significant decrease in the oocyst burden and number of viable oocysts in stool and a statistically significant increase in serum gamma interferon in comparison to the corresponding group receiving the standard treatment and to the infected non-treated control group. Scanning electron microscopic examination revealed mutilated oocysts with irregularities, poring and perforations. Biochemical results showed no evidence of toxicity of silver nanoparticles, as the sera of the mice showed a statistically non-significant decrease in liver enzymes in immunocompetent subgroups, and a statistically significant decrease in immunosuppressed subgroups. Furthermore, a statistically non-significant decrease in urea and creatinine was recorded in all subgroups. Thus, silver nanoparticles proved their effectiveness against Cyclospora infection, and this will draw the attention to its use as an alternative to the standard therapy.

Antiprotozoal activity of magnesium oxide (MgO) nanoparticles against Cyclospora cayetanensis oocysts.

Outbreaks of Cyclospora cayetanensis infection have been linked to consumption of food and water contaminated by oocysts that can survive both physical and chemical disinfectants. Magnesium oxide (MgO) nanoparticles (NPs) can be potentially used in food as bactericides. In this study, C. cayetanensis pre- and post-sporulated oocysts were exposed to MgO NPs with different doses ranging from 1.25-25 mg/ml. With comparison to control, the antiprotozoal activity of MgO NPs was evaluated by identifying the median effective concentration dose (EC50), lethal concentration dose (LC90), microscopically changes on treated oocysts and rates of sporulation. Among pre- and post-sporulated oocysts, MgO NPs ≥ EC50 was observed after 24 h at concentrations 10 and 12.5 mg/ml, respectively, while ≥ LC90 was observed after 24 h, 48 h and 72 h at concentrations 15, 12.5 and 10 mg/ml, respectively. MgO NPs treated oocysts showed abnormal morphological changes such as an increase in size, wall injury, deposition of vacuolated homogenous particles in the cytoplasm, evacuation of oocyst's contents, and collapse. Sporocysts of treated oocysts were noticed to be peripherally shifted. Sporulation failure of treated oocysts achieved ≥90% after 24 h and 72 h of incubation with 15 and 12.5 mg/ml, respectively, while it was 10.1% among non-treated. All the differences were statistically significant. Our results demonstrated that MgO NPs has a significant anti-Cyclospora effect on both unsporulated and sporulated oocysts, especially considering that it could be biologically synthesized, that way it can be used safely as a preventive agent in food and water disinfectant treatment.

Efficacy of wash solutions in recovering Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil.

Parasitic diseases can be acquired by ingestion of contaminated raw or minimally processed fresh produce (herbs and fruits). The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash solutions in removing them from suspect samples. In this study, six wash solutions (sterile E-Pure water, 3% levulinic acid-3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate-buffered saline, 0.1% Alconox, and 1% HCl-pepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil. One hundred or 1,000 oocysts of these parasites were inoculated onto the adaxial surfaces of 25 g of basil leaves, placed in stomacher bags, and stored for 1 h at 21°C or 24 h at 4°C. Leaves were hand washed in each wash solution for 1 min. DNA was extracted from the wash solutions and amplified using PCR for the detection of all parasites. Oocysts inoculated at a concentration of 1,000 oocysts per 25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts per 25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl-pepsin wash solution. Oocyst recovery rates were higher at 1 h than at 24 h postinoculation. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. The observations in this study provide guidance concerning the selection of wash solutions giving the highest retrieval of parasite oocysts.

Efficacy of gaseous chlorine dioxide as a sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on produce.

The efficacy of gaseous chlorine dioxide to reduce parasite and bacterial burden in produce was studied. Basil and lettuce leaves were inoculated with Cryptosporidium parvum and Cyclospora cayetanensis oocysts, Encephalitozoon intestinalis spores, and a cocktail of two isolates of nalidixic acid-resistant Escherichia coli O157:H7. The inoculated samples were then treated for 20 min with gaseous chlorine dioxide at 4.1 mg/liter. Cryptosporidium had a 2.6 and 3.31 most-probable-number log reduction in basil and lettuce, respectively. Reduction of Encephalitozoon in basil and lettuce was 3.58 and 4.58 CFU/g respectively. E. coli loads were significantly reduced (2.45 to 3.97 log), whereas Cyclospora sporulation was not affected by this treatment.

High hydrostatic pressure and UV light treatment of produce contaminated with Eimeria acervulina as a Cyclospora cayetanensis surrogate.

The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (10(6) oocysts) and low (10(4) oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40 degrees C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (10(4) oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (10(4) oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.

Six human cyclosporiasis: with general review.

Cyclospora cayetanensis is an emerging coccidian pathogen known in animal since a long time, but recognized in human in last two decade. The present study of cyclosporiasis specified its characteristic features. An epidemiological, clinical and biological retrospective study was carried out on 6 patients, 24 to 67 years old, who complained of diarrhea and weight loss, after a travel abroad. The only pathogenic agent found in every patient was Cyclospora cayetanensis. The patients returned back from Asian countries and from Madagascar. They were cured by Trimethoprim-sulfamethoxazole (TMZ). Diarrhoea was the main symptom of infection by C. cayetanensis. Undoubtedly, transmission occurred by the oro-fecal route, and/or by consumption of infected water or foods. Diagnosis was asserted, recovery of this protozoan parasite in the stools samples. TMZ proved to be the most effective and possibly replaced Ciprofloxacine. The prophylaxis recommended control measure of water supply and foods. Cyclospora should be considered in the assessments of patients with unexplained prolonged diarrhoea, if the investigation for bacterial and parasitological diarrhoea or the traveler's diarrhoea were negative.

Inactivation of protozoan parasites in food, water, and environmental systems.

Protozoan parasites can survive under ambient and refrigerated storage conditions when associated with a range of substrates. Consequently, various treatments have been used to inactivate protozoan parasites (Giardia, Cryptosporidium, and Cyclospora) in food, water, and environmental systems. Physical treatments that affect survival or removal of protozoan parasites include freezing, heating, filtration, sedimentation, UV light, irradiation, high pressure, and ultrasound. Ozone is a more effective chemical disinfectant than chlorine or chlorine dioxide for inactivation of protozoan parasites in water systems. However, sequential inactivation treatments can optimize existing treatments through synergistic effects. Careful selection of methods to evaluate inactivation treatments is needed because many studies that have employed vital dye stains and in vitro excystation have produced underestimations of the effectiveness of these treatments.

Effects of pesticides on sporulation of Cyclospora cayetanensis and viability of Cryptosporidium parvum.

Disease outbreaks caused by the coccidian parasite Cyclospora cayetanensis in food have been linked to consumption of raspberries that may have been contaminated through exposure to water mixed with insecticides and fungicides that may have been sprayed onto the berries. Three different fungicides (captan 50% W.P., benomyl 50% W.P., and zineb 75% W.P.) and two different insecticides (malathion 25% W.P. and diazinon 4E 47.5%) were evaluated at five different concentrations and for exposure times of 30 min to 1 week. Sporulation of C. cayetanensis did not decrease with use of any of the pesticides from time periods of 30 min to 24 h at all concentrations. Sporulation percentage was reduced with the fungicide benomyl at 1 week of exposure. The growth of the parasite Cryptosporidium parvum was also evaluated using captan 50% W.P., benomyl 50% W.P., and diazinon 4E 47.5%. Oocyst infectivity was reduced only after 7 days of exposure. These results indicate that these pesticides used at recommended concentration levels do not affect the sporulation of Cyclospora.

Coccidial contamination of raspberries: mock contamination with Eimeria acervulina as a model for decontamination treatment studies.

Numerous outbreaks have been reported since 1995 in the United States and Canada that were linked to the consumption of imported fresh raspberries contaminated with Cyclospora. Because Cyclospora has no laboratory animal hosts, Eimeria acervulina, a common chicken coccidium similar in characteristics to Cyclospora, was used as a surrogate to test decontamination treatments. Raspberries were mock contaminated with E. acervulina-sporulated oocysts in a water suspension, then exposed to washing, freezing, heat, or irradiation before they were fed to chicks. The presence of oocysts in the contaminated raspberries was confirmed either by duodenal lesions or oocysts in cecal contents 5 days postinoculation (PI) or in fecal contents 6 days PI, after 24 h of fecal collection. Washing of raspberries was generally not adequate in removing coccidial contamination, but freezing and heat treatment appeared effective. Gamma irradiation of E. acervulina-sporulated oocysts at a dose of 0.5 kGy was partially effective, but it was completely effective at 1.0 kGy and higher. We suggest that E. acervulina, for mock contamination of raspberries and subsequent decontamination treatments, is easy to handle, safe, and economical to study.