Foodborne illness associated with Cryptosporidium and Giardia from livestock

Waterborne outbreaks caused by Cryptosporidium and Giardia are well documented, while the public health implications for foodborne illness from these parasites have not been adequately considered. Cryptosporidium and Giardia are common in domestic livestock, where young animals can have a high prevalence of infection, shedding large numbers of oocysts and cysts. Molecular epidemiological studies have advanced our knowledge on the distribution of Cryptosporidium and Giardia species and genotypes in specific livestock. This has enabled better source tracking of contaminated foods. Livestock generate large volumes of fecal waste, which can contaminate the environment with (oo)cysts. Evidence suggests that livestock, particularly cattle, play a significant role in food contamination, leading to outbreaks of cryptosporidiosis. However, foodborne giardiasis seems to originate primarily from anthroponotic sources. Foodborne cryptosporidiosis and giardiasis are underreported because of the limited knowledge of the zoonotic potential and public health implications. Methods more sensitive and cheaper are needed to detect the often-low numbers of (oo)cysts in contaminated food and water. As the environmental burden of Cryptosporidium oocysts and Giardia cysts from livestock waste increases with the projected increase in animal agriculture, public health is further compromised. Contamination of food by livestock feces containing Cryptosporidium oocysts and Giardia cysts could occur via routes that span the entire food production continuum. Intervention strategies aimed at preventing food contamination with Cryptosporidium and Giardia will require an integrated approach based on knowledge of the potential points of entry for these parasites into the food chain. This review examines the potential for foodborne illness from Cryptosporidium and Giardia from livestock sources and discusses possible mechanisms for prevention and control.     

Passage of a coccidial parasite (Eimeria acervulina) through the Eastern oyster (Crassostrea virginica)

Human illness resulting from the consumption of raw oysters is well documented for bacterial and viral pathogens but not for coccidial parasites. This study explores the passage of coccidial parasites through and the viability of these parasites in the Eastern oyster, Crassostrea virginica. Because both Cryptosporidium and Toxoplasma are human parasites and are not safe to handle, we chose to work with a close relative, Eimeria acervulina, as a surrogate. This parasite was analyzed in chickens. Oysters were found to concentrate coccidial oocysts within 6 h of exposure in a seawater tank. After 24 h, oysters still contained viable oocysts, but by 48 h, few oysters contained viable oocysts. No oysters were found to harbor oocysts 72 or 96 h after exposure to oocysts. After oysters had been exposed to oocysts for 24 h in one saltwater tank and then transferred to a clean saltwater tank for 48 h, their feces tested positive for viable oocysts. We conclude that coccidial parasites are not pathogenic to oysters, but move through oysters in just 1 day. Unless contaminated waters continuously carry oocysts, raw oysters are unlikely to pose a threat to human health through the carriage of coccidial parasites.     

Comparison of Rapid Methods for Detection of Giardia spp. and Cryptosporidium spp. (Oo)cysts Using Transportable Instrumentation in a Field Deployment

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.     

Occurrence of parasites on fruits and vegetables in Norway.

Between August 1999 and January 2001, samples of various fruits and vegetables obtained within Norway were analyzed by published methods for parasite contamination. Neither Cyclospora oocysts nor Ascaris (or other helminth) eggs were detected on any of the samples examined for these parasites. However, of the 475 samples examined for Cryptosporidium oocysts and Giardia cysts, 29 (6%) were found to be positive. No samples were positive for both parasites. Of the 19 Cryptosporidium-positive samples. 5 (26%) were in lettuce, and 14 (74%) in mung bean sprouts. Of the 10 Giardia-positive samples, 2 (20%) were in dill, 2 (20%) in lettuce, 3 (30%) in mung bean sprouts, 1 (10%) in radish sprouts, and 2 (20%) in strawberries. Mung bean sprouts were significantly more likely to be contaminated with Cryptosporidium oocysts or Giardia cysts than the other fruits and vegetables. Concentrations of Cryptosporidium and Giardia detected were generally low (mean of approximately 3 [oo]cysts per 100 g produce). Although some of the contaminated produce was imported (the majority, if sprouted seeds are excluded), there was no association between imported produce and detection of parasites. Crvptosporidium oocysts and Giardia cysts were also detected in water samples concerned with field irrigation and production of bean sprouts within Norway. This is the first time that parasites have been detected on vegetables and fruit obtained in a highly developed. wealthy country, without there being an outbreak situation. These findings may have important implications for global food safety.     

Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process.

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments world wide. Pathogenic strains of these bacteria are often transmitted to humans through consumption of raw oysters, which flourish in the same estuaries. Previous studies reported the effective use of hot water pasteurization followed by cold shock to eliminate from raw oysters naturally and artificially incurred environmental strains of V. vulnificus and V. parahaemolyticus common to the Gulf of Mexico. The present study focused on the use of the same pasteurization method to reduce a highly process resistant Vibrio strain, V. parahaemolyticus O3:K6 to non-detectable levels. Oysters were artificially contaminated with 10(4) and 10(6) V. parahaemolyticus 03:K6 cfu g(-1) oyster meat. Contaminated oysters were pasteurized between 50 and 52 degrees C for up to 22 min. Samples of processed oysters were enumerated for V. parahaemolyticus O3:K6 at 2-min intervals beginning after the 'come-up time' to achieve an oyster internal temperature of at least 50 degrees C. The D value (D(52)deg C) was 1.3-1.6 min. V. parahaemolyticus O3:K6 proved more process resistant than non-pathogenic environmental strains found in Gulf of Mexico waters. A total processing time of at least 22 min at 52 degrees C was recommended to reduce this bacterium to non-detectable levels (< 3 g(-1) oyster meat).     

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.     

Detection of protozoan parasites and microsporidia in irrigation waters used for crop production

The occurrence of human pathogenic parasites in irrigation waters used for food crops traditionally eaten raw was investigated. The polymerase chain reaction was used to detect human pathogenic microsporidia in irrigation waters from the United States and several Central American countries. In addition, the occurrence of both Cryptosporidium oocysts and Giardia cysts was determined by immunofluorescent techniques. Twenty-eight percent of the irrigation water samples tested positive for microsporidia, 60% tested positive for Giardia cysts, and 36% tested positive for Cryptosporidium oocysts. The average concentrations in samples from Central America containing Giardia cysts and Cryptosporidium oocysts were 559 cysts and 227 oocysts per 100 liters. In samples from the United States, averages of 25 Giardia cysts per 100 liters and <19 (average detection limit) Cryptosporidium oocysts per 100 liters were detected. Two of the samples that were positive for microsporidia were sequenced, and subsequent database homology comparisons allowed the presumptive identification of two human pathogenic species, Encephalitozoon intestinalis (94% homology) and Pleistophora spp. (89% homology). The presence of human pathogenic parasites in irrigation waters used in the production of crops traditionally consumed raw suggests that there may be a risk of infection to consumers who come in contact with or eat these products.     

Foodborne protozoan parasites

This report addresses Cryptosporidium, Giardia, Cyclospora, and more briefly, Toxoplasma as the main parasitic protozoa of concern to food production worldwide. Other parasitic protozoa may be spread in food or water but are not considered as great a risk to food manufacture. The protozoan parasites Cryptosporidium, Giardia, and Cyclospora have proven potential to cause waterborne and foodborne disease. Toxoplasma gondii has been considered a risk in specific cases, but humans are not its primary host. Cryptosporidium and Giardia are widespread in the environment, particularly the aquatic environment, and major outbreaks of cryptosporidiosis and giardiasis have occurred as a result of contaminated drinking water. Large outbreaks of waterborne cyclosporiasis have not been identified. Cryptosporidium, Giardia, and Cyclospora have potential significance in the preparation and consumption of fresh produce and in catering practice, in which ready-to-eat foods may be served that have not received heat treatment. None of the three organisms Cryptosporidium, Giardia, and Cyclospora has been shown to be a problem for heat processed food or tap water that has undergone appropriate treatment at a water treatment works. All three are sensitive to standard pasteurisation techniques. Although humans are not a primary host for T. gondii, the potential exists for both waterborne and foodborne toxoplasmosis. Parasitic protozoa do not multiply in foods, but they may survive in or on moist foods for months in cool, damp environments. Their ecology makes control of these parasites difficult. For general control of parasitic protozoa in the food chain, the following steps are necessary: - Follow good hygienic practice in food service and catering industries.- Minimise dissemination of cysts and oocysts in the farming environment and via human waste management.- Include these microorganisms in Hazard Analysis Critical Control Point (HACCP) plans of water suppliers, industries or sectors that use fresh produce, and operations in which contaminated process or ingredient water could end up in the product (e.g., where water supplies may become contaminated).     

The effect of waste water reuse in irrigation on the contamination level of food crops by Giardia cysts and Ascaris eggs.

In Marrakech, raw sewage has been used for farming purposes for several decades for many types of crops. This study aimed to determine the contamination level of Giardia cysts and Ascaris eggs for crops designated for human consumption. Collected crops in irrigated fields were turnip, marrow, squash, potatoes, pepper and eggplant. Field trials were also carried out on four crops, coriander, carrots, mint and radish, using three water types for irrigation, i.e. raw waste water, treated waste water (sedimentation and 16 days retention) and fresh water. Giardia cysts were detected at a level of 5.1 cysts/kg in potatoes, while Ascaris eggs were observed in numbers varying between 0.18 eggs/kg in potatoes and 0.27 eggs/kg in turnip. Field trials confirmed that irrigation of crops by raw waste water leads to contamination. Giardia and Ascaris were isolated in coriander at concentrations of 254 cysts/kg and 2.7 eggs/kg, respectively; mint was also highly contaminated with numbers reaching 96 cysts/kg and 4.63 eggs/kg. Carrots and radish were contaminated and respective numbers observed for Giardia were 155 and 59.1 cysts/kg; Ascaris was discovered in numbers of 0.7 and 1.64 eggs/kg, respectively. However, cultures irrigated with treated waste water and fresh water were free from contamination. Cysts and eggs on coriander persisted for a maximum of 8 days.     

The response of Campylobacter jejuni and Campylobacter coli in the Sydney rock oyster (Crassostrea commercialis), during depuration and storage

Sydney Rock Oysters, when allowed to feed in waters containing approximately 10(4) cfu of Campylobacter cells per ml, concentrated between 10(2) and 10(3) cfu of the organism per g of oyster tissue, within 1 h. When these contaminated oysters were subjected to depuration, they were effectively cleaned in 48 h. The survival of Campylobacter jejuni and Campylobacter coli was also investigated. Oysters contaminated by feeding and injection were processed as half shells and bottled oysters and were held at 3 and 10 degrees C. Half shells were also stored at -20 to -24 degrees C. At all these temperatures the organism survived for periods varying between 8 to 14 days and in oysters contaminated by feeding, the survival was substantially greater. Survival was better at 3 than at 10 degrees C in half-shelled oysters. Campylobacter survived better in bottled oysters than in half shells stored at the same temperature. In frozen half shelled oysters previously contaminated by feeding, the organisms were viable for months. In contaminated unopened oysters stored at 20 and 30 degrees C, C. jejuni and C. coli failed to multiply as expected. They survived for periods varying from 2 to 9 days.     

Multiple Clusters of Norovirus among Shellfish Consumers Linked to Symptomatic Oyster Harvesters

We describe the investigation of a norovirus outbreak associated with raw oyster consumption affecting 36 people in British Columbia, Canada, in 2010. Several genotypes were found in oysters, including an exact sequence match to clinical samples in regions B and C of the norovirus genome (genogroup I genotype 4). Traceback implicated a single remotely located harvest site probably contaminated by ill shellfish workers during harvesting activities. This outbreak resulted in three recalls, one public advisory, and closure of the harvest site.     

Managing the risk of Vibrio parahaemolyticus infections associated with oyster consumption: A review

Vibrio parahaemolyticus is a Gram‐negative bacterium that is naturally present in the marine environment. Oysters, which are water filter feeders, may accumulate this pathogen in their soft tissues, thus increasing the risk of V. parahaemolyticus infection among people who consume oysters. In this review, factors affecting V. parahaemolyticus accumulation in oysters, the route of the pathogen from primary production to consumption, and the potential effects of climate change were discussed. In addition, intervention strategies for reducing accumulation of V. parahaemolyticus in oysters were presented. A literature review revealed the following information relevant to the present study: (a) managing the safety of oysters (for human consumption) from primary production to consumption remains a challenge, (b) there are multiple factors that influence the concentration of V. parahaemolyticus in oysters from primary production to consumption, (c) climate change could possibly affect the safety of oysters, both directly and indirectly, placing public health at risk, (d) many intervention strategies have been developed to control and/or reduce the concentration of V. parahaemolyticus in oysters to acceptable levels, but most of them are mainly focused on the downstream steps of the oyster supply chain, and (c) although available regulation and/or guidelines governing the safety of oyster consumption are mostly available in developed countries, limited food safety information is available in developing countries. The information provided in this review may serve as an early warning for managing the future effects of climate change on the safety of oyster consumption.     

Effects of refrigeration and alcohol on the load of Aeromonas hydrophila in oysters

Members of the bacterial genus Aeromonas are widely distributed throughout the environment and are readily cultured from a variety of foods. One member of this genus, Aeromonas hydrophila, has been reputed to be a significant cause of gastrointestinal disease. In this study, we examined the effects of refrigeration and alcohol on the level of A. hydrophila in oysters. Specifically, vodka was examined because it is used by the food service industry in preparation of Oysters Romanoff. One set of oysters was shucked on receipt, whereas others were refrigerated intact for 7 days at 5 degrees C. The oysters were blended and the numbers of A. hydrophila present determined using starch ampicillin agar. Oysters were also shucked and placed on the half shell with 5 ml of vodka for 10 min. The oysters were then washed and presumptive A. hydrophila levels determined in both the washate and homogenate. On the day of purchase, the average number of presumptive A. hydrophila found was 7.6 x 10(4) CFU/g of oyster meat. After 7 days of refrigeration, the average number had increased to 3.2 x 10(5) CFU/g of oyster meat. In the oysters treated with vodka, the average number of A. hydrophila present internally was 9.9 x 10(4) with high numbers (10(3) to 10(4)) isolated from the oyster surface. From these data, it is clear that refrigeration and alcohol treatment are not sufficient to reduce loads of A. hydrophila in or on oysters.     

Development and use of a pepsin digestion method for analysis of shellfish for Cryptosporidium oocysts and Giardia cysts

Investigation of shellfish for Cryptosporidium oocysts and Giardia cysts is of public health interest because shellfish may concentrate these pathogens in their bodies, and because shellfish are frequently eaten raw or lightly cooked. To date, the methods used for the analysis of shellfish for these parasites are based on those originally designed for water concentrates or fecal samples; the reported recovery efficiencies are frequently relatively low and the amount of sample examined is small. Here, we describe the development and use of a pepsin digestion method for analyzing shellfish samples for these parasites. The conditions of the isolation method did not affect subsequent parasite detection by immunofluorescent antibody test, and allowed examination of 3-g samples of shellfish homogenate, with recovery efficiencies from blue mussel homogenates of between 70 and 80%, and similar recoveries from horse mussel and oyster homogenates. Although exposure of the parasites to the conditions used in the technique affected their viability, as assessed by vital dyes, the maximum reduction in viability after 1-h incubation in digestion solution was 20%. In a preliminary survey of shellfish collected from the Norwegian coast, Cryptosporidium oocysts were detected in blue mussel homogenates in 6 (43%) of 14 batches and Giardia cysts in 7 (50%) of these batches. However, this relatively high occurrence, compared with other surveys, may be due to the higher recovery efficiency of the new method, and the relatively large sample size analyzed. A more comprehensive study of the occurrence of these parasites in shellfish would be of pertinence to the Norwegian shellfish industry.     

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter‐feeding molluscan shellfish exposed to sewage‐contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male‐specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log₁₀ genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.     

Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters

Consumption of raw oysters, whether wild-caught or aquacultured, may increase health risks for humans. Vibrio vulnificus and Vibrio parahaemolyticus are two potentially pathogenic bacteria that can be concentrated in oysters during filter feeding. As Vibrio abundance increases in coastal waters worldwide, ingesting raw oysters contaminated with V. vulnificus and V. parahaemolyticus can possibly result in human illness and death in susceptible individuals. Depuration is a postharvest processing method that maintains oyster viability while they filter clean salt water that either continuously flows through a holding tank or is recirculated and replenished periodically. This process can reduce endogenous bacteria, including coliforms, thus providing a safer, live oyster product for human consumption; however, depuration of Vibrios has presented challenges. When considering the difficulty of removing endogenous Vibrios in oysters, a more standardized framework of effective depuration parameters is needed. Understanding Vibrio ecology and its relation to certain depuration parameters could help optimize the process for the reduction of Vibrio. In the past, researchers have manipulated key depuration parameters like depuration processing time, water salinity, water temperature, and water flow rate and explored the use of processing additives to enhance disinfection in oysters. In summation, depuration processing from 4 to 6 days, low temperature, high salinity, and flowing water effectively reduced V. vulnificus and V. parahaemolyticus in live oysters. This review aims to emphasize trends among the results of these past works and provide suggestions for future oyster depuration studies.     

Prevalence and characterization of Salmonella serovars isolated from oysters served raw in restaurants

To determine if Salmonella-contaminated oysters are reaching consumer tables, a survey of raw oysters served in eight Tucson restaurants was performed from October 2007 to September 2008. Salmonella spp. were isolated during 7 of the 8 months surveyed and were present in 1.2% of 2,281 oysters tested. This observed prevalence is lower than that seen in a previous study in which U.S. market oysters were purchased from producers at bays where oysters are harvested. To test whether the process of refrigerating oysters in restaurants for several days reduces Salmonella levels, oysters were artificially infected with Salmonella and kept at 4°C for up to 13 days. Direct plate counts of oyster homogenate showed that Salmonella levels within oysters did not decrease during refrigeration. Six different serovars of Salmonella enterica were found in the restaurant oysters, indicating multiple incidences of Salmonella contamination of U.S. oyster stocks. Of the 28 contaminated oysters, 12 (43%) contained a strain of S. enterica serovar Newport that matched by pulsed-field gel electrophoresis a serovar Newport strain seen predominantly in the study of bay oysters performed in 2002. The repeated occurrence of this strain in oyster surveys is concerning, since the strain was resistant to seven antimicrobials tested and thus presents a possible health risk to consumers of raw oysters.     

High‐pressure processing with hot sauce flavouring enhances sensory quality for raw oysters (Crassostrea virginica)

This study evaluated flavouring raw oysters by placing them under pressure in the presence of a commercially available hot sauce. Hand‐shucked raw oysters were processed at high pressure (600 MPa), in the presence or absence of hot sauce flavouring and evaluated by an experienced sensory panel 3 and 10 days after postharvest processing. The sensory panel evaluated high‐pressure‐processed oysters, with and without flavouring, for eleven flavours and three texture characteristics using an 11‐point intensity scale. Oysters were plump and characterised as moderately chewy and firm. Most oyster flavour characteristics were low in intensity with moderate intensity for briny and umami attributes. Flavoured oysters had a moderately intense tangy flavour and aftertaste. Flavouring a raw oyster by high‐pressure processing provides the potential to create a microbiologically safe product with unique sensory characteristics, which may influence consumer acceptance and marketability.