Red meat is derived from a number of animal species (e.g. cattle, sheep, goat, ..... cattle during the first 18 days of entering a feed-lot from a market. ..... O:3 is common in pigs in Eastern Canada, it is rare in Western Canada where O:5,27 ...... occur on high-pH meat and at higher temperatures (Dainty et al., 1979; Palumbo,.
1 Meat and meat products
I Introduction Red meat is derived from a number of animal species (e.g. cattle, sheep, goat, camel, deer, buffalo, horse, and pig). Total world production of red meats and quantities in international trade can be obtained from http://apps.fao.org/page/collections?subset=agriculture, a part of http://www.fao.org. Red meat has the potential to carry pathogenic organisms to consumers. In the past, the main public health problem was caused by the classical zoonoses, i.e. diseases or pathogens that can be transmitted from animals to human beings, such as bovine tuberculosis, and also produce pathological changes in animals. However, the measures introduced by classical meat inspection (inspection, palpation, and incision) have proved highly effective against them. Thus, tuberculosis shows very typical changes of the lymph nodes (granulomatous lymphadenitis); they can be reliably detected by incision of the nodes during meat inspection. However, today, the main problem is latent zoonoses. These pathogens occur as a reservoir in healthy animals, where they produce no pathological conditions or changes. However, they can contaminate the food chain in meat production, for instance during slaughtering. The slogan “healthy animals, healthy food” is not true from this point of view. Strict maintenance of good practices of slaughter hygiene in meat production is of central importance, because microbiological hazards are not eliminated in the slaughtering process. Bacteria able to cause food-borne disease, and which can constitute a hazard in at least some meat products, include Salmonella spp., thermophilic Campylobacter spp., enterohemorrhagic Escherichia coli (e.g. serogroup O157; EHEC), some serovars of Yersinia enterocolitica, Listeria monocytogenes, Clostridium perfringens, Staphylococcus aureus, Cl. botulinum, and Bacillus cereus. Meats are also subject to microbial spoilage by a range of microorganisms including Pseudomonas spp., Shewanella, Enterobacteriaceae, Brochothrix thermosphacta, lactic acid bacteria (LAB), psychrotrophic clostridia, yeasts, and molds. In recent years, bovine spongiform encephalopathy (BSE) (“mad cow disease”) has attracted public health attention. The first cases of BSE were reported in Great Britain in November 1986. It appears probable that the disease can be transmitted to humans by food. The prions that cause the disease are very resistant to chemical and physical influences, i.e. to heat, UV, and ionizing radiations and disinfectants. Prions are sensitive to certain alkaline substances and moist heat under high pressure. An effective disinfectant measure is steam sterilization at 133◦ C and 3 bar pressure for 20 min. On the basis of current knowledge, the cause of the BSE epidemic was animal feed (meat- and bone-meal and the like) containing brain, eyes or spinal cord of infected animals, and other tissues that had been inadequately heated during the production process. To protect human health, the use of certain bovine organs (so-called specified risk materials: brain, eyes, spinal cord, spleen, thymus (sweetbread), bovine intestines of cattle >6 months old, visible lymph and nerve tissue, as well as lymph nodes) is prohibited for manufacturing foodstuffs, gelatine, tallow, drugs or cosmetics. More information and actual data can be obtained from the following web-sites: http://www.oie.int/eng/en index.htm; http://www.who.int/mediacentre/factsheets/fs113/en/; http://www.defra.gov.uk/animalh/bse/index.html; http://www.aphis.usda.gov/oa/bse/; http://www.tseandfoodsafety.org/; http://www.unizh.ch/pathol/neuropathologie/. This chapter, however, mainly describes the microorganisms that contaminate red meats and meat products, and factors and operations that increase or decrease the numbers or spread of microorganisms
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during processing, storage, and distribution. It also contains sections on the microbiology of froglegs and snails as foods. A Definitions Red meat is primarily the voluntary striated skeletal muscular tissue of “red” meat animals. The muscle is made up of contractile myofibrillar proteins, soluble sarcoplasmic proteins (e.g. glycolytic enzymes and myoglobin) and low molecular weight soluble organic and inorganic compounds. Connective tissue is in intimate association with muscle cells and can constitute up to 30% of total muscle protein. Fat cells occur subcutaneously and both within and surrounding the muscle. Within a muscle, fat cells are located in the perimysial space. Up to one-third of the weight of some muscles may be fat. Muscle tissues also contain 0.5–1% phospholipid. Meat as legally defined commonly includes various organs (“variety meats” or “offals”). The organs and other parts of the carcass that are regarded as edible vary between countries. The heart has some similarities to skeletal muscle and is composed of striated involuntary muscle, connective tissue, and some lipid. The liver contains uniform liver cells with a network of blood vessels and epithelial-lined sinusoids. In the kidney, there is a meshwork of connective tissue that supports renal tubules, small veins, and arteries. B Important properties Meat has a high water and protein content, is low in carbohydrates and contains a number of low molecular weight soluble constituents (Table 1.1). The vitamin content (µg/g) of muscle is approximately: thiamine, 1; riboflavin, 2; niacin, 45; folic acid, 0.3; pantothenic acid, 10; B6 , 3; B12 , 0.02 and biotin, 0.04 (Schweigert, 1987). The concentrations of vitamins vary with species, age, and muscle. Pork muscle has 5–10 times more thiamine than is found in beef or sheep muscle. Vitamins tend to be higher in organs (e.g. liver and kidney) than in muscle. Meat is a nutritious substrate with an aw (0.99) suitable for the growth of most microorganisms. Growth is primarily at the expense of low molecular weight materials (carbohydrates, lactate, and amino acids). Microbial proteolysis of structural proteins occurs at a very late stage of spoilage (Dainty et al., 1975).
Table 1.1 Approximate composition of adult mammalian muscle after rigor mortis Component Water Protein Lipid Glycogena Glucosea,b and glycolytic intermediatesa Lactic acida Inosine monophosphateb Creatineb Amino acidsb Dipeptides (carnosine and anserine)b pHa Lawrie (1985). a Varies between muscles and animals. b Varies with time after rigor mortis.
% Wet weight 75 19 2.5 0.1 0.2 0.9 0.3 0.6 0.35 0.35 (5.5)
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During death of the animal when the oxygen supply to the muscle is cut off, anaerobic glycolysis of stored glycogen to lactic acid lowers the pH. Post-mortem glycolysis continues as long as glycogen is available or until a pH is reached which inhibits the glycolytic enzymes. In typical muscles this pH is 5.4–5.5. In some muscles (e.g. beef sternocephalicus muscle), glycolysis ceases at a pH near 6 even though considerable glycogen remains. The ultimate pH varies between muscles of the same animal and between animals, and is determined by the glycogen content of the muscle and the accessibility of glycogen to glycolysis. The pH of post-rigor muscle can vary from 5.4–5.5 (lactate content close to 1%) to 7.0 (very little lactate present). The lactate content of muscle is inversely proportional to its pH. On the surfaces of beef and sheep carcasses, the availability of oxygen permits aerobic metabolism to continue, and much of the exposed surface tissue has a pH >6 (Carse and Locker, 1974), which facilitates microbial growth. In the live animal, the glycogen concentration of muscle averages 1%, but varies considerably. Glycogen in pig muscle is readily depleted by starvation and moderate exercise, whereas glycogen in the muscles of cattle is more resistant to starvation and exercise. In both species, pre-slaughter stress (e.g. excitement and cold) depletes muscle glycogen. Glycogen is more concentrated in liver (2–10%) than in muscle, and its content is also affected by pre-slaughter conditions. A low concentration of glycogen in muscles results in a high ultimate pH, which gives rise to “dark-cutting” beef or dark, firm and dry meat (DFD). The amount of glucose in post-rigor muscle varies with pH (Newton and Gill, 1978) being virtually absent in muscle of pH > 6.4. In normal-pH (5.5–5.8) muscle, glucose is present at about 100–400 µg/g (Gill, 1976). Liver has a high glucose content (3–6 mg/g), which appears to be independent of pH (Gill, 1988). By the time the ultimate pH is reached, adenosine triphosphate has largely broken down to inosine monophosphate (IMP). During the storage of meat, IMP and inosine continue to degrade to hypoxanthine, ribose, and ribose phosphate. Ribose, inosine, and IMP can be used as energy sources by a number of fermentative Gram-negative bacteria, and ribose by Broch. thermosphacta, and a number of lactic acid bacteria. Fatty tissue contains less water than muscle, has a pH near neutrality with little lactate, and contains low molecular weight components (glucose and amino acids) from serum (Gill, 1986). Consequently, microbial growth on fat is slower than on the surface of muscle. C Methods of processing and preservation Animals are raised on farms where some are grazed and some are raised under intensive or almost industrial conditions. The microflora in the intestinal tract or on the external surfaces of the animals may vary with the systems of animal production (e.g. more fecal material on the hides of feed-lot cattle). Animals may be slaughtered when young (e.g. calves at 3–4 weeks of age), or when 1 or 2, or several, years old (e.g. cattle and sheep). At the abattoir, the skin of cattle and sheep is removed, the skin of pigs is usually scalded (although it is removed in some plants), then the intestinal tract and viscera are removed. The carcass may then be washed, where regulations permit it, or not, and then chilled. Spoilage organisms grow rapidly on meat, which is a highly perishable commodity. Thus, trade in meat, even at the local level, depends on some degree of preservation that controls the spoilage flora. The most important means of preservation are chilling or freezing, cooking (includes canning), curing, drying, and packaging. Packaging affords extension of shelf-life. Several procedures to reduce microbial growth are often combined. Chilled temperature storage enables fresh meat to be held for only a limited time before spoilage ensues. However, by vacuum-packaging chilled meat in films of low permeability to gases, or by packaging in modified atmospheres, storage-life may be extended for up to at least 12 weeks.
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D Types of meat products Red meats are traded as chilled or frozen carcasses, large primal pieces or retail size portions, chilled or frozen offals, chilled vacuum-packed meat, dried meats, fermented meat, raw or cooked cured products, cooked uncured meat and cooked canned products.
II Initial microflora A Ruminants At birth, the digestive tract of a ruminant is physiologically that of a monogastric. The rumino-reticulum complex develops quickly between 2 and 6 weeks of age when the animals are fed roughage. Initially, large numbers of E. coli, Cl. perfringens and streptococci are in the gut and are shed in feces (107 – 108 cfu Cl. perfringens/g, 109 cfu E. coli/g). After about 2 weeks, Cl. perfringens declines to about 104 cfu/g and E. coli to ca. 106 cfu/g at about 3 months of age. When comparing fecal excretion of coliforms, the mean count for eight calves between 3 and 8 weeks of age was log10 7.2 cfu/g and for adult cows was log10 4.9 cfu/g (Howe et al., 1976). Invasive serotypes of salmonellae, such as Salmonella Typhimurium and S. Enteritidis, are more difficult to control in the live animal than serovars occasionally found in feed. In the first few days of life, young ruminants are more susceptible to salmonellae. Calves dosed with S.Typhimurium prior to 3 days of age were more easily infected, and excreted salmonellae for longer periods and in greater numbers, than calves inoculated at 18 days (Robinson and Loken, 1968). At slaughter, salmonellae were also detected more frequently in mesenteric and cecal lymph nodes from the younger animals. Young calves that are surplus to dairy farm requirements may be sold through markets and dealers to rearing farms. In England, salmonellae have been found in 3.7% of environmental samples taken at calf markets and in 20.6% of swab samples from vehicles used to transport calves (Wray et al., 1991). Salmonellae have also been detected on the walls (7.6% of swabs) and floors (5.3% of swabs) at dealers’ premises (Wray et al., 1990). The mixing of young susceptible calves and their subsequent transport to rearing farms disseminates salmonellae. On arrival at rearing farms, the prevalence of salmonellae in calf feces is relatively low but can increase rapidly. When fecal samples were taken from 437 calves within 2 days of arrival at a rearing farm, salmonellae were detected in 5.3% (Hinton et al., 1983). After about 2 weeks on the farm, salmonellae were found in 42.2% of 491 animals sampled. The shedding rate of salmonellae peaked at 2–3 weeks and then declined; this is possibly associated with the development of a more adult-type intestinal flora. The high concentration of volatile fatty acids and the pH of the fluid in the developed rumen of the well-fed animal provide some protection to infection with salmonellae and verotoxin-producing E. coli (often of the serogroup O157; VTEC) (Chambers and Lysons, 1979; Mattila et al., 1988). Viable cells of these organisms disappear from rumen fluid at a rate faster than expected from wash-out. Starved or intermittently fed ruminants are more susceptible to infection as salmonellae and VTEC O157 can then grow in the rumen. This probably influences the percentage of infected animals on farms during periods of low feed intake (e.g. drought, mustering, shearing or dipping and high stocking densities). On farms, the prevalence of salmonellae in the intestinal tract varies (Edel and Kampelmacher, 1971). Outbreaks of clinical bovine salmonellosis tend to show seasonal patterns. In the UK, most incidents of bovine salmonellosis occur in summer–autumn and peak near the end of the grazing season (Williams, 1975). Peaks of clinical salmonellosis in sheep in New Zealand during summer–autumn have been associated with movement and congregation of sheep for shearing and dipping. In a study of the prevalence of salmonellae in cow–calf operations (Dargatz et al., 2000), of 5 049 fecal samples collected from 187 beef cow–calf operations, salmonellae were recovered from 1 or more
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fecal samples collected on 11.2% (21 of 187) of the operations. Overall 78 salmonellae representing 22 serotypes were isolated from 1.4% (70 of 5 049) of samples, and multiple serotypes from eight samples from a single operation. The five most common serotypes were S. Oranienburg (21.8% of isolates) and S. Cerro (21.8%), followed by S. Anatum (10.3%), S. Bredeney (9.0%) and S. Mbandaka (5.1%). Although it is broadly accepted that human salmonellosis is derived from foods, especially meat and poultry, firm proof is elusive. Sarwari et al. (2001) concluded from US data for 1990–1996, that there was a significant mismatch between the distribution of Salmonella species isolated from animals at the time of slaughter and that of isolates found in humans. This questions the validity of assumptions that raw animal products are the primary source for human salmonellosis, or whether there are methodological reasons for the difference. The increased susceptibility to infection resulting from changes in the rumen can also affect the prevalence of salmonellae in cattle and sheep during transport from farm to slaughter, or in long transport from farm to farm when feeding patterns and type of feed are changed. Frost et al. (1988) reported a high prevalence of salmonellae in the mesenteric lymph nodes and rumen fluid of adult cattle during the first 18 days of entering a feed-lot from a market. After 80 days in the feed-lot, there was little evidence of salmonellae infection. Some of the deaths of sheep during sea-shipment from Australia to Singapore and the Middle East have been due to salmonellosis, which was associated with empty gastrointestinal tracts, loss of appetite and poor adjustment from grazing green pastures to dry feed. Although healthy cattle may excrete thermophilic campylobacters in their feces, numbers are generally low (NACMCF, 1995). While thermophilic campylobacters are frequently found in the lower intestinal tract of ruminants (prevalence range 0–54%), it is usually present in numbers 50% of the spoilage flora with Ps. fragi (clusters 1 and 2), Ps. lundensis and Ps. fluorescens being the most important species. Broch. thermosphacta and psychrotrophic Enterobacteriaceae usually form only a small proportion of the spoilage flora but appear to be more prevalent on the fat surfaces of sheep and pork. Storage at 5◦ C rather than at 0–1◦ C tends to favor their growth (Dainty and Mackey, 1992). At high ambient storage temperatures (25–30◦ C), Enterobacteriaceae and Acinetobacter spp. dominate the spoilage flora (Gill and Newton, 1980; Rao and Sreenivasamurthy, 1985). When storage has caused the surfaces to remain dry, colonies of micrococci, yeasts, and molds may appear. Growth is uneven over the carcass mostly owing to variations in surface aw . Limited information on the bacterial flora of horses and goats has been reported (Cantoni, 1977; Sinha and Mandar, 1977). D Pathogens Salmonella. The prevalence of salmonellae on beef, sheep, and pig carcasses varies widely. Sometimes salmonellae are rarely found (Biemuller et al., 1973). Sometimes they can be found on close to half of the carcasses (Oosterom and Notermans, 1983), and at other times on all carcasses from a herd (Grau and Smith, 1974). In a large survey in the United States, salmonellae were found on 1% of excised 25 g samples of brisket from 3 075 chilled carcasses of steers, heifers, bulls, and cows and on 5% of samples from 397 calves (Hogue et al., 1993). A more recent US survey (Tables 1.15 and 1.16), also detected salmonellae on 1% of samples excised from chilled steer and heifer carcasses (USDA, 1994). In Canada, salmonellae were detected on 11.2% of 596 pork carcasses, on 4.1% of 267 veal and on 1.7% of 666 beef carcasses when neck muscle samples, excised from carcasses before chilling, were examined (Lammerding et al., 1988). Contamination rates for animals in Eygptian abattoirs were: buffalos 1–2% (Lotfi and Kamel, 1964; El Moula, 1978); camels up to 44% (El Moula, 1978); sheep 3–4% (Lotfi and Kamel, 1964); a wide range of serotypes were found. The extent of carcass contamination is strongly influenced by the prevalence and concentration of salmonellae in the intestinal tract and, for sheep and cattle, by the contamination of the fleece and hide. It is also influenced by the care taken in slaughter and dressing. The salmonellae status of animals at slaughter is determined by contamination acquired at the farm and by holding conditions before slaughter. Salmonellae can be found in the internal tissues of liver and spleen from apparently normal animals (Tables 1.6 and 1.7). Normally, only small numbers of salmonellae are on carcass or offal meats. However, inadequate chilling, storage or transport, at temperatures above about 7◦ C, can permit growth. Outbreaks of salmonellosis can follow from inadequate cooking, mishandling, and recontamination. Raw meats can act as a source of cross-contamination of cooked meats, or other foods, in the kitchen or in meat processing plants. Escherichia coli O157:H7. Generally a small percentage of cattle carry EHEC O157:H7 in the intestinal tract at slaughter (Table 1.4), but occasionally the percentage is high. Care taken during evisceration and hide removal can limit, but not entirely prevent, contamination of the carcass. Growth of the organism can occur if chilling, storage or transport conditions of the carcass are inadequate (temperature above about 7◦ C). Inadequately cooked ground beef, contaminated with E. coli O157:H7, has caused a number of outbreaks of bloody diarrhea (HC) and hemolytic uraemic syndrome (Doyle, 1991; Griffin
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and Tauxe, 1991). Cooking hamburgers to an internal temperature of 68◦ C has been recommended (Meng et al., 1994). Undercooking of ground beef or hamburger patties is a common cause of outbreaks attributed to E. coli O157:H7. A large outbreak in the United States (1993) affected 732 people, undercooked hamburgers were the cause. Ground beef has also been identified as carrying E. coli O157, published prevalences varying from 0 (Willshaw et al., 1993; Lindqvist et al., 1998; Tarr et al., 1999) to 0.7 (Doyle and Schoeni, 1987), 1.3% (Kim and Doyle, 1992) and 2.4% (Sekla et al., 1990). Quantitative analysis of some of the positive lots conducted by FSIS and CDC resulted in MPN values in the range of 1–4 cfu/g, with a single high value of 15 cfu/g (FSIS, 1993). In an Argentine study 11 E. coli O157:H7 isolates were detected in 6 (4%) out of 160 ground beef samples produced directly by retailers from different combinations of cuts and trimming of boneless beef (Chinen et al., 2001). In the same study, 4 (8%) out of 83 fresh sausages were positive for E. coli O157:H7. In a Swiss study, a total of 400 minced meat samples from 240 small butcheries were collected and analyzed for the presence of STEC and L. monocytogenes (Fantelli and Stephan, 2001). The samples comprised 211 samples of minced beef and 189 samples of minced pork. STEC was isolated from 7/400 (1.75%) samples. In particular, 5/211 (2.3%) minced beef samples and 2/189 (1%) minced pork samples were contaminated. Serotyping of the seven strains yielded five different serotypes, but none of the strains was O157:H7. Two STEC strains harbored stx1 and stx2 and five strains harbored stx2c genes. Furthermore, four strains harbored one or more additional virulence factors. However, none of the strains was positive for eae. L. monocytogenes was isolated from 43/400 (10.75%) samples. Nineteen of the 43 strains belonged to serotype 1/2a, two to serotype 1/2b, 12 to serotype 1/2c, and 10 to 4b. Forty-two strains harbored the Lhly and 43 strains the plcA genes. Macrorestriction analysis of the L. monocytogenes strains using SmaI yielded 12 different PFGE-patterns. The predominating pattern G was associated to the serotype 1/2c. Ground beef patties inoculated with ca. 106 E. coli O157:H7 and frozen at −20◦ C for 24 h and then thawed at 4◦ C for 12 h, at 23◦ C for 3 h, or using microwave heating for 120 s at 700 W, showed variable destruction depending upon the strain, the recovery method and the thawing regimen (Sage and Ingham, 1998). Podolak et al. (1995) reported fumaric acid (1% and 1.5%) to be more effective than 1% lactic or acetic acid in reducing populations of E. coli O157:H7 in vacuum-packaged ground beef patties. According to guidelines of the UK Advisory Committee on the Microbiological Safety of Food (1995), however, target/temperature of 70◦ C for 2 min is recommended to ensure safety of minced meat products. Campylobacter jejuni. After slaughter, thermophilic campylobacters have been found from 19% to 70% of sheep carcasses, from 2% to 32% of adult cattle carcasses and from 20% to 97% of calf carcasses. Thermophilic campylobacters (mostly C. coli) have been found on 20–60% of pork carcasses. In a Canadian survey (Lammerding et al., 1988), campylobacters were found on 12% of pork, 15% of beef, and 35% of beef neck samples taken from carcasses before chilling. Fecal samples from 94 dairy cows and 42 calves in three different herds were examined by a variety of techniques for campylobacters (Atabay and Corry, 1998). Seventy-nine percent of cattle in herd A carried campylobacters, 40% in herd B and 37.5% in herd C. Most animals carried only one species of Campylobacter: C. hyointestinalis was isolated most frequently (32% animals positive) with C. fetus subsp. fetus detected in 11% of animals and C. jejuni subsp. jejuni 7%. In addition, a novel biotype of C. sputorum was isolated from 60% of 47 cows tested in herd A. During carcass chilling, there is a significant reduction in the prevalence and number of viable campylobacters, and, even on originally relatively heavily contaminated carcasses, numbers are usually
