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Abstract: Survival and growth of Salmonella and Shiga toxin-producing Escherichia coli (STEC) were investigated in kombucha prepared from four brands of commercially available kombucha kits intended for use by home brewers. Changes in populations of the indigenous microbiota responsible for fermentation of kombucha were also determined. An initial population of Salmonella (6.77 log CFU/mL) decreased to below the detection limit (0.30 log CFU/mL) within 10 days in kombucha prepared from two of the test brands. Populations of 1.85 and 1.20 log CFU/mL were detected in two brands fermented for 14 days. An initial STEC population of 7.02 log CFU/mL decreased to <0.30 log CFU/mL in two brands within 14 days; 0.77 and 0.87 log CFU/mL were detected in kombucha prepared from the other two brands. Salmonella and STEC increased within 1 day in three brands of base tea used to prepare kombucha and were stable throughout 14 days of incubation. Both pathogens steadily declined in base tea prepared from one brand of kombucha kit. Inactivation of the pathogens occurred as the pH of the kombucha decreased, but a clear correlation between rates of inactivation among different brands of kits and decrease in pH was not evident. Growth and peak populations of mesophilic aerobic microorganisms, yeasts, lactic acid bacteria, and acetic acid bacteria varied depending on the kombucha kit brand. No strong evidence was found of a correlation between the behavior of Salmonella or STEC and that of any of these groups of indigenous microbiota. Results of this study show that survival of Salmonella and STEC in kombucha and base tea used to prepare kombucha is dependent on inherent differences in commercially available kombucha kits intended for use in home settings. Strict application of hygienic practices is essential for preventing contamination with Salmonella or STEC and reducing the risk of illness associated the consumption of kombucha., (Copyright ©, International Association for Food Protection.)

Background: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant's antimicrobial content., Results: Internalized Salmonella was detected in cabbage within 24 h with prevalence ranging from 62% (16 of 26) for the 'Super Red 80' cultivar to 92% (24 of 26) for the 'Red Dynasty' cultivar. Surface survival of pathogens on small cabbage plants over nine days was significantly affected by cultivar with both pathogens surviving the most on the 'Farao' cultivar and Salmonella and O157 surviving the least on the 'Super Red 80' and 'Capture' cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen's fate. An inverse relationship existed between antimicrobial levels and the pathogen's surface survival on cabbage heads (P < 0.05)., Conclusions: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Outbreaks of salmonellosis have been associated with consumption of high-sugar, low-water activity (a w ) foods. The study reported here was focused on determining the effect of storage temperature (5 and 25°C) on survival of initially high and low levels of Salmonella in dry-inoculated sucrose (a w 0.26 ± 0.01 to 0.54 ± 0.01) and wet-inoculated sucrose (a w 0.24 ± 0.01 to 0.44 ± 0.04) over a 52-week period. With the exception of dry-inoculated sucrose at a w 0.26, Salmonella survived for 52 weeks in dry- and wet-inoculated sucrose stored at 5 and 25°C. Retention of viability was clearly favored in sucrose stored at 5°C compared with 25°C, regardless of level or type of inoculum or a w . Survival at 5°C was not affected by a w . Initial high-inoculum counts of 5.18 and 5.25 log CFU/g of dry-inoculated sucrose (a w 0.26 and 0.54, respectively) stored for 52 weeks at 5°C decreased by 0.56 and 0.53 log CFU/g; counts decreased by >4.18 and >4.25 log CFU/g in samples stored at 25°C. Inactivation rates in wet-inoculated sucrose were similar to those in dry-inoculated sucrose; however, a trend toward higher persistence of Salmonella in dry- versus wet-inoculated sucrose suggests there was a higher proportion of cells in the wet inoculum with low tolerance to osmotic stress. Survival patterns were similar in sucrose initially containing a low level of Salmonella (2.26 to 2.91 log CFU/g). The pathogen was recovered from low-inoculated sucrose stored at 5°C for 52 weeks regardless of type of inoculum or a w and from dry-inoculated sucrose (a w 0.54) and wet-inoculated sucrose (a w 0.24) stored at 25°C for 12 and 26 weeks, respectively. Results emphasize the importance of preventing contamination of sucrose intended for use as an ingredient in foods not subjected to a treatment that would be lethal to Salmonella.

Background: The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages., Methodology/principal Findings: T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development--days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15-30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages., Conclusions/significance: This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite's immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.

Poor hygiene and improper food preparation practices in consumers' homes have previously been demonstrated as contributing to foodborne diseases. To address potential cross-contamination by kitchen utensils in the home, a series of studies was conducted to determine the extent to which the use of a knife or grater on fresh produce would lead to the utensil's contamination with Escherichia coli O157:H7 or Salmonella enterica. When shredding inoculated carrots (ca. 5.3 log CFU/carrot), all graters became contaminated and the number of E. coli O157:H7 present on the utensil was significantly greater than Salmonella (p < 0.05). Contamination of knives after slicing inoculated produce (4.9-5.4 log CFU/produce item) could only be detected by enrichment culture. After slicing tomatoes, honeydew melons, strawberries, cucumbers, and cantaloupes, the average prevalence of knife contamination by the two pathogens was 43%, 17%, 15%, 7%, and 3%, respectively. No significant increase in the incidence or level of contamination occurred on the utensils when residues were present (p > 0.05); however, subsequent contamination of 7 produce items processed with the contaminated utensils did occur. These results highlight the necessity of proper sanitization of these utensils when used in preparation of raw produce., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Consumers are being advised to increase their consumption of fruits and vegetables to reduce their risk of chronic disease. However, to achieve that goal, consumers must be able to implement protocols in their kitchens to reduce their risk of consuming contaminated produce. To address this issue, a study was conducted to monitor the fate of Escherichia coli O157:H7 and Salmonella on produce (cantaloupe, honeydew melon, carrots, and celery) that were subjected to brushing or peeling using common kitchen utensils. Removal of similar levels of Salmonella from carrots was accomplished by peeling and by brushing, but significantly greater removal of E. coli O157:H7 from carrots was accomplished by peeling than by brushing under running water (P < 0.05). Brushing removed significantly fewer pathogens from contaminated cantaloupes than from other produce items (P < 0.05), suggesting that the netted rind provided sites where the pathogen cells could evade the brush bristles. A Sparta polyester brush was less effective than a scouring pad for removing Salmonella from carrots (P < 0.05). In all cases, brushing and peeling failed to eliminate the pathogens from the produce items, which may be the result of contamination of the utensil during use. High incidences of contamination (77 to 92%) were found among peelers used on carrots or celery, the Sparta brush used on carrots, and the scouring pad used on carrots and cantaloupe. Of the utensils investigated, the nylon brush had the lowest incidence of pathogen transference from contaminated produce (0 to 12%). Transfer of pathogens from a potentially contaminated Sparta brush or peeler to uncontaminated carrots did not occur or occurred only on the first of seven carrots processed with the utensil. Therefore, risk of cross-contamination from contaminated utensils to uncontaminated produce may be limited.

Cyclospora cayetanensis, a coccidian parasite, can cause gastrointestinal illness in humans and is characterized by watery and persistent diarrhea and abdominal pain. Cyclosporiasis has been associated with traveler's diarrhea. The infection is acquired through food and waterborne transmission, particularly by consumption of contaminated fresh fruits and vegetables. In the present study, stool samples from 8,877 children were examined for ova and parasites at the Pediatric Hospital of Morelia in Michoacán, Mexico, during 2000-2009. Sixty children (0.67%) had Cyclospora in their stools. Diarrhea (45.8%), abdominal pain (39.6%), and vomiting (18.8%) were the most frequent symptoms of cases with cyclosporiasis. Most of the cases (93.3%) were observed during June-August, the rainy season. In 45 children, Cyclospora was the only parasitic pathogen detected (75%); 15 children were co-infected with commensal, pathogenic, or both groups of parasites. Our findings suggest that C. cayetanensis is endemic to Michoacán and shows characteristically temporal patterns., (© The American Society of Tropical Medicine and Hygiene.)

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.

Globalisation is a manmade phenomenon encompassing the spread and movement of everything, animate and inanimate, material and intangible, around the planet. The intentions of globalisation may be worthy--but may also have unintended consequences. Pathogens may also be spread, enabling their establishment in new niches and exposing new human and animal populations to infection. The plethora of foodborne parasites that could be distributed by globalisation has only recently been acknowledged and will provide challenges for clinicians, veterinarians, diagnosticians, and everyone concerned with food safety. Globalisation may also provide the resources to overcome some of these challenges. It will facilitate sharing of methods and approaches, and establishment of systems and databases that enable control of parasites entering the global food chain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human noroviruses and hepatitis A virus (HAV) are commonly associated with outbreaks occurring in restaurant establishments and catered events. Food handlers are major contributing factors to foodborne illnesses initiated in the kitchen setting. In this study, transfer of HAV and murine norovirus (MNV-1), a human norovirus surrogate, between produce (cucumbers, strawberries, tomatoes, cantaloupes, carrots, and honeydew melons) and common kitchen utensils (graters and knives) was investigated. The extent of virus transfer to produce during utensil application, in the presence and the absence of food residue, and the impact of knife surface properties (sharp, dull, serrated) was also investigated. Transfer of MNV-1 and HAV from produce items, initially contaminated with ~5.5 log PFU, to knives and graters during application ranged from 0.9 to 5.1 log PFU. MNV-1 transfer to knives was the greatest for cucumbers, strawberries, and tomatoes, and the least for honeydew melons, while transfer of HAV to knives was greater for tomatoes and honeydew melons than strawberries, cantaloupes, and cucumbers. After preparation of a contaminated produce item, knife cross-contamination easily occurred as viruses were detected on almost all of the seven produce items successively prepared. Produce residues on utensils often resulted in less virus transfer when compared to utensils without residue accumulation. Knife surface properties did not impact virus transfer. The ease of virus transfer between produce and utensils demonstrated by the current study highlights the importance of efforts aimed toward preventing cross-contamination in the kitchen environment.

The association of wealth and infections with Giardia, Cryptosporidium, Cyclospora, and microsporidia were examined in a longitudinal cohort conducted in Peru from 2001 to 2006. Data from 492 participants were daily clinical manifestations, weekly copro-parasitological diagnosis, and housing characteristics and assets owned (48 variables), and these data were used to construct a global wealth index using principal component analysis. Data were analyzed using continuous and categorical (wealth tertiles) models. Participant's mean age was 3.43 years (range = 0-12 years), with average follow-up of 993 days. Univariate and multivariate analyses identified significant associations between wealth and infections with Giardia and microsporidia. Participants with greater wealth indexes were associated with protection against Giardia (P < 0.001) and persistent Giardia infections (> 14 days). For microsporidia, greater wealth was protective (P = 0.066 continuous and P = 0.042 by tertiles). Contrarily, infections with Cryptosporidium and Cyclospora were independent of wealth. Thus, subtle differences in wealth may affect the frequency of specific parasitic infections within low-income communities.

Foodborne parasites are characterized as being highly resistant to sanitizers used by the food industry. In 2009, a study reported the effectiveness of levulinic acid in combination with sodium dodecyl sulfate (SDS) in killing foodborne bacteria. Because of their innocuous properties, we studied the effects of levulinic acid and SDS at various concentrations appropriate for use in foods, on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis. The viability of Cryptosporidium and E. intestinalis was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines, respectively. Two Escherichia coli O157:H7 isolates were also used in the present study: strain 932 (a human isolate from a 1992 Oregon meat outbreak) and strain E 0018 (isolated from calf feces). Different concentrations and combinations of levulinic acid and SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10(5)), E. intestinalis spores (10(6)), and E. coli O157:H7 (10(7)/ml) when in suspension. Microsporidian spores were treated for 30 and 60 min at 20 ± 2°C. None of the combinations of levulinic acid and SDS were effective at inactivating the spores or oocysts. When Cryptosporidium oocysts were treated with higher concentrations (3% levulinic acid-2% SDS and 2% levulinic acid-1% SDS) for 30, 60, and 120 min, viability was unaffected. E. coli O157:H7, used as a control, was highly sensitive to the various concentrations and exposure times tested. SDS and levulinic acid alone had very limited effect on E. coli O157:H7 viability, but in combination they were highly effective at 30 and 60 min of incubation. In conclusion, Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid-1% SDS or 3% levulinic acid-2% SDS at 20°C, suggesting that this novel sanitizer cannot be used to eliminate parasitic contaminants in foods.

Objective: A higher incidence of infectious disease has been documented in U.S. regions bordering Mexico compared with non-border areas. We assessed the prevalence of important gastrointestinal infections in Ciudad Juarez, Mexico, and El Paso, Texas, the largest binational community along the U.S.-Mexico border., Methods: Fecal specimens from a sample of the asymptomatic population representing all ages were tested for Helicobacter pylori (H. pylori), Cryptosporidium spp., Giardia spp., and other intestinal parasitic pathogens using flotation, immunoassays, and/or polymerase chain reaction. We also measured indicators of microbiological contamination of drinking water, hands of food preparers, and kitchen surfaces., Results: Overall, of the 386 participants, H. pylori was present in 38.2%, Taenia spp. in 3.3%, Giardia spp. in 2.7%, Cryptosporidium spp. in 1.9%, Entamoeba dispar in 1.3%, and Ascaris lumbricoides and Necator americanus in 0.3% of the study subjects; Cyclospora spp. and Entamoeba histolytica were not found. H. pylori infection was associated with handwashing (prevalence ratio [PR] = 1.3, 95% confidence interval [CI] 1.0, 1.8). Taenia spp. was found more often on the U.S. side (PR=8.6, 95% CI 2.3, 30.8). We did not find an association between these infections and the occurrence of total coliforms or fecal coliforms on kitchen surfaces. In addition, Escherichia coli was not found in any drinking water sample., Conclusion: The study results indicated that H. pylori and Taenia spp. infections may be highly prevalent along the U.S.-Mexico border. Additional research is necessary to adequately characterize the prevalence, as well as determine whether interventions that reduce these infections are warranted.

The coccidian parasite Cyclospora cayetanensis is recognized as an emerging pathogen that causes protracted diarrhea in humans. The first cases of Cyclospora infection were reported in the late 1970s and were observed among expatriates and travelers in regions where infections are endemic. Since then, Cyclospora has been considered a cause of traveler's diarrhea. Epidemiological investigations were reported and examined in areas of endemicity even before the true identity of Cyclospora was elucidated. Cyclospora was fully characterized in the early 1990s, but it was not until the 1995 Cyclospora outbreak in the United States and Canada that it caught the attention of the public and physicians. The biology, clinical presentation, epidemiology, diagnosis, treatment, and control of cyclosporiasis are reviewed, with a focus on diagnostic assays currently being used for clinical and environmental samples. Challenges and limitations in working with Cyclospora are also discussed.

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.

Cyclospora cayetanensis infection continues to be a major cause of diarrhea particularly in immunosuppressed patients. The emergence of new related parasite pathogens, is an issue of public health concern. We report here a case where a Cyclospora-like organism was the cause of diarrhea in an immunocompetent patient from Venezuela.

Neospora caninum is known to cause abortion in cattle. This study demonstrated the presence of specific IgG to Neospora in milk and serum samples obtained from three dairy farms in Georgia and two in Texas. Samples from four hundred fourteen dairy cows were examined using a western blot assay of which 362 were milk and 87 were serum. Samples with antibodies to Neospora were identified in 32.1% (105/327) of the examined animals in Georgia, whereas in Texas it was identified in 10.3% (9/87). Positive Georgia samples were found in 24.4% from farm A (28/115), 21.6% from farm B (30/139), and 64.4% from farm C (47/73). In Texas, 13.5% (7/52) of animals in farm D and 5.71% (2/35) from farm E also had specific antibodies to Neospora. The number of animals from Georgia dairy farms with antibodies to Neospora was significantly higher than the Texas dairy farms. This may be related to the age of the animals examined in this study (more than 2 years old). Antibodies present in sera had excellent agreement with the antibodies present in milk. Collection of milk samples for serological testing is easier and less invasive than obtaining bovine sera, therefore offering an alternative for animal testing.

The order Microsporidia contains a number of ubiquitous pathogens that can infect various animals, including humans. Enterocytozoon bieneusi and Encephalitozoon intestinalis have been associated with gastrointestinal illness in humans. The effect of four disinfectants--ammonium hydroxide, hydrogen peroxide, and two commercial disinfectants containing peroxyacetic acid (Tsunami) and N-alkyl dimethyl benzyl ammonium chloride (Timsen)--on E. intestinalis spores was examined using exposure times of 1, 5, and 15 min. Spore viability was determined in vitro with RK-13 cells. Hydrogen peroxide was most efficient at inactivating microsporidial spores at all tested concentrations and treatment times, whereas ammonium hydroxide was effective only at the highest concentration at all exposure times. Tsunami (40 microg/ml) and Timsen (200 and 400 ppm) could inactivate spores when incubated for 5 and 15 min.

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.

The protozoan parasite Neospora caninum is a major cause of abortion in cattle throughout the world. In the process of propagating Neospora in vitro and producing specific antibodies for development of diagnostic assays in the food supply, our laboratory identified the presence of bovine antibodies to N. caninum in fetal bovine sera. The sera were produced commercially and preferentially recommended for tissue culture use and monoclonal antibody production. Seventeen different fetal bovine serum samples of different grades and from four different companies were examined for the presence of total IgG, IgG1, IgG2, and IgM specific for N. caninum. All of the tested serum samples recognized N. caninum specific bands on Western blot. Low IgG serum also recognized these antigens but with lower intensity. Antibody response was also evaluated using a commercially available ELISA kit for N. caninum.

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23 degrees C for 2 weeks, and sporulation rates were then determined. The 4',6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80 degrees C or higher were reached in the microwave ovens.

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.

Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2-4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.

Twenty-two coprolites of human origin, collected from excavations along the north-central coast of Peru, were examined using fluorescent microscopy for the presence of fecal parasites, with emphasis on Cryptosporidium sp., Giardia sp., and Cyclospora sp. Three samples were positive. One coprolite dated between ca. 2,375 and 1,525 BC contained Giardia sp. cysts. This coprolite corresponded to the Peruvian preceramic period. Another positive coprolite ca. AD 770-830 corresponded to Epoch 3 of the Middle Horizon and contained Cryptosporidium sp. oocysts. The third positive coprolite (corresponding to the Middle Horizon. ca. AD 500-900) contained Giardia sp. cysts. This report demonstrates that Giardia sp. and Cryptosporidium sp. were present in Peruvian coastal populations for at least 4,300 and 1,100 BP.

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis. The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis. In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

To evaluate enteropathogens and other factors associated with severe disease in children with diarrhea, 381 children <5 years of age with diarrhea and moderate to severe dehydration (in-patients) and 381 age-, sex-, and date-of-visit-matched children with mild diarrhea (out-patients) presenting to a hospital in Peru, were studied. Rotavirus was detected in 52% of the in-patients and 35% of the out-patients (odds ratio [OR]=2.3, 95% confidence interval [95% CI]= 1.6-3.2); 95% of the rotaviruses among in-patients were of serotypes G1-G4. The risk of severe diarrhea was particularly great in children who were not exclusively breast-fed in early infancy and who also lacked piped water in their homes (for children with both characteristics OR=6.8, 95% CI=3.6-12.8). The high prevalence of rotavirus and its association with severe diarrhea underscores the need for rotavirus vaccines. Interventions to educate mothers and improve access to safe water should augment the impact of rotavirus vaccines in preventing severe diarrhea.

Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously.

In part, Cyclospora cayetanensis owes its recognition as an emerging pathogen to the increased use of staining methods for detecting enteric parasites such as Cryptosporidium. First reported in patients in New Guinea in 1977 but thought to be a coccidian parasite of the genus Isospora, C. cayetanensis received little attention until it was again described in 1985 in New York and Peru. In the early 1990s, human infection associated with waterborne transmission of C. cayetanensis was suspected; foodborne transmission was likewise suggested in early studies. The parasite was associated with several disease outbreaks in the United States during 1996 and 1997. This article reviews current knowledge about C. cayetanensis (including its association with waterborne and foodborne transmission), unresolved issues, and research needs.

Asian freshwater clams (Corbicula fluminea) were exposed for 24 hr in 38 liters of water contaminated with 1.0 x 10(5) Cyclospora cayetanensis oocysts (2.6 x 10(3) oocysts/L). The hemolyph and gill smears of 30 clams were examined by acid-fast stain on days 1, 3, 5, 7, 10, 13, and 18 postexposure (PE). Since no oocysts were detected in the water 24 hr after contamination by the membrane filter-dissolution method, the oocyst retention rate was 4.6 X 10(2) oocysts/clam. The prevalence of oocyst-positive clams significantly decreased (P < 0.01) from 93% to 47% during 13 days PE. None of the clams contained oocysts on day 18 PE; no oocysts were detected in the clam feces. The numbers of oocysts recovered from six clam size classes varied and significantly decreased with smaller clam size (P < 0.01). The lowest prevalence values of oocyst-positive clams, 45% and 34%, were observed in the two lowest size classes: 12.1-14.0 mm and 14.1-16.0 mm, respectively. The prevalence values in the remaining four classes ranged from 84% to 100%. The sampling program demonstrated that the population of 180 clams examined during the study up to 13 day PE could be assessed for C. cayetanensis positivity by random testing of a minimum of 75 clams (42%). When the two lowest clam size classes are eliminated, the population of 114 clams could be assessed by sampling a minimum of 32 clams (28%). The results demonstrate that Corbicula fluminea can recover waterborne oocysts of C. cayetanensis, and could be used as biological indicators of contamination of water with C. cayetanensis oocysts.

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.

Cyclospora cayetanensis is a coccidian pathogen in humans. Cyclosporiasis is characterized by mild to severe nausea, anorexia, abdominal cramping, and watery diarrhea. Cyclospora has now been described from patients with protracted diarrheal illness in North, Central and South America, the Caribbean, Africa, Bangladesh, south-east Asia, Australia, England, and eastern Europe, and is characterized by marked seasonality. Routes of transmission are still unknown, although the fecal-oral route, either directly or via water, is probably the major one. A recent outbreak in the USA suggested transmission of Cyclospora by ingestion of contaminated berries. Cyclospora oocysts can be detected by phase contrast microscopy, modified acid-fast staining, autofluorescence, and amplification by the polymerase chain reaction. Oocysts are not sporulated when excreted in the feces, and sporulated oocysts are needed for infection. Each sporulated oocyst contains two sporocysts and each sporocyst contains two sporozoites. Humans seem to be the only host for this parasite. Histopathological examination of jejunal biopsies from infected individuals showed mild to moderate acute inflammation of the lamina propria and surface epithelial disarray. Parasitophorous vacuoles containing sexual and asexual forms of Cycl. cayetanensis were located in the cytoplasm of epithelial cells. Cyclospora infections can be treated successfully with trimethoprim-sulfamethoxazole.

Cyclospora cayetanensis has been observed in the feces of persons with prolonged diarrhea. A description of the symptoms and histopathologic findings for patients with cyclosporiasis is presented. The intracellular life-cycle stages of these parasites in the enterocytes of patients will also be described. Seventeen Peruvian patients positive for Cyclospora organisms were surveyed and underwent endoscopy, and their symptoms were recorded. Patients presented with gastrointestinal symptoms, including diarrhea, flatulence, weight loss, abdominal discomfort, and nausea. Jejunal biopsies showed an altered mucosal architecture with shortening and widening of the intestinal villi due to diffuse edema and infiltration by a mixed inflammatory cell infiltrate. There was reactive hyperemia with vascular dilatation and congestion of villous capillaries. Parasitophorous vacuoles contained sexual and asexual forms. Type I and II meronts, with 8-12 and 4 fully differentiated merozoites, respectively, were found at the luminal end of epithelial cells. These findings demonstrate the complete developmental cycle associated with host changes due to Cyclospora organisms.

Cryptosporidium parvum and Cyclospora cayetanensis are protozoan pathogens that cause prolonged diarrhea in both immunocompetent and immunocompromised hosts. Cryptosporidium parvum can be transmitted via the fecal-oral route, while the exact mechanisms of transmission of Cyclospora cayetanensis have not been fully determined. Humans appear to be the sole host for the latter and a distinct seasonality has been observed in endemic areas around the world. Samples of vegetables were collected at several small markets in a periurban slum in Peru during the seasons of high and low incidence. The vegetables were washed, the supernatants were collected and centrifuged, and the pellets were resuspended in a solution of 2.5% potassium dichromate. Pellets were examined using direct microscopic observation, acid-fast staining, and immunofluorescent assays for C. parvum and Cyclospora cayetanensis oocysts. Samples were collected during three time periods: the season of low incidence, the beginning of the season of high incidence, and end of the season of high incidence. Of the total vegetables examined, 14.5% contained C. parvum oocysts and 1.8% had Cyclospora oocysts. Thus, market vegetables may provide a route by which Cryptosporidium and Cyclospora can be transmitted. Our study also suggests that washing vegetables does not completely remove Cryptosporidium and Cyclospora oocysts.

A new coccidian parasite has been found in stool specimens of humans with and without diarrhea. The oocyst of this parasite measures 8.6 microns in diameter (7.7-9.9 microns), with ovoid sporocysts 4.0 x 6.3 (3.3-4.4 x 5.5-7.1) microns. Each oocyst has 2 sporocysts and each sporocyst contains 2 sporozoites. Based on these characteristics and the structures observed by electron microscopy, this parasite has been classified in the genus Cyclospora. We propose the name Cyclospora cayetanensis n. sp. for this new human parasite.

Background: Organisms referred to as "cyanobacterium-like bodies" have now been identified worldwide in the feces of both immunocompetent and immunocompromised patients with diarrhea. Organisms with a similar appearance have been isolated from Peruvian patients since 1985. From 1988 to 1991 we studied prospectively two cohorts of infants and young children infected with this organism. We now attempt to identify it., Methods: Fecal samples were collected weekly from the children and examined with the use of acid-fast staining and staining with a monoclonal antibody specific for cryptosporidium. Stools positive for cyanobacterium-like bodies were preserved in potassium dichromate and exposed to conditions allowing coccidian sporulation and excystation. Both unsporulated and sporulated oocysts were fixed by freeze-substitution techniques and then examined by electron microscopy., Results: Organisms isolated from the feces of Peruvian patients and two patients from the United States were identified as belonging to the coccidian genus cyclospora, after sporulation and excystation of the oocysts according to standard techniques. Complete sporulation occurred within 5 to 13 days in oocysts maintained in potassium dichromate at 25 or 32 degrees C. Complete excystation resulted in the liberation of two sporozoites from the two sporocysts within each oocyst (cryptosporidia have four naked sporozoites within each oocyst). The presence of organelles characteristic of coccidian organisms was confirmed by electron microscopy., Conclusions: We have identified organisms of the genus cyclospora that are remarkably similar to cryptosporidia in their morphologic features and the diarrheal disease that they produce in humans. The complete life cycle and epidemiology of this new protozoan parasite remain to be described.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.