3. HAZARD CHARACTERIZATION OF SALMONELLA

3.1 Summary

This section reviews the basic characteristics of the organism, human host factors, and composition factors of the food matrix that influence the outcome of exposure to non-typhoidal Salmonella enterica. Human volunteer feeding trial data for various Salmonella serotypes and dose-response models that have been developed based on those studies are reviewed. Limitations in the results from human feeding trials are discussed. Additional data were collected from salmonellosis outbreak reports that provided detailed information on parameters such as the numbers of the pathogen in the contaminated food, approximate amount of food eaten, numbers of people who consumed the food, numbers of people exposed who developed the clinical symptoms of acute gastroenteritis, age information, and, in some cases, prior health information.

The existing dose-response models were compared with the outbreak data as a validation step. These models failed to adequately represent the observed outbreak data. Consequently, a new dose-response model was developed, based on the outbreak data, and was used with exposure assessment information for eggs and broiler chickens to derive the risk estimates. In addition, an analysis of the outbreak data was done to attempt to derive quantitative estimates for the effect of host age and Salmonella serotype on the probability of acute gastroenteritis. No differentiation could be made on the basis of the dose-response outbreak data available at this time. The dose-response relationship derived from the outbreak data measured the host response in terms of acute gastroenteritis. Follow-up patient information on progression of the primary illness to more severe consequences was not detailed in the outbreak reports; in addition, the severity of illness - i.e. severity characterized by hospitalization, bacteraemia, reactive arthritis, other symptoms or death - is often complicated by factors that are difficult to quantify, and hence the corresponding risk estimates were not calculated.

3.2 Organism, host and matrix characteristics

3.2.1 Characteristics of the organism

In order for infection with a non-typhoid Salmonella to occur, the organism must survive a rather hostile environment. It must adapt to differences in growth conditions between the outside environment and the host, and within highly variable microenvironments within the host. The invasive journey towards illness in the host must negotiate distinct temperature differences, osmolarity, oxidation-reduction potentials, environmental iron concentrations, pH and organic and inorganic nutrient environments (Slauch, Taylor and Maloy, 1997). An infective Salmonella must then survive peristalsis, the epithelial surface and the host immune response.

Non-typhoid salmonellae possessing certain adaptive characteristics are more likely to produce foodborne disease. First, they must be acid tolerant to survive the pH of the stomach. They must also be able to attach themselves to and invade the intestinal epithelia and Peyer’s patches (D’Aoust, 1997). Bacterial virulence factors include those that promote adhesion to host cells in the intestines: specific fimbriae, chromosome-coded bacterial surface adhesins, haemagglutinins, and epithelial cell induction of bacterial polypeptides that can promote colonization and adhesion.

Resistance of Salmonella to lytic action of complement varies with the length of the O side chains of lipopolysaccharide (LPS) molecules (D’Aoust, 1991). Smooth varieties are more resistant than rough types. The O side chains of the lipopolysaccharide molecules have also been shown to affect invasiveness and enterotoxin production (Murray, 1986).

Siderophores, which chelate iron, are necessary for the accumulation of sufficient environmental iron to allow growth of Salmonella. Siderophores include hydroxamate, phenolate and catechol types. Porins are hydrophobic bacterial cell proteins that enhance the virulence of Salmonella by repression of macrophage and polymorphonuclear-dependent phagocytosis. Salmonella porins may, however, have a limited importance in pathogenicity. Chromosomal determinants include specific virulence genes whose potential for action is tightly controlled by regulatory genes. Gene expression is determined by the environment and invasion occurs by the two-component regulatory system PhoPQ, which enables survival of Salmonella within the hostile environment of phagocytes (Slauch, Taylor and Maloy, 1997).

Virulence plasmids in the range of 50-100 kilobases been associated with the ability to spread after colonization, invasion of the intestine, ability to grow in the spleen, and a general suppression of the host immune response (Slauch, Taylor and Maloy, 1997). The presence of virulence plasmids in Salmonella is limited. Chiu, Lin and Ou (1999) studied virulence plasmids in 436 clinical human samples in Taiwan: 287 isolates were from faeces, 122 from blood and the remaining were isolated from other sites. Of the non-faecal isolates, 66% contained a virulence plasmid, compared with 40% of the faecal isolates. All the isolates (n=50) of the three highly invasive serotypes - S. Enteritidis, S. Dublin and S. Choleraesuis contained virulence plasmids. Virulence plasmids have also been confirmed in S. Typhimurium, S. Gallinarum-pullorum and S. Abortusovis, but are notably absent in S. Typhi, which is host-adapted and highly infectious.

Other factors that affect the ability of the organism to cause disease include the presence of cytotoxins and diarrhoeagenic enterotoxins. The enterotoxin is released into the lumen of the intestine and results in the loss of intestinal fluids (D’Aoust, 1991).

Antimicrobial resistance can have two effects on the outcome of exposure: there can be an accompanying change in the virulence of the organism, or there can be a poorer response to treatment because of the empirical choice of an antimicrobial to which the organism is resistant (Travers and Barza, 2002). An increase in virulence could result from linkage of resistance factors to other virulence genes, such as those for adherence, invasion and toxin production. A study by the United States Centers for Disease Control and Prevention (CDCs) (Lee et al., 1994) revealed that subjects with infections caused by antimicrobial-resistant Salmonella were significantly more likely to be hospitalized than those with antimicrobial-susceptible infections (35% vs 27%, P = 0.006) and this difference persisted even after correction for underlying illness. Patients infected with resistant strains also tended to be ill longer (median: 10 vs 8 days) and hospitalized longer (median: 5 vs 4 days). Most subjects were treated with an agent to which the organism was susceptible, and therefore the difference in hospitalization rates probably reflected increased virulence of the infecting organism rather than inappropriate choice of treatment. Thus, the data suggest that antimicrobial-resistant strains are somewhat more virulent than susceptible strains, in that they cause more prolonged or more severe illness than do antimicrobial-susceptible strains (Travers and Barza, 2002).

Two potentially confounding factors in the study were the host susceptibility in terms of age, and potential differences in virulence between serotypes. Neither factor was controlled for in the study (Travers and Barza, 2002). Black race and less than one year of age appeared to be host characteristics associated with a resistant infection, although differences in the distribution of infecting serovars among ethnic and age groups contributed to the occurrence of such effects. Varying food preferences or methods of food preparation might have been at the basis of different serovar distribution. The same consideration may explain the results of an earlier study, which associated infection with S. Heidelberg, penicillin intake, Hispanic origin, more than 60 years of age and antacid use to infection with a multi-resistant Salmonella (Riley et al., 1984). The conclusion of this study - that multi-resistant organisms are more dependent on host characteristics than sensitive organisms to cause disease - should be qualified accordingly.

3.2.2 Host characteristics

Literature tends to be biased towards reporting statistically significant and positive results. This review can only reflect such a bias, and the focus is evidently on host factors for which a statistically significant association to salmonella gastroenteritis and related complications has been reported. Where clear indication of a non-significant finding is made in the original study, such a finding is also reported. In addition, since not all studies considered the same factors, the significance of one factor in a given study may merely depend on the presence or absence of other ones. For instance, while a Swiss study considered travel abroad an important source of resistant Salmonella (Schmid et al., 1996), such an association was not seen in a United States of America study (Lee et al., 1994). Such apparent inconsistencies may have various explanations, but their discussion is beyond the scope of this review.

Host factors that can affect the outcome of exposure to the pathogen by ingestion, and which are considered in this review, are the following:

Demographic and socioeconomic factors

The following factors are considered in this section: age; gender; race and ethnicity; nutritional status; socioeconomic and environmental factors; and travel abroad.

Age

A common observation is that the age of patients with Salmonella infections is distributed according to a bimodal distribution with peaks in children and elderly. In a Belgian hospital-based study covering isolates for a 20-year period (1973-1992), S. Typhimurium and S. Enteritidis were mainly isolated in children under 5 years of age (Le Bacq, Louwagie and Verhagen, 1994). The age distribution was, however, less accentuated for S. Enteritidis than for S. Typhimurium. Both serovars were more likely to lead to bacteraemia in middle and older age groups than in those younger than 5 years of age (Le Bacq, Louwagie and Verhagen, 1994), confirming a previous observation made in the United States of America (Blaser and Feldman, 1981). Another study reports on Salmonella isolates from a Hong Kong hospital for the period 1982-1993 (Wong et al., 1994). Among both intestinal and extra-intestinal isolates, S. Typhimurium, S. Derby and S. Saintpaul predominated in infants. In patients older than 1 year of age, S. Derby and S. Typhimurium were the most common intestinal isolates, while S. Typhi, S. Typhimurium and S. Enteritidis were the most common extra-intestinal isolates. In a British population-based study, highest age-specific isolation rates for S. Enteritidis were observed in children aged under 2 years, and S. Typhimurium in those under 1 year (Banatvala et al., 1999).

In children in their first year, the peak incidence is generally observed in the second and third months (Ryder et al., 1976; Davis, 1981). The study from Hong Kong showed, however, a peak at 12 months of age (Wong et al., 1994). In a study of Peruvian children, the IgG and IgM titres against Salmonella serogroups AO, BO and DO were higher at 12 months of age than at 2 or 3 months of age, which was interpreted as an indication of acquired immunity (Nguyen et al., 1998). In the United States of America, infants under the age of 1 year have the highest reported incidence rate of salmonellosis, with the highest rate in infants 2 months of age, and an abrupt decrease after infancy (Olsen et al., 2001). Most cases are relatively mild. However, as with the immunocompromised and the elderly, children also face a relatively higher rate of severe outcomes, including death, than other demographic categories. Olsen et al. (2001) note a 4-13-fold higher rate of invasive disease in young children than other age groups. Buzby (2001) noted that most children who contract salmonellosis are believed to have been infected from contaminated food, as outbreaks in childcare facilities are rare. However, a matched case-control study among children in France found that cases were more likely to report a case of diarrhoea in the household 3-10 days before onset of illness, particularly in the age group less than 1 year old, indicating a role of person-to-person transmission of salmonellosis in infants (Delarocque-Astagneau et al., 1998).

It is noted that age associations may be influenced by other factors. In the very young, this includes increased susceptibility upon first exposure, but also that medical care is quickly sought for infants and incidents reported, and they are also more likely to be tested than adults with foodborne illness. Similarly, the very elderly with diarrhoea may also be expected to be more frequently cultured than other age groups (Banatvala et al., 1999). As mentioned earlier, differences in the distribution of infecting serovars among age groups was considered the reason for an apparent increased risk of resistant Salmonella infection in infants (Lee et al., 1994). When exposed to the same contaminated food in an outbreak, with the assumption that the individuals involved were exposed to a similar dose, no significant age-related difference was observed between those who became ill and those who remained healthy (range: 1-61 years old; median, 30; 12 children under the age of 15 years, 4 of whom became ill) in an outbreak investigated by Rejnmark et al. (1997). Similarly, no age-related association with hospitalization was noted in that investigation. Cowden and Noah (1989) postulated that the popularity of eggs and egg dishes in the diets of weaned and older children poses a serious problem. This suggests an increased rate of exposure to S. Enteritidis. Moreover, age association may reflect behavioural characteristics. For instance, eating snow, sand, or soil - a behaviour more likely in children - was found to be associated with infection by S. Typhimurium O:4-12 (Kapperud, Stenwig and Lassen, 1998). Handling pets, including reptiles, and farm animals, followed by hand-to-contact without washing increases exposure opportunities.

Gender

In terms of number of isolates, several studies indicate that men seem to be generally more affected than are women. A male-to-female ratio of 1.1 has been reported on various occasions (Blaser and Feldman, 1981; Le Bacq, Louwagie and Verhagen, 1994; Wong et al., 1994). However, in other studies, the isolation rate for women exceeded that for men between the ages of 20 and 74 years, although boys 15 years or under had a slightly higher age-specific isolation rate than girls (Olsen et al., 2001). The significance of such a findings does not appear to have been addressed. Several factors may play an important role, such as proportion of the two genders, as well as different age distributions for males and females within a country or hospital catchment area. In the evaluation of a single study, it should be pointed out that the occurrence of other factors, e.g. pregnancy or use of antacids, relates to one gender more often or exclusively, and gender may thus have the effect of a confounder. Furthermore, differences in food handling practices and hygiene during food preparation, and amount of food consumed, may also be contributors to any apparent gender differences.

Race and ethnicity

The potential role of race and ethnicity has seldom been considered. As mentioned above, an association with black race and Hispanic origin was reported for resistant Salmonella infections (Lee et al., 1994; Riley et al., 1984). In the former case, the association was explained by differences in the distribution of infecting serovars among ethnic groups, which in turn depended on varying food preferences or methods of food preparation.

Nutritional status

An association between altered nutritional status and acute gastroenteritis has been shown in AIDS patients (Tacconelli et al., 1998). Apart from this report, no direct reference to the role of nutritional status was found in the literature.

Social, economic and environmental factors

Isolation rates of several Salmonella serovars among groups of different socioeconomic extraction have been compared on the basis of the Townsend score, an index for deprivation (Banatvala et al., 1999). While isolation rates for S. Typhimurium were not related to the Townsend score, the highest isolation rates of S. Enteritidis were observed in more prosperous areas. It was advanced that populations living in such areas more frequently ingested vehicles harbouring S. Enteritidis.

Sanitation deficiencies have been associated with high rates of enteric disease but direct reference to the potential role of Salmonella is scarce. In the 1950s, lack of sanitation, poor housing, limited water supply and poor personal hygiene were associated with high Shigella rates in Guatemala (Beck, Muñoz and Scrimshaw, 1957). A similar observation was made in the United States of America where, in areas of inadequate sanitary facilities, poor housing and low income, Shigella infections were the major causes of diarrhoeal disease. In particular, there were nearly twice as many cases of diarrhoea among persons living in dwellings having outhouses than among those whose houses had indoor lavatories (Schliessmann et al., 1958). In certain Guatemalan villages, the habits of the people and the density of the population were found to be more important determinants than type of housing (Bruch et al., 1963). In a study conducted in Panama, six representative types of dwellings were considered as an index of social and economic influences on the prevalence of enteric pathogens among infants with diarrhoeal disease (Kourany and Vasquez, 1969). Each dwelling type differed characteristically from one another but five of the six types were considered substandard and their occupants were of low socioeconomic status. Infection rates for enteropathogenic Escherichia coli, Shigella and Salmonella among infants from the various groups of substandard dwellings ranged from 6.0 to 10.2%, in contrast to the zero infection rate observed in infants from the better-type housing. It is worth noting that the literature on sanitation and housing was mainly published in the 1950s and 1960s. It is possible that safety improvement in the water supply consequent to economic development has sensibly diminished the importance of those factors in several countries.

A French study on sporadic S. Enteritidis infections in children investigated the influence of diarrhoea in another household member in the 3 to 10 days before a child shows clinical symptoms. The strength of the association with such a factor appeared stronger for cases in infants (1 year of age or less) compared with cases in children between 1 and 5 years of age (Delarocque-Astagneau et al., 1998). On the basis of this observation, as well as other results of the study, it was postulated that S. Enteritidis infection in children of less than 1 year of age may arise from person-to-person contact, while children between 1 and 5 years of age contract the infection by consuming raw or undercooked egg products or chicken.

A seasonal pattern in isolations, which generally shows increased rates during hotter months, has been documented. For instance, increased isolation rates for S. Enteritidis, S. Typhimurium, S. Virchow and S. Newport were observed in summer in a British study (Banatvala et al., 1999). The French study mentioned in the previous paragraph noted that the association between S. Enteritidis infection and prolonged storage of eggs was stronger during the summer period.

Travel abroad

Travel abroad is a risk factor for Salmonella gastroenteritis that has been consistently demonstrated in both North America and Europe. For California residents, Kass et al. (1992) demonstrated an association between sporadic salmonellosis and travel outside the United States of America within 3 weeks prior to the onset of illness. Possible variations related to serovar in sporadic salmonellosis were indicated by a study concerning residents of Switzerland (Schmid et al., 1996). Having been abroad within three days prior to clinical onset of the illness was found to be associated with both S. Enteritidis and serovars other than Enteritidis, although to a greater extent for the latter case. Little difference was seen between the results of all S. Enteritidis phage types (PTs) and of S. Enteritidis PT4. While most patients with S. Enteritidis were more likely to have travelled within Europe, the majority of non-Enteritidis infections might have been imported from outside Europe. Individuals of a British region with Salmonella infection were more likely to have reported travel abroad in the week before the onset of illness (Banatvala et al., 1999). Frequency of overseas travel between patients with S. Enteritidis or S. Typhimurium was not different, but it was among patients with other serovars. Indication of how travel abroad may lead to salmonellosis can be found in a study referring to residents of Norway (Kapperud, Lassen and Hasseltvedt, 1998). This study suggested that about 90% of the cases from whom a travel history was available had acquired their infection abroad, but failed to show an association to either foreign travel among household members or consumption of poultry. However, consumption of poultry purchased abroad during holiday visits to neighbouring countries was the only risk factor considered by the study that remained independently associated with the disease. Only cases of S. Typhimurium allowed for a separate analysis that showed an association with both poultry purchased abroad and foreign travel among household members.

Genetic factors

As far as acute gastroenteritis caused by Salmonella is concerned, no host genetic factors have been reported. Reports concerning race and ethnicity should be considered in the light of eating habits.

The putative association of the gene Human Leukocyte Antigen B27 (HLA-B27) for patients with spondyloarthropathies, in particular reactive arthritis and Reiter’s syndrome, has been described. The HLA-B27 gene has a very high prevalence among the native peoples of the circumpolar arctic and sub-arctic regions of Eurasia and North America, and in some regions of Melanesia. In contrast, it is virtually absent among the genetically unmixed native populations of South America, Australia, and among equatorial and southern African Bantus and Sans (Bushmen) (Khan, 1996). Fifty percent of Haida Indians living on the Queen Charlotte Islands of the Canadian province of British Columbia have the HLA-B27 gene, which is the highest prevalence ever observed in a population. The prevalence among Americans of African descent varies between 2 to 3%, while 8% of the Americans of European descent posses the gene (Khan, 1995).

Health factors

Immune status

The host immune status is, as in any other infectious disease, a very important factor in determining both infection and clinical illness. In general terms, its importance does not seem to have been the direct goal of any formal work and has thus to be indirectly assessed though other factors, such as age or acquired immunodeficiency. Evidence for the development of immunity against non-typhoidal S. enterica was recognized in human volunteer experiments (McCullough and Eisele, 1951b). When subjects who became ill on the first challenge were later re-challenged, if they became ill again the severity of the illness was usually less than that of the initial illness, despite higher challenge doses being used. This is in contrast to experiments with typhoid, where vaccines gave protection against low- but not high-challenge doses, and once clinical disease occurred, the severity was not altered by previous vaccination. Evidence that immunity is partially serotype specific is suggested by the increased incidence of salmonellosis amongst people who have travelled, and are presumably exposed to different serotypes and strains of Salmonella in food and water in other countries. There is a need to examine country- or region-specific population immunities in general to better understand the applicability of dose-response models to populations, countries and regions other than those where dose-response data were acquired.

Concurrent infections

Persons infected with Human Immunodeficiency Virus (HIV) tend to have recurrent enteric bacterial infections. Such infections are often virulent and associated with extraintestinal disease (Smith et al., 1988; Angulo and Swerdlow, 1995). Six risk factors for enteric salmonellosis have been identified in HIV-infected patients: increasing value on the prognostic scoring system APACHE II (Acute Physiology and Chronic Health Evaluation); altered nutritional status; previous antibiotic therapy; ingestion of undercooked poultry or eggs, or of contaminated cooked food; previous opportunistic infections; and stage C of HIV infection (Tacconelli et al., 1998).

Underlying diseases

The significance of Acquired Immunodeficiency Syndrome (AIDS) has been discussed in the previous paragraph. The risk represented by other underlying conditions was evaluated in a large nosocomial foodborne outbreak of S. Enteritidis that occurred in 1987 in New York (Telzak et al., 1991). Gastrointestinal and cardiovascular diseases, cancer, diabetes mellitus and alcoholism as well as use of antacids and antibiotics were the factors considered. However, diabetes was the only condition that was independently associated with infection after exposure to the contaminated meal. Although diabetic cases were more likely to develop symptomatic illness compared with non-diabetic, the difference was not statistically significant. Decreased gastric acidity and autonomic neuropathy of the small bowel (which leads to reduced intestinal motility and prolonged gastrointestinal transit time) are the two biologically plausible mechanisms for the increased risk of S. Enteritidis infection among diabetics. Among patients with sporadic salmonellosis in Northern California, diabetes mellitus and cardiac disease were both associated with clinical illness (Kass et al., 1992). This study contemplated 14 health conditions. Non-gastrointestinal medical conditions and, to a larger extent, a recent history of gastrointestinal disorder were associated with sporadic S. Typhimurium O:4-12 infection in Norway (Kapperud, Stenwig and Lassen, 1998). It was, however, noted that physicians are more likely to require a stool culture from patients with preceding illness. In a British epidemiological study, cases of Salmonella infection were more likely to report a long-term illness (including gastroduodenal conditions) than controls (Banatvala et al., 1999). All individuals with diabetes mellitus, malignancy or immunodeficiency were cases.

Concurrent medications

A number of investigations have examined the effects of antacids and prior or concurrent antimicrobial usage as factors influencing likelihood of contracting salmonellosis or affecting the severity of the outcome. The evidence found in the literature concerning their association with human salmonellosis is contrasting. While some studies have shown an association with antacid use (Banatvala et al., 1999), others have failed to do so (Telzak et al., 1991; Kapperud, Stenwig and Lassen, 1998). A similar situation is found for the use of antibiotics in the weeks or days preceding the infection or disease onset: some studies have demonstrated an association (Pavia et al., 1990; Kass et al., 1992; Bellido Blasco et al., 1998) but others have not (Telzak et al., 1991; Kapperud, Stenwig and Lassen, 1998; Banatvala et al., 1999). Having a resistant Salmonella infection has been associated with previous antibiotic use (Lee et al., 1994). A delay between antimicrobial use and onset of symptoms suggests that the effect may be due to prolonged alteration of the colonic bacterial flora, resulting in decreased resistance to colonization (Pavia et al., 1990).

Among the 11 different medical therapies considered by a North California, USA, study on sporadic clinical salmonellosis, which included antacids and antibiotics, only hormonal replacement therapy (principally conjugated estrogen) in older women was found to be associated with clinical salmonellosis (Kass et al., 1992). An association between serovars other than S. Enteritidis and intake of medications other than antacids was shown in Switzerland (Schmid et al., 1996). Regular use of medications was a risk factor for S. Typhimurium O:4-12 infection in Norway (Kapperud, Stenwig and Lassen, 1998). In the same study, use of antacids and antibiotics were not risk factors.

Pregnancy

There is a little information concerning the effect of salmonellosis specifically on pregnant women and foetuses or neonates. No studies were found to indicate that pregnant women are at an increased risk for Salmonella-induced enteritis. However, when a pregnant woman suffers from foodborne infection the foetus or neonate may also be affected. A recent review by Smith (2002) of Campylobacter jejuni infection during pregnancy summarizes the small amount of available data on the consequences of maternal C. jejuni enteritis or bacteraemia, or both. Outcomes may include abortion, stillbirth, premature labour, bacteraemic newborn infants, and neonates with diarrhoea or bloody diarrhoea. Similar outcomes might be expected for some cases of salmonellosis in pregnant women.

3.2.3 Factors related to the conditions of ingestion

Empirical observation, mainly from outbreak investigations, shows that foodborne salmonellosis can be related to a variety of food items. Table 3.1 lists major foodborne outbreaks of human salmonellosis and shows the wide range of foods implicated in these outbreaks (D'Aoust, 1997).

Table 3.1. Major foodborne outbreaks of human salmonellosis and the food items implicated (Adapted from D'Aoust, 1997)

Gastric acidity is recognized as an important defence against foodborne pathogens. Pathogen, host and food factors interact in determining whether sufficient bacteria are able to withstand stomach acidity and go on to colonize the gut. Such an interaction appears extremely dynamic. Although Salmonella prefer to grow in neutral pH environments, they have evolved complex, inducible acid survival strategies that allow them to face the dramatic pH fluctuations encountered in nature and during pathogenesis (Bearson, Bearson and Foster, 1997). While the human stomach is normally pH 2, several host factors may cause decreased gastric acidity. Examples reported in the previous section are older age, diabetes mellitus, and use of antacid drugs. As for factors specifically related to food, it appears that a systematic treatment of this topic has not yet been carried out. Circumstantial evidence suggests that the following elements are of particular relevance: amount of food ingested; nutrient composition, including fat content of the food; buffering capacity of the food at the time of the meal; and nature of contamination. The reference to "food" rather than to "food item" emphasizes the importance of considering the whole meal.

In an S. Typhimurium outbreak, it was observed that persons who had eaten two or more pieces of chicken tended to have shorter incubation periods. However, both attack rate and illness severity did not appear to be a function of the amount of chicken consumed. It was concluded that the amount of food consumed provides only a crude estimate of dose because a homogenous distribution of the pathogen among the chicken pieces is unlikely (Glynn and Palmer, 1992). This also means that since infectivity is not uniformly distributed within a food, a larger meal may increase the chances of ingesting an infected portion. D’Aoust (1985) noted that in foodborne outbreaks involving fatty vehicles, relatively low doses can lead to substantial numbers of illness (chocolate: <100 cells of S. Eastbourne, 50 cells of S. Napoli; cheddar cheese: 100-500 cells of S. Heildelberg, 1-6 cells of S. Typhimurium). Microorganisms trapped in hydrophobic lipid moieties may survive the acidic conditions of the stomach and thus the fat content of contaminated foods may play a significant role in human salmonellosis. In contrast, experimental evidence in rats shows that Salmonella infection is not affected by milk fat (Sprong, Hulstein and van der Meer, 1999). Salmonella were actually protected from acid killing when inoculated onto boiled egg white - a food source high in protein and low in fat (Waterman and Small, 1998). The same study shows that the pH of the microenvironment occupied by the bacteria on the surface of a food source is critical to their survival.

The effect of substrate was studied in volunteers challenged with Vibrio cholerae fed in a medium with buffering capacity (Cash et al., 1974). The group of subjects that overcame the effect of a bicarbonate vehicle in less than 30 minutes (approximately half of the challenged individuals) experienced a lower attack rate than the group experiencing a prolonged buffering effect. Ingestion of low numbers of Salmonella between meals, i.e. on an empty stomach, was associated with an increased attack rate (Mossel and Oei, 1975). It was postulated that at such moments the pyloric barrier would initially fail. The authors also speculated that some food items, such as chocolate and ice cream, are more likely to be ingested between meals and thus lead to illness even with only a few organisms. A protective effect of alcoholic beverages was observed in an S. Enteritidis outbreak (Bellido Blasco et al., 1996). Besides the direct effect of ethanol on bacteria, alcohol may stimulate secretion of gastric acid. Last, but not least, an important factor in determining the survival of bacteria in the stomach may be how uniformly a food is contaminated. Although a uniform distribution is usually assumed, the very nature of bacterial growth in colonies would suggest that agglomerations of bacteria occur within the food. It can be speculated that the outer layers of bacteria would protect the inner ones, allowing some pathogen to survive the gastric passage.