**6.** *Salmonella* **in meat and meat products**

Meat includes all the edible parts of slaughtered warm-blooded animals, fit for human consumption. According to the EC Regulation 853/2004, this includes domestic ungulates (cattle, pigs, sheep and domestic equines), poultry, farmed lagomorphs (rabbits, hares), farmed game and hunted venison. It is called *fresh* meat if it has not undergone any treatment to extend its shelf life, except for the use of cold (refrigeration, freezing, deep freezing). Vacuum-packed meat and meat packed in a protective atmosphere are also considered fresh. Due to its chemical composition and to its intrinsic characteristics (Aw above 0.99, pH between 5.5 and 5.8), fresh meat is a good substrate for microbial growth. For this reason, cooling after slaughter is a critical point because it determines the microbiological quality of the product and must occur as fast as possible (internal temperature ≤ 7 °C, within 24-30 hours following slaughter). The flesh of healthy and unstrained animals is devoid of microorganisms in depth; but due to stress before slaughter, disease, or weakness, microbial contamination can occur and is defined as endogenous: pathogens, in particular starting from the intestine, spread into the blood due to the failing

**Year Country Product Serovar Cases**<sup>a</sup> **Deaths**  1976 Spain Egg salad Typhimurium 702 6

1988 Japan Cooked eggs *Salmonella* spp. 10476 NS 1993 France Mayonnaise Enteritidis 751 0

2002 England Bakery products Enteritidis PT 14 >150 1

2003 United States Egg salad kit Typhimurium 18 0

Cake/raw egg

Table 4. Examples of outbreaks of human salmonellosis from eggs and egg products

Meat includes all the edible parts of slaughtered warm-blooded animals, fit for human consumption. According to the EC Regulation 853/2004, this includes domestic ungulates (cattle, pigs, sheep and domestic equines), poultry, farmed lagomorphs (rabbits, hares), farmed game and hunted venison. It is called *fresh* meat if it has not undergone any treatment to extend its shelf life, except for the use of cold (refrigeration, freezing, deep freezing). Vacuum-packed meat and meat packed in a protective atmosphere are also considered fresh. Due to its chemical composition and to its intrinsic characteristics (Aw above 0.99, pH between 5.5 and 5.8), fresh meat is a good substrate for microbial growth. For this reason, cooling after slaughter is a critical point because it determines the microbiological quality of the product and must occur as fast as possible (internal temperature ≤ 7 °C, within 24-30 hours following slaughter). The flesh of healthy and unstrained animals is devoid of microorganisms in depth; but due to stress before slaughter, disease, or weakness, microbial contamination can occur and is defined as endogenous: pathogens, in particular starting from the intestine, spread into the blood due to the failing

eggs

dressing Enteritidis PT 4 2865 0

pastry Enteritidis PT 6 1433 0

mayonnaise *Salmonella* spp. >106 1

topping Enteritidis 197 NS

Enteritidis 688 0

Enteritidis PT 4 19 0

Enteritidis PT 6 68 0

China Egg drink Typhimurium 1113 NSb

Scotland Egg sandwiches Bareilly 186 NS

1977 Sweden Mustard

2001 United States Tuna salad with

2001 Latvia Cake/raw egg

2002 Spain Custard-filled

2003 Australia Raw egg

2005 England Imported shell

**6.** *Salmonella* **in meat and meat products** 

eggs

sauce

<sup>1987</sup>People's Republic of

<sup>2003</sup>England, Wales,

<sup>2004</sup>People's Republic of China

b Not stated

a Confirmed cases, unless stated otherwise.

(Adapted from: D'Aoust. J.Y. & Maurer J., 2007)

immune system, and reach the muscles, lymph nodes and internal organs. Among these microorganisms, there may also be *Salmonella*, if it is present in the intestinal contents. In this case, after the analytical laboratory investigations requested by the Veterinary Inspector, carcasses must be confiscated and destroyed, as they represent a potential danger to the consumer. On the other hand, the main microbial contamination occurs during the various stages of butchering and cutting, as well as in the following stages, such as the preparation one (minced meat, sausages, kebabs, etc.) and processing (salami mixture), until the purchase and the preservation of meat products before consumption. This contamination, defined as exogenous, is inevitable, but, by applying good manufacturing practices, it can be successfully controlled. The slaughtering stage which can lead to greater contamination by *Salmonella* is the gutting, where the release of feces even if it is limited (from 1010 to 1012 cfu/g microorganisms depending on the animals) results in the contamination of more or less large parts of the carcass. The main animal species that can host *Salmonella* spp. in their intestine, in descending order, are farmed birds, pigs and cattle. Meat is no doubt the food that undergoes the greatest number of tests, imposed by strict rules: on-farm veterinary visits, certificates accompanying the animals during its transport to slaughter, *ante mortem* and *post mortem* inspections, the scalding of the carcass (domestic ungulates and big game) that makes it fit for human consumption; followed by tests in the next stages (butchering facilities, butcher shops, supermarkets, meat-processing facilities, etc.) on meat and internal organs (heart, liver, stomach, etc.). Nevertheless, to restrict the *Salmonella* issue in meat, it is important to act upstream of the chain of production, during primary production. Ever since the 1990s, for poultry, the WHO (1994a) indicated guidelines to follow in order to identify the infected farms, to keep the vectors that carry the infection under control (WHO, 1994b) and to apply prevention methods (WHO, 1994c). In more recent years, the EU has released surveillance systems with specific control programs to significantly reduce the problem of *Salmonella* on farms rearing breeding poultry, egg-laying hens, broilers, turkeys and pigs, both for breeding and for meat (EC Regulation 2160/2003, Appendix 1). In these farming facilities, it is necessary to keep under control the hygienic characteristics of raw materials and animal feed, environmental hygiene, rodents, overcrowding, animal welfare, etc. The contamination of food for animal feed can occur in the factory as well as on the farm by cross contamination (not properly sanitized utensils) or by means of vectors (rodents, insects). Against the spread of *Salmonella* Enteritidis on poultry farms, it is effective to use antimicrobial agents in the feed, such as organic acids; as well as adding to the drinking water mixtures of probiotic bacteria in the early weeks of life, during which the intestinal colonization by potentially pathogenic microorganisms is most likely. Another difficulty resides in the elimination of the pathogen from the environment through cleaning and disinfection carried out after sending the animal to slaughter and before the arrival of the next cycle. Therefore, a good approach for controlling infection on the farm is definitely that of adopting prophylactic measures with serological monitoring and an accurate microbiologic testing of environment and faeces, trying to avoid the overuse of antibiotics in animals, which, on the other hand, can decrease their own organic resistance against *Salmonella*. In pigs, *Salmonella* can also be found very frequently in the tonsils, contaminated orally together with the intake of food. According to Griffith *et al*. (2006), it is possible to detect it in the oropharyngeal secretions and transmission between animals can happen nasally, especially in case of overcrowding on the farm or in transport. In cattle, the increased susceptibility to infection may arise from errors in the formulation of the food that changes the rumen flora, thus favouring the development of *Salmonella*. The EFSA report on

Food as Cause of Human Salmonellosis 61

Snails, however, although they are very often contaminated, rarely cause food-poisoning episodes as the way they are cooked ensures they are sanitized; nevertheless, they represent an excellent means for the germ to spread (Tiecco, 2000). Possible contaminations can also occur when the meat is subjected to various types of processing, such as "minced meat", defined as meat meant to be minced and to be sold as such, with the addition of salt up to a maximum of 1%, and "meat preparations", i.e. meat products to which seasonings or permitted food additives have been added or any treatment that does not alter the cellular structure of the core of the meat and does not change its characteristics as fresh meat. In both cases one can easily verify microbial contamination due either to the physical characteristics of the products, or to whether other potentially *Salmonella* carrying ingredients (vegetables, eggs, spices, etc.) are added. It is therefore important to keep the temperature constantly below 4°C, staff hygiene rules must be respected, as some staff members may be asymptomatic carriers of *Salmonella* and, most importantly, cross contamination must be prevented throughout the production chain and after cooking the meat, because this is the major problem for *Salmonella*. The processes of salting, curing, cold smoking, antimicrobial preserving additives and lactic fermentation the meat undergoes for becoming cold cuts are not able to completely eliminate *Salmonella*, but do cause the numbers to fall, sometimes substantially, which protects the consumer from food poisoning (<100 CFU/g). Heat (pasteurization, cooking, hot smoking), on the other hand, if it is done properly, is a good clearing method. But, without respecting basic technological principles, the previous operations will be completely useless. Many researchers agree that *Salmonella* usually contaminates food with a very low charge, mostly <10 cfu/g, but in the case of a human in normal immunological conditions, in order to be effective, an infection requires the ingestion of a fairly large charge, more than 104 cfu/g of food. As a result, it is necessary for the bacteria to find adequate conditions in the substrate to duplicate more or less rapidly. The "hygiene package" regulations introduced the obligation for the owners of manufacturing and processing factories to arrange and implement self-control procedures, to perform microbiological tests on the finished product and to indicate on the label if such meat, other than poultry, is to be subjected to adequate cooking before

Researches in EU in 2009 (EFSA, 2011) showed that *Salmonella* was found most frequently in raw turkey meat (8.7%), chicken (5.4%), pork (0.7%) and cattle (0.2%). In general, at the pig slaughterhouse, *Salmonella* was detected between 0 and 14%, with a higher incidence in Belgium where a very meticulous sampling method is carried out. According to the EFSA, many episodes of human food-borne illnesses are attributable to pork. Minced meat and meat preparations, especially those intended to be eaten raw, are most often contaminated by bacteria (5.5% positive) and therefore do not meet European sanitary standards. In retail shops *Salmonella* was found in 3.5% of the analyzed samples, a decrease compared with the previous year. Finally, the analysis of the results shows that there is a prevalence of serovars

In conclusion, the major risk factors of salmonellosis due to meat are to be found in insufficient cooking, the consumption of raw meat (pork sausages) which was not processed properly (minced horse meat, carpaccio, tartare, etc.), storage at inadequate temperatures, and cross contamination, if the product ready for consumption is in contact with other raw foods or dirty utensils. On the other hand, the staff's role, whether infected or carrier, appears to be less important, as it has been proven that, if Good Manufacturing Practice (GMP) is applied to food, such as careful hand washing, the risk of conveying infection is

consumption.

Typhimurium and Enteritidis.

kept well under control.

the progress of zoonoses and foodborne diseases (EFSA, 2011) shows that the verification of *Salmonella* in intensive European poultry farming facilities went down in 2009 compared to previous years. Greece (7%), the Czech Republic (11%) and Hungary (32%) were the states where the detections were greater, respectively for roosters, egg-laying hens and broilers. In the first 2 types of farming facilities, the most detected serotypes were: *S*. Enteritidis, *S*. Typhimurium, *S*. Infantis, *S*. Virchow and *S*. Hadar. On pig farms, the data collected among the member states are not homogeneous about sampling time in the production chain (faeces on the farm, lymph nodes in the slaughterhouse): the only countries that submitted full data were Estonia (0.9% on the farm and 8% positive in the slaughterhouse) and Norway (0% in both cases). However, the EU estimated that the presence of *Salmonella* in intensively farmed pigs in 2009 varied from 0 to 64% (on average between 26 and 31%). S. Derby was the most detected serotype. For all other animals, although gathered data are few, the presence of *Salmonella* is low both on the farm and in the slaughterhouse. Finally, *Salmonella* was found in animal feed with a low incidence, ranging on average from 0.4% (for cattle) to 1% (for poultry), and the most contaminated products were meat and bone meal (1.4%). In later farming stages it is necessary to ensure that the animals arrive at the slaughterhouse in the best conditions possible (avoiding overcrowding, dirt on skin and feathers); it is also important to check, in addition to the above, the fasting time before slaughter, the temperature and the renewal of scalding water (poultry, pork) in order to limit the load of organic material and reduce the adhesion of *Salmonella* on the skin; it is also essential to check that the cleaning procedures, the maintenance of facilities and equipment are performed in optimal conditions. These checks should be supported by microbiological tests, in accordance with EC Regulation 2073/2005, to make sure that the slaughter took place in full respect of hygiene conditions and, if not, to review the process. Moreover, EFSA (2006) recommends that swine slaughter monitoring should provide for the research of the pathogen in the ileocaecal lymph nodes or in the meat juice. Decontamination of carcasses after slaughter can be useful for controlling pathogens, including *Salmonella*, but it can absolutely not replace poor hygiene during the slaughter. Some methods put forward, but not always accepted by the regulations, are the use of organic acids, such as acetic acid, which cannot be used for obvious reasons, and lactic acid, which, if used at concentrations of 1% v/v, affects S. Typhimurium well without affecting the colour or the flavour of the meat too much; also ozone mixed with the water used for showering poultry provides excellent results regarding the reduction of microbial load. In the USA, trisodium phosphate (TSP) can be used, an alkaline composite (pH 11) able to diminish the pathogen by 2 logarithmic cycles and bring down the contamination of poultry carcasses to under 5% (Gudmundsdottir *et al*., 1993), but its disposal after use is a problem.

A very powerful physical method to reduce microbial load is γ-ray irradiation, which is not licensed in Europe. Doses of 3-5 kGy are employed, effective for the decontamination of fresh meat, the Enterobacteriaceae loads ensuing in a fall of 6 logarithmic degrees (WHO, 1994), without causing significant deterioration of the sensory quality of the treated products. As regards the meat of hunted wild game, the microbiological quality depends on where skinning and handling is carried out: on average, it was stated that the total viable count as well as that of fecal coliform are about 2 logarithmic higher in game eviscerated outdoor than the similar value of game eviscerated in slaughterhouse (105-108 vs. 103-106 cfu/g). Anyway, *Salmonella* is rarely found, especially if the wild game does not come into contact with domestic animals. Frogs imported from various countries are frequently contaminated with *Salmonella* (20-30%) belonging to most diverse serotypes and often exotic ones, according to the importing country.

the progress of zoonoses and foodborne diseases (EFSA, 2011) shows that the verification of *Salmonella* in intensive European poultry farming facilities went down in 2009 compared to previous years. Greece (7%), the Czech Republic (11%) and Hungary (32%) were the states where the detections were greater, respectively for roosters, egg-laying hens and broilers. In the first 2 types of farming facilities, the most detected serotypes were: *S*. Enteritidis, *S*. Typhimurium, *S*. Infantis, *S*. Virchow and *S*. Hadar. On pig farms, the data collected among the member states are not homogeneous about sampling time in the production chain (faeces on the farm, lymph nodes in the slaughterhouse): the only countries that submitted full data were Estonia (0.9% on the farm and 8% positive in the slaughterhouse) and Norway (0% in both cases). However, the EU estimated that the presence of *Salmonella* in intensively farmed pigs in 2009 varied from 0 to 64% (on average between 26 and 31%). S. Derby was the most detected serotype. For all other animals, although gathered data are few, the presence of *Salmonella* is low both on the farm and in the slaughterhouse. Finally, *Salmonella* was found in animal feed with a low incidence, ranging on average from 0.4% (for cattle) to 1% (for poultry), and the most contaminated products were meat and bone meal (1.4%). In later farming stages it is necessary to ensure that the animals arrive at the slaughterhouse in the best conditions possible (avoiding overcrowding, dirt on skin and feathers); it is also important to check, in addition to the above, the fasting time before slaughter, the temperature and the renewal of scalding water (poultry, pork) in order to limit the load of organic material and reduce the adhesion of *Salmonella* on the skin; it is also essential to check that the cleaning procedures, the maintenance of facilities and equipment are performed in optimal conditions. These checks should be supported by microbiological tests, in accordance with EC Regulation 2073/2005, to make sure that the slaughter took place in full respect of hygiene conditions and, if not, to review the process. Moreover, EFSA (2006) recommends that swine slaughter monitoring should provide for the research of the pathogen in the ileocaecal lymph nodes or in the meat juice. Decontamination of carcasses after slaughter can be useful for controlling pathogens, including *Salmonella*, but it can absolutely not replace poor hygiene during the slaughter. Some methods put forward, but not always accepted by the regulations, are the use of organic acids, such as acetic acid, which cannot be used for obvious reasons, and lactic acid, which, if used at concentrations of 1% v/v, affects S. Typhimurium well without affecting the colour or the flavour of the meat too much; also ozone mixed with the water used for showering poultry provides excellent results regarding the reduction of microbial load. In the USA, trisodium phosphate (TSP) can be used, an alkaline composite (pH 11) able to diminish the pathogen by 2 logarithmic cycles and bring down the contamination of poultry carcasses to under 5%

(Gudmundsdottir *et al*., 1993), but its disposal after use is a problem.

A very powerful physical method to reduce microbial load is γ-ray irradiation, which is not licensed in Europe. Doses of 3-5 kGy are employed, effective for the decontamination of fresh meat, the Enterobacteriaceae loads ensuing in a fall of 6 logarithmic degrees (WHO, 1994), without causing significant deterioration of the sensory quality of the treated products. As regards the meat of hunted wild game, the microbiological quality depends on where skinning and handling is carried out: on average, it was stated that the total viable count as well as that of fecal coliform are about 2 logarithmic higher in game eviscerated outdoor than the similar value of game eviscerated in slaughterhouse (105-108 vs. 103-106 cfu/g). Anyway, *Salmonella* is rarely found, especially if the wild game does not come into contact with domestic animals. Frogs imported from various countries are frequently contaminated with *Salmonella* (20-30%) belonging to most diverse serotypes and often exotic ones, according to the importing country. Snails, however, although they are very often contaminated, rarely cause food-poisoning episodes as the way they are cooked ensures they are sanitized; nevertheless, they represent an excellent means for the germ to spread (Tiecco, 2000). Possible contaminations can also occur when the meat is subjected to various types of processing, such as "minced meat", defined as meat meant to be minced and to be sold as such, with the addition of salt up to a maximum of 1%, and "meat preparations", i.e. meat products to which seasonings or permitted food additives have been added or any treatment that does not alter the cellular structure of the core of the meat and does not change its characteristics as fresh meat. In both cases one can easily verify microbial contamination due either to the physical characteristics of the products, or to whether other potentially *Salmonella* carrying ingredients (vegetables, eggs, spices, etc.) are added. It is therefore important to keep the temperature constantly below 4°C, staff hygiene rules must be respected, as some staff members may be asymptomatic carriers of *Salmonella* and, most importantly, cross contamination must be prevented throughout the production chain and after cooking the meat, because this is the major problem for *Salmonella*. The processes of salting, curing, cold smoking, antimicrobial preserving additives and lactic fermentation the meat undergoes for becoming cold cuts are not able to completely eliminate *Salmonella*, but do cause the numbers to fall, sometimes substantially, which protects the consumer from food poisoning (<100 CFU/g). Heat (pasteurization, cooking, hot smoking), on the other hand, if it is done properly, is a good clearing method. But, without respecting basic technological principles, the previous operations will be completely useless. Many researchers agree that *Salmonella* usually contaminates food with a very low charge, mostly <10 cfu/g, but in the case of a human in normal immunological conditions, in order to be effective, an infection requires the ingestion of a fairly large charge, more than 104 cfu/g of food. As a result, it is necessary for the bacteria to find adequate conditions in the substrate to duplicate more or less rapidly. The "hygiene package" regulations introduced the obligation for the owners of manufacturing and processing factories to arrange and implement self-control procedures, to perform microbiological tests on the finished product and to indicate on the label if such meat, other than poultry, is to be subjected to adequate cooking before consumption.

Researches in EU in 2009 (EFSA, 2011) showed that *Salmonella* was found most frequently in raw turkey meat (8.7%), chicken (5.4%), pork (0.7%) and cattle (0.2%). In general, at the pig slaughterhouse, *Salmonella* was detected between 0 and 14%, with a higher incidence in Belgium where a very meticulous sampling method is carried out. According to the EFSA, many episodes of human food-borne illnesses are attributable to pork. Minced meat and meat preparations, especially those intended to be eaten raw, are most often contaminated by bacteria (5.5% positive) and therefore do not meet European sanitary standards. In retail shops *Salmonella* was found in 3.5% of the analyzed samples, a decrease compared with the previous year. Finally, the analysis of the results shows that there is a prevalence of serovars Typhimurium and Enteritidis.

In conclusion, the major risk factors of salmonellosis due to meat are to be found in insufficient cooking, the consumption of raw meat (pork sausages) which was not processed properly (minced horse meat, carpaccio, tartare, etc.), storage at inadequate temperatures, and cross contamination, if the product ready for consumption is in contact with other raw foods or dirty utensils. On the other hand, the staff's role, whether infected or carrier, appears to be less important, as it has been proven that, if Good Manufacturing Practice (GMP) is applied to food, such as careful hand washing, the risk of conveying infection is kept well under control.

Food as Cause of Human Salmonellosis 63

Czech Republic (135 samples), Germany (980 samples), Greece (26 samples), Hungary (85 samples) and Romania (57 samples). Again, none of the samples tested contained *Salmonella*. Italy reported that out of a total of 928 samples of cow's milk, 3 were positive for *Salmonella* and that 5 samples out of a total of 5,799 samples of milk from "other unspecified species" also tested positive for the pathogen. 11 member countries supplied EFSA with results of their investigations on cheeses, for a total of 23,023 samples analyzed. In the great majority of cases, the cheese samples proved to be negative for *Salmonella*, with the exception of Spain (4 positive samples out of a total of 424 samples tested), Portugal (2 positive samples out of 181 analyzed) and Italy (2 positive out of a total of 1,879 samples tested). As far as we know from the EFSA report, all the cheeses tested positive were semi-hard cheeses, and only semimature, and made from raw or thermised milk (i.e. heated to a temperature between 45 °C and 54 °C, no more). As far as butter is concerned, 7 member states communicated the results of their inspections; no case revealed the presence of *Salmonella*. Besides cheese, the only other product derived from milk which pointed out the presence of *Salmonella* was ice cream. Spain, Hungary and Germany reported the presence of the bacterium respectively in 13 samples out of 305 samples analyzed, 1 out of 140 and 1 in 2,626 samples, always taken in the production facilities. The presence of salmonellae in raw milk is widely documented in the literature, both in the collection tanks on the farms, and in the storage tanks in the food factories (Donaghy *et al*., 2004; Tondo *et al*., 2000). *Salmonella* may be present in raw milk ever since milking because the bacterium is present in the mammary gland, but this occurs very rarely. Mastitis due to *Salmonella* is a very rare condition in dairy cows, but it is reported. We know that different *Salmonella* serotypes can colonize the mammary gland and lead to the excretion, at the same time as the milk, of bacterial loads that can extend up to 3.3 log10 cfu/ml (Fontaine *et al*., 1980). Furthermore, *Salmonella* can pass from animal to animal at the time of milking, both through the milker's hands, and through polluted parts of the milking machines (Bergonier *et al*., 2003; Vautor *et al*., 2003; Zadoks *et al*., 2002; Zschöck *et al*., 2000). Much more often, however, salmonellae contaminate raw milk in the stages that follow the milking process, because the bacteria may be present on the various surfaces that come into contact with the milk being collected. In particular, *Salmonella* (such as *Listeria monocytogenes* and verotoxigenic strains of *E. coli*) can enter the milk through the traces of animal faeces in the environment (Van Kessel *et al*., 2004). This factor of pollution, in turn, is influenced by the prevalence among dairy cows of *Salmonella* healthy carriers, which can evacuate various loads of the pathogen more or less frequently. In this regard, it is estimated that the U.S. dairy cows can be healthy carriers of *Salmonella* in their faeces with a prevalence that ranges from a minimum of 2% to a maximum of 27.5% of the animals tested (Kabagambe *et al*., 2000; Losinger *et al*., 1995, Wells *et al*., 2001). What can be the prevalence of a batch of raw milk tested positive for *Salmonella* ever since the milking phase? In view of the data that we can gather from the literature, we can estimate that the batches of raw milk straight after milking can be positive for *Salmonella* from a minimum of 2.6% to a maximum of 25.3% (Jayarao & Henning, 2001; Murinda *et al*., 2002; Zhao *et al*. 2002). Compared to other pathogenic microorganisms such as *L. monocytogenes*, salmonellae are not very resistant in the outside, so it is not very frequent for the work surfaces in the production plants to transfer salmonellae to the product. Nevertheless, in theory, *Salmonella* can survive for long on any work surface and then pollute the cheese curd which is meant to become cheese. This justifies the episodes of foodborne infection caused by processed dairy products, such as
