**4.** *Salmonella* **in vegetables**

Compared to foods of animal origin, which are usually consumed once cooked, fruit and vegetables are mostly eaten raw and therefore a significant part of foodborne outbreaks due to the consumption of raw vegetables has been attributed to *Salmonella* (Cantoni & Bersani, 2010). Animal faeces and irrigation water are the main ways for *Salmonella* to spread to crops (Islam *et al*., 2004). The water can contaminate the food if it is used for irrigation, for washing or for handling it (Rondanelli *et al*., 2005). The salmonellae present in not perfectly ripened manure or in irrigation water invade the plants by gripping to the roots or contaminating the leaves (Cantoni & Bersani, 2010). Studies headed by Professor

there cannot be any growth, but rather a sublethal stress which leads to an adaptation of the pathogen (Jasson *et al*., 2011). In the presence of NaCl concentrations between 3 and 4%, the development of *Salmonella* is usually inhibited. However, it appears that the inhibitory action of salt increases with increasing storage temperature. Variations depending on the

The minimum pH is 3.80, but it all depends on the type of acid used, among which acetic acid seems to be more effective. Over the past twenty years, the survival of *Salmonella* under varying conditions of acid stress (Acid Tolerance Response ATR) has been extensively studied, especially regarding sublethal exposure with organic acids, which make the pathogen adapt to the acid used. The complex molecular mechanisms and environmental factors involved in ATR have been studied. An interesting discussion on this topic can be found in the article by Álvarez-Ordóñez *et al*. (2011). The increase in resistance to acids is very consistent, not only for the chances of survival of *Salmonella* in food, but also because it can lead the pathogen to resist to gastric pH (<1.5) and thus pass through the intestine unharmed. Generally speaking, we can say that *Salmonella* is very sensitive to acetic acid and lactic acid, while it is much more resistant to citric acid, used to acidify foods. In turn, these acids are more active if storage or treatment temperatures are close to the pathogen's minimum or maximum values of growth. Finally, we also have to underline that the acidification and/or heat treatment should not be applied to food in sublethal conditions, in order to avoid adaptation phenomena of pathogenic strains to the same treatment or even to different treatments (salt, water activity, etc.). Leyer & Johnson (1993) tested a strain of adapted to acid *S*. Typhimurium by constantly lowering the pH, finding out that the adaptation was not only due to the rebalance of intracellular pH, but also to a change in

An incorrect method of disinfection and sanitization can make *Salmonella* persist on tools and utensils used in the food industry and kitchens, with the ability to form *biofilm* and,

Møretrø *et al*. (2009), using a treatment with a concentration of 100 ppm chlorine or 50 ppm of iodine for 15 minutes, noticed a biofilm can be completely removed, while with sodium hypochlorite (approximately 400 ppm) or cationic surfactants (benzalkonium chloride) for 5 minutes, *Salmonella* biofilm can resist on stainless steel surfaces. 70% ethanol for 5 min. is

Compared to foods of animal origin, which are usually consumed once cooked, fruit and vegetables are mostly eaten raw and therefore a significant part of foodborne outbreaks due to the consumption of raw vegetables has been attributed to *Salmonella* (Cantoni & Bersani, 2010). Animal faeces and irrigation water are the main ways for *Salmonella* to spread to crops (Islam *et al*., 2004). The water can contaminate the food if it is used for irrigation, for washing or for handling it (Rondanelli *et al*., 2005). The salmonellae present in not perfectly ripened manure or in irrigation water invade the plants by gripping to the roots or contaminating the leaves (Cantoni & Bersani, 2010). Studies headed by Professor

membrane proteins and not in the lipopolysaccharidic component.

therefore, enable the spread of the pathogen.

unable to remove the biofilm (Ramesh *et al*., 2002).

**4.** *Salmonella* **in vegetables** 

serotype can be noticed (ICSFM, 1996).

**pH value** 

**Disinfectants** 

Gadi Frankel (2008) of the Imperial College, London, UK, have revealed how salmonellae use their flagella to stick to salad and basil leaves. The results were presented at the 21st ICFMH International Symposium "Food Micro 2008" held in Aberdeen. This ability to attach itself to vegetables is described for a certain strain, *S. enterica* ser. Senftenberg, but not for *S.* Typhimurium (Frankel, 2009). Increased understanding of the mechanism that pathogens such as *Salmonella* use to adhere to vegetables is important if scientists are to develop new methods to prevent this type of contamination and the disease it causes (Berger *et al*., 2010). Schikora *et al*. (2008) have shown that *S.* Typhimurium, until now considered dangerous only for animals, can be a real danger for the vegetable kingdom too. Like any other plant pathogen, *S.* Typhimurium triggers the plant's immune defences and does not just cover the root surface, but enters physically into the plant's cells. The researchers attached a fluorescent probe to the bacterium and injected it, following its route: in just 17 hours the root cells were infected. Moreover, the infection later occurred simply by placing the plant (*Arabidopsis*) and the bacterium in the same liquid. *Salmonella* strains were detected in: aubergines, green salads, fennel, lettuce, onions, mustard, orange juice, pepper, parsley, spinach, strawberries, tomatoes, watermelons, coconuts, cereals, barley, chocolate and soy sauce (Cantoni & Bersani, 2010; Cantoni & Ripamonti, 1998). Today more and more ready-to-eat (RTE) vegetables are available in supermarket fridges because they are offered to the consumer as a convenience food, every part can be used, and, being already washed, peeled and chopped, they are quick and easy to prepare (Catellani *et al*., 2005). For their packing, various techniques are used, such as modified atmosphere packaging (MAP) – the gaseous composition of which varies according to the vegetable –, vacuum packaging, and ordinary atmosphere packaging. For the first two methods, the product should be packaged at refrigeration temperature, while with ordinary atmosphere it just needs to be kept cool. CO2 has the function to slow the breathing and the appearance of rotting, to inhibit pectinolytic enzymes and the development of *Pseudomonas* and other Gram-negative bacteria (Galli & Franzetti, 1998). Manvell & Ackland (1986) show that RTE vegetables can host various saprophytic microbial forms: 80-90% are Gram negative spoiling bacteria (including *Pseudomonas* spp, *Enterobacter* spp, *Erwinia* spp) and the rest are yeasts and moulds. If Good Manufacture Practices are respected, pathogens (*Salmonella* spp., *L. monocytogenes*, *E. coli* O157, enterovirus) or protozoa (*Giardia*, *Entamoeba, Cryptosporidium*) are detected only occasionally (Catellani *et al*., 2005). The study of *Salmonella* has dramatically contributed to the knowledge of the epidemiology of these infections. Large-scale distribution, particularly of fruit and vegetables, sets the conditions for events that touch a very wide area, involve the exposure of a big number of individuals, and that are difficult to recognize for lack of sophisticated surveillance systems that should involve international collaboration networks.

#### **5.** *Salmonella* **in eggs and egg products**

Eggs laid by healthy animals are generally safe to eat because if they kept in sound hygiene conditions they can be considered almost sterile inside, especially as regards bacterial agents of food diseases (Bozzo, 2008; Galli & Neviani, 2005). Nevertheless, *Salmonella* spp. is the most important pathogen transmitted by eggs (ICMSF, 1998). The natural defence factors that may affect the egg's infection by microbiological contaminations are:


Food as Cause of Human Salmonellosis 57

having been to several European countries with different *Salmonella* prevalence. The research seems to confirm the existence of a clear causal link between the presence of salmonellae in the egg production chain and the human disease. In Spain, Soler Crespo *et al*. (2005) focused on foodborne infections associated with the intake of eggs and egg products between 2002 and 2003. These outbreaks alone would account for 41% of all the episodes of food poisoning recorded throughout the duration of the study. The risk factors most often identified by the authors are the storage of the products at excessively high temperatures, the consumption of raw foods and a too long wait between the preparation and the consumption of the food. **Table 4** shows some events in epidemic proportions observed in recent decades in various parts of the world. These epidemics serve as a constant reminder of the fact that food technology cannot always protect against infectious diseases that may result in large-scale epidemics (multistate outbreaks) (Winn *et al*., 2009). In the United States, however, the introduction of a program for egg safety and quality (egg quality assurance programs [EQAPs]) plays an important role in

reducing disease by *S.* Enteritidis transmitted from eggs (Mumma *et al*., 2004).

Poultry import

Reproducers

Poultry Feed Man Infectious animals Water Means of transport equipment

Horrizontal transmission

Shell contamination endogenous contamination contamination among chicks

Horrizontal transmission

Cross contamination of carcasses

Incubators

Hen and broiler farms

Slaughterhouse

Table 3. *Salmonella* infection cycle in poultry farming.


In addition to the factors mentioned above, we must add the environmental protection factors that are related to hygiene: egg-laying place, litter, surfaces, air, handlings, shell, duration and means of storage. The eggs can, however, be infected transovarianly with *Salmonella* by sick hens or healthy carriers (Cantoni & Ripamonti, 1998). There are many cases reported in the literature in which *Salmonella* was detected in eggs laid by hens with ovarian localization of this pathogen (in this case we speak about "endogenous contamination") (Bozzo, 2008). Through good hygiene practices in breeding facilities, it is possible to limit the number of microorganisms on the shell, as more than 90% of the contaminations of various origins occur after egg laying (Gandini, 1993). These *exogenous contaminations* of the egg can occur at different times: during transport or packaging or during the shelling (Bozzo, 2008). There is evidence (EFSA, 2009b) to indicate that crosscontamination between egg shells may occur during the manufacturing processes (sorting of the eggs, packing, etc.). The probability of this cross-contamination depends on the percentage of eggs contaminated with *Salmonella*, and the prevalence of eggs tested positive for *Salmonella* is also affected by the type of technology used and hygiene practices applied. However, the authors argue that we lack sufficient data to evaluate the occurrence of penetration through the shell and the proliferation of *Salmonella* due to cross- contamination during processing and, therefore, to assess the risks for consumers. The factors that influence the passage of microorganisms into the egg are: dampness, the shell's degree of contamination, the age-related decline in physical defences of the hen and the type of dirt that causes changes in surface tension (Galli & Neviani, 2005). Table eggs are pointed at as a major source of *Salmonella* and egg refrigeration has been recommended as one of many possible measures along the food chain to reduce the incidence of salmonellosis in the human population (EFSA, 2009b). The panel of experts on biological hazards states that refrigerating table eggs to temperatures at or below 7 °C limits the multiplication of pathogens such as *Salmonella*. If the cold chain is maintained, starting the refrigeration already on the farm is the measure with the highest positive effect in order to limit the proliferation of *Salmonella*. Table egg refrigeration is another safety measure together with other steps taken on the farm and during the processing as part of an integrated approach. Interruption of the cold chain is a factor that increases the risk of condensation and this may increase the penetration of bacteria into the egg (Ricci, 2005). The Salmonella infection cycle in poultry farms is summarized in **Table 3**. As stated in the EU Summary Report on trends and sources of zoonoses, zoonotic agents and resistance to antimicrobiotics (EFSA, 2007), the reported cases of human salmonellosis in the EU, respectively amounted to 154,099 and 31.1/100,000 inhabitants. The report also indicates that the prevalence of *Salmonella* in table eggs was 0.8%. According to the opinion of the European Scientific Committee on veterinary measures related to Public Health on *Salmonella* in food products in 2003, eggs and food produced with raw eggs (unpasteurized) are among the food categories most likely to cause cases of human salmonellosis (EFSA 2009). In Sweden, de Jong & Ekdhal (2006) compared the EFSA data on the prevalence of *Salmonella* on European egg-laying hen farms and the prevalence of human salmonellosis, revealing a high linear correlation between the two aspects. The same study analyzed the cases of salmonellosis in travellers returning to Sweden after


In addition to the factors mentioned above, we must add the environmental protection factors that are related to hygiene: egg-laying place, litter, surfaces, air, handlings, shell, duration and means of storage. The eggs can, however, be infected transovarianly with *Salmonella* by sick hens or healthy carriers (Cantoni & Ripamonti, 1998). There are many cases reported in the literature in which *Salmonella* was detected in eggs laid by hens with ovarian localization of this pathogen (in this case we speak about "endogenous contamination") (Bozzo, 2008). Through good hygiene practices in breeding facilities, it is possible to limit the number of microorganisms on the shell, as more than 90% of the contaminations of various origins occur after egg laying (Gandini, 1993). These *exogenous contaminations* of the egg can occur at different times: during transport or packaging or during the shelling (Bozzo, 2008). There is evidence (EFSA, 2009b) to indicate that crosscontamination between egg shells may occur during the manufacturing processes (sorting of the eggs, packing, etc.). The probability of this cross-contamination depends on the percentage of eggs contaminated with *Salmonella*, and the prevalence of eggs tested positive for *Salmonella* is also affected by the type of technology used and hygiene practices applied. However, the authors argue that we lack sufficient data to evaluate the occurrence of penetration through the shell and the proliferation of *Salmonella* due to cross- contamination during processing and, therefore, to assess the risks for consumers. The factors that influence the passage of microorganisms into the egg are: dampness, the shell's degree of contamination, the age-related decline in physical defences of the hen and the type of dirt that causes changes in surface tension (Galli & Neviani, 2005). Table eggs are pointed at as a major source of *Salmonella* and egg refrigeration has been recommended as one of many possible measures along the food chain to reduce the incidence of salmonellosis in the human population (EFSA, 2009b). The panel of experts on biological hazards states that refrigerating table eggs to temperatures at or below 7 °C limits the multiplication of pathogens such as *Salmonella*. If the cold chain is maintained, starting the refrigeration already on the farm is the measure with the highest positive effect in order to limit the proliferation of *Salmonella*. Table egg refrigeration is another safety measure together with other steps taken on the farm and during the processing as part of an integrated approach. Interruption of the cold chain is a factor that increases the risk of condensation and this may increase the penetration of bacteria into the egg (Ricci, 2005). The Salmonella infection cycle in poultry farms is summarized in **Table 3**. As stated in the EU Summary Report on trends and sources of zoonoses, zoonotic agents and resistance to antimicrobiotics (EFSA, 2007), the reported cases of human salmonellosis in the EU, respectively amounted to 154,099 and 31.1/100,000 inhabitants. The report also indicates that the prevalence of *Salmonella* in table eggs was 0.8%. According to the opinion of the European Scientific Committee on veterinary measures related to Public Health on *Salmonella* in food products in 2003, eggs and food produced with raw eggs (unpasteurized) are among the food categories most likely to cause cases of human salmonellosis (EFSA 2009). In Sweden, de Jong & Ekdhal (2006) compared the EFSA data on the prevalence of *Salmonella* on European egg-laying hen farms and the prevalence of human salmonellosis, revealing a high linear correlation between the two aspects. The same study analyzed the cases of salmonellosis in travellers returning to Sweden after

protein molecules (ovostatin, ovomucoid, cystatin, etc.).

having been to several European countries with different *Salmonella* prevalence. The research seems to confirm the existence of a clear causal link between the presence of salmonellae in the egg production chain and the human disease. In Spain, Soler Crespo *et al*. (2005) focused on foodborne infections associated with the intake of eggs and egg products between 2002 and 2003. These outbreaks alone would account for 41% of all the episodes of food poisoning recorded throughout the duration of the study. The risk factors most often identified by the authors are the storage of the products at excessively high temperatures, the consumption of raw foods and a too long wait between the preparation and the consumption of the food. **Table 4** shows some events in epidemic proportions observed in recent decades in various parts of the world. These epidemics serve as a constant reminder of the fact that food technology cannot always protect against infectious diseases that may result in large-scale epidemics (multistate outbreaks) (Winn *et al*., 2009). In the United States, however, the introduction of a program for egg safety and quality (egg quality assurance programs [EQAPs]) plays an important role in reducing disease by *S.* Enteritidis transmitted from eggs (Mumma *et al*., 2004).

Table 3. *Salmonella* infection cycle in poultry farming.

Food as Cause of Human Salmonellosis 59

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


a Confirmed cases, unless stated otherwise.

b Not stated

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

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