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Journal of Food Protection, Vol. 62, No. 9, 1999, Pages 1059±1070 Copyright ᮊ, International Association of Milk, Food and Environmental Sanitarians

Review Giardia, , and Cyclospora and Their Impact on Foods: A Review

JOAN B. ROSE AND THERESA R. SLIFKO*

Department of Marine Science, University of South Florida, 140 7th Avenue S., St. Petersburg, Florida 33701, USA

MS 98-342: Received 23 December 1998/Accepted 10 May 1999 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 ABSTRACT While the risk from pathogenic microorganisms in foods has been recognized for hundreds of years, bacterial agents are generally implicated as the contaminants. Although many outbreaks of gastroenteritis caused by protozoan pathogens have occurred, it is only in the last 3 years that attention has focused on protozoan association with foodborne transmission. Recognized as waterborne parasites, Giardia, Cryptosporidium, and Cyclospora have now been associated with several food- borne outbreaks. The oocysts and cysts of these organisms can persist and survive for long periods of time both in water and on foods. While Cyclospora oocysts require a maturation period, Cryptosporidium oocysts and Giardia cysts are immediately infectious upon excretion from the previous host. As a result, these parasites have emerged as public health risks and have become a concern to the food industry. More than 200 cases of foodborne giardiasis (seven outbreaks) were reported from 1979 to 1990. Four foodborne Cryptosporidium outbreaks (with a total of 252 cases) have been documented since 1993. Cyclospora caused a series of sporadic outbreaks of cyclosporasis throughout North America that have affected over 3,038 people since 1995. Control and prevention of protozoan foodborne disease depends upon our ability to prevent, remove, or kill protozoan contaminants. This review will address the biology, foodborne and waterborne transmission, survival, and methods for detection and control of Giardia, Cryptosporidium, and Cyclospora.

Threats to global food safety have received increasing gate parasites (they require a host to reproduce) that are attention within the food industry. These threats are associ- transmitted to humans through ingestion of an environmen- ated with the changing world, and several key points must tal stage. Environmental stages can be ingested by the fecal/ be considered with regard to these threats: (i) the number of oral route or through ingestion of undercooked meat that susceptible populations, including the elderly and the im- contains the tissue stages (i.e., helminths). In the United munocompromised (persons with AIDS, transplant recipi- States, inspection requirements include helminth screening ents, and cancer patients), is growing; (ii) trade barriers have to detect ova or worms in meats such as beef, pork, and been relaxed, and food is coming from an international mar- ®sh. ket; (iii) food-processing technology is advancing; and (iv) Clinically signi®cant intestinal protozoa are taxonom- national and international policy changes are affecting food ically divided into the amoebae (i.e., Entamoeba sp.), the safety. As a consequence, a greater need for assessing, de- ¯agellates (Giardia), and the (those that are glob- veloping, and protecting those programs that address food ular in shape; Cryptosporidium and Cyclospora) (31). Tox- safety among producers, wholesalers, and retailers has de- oplasma gondii, a protozoan parasite that causes toxoplas- veloped. Foodborne disease may be the most notable con- mosis (which affects the body's organs and other tissues), temporary public health problem, and it has the potential to is of particular concern in human fetal development. Toxo- create the most signi®cant economic impact. As new threats plasmosis is estimated to occur in between 407 and 9,500 emerge, it is dif®cult to know what risks should be addressed congenital cases per year in the United States (64). The ®rst. Clearly, one of the most notable emerging risks is as- intestinal protozoa, however, are an emerging concern in sociated with intestinal protozoa. Regardless of whether the area of foodborne disease, and this review will focus these parasites are new or are newly recognized, there is a on three of the newly recognized threats to food safety. growing interest in their association with food and in various The major protozoan species that affect humans are approaches designed to ensure food protection. Entamoeba histolytica, Giardia lamblia, Cryptosporidium DESCRIPTION AND HISTORY OF GIARDIA, parvum, and Cyclospora cayentanensis. E. histolytica is CRYPTOSPORIDIUM, AND CYCLOSPORA found only in humans, and while contamination of foods Parasites associated with foodborne disease fall into can occur, foodborne transmission in the United States is three main categories: intestinal protozoa, tissue protozoa, very rare. Although several other species of Giardia, Cryp- and tissue helminths (Table 1). These organisms are obli- tosporidium, and Cyclospora exist, only those that affect humans will be discussed in this review. These protozoa * Author for correspondence. Tel: 727-553-3930; Fax: 727-553-1189; are single-celled, microscopic animals that reproduce in the E-mail: [email protected]. intestinal tract of the host and, as a result of their life cycle, 1060 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9

TABLE 1. Parasites associated with foodborne diseasea Type of parasite Genera Disease Transmission

Intestinal protozoa Cryptosporidium parvum Cause intestinal infections and diar- Fecal±oral transmission from inges- rhea. tion of oocysts and cysts; excret- Giardia lamblia ed by humans and in some cases Entamoeba histolytica by animals. Tissue protozoa Can cause fetus malformations; in Infection can come from ingestion adults, may cause fever, head- of oocysts from cat feces and ache, and lesions in organs; prob- from consumption of under- lem in immunosuppressed pa- cooked meat, where the parasitic tients. life stages can be found in the meat and organs.

Tissue helminthsb Anisakis sp. Intestinal worms (i.e., causes diar- Infection comes from ingestion of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 Ascaris lumbricoides rhea and infection of organs. undercooked meats (beef, ®sh, Diphyllopothrium latum and pork) containing the tissue Gnathostoma spinigerum stages; in some cases caused by Metagonimus yokogawai the direct ingestion of the eggs. Taenia solium Trichinella spiralis a Source: (31). b Meat inspection required in the United States to examine for tissue stages. produce environmental stages (cysts or oocysts) that are through the stomach cause the cyst or oocyst to open, at excreted in the feces. Table 2 shows the major character- which time the infectious trophozoites or sporozoites, re- istics that distinguish the three genera. Transmission occurs spectively, are released. This process is known as excys- during the infectious cyst (G. lamblia) or oocyst (C. parvum tation. Excystation can also be accomplished in vitro in the and C. cayetanensis) stages via the fecal±oral route. Con- laboratory (with trypsin, bile salts, and an incubation tem- taminated hands, surfaces, water, and foods can transmit perature of 37ЊC). In vitro excystation is used as a measure these protozoa (13, 16, 18, 51, 52, 57, 59, 60, 62, 66, 72, of viability. 78). Waterborne transmission has been documented for The parasite G. lamblia exists in the environment as a these organisms; however, it is becoming clear that fecally cyst with dimensions of about 12 by 6 ␮m. The cyst wall contaminated food is an emerging risk. acts as a protective outer coating; it contains a chitinlike The life cycles of Giardia, Cryptosporidium, and Cy- substance that makes the wall resistant to environmental clospora sp. are similar. The initial stage occurs during in- conditions. Once ingested, the cyst undergoes excystation gestion of the cyst or oocyst. Body temperature and passage and releases two trophozoites that can attach to and grow

TABLE 2. Characteristics of the intestinal protozoaa Infectious and Major species associated environmental Cross-infectivity between Protozoan Classi®cation with human disease stageb Symptoms animals and humansc

Cryptosporidium Coccidian parvum Oocyst 7 to 14 days of watery Yes; associated with cat- diarrhea, fever, and tle and possibly other muscle aches. domestic and wild an- imal wastes. Cyclospora Coccidian cayetanensis Oocyst Range from none to ab- No; cross-transmission dominal cramps, nau- has not been docu- sea, vomiting, and fe- mented. ver, 3 to 25 days. Giardia Flagellate intestinalis and duode- Cyst Acute diarrhea may last Yes; beavers have been nalis; also referred to days to weeks; chron- implicated in water as lamblia in the older ic infections can occur outbreaks. literature. for months. a Sources: (25, 31, 32, 50, 66). b Egglike structure produced as a result of the life cycle, excreted in the feces: the cyst is produced as a result of asexual reproduction, contains chitinlike substance, and houses two trophozoites (the stage that attaches and grows in the intestinal tract), whereas the oocyst is produced as a result of sexual reproduction, contains complex carbohydrates and lipids, and houses four sporozoites (the stage that initiates infection in the intestinal cell). c Only Cryptosporidium and Giardia are associated with cross-transmission from animals to humans. J. Food Prot., Vol. 62, No. 9 FOODBORNE AND WATERBORNE ENTERIC PROTOZOA 1061 within the intestinal tract through asexual reproduction. As Isolates of Giardia are associated with disease in hu- the trophozoites begin to cover the wall of the intestinal mans and animals, and there is evidence that animals can tract, diarrhea can result. When the trophozoites are passed become infected with human Giardia (25). Thus, the sourc- through the bowel, some encyst (or form cysts) and are es of environmental contamination for Giardia are similar excreted in the feces (25). to those associated with Cryptosporidium. The cysts are C. parvum and C. cayetanensis both exist in the en- immediately infectious, and person-to-person transmission vironment as oocysts. C. parvum oocysts are about 5 ␮m has been documented (50). in diameter, and they house four sporozoites. After inges- Cryptosporidium. Cryptosporidium was ®rst described tion and excystation, the sporozoite stage enters into the in 1908 in mice and was subsequently noted as a cause of intestinal cell and begins the infection process. Multiple life diarrhea in turkeys, lambs, and calves. It was not diagnosed stages of the parasite form during the reproductive process in humans until 1976, and since that time, C. parvum has within the host cells. One stage (the meront) initiates an been well recognized as a cause of diarrhea in humans autoreinfective process, and other cells become infected. As Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 worldwide (22, 27). The disease called more cells become infected, illness begins, and oocysts are has been described as one that is cholera-like, and its symp- excreted in high numbers (about 100,000 oocysts/g of fe- toms include large volumes of ¯uid loss, fever, and abdom- ces). Oocysts are produced as a result of sexual reproduc- inal pain. The incidence of cryptosporidiosis in the popu- tion (22, 27). lation has been reported to range from 0.6 to 20%, C. cayetanensis oocysts are about 10 ␮m in diameter depending upon the geographic locale. Incidences of cryp- and contain complex carbohydrates and lipids that enable tosporidiosis are more prevalent in populations in Asia, them to be ``acid fast.'' Acid-based stains are used in the Australia, Africa, and South America, while Cryptosporid- clinical laboratory to detect oocysts in feces. C. cayetanen- iosis is associated with 0.4 to 1% of cases of diarrhea in sis oocysts each contain two sporocysts (two oblong struc- the United States. However, based on the seroprevalence of tures in the oocyst). Each sporocyst contains two sporo- antibodies in the blood, about 30% of the population has zoites. These sporocysts are not formed until the oocyst probably been exposed (66). undergoes maturation in the environment; this process is Cryptosporidium is of particular concern for four rea- called sporulation. After sporulation, the oocyst is referred sons: (i) the oocyst is extremely resistant to disinfection and to as ``sporulated,'' and only then is it considered infec- cannot be killed with routine water-disinfection procedures; tious. The life cycle of C. cayetanensis is not as well de- (ii) the disease is not treatable with antibiotics; (iii) the risk scribed as that of C. parvum. After ingestion and excysta- of mortality ranges between 50 and 60% in the immuno- tion, the life cycle appears to proceed in a manner similar compromised population (66); and (iv) animal and human to that of Cryptosporidium (32). fecal wastes are associated with transmission of the disease Giardia. The ®rst description of Giardia is credited to to humans. Van Leeuwenhoek in 1632; he described what he saw as An emerging protozoan: Cyclospora. C. cayetanensis he examined his own stool under the microscope. The par- (previously termed cyanobacteriumlike body) is a single- asitologist Lambl scienti®cally and formally described cell coccidian protozoan that has been implicated as an eti- Giardia in 1859. The cyst stage was not associated with ologic agent in cases involving prolonged, watery diarrhea, the ¯agellate until about 1881. Dobell is credited with de- fatigue, and anorexia in humans and other primates (56). lineating the intestinal tract as this protozoa's natural habitat The organism was ®rst described as early as 1977 in hu- for reproduction and growth in 1932 (25). The infection mans (3) and has been reported with increased frequency called giardiasis presents itself in this manner: a high per- since the mid-1980s (73). centage of individuals remain asymptomatic and exhibit no C. cayetanensis is now known to be an obligate para- speci®c ill effects. Therefore, giardiasis was not recognized site of both immunode®cient and immunocompetent hu- as a cause of disease for many years, and it was not until mans (56). The infection is much less severe in the im- the late 1950s and early 1960s that the medical community munocompetent, who may be asymptomatic or who may began to accept Giardia as a cause of diarrheal disease. have symptoms, including abdominal cramps, nausea, vom- The most common symptoms of giardiasis are diarrhea iting, and fever, which can last from 3 to 25 days. The followed by ¯atulence, foul-smelling stools, and cramps. parasite can cause profuse, watery diarrhea that can last for An acute stage can last from a few days to a week or, on several months in an immunocompromised host. However, rare occasions, months. A chronic stage may exhibit itself the infection is treatable (32). with milder symptoms, sporadic episodes, and weight loss, Cyclospora has been described in patients from North, and may last 10 days or more (and, on the rare occasion, Central, and South America; Europe; Asia; and North Af- up to a year). However, giardiasis is treatable. Giardiasis is rica; however, the true prevalence of this parasite in any the most common parasitic infection in the United States population is unknown (73). The cases identi®ed in the and, indeed, worldwide. In a national survey, Giardia ac- United States prior to 1995 were all thought to be imported counted for anywhere from 1 to 19% of the positive stools and were associated with people who had traveled to areas sent to state health laboratories (10), whereas most other where the disease was endemic (i.e., India and South Amer- parasites were detected in less than 1% of the specimens ica). received at these laboratories. Although Cyclospora is very similar to Cryptosporid- 1062 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9

TABLE 3. Comparison of Cryptosporidium and Cyclosporaa Attribute Cryptosporidium Cyclospora

Taxonomy Intestinal coccidian Intestinal coccidian Infective unit Oocyst 4 ␮m±5 ␮m immediately infectious upon Oocyst 8 to 10 ␮m requires sporulation in the en- excretion. vironment; not immediately infectious upon ex- cretion.b Animal reservoirs C. parvum found in most mammals; can cross spe- C. cayetanensis documented in humans and ba- cies barriers. boons. Waterborne disease Twelve outbreaks in North America since 1985. One outbreak in Chicago, one in Nepal. Largest outbreak in the United States with 400,000 ill, in Milwaukee in 1993. Foodborne disease Four outbreaks since 1993. Apple cider (two of Eighty separate clusters in outbreaks in 1995,

four), chicken salad, and green onions implicat- 1996, and 1997 affecting 20 states; associated Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 ed. with raspberries, basil, and lettuce. a Sources: (2, 32). b Sporulation is a process by which the oocyst undergoes maturation in the environment before becoming infectious; unlikely that direct person-to-person transmission can occur. ium, there are several major biological distinctions that can infection (33, 67). Most feces that are carrying cysts and be made between the two (Table 3). Two of the major dif- oocysts end up in the environment and can be spread to ferences are related to oocyst transmission. Whereas Cryp- foods, such as produce, by irrigation or by direct contact. tosporidium oocysts are immediately infectious upon ex- Routine wastewater treatment eliminates only a small frac- cretion, the oocysts of Cyclospora are immature when ex- tion of the oocysts and cysts (45). creted and mature, through a process known as sporulation, in the environment. Therefore, it is easy to understand why Foodborne transmission. Parasites contributed to 2% Cryptosporidium may be much more likely to be transmit- of the foodborne outbreaks between 1988 and 1992, ac- ted by direct person-to-person transmission. Cyclospora is cording to data compiled by the Centers for Disease Control not likely to be transmitted in this manner. In addition, to and Prevention (Table 4) (13). Of the 17 total parasitic out- date no animals have been identi®ed to be a reservoir of breaks, Trichinella was associated with 59%, whereas Giar- human infection for Cyclospora. dia caused the other 41%. However, foodborne transmis- sion of Giardia is not as well documented as is waterborne TRANSMISSION transmission. The ®rst foodborne outbreak of giardiasis in Fecal±oral transmission entails many potential delivery the United States was described in 1979. Most foodborne routes. One might say that where the feces go, so do the outbreaks of giardiasis have been related to contamination protozoa. Transmission through the environment is facili- by food handlers and preparers (Table 5) (52, 57, 59, 60, tated by several factors. The oocysts and cysts are stable 61, 72, 78). and can survive for weeks to months in the environment. Whereas Cryptosporidium also appears more likely to Second, small numbers of cysts and oocysts are capable of be associated with waterborne transmission, suspected initiating infection. Human infectious dose studies and foodborne outbreaks have been reported from travelers who models demonstrate that for Cryptosporidium and Giardia, visited Mexico, the United , and Australia. Sus- one oocyst or cyst carries some probability of causing an pect foods included salad, raw milk, sausage, and tripe (72). The ®rst foodborne outbreak of cryptosporidiosis in the United States was reported to be related to apple cider and TABLE 4. Foodborne parasitic outbreaks reported in the CDC, occurred in 1993. Three other outbreaks have since been a 1988±1992 reported, one was associated with chicken salad, one (pos- Out- sibly) with green onions, and another with apple cider (Ta- breaks/ ble 6) (11, 16, 18, 51). Year Parasite cases Food (when identi®ed) The ®rst reported foodborne outbreak of Cyclospora 1988 Trichinella 3/34 Meats and stews that was not associated with travel outside the United States 1989 Giardia 1/21 Multiple vehicles occurred in 1995 in eastern Florida. At that time, the out- Trichinella 4/15 Sausage and meats break appeared to be linked to the consumption of straw- 1990 Giardia 3/129 Fruits, vegetables, and other salads berries. It was suspected that ¯ooding in Monterey County, Trichinella 2/105 Pork California, that year could have resulted in water contam- 1991 Giardia 2/32 Fruits and vegetables inated with sewage that contained Cyclospora. The Mon- Trichinella 1/41 Sausage terey County agricultural commissioner estimated that up 1992 Giardia 1/2 Unknown to 35% of the strawberry crop was damaged that year by Total 17/379 ¯ooding (12, 58). a Source: (13). In 1996, a total of 1,465 cases of cyclosporasis were J. Food Prot., Vol. 62, No. 9 FOODBORNE AND WATERBORNE ENTERIC PROTOZOA 1063

TABLE 5. Some foodborne outbreaks of giardiasis in the United Statesa Number of cases/total Date Locale exposed Food Venue and contributing factors

December 1979 Minnesota 29/60 Salmon School; food prepared by an individual caring for a 1-year-old child who was excreting cysts. July 1985 Connecticut 13/16 Noodle salad Picnic; food preparer became ill and had asymptomatic children excreting cysts. March 1986 New Jersey 10/25 Fruit salad Family party; individual preparing the salad had asymptomatic 2-year-old child. Pet rab- bit was also positive for Giardia.

June 1986 Minnesota 88/312 Sandwiches Nursing home; 57% of the sandwich preparers Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 were excreting cysts. 1989 New Mexico 21/108 Lettuce, onions, tomatoes Church dinner; no obvious contamination. July 1990 Washington 27/36 Ice Restaurant, daylong meeting; of employees serving the ice, one was asymptomatic but excreting cysts, and one had a child excret- ing cysts. 1990 Not known; Sliced raw vegetables Of®ce; infected food handler. 27 cases by passive sur- veillance. a Sources: (11, 52, 57, 59, 60, 61, 72, 78). reported in 20 states in the United States (36). Some 55 outbreaks of cryptosporidiosis from 1985 to 1994 (45). In events were identi®ed as being a part of this nationwide Milwaukee, Wisconsin, in 1993, the largest waterborne out- outbreak. Cases were documented in Colorado, Connecti- break ever documented in the United States occurred, and cut, the District of Columbia, Florida, Georgia, Illinois, it was attributed to Cryptosporidium (38, 46). It is estimated Iowa, Maine, Maryland, Massachusetts, New Hampshire, that 400,000 people became ill, and approximately 100 New Jersey, New York, Ohio, Pennsylvania, Rhode Island, died. South Carolina, Texas, Vermont, Virginia, and Wisconsin The cysts of Giardia and the oocysts of Cryptosporid- and in Ontario and Quebec, Canada (36). The suspected ium are commonly found in surface waters, with 0.1 to vehicle of transmission for these outbreaks was Guatemalan 10,000 cysts and oocysts/100 liters in 4 to 100% of samples raspberries. In retrospect, it is believed that the 1995 out- examined, depending on the impact from sewage works and break may also have been due to the consumption of con- animals (45). Cryptosporidium oocysts can be found in 17 taminated raspberries and not to strawberries. The contam- to 26.8% of treated drinking-water samples, at densities of ination of the raspberries has not been de®nitively 0.005 to 0.017 oocysts per liter (71). Although groundwater described. Because of the requirements for infection, the was thought to be a source that is more often protected from oocysts must have been sporulated either prior to contam- the large oocysts (5 ␮m) and cysts (7 by 15 ␮m), recent ination or directly upon the berries. After harvest, the ber- data have shown that between 9.5 and 22% of groundwater ries are stored and shipped under refrigeration at tempera- samples were positive for Cryptosporidium (34). tures that would inhibit the sporulation process but that Cyclospora has also been implicated in waterborne would maintain Cyclospora viability. transmission, though not as often as are Cryptosporidium Cyclospora reappeared in 1997 (14, 15, 17). Twenty- and Giardia. In June 1994, several cases of diarrhea were ®ve clusters of outbreaks have occurred, resulting in 1,450 detected among British soldiers and dependents stationed cases in nine states. The foods associated with these out- in a small military detachment in Pokhara, Nepal (62). The breaks include raspberries, basil, and lettuce. Some of these drinking water for the camp was a mixture of river and foods were traced to Central Guatemala (Table 7). In 1998, municipal water that was treated with chlorination. A can- few cases were identi®ed in the United States; however, an dle ®ltration system was also used to remove particulates, outbreak did occur in Canada (19). but this system was not guaranteed to ®lter Cyclospora- Waterborne transmission. Cryptosporidium and sized particles (8 to 10 ␮m). Cyclospora was detected in Giardia are the most signi®cant causes of waterborne dis- 75% of the diarrhea samples examined, and a water sample ease in the United States today. The Centers for Disease processed by membrane ®ltration taken from the camp also Control and Prevention have estimated that 60% of all giar- revealed the presence of Cyclospora oocysts. diasis cases are associated with contaminated water (5). In the United States, 21 cases of prolonged diarrhea in There have been 117 waterborne outbreaks of giardiasis in employees and staff physicians were noted on 9 July 1990 the United States from 1960 to 1994, and 7 waterborne in a Chicago hospital (39). Upon investigation, Cyclospora 1064 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9

TABLE 6. Foodborne outbreaks of cryptosporidiosis in the United Statesa Number of cases/total Date Locale exposed Food Venue and contributing factors

October 1993 Maine 154/284 Apple cider Local fair; apples contaminated: dropped apples from farm with livestock were used in cider prep- aration. September 1995 Minnesota 15/26 Chicken salad Social event; hostess ran a home day-care facility. No specimens were submitted for examination from hostess or children. October 1996 New York 31/?b Apple cider Community outbreak associated with apple cider from a mill that purchased only picked apples, as

opposed to dropped apples. Dairy livestock were Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 nearby but not in the orchard. Well water tested positive for coliforms and E. coli. Apples were washed and brushed prior to pressing. December 1997 Washington 54/62 Possibly green Dinner banquet; no single food item (out of 18) had onions a strong association with the illness. Two of the workers at the banquet also tested positive and had consumed the food. Green onions had not been washed. a Sources: (12, 16, 18, 51). b Total number exposed is unknown. oocysts were identi®ed in the stools, and epidemiologic in- ic link to outbreaks has been noted, over 100 products (i.e., vestigations implicated the tap water in the physician's dor- cottage cheese) were withheld from the market during the mitory. It was speculated that the storage tank had become Milwaukee waterborne outbreak, and beverages such as contaminated. Although this outbreak has been identi®ed as fountain soda drinks were not served (40). Contamination a waterborne one, a plausible scenario for the contamina- of irrigation waters, fertilizers, and soils with human and/ tion of the water has not yet been developed. or animal wastes was also a suspected vehicle for pathogen transmission. Risks associated with produce and agriculture. Con- The potential for and the level of contamination are taminated agricultural products, such as raw fruits and veg- often dif®cult to predict; therefore, prevention approaches etables, may be an underappreciated emerging risk. Risk must include a variety of routine sanitary practices. Wash- may be associated with the use of untreated wastewater for ing (hands, surfaces, and produce) with and without sani- crop irrigation in various countries. Consequently, the in- tizers has been common practice, although in many cases, ternational market may become a global environmental very little information is available regarding the ef®cacy of health risk. Giardia cysts have been isolated from lettuce this approach when it comes to the enteric protozoa. Dis- in Rome; 48 of 64 heads were found to be contaminated infection has been used as another approach; however, re- (4). Viable cysts were also isolated in strawberries from sistance of the cysts and oocysts means that greater con- Poland. Cryptosporidium oocysts and bacterial pathogens centrations of disinfectant may be needed, as perhaps are have also been detected on fruits and vegetables (Fig. 1) longer contact times, if the disinfectant is to work. Destroy- (6, 53). Fresh vegetables from Costa Rica were found to be ing the oocysts and cysts by varying temperature (i.e., boil- contaminated with oocysts in 1 to 8% of the samples ex- ing contaminated tap water) has also been used as an ap- amined. Widespread studies on the occurrence of these pro- proach. The experimental data on the control of Crypto- tozoa on fruits and vegetables have been limited by a lack sporidium, Cyclospora, and Giardia are minimal, and in of adequate methods for recovery and detection. many cases, data are nonexistent. More studies have been CONTROL focused on waterborne (speci®cally for Cryptosporidium) than on foodborne transmission. However, some conclu- The control and prevention of protozoan foodborne sions can be drawn based on the current state of knowledge. disease depends on two main approaches: the prevention of contamination and/or the removal or extermination of the Die-off and maturation under environmental con- oocysts and cysts. As previously mentioned, contamination ditions. Once feces are excreted into the environment, the can occur directly through food handlers, as was the case oocysts of Cryptosporidium and the cysts of Giardia are for Cryptosporidium and Giardia. In addition, contamina- immediately infectious and begin an aging process that tion of the food product could arise in the environment, as leads to the die-off of some proportion of the population. was reported for Cryptosporidium and Cyclospora. The This process is dependent on time, temperature, and other contamination of the tap water used in food processing environmental conditions, such as water activity (21, 25, comprises one possible scenario. Although no epidemiolog- 26, 65, 68). The scenario is slightly different for Cyclo- J. Food Prot., Vol. 62, No. 9 FOODBORNE AND WATERBORNE ENTERIC PROTOZOA 1065

TABLE 7. Foodborne outbreaks of in the United Statesa Total cases/ Date Locale clusters Food Description and contributing factors

May to August Florida 123/?b Strawberries or Initial investigation identi®ed strawber- 1995 raspberries ries that were traced to California, where there had been ¯ooding and potential contamination. May to June 1996 Colorado, Connecticut, District of Co- 1,465/55 Raspberries At 21 of the dinners associated with lumbia, Florida, Georgia, Illinois, clusters, raspberries shipped from Iowa, Maine, Maryland, Massachu- Guatemala were served; irrigation setts, New Hampshire, New Jersey, water, water used for pesticide mix- New York, Ohio, Pennsylvania, ture, or workers' hands could have

Rhode Island, South Carolina, Texas, been the source. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 Vermont, Virginia, Wisconsin; and Ontario and Quebec, Canada March to May California, Florida, Maryland, Nebras- 1,450/25 Raspberries, Clusters associated with raspberries in 1997 ka, Nevada, New York, Rhode Is- mixed mesclun 19 of 21 events. Guatemala one pos- alnd, Texas (®eld greens or sible source of the fruit. In Florida, baby greens) one outbreak on a cruise ship. July 1997 Maryland, Virginia ?b Basil Basil left at room temperature allowed for sporulation. Food handler could have been source. a Sources: (12, 14, 15, 17, 19, 36, 58). b Numbers are unknown. spora oocysts. The oocysts need an environment that is from desiccation, prolonging the oocyst's viability within hospitable to the maturation or sporulation of the oocysts. the environment. Given that one million oocysts or cysts This process is also time and temperature dependent. Once can be found per gram of feces or per 100 liters of waste- maturation has occurred, then some die-off may begin. water, signi®cant numbers of oocysts and cysts would re- Survival within the given environment is likely once main viable, even after several months. contamination occurs. Cryptosporidium oocysts survived Interestingly, Cyclospora may have a seasonality that up to 176 days in drinking water or river water, with in- is associated with the environment and with the ability of activation of between 89 and 99% of the population (65) the oocyst to mature to the infectious stage (i.e., ability to (Table 8). Similarly, Giardia cysts were shown to remain sporulate). Early studies in India showed that the disease viable for up to 56 days in river water, with between 75 peaked within the population in May, June, July, and Au- and 99.9% inactivation (21). Oocysts also survive in human gust. More recently, in Peru, increases in infections in chil- and cattle feces, as the fecal material protects the oocyst dren were associated with air temperatures above 20ЊC (37, 47). This corroborates with laboratory studies that showed that the greatest maturation of the oocysts occurred at tem- peratures of 30ЊC, when compared with maturation at 4 and 37ЊC (Fig. 2) (70). Temperature sensitivity. The protozoa are sensitive to extreme temperatures (created by heating or freezing) that destroy the viability of oocysts and cysts. Data are not available for Cyclospora; however, its sensitivity may be similar to that of Cryptosporidium (Table 9). Fayer reported that oocysts maintained at temperatures of up to 60ЊC for 1 min infected 6 of 6 mice in his study (26). Oocysts main- tained at 59.7ЊC for 5 min infected only 1 of 6 mice. No mice became infected with oocysts that had been subjected to temperatures above 67.5ЊC for 5 min, and at least 99.9% inactivation was achieved when oocysts were subjected to 70ЊC for 1 min. All mice (6 of 6) were infected in a control group with 150,000-oocyst inoculum (26). Studies on pas- FIGURE 1. Cryptosporidium contamination of fresh produce. teurization of oocysts in milk yielded no reportable infec- Staphylococcus aureus, Listeria monocytogenes, or Salmonella tion in mice inoculated with 100 million oocysts treated detected (overall) in 86% of the fruit and vegetable samples test- with a temperature of 71.1ЊC for 5 s (35). ed. Sources: (6, 53). Low temperatures were shown to have a similar det- 1066 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9

TABLE 8. Inactivation of Cryptosporidium oocysts and Giardia cysts in envirnomental waters and fecesa Genera Suspending medium Temperature Time (days) Inactivation (%)

Giardia lamblia River water 12 to 20ЊC 3to8 90 River water 2to5ЊC 14 to 143 90 Cryptosporidium parvum Human stools 4ЊC 178 41 to Ͼ99 Cow feces 5to10ЊC 176 60 to 72 Tap water 5to10ЊC 176 96 to 99 River water 5to10ЊC 176 89 to 99 a Sources: (21, 65).

rimental effect on oocyst viability (Table 9). Fayer and Ner- weeks were required to achieve this same level of die-off Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 ad (28) found that oocysts remained infectious after 7 days when oocysts were incubated at 7ЊC (when sporozoite yield at refrigeration temperatures (5ЊC) and after freezing tem- became 0.08 sporozoites/oocyst). Infectivity in cell culture Ϫ Њ peratures of 10 C (6 of 6 mice were infected with an was similarly reduced. Under aw conditions of 0.85, inoculum of 1 million oocysts). Below Ϫ15ЊC, die-off be- Ն99.9% of the oocysts were found to be noninfectious by gan after 1 day, and no infection was noted by 7 days. At 24 h and by 1 week when stored at 28 and 7ЊC, respec- Ϫ20 and Ϫ70ЊC, the majority of the oocysts were rendered tively. Oocyst infectivity was reduced by Ն99.9% after Њ noninfectious after 8 and 1 h, respectively. At least 99.99% storage in aw 0.95 after 1 and 2 weeks at 28 and 7 C, inactivation could be shown using this experimental design. respectively (68). Data on the other enteric protozoa are not available. Short-term survival studies (24 h) of Cryptosporidium oocysts in beverages demonstrated that temperature and a Survival in foods and beverages. Survival data on the combination of carbonation and low pH affected die-off enteric protozoa in foods and beverages are limited to Cryp- (30). Morphologic changes and vital dye stains were used tosporidium (30, 68). The survival of oocysts was exam- ined in two model food products that had water activity to assess viability. After 24 h, 85% of the oocysts stored in cola were empty or partially empty of sporozoites when (aw) values of 0.85 (pancake syrup) and 0.95 (9.0% sodium chloride and 2% sodium dextrose, to simulate bread dough) examined under the microscope. Oocysts stored in beers (3 at temperatures of 28 and 7ЊC. Infectivity was evaluated to 4% alcohol) were 52 to 80% empty or partially empty. using the cell culture foci detection method (68, 69). Ex- However, oocysts stored in juice and baby formula were Ј Ј cystation and staining procedures were used as conservative only 35 and 11% empty or partially empty. DAPI/PI (4 ,6 - measures of oocyst viability. The results showed that both diamidino-2-phenylindole/propidium iodide) vital dye water activity and temperature had a dramatic effect on oo- stains determined that 59 to 70% of the oocysts remained cyst viability (68). Oocysts suspended in sample media re- viable when stored in water; 89% remained viable when mained intact. Sporozoites were visible inside each oocyst stored in baby formula; and 65% remained viable when under the microscope. Upon in vitro excystation, the oo- stored in orange juice. Oocyst viability decreases 7 to 20% cysts were capable of excysting, but only the controls re- when stored in beer; 14 to 21% when stored at pH 4.0; 13 leased detectable sporozoites (2.5 to 4 sporozoites per oo- to 17% when stored in 1.3 to 4.3% ethanol; and 15% when stored in cola. cyst). Sporozoite ratios for aw 0.85 were 0.07 sporozoites/ oocyst by 24 h when incubated at 28ЊC. The same level of These few studies suggest that if tap water contami- Ͻ die-off was seen after 72 h when oocysts were incubated nates products with short shelf lives ( 48 h), natural oocyst Њ die-off cannot be assumed and cannot be used to render the at 7 C. Oocysts exposed to an aw of 0.95 had sporozoite ratios of 0.09 sporozoites/oocyst after 48 h at 28ЊC. Two product safe. However, temperature, water activity, carbon- ation, pH, and alcohol content decrease oocyst viability and subsequently dramatically increase the die-off rate (com- pared with that of oocysts stored in water). Products that contain protein (i.e., baby formula) and that require refrig- eration appear to enhance the survival rate of Cryptospo- ridium oocysts. Process control. Disinfection of water, surfaces, hands, and equipment has been used to prevent the spread of in- fectious disease. Table 10 compares various disinfectants based on their ability to kill enteric viruses, bacteria, and protozoa. Cryptosporidium remains one of the most resis- tant microorganisms. Routine water chlorination is not ef- fective against the oocysts of Cryptosporidium. Even with FIGURE 2. Sporulation of Cyclospora oocysts related to temper- a powerful disinfectant such as ozone, 4.5 min of contact ature. Source: Smith et al. (71). time with a 1 mg/liter concentration is needed to kill 99% J. Food Prot., Vol. 62, No. 9 FOODBORNE AND WATERBORNE ENTERIC PROTOZOA 1067

TABLE 9. Inactivation of Cryptosporidium oocysts under various conditionsa Condition Temperature Time Inactivation (%)b

Cleaned preparation of oocysts in distilled water 60ЊC 1 min Ͼ99.9 Cleaned preparation of oocysts in deionized water Ϫ15ЊC 7 days Ͼ99.99 Ϫ20ЊC 8h Ͼ99.99 Ϫ70ЊC 1h Ͼ99.99 Cleaned preparation of oocysts in whole milk 71.7ЊC5s Ͼ99.9999 a Sources: (26, 28, 35). b Infectivity was determined using neonatal mice. of the oocysts (66). Giardia is also more resistant than bac- has been shown to destroy Toxoplasma oocysts (23). Un- teria; however, given suf®cient concentrations and contact sporulated T. gondii oocysts irradiated at Ն0.4 to 0.8 kGy Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 times, the cysts can be killed. Ultraviolet light was initially sporulated but were not infective to mice. Sporulated oo- shown to be ineffective, unless doses of greater than 180 cysts irradiated at 0.4 kGy were not capable of multipli- and 8,748 mJ cmϪ2 were used for cysts and oocysts, re- cation in mice. Although irradiation does not render foods spectively (7, 42). New C. parvum data show that the oo- sterile, it may effect a reduction in the levels of contami- cysts remain intact after treatment and will excyst in vitro nation. More studies are needed to examine this process for but will not infect mice (0 of 25 mice were infected for a Giardia, Cryptosporidium, and Cyclospora. 1 ϫ 105 inoculum) using a medium pressure lamp at ultra- Currently, no data are available to support or quantify violet doses of 66 mJ cmϪ2 (8). DNA damage as a result cyst or oocyst reduction caused by washing fruits and veg- of ultraviolet treatment may prevent the oocysts from in- etables. However, studies have examined the removal of fecting, even though they can still excyst and maintain oo- Giardia cysts from hands using liquid and bar soap (49). cyst wall integrity. For this study, approximately 10,000 cysts were added to Commercially available disinfectants (phenol-based the palm of the hand; 100 cysts remained after washing. products such as Pine Glo, ammonia, and Dettol [Reckitt Because as many as 10,000 cysts may be excreted in 1 g and Coleman, Berkshire, UK]) (43) were shown to inacti- of feces, hands may be readily contaminated. Even after vate 99% of Giardia cysts and no infection was seen in hand washing, the numbers of remaining cysts would be studies involving gerbils. This occurred when 1-min dis- suf®cient to initiate infections if these cysts were trans- infectant contact times were used. Disinfectants such as ferred to foods. Pine Sol (Clorox, Oakland, Calif.) and vinegar were less The physical removal of the oocysts and cysts can be effective. Cryptosporidium oocysts were more resistant achieved during drinking-water processes via sand ®ltration (75). Formol/dimethoxymethane, iodophor/phosphoric acid, using coagulant aids (i.e., alum, ferric, and polymers). Stud- isopropanol, quaternary ammonium, and chlorhexidine did ies have shown that on average, 99.4 to 99.8% of Crypto- not affect the ability of the oocyst to excyst (excystation sporidium oocysts and Giardia cysts can be removed with after exposure was shown to be similar to that in the con- the optimization of the process (55). However, this per- trol; only 12% failed to excyst). On the other hand, am- centage can drop to as low as 94%. No studies have been monia (3.1%), bleach (4.7%), phenol/isopropanol, and undertaken to examine the removal of Cyclospora oocysts; Creolin (tar acids) inhibited excystation by 97, 89, 61, and however, given that the oocysts are similar in size to Giar- 58%, respectively (75). dia cysts, similar removals might be expected. Point-of-use Irradiation (i.e., ionizing gamma irradiation) of foods and point-of-entry devices may also remove the large oo-

TABLE 10. Ct values for inactivating Cryptosporidium oocysts and Giardia cysts compared to virusesa Disinfectant (Ct)a

Microorganism UV (mJ cmϪ2) Chlorine Chloramine Chlorine dioxide Ozone

Cryptosporidium parvumb 19c 7,200 7,200d 78d 5to10 Giardiab 80e 93 to 121e,f 1,470 f 17f 1.3e,f Enterovirusesg 20 to 35b 4 857 6 0.6 a Sources: (8, 9, 42, 66, 74). ``Ct'' refers to concentration ϫ contact time needed for 99% inactivation (water temperature 5ЊC, with pH of 7.0). b Infectivity was determined using animals and/or excystation. c Achieved 99.987% inactivation. d Achieved only 90% inactivation. e Assayed using G. lamblia. f Assayed using G. muris. g Viability was determined using cell culture. h Achieved 99.9% inactivation. 1068 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9 cysts and cysts. An absolute 1-␮m pore-size membrane ®l- fore, waters with algae, turbidity, suspended solids, and oth- ter or smaller has been recommended by the Centers for er materials interfere with large volume collection (greater Disease Control and Prevention for removal of the proto- than 10 liters) and the detection process. Whereas density zoan cysts and oocysts. Standards developed by the Na- gradients, such as sucrose±percoll, have been applied to the tional Sanitation Foundation (54) and the United States En- clari®cation of the sample, a more promising technique is vironmental Protection Agency's standard guide and pro- immunomagnetic separation (7, 41). Immunomagnetic sep- tocol (76) suggest that at least 99.9% removal of oocysts aration uses antibodies tagged to silicon-coated iron-oxide and cysts be demonstrated. Removals of greater than 99.9% beads and a magnetic system to pull the target oocysts and of cysts and oocysts have been shown using these proce- cysts from the suspension. This has applications for both dures with an activated carbon block ®lter (1). Although microscopic detection and polymerase chain reaction (20, the system tested used ultraviolet disinfection, no cysts or 41). Several immunomagnetic separation kits are now avail- oocysts were recovered post®ltration. able for Cryptosporidium (Dynal, Lake Success, N.Y.;

ImmuCell, Crypto-Scan, Portland, Maine). Since no anti- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 Approaches for control and management. From the body exists for Cyclospora, immunomagnetic separation farm to the table, there are a number of methods that may does not have an application at this time. be implemented to prevent or reduce the level of contam- Recovery methods from produce have been relatively ination: (i) use of noncontaminated irrigation and process simplistic and involve washing the oocysts and cysts from water (shallow wells may be more likely to be contami- the surfaces of the fruits and vegetables with a detergent nated); (ii) improvement of drinking-water treatment; (iii) solution. These methods do not appear very ef®cient for implementation of hygienic practices during harvesting and several reasons. The ®rst lies in our inability to wash the packaging; (iv) use of hygienic practices for food handlers cysts and oocysts off the produce (they appear to have some (hand washing and gloves); (v) pasteurization of juices; and stickiness). Second, only a small percentage of the crop or (vi) irradiation of produce. batch can be sampled. It may be more appropriate to sample In order to evaluate the various management strategies, irrigation waters or to develop a large batch-rinse technique methods are needed to manage the production of oocysts and sample the rinse waters. Better methods are needed for and cysts as a supply for various laboratory and ®eld stud- recovering oocysts and cysts from strawberries, raspberries, ies. Methods are also needed for the recovery, isolation, lettuce, and other produce. detection, and viability assessment of cysts and oocysts. Molecular methods are used to detect the nucleic acid Without better procedures and new approaches, signi®cant within the cysts and oocysts and are rapid, sensitive, and data gaps will remain. speci®c to the species. The polymerase chain reaction and Methods. New methods are now available for deter- ¯uorescent in situ hybridization have been used to identify mining the level of oocyst and cyst contamination. Molec- Cryptosporidium (41, 44); these techniques are relatively ular- and immunology-based methods perhaps show the new but offer a signi®cant application for detection, be- greatest promise when it comes to the rapid detection and cause the probe binds to the nucleic acid within the oocyst, identi®cation of the enteric protozoa. Antibody-based meth- and both microscopy and the speci®city of the probe can ods have been used for detecting Cryptosporidium and be used for identi®cation and detection. Polymerase chain Giardia (77). Commercially available monoclonal antibod- reaction techniques are also available for Giardia and Cy- ies speci®c to the cyst and oocyst walls have been tagged clospora (48, 63). with ¯uorescein±isothiocyanate and used with microscopy Risk-assessment methods have been used for risk man- (examining ¯uorescence, size, shape, and presence of in- agement in water and food, and models are now available ternal features, such as trophozoites or sporozoites) for de- for Cryptosporidium and Giardia (33, 67). Quantitative in- tection from water. No antibody is available for Cyclospora; formation provides a more useful database that can be used light microscopy, acid-fast staining, morphology (oocyst to evaluate speci®c key risks associated with priority mi- wall appearance, size, and shape), and measurement of the croorganisms and that can allow for the development of ability to sporulate have been used for identi®cation. How- scienti®cally based prioritization based on public health ever, these approaches have limited application to environ- bene®ts. This type of data will improve the decisions made mental samples. The oocyst of Cyclospora does have one with regard to wastewater management and reuse, protec- unique featureÐthe ability to auto¯uoresce (24). The oo- tion and treatment of water, and improved safety of the food cyst produces a distinct blue ¯uorescence around the out- supply. If these data are not available, then it will not be side wall under epi¯uorescent illumination (excitation 375 possible to know whether the risks are severe or whether nm; emission Ͼ420 nm) and can easily be seen at ϫ200 they can be easily controlled. Many groups have made im- magni®cation (2). proved microbiological assessment recommendations. Most Because they occur in relatively small numbers within recently, a work group of experts organized by the Amer- the environment, cyst and oocyst detection relies upon con- ican Academy of Microbiology (29) clearly stated this key centration techniques. Membrane ®ltration, cartridge ®ltra- message: ``Reliance on the coliform indicator system alone tion, and centrifugation all have application to surface, ir- is no longer adequate for the protection of public health rigation, and drinking water. However, these procedures are and determination of microbiological quality.'' The types plagued by similar problems; they concentrate the protozoa of data that are needed include the following: (i) health as well as other types of particles indiscriminately. There- surveillance in the community; (ii) environmental monitor- J. Food Prot., Vol. 62, No. 9 FOODBORNE AND WATERBORNE ENTERIC PROTOZOA 1069 ing data and development of an occurrence database to de- break of cryptosporidiosisÐSpokane, Washington, 1997. MMWR termine exposure; and (iii) evaluation of water systems used 47:27. 19. Centers for Disease Control and Prevention. 1998. Outbreak of cy- for irrigation (coliforms can no longer serve as the sole closporaisisÐOntario, Canada, May 1998. MMWR 47:38. indicator of microbial safety). These data can then be used 20. Deng, M. Q., D. O. Cliver, and T. W. Mariam. 1997. Immunomag- to improve and/or implement (i) the risk-assessment meth- netic capture PCR to detect viable Cryptosporidium parvum oocysts odology (by providing better databases); (ii) communica- from environmental samples. Appl. Environ. Microbiol. 63:3134± tion through educational programs for industry, community, 3138. and government; and (iii) the development of global ap- 21. DeRegnier, D., L. Cole, D. G. Schupp, and S. Erlandsen. 1989. Vi- ability of Giardia cysts suspended in lake, river and tap water. Appl proaches for creating opportunities for interface between Environ. Microbiol. 55:1223±1229. policies at the local, state, national, and international levels. 22. Dubey, J. P., C. A. Speer, and R. Fayer (ed.). 1990. Cryptosporidiosis of man and animals. CRC Press, Boca Raton, Fla. ACKNOWLEDGMENTS 23. Dubey, J. P., D. W. Thayer, C. A. Speer, and S. K. Shen. 1998. Effect of gamma irradiation on unsporulated and sporulated Toxoplasma We thank Dr. Isabel Walls of the National Food Processors Associ- gondii oocysts. Int. J. Parasitol. 28:367±375. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 ation and Erin Lipp of the University of South Florida for their time and 24. Dytrych, J. K., and R. P. D. Cooke. 1995. Auto¯uorescence of Cy- expert assistance in reviewing the manuscript. clospora. Br. J. Environ. Sci. 52:76. 25. Erlandsen, S. L., and E. A. Meyer (ed.). 1984. Giardia and giardiasis REFERENCES biology, pathogenesis, and epidemiology. Plenum Press, New York. 26. Fayer, R. 1994. Effect of high temperature on infectivity of Cryp- 1. Abbaszadegan, M., M. N. Hasan, C. P. Gerba, P. F. Roessler, B. R. tosporidium parvum oocysts in water. Appl. Environ. Microbiol. 60: Wilson, R. Kuennen, and E. Van Dellen. 1997. The disinfection ef- 2732±2735. ®cacy of a point-of-use water treatment system against bacterial, 27. Fayer, R. (ed.). 1997. Cryptosporidium and cryptosporidiosis. CRC viral and protozoan waterborne pathogens. Wat. Res. 31:574±582. Press, Boca Raton, Fla. 2. American Water Works Association Microbial Contaminants Re- 28. Fayer, R., and T. Nerad. 1996. Effects of low temperatures on via- search Committee. Emerging pathogens: names to know and the bugs to watch out for. J. AWWA, in press. bility of Cryptosporidium parvum Oocysts. Appl. Environ. Micro- 3. Ashford, R. W. 1979. Occurrence of an undescribed coccidian in biol. 62:1431±1433. man in Papua New Guinea. Ann. Trop. Med. Parasitol. 73:497±500. 29. Ford, T. E., and R. R. Colwell. 1996. A global decline in microbi- 4. Barnard, R. J., and G. J. Jackson. 1984. Giardia lamblia: the transfer ological safety of water: a call for action. American Academy of of human infections with foods, p. 365±378. In S. L. Erlandsen and Microbiology, Washington, D.C. E. A. Meyer (ed.). Giardia and giardiasis biology, pathogenesis, and 30. Friedman, D. E, K. A. Patten, J. B. Rose, and M. C. Barney. 1997. epidemiology. Plenum Press, New York. The potential for Cryptosporidium parvum oocyst survival in bev- 5. Bennett, J. V., S. D. Homberg, and M. F. Rogers. 1987. Infectious erages associated with contaminated tap water. J. Food Saf. 17:25± and parasitic diseases. Am. J. Prev. Med. 3:102±114. 132. 6. Beuchat, L. R. 1996. Pathogenic microorganisms associated with 31. Garcia, L. S., and D. A. S. Bruckner (ed.). 1988. Diagnostic medical fresh produce. J. Food Prot. 59:204±216. parasitology. Elsevier Science Publishing Co., Inc., New York. 7. Bilfulco, J. M., and F. W. Schaeffer, III. 1993. Antibody-magnetite 32. Goodgame, R. W. 1996. Understanding intestinal spore-forming pro- method for selective concentration of Giardia lamblia cysts from tozoa: Cryptosporidia, Microsporidia, , and Cyclospora. water samples. Appl. Environ. Microbiol. 59:772. Ann. Intern. Med. 124:429±441. 8. Buchari, Z., T. M. Hargy, J. R. Bolton, B. Dussert, and J. L. Clancy. 33. Haas, C. N., C. S. Crockett, J. B. Rose, C. P. Gerba, and A. M. Fazil. 1999. Medium-pressure UV for oocyst inactivation. J. AWWA 91: 1996. Assessing the risk posed by oocysts in drinking water. J. 86±94. AWWA 88:131±136. 9. Campbell, A. T., L. S. Robertson, M. R. Snowbal, and H. V. Smith. 34. Hancock, C. M., J. B. Rose, and M. C. Callahan. 1998. Crypto and 1995. Inactivation of oocysts of Cryptosporidium parvum:UVra- Giardia in US groundwater. J. AWWA 90:58±61. diation. Appl. Environ. Microbiol. 53:375±378. 35. Harp, J. A., R. Fayer, B. A. Pesch, and G. J. Jackson. 1996. Effect 10. Centers for Disease Control and Prevention. 1991. Results of testing of pasteurization on infectivity of Cryptosporidium parvum oocysts for intestinal parasites by state diagnostic laboratories, United States, in water and milk. Appl. Environ. Microbiol. 62:2866±2868. 1987. MMWR 40:25±45. 36. Herwaldt, B. L., M.-L. Ackers, and T. C. W. Group. 1997. An out- 11. Centers for Disease Control and Prevention. 1996. Foodborne out- break in 1996 of cyclosporiasis associated with imported raspberries. break of diarrheal illness associated with Cryptosporidium parvum, N. Engl. J. Med. 336:1548±1556. Minnesota, 1995. MMWR 45:783. 37. Hoge, C. W., D. R. Shlim, R. Rajah, J. Triplett, M. Shear, J. G. 12. Centers for Disease Control and Prevention. 1996. Outbreaks of Cy- Rabold, and P. Echeverria. 1993. Epidemiology of diarrhoeal illness clospora cayetanensis infectionÐUnited States, 1996. MMWR 45: associated with coccidian-like organism among travellers and foreign 549±551. residents in Nepal. Lancet 341:1175±1179. 13. Centers for Disease Control and Prevention. 1996. Surveillance for 38. Hoxie, N. J., J. P. Davis, J. M. Vergeront, R. D. Nashold, and K. A. foodborne-disease outbreaksÐUnited States, 1988±1992. MMWR Blair. 1997. Cryptosporidiosis-associated mortality following a mas- 45:1±65. sive waterborne outbreak in Milwaukee, Wisconsin. Am. J. Public 14. Centers for Disease Control and Prevention. 1997. Outbreaks of cy- Health 87:2032±2035. closporiasisÐNorthern Virginia±Washington, D.C.±Baltimore, 39. Huang, P., J. T. Weber, D. M. Sosin, P. M. Grif®n, E. G. Long, J. J. Maryland Metropolitan area, 1997. MMWR 46:30. Murphy, F. Kocka, C. Peters, and C. Kallick. 1995. The ®rst reported 15. Centers for Disease Control and Prevention. 1997. Outbreaks of cy- outbreak of diarrheal illness associated with Cyclospora in the Unit- closporiasisÐUnited States, 1997. MMWR 46:451. ed States. Ann. Intern. Med. 123:409±414. 16. Centers for Disease Control and Prevention. 1997. Outbreaks of 40. Jackson, G. Personal communication. Escherichia coli O157:H7 infection and cryptosporidiosis associated 41. Johnson, D. W., N. J. Pieniazek, D. W. Grif®n, L. Misener, and J. with drinking unpasteurized apple ciderÐConnecticut and New B. Rose. 1995. Development of a PCR protocol for sensitive detec- York, October 1996. MMWR 46:4±8. tion of Cryptosporidium oocysts in water samples. Appl. Environ. 17. Centers for Disease Control and Prevention. 1997. Update: outbreaks Microbiol. 61:3849±3855. of cyclosporiasis. MMWR 46:521. 42. Karanis, P., W. A. Maier, H. M. Seitz, and D. Schoenen. 1992. UV 18. Centers for Disease Control and Prevention. 1998. Foodborne out- sensitivity of protozoan parasites. J. Wat. SRT-Aquat. 451:95±100. 1070 ROSE AND SLIFKO J. Food Prot., Vol. 62, No. 9

43. Lee, M. B. 1992. The effectiveness of commercially available dis- Restaurant-associated outbreak of giardiasis. J. Infect. Dis. 166:673± infectants upon Giardia lamblia cysts. Can. J. Public Health 83:171± 676. 172. 62. Rabold, J. G., C. W. Hoge, D. R. Shlim, C. Kefford, R. Rajah, and 44. Lindquist, J. A. D. 1997. Probes for the speci®c detection of Cryp- P. Echeverria. 1994. Cyclospora outbreak associated with chlorinated tosporidium parvum. Wat. Res. 31:2668±2671. drinking water. Lancet 344:1360±1361. 45. Lisle, J. T., and J. B. Rose. 1995. Cryptosporidium contamination of 63. Relman, D. A., T. M. Schmidt, A. Jajadhar, M. Sogin, J. Cross, K. water in the USA and UK: a mini-review. J. Wat. SRT-Aquat. 44: Yoder, O. Sethabutr, and P. Echeverria. 1995. Molecular phylogenetic 103±117. analysis of Cyclospora, the human intestinal pathogen, suggests that 46. MacKenzie, W. R., N. J. Hoxie, M. E. Proctor, M. S. Gradus, K. A. it is closely related to species. J. Infect. Dis. 173:440±445. Blair, D. E. Peterson, J. J. Kazmierczak, D. G. Addiss, K. R. Fox, 64. Roberts, T., and J. K. Frenkel. 1990. Estimating income losses and J. B. Rose, and J. P. Davis. 1994. A massive outbreak in Milwaukee other preventable costs caused by congenital toxoplasmosis in people of Cryptosporidium infection transmitted through the public water in the United States. JAVMA 196:249±256. 65. Robertson, L. J., A. T. Campbell, and H. V. Smith. 1992. Survival supply. N. Engl. J. Med. 331(3):161±167. of Cryptosporidium parvum oocysts under various environmental 47. Madico, G., J. McDonald, R. H. Gilman, L. Cabrera, and C. Sterling. pressures. Appl. Environ. Microbiol. 55:1519±1522. 1997. Epidemiology and treatment of Cyclospora cayetanensis in- 66. Rose, J. B. 1997. Environmental ecology of Cryptosporidium and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1059/2007718/0362-028x-62_9_1059.pdf by guest on 29 September 2021 fection in Peruvian children. Clin. Infect. Dis. 24:977±981. public health impacts. Ann. Rev. Public Health 18:135±161. 48. Mahbubani, M. H., A. K. Bej, M. Perlin, F. W. Schaefer, W. Jaku- 67. Rose, J. B., C. N. Haas, and S. Regli. 1991. Risk assessment and bowski, and R. M. Atlas 1991. Detection of Giardia cysts by using control of waterborne giardiasis. Am. J. Public Health Assoc. 81: the polymerase chain reaction and distinguishing live from dead 709±713. cysts. Appl. Environ. Microbiol. 57:3456±3461. 68. Slifko, T.R., D. E. Friedman, J. H. Fraser, and J. B. Rose. 1997. 49. Manthriratna, G. A. 1989. Ef®cacy of handwashing as an aid in the Survival of Cryptosporidium in food products, p. 444. In 97th Gen- control of Rotavirus and Giardia transmission. Thesis. University of eral Meeting, ASM; Miami Beach May 4±8, 1997. Abstracts. ASM Arizona, Tucson. Press, Washington, D.C. 50. Meyer, E. A. (ed.). 1990. Giardiasis. Elsevier, New York. 69. Slifko, T. R., D. E. Friedman, J. B. Rose, S. Upton, and W. Jaku- 51. Millard, P. S., K. F. Gensheimer, D. G. Addiss, D. M. Sosin, G. A. bowski. 1997. An in vitro method for detection of infectious Cryp- Beckett, A. Houck-Jankoski, and A. Hudson. 1994. An outbreak of tosporidium oocysts using cell culture. Appl. Environ. Microbiol. 63: cryptosporidiosis from fresh-pressed apple cider. J. Am. Med. Assoc. 3669±3675. 272:592±1596. 70. Smith, H. V., C. A. Paton, M. M. A. Mitambo, and R. W. A. Gird- 52. Mintz, E. D., M. Huson-Wragg, P. Mshar, M. L. Cartter, and J. L. wood. 1997. Sporulation of Cyclospora sp. oocysts. Appl. Environ. Hadler. 1993. Foodborne giardiasis in a corporate of®ce setting. J. Microbiol. 63:1631±1632. Infect. Dis. 167:250±253. 71. Smith, H. V., and J. B. Rose. 1998. Waterborne cryptosporidiosis: 53. Monge, R., and M. Chinchilla. 1996. Presence of Cryptosporidium current status. Parasitol. Today 14:14±22. oocysts in fresh vegetables. J. Food Prot. 59:202±203. 72. Smith, J. L. 1993. Cryptosporidium and Giardia as agents of food- 54. National Sanitation Foundation. 1991. Standard 53: drinking water borne disease. J. Food Prot. 56:451±461. treatment unitsÐhealth effects. NSF Joint Committee on Drinking 73. Soave, R., and W. D. Johnson. 1995. Cyclospora: conquest of an Water Treatment Units, Ann Arbor, Mich. emerging pathogen. Lancet 345:667±668. 55. Nieminski, E. C., and J. E. Ongerth. 1995. Removing Giardia and 74. Sobsey, M. D. 1989. Inactivation of health-related microorganisms in water by disinfection processes. Wat. Sci. Technol. 2:179±195. Cryptosporidium by conventional and direct ®ltration. J. AWWA 87: 75. Sundermann, C. A., D. S. Lindsay, and B. L. Blagburn. 1987. Eval- 96±106. uation of disinfectants for ability to kill avian Cryptosporidium oo- 56. Ortega, Y. R., C. R. Sterling, R. H. Gilman, C. A. Vitaliano, and F. cysts. Companion Anim. Pract. 1:36±39. Diaz. 1993. Cyclospora speciesÐa new protozoan pathogen of hu- 76. United States Environmental Protection Agency Task Force. 1987. mans. N. Engl. J. Med. 328:1308±1312. Guide standard and protocol for testing microbiological water puri- 57. Osterholm, M. T., J. C. Forfang, R. L. Ristinen, A. G. Dean, J. W. ®ers. U.S. EPA Registration Division, Of®ce of Pesticide Programs Washburn, J. R. Godes, R. A. Rude, and J. G. McCullough. 1981. and Criteria and Standards Division, Of®ce of Drinking Water, An outbreak of foodborne giardiasis. N. Engl. J. Med. 304:24±28. Washington, D.C. 58. Palm Beach Post. 4 November 1995. 77. United States Environmental Protection Agency. 1994. National pri- 59. Petersen, L. R., M. L. Cartter, and J. L. Hadler. 1988. A food-borne mary drinking water regulations; enhanced surface water treatment outbreak of Giardia lamblia. J. Infect. Dis. 157:846±848. requirements; proposed rule. Fed. Reg. 54:38832±38858. 60. Porter, J. D. H., C. Garney, D. Heymann, and W. Parkin. 1990. Food- 78. White, K. E., C. W. Hedberg, L. M. Edmonson, D. B. W. Jones, M. borne outbreak of Giardia lamblia. Am. J. Public Health 80:1259± T. Osterholm, and K. L. MacDonald. 1989. An outbreak of giardiasis 1260. in a nursing home with evidence for multiple modes of transmission. 61. Quick, R., K. Paugh, D. Addiss, J. Kobayashi, and R. Baron. 1992. J. Infect. Dis. 16:298±303.