An Evaluation of the Role of Microbiological Criteria for Foods and Food Ingredients (1985)

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4 Selection of Pathogens as Components of Microbiological Criteria INTRODUCTION Microorganisms as components of microbiological criteria for foods may be grouped into two categories: pathogens, including their harmful toxins, and indicator organisms. Pathogens suitable as components of a criterion are those likely to be found in the food or ingredient which thereby becomes a potential vehicle for its transmission to consumers. Suitable indicator organisms or agents are those whose presence in the food or ingredient indicates: (1) the likelihood that a pathogenks) or harm- ful toxints) of concern also may be present; (2) the likelihood that faulty practices occurred during production, processing, or distribution that may adversely affect the safety and/or the shelf-life of the product; or (3) that the food or ingredient is unsuited for an intended use. Furthermore, ad- equate and practical methods must be available for detection and/or enu- meration of selected pathogens or indicator organisms. Other ancillary factors that relate to the selection of contaminants, as well as other com- ponents of microbiological criteria, are presented elsewhere (see Chapters 2, 3, and 6~. Indicator organisms that may be useful for the purposes indi- cated above are treated in Chapter 5. This chapter deals with the various foodborne pathogens that are of particular concern. Each pathogen is grouped in one of three categories according to the severity of the hazard it may present. The discussion of each organism or group of organisms includes its relative importance, the status of the methodist available for its detection and/or enumeration, and conclusions on suitability of the organism as a component of a micro- biological criterion. 72

SELECTION OF PATHOGENS 73 PATHOGENS An extensive listing of foodborne diseases and the agents that cause them has been prepared by Bryan (19821. Included in this manual are brief but pertinent summaries of the nature of the organism, incubation period and symptoms of the disease, likely sources and reservoirs of the organisms, the foods most likely involved, and the most useful specimens for laboratory examination. Similar listings with emphasis on only the more commonly occurring and important foodborne diseases also are avail- able (Bryan, 1980a; Sanders et al., 19841. Although the list of foodborne diseases of microbial etiology is long, only about 20 are known to be transmitted by foods with a consequence serious and frequent enough to cause concern relative to microbiological criteria (see listing below). As for the others, either proof of their trans- mission by foods is inconclusive or suspect or they are so infrequently foodborne that their involvement is remote. Table 4- 1 presents a summary of reported foodborne disease outbreaks in the United States during the years 1977-1981 (CDC, 1983~. Similar data are available for Japan (Okabe, 1974), the United Kingdom (Vernon, 1977), and Canada (Health and Welfare Canada, 19811. The number of foodborne disease organisms required to produce disease has been investigated to some extent, largely through feeding studies using healthy adult volunteers. A summary of a series of such studies is given in Table 4-2. Except for Shigella dysenteriae, the results indicate that large numbers were necessary to cause illness in the volunteers. However, it would be imprudent to conclude that small dosages are relatively harm- less. Host parasite susceptibility varies, the more susceptible being infants, the aged, and debilitated persons. Studies in connection with investigations of certain Salmonella foodborne outbreaks indicate that relatively few cells of several serotypes caused illness. A summary of these studies in which the number of cells found in the causative vehicles were quantitated is shown in Table 4-3. Compared to those given in Table 4-2, the numbers are surprisingly low. Assuming that a reasonable serving was consumed, e.g., approximately 3 ounces of chocolate, the number causing illness would approximate 0.6-6 cells of Salmonella eastbourne. A 3-ounce serv- ing of cereal would have contained approximately 600-1,200 cells of Salmonella muenchen. It must be recognized that investigation and reporting of foodborne disease outbreaks are grossly incomplete. For example, about 400-500 outbreaks and 10,000-20,000 cases are reported annually in the United States. The actual incidence, however, may be 100 times or more the reported figures (CDC, 1981a; Hauschild and Bryan, 19801. Although

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76 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA TABLE 4-2 Summary of Various Organisms Found to Induce Illness in Volunteer Feeding Studiesa Number Causing Organism Illnessb Reference Campylobacter jejuni lob 5x 1o2 Steele and McDermott, 1978 Robinson, 1981 Clostridium perfringens type A, heat resistant 108-109 Dische and Elek, 1957 type A, heat sensitive 109 Hauschild and Thatcher, 1967 Escherichia cold 106-10~° DuPont et al., 1971; Ferguson and June, 1952; June et al., 1953; Kirby et al., 1950 Salmonella anatum 105- 108 McCullough and Eisele, 195 la,b Salmonella derby 107 McCullough and Eisele, l951c Salmonella bareilly 105- 1 o6 McCullough and Eisele, 1951 c Salmonella meleagridis 1 o6- 107 McCullough and Eisele, l951a,b Salmonella newport 106 McCullough and Eisele, l951c Salmonella pullorum 109-10 'A McCullough and Eisele, 195 id Salmonella typhi 104- 1 O9 Hornick et al., 1970a,b; DuPont et al., 1970 Shigella flexneri lo2-109 Shaughnessy et al., 1946; DuPont et al., 1969, 1972 Shigella dysenteriae 1o'-104 Levine et al., 1973 Streptococcus faecalis 109- 10~° Sedova, 1970 subsp. Iiquefaciens Vibrio cholerae (unbuffered) 1 08- 1 0 ~ ~Hornick et al., 1 97 1 (buffered) 103-104 Hornick et al., 1971 Vibrio parahaemolyticus (buffered) 1oS-7 Sanyal and Sen, 1974 Yersinia enterocolitica 109 Szita et al., 1973 aAdapted from Bryan (1977). bIn some studies no attempt was made to determine lowest levels. In others, such attempts were made for C. perfringens, V. cholerae., S. typhi, S. anatum, S. meleagridis, S. derby, E. coli, S. faecalis subsp. Iiquefaciens, Y. enterocolitica. Adults were subjects for all studies except for E. cold studies in which some nonadults were included. incomplete, the reporting at current levels through the efforts of the Centers for Disease Control (CDC) and other federal and state agencies does help to provide a continuing assessment of trends in etiologic agents and food vehicles. Furthermore, such efforts contribute to (1) identification and  removal of contaminated products from the market, (2) correction of faulty food-handling practices, (3) identification and treatment of human car- riers, and (4) detection of newly emerging agents of foodborne disease. Items (1) and (2) are of particular significance with respect to microbiol- ogical criteria. Many sources of information, those referred to above and others (Bryan, 1972; Riemann and Bryan, 1979; Sours and Smith, 1980), amply justify

SELECTION OF PATHOGENS 77 TABLE 4-3 Number of Various Organisms Found in Foods Involved as Vehicles in Foodborne Outbreaks Vehicle Strain/Serotype Number Reference Ham S. infantis 23,000/g Angelotti et al., 1961 Frozen egg yolk S. typhimurium 0.6/g CDC, 1967 Carmine dye S. cubana 30,000/0.3 g Lang et al., 196i Protein dietary supplement Spice flavored S. minnesota 11/100 g Andrews and Wilson, 1976 Apricot flavored S. minnesota 13/100 g Chocolate S. eastbourne 1.7/100 g Deibela 0.6/100 g 6/100 g 2/100 g 2.5/g 0.2-0.9/g 7-14/g 113/75 g Cereal S. muenchen Frozen dessert S. typhimurium and (Chiffonade) S. braenderup Raw hamburger S. newport 60-2300/100 g Craven et al., 1975 D'Aoust et al., 1975 Silverstolpe et al., 1962 Armstrong et al., 1970 Fontaine et al., 1978 aR. H. Deibel, Department of Bacteriology, University of Wisconsin, Madison, personal com- munication, 1974. the listing of the organisms and/or toxins given below as being of concern as causative agents of foodborne disease in the United States. The agents are grouped according to the seriousness of the hazard they present when found in food. The grouping parallels that of the International Commission on Microbiological Specifications for Foods (see Chapter 4 and Table 7 of ICMSF, 19741. Some rearrangement within categories was necessary as the scope of the current report is confined to the United States. Severe Hazards: Clostridium botulinum, Shigella, Vibrio cholerae, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B. Sal-  monella paratyphi C, Salmonella sendai, Salmonella cholerae-suis, Bru- cella abortus, Brucella melitensis and Brucella suds, Mycobacterium bovis, hepatitis A virus, fish and shellfish toxins, and certain mycotoxins. C. botulinum causes botulism, which frequently results in death. Shiga dysentery, caused by S. dysenteriae, is an example of a disease that, without specific treatment, may result in high mortalities (ICMSF, 19781. While the illness caused by the other three species of Shigella (S. flexneri, S. boydii, S. sonneiJ is usually less severe, considerable variation in se- verity occurs; hence, these species are included. Cholera may be severe and result in death if untreated; however, mild to asymptomatic infections are common. Salmonellae more or less strictly adapted to humans are

78 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA S. typhi, S. paratyphi A, S. paratyphi B. S. paratyphi C, and S. sendai. These species and S. cholerae-suis (primary host, swine) generally pro- duce a systemic syndrome as opposed to the gastroenteritis caused by other Salmonella (NRC, 19691. Brucellosis and tuberculosis, although now rarely foodborne in the United States, result in serious consequences for those affected. The effects of hepatitis A virus infection may range from asymptomatic to acute liver disease resulting in severe debilitation and even death. Paralytic shellfish poisoning is a severe intoxication that may result in respiratory failure and death. Ciguatera fish poisoning results in gastrointestinal and neurological symptoms with disability lasting sev- eral days, several months, or longer. The disease implications of myco- toxins for man are still not clear; however, aflatoxin is a known carcinogen. Moderate Hazards with potentially extensive spread: Salmonella. pathogenic Escherichia cold (PEC), and Streptococcus pyogenes. Moderate Hazards with limited spread: Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, Vibrio parahaemolyticus, Cox- iella burnetii, Yersinia enterocolitica, Campylobacter fetus subsp. jejuni, Trichinella spiralis, and histamine. The agents listed as "moderate" hazards generally cause illnesses milder than those caused by the agents listed as "severe" hazards. Agents with potential for extensive spread are often initially spread by specific foods; however, secondary spread to other foods commonly occurs from envi- ronmental contamination and cross-contamination within processing plants and food preparation areas including homes. The illness dose for these agents may be low. Agents in the lowest risk group (moderate hazards, limited spread) are found in many foods, usually in small numbers. Generally, illness is caused only when ingested foods contain large numbers of the pathogens, e.g., C. perfringens, or have at some time contained large enough numbers to produce sufficient toxin to cause illness, e.g., S. aureus. Outbreaks are usually restricted to consumers of a particular meal or a particular kind of food, e.g., milk in the instance of C. burnetii. Y. enterocolitica and C. fetus subsp. jejuni are included as moderate hazards although the full importance of these species is not clear, nor are the factors governing secondary spread to other foods. Severe Hazards ClostridFium botulinum The importance of the botulism hazard in foods is well known and needs no further elaboration here. Food containing botulinal toxin in any amount

SELECTION OF PATHOGENS 79 is unacceptable. Excellent methods are available for detection of C. botulinum and its toxins (AOAC, 19801. They are invaluable for the examination of foods implicated in botulism outbreaks and for other investigational pur- poses. However, the expertise required in application of the methods and in the interpretation of results precludes their use in most laboratories that routinely analyze food. Sampling and testing for purposes of routine sur- veillance of the botulism hazard in foods is ill advised. The probability that the examination of any reasonable size sample of a low-acid canned food contaminated with C. botulinum would result in~detection of the organism is too low to assure the level of safety necessary. Safety of low- acid canned foods must depend primarily on instrumentation and other process assurance mechanisms to be certain that processing has been adequately accomplished and that container integrity has been maintained (FDA, 19791. Control of the botulism hazard in perishable foods must be based on adherence to food-handling practices that will prevent growth of C. botulinum. Shigella The importance and epidemiology of shigellosis as a foodborne disease has been reviewed by Morris (1984) and Bryan (19791. Briefly, Shigella is host-adapted to humans and higher primates. Shigellosis is an infectious disease transmitted most commonly by close person-to-person contact via the fecal-oral route. Poor personal hygiene, inadequate sanitation, and crowded living conditions are important factors in the spread of this dis- ease. Migrant workers often are victims and outbreaks among younger children and others confined in hospital wards are not uncommon. Pro- cessed foods are rarely involved as vehicles of transmission; however, food may be easily contaminated during preparation by infected food handlers, particularly by convalescent carriers. The infectious dose can be as low as 10 organisms. The mere presence of Shigella in a food that will not be subsequently cooked presents a serious hazard. Detection of Shigella in foods is usually done by an enrichment pro- cedure followed by subculturing to a variety of selective/differential media (Morris, 19841. The procedures are useful for the examination of suspect foods in the investigation of outbreaks and should be applied when in- dicated. The method for their detection in foods is not sensitive and quantitation is rarely done. Because processed foods are rarely involved and analytical procedures are relatively insensitive and complicated, routine sampling and testing for Shigella would not be practical in routine surveillance programs. San- itary handling of food prior to serving, strict adherence to good personal

80 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA hygiene practices, and proper temperature control should be relied upon as effective preventive measures. Vibrato cholerae Cholera is an acute diarrhea! disease caused by V. cholerae. This species includes the strains that cause cholera epidemics (V. cholerae O group 1) and others that are similar to the epidemic strains but have not been associated with epidemic disease; these organisms, now referred to as non- O1 V. cholerae, were formerly referred to as nonagglutinating vibrios (NAGs) or noncholera vibrios (NCVs) (CDC, 1979a). The number of V. cholerae Of and non-O1 infections found in the United States has increased in recent years. Recent outbreaks have been reported from sev- eral states (Blake et al., 1980; CDC, 1979a,b, 19801. Raw oysters and steamed crabs were often epidemiologically associated with infections. V. cholerae O 1 produces an enterotoxin that causes excretion and severe loss of fluid and electrolytes from the body. The mortality rate for severe cases where rapid replacement of fluid and electrolytes is not provided may reach 30-40% (WHO, 19761. Non-O1 V. cholerae infections are characterized by diarrhea, nausea and vomiting, abdominal cramps and fever (Morris et al., 1981), and the symptoms may range from mild to severe. Certain of the non-O1 strains produce a toxin similar to that of Of strains. The pathogenic mechanism for the nontoxigenic strains, how- ever, is not well defined. Detection of V. cholerae in foods (Twedt et al., 1984) requires primary enrichment. Subsequently, subculturing usually is done in selective media. Suspect colonies are confirmed by biochemical and serological procedures and other tests, including a mouse adrenal cell assay for cholera toxin. The method has proven useful in the examination of Moore swabs and gauze filters used in investigations of suspect water areas for presence of V. cholerae (CDC, 1979b). However, it is too insensitive and time-con- suming for routine application to oysters, crabs, and other seafoods in surveillance programs. Further, experience has shown that testing crabs from suspect waters was not an effective and beneficial means of moni- toring such areas (CDC 1979b). Over 2,000 crabs were examined in one such program and V. cholerae was found in none. Thus, microbiological criteria with V. cholerae as the designated contaminant are not indicated. Strict adherence to measures specified under the National Shellfish San- itation Program should be relied upon as the principal means of minimizing the risk of raw oysters, clams, and mussels contaminated with V. cholerae from reaching the consumer. Furthermore, adequate heat treatment of crabmeat before eating and use of good sanitary practices during the

SELECTION OF PATHOGENS 81 handling of crabmeat before and after heat treatment (especially avoidance of cross-contamination between raw and heated product) should prevail. Salmonella With respect to the establishment and application of microbiological criteria for Salmonella in foods, there is little if any present justification to consider the human-adapted species and S. cholerae-suis mentioned above (see "Pathogens, severe hazards") differently from the other Sal- monella. All are considered pathogenic for human beings and methods for their detection and enumeration are equally applicable; therefore, see the discussion of Salmonella below under "Moderate Hazards." Brucella abortus, Brucella melitensis, Brucella suds, and Mycobacter~um basis Brucellosis and tuberculosis in man are serious diseases often resulting in long-term illness and serious complications. Brucellosis is primarily an occupational disease of workers in the meat-processing and livestock in- dustries. However, since the organisms may be shed in milk from infected animals, the disease may be acquired by drinking raw milk, usually from infected cattle and goats, or by eating fresh cheese made from such raw milk. Similarity M. bovis may be shed in milk from infected cattle and goats and be transmitted to human beings through the milk. The organisms, however, do not survive commercial milk pasteurization processes. The methods for detection of Brucella and Mycobacterium in foods are insensitive and lengthy and are unsuited for routine application to milk or other foods. The likelihood of Brucellosis in man being acquired from milk is remote for several reasons. First, availability of pasteurized milk in the United States is virtually universal. Second, the National Cooperative State/Fed- eral Brucellosis Eradication Program in cattle (USDA, 1982), which has been operative over a period of many years, has reduced the incidence of this disease in cattle to an extremely low level. For example, 11 states are now certified as brucellosis-free and all but 4 of the remaining states are nearly brucellosis-free. Finally, the public has been repeatedly warned of the risk of acquiring milkborne diseases by consuming raw milk. Never- theless a few, some perhaps in ignorance, persist in ignoring the hazards involved. Others, overly impressed by often unwarranted emphasis on attributes of so-called "natural foods", continue to seek out raw milk for their fluid milk needs. Consequently, occasional outbreaks of diseases, including Brucellosis and tuberculosis, do occur through the consumption

82 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA of raw milk and products such as unripened cheese prepared from raw milk. As part of the brucellosis eradication program, serological techniques are used in testing the milk of dairy cattle to detect Brucella infections. In this circumstance, a microbiological criterion is applicable, effective, and widely applied. Milkborne tuberculosis in the United States is rare, largely due to the reasons stated above for brucellosis. The State/Federal Tuberculosis Erad- ication Program for cattle (USDA, 1981a) has practically eliminated the disease from milking animals. Milk pasteurization has been an effective measure. At one time, the mandatory time-temperature requirements for milk pasteurization were based on those necessary for the destruction of M. tuberculosis; present requirements are even more stringent. Also, ed- ucational programs warning of the hazards of raw milk consumption have contributed to reducing the exposure to infection. While serological methods are used in testing cattle for tuberculosis infection, neither these tests nor cultural methods are applied routinely to milk available for purchase by the public. However, suspect tuberculosis lesions found at time of carcass inspection at slaughter are routinely cul- tured for Mycobacterium. This is an important part of the state/federal eradication program. In this circumstance, a microbiological criterion is appropriate, effective, and routinely applied. viruses Viruses as agents of foodborne disease have been reviewed extensively by Cliver (1979) and Cliver et al. (19841. Hepatitis A, the cause of in- fectious hepatitis, is a virus frequently transmitted to humans by food. The consequences of infection range from asymptomatic Unapparent in- fection) to severe liver involvement and damage, and even death. Infected persons shed large numbers of the virus in the feces several days before onset of symptoms and during the acute stage of infection. Raw or in- adequately cooked shellfish that have been harvested from sewage-polluted waters have been the most frequent food vehicles for transmission. Con- tamination of foods may occur during handling and processing by infected persons during the incubation period of the disease. Viral diarrhea may be transmitted through foods. The agents most com- monly involved resemble the "Norwalk" virus. The severity of the disease however is less than for hepatitis A. Poliomyelitis also may be foodborne. Viruses are detected on the basis of their infectivity; therefore, a sus- ceptible living host must be available for testing. Cell cultures are most frequently used. However, some viruses are difficult to isolate and others

SELECTION OF PATHOGENS 83 have not been successfully grown in the laboratory. Only recently has hepatitis A been grown in cell culture. Poliomyelitis virus can be readily isolated and cultured. Microbiological criteria for viruses in foods are not practical at present because of the lack of readily applied analytical methods. Control measures for hepatitis A must depend primarily upon surveillance of shellfish grow- ing waters (see Chapter 5 on fecal coliforms). Prevention of contamination of other foods, including shellfish, with hepatitis A and the diarrhea! viruses must depend upon the application of personal hygiene and sanitary handling practices in processing plants and food preparation and serving areas. Poliomyelitis is effectively controlled by universal vaccination. Fish and Shellfish Toxins Poisonings through eating toxic fish and shellfish are significant causes of human illness. Outbreaks are due primarily to two types of poisoning: ciguatera poisoning and paralytic shellfish poisoning. Ciguatera. This is a type of human poisoning caused by eating fish containing ciguatoxin and perhaps other closely related toxins. The toxinfs) istare) synthesized by the dinoflagellate Gambierdiscus toxicus and pos- sibly by certain other dinoflagellate species (Bagnis et al., 1980~. Nu- merous species of fish may feed on these organisms and thereby accumulate the toxints) in their tissues. Toxicity may be magnified by transfer through the fish food chain, e.g., large carnivores tend to be more toxic than herbivores and small carnivores. Between 1977 and 1981 ciguatera fish poisoning was the most common foodborne disease in the United States associated with eating fish (Bryan, 1980b; CDC, 19831. Much confusion has been associated with ciguatoxin poisoning. Withers (1982) has presented a comprehensive review of this problem. Until re- cently, it was thought that eating fish containing the toxints) produced by the dinoflagellate Ptychodicus breve (formerly named Gymnodinium breve) was the cause of ciguatoxin poisoning of humans. Extensive fish kills in Gulf coastal waters are due to ingestion of this organism by fish. The organism is often present in massive numbers in the "red tides" that occur sporadically in Gulf coastal waters. It has recently become apparent that the toxinfs) of Ptychodicus breve istare) not the cause of ciguatoxin poi- soning in humans. Rather it is the toxinfs) of G. toxicus that now appear to be the principal cause of the fish poisoning in humans. The factors that trigger the buildup of toxic concentrations of the toxinfs) of G. toxicus and possibly those of other dinoflagellate species are not clear. Further, the toxins, other than ciguatoxin, are not well defined and assay methods for them are neither precise nor specific. Because of the

84 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA lack of sufficient knowledge in these areas there are no federal or state surveillance programs for preventing the occurrence of ciguatoxin poi- soning. At present, there is no basis for establishment and application of a microbiological criterion. However, research is under way to determine the conditions that lead to the occurrence of toxic fish and the nature of the toxinks) involved, and to develop methods for quantitation of the toxins. For further details see the reviews by Withers (1982) and DeSilva and Poll (19821. Paralytic Shellfish Poisoning. Paralytic shellfish poisoning (PSP) is one of the most toxic forms of food poisoning. Certain species of dino- flagellates, most notably Gonyaulax catenella and Gonyaulax tamarensis, produce saxitoxin, which is known to cause PSP. However, recent studies (Oshima et al., 1977; Shimizu, 1979) have shown that toxic shellfish may contain multiple toxins and that saxitoxin represents only a part of the total toxicity. These newly discovered toxins are related to saxitoxin and their pharmacological action seems to be similar to that of saxitoxin. Shellfish ingest the dinoflagellates and concentrate the toxins in their tissues. Ingestion of toxic shellfish of sufficient toxicity causes acute toxicity in humans. Quantitation of saxitoxin and related toxins is done by mouse bioassay (APHA, 19701. There are medical records of over 1,650 cases of PSP worldwide that have resulted in at least 300 fatalities (Dale and Yentsch, 19781. Out- breaks, although infrequent, occur sporadically along the Atlantic and Pacific coasts of the United States (CDC, 19831. Shellfish involved most frequently are mussels, clams, soft-shelled clams, butter clams, and oc- casionally, scallops. Routine sampling and testing for saxitoxins in shellfish obtained at wholesale or retail markets is not practiced, nor is it necessary. However, a microbiological criterion for PSP is applied, as state authorities regularly assay representative samples of shellfish from growing areas. If toxin content reaches 80 ,ug per 100 g of edible portion of raw shellfish meat, the area is closed to harvesting of the species involved. These actions and other preventive measures are undertaken in accordance with the National Shellfish Sanitation Program (NSSP), a federal/state/ industry cooperative program described in an operations manual (USDHEW, 1965~. Although cases of PSP continue to occur, undoubtedly the incidence would be much higher in the absence of this program. Mycotoxins A brief and pertinent review of the problem of foodborne mycotoxicosis was prepared by Stoloff (19841. There is serious concern about the pos

SELECTION OF PATHOGENS 85 sibility that cancer or delayed organ damage can result from repeated ingestion of subacute levels of mycotoxins. The most intensive effort to determine toxicologic significance has been concentrated on the aflatoxins because of their known widespread natural occurrence in foods and feeds. Yet after two decades of intensive research, the implication of aflatoxins for man is still not clear. Accordingly, control efforts are based more on prudence than on demonstrated danger. Other mycotoxins found in foods or feeds that have prompted toxico- logical studies are ochratoxin, patulin, penicillic acid, and zearalenone. Results of such studies provide no cause for concern about the incidence and levels of these toxins that have been encountered in foods in the United States. Mold contamination of raw farm commodities is not completely pre- ventable. Nevertheless, measures to reduce contamination and subsequent growth are effective in reducing the levels of mycotoxins likely to be found in such commodities and subsequently in processed products. Chemical methods are used for the detection and quantitation of my- cotoxins. The development and validation of methods is achieved through a Joint Mycotoxin Committee representing the Association of Official Analytical Chemists (AOAC), the American Association of Cereal Chem- ists, the American Oil Chemists Society, and the International Union of Pure and Applied Chemistry. All methods validated to date under the aegis of the Joint Mycotoxin Committee have been adopted by AOAC (AOAC, 1980). At present, FDA has set tolerance levels only for aflatoxins (20 ppb, aflatoxin Be wherever encountered; and 0.05 ppb, aflatoxin Ma in milk). Susceptible foods and feeds are monitored regularly. Thus, in this instance, a microbiological criterion is applicable and widely applied. Moderate Hazards, Potentially Extensive Spread Salmonella Salmonella continues to rank among the three most frequent causative agents of foodborne disease in the United States. As shown in Table 4- 1, Salmonella was the most frequently reported etiologic agent in the 5- year period 1977-1981. The problem of foodborne salmonellosis in the United States was ex- tensively evaluated by the Salmonella Committee of the National Research Council (NRC, 19691. The committee was primarily concerned with hu- man health; thus, the review was organized around two viewpoints: (1) the disease: its importance, causal organism, and means by which humans become infected; and (2) how humans can be protected. Means of pro- tection included minimizing exposure by reducing the incidence of animal

86 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA salmonellosis, avoiding contamination of raw and processed foods, and bactericidal treatment and prevention of growth; surveillance, investiga- tion, and control by industry and regulatory agencies; education; and research. Fifty-five recommendations and suggestions for implementation were made by the committee. Because Salmonella may be found in many foods (some more sensitive to contamination than others), two of the recommendations are of particular significance relative to the establish- ment and application of microbiological criteria. They are: (1) development of a realistic assessment of the degree of hazards imposed by various foods and drugs; and (2) development of sampling plans commensurate with the likely degree of hazard inherent in the food and based on the probability that Salmonella are present at less than a statistically defined level. The committee proposed such a classification and sampling system. Sampling plans for raw meat, poultry, and fish were not included. The plans sug- gested by the committee were not designed to replace testing or other routine surveillance operations of food manufacturers or government agen- cies. They were intended for use in arriving at a final decision on whether to accept or reject a particular lot in question. In an interesting departure from this principle, FDA has taken the position that the sampling plan, including the acceptance criterion, is applicable to any lot of product tested in connection with any of FDA's surveillance or compliance programs and is not restricted to use with questionable lots. The current state of analytical methods is no barrier to the designation of Salmonella in microbiological criteria. An AOAC official method exists and it is used successfully by many. Also, because of coordination of the efforts of AOAC, the International Standards Organization (ISO), and the International Dairy Federation (IDF), the methods issued by each for the detection of Salmonella are essentially the same. In spite of implementation of some of the National Research Council committee's recommendations and other ancillary control programs, there has been little evidence of change in the magnitude of the Salmonella problem over the last 10-15 years (Gangarosa, 1978; Silliker, 19801. Nevertheless, containment of the problem within its present dimension has been a significant accomplishment; for it to remain so or be improved will continue to require major effort. From the above, it is evident that microbiological criteria in which Salmonella is the designated contaminant are appropriate for inclusion with other control measures to reduce the hazard of Salmonella in foods, provided that sampling plans are based on a realistic assessment of the hazard the candidate food may present. The Salmonella committee pro- vided principles and guidance for such assessment that currently are fully applicable (NRC, 19691.

SELECTION OF PATHOGENS Pathogenic Escherichia cold (PEC) 87 Certain biotypes of E. cold cause gastrointestinal illness in man and in several other animals. The biotypes include toxigenic and invasive bio- types and others that appear, on the basis of tests for these attributes, to be neither toxigenic nor invasive. While the importance of E. cold in diarrhea! disease is well established, outbreaks of E. cold foodborne illness in the United States are rare, only three outbreaks having been reported to CDC. One, due to imported soft cheese, occurred in several incidents during late 1971 and early 1972 (Marier et al., 19731. The second, in which the vehicle was beef, occurred in 1978 (CDC, 1981b3. Recently, sporadic cases of hemorrhagic colitis due to E. cold 0157:H7 were reported in the United States (CDC, 1982b; Riley et al., 1983; Wells et al., 1983) and two outbreaks were reported in Canada (Health and Welfare Canada, 1983a,b). Inadequately cooked hamburger was the vehicle in certain of these cases. It is quite possible that the incidence of PEC outbreaks is underestimated for several reasons, primarily because of the complexities involved in recovery and recognition of PEC from foods. Methods for recovery of E. cold and pathogenic biotypes of E. cold are available (Mehlman, 19841. Briefly, the standard enrichment and plating procedures for enumeration of E. cold that are so routinely applied in food laboratories neither quantitatively recover pathogenic biotypes nor differ- entiate them from other E. colt. Recognition of pathogenic E. cold requires a regimen of serological, pathological (requiring laboratory animals), and biochemical-physiological tests that are costly and require expertise not common to most laboratories that routinely examine foods. Furthermore, these tests, in lieu of controlled primate feeding studies, while highly indicative of human pathogenicity, are not unequivocal. However, the current interest in this foodborne illness might well result in more rapid and specific techniques for recovery and identification of PEC types. Microbiological criteria for E. cold are widely applied (see Chapter 51. However, in view of the lack of procedures that can be routinely applied for detection and enumeration of pathogenic biotypes and the likely low incidence of foodborne PEC illness, establishment of microbiological cri- teria specifically for these organisms in foods is presently impractical. Streptococcus pyogenes (Lancef~eld Group A, beta hemolytic) Prior to widespread acceptance and availability of pasteurized milk, raw milk was responsible for most of the many outbreaks of scarlet fever and septic sore throat caused by S. pyogenes. Cross-infection of cows via

88 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA milking personnel often resulted in mastitis and subsequent shedding of S. pyogenes in the milk from infected udders. Infected food handlers, including carriers, may contaminate foods when preparing them for serv- ing. However, S. pyogenes infection is currently infrequently foodborne, and only a few outbreaks were reported during the 5-year period 1977- 1981 (see Table 4-11. Methods that are sufficiently selective and quantitative for routine ex- amination of foods are not available. Accordingly, lack of an adequate method and the infrequency of S. pyogenes foodborne outbreaks preclude establishment and application of microbiological criteria as a control mea- sure. Pasteurization of milk, adequate heating of other sensitive foods and sanitary food handling practices are effective measures of preventing food- borne S. pyogenes infections. Moderate Hazards, Limited Spread Staphylococcus aureus Staphylococcal food poisoning outbreaks usually are the second most frequently reported causes of foodborne illness in the United States (see Table 4-11. Illness is caused by one or more of several heat-stable enter- otoxins produced by certain strains of this species. Bergdoll (1979) pro- vided a comprehensive review of the organism, its nature, the disease it causes, its epidemiology, applicable methodology, and preventive mea- sures for control of the disease. The number of S. aureus required to produce detectable amounts of enterotoxin exceeds one million per gram. The presence of enterotoxin in food, rather than the organism per se, is the principal concern. Large numbers of organisms without associated toxin may be tolerated via inges- tion. Methods for detection and enumeration of S. aureus are adequate and practical for purposes of microbiological criteria (Tatini et al., 19841. Certain of these methods provide for resuscitation of injured cells that otherwise would not be detected. Application of microbiological criteria with S. aureus as the designated contaminant is feasible either (1) to indicate the possible presence of detectable enterotoxin in a susceptible food, or (2) to indicate faulty san- itary or production practices in the preparation of food. Small numbers of S. aureus are to be expected in foods that have been exposed to or handled by food handlers. Small numbers do not, however, assure safety

SELECTION OF PATHOGENS 89 because the organism can grow and produce enterotoxin and then die off during storage or be killed during subsequent processing (usually heat) of the food; preformed toxin, however, usually will remain in the food. For application of the thermonuclease test to detect possible presence of staph- ylococcal enterotoxin in food, see Chapter 5 and Chapter 9, Part A. Clostridium perfringens C. perfringens enteritis is consistently among the three most frequently reported causes of foodborne illnesses that occur in the United States. Although the presence of small numbers of C. perfringens in many foods is unavoidable, large numbers may be indicative of mishandling, including temperature abuse. Several hundred thousand or more per gram of food are usually found in foods involved in outbreaks. An enterotoxin released in the intestine by sporulating cells causes the illness. Hobbs (1979) has provided a comprehensive review of this organism as a foodborne agent. An adequate and official AOAC method is available for detection and enumeration of C. perfringens in foods (Harmon and Duncan, 19841. However, several factors limit its application in routine surveillance of foods. One of these is that vegetative cells of C. perfringens lose viability in foods that are frozen or held under refrigeration such as may occur during shipment of samples to laboratories. The presence of C. perfringens in low numbers in many foods usually is unrelated to faulty sanitary practices, and only relatively large numbers in a food are cause for concern. Therefore, the use of C. perfringens microbiological criteria in routine surveillance programs would not con- tribute significantly to prevention of outbreaks of this foodborne illness. Prompt and proper refrigeration and adequate reheating of "leftovers" of sensitive foods should be relied upon as control measures. It is emphasized, however, that foods that are suspect vehicles in gastroenteritis outbreaks and in which C. perfringens is likely to be found should be examined quantitatively for this organism. Analysis of foods for this purpose is well within the capabilities of most microbiological laboratories. Also, such foods, especially when their temperature history is unknown, should be tested for the presence of the alpha toxin of C. perfringens (Harmon and Duncan, 19841. A detectable quantity of alpha toxin is produced when cell numbers reach about 106 C. perfringens cells per gram, and the titer or amount of alpha toxin increases as the numbers increase. Since the alpha toxin is little affected by freezing or refrigerated storage of foods, its presence in any detectable amount indicates that the C. perfringens population probably was sufficient to cause illness.

90 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA Bacillus cereus Only a few confirmed outbreaks and cases of B. cereus foodborne illness have been reported in the United States in recent years (Table 4-11. The lack of greater incidence of reported outbreaks may be due to several factors. The organism is so widely distributed in many environments and foods that it is commonly viewed as relatively harmless under most cir- cumstances. Until recently, methods for its detection and enumeration were somewhat lacking in specificity. As a consequence, the organism was not sought regularly in analysis of foods involved in foodborne out- breaks. In addition, two syndromes of B. cereus illness have been rec- ognized; one closely resembles that caused by S. aureus and the other resembles that caused by C. perfringens. Thus, failure to look for B. cereus in suspect foods and the similarity of its disease syndromes to those of S. aureus and C. perfringens may in large measure account for the infrequent recognition of B. cereus foodborne disease. For a detailed discussion of the nature and epidemiology of B. cereus gastroenteritis, and in particular its occurrence in the United Kingdom, reference is made to the review by Gilbert (1979~. Currently, the greatest need for sampling and testing for B. cereus is in application to suspect foods associated with disease outbreaks. If such analyses are routinely done, a more accurate estimate of the incidence of B. cereus in foodborne diseases soon would be obtained, and the more common food vehicles for its transmission would be identified. At present, B. cereus can be enumerated in foods by a plating procedure (Harmon and Goepfert, 19841. However, considerable difficulty may be encountered in obtaining quantitative counts of B. cereus in foods con- taining large numbers of competitive organisms. Colonies of such organ- isms may overgrow the plates, making B. cereus colonies difficult to discern or masking them completely. This overgrowth and other factors largely restrict use of the method to foods that do not contain a large competitive flora. A presumptive plate count can be obtained in 24 hours. If necessary, uncomplicated confirmatory tests can be applied to selected colonies. Generally, presumptive counts would be sufficient for use in surveys to determine population levels likely to be encountered in foods. Similarly, presumptive counts in foods suspect as causes of outbreaks are useful to determine whether or not B. cereus was the likely causative agent. As B. cereus is found in low numbers in many foods, it is questionable that application of B. cereus microbiological criteria for purposes of rou- tine surveillance of foods would be worthwhile; however, for those foods such as cooked rice products that become more clearly identified as fre

SELECTION OF PATHOGENS 91 quent vehicles in B. cereus food poisoning outbreaks, a microbiological criterion may be useful. Vibrato parahaemolytnicus The first reported outbreak outside Japan of foodborne disease caused by this species occurred in Maryland in 1971. Since then 11 confirmed and 8 suspected outbreaks have occurred through 1978 (Bryan, 1980b). In Japan raw fish is the principle vehicle for transmission; in contrast, cooked seafoods (crustaceans) were vehicles in the United States, except for one outbreak in which raw oysters were suspect. It appears that foodborne disease outbreaks caused by V. parah~zemolyticus do not occur frequently in the United States. Also, the method for enu- meration of V. parahaemolyticus is time-consuming and the method for identifying pathogenic strains (Twedt et al., 1984) is complicated. There- fore, microbiological criteria would not be justified in the United States. However, various methods have been helpful in investigations of outbreaks and surveys of susceptible foods. All confirmed outbreaks in the United States resulted from gross cross-contamination of cooked product from contaminated raw product or seawater coupled with subsequent temper- ature abuse. Therefore, sanitary handling, especially of the cooked prod- uct, and refrigerated storage are the most productive preventive measures. Other Vibrio species such as V. alginolyticus, V. vulnificus, v. fuvialis, V. mimicus, V. metschnikovii, and V. hollisae are associated with human disease (Hickman et al., 19821. More needs to be learned about their ecology, epidemiology, and role in human disease before they can be firmly associated with causes of foodborne illness. Coxielia burnetii C. burnetii causes the rickettsial disease Q-fever in man. In infected cattle, sheep, and goats, the organism may be found in large numbers in placental tissues and fluids and feces and also may be shed in the milk. Q-fever infection in man is usually acquired via the respiratory route as a result of casual or occupational exposure to infected animals and contaminated premises, and can also be acquired through consumption of raw milk. Outbreaks of Q-fever acquired through milk are rare, primarily due to the widespread use of pasteurized milk. No cases were reported to CDC during 1977-1981 (Table 4-11. Present pasteurization time-temper- ature requirements (USPHS/FDA, 1978) are based on those necessary for the destruction of C. burnetii (Enright, 1961; Enright et al., 19561. C. burnetii is usually detected in milk by either guinea pig inoculation

92 EVALUATION OF THE ROLE OF MlCROBIOLOGlCAL CRITERIA or agglutination tests (Ormsbee, 19801. The former test requires several weeks before results are obtained; the latter is a simple and rapid procedure that is useful in surveys to determine the infection status of dairy animals. Routine application of microbiological criteria for C. burnetii in milk (via serological tests) would not be a practical measure for preventing Q- fever in man in view of the effectiveness, ready availability, and wide- spread use of pasteurized milk. Histamine Poisoning This is the second most frequently-reported fishborne illness in the United States (CDC, 1981a). Illness results from eating fish, usually of the Scombridae family, which have become toxic after having undergone some microbial decomposition, although overt signs of spoilage may not be evident. Symptoms of scombroid poisoning include flushing, rapid pulse, headache, dizziness, nausea, and diarrhea. With the exception of cheese (see below), foods other than fish are rarely involved. The occurrence, mechanism of formation, and catabolism of biologi- cally active amines in foods were reviewed by Rice et al. (1976), Voigt and Eitenmiller (1978), and Voigt et al. (19741. Histamine, formed by decarboxylation of histidine, is the apparent principal toxic agent. Scom- broid fish normally have a high concentration of the amino acid histidine in their tissues. Many species of bacteria have been associated with his- tamine formation in various scombroid fish. These include Proteus mor- ganii, Klebsiella pneumonias, C. perfringens, certain coliforms, and others. Toxic concentrations of histamine and tyramine occasionally occur in certain cheeses (Edwards and Sandine, 19811. Tyramine is the decarbox- ylation product of tyrosine, which is common in aged cheese including Cheddar, Swiss (Emmenthaler), Gruyere, Roquefort, and certain others. Tyramine can cause critical increases in blood pressure, especially in persons receiving monoamine oxidase inhibiting (MAGI) drugs. Bacterial isolates from cheese that produce amines include certain coliforms, en- terococci, and lactobacilli. An assay method for histamine is available (AOAC, 1980) and is ap- plicable for routine use in a well-equipped and -staffed laboratory. It is common commercial practice within the fisheries industry to organolept- ically inspect scombroid-type fish at point of receipt and in-plant at time of evisceration for off-odors and other evidence of mishandling that could result in histamine formation; also, routine examination for histamine is common even though decomposition is not evident. Scombroid fish, usu- ally tuna, offered at import frequently are inspected for evidence of de- terioration and are analyzed for histamine content by FDA. Canned albacore,

SELECTION OF PATHOGENS 93 skipjack, and yellow fin tuna with histamine levels of 20 mg or more per 100 g are subject to regulatory action by FDA as being suspect of dete- rioration. The agency will consider regulatory action against any tuna found to contain between 10 and 20 mg of histamine per 100 g when a second indication of decomposition is present (FDA, 1982a). Furthermore, FDA has established on an interim basis a level of 50 mg of histamine per 100 g of tuna as the level of histamine in tuna that it considers a health hazard. This value may be changed after evaluation of additional data (FDA, 1982b). Accordingly, a microbiological criterion with histamine as the designated contaminant is useful when applied as indicated above to prevent deteriorated fish as well as toxic fish from reaching the processor or consumer. The histamine problem in cheese is not of sufficient incidence to justify routine testing for histamine or tyramine. Yersin~a enterocolitica The most common symptoms of Y. enterocolitica infections are gastro- enteritis and terminal ileitis. This organism has been isolated from a wide variety of foods and animals and therefore is of concern as a cause of foodborne illness. Reviews by Lee (1977), Stern and Pierson (1979), and Swaminathan et al. (1982) provide details on Y. enterocolitica as a food- borne pathogen. Current methods for its recovery from food and tests for pathogenicity are provided by Feeley and Schiemann (19841. Currently, members of the species Y. enterocolitica include biochem- ically and serologically diverse strains. In addition to the typical strains of Y. enterocolitica, there are closely related species often referred to as Y. enterocolitica-like bacteria. The species also includes nonpathogenic strains. Recently, it has been proposed to separate the group into four species, i.e., Y. enterocolitica, Y. intermedia, Y. frederiksenii, and Y. kristensenii (Bercovier et al., 1 980; Brenner, 1 9804. Although frequently found in swine and other food animals and often reported as causing illness in humans in many countries, only three doc- umented outbreaks of foodborne illness due to Y. enterocolitica have been reported in the United States. One was due to recontaminated chocolate milk (Black et al., 1978), another to tofu (soybean curd packed in water) (Aulisio et al., 1983), and the third to recontaminated pasteurized milk (CDC, 1982a). Methods for recovery of Y. enterocolitica from foods have been im- proved in recent years; however, no single method is suitable for recovery of all types of this species from various foods. Since not all strains are pathogenic, isolates must be tested for pathogenicity. Primary and sec- ondary or selective enrichment procedures, followed by determination of

94 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA biochemical and serological characteristics of cultural isolates, are re- quired. Recently, however, a simple method based on reaction of Y. enterocolitica with Congo red has been proposed to differentiate path- ogenic from nonpathogenic strains (Prpic et al., 1983, 19841. If confirmed, this finding will fulfill an important need. In view of the limited occurrence of known foodborne outbreaks of Y. enterocolitica infections and the complexities of recovery methods, the application of microbiological criteria would currently be of little value. Methods, however, are adequate and their use should be encouraged for investigational purposes whenever foodborne outbreaks occur and in sur- veys to determine the incidence of potential pathogenic strains in suspect foods. Campylobacter The nature and significance of Campylobacter as a cause of illness in man were presented in reviews by Doyle (1981) and Bokkenheuser and Mosenthal (19811. The two species pathogenic for humans are C. fetus subsp. jejune and C. fetus subsp. intestinalis. Infections with the sub- species intestinalis rarely are foodborne and have been almost entirely limited to compromised patients such as those afflicted with cirrhosis, diabetes, or cancer. On the other hand, C. fetus subsp. jejuni is recognized as a common cause of gastroenteritis in humans. It is commonly found as a commensal or pathogen in cattle, sheep, fowl, swine, and rodents. In spite of the widespread animal reservoir, few foodborne outbreaks of Campylobacter enterocolitis have been reported in the United States. In those few, raw milk and perhaps undercooked poultry were the principal vehicles of transmission (Bryan, 1983; Park et al., 19841. C. fetus subsp. jejuni has been inappropriately termed thermophilic, as it is neither thermophilic nor heat resistant. However, an outstanding characteristic of C. fetus subsp. jejuni is its growth at 42-43°C (107.6- 109.4°F) with poorer growth at 37°C (98.6°F) and no growth at 25°C (77°F). Also, the organism is microaerophilic and requires an atmosphere of reduced oxygen for growth. Because of its sensitivity to air and the relatively high temperature required for growth, growth of C. fetus subsp. jejuni in foods would be unlikely under ordinary conditions of food han . . ~Taxonomic uncertainty existed over the nomenclature for members of this species. In the eighth edition of "Bergey's Manual" (Buchanan and Gibbons, 1974), they are classified as a single species, C. fetus subsp. jejuni. In the classification by Veron and Chatelain (1973) and in the Approved Lists of Bacterial Names of the International Committee on Systematic Bac- teriology, they are divided into the two species C. jejuni and C. coli.

SELECTION OF PATHOGENS 95 cling. Until recently, C. fetus subsp. jejuni was not usually sought in foods suspect in gastroenteritis outbreaks. Also, while the animal reser- voirs are well known, little is known of the incidence of this organism in food supplies in general. Methods for detecting C. fetus subsp. jejuni in foods are available (Park et al., 1984~. The following conditions are required for accurate analysis: (1) large sample size, (2) suitable microaerophilic condition, (3) selective broth enrichment, (4) filtration to separate the small filterable campylo- bacters from other nonfilterable organisms in the enrichment cultures, and (5) selective plating agar. Routine analysis of foods for the presence of C. fetus subsp. jejuni at the present is not practical because procedures for its recovery from foods are in a state of rapid development. While published data on the incidence of foodborne Campylobacter enteritis in the United States are limited, unofficial information appears to indicate that campylobacteriosis is more widespread. Sanitary handling, proper cooking, and prevention of tem- perature abuse of sensitive foods and avoidance of consumption of raw milk would be far more effective control measures. While microbiological criteria would not be applicable, surveys to ascertain the incidence of this organism in the general food supply should be encouraged and investi- gations of foodborne gastroenteritis outbreaks should include examination of suspect food for the presence of C. fetus subsp. jejuni. Tr~chinella spiralis This nematode, which infects many species of animals, causes a fre- quently severe parasitic disease, trichinellosis, in humans. The most im- portant source of human infection is inadequately cooked pork. Human infection occurs only when the meat of host animals is eaten raw or in an undercooked state. Several outbreaks of trichinellosis are reported an- nually in the United States. Forty-four outbreaks were reported from 1977 through 1981 (see Table 4-11. For a brief summary of the sources of the parasite and the nature and consequences of infection see Healy and Jur- anek (19791. Symptoms of trichinellosis in humans that occur at time of initial in- vasion include vomiting, diarrhea, and fever. Later, during the phase of parasite distribution to various tissues, joint manifestations, edema, and even myocarditis, encephalitis, and neuritis may occur. The larvae of T. spiralis (trichinellae) in meat can be detected by several methods, including microscopic examination of fresh or digested meat and by enzyme-linked immunosorbent assay (ELISA). While larvae de- tection methods are routinely used in many countries for examination of

96 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA swine after slaughter, they are not used in the United States as a means of preventing infected meat from reaching the consumer. Rather, the processing procedures specified in USDA regulations for destroying tri- chinellae in infected meat are emphasized by regulatory agencies and others. These procedures thorough cooking of pork (to at least 58°C [137°Fl), proper meat curing conditions, and specified freezing conditions (USDA, 1973, 1981b) are considered more effective in preventing hu- man infection as well- as less expensive than is routine examination of meat by larvae detection methods. REFERENCES Andrews, W. H., and C. R. Wilson 1976 Salmonella contamination in a protein dietary supplement. Pp. 219-220 in FDA Byliner No. 5. Washington, D.C.: Food and Drug Administration. Angelotti, R., G. C. Bailey, M. J. Foter, and K. H. Lewis 1961 Salmonella infantis isolated from ham in food poisoning incident. Publie Health Reports 76:771-776. AOAC (Association of Offieal Analytical Chemists) 1980 Official Methods of Analysis. 13th Ed. Washington, D.C.: AOAC. APHA (American Publie Health Association) 1970 Recommended Procedures for the Examination of Sea Water and Shellfish. 4th Ed. Washington, D.C.: APHA. Armstrong, R. W., T. Fodor, G. T. Curlin, A. B. Cohen, G. K. Morris, W. T. Martin, and J. Feldman 1970 Epidemic Salmonella gastroenteritis due to contaminated imitation ice cream. Am. J. Epidemiol. 91:300-307. Aulisio, C.C.G., J. T. Stanfield, S. W. Weagant, and W. E. Hill 1983 Yersiniosis associated with tofu consumption. Serological, biochemical and patho- genieity studies of Yersinia enterocolitica isolates. J. Food Prot. 46:226-230, 234. Bagnis, R., S. Chanteau, E. Chungue, J. M. Hurtel, T. Yasumoto, and A. Inoue 1980 Origins of ciguatera fish poisoning: a new dinoflagellate, Gambierdiscus toxicus Adaehi and Fukuyo, definitely involved as a causal agent. Toxieon 18:199-208. Bereovier, H., D. J. Brenner, J. Ursing, A. G. Steigerwalt, G. R. Fanning, J. M. Alonzo, G. P. Carter, and H. H. Mollaret 1980 Characterization of Yersinia enterocolitica sensu stricto. Curr. Microbiol. 4:201-206. Bergdoll, M. S. 1979 Staphylococcal intoxications. Pp. 443-494 in Food-borne Infections and Intoxica- tions. 2nd Ed. H. Riemann and F. L. Bryan, eds. New York: Academic Press. Black, R. E., R. J. Jackson, T. Tsai, M. Medevesky, M. Shayegani, J. C. Feeley, K.J.E. McLeod, and A. W. Wakelee 1978 Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. N. Engl. J. Med. 298(2):76-79. Blake, P. A., D. T. Allegra, J. D. Snyder, T. J. Barrett, L. McFarland, C. T. Caraway, J. C. Feeley, J. P. Craig, J. V. Lee, N. D. Puhr, and R. A. Feldman 1980 Cholera a possible endemic focus in the United States. N. Engl. J. Med. 302:305-  309. Bokkenheuser, V. D., and A. C. Mosenthal 1981 Campylobacteriosis: A foodborne disease. J. Food Safety 3:127-143.

SELECTION OF PATHOGENS 97 Brenner, D. J. 1980 Classification of Yersinia enterocolitica. In Yersinia enterocolitica. E. J. Bottone, ed. Boca Raton, Fla.: CRC Press. Bryan, F. L. 1972 Emerging foodborne diseases. I. Their surveillance and epidemiology. J. Milk Food Technol. 35:618-625. 1977 Diseases transmitted by foods contaminated by wastewater. J. Food Prot. 40:45-56. 1979 Infections and intoxications caused by other bacteria. Pp. 211-297 in Food-borne Infections and Intoxications. 2nd Ed. H. Riemann and F. L. Bryan, eds. New York: Academic Press. 1980a Procedures to use during outbreaks of foodborne disease. Pp. 40-51 in Manual of Clinical Microbiology. 3rd Ed. E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant, eds. Washington, D.C.: American Society for Microbiology. 1980b Epidemiology of foodborne diseases transmitted by fish, shellfish and marine crus- taceans in the United States, 1970-1978. J. Food Prot. 43:859-876. 1982 Diseases transmitted by foods A classification and summary. 2nd Ed. Atlanta: Centers for Disease Control. 1983 Epidemiology of milk-borne diseases. J. Food Prot. 46:637-649. Buchanan, R. E., and N. E. Gibbons 1974 Bergey's Manual of Determinative Bacteriology. 8th Ed. Baltimore, Md.: Williams and Wilkins. CDC (Center for Disease Control) 1967 Salmonella Surveillance Report No. 61 (and supplement dated Oct. 23, 1967). Atlanta: Center for Disease Control. 1979a Non-O1 Vibrio cholerae infections Florida. Morb. Mort. Weekly Rpt. 28:571-572, 577. 1979b Vibrio cholerae O-group 1 infections in Louisiana, 1978. EPI-78-102-2. Atlanta: Center for Disease Control. CDC (Centers for Disease Control) 1980 Cholera Florida. Morb. Mort. Weekly Rpt. 29:601. 1981a Foodborne disease outbreaks. Annual Summary 1979. Atlanta: Centers for Disease Control. 1981b Foodborne disease surveillance. Annual Summary 1978 (revised). Reissued February 1981. Atlanta: Centers for Disease Control. 1982a Multi-state outbreak of yersiniosis. Morb. Mort. Weekly Rpt. 31:505-506. 1982b Isolation of E. cold 0157:H7 from sporadic cases of hemorrhagic colitis United States. Morb. Mort. Weekly Rpt. 31:580, 585. 1983 Foodborne disease outbreaks. Annual Summary 1981. Atlanta: Centers for Disease Control. Cliver, D. O 1979 Viral Infections. Pp. 299-342 in Food-Borne Infections and Intoxications, 2nd Ed., H. Riemann and F. L. Bryan, eds. New York: Academic Press. Cliver, D. O., R. D. Ellender, and M. D. Sobsey 1984 Foodborne Viruses. In Compendium of Methods for the Microbiological Examination of Foods. 2nd Ed. M. L. Speck, ed. Washington, D.C.: American Public Health Association.  Craven, P. C., W. B. Baine, D. C. Mackel, W. H. Barker, E. J. Gangarosa, M. Goldfield, H. Rosenfeld, R. Altman, G. Lachapelle, J. W. Davies, and R. C. Swanson 1975 International outbreak of Salmonella eastbourne infection traced to contaminated chocolate. Lancet 1:788-793, April 5. Dale, B., and C. M. Yentsch 1978 Red tide and paralytic shellfish poisoning. Oceans 21:41-49.

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