Foodborne Animal Parasites 683 of stools from patients Later symptoms consist of pronounced abdominal pain, fever, severe diarrhea, vomiting, and lumbago, and somewhat resemble those of shigellosis Weight loss is common, and all patients have heme-positive stools According to Jackson,48 fulminating amebiasis with ulceration of the colon and toxicity occur in 6–11% of cases, especially in women stressed by pregnancy or nursing Masses of amebae and mucus may form in the colon, leading to intestinal obstruction Amebiasis may last in some individuals for many years, in contrast to giardiasis, where disease symptoms rarely exceed months.3 Under some conditions, amebiasis may result from a synergistic relationship with certain intestinal bacteria Amebiasis is diagnosed by demonstrating trophozoites and cysts in stools or mucosal scrapings Immunological methods such as indirect hemagglutination, indirect immunofluorescence, latex agglutination, and ELISA are useful The sensitivity of these tests is high with extraintestinal amebiasis, and a titer of 1:64 by indirect hemagglutination is considered significant This syndrome can be treated with the amebicidal drugs metronidazole and chloroquine Resistance is mediated by cell immunity Lymphocytes from patients in the presence of E histolytica antigens have been shown to produce γ -interferon, which activates macrophages that display amebicidal properties.90 Toxoplasmosis This disease is caused by Toxoplasma gondii, a coccidian protozoan that is an obligate intracellular parasite The generic name is based on the characteristic shape of the ameba stage of the protozoan (Gr toxo, “arc”) It was first isolated in 1908 from an African rodent, the gondi—hence, its species name In most instances, the ingestion of T gondii oocysts causes no symptoms in humans, or the infection is self-limiting In these cases, the organism encysts and becomes latent However, when the immunocompetent state is abated, life-threatening toxoplasmosis results from the breaking out (recrudescence) of the latent infection Domestic and wild cats are the only definitive hosts for the intestinal or sexual phase of this organism, making them the primary sources of human toxoplasmosis Normally, the disease is transmitted from cat to cat, but virtually all vertebrate animals are susceptible to the oocysts shed by cats As few as 100 oocysts can produce clinical toxoplasmosis in humans, and the oocysts can survive over a year in warm, moist environments.33 Pigs are the major animal food source to humans Symptoms, Diagnosis, and Treatment In most individuals, toxoplasmosis is symptomless, but when symptoms occur, they consist of fever with rash, headache, muscle aches and pain, and swelling of the lymph nodes The muscle pain, which is rather severe, may last up to a month or more At times, some of the symptoms mimic infectious mononucleosis The incubation period in adults is 6–10 days while in infants it is congenital The disease is initiated upon the ingestion of oocysts (if from cat feces), which pass to the intestine where digestive enzymes affect the release of the eight motile sporozoites Oocysts are ovoid shaped, measure 10–12 µm in diameter, and possess a thick wall Sporozoites are crescent shaped and measure about × µm They cannot survive for long outside animal host tissues, nor can they survive the activities of the stomach When freed in the intestines, these forms pass through intestinal walls and multiply rapidly in many other parts of the body, giving rise to clinical symptoms The most rapidly multiplying forms are designated tachyzoites (Gr tachy, “rapid”), and in immunocompetent individuals, they eventually give rise to clusters that are surrounded by a protective wall This is a tissue cyst, and the protozoa inside are designated bradyzoites (Gr bradus, “slow”) These cysts are 684 Modern Food Microbiology 10–200 µm in diameter, and the bradyzoites are smaller in size than the more active tachyzoites Bradyzoites may persist in the body for the lifetime of an individual, but if the cysts are mechanically broken or break down under immunosuppression, bradyzoites are freed and begin to multiply rapidly as tachyzoites and thus bring on another active infection The development of a cyst wall around bradyzoites coincides with the development of permanent host immunity The cysts are normally intracellular in host cells T gondii infections are asymptomatic in the vast majority of human cases (immunocompetents), but in congenital infections and in immunocompromised hosts, such as patients with acquired immunodeficiency syndrome (AIDS), the disease is much more severe In pregnant mothers with newly contracted toxoplasmosis, the tachyzoites are reported to cross the placenta about 45% of the time Unlike certain other intestinal protozoal diseases, toxoplasmosis cannot be diagnosed by demonstrating oocysts in stools, as these forms occur only in cat feces Various serological methods are widely used to diagnose acute infection A fourfold rise in immunoglobulin G (IgG) antibody titer between acute and convalescent serum specimens is indicative of acute infection A more rapid confirmation of acute infection can be made by the detection of immunoglobulin M (IgM) antibodies, which appear during the first week of infection and peak during the second to fourth weeks.80 Among other diagnostic methods are the methylene blue dye test, indirect hemagglutination, indirect immunofluorescence, and immunoelectrophoresis With the indirect hemagglutination test, antibody titers above 1:256 are generally indicative of active infection Although toxoplasma infection induces protective immunity, it is, in part, cell mediated In many bacterial infections where phagocytes ingest the cells, their internal granules release enzymes that destroy the bacteria During this process, aerobic respiration gives way to anaerobic glycolysis, which results in the formation of lactic acid and the consequent lowering of pH The latter contributes to the destruction of the ingested bacteria along with the production of superoxide, which, at the acid pH, yields singlet oxygen (1 O2 ) The latter is quite toxic T gondii tachyzoites are unusual in that once they are phagocytosed, the production of H2 O2 is not triggered, and neither the acid pH nor the singlet oxygen events occur Also, they reside in vacuoles of phagocytes that not fuse with preexisting secondary lysosomes Thus, it appears that their mode of pathogenicity involves an alteration of phagocyte membranes in such a way that they fail to fuse with other endocytic or biosynthetic organelles, in addition to the other events noted.55 T cells play a role in immunity to T gondii, and this has been demonstrated by use of nude rats, where T cells from T gondiiinfected normal rats conferred to nude rats the ability to resist infection by a highly virulent strain of T gondii.29 Antimicrobial therapy for toxoplasmosis consists of sulfonamides, pyrimethamine, pyrimethamine plus clindamycin, or fluconazole Pyrimethamine is a folic acid antagonist that inhibits dihydrofolate reductase Distribution of T gondii Toxoplasmosis is regarded as a universal infection, with the incidence being higher in the tropics and lower in colder climes It is estimated that 50% of Americans have circulating antibodies to T gondii by the time of adulthood.80 In a study of U.S Army recruits, 13% were positive for toxoplasma antibodies.36 In the United States, it is estimated that over 3000 babies are infected each year with T gondii because their mothers acquire the infection during pregnancy.33 Fetal infections occur in 17% of first-trimester and 65% of third-trimester cases, with the first-trimester cases being more severe.80 Among 3000 pregnant women tested for T gondii antibodies, 32.8% were positive.58 Foodborne Animal Parasites 685 Table 29–1 Estimated Number of Clinically Significant Cases of Protozoal Infections in the United States, 1985 Infections Cases Amebiasis Cryptosporidiosis Giardiasis Toxoplasmosis∗ ∗ Excluding 12,000 50 120,000 2,300,000 congenital Source: From Bennett et al.7 Extensive surveys of T gondii antibodies in meat animals have been reviewed by Fayer and Dubey33 who reported that of more than 16,000 cattle surveyed, an average of 25% contained antibodies, and infectious cysts administered from cats persisted as long as 267 days, with most being found in the liver In more than 9000 sheep, an average of 31% had antibodies, and oocysts administered persisted 173 days, with most protozoa found in the heart Similarly for pigs, 29% had antibodies, and oocysts persisted for 171 days, with most in the brain and heart, whereas for goats, oocysts persisted in the animals for 441 days, with most being found in skeletal muscles Because the meat animals noted are herbivores, Fayer and Dubey33 concluded that the contamination of feed and water with oocysts from cat feces must be the ultimate source of infection, aided by the practice on some farms of keeping cats to kill mice Food-Associated Cases The number of cases of toxoplasmosis that are contracted from foods is unknown, but the estimated number in the United States from all sources for 1985 has been put at 2.3 million (Table 29–1) This estimated number far exceeds the recorded cases for the total of all other protozoal diseases Fresh meats may contain toxoplasma oocysts As early as 1954, undercooked meat was suspected to be the source of human toxoplasmosis.51 In a study in 1960 of freshly slaughtered meats, 24% of 50 porcine, 9.3% of 86 ovine, but only of 60 bovine samples contained oocysts.53 T gondii is more readily isolated from sheep than other meat animals.51 The following cases have been proved or suspected: In France in the early 1960s, 31% of 641 children in a tuberculosis hospital became seropositive for T gondii after admission When two additional meals per day of undercooked mutton were served, toxoplasmosis cases doubled The investigators concluded that the custom of this hospital to feed undercooked meat was the cause of the high number of infections.28 At an educational institution, 771 mothers were questioned about their preferences for meat Of those who preferred well-done meat, 78% had toxoplasma antibody; of those who liked less well-done meats, 85% were antibody positive; and of those who ate meat rare or raw, 93% had toxoplasma antibodies.28 The investigators were unable to make distinctions among beef, mutton, or horse meat They further noted that 50% of children in France are infected with T gondii before age and believe this is due to the consumption of undercooked meats 686 Modern Food Microbiology Eleven of 35 medical students in New York City in 1968 had an increase in toxoplasma antibodies following the consumption of hamburger cooked rare at the same snack bar, and contracted clinical toxoplasmosis.58 In 1974, a 7-month-old infant who consumed unpasteurized goat’s milk developed clinical toxoplasmosis Although T gondii could not be recovered from milk, some goats in the herd had antibody titers to T gondii as high as 1:512, and the child had a titer of over 1:16,000.83 In 1978, 10 of 24 members of an extended family in northern California contracted toxoplasmosis after drinking raw milk from infected goats.89 In S˜ao Paulo, Brazil, 110 university students suffered acute toxoplasmosis after eating undercooked meat.19 Since most of the above cases have been traced to meats, the consumption of raw or undercooked meats carries the risk of this infection Other documented meatborne outbreaks have been reviewed.95 Control Toxoplasmosis in humans can be prevented by avoiding environmental contamination with cat feces (from cat litter boxes, for example) and by avoiding the consumption of meat and meat products that contain viable tissue cysts The cysts of T gondii can be destroyed by heating meats above 60◦ C or by irradiating at a level of 30 krad (0.3 kGy) or higher.33 The organism may be destroyed by freezing, but because the results are variable, freezing should not be relied on to inactivate oocysts See reference for a detailed review of the life cycle of Toxoplasma gondii Sarcocystosis Of more than 13 known species of the genus Sarcocystis, two are known to cause an extraintestinal disease in humans One of these is obtained from cattle (S hominis) and the other from pigs (S suihominis) Humans are the definitive hosts for both species: the intermediate host for S hominis is bovines, and pigs for S suihominis When humans ingest a sarcocyst, bradyzoites are released and penetrate the lamina propria of the small intestine, where sexual reproduction occurs that leads to sporocysts The latter pass out of the bowel in feces When sporocysts are ingested by pigs or bovines, the sporozoites are released and spread throughout the body They multiply asexually and lead to the formation of sarcocysts in skeletal and cardiac muscles In this stage, they are sometimes referred to as Miescher’s tubules The bradyzoite-containing sarcocysts are visible to the unaided eye and may reach cm in diameter.19 Several studies have been conducted to determine the relative infectivity of Sarcocystis spp Of 20 human volunteers in five studies who ate raw beef infected with S hominis, 12 became infected and shed oocysts, but only had clinical illness.34 Symptoms occurred within 3–6 hours and consisted of nausea, stomachache, and diarrhea In 15 other volunteers who ate raw pork infected with S suihominis, 14 became infected and shed oocysts, and 12 of these had clinical illness 6–48 hours after eating the pork.34 Six who ate well-cooked pork did not contract the disease In another study of another species of Sarcocystis, dogs did not become infected when fed beef cooked medium (60◦ C) or well done (71.1–74.4◦ C), but the beef was infective when fed raw or cooked rare (37.8–53.3◦ C) Dogs fed the same raw beef after storage for week in a home freezer did not become infected.35 In another study, two human volunteers passed sporocysts for 40 days after eating 500 g of raw ground beef diaphragm muscle infected with Sarcosporidia.85 Foodborne Animal Parasites 687 Because bovine and porcine animals serve as intermediate hosts for these parasites, their potential as foodborne pathogens to humans is obvious Cryptosporidiosis The protozoan Cryptosporidium parvum was first described in 1907 in asymptomatic mice, and for decades now it has been known to be a pathogen of at least 40 mammals and varying numbers of reptiles and birds Although the first documented human case was not recorded until 1976, this disease has a worldwide prevalence of 1–4% among patients with diarrhea,104 and it appears to be increasing In England and Wales for the years 1985–1989, the numbers of identified cases were 1874, 3694, 3359, 2838, and 7769, respectively.2 This disease was the fourth most frequent cause of diarrhea during the period noted It is estimated to cause infections in from to 38% of AIDS patients in some hospitals.104 The prevalence of C parvum in diarrheal stools is similar to that of Giardia lamblia.104 In humans, the disease is self-limiting in immunocompetent individuals, but it is a serious infection in the immunocompromised, such as AIDS patients The protozoan is known to be present in at least some bodies of water (see below) and thus exists the potential for food transmission The fecal–oral route of transmission is the most important, but indirect transmission by food and milk is known to occur C parvum is an obligate intracellular coccidian parasite that carries out its life cycle in one host Following ingestion of the thick-walled oocysts, they excyst in the small intestine and free sporozoites and penetrate the microvillous region of host enterocytes, where sexual reproduction leads to the development of zygotes They invade host cells by disrupting their own membrane as well as that of the host Host cell actin polymerization at the interface between the parasite and the host cell cytoplasm has been found to be necessary for infection.30 About 80% of the zygotes form thick-walled oocysts that sporulate within host cells.24 The environmentally resistant oocysts are shed in feces, and the infection is transmitted to other hosts when they are ingested The oocysts of C parvum are spherical to ovoid and average 4.5–5.0 µm in size Each sporulated oocyst contains four sporozoites The oocysts are highly resistant in the natural environment and may remain viable for several months when kept cold and moist.24 They have been reported to be destroyed by treatments with 50% or more ammonia and 10% or more formalin for 30 minutes.24 The latter investigator has reported that temperatures above 60◦ C and below −20◦ C may kill C parvum oocysts The organism is destroyed by high-temperature, short time (HTST) milk pasteurization Holding oocysts at 45◦ C for 5–20 minutes has been reported to destroy their infectivity.1 In one study, infectivity was lost after months when cysts were stored in distilled water or at 15–20◦ C within weeks or at 37◦ C in days.92 In the latter study, cysts did not survive freezing even when stored in a variety of cryoprotectants The survival of C pavum oocysts of human and ovine origins in still natural mineral waters was assessed in a study in Scotland When added to mineral waters and held at 20◦ C, a progressive decline in viability of both types was noted, but viability remained unaltered when stored for 12 weeks at 4◦ C.73 Commonly used disinfectants are ineffective against the oocysts,10 and this has been demonstrated for ozone and chlorine compounds For a 90% or more inactivation of C parvum oocysts, ppm ozone required minutes, 1.3 ppm chlorine dioxide required 60 minutes, and 80 ppm each of chlorine and monochloramine required about 90 minutes.62 The oocysts were 14 times more resistant to ClO2 than Giardia cysts, and these investigators suggested that disinfection alone should not be relied upon to inactivate C parvum oocysts in water Human cryptosporidiosis may be acquired by at least one of five known transmission routes: zoonotic, person-to-person, water, nosocomial (hospital acquired), or food Zoonotic transmission ... further noted that 50% of children in France are infected with T gondii before age and believe this is due to the consumption of undercooked meats 686 Modern Food Microbiology Eleven of 35 medical... dogs did not become infected when fed beef cooked medium (60◦ C) or well done (71.1–74.4◦ C), but the beef was infective when fed raw or cooked rare (37.8–53.3◦ C) Dogs fed the same raw beef after... additional meals per day of undercooked mutton were served, toxoplasmosis cases doubled The investigators concluded that the custom of this hospital to feed undercooked meat was the cause of the high