<b>
<i>Salmonella</i>
</b> Epidemiology and Pathogenesis in Food-Producing Animals
TIMOTHY S. WALLIS AND PAUL A. BARROW*
[SECTION EDITOR: GORDON DOUGAN]
Posted July 25, 2005
Division of Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, United Kingdom
*Corresponding author. Phone: +44 1635 578411, Fax: +44 1635 577243, E-mail:
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The bacterial species Salmonella enterica subspecies enterica can be divided into over 2,400 antigenically distinct serovars, and the pathogenicity of most of these serovars is undefined. On the basis of pathogenesis, serovars can be classified by association with production of at least three distinct types of infection. A small number of serovars are capable of producing severe systemic disease in immunologically and physiologically healthy, outbred, healthy adult individuals of a narrow range of animal species. In general, transmission is by the fecal-oral route and bacterial multiplication is widely considered to take place primarily in the cells of the macrophage monocyte lineage. In the target species, the alimentary tract becomes involved pathologically only in the later stages of the disease, and thus, in the absence of disease little intestinal colonization takes place. Such infections are associated with S. enterica serovars Typhi and Paratyphi A and some strains of Paratyphi B, which produce typhoid in humans, and with serovar Gallinarum, which infects poultry and probably other birds. A second group of serovars is associated with systemic infections with a focus of infection frequently involving the reproductive tract when animals are pregnant (mammals) or in lay (birds) and possibly involving more extensive systemic multiplication in very young animals. These include serovar Dublin in cattle, serovar Abortusovis in sheep, serovar Abortusequi in horses, and serovar Pullorum. The vast majority of the remaining serovars are unable to produce systemic infections in healthy adult animals. They are, however, able to colonize the alimentary tract of a range of animals and may cause acute enteritis or subclinical infections. The molecular basis of Salmonella-induced enteropathogenesis is only now becoming better understood. As a consequence of intestinal colonization and high levels of fecal shedding, particularly in food animals, bacteria can enter the human food chain, infect the gut, and cause gastroenteritis.
A classification of Salmonella may also be made on the basis of host prevalence in which the serovars may be divided into three groups. Host-specific serovars typically cause systemic disease in a limited number of phylogenetically related species, as indicated previously. Thus, serovar Typhi, serovar Gallinarum, and serovar Abortusovis are almost exclusively associated with systemic disease in humans, fowl, and sheep, respectively. Host-restricted strains, while primarily associated with one or two closely related host species, may also infrequently cause disease in other hosts. For example, serovar Dublin and serovar Choleraesuis are generally associated with severe systemic disease in ruminants and pigs, respectively. In the United Kingdom between 1958 and 1967 (the most recent years for which meaningful epidemiological data are available), analysis of the incidence of salmonellosis in food animals demonstrated that 99% of all serovar Choleraesuis incidents were associated with pigs and 95% of all serovar Dublin incidents were associated with cattle (153). In contrast, the ubiquitous serovars, such as serovar Typhimurium and serovar Enteritidis, usually induce gastroenteritis in a broad range of unrelated host species.
Clearly the nature and severity of Salmonella infections in different animal species vary enormously and are influenced by many factors, including the infecting Salmonella serovar, strain virulence, infecting dose, host animal species, age and immune status of the host, and geographical region. All of these factors are likely to interrelate, and the discussion of all aspects of this is beyond the scope of this review. The following sections review the pathogenesis of different phases of Salmonella infections. However, this is not a simple task because pathogenesis varies enormously when different serovars infect different host species. Thus, simply because one serovar induces a particular response with a particular virulence factor does not necessarily hold true for other serovars/host combinations. In an attempt to illustrate this, the nature of Salmonella infections in several domesticated animal species is described to highlight differences in the epidemiology and pathogenesis of salmonellosis in different hosts. The biology of Salmonella serovar host specificity will be discussed in the context of our current understanding of the molecular basis of pathogenesis and the potential impact of different virulence determinants on Salmonella natural history.
The ability to colonize the intestine, as evidenced by the shedding of relatively large numbers of bacteria in the feces over a long period, is shared unequally by Salmonella serovars. The definition of colonization, to avoid inclusion of microorganisms passing through, together with other interactions with the host, has been discussed by previous authors (15). The phenotype has been extensively studied in poultry, because disease-free colonization with fecal shedding is a central component of the infection process that results in carcass contamination and human food poisoning. However, the biology is poorly understood or misunderstood and the molecular basis in Salmonella is only now being studied.
Understanding intestinal colonization in each of the major food animals is complicated by the fact that the gastrointestinal anatomy is so different in each species and influenced by the developmental age of the animal.
Cattle and sheep are ruminants in which, in the adult animal, orally ingested bacteria, including Salmonella, are required to pass through the reticulum and rumen, which has an enormous microbial load that exerts a large excluding effect, demonstrated by the very large infectious doses of Salmonella required to infect adult cows (67). In calves, the rumen is rudimentary and the esophageal groove allows liquids to bypass this organ and gain direct entry to the true stomach. The distal end of the small intestine contains large areas of confluent Peyer’s patch tissue. Pigs are monogastric and have a large cecum and spiral colon. The distal end of the small intestine again comprises confluent Peyer’s patch tissue rich in domed epithelium. By contrast, the chicken and other gallinaceous birds have a very different alimentary tract. Proximal to the true stomach is a storage organ, the crop, where a microbial lactic fermentation occurs. There are two large ceca where the highest Salmonella counts are found. There is a single small Peyer’s patch in the small intestine, but at the ileo-cecal junction there are also two clusters of lymphoid tissue, the cecal tonsils, where bacteria counts are also high. The high bacteria count suggests that these organs are important for transmission to the tissues, in particular the spleen, since chickens and other birds do not have lymph nodes.
How far colonization is an integral initial stage of systemic disease is perhaps currently more of a philosophical discussion. There is microbiological evidence that those serovars that typically produce systemic infections and are host-specific are poor colonizers of the intestine. All the evidence in the literature suggests that serovar Typhi in humans infects orally, but, soon after infection, the organism disappears from the intestine until clinical disease occurs when it reappears in the gut and is shed in the feces (49, 182). There is direct evidence that this also takes place with serovar Gallinarum in chickens (151a) and that, in the absence of clinical disease, the organism simply disappears from the gut soon after infection. That these serovars must survive in the intestine long enough for them to interact with appropriate cells of the epithelium that will facilitate their uptake is obvious; with large experimental doses, most of the inoculum is destroyed in the upper intestines with just a minority required to produce infection. Thus, the oral LD50 for serovar Gallinarum in chickens is 104 CFU, whereas it is less than 10 bacteria by any parenteral route, indicating that it is necessary for only a small number of bacteria to reach and penetrate the intestinal tissues. Where persistent colonization occurs, a combination of an understanding of the nature of the interaction between pathogen and host and the metabolic processes that enable the bacteria to survive and multiply in the intestine is required.
After early studies in the 1970s on the role of adhesion in colonization of the intestine by pathogens such as enterotoxigenic Escherichia coli, it was suggested that adhesion directly to mucosa was an important component in colonization of the chicken ceca by Salmonella. However, there is little ecological rationale for a requirement for adhesion to the mucosa in an organ where the flow rate of contents is very low (poultry ceca empty two to four times a day) and there is no microscopic evidence for this. There is evidence for association with the mucosa in much lower numbers than expected, and this seems to reflect an invasive process (18). The suggestion that colonization of the ceca is primarily a metabolic function of the bacteria may, however, be overly simplistic.
It seems likely that one or some of the fimbrial structures possessed by Salmonella are involved in the initial interaction between the bacteria and the target cell in the gut, but the evidence remains elusive. This may differ depending on whether the target cell is an M cell or an absorptive epithelial cell. Given the large number of fimbrial types possessed by S. enterica and the increasing evidence of variety between serovars showing different pathotypes, it is perhaps not surprising that little headway has been made in determining the contribution of these organelles to infection and colonization (for a review, see chapter Structure of the Ribosome).
Studies probing the molecular basis of Salmonella intestinal colonization have been carried out by screening random transposon mutant banks of serovar Typhimurium in a range of avian and mammalian species (112, 162). Such studies have, not surprisingly, shown a requirement for functional expression of lipopolysaccharide (LPS). The role of LPS in adherence and intestinal colonization may be explained by its involvement in cell envelope stability and associated resistance to bile salts, cell surface hydrophibicity, and the correct insertion and folding of membrane proteins. The importance of other cell surface polysaccharides is suggested as mutants carrying insertions in colanic acid biosynthesis (wcaE) and enterobacterial common antigen biosynthesis (wecE) were attenuated in both chicken and mammalian intestinal colonization models (112).
Very recent studies (11) analyzing gene transcription by whole-genome microarray in serovar Typhimurium organisms harvested directly from the intestine suggest that most bacterial growth takes place at the interface between gut contents and mucosa, where nutrients and electron acceptor concentrations are likely to be higher. Most of the bacteria in the lumen show very little bacterial growth, although characteristic patterns of gene expression can be observed in comparison with bacteria grown in vitro, including up-regulation of several fimbrial genes and operons associated with propanediol utilization.
The involvement of the host in defining colonization remains obscure. In systemic disease, interaction with the host must take place, and there is now ample evidence for the role of fimbrial antigens in this. The role of physical interaction with the host in colonization in the absence of disease is unclear. Recent studies have shown that the host genetic background can exert considerable influence not only on the course of systemic disease in the chicken (31, 32) and pig (46, 165) but also on the extent of intestinal colonization and fecal shedding (12, 46). This effect is not related to immune responsiveness and is expressed within hours of infection, but the mechanism is unknown.
The molecular mechanisms by which Salmonella invade eukaryotic cells have been studied intensively in vitro using cultured cells. A virulence locus was found at centisome 63 on the serovar Typhimurium chromosome, which was identified as the first Salmonella pathogenicity island (SPI-1). SPI-1 encodes a type three secretion system termed TTSS-1, which has a major role in the invasion process (unpublished data). Molecular genetic approaches are now being applied to study intestinal invasion in vivo. Mutations in genes that encode essential structural proteins of TTSS-1 act to block the delivery of translocated effector proteins and also block Salmonella invasion of intestines in a range of animal species including cattle (172), sheep (164), and poultry (35).
The respective roles of the TTSS-1-dependent translocated effector proteins in intestinal invasion are not clear. Mutation of sopE1 was found to reduce invasion of cultured cells (185), although this reduction was relatively minor when compared with mutations that entirely disrupt the function of TTSS-1 and therefore block the delivery of all TTSS-1-dependent effectors. SopE1 was found to activate small Rho GTPases, which influence cytoskeletal rearrangements, thus promoting epithelial cell invasion (72). The sopE1 gene is not present in many Salmonella strains, which, together with the weak invasion phenotype of the sopE1 mutant, suggests that SopE1 may have functions other than those that influence invasion and, consequently, that other secreted invasins await characterization. One such invasin is the SopE2 protein, which is highly homologous to SopE1. Not only is sopE2 more prevalent than sopE1 in Salmonella strains, but also mutation of sopE2 appears to have a major effect on the invasion phenotype (9). SopE1 is carried by the SopEΦ bacteriophage and epidemiological evidence shows that the sopE1 gene is associated with serovar Typhimurium phage types that cause epidemics in cattle. Horizontal transfer of the sopE1 gene by lysogenic conversion with the SopEΦ was shown to increase the enteropathogenicity of serovar Typhimurium in the bovine ligated ileal loop model (190). These observations support the hypothesis that phage-mediated horizontal transfer of the sopE1 gene contributes to the emergence of epidemic cattle-associated serovar Typhimurium clones.
The extent to which active invasion utilizing TTSS-derived effector proteins is required for initiating systemic disease in adult animals, where systemic disease is a common consequence of infection, as opposed to infection in young animals, which frequently is manifest primarily as diarrhea and dysentery, is totally unknown. It may be that, as appears to occur with serovar Gallinarum (89) and serovar Pullorum (177) infections in chickens, SPI1-associated functions are not required, or contribute in a relatively minor way, at least to establishment of systemic disease (see below).
During the invasion process in calves Salmonella can be seen within M cells, enterocytes, gut macrophages (55), and dendritic cells (115) in the intestinal mucosa. As a consequence, in general, it is believed that Salmonella disseminate from the gut within an intracellular niche. Thus, if Salmonella are to successfully infect systemic tissues, they must survive this initial interaction with hostile cells. Salmonella can kill macrophages in vitro, and TTSS-1 is involved in this process (36, 65, 170). Several groups have reported that macrophage killing is mediated by Salmonella-induced apoptosis (36, 110), and this occurs by SipB-mediated activation of caspase-1 (74). However, more recently it has been shown that Salmonella can kill macrophages by TTSS-1-dependent mechanisms that do not resemble apoptosis (171) and are instead termed pyroptosis (29). Despite the cytotoxicity of Salmonella for macrophages, no correlation has been found between host specificity and the magnitude of Salmonella-induced lysis of porcine (171) and avian (35) macrophages, at least in vitro.
Having traversed the intestinal mucosa of mammalian species, the interactions of Salmonella with cells in the mesenteric lymph nodes (MLN) are also likely to influence whether a systemic infection results. The precise route and mechanism by which pathogenic Salmonella disseminate to extraintestinal sites or tissues remains poorly defined. This is due in part to the experimental difficulty in assessing such parameters of the infectious process in vivo. Translocating Salmonella can be found in high numbers within MLN following infection of ligated ileal loops (174) and after oral inoculation of different host species, including calves (167, 169, 170). As such, it is likely that Salmonella disseminate from the intestinal mucosa via the draining lymphatics. Conversely, the venous drainage represents an alternative means of dissemination because bacteria may gain access to blood vessels if damage to the intestinal mucosa is extensive. Recently a model has been developed with which to study the route and mechanisms of bacterial translocation from the intestinal mucosa to systemic tissues in vivo. By separately cannulating both venules and lymphatics that drain from infected mucosa and MLN, it was possible to show that serovar Dublin primarily disseminates from the bovine gut in lymph, rather than blood, at early stages of infection. Furthermore, the interactions between Salmonella and the host within the MLN appear pivotal in determining the outcome of pathogenesis, because serovar Gallinarum, which does not cause disease in calves, passed through the MLN in significantly lower numbers than the virulent serovar Dublin (122).
The mechanisms of survival and persistence of Salmonella within the liver and spleen of an infected host have received a lot of study, in particular, in the murine model of infection. At present comparatively little is known about the specific niches in which bacterial replication takes place. Results from detailed histological and microscopic studies suggest that serovar Typhimurium is likely to reside within murine splenic or hepatic polymorphonuclear neutrophils (PMNs), hepatocytes, and/or Kupffer cells during the early stages of disease (38, 47) and within macrophages during the later stages of the infectious process (134). Furthermore, it has been suggested that the ability of specific serovars to persist within particular host macrophages may correlate with serovar-host specificity. For example, a comparison of the uptake and persistence of serovar Typhi in human and murine macrophages correlates with the virulence of this serovar in humans but not in mice (65, 85). Furthermore, serovar Typhimurium has been shown to persist in higher numbers than serovar Typhi in primary murine macrophages in vitro (168). Similar results have been found with serovar Typhimurium and serovar Gallinarum in murine and avian macrophages (P. Wigley, unpublished results). However, in contrast, no correlation was found between the virulence of different Salmonella serovars for cattle and pigs with factors such as bacterial uptake, intracellular persistence, killing or production of cytokines in bovine or porcine macrophages in vitro (171, 173). Thus the role of Salmonella-macrophage interactions in determining host range and the severity of systemic disease remains unclear.
A second type three secretion system (TTSS-2) encoded on Salmonella pathogenicity island 2 (SPI-2) was shown to be pivotal in influencing systemic disease in mice (148). Whereas TTSS-1 is expressed by extracellular bacteria, TTSS-2 is expressed by intracellular Salmonella, and TTSS-2-dependent secreted effectors are thought to modulate vesicular trafficking inside Salmonella-infected cells (unpublished data). Through modulation of different cellular compartments TTSS-2-secreted effectors are thought to stabilize the membranes surrounding intracellular bacteria (25), promote intracellular replication rates in epithelial cells (147), and block oxygen-dependent killing mechanisms by macrophage (166). It is becoming increasingly clear that SPI-2 is a major virulence factor during infection of food-producing animals, including cattle (27) and poultry (177).
A recent study using multicolor fluorescence microscopy to visualize individual serovar Typhimurium bacteria within the livers and spleens of mice enabled the study of the interactions between different bacterial populations within the same animal (149). The results demonstrated that an increase in bacterial load within an organ could be attributed to the establishment of new foci of infection, rather than to increased numbers of bacteria per phagocyte. This suggests that Salmonella are not replicating freely within phagocytes in systemic tissues and highlights how little we know about the very fundamental aspects of Salmonella pathogenesis. Only through the application of modern molecular genetics to strains of defined virulence in conjunction with infection studies in natural target animal species will the complex biology of Salmonella serovar host specificity be unraveled.
Epidemiology.
Salmonellosis occurs worldwide in cattle and is associated primarily with serovars Dublin and Typhimurium. Although other serovars are sporadically associated with bovine infections, during the past 20 years approximately 100 serovars other than serovar Dublin and serovar Typhimurium accounted for less than 10% of incidents in the United Kingdom. Salmonellosis reached a peak in the British cattle industry in the 1960s with more than 4,000 incidents in 1969 (154), most of these being associated with infections in young calves, predominantly involving serovar Dublin. More recently there has been a steep decline in the number of Salmonella outbreaks and over the past 5 years there have been only 400 to 500 incidents per annum with similar numbers of incidents caused by serovar Dublin and its replacement by serovar Typhimurium (74). Serovar Dublin and serovar Typhimurium are endemic in northern Europe, although the distributions of these serovars differ. In the United Kingdom serovar Typhimurium occurs in all geographical regions, whereas serovar Dublin is predominately found in north- and southwestern England and Wales. In the United States serovar Typhimurium is endemic in cattle throughout the country. In contrast, serovar Dublin, which before the 1980s occurred only to the west of the Rocky Mountains, has recently spread eastward to other states and north into Canada from where it had not previously been isolated (135).
Epidemiological analysis of serovar Dublin by electrophoresis indicated three phenotypes, one of which is global in distribution and in which, in the United States, nonmotile strains are common (146). Vi-producing strains were restricted to one of the taxa, supposedly isolated from the United Kingdom and France only, although these strains are not currently isolated. Phage typing of serovar Dublin also indicates that a small number of types predominate. This, together with the fact that most infections are limited to within herds, may to some extent account for the absence of multiple resistance in serovar Dublin while it is frequent in serovar Typhimurium.
In serovar Typhimurium, a small number of strains also tend to predominate and to be multiresistant: PT29 in the 1960s, 204c in the 1990s, and DT104 at present.
The source of most outbreaks of salmonellosis in cattle is probably fecal-to-oral contact. Infected cattle may excrete up to 108 CFU Salmonella per g of feces, and contamination of the environment in the proximity of other animals is thus a potent source of infection. Subclinical excretion of Salmonella exacerbates the problem of dissemination. Cattle that carry an active infection of Salmonella but display no clinical symptoms (often convalescing animals) are known as "active carriers." These may excrete Salmonella continuously in concentrations greater than 105 CFU/g of feces and thus can be detected by routine bacteriological examination. Active carriage is usually the sequel to clinical enteritis or systemic infection, and infected animals may excrete Salmonella for years or even for life. "Passive carriers" are immune animals that ingest Salmonella with feed and subsequently pass them in their feces with no active infection of the intestines. Consequently, when removed from an infected environment these animals will stop excreting Salmonella. In some animals, known as "latent carriers," Salmonella persist subclinically in the tissues but are only intermittently excreted in feces. Excretion may be activated by stress, for example, at parturition or after Fasciola hepatica or Babesia infection. Understanding the biology of this true "carrier state" is likely to be key to ultimately controlling this important pathogen in cattle and may also provide insight into, for example, the asymptomatic carriage of serovar Typhi by humans.
Salmonellosis in Cattle.
As in other animal species the nature and extent of the course of infection depends on factors such as the infecting serovar, the age of the animal, and the route of infection (34, 53, 186, 189). Cattle are most likely to be infected by the oral route, although respiratory and conjunctival infection may also occur. The immune status of the animal is also important; calves that are deprived of colostrum are particularly susceptible to infection. In such animals, as with E. coli infection, a combination of infection by particular serovars soon after birth and low levels of circulating specific immunoglobulin G (IgG) may lead to a rapid septicemia (see below).
Salmonellosis in Calves.
In calves clinical disease is most common at 2 to 6 weeks of age. The peak incidence of disease with serovar Typhimurium is at 3 weeks of age, whereas with serovar Dublin it is at 4 weeks of age. Several other serotypes are occasionally isolated from diseased calves, and serovar Choleraesuis can be demonstrated to be fully virulent under experimental conditions (151). Clinical signs vary, but typically the enteric form of disease predominates, which is characterized by pyrexia, dullness, and anorexia, followed by diarrhea that may contain fibrin and mucous. Pneumonia may also occur. The feces may become blood stained and "stringy" because of the presence of pseudomembrane formation, shreds of necrotic intestinal mucosa, and undigested milk. Infected calves rapidly become weak and dehydrated and, unless treated, usually die 5 to 7 days after the onset of disease. At this stage the organism has become systemic, largely probably as a result of reduced innate immunity, and may be isolated from a variety of tissues, including the blood (53, 151). Calves that recover from infection do not typically remain carriers.
Salmonellosis is very variable and in some animals, in particular the very young, rapid multiplication occurs both in the intestine and systemically, associated with poor absorption of specific IgG from colostrum or with calves receiving insufficient or no colostrum. This may be accompanied by true septicemia, although this has never been accurately determined, and animals may die in a comatose state after 3 to 5 days in the absence of diarrhea.
The breed of animal can also affect the outcome, with some breeds, such as Jersey, being far more susceptible than Friesian and beef breeds (188). Similarly, preinfection with parasites, such as F. hepatica (4), in experimental rat infections with this parasite or the nematode Nippostrongylus brasiliensis (3) increases the severity of disease. Release of iron in the host and other nonspecific effects on immunity may thus increase susceptibility where these parasites are common.
Salmonellosis in Adult Cattle.
In adult cattle both acute and subacute forms of disease are recognized, caused by serovar Dublin or, less frequently, serovar Typhimurium or other serovars, including serovar Enteritidis. The onset of the severe, acute form of disease is sudden and typically accompanied by pyrexia, dullness, anorexia, and reduced milk yield. Severe diarrhea follows, which may contain blood, mucous, and necrotic intestinal mucosa. Early in infection Salmonella may be isolated from blood and milk in addition to the feces. Pregnant animals, in general, abort after serovar Dublin infection. High temperatures usually persist for several days and typically drop precipitously in the 24 h just before death, which may occur between 1 to 5 days after infection. The subacute form of disease is less severe, though it may be more protracted. Animals may become emaciated and dehydrated and show signs of abdominal pain. Abortion may occur in the absence of other clinical signs, and this is particularly the case with serovar Dublin infections (76). Abortion is usually preceded by pyrexia (68) when bacterial multiplication is likely to be occurring in the placenta. It is thought that this leads to placental tissue destruction with associated hormonal changes (69). However, these studies used intravenous inoculation, and the spread of serovar Dublin to reproductive tissues is not well understood and may originate either from a systemic infection or possibly from fecal contamination of the vagina. Experimental intrarumenal inoculation leads to ileal and colonic establishment of infection with invasion as far as the local lymph nodes (70).
Adult survivors of serovar Dublin infections generally become carriers, a state that may last for life. The organism may be transmitted from carrier animals to their calves, either in embryo or postnatally. The outcome of infection with other serovars seldom results in the latent carrier state, although active excretion may continue for years. The reasons for this remain unclear. Persistent carriers may also excrete the organism in the milk. Chronic shedding of serovar Dublin in milk has been reported (119, 135, 156) as has shedding of other serotypes (117, 156). Between 101 and 105 CFU of serovar Dublin can be shed in the milk, accompanied by increases in titer of specific circulating or milk IgG (150).
Molecular Basis of Pathogenesis of Salmonella in Cattle.
Intestinal Invasion.
Invasion of intestinal mucosa is a characteristic feature of Salmonella pathogenesis in the calf. Within minutes of contact with intestinal mucosa in ligated ileal loops, Salmonella can be seen to invade both M cells and enterocytes that overlie domed villi associated with lymphoid follicles and absorptive villi, respectively (55). Subsequently, Salmonella elicit membrane ruffles in the apical membranes of both M cells and enterocytes, which result in the uptake of bacteria into membrane-bound vesicles. Thereafter, Salmonella can be seen associated with reticuloendothelial cells and PMNs, which are recruited to the foci of infection.
Disruption of the function of TTSS-1 acts to block the delivery of translocated effector proteins and also to block Salmonella invasion of intestines in cattle (172), an indication that SPI-1 is a major virulence factor for serovars Dublin and Typhimurium in cattle. In general, it is assumed that Salmonella invasiveness is important in virulence. However, the actual requirement for intestinal invasion in the induction of enteritis in the calf or systemic disease in the adult has not been demonstrated. In orally infected calves, extracellular bacteria are present in the intestinal lumen in high numbers. Similarly, in ligated ileal loop studies, >90% of the inoculum remains gentamicin sensitive, suggesting that bacteria are primarily located in an extracellular niche (172). Extracellular bacteria deliver proteins, via TTSS-1, into epithelial cells, and it is these effector proteins that are required for enteritis. This questions the requirement for invasion of Salmonella into intestinal mucosa for the induction of enteritis. To date it has not been possible to directly assess the role of invasion in enteropathogenesis because mutation of TTSS-1 results in pleiotropic effects. However, the observation that different but equally invasive serovars of Salmonella induce different levels of enteropathogenic response (108, 170, 172) suggests that there is no direct correlation between the magnitude of invasion and enteropathogenicity. More recently it was shown that invasion of epithelial cells can be uncoupled from Salmonella-induced PMN transmigration in vitro (61), a fact which supports this observation.
Salmonella-Induced Enteritis in Cattle.
In addition to cellular invasion, TTSS-1 also has a major role in the induction of enteropathogenesis. Disruption of TTSS-1 abolishes enteropathogenicity in bovine ligated ileal loops (2, 170) and orally infected calves (161, 170). Some of the translocated effector proteins have important roles in the induction of intestinal inflammation and fluid secretion. The SopB protein of serovar Dublin (also known as SigD in serovar Typhimurium [77]) is translocated into eukaryotic cells via a TTSS-1-dependent mechanism (57). Inactivation of sopB has little or no effect on intestinal invasiveness (57, 133), yet it significantly reduces enteropathogenesis (57, 143). SopB was shown to be an inositol phosphate phosphatase capable of hydrolyzing several inositol phosphates (116). This is of particular interest since it is known that Salmonella infection of intestinal epithelial cells in vitro results in elevated intracellular levels of D-myo-inositol 1,4,5,6-tetraphosphate [Ins(1,4,5,6)P4], which in turn can antagonize the closure of chloride channels (48), influencing electrolyte and thus fluid secretion in intestinal mucosa. The increase in Ins(1,4,5,6)P4 levels in Salmonella-infected cells could be directly or indirectly attributed to SopB activity (116). Together these observations implicate SopB as a novel bacterial enterotoxin.
Unlike mutations that disrupt TTSS-1 which abolish enteropathogenesis entirely, mutation of sopB only reduces enteropathogenesis (57). This implicates other TTSS-1-secreted effector proteins in the induction of enteritis, and indeed, several have already been demonstrated to be involved in this process (90, 191).
Role of Virulence Genes in Systemic Pathogenesis.
TTSS-2 also influences the pathogenesis of Salmonella in cattle. A mutation in sseD, which encodes a putative TTSS-2 translocon protein, thought to mediate the translocation of other effector proteins through the target cell membrane, was shown to attenuate serovar Dublin in calves following infection either by the intravenous route or by the oral route (27). The sseD mutant was fully invasive for bovine intestinal mucosa but was unable to proliferate to the same extent as the parental strain in vivo. The sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that TTSS-2 is required by serovar Dublin for the induction of both systemic and enteric phases of salmonellosis in cattle.
Other genes have been identified that also directly affect Salmonella enteropathogenicity. The region around sopB in serovar Dublin contains a Salmonella-specific DNA fragment, termed Salmonella pathogenicity island 5 (SPI-5) (184). Mutations in SPI-5-encoded pip genes (pathogenicity island-encoded proteins) produced a phenotype similar to that of a sopB/sigD mutant, that is, partial attenuation of enteropathogenicity in bovine ligated ileal loops and no effect on systemic virulence in mice. PipC (SigE), was identified as a SopB/SigD-specific chaperone (77). However, the functions and sites of action of other Pips are ill-defined. Recently it was shown that SPI-5-encoded effectors are induced by distinct regulatory cues and targeted to different TTSSs. As stated previously, SPI-5 encodes the TTSS-1-translocated effector SopB/SigD. In contrast, an adjacently encoded effector PipB is part of the Salmonella pathogenicity island 2 (SPI-2) regulon (see chapter Salmonella Intestinal Infections). PipB is translocated by TTSS-2 to the Salmonella-containing vacuole and Salmonella-induced actin filaments (93). SPI-5 is not conserved in all Salmonella spp. Although sopB/sigD is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional TTSS-2. Thus there appears to be functional and regulatory cross-talk between three chromosomal pathogenicity islands, SPI-1, SPI-2 and SPI-5; this has significant implications for the evolution of bacterial pathogenesis and host specificity (see below).
Plasmid-Mediated Virulence Genes.
Large-molecular-weight plasmids have been shown to influence the systemic virulence in mice of several mammalian Salmonella serovars including serovars Typhimurium (88), Enteritidis (114), Dublin (8, 157), Choleraesuis (40), Abortusequi (5), and, through the intestinal phase, Abortusovis (163). However, the role of plasmid genes in Salmonella pathogenesis in cattle is somewhat controversial. In one study the virulence of plasmid-bearing and plasmid-cured strains of serovar Dublin was compared in calves. The plasmid-bearing strains were highly virulent, causing severe enteric and systemic disease with high mortality. In contrast, the plasmid-cured strain caused diarrhea but only low mortality, suggesting that a systemic phase contributes to the final stages of the disease. The strains were equally invasive for intestinal mucosa and elicited comparable secretory and inflammatory responses in ligated ileal loops. It was concluded that virulence plasmid genes are not involved in either the enteric phase of infection or the systemic dissemination of serovar Dublin but probably mediate the persistence and multiplication of serovar Dublin at systemic sites (169). More recently a second study assessed the role of the virulence plasmid-encoded spv operon in the induction of salmonellosis in cattle by serovar Dublin. SpvR is the transcriptional regulator required for expression of the spvABCD operon. The virulence of wild type, an isogenic spvR knockout mutant and a strain in which spvR was transcomplemented, cloned in pSC101, was compared in calves. As in the previous study, calves that were infected with the spvR mutant showed little or no clinical signs of systemic salmonellosis. However, in contrast to the previous study, calves developed only mild diarrhea compared with those infected with the wild type. The intracellular survival and growth of the wild-type strain and the spvR mutant were determined by using blood-derived bovine monocytes. Wild-type serovar Dublin survived and grew inside cells, whereas the spvR mutant did not proliferate. These results suggest that the spv genes of serovar Dublin promote intracellular proliferation in intestinal tissues and at extra intestinal sites in calves (97). The reasons for the contradictions of the two studies remain unclear but are possibly strain related.
Epidemiology.
Compared with bovine salmonellosis the general incidence of disease in sheep in most countries is low, reflecting their generally less intensive management. In many countries, including Mediterranean countries, the host-adapted strain serovar Abortusovis remains an important economic problem. In many other countries, it is a relatively minor problem and other serovars, such as serovar Typhimurium, are predominant. Serovar Dublin may also produce disease in sheep. In the United Kingdom it has less economic importance than bovine salmonellosis, since only a few hundred incidents occur and the sheep population is large. Serovar Abortusovis used to be widespread in the western United Kingdom, but it has not been isolated for some years. Currently, the most frequently isolated serovars are serovar Typhimurium (primarily DT104), S. enterica subsp. arizonae (O61:k:1,2,7), serovar Derby, and serovar Montevideo, but the list is by no means restricted to these. Serovar Montevideo has been associated with abortion in ewes for a number of years. This is caused largely by a single biotype, which is different from that causing infections in humans, cattle, and poultry. In the United States, Canada, and some European countries S. enterica subsp. arizonae (O61:k:1,5,7) is an important serovar (187).
In comparison with bovine salmonellosis, there is much less solid information on the routes of spread and a good deal of controversy. Serovar Abortusovis is highly specific and is undoubtedly introduced from imported sheep, whereas other serovars may arise from many potential sources. Serovar Abortusovis may be isolated from the genitalia of both female and male sheep, but the extent to which transmission occurs by this route and is responsible for infection is disputed (20, 86, 87, 142, 155). Infection resulting in abortion is difficult to establish by oral administration but has been established by conjunctival infection (141). In the case of S. enterica subsp. arizonae nasal carriage also appears to be an important aspect of the epidemiology, and the extent to which this also occurs with other serovars is unknown.
As with serovar Dublin infection in cattle, other factors may precipitate disease in latent carriers. These include concurrent Chlamydia infection, chemoprophylaxis, nutritional factors, including drought, and other factors inducing stress, such as cold, transport, and overcrowding.
Pathogenesis of Salmonellosis in Sheep.
The course of infection is similar with serovar Abortusovis and serovar Montevideo, and mortality varies between 10 and 75%. In adult animals signs vary and may be few prior to abortion. Profuse growth of serovar Abortusovis may be obtained from several embryonic tissues. In general, this does not seem to greatly affect the ewe, which may lamb normally in the next season and after abortion; excretion is short lived. Lambs may also be stillborn or may die soon after birth; septicemia may occur during the first few weeks of life, although little is known of the pathogenesis of these infections. In addition, as might be expected, serovar Montevideo may also be excreted in the feces without abortion, and nonpregnant flocks may be infected with no apparent disease at all.
Serovar Typhimurium and serovar Dublin may produce largely enteric infections, with systemic involvement, depending on several additional factors, such as age, nutrition, and stress. Serovar Dublin-induced abortion may also occur as a result of systemic infection in pregnant ewes. The published results of experimental infection have added little to this in terms of the route of transfer of the organism from the site of inoculation to the reproductive tract (187).
Serovar Abortusovis is unusual among serovars that characteristically induce systemic disease, in that the virulence-associated plasmid appears to be required solely for the intestinal phase of the infection, in mice at least, and is not required for the systemic phase (163). Serovar Abortusovis has been shown to be invasive for epithelial cells and in vitro (138) and in vivo, which is TTSS-1 dependent (164). The colonization of ovine intestinal and systemic tissues by S. enterica serovars with different host specificities has been determined in 1- to 2-month-old lambs. After oral inoculation, serovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than those of the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites after intravenous inoculation was similar. Intestinal invasion was quantified in ovine ligated ileal loops; serovars Dublin, Gallinarum, and Typhimurium were recovered in comparable numbers, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Serovar Typhimurium and serovar Dublin induced intestinal inflammatory responses, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest that Salmonella serovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the host's intestinal inflammatory response may contribute to but not determine the specificity of serovar Abortusovis for sheep. In common with some other systemic serovars, cytokine profiles differ from those of animals infected with serovars that are not host specific. Thus, in contrast to serovar Dublin, tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12) transcription is reduced in the spleen and draining lymph node after subcutaneous challenge with serovar Abortusovis (111). Sheep mount a strong cell-mediated response against subcutaneous infection with serovar Abortusovis, as detected by T-cell proliferation studies (45).
Epidemiology.
The serovars of Salmonella associated with clinical disease in pigs can be divided into two groups: the host-restricted serovars typified by serovar Choleraesuis and the ubiquitous serovars typified by serovar Typhimurium. In the 1950s and 1960s serovar Choleraesuis was the predominant serovar isolated from pigs in the United Kingdom and represented a major problem for the pig industry, affecting pig health and welfare (154). Since then the occurrence of serovar Choleraesuis has fallen dramatically, and it is now isolated only sporadically (7). In contrast, serovar Choleraesuis infections remain a major threat to the pig industry in the United States (178). The decline of this serovar in the United Kingdom was not associated with any specific intervention measure. The reasons for this decline, therefore, are unclear. Serovar Typhimurium is the most common serovar isolated from pigs both in Europe and in the United States. Likewise, serovar Derby has a strong association with pigs on both sides of the Atlantic Ocean, and for the past 15 years it has been the second most prevalent serovar in pigs in the United Kingdom (7). A recent national survey of healthy pigs at slaughter in abattoirs in the United Kingdom showed 23% of pigs had Salmonella culture-positive cecal contents in which serovars Typhimurium and Derby were the predominant serovars (41).
Oral ingestion is thought to be an important route of infection because Salmonella are shed in high numbers in the feces of clinically infected pigs. In experimental infections, high doses of between 108 and 1011 CFU are required to reproducibly cause disease in pigs via the oral route (62, 63). Reproducible results are only obtained by using a lower dose if the gastric pH is first neutralized with antacids (173). This demonstrates that the low pH of the stomach is an effective barrier to infection by Salmonella. Inhalation of infected material into the upper respiratory tract is another potential route of infection. Pneumonia is a common feature of serovar Choleraesuis infections in pigs (21), and several studies have shown that pigs can be experimentally infected by intranasal inoculation. Pigs infected with serovar Choleraesuis via the intranasal route develop more severe clinical signs than those infected via the oral route (62). Intranasal inoculation with serovar Typhimurium results in rapid dissemination of Salmonella to intestinal sites, even in pigs in which an esophagotomy had been performed (52). Together these observations suggest that the tonsils and lungs are likely to be important sites of invasion.
Salmonellosis in Pigs.
The capacity of Salmonella to cause disease in pigs depends on numerous factors, including the infecting serovar and the age of the pig. Clinical salmonellosis in pigs is typically of two forms: septicemia caused by host-restricted serovars such as Choleraesuis and enterocolitis caused by broad-host-range serovars such as serovar Typhimurium. Not surprisingly, weaned pigs that are intensively reared are most frequently affected by Salmonella infections. Like other host-specific serovars, serovar Choleraesuis has the capacity to cause disease in both young and older animals, whereas serovar Typhimurium typically causes disease in pigs aged between 6 and 12 weeks but rarely does so in adult animals. In older animals subclinical infections with serovar Typhimurium are frequent, leading to high transmission rates if active carrier animals are not detected.
Infections Caused by Serovar Choleraesuis.
Serovar Choleraesuis typically causes systemic forms of infection. Affected pigs are lethargic and pyrexic, and they often have respiratory symptoms, including coughing. Diarrhea may or may not be present, and cyanosis of the extremities is common. In most cases mortality is high. Details of the pathology have been described elsewhere (179). Gross lesions typically include colitis, swollen mesenteric lymph nodes, splenomegaly, hepatomegaly, and lung congestion. Foci of necrosis are often seen on the liver.
Serovar Choleraesuis is an unusual serovar among the host-restricted types in that it has the capacity to produce severe systemic disease in a relatively wide range of animals, including pigs, calves, rabbits, guinea pigs, and humans (13, 33, 122)
Infections Caused by Serovar Typhimurium and Other Serovars.
Serovar Typhimurium typically causes enterocolitis. Here, watery diarrhea is the initial clinical sign of infection. Pigs become anorexic, lethargic, and febrile, but mortality is typically low. Details of the pathology have been described elsewhere (179). Gross lesions typically include necrotic colitis and pseudomembranous typhlitis. Mesenteric lymph nodes are typically swollen. Intestinal necrosis is often seen as distinct button ulcers. Pigs are frequently associated with acute and subclinical infections caused by other serovars of Salmonella.
Salmonella
Pathogenesis in Pigs.
Our understanding of the mechanisms that Salmonella utilize to cause disease in pigs is limited because of the relatively few studies carried out using pigs as an experimental infection model. Furthermore, insights into pathogenesis are complicated by the different forms of infection caused by the different serovars. Several studies have attempted to gain further understanding of pathogenesis by comparing and contrasting the pathogenic process of different serovars in experimental infections of pigs. The distinct forms of salmonellosis can be reproduced in experimental models of infection. Inoculation of pigs with serovar Typhimurium results in acute enterocolitis, whereas inoculation with serovar Choleraesuis initially produces a systemic disease with septicemia followed by necrosis of the colonic mucosa (132, 173). There is some evidence that serovars Typhimurium and Choleraesuis may invade intestinal mucosa by distinct routes. After oral infection, serovar Typhimurium had a low tendency to invade the enteric mucosa and did not reveal any tropism for a specific intestinal location. However, in the same experiment serovar Choleraesuis was found predominantly in colon and on the luminal surface of ileal M cells of Peyer’s patches, and it had a tendency to invade epithelial cells (127). In contrast, a study using a polarized in vitro organ culture system demonstrated that serovars Typhimurium and Choleraesuis invaded ileal mucosa, with and without Peyer’s patches, in equal numbers (28). More recently it was reported that serovars Choleraesuis and Typhimurium invaded enterocytes, goblet cells, and M cells in porcine ileal mucosa but that serovar Choleraesuis was found more frequently within M cells than serovar Typhimurium. In addition, serovar Choleraesuis appeared to induce less damage to mucosa than serovar Typhimurium (109), which is consistent with the theory that host-specific serovars cause systemic disease by a strategy of stealth (see below). This study needs to be repeated with additional strains of serovars Typhimurium and Choleraesuis of defined virulence to confirm that the observations are serovar specific and not strain specific.
It has long been understood that stressed animals and, in particular, pigs are more susceptible to diarrheal diseases such as salmonellosis (181). Evidence that bacterial pathogens are able to respond to the host environment by detecting host-derived neurotransmitters is increasing (105). Recently it was reported that serovar Choleraesuis invasion of porcine Peyer’s patches could be promoted by the stress hormone norepinephrine and that this phenotype was blocked by the α-adrenergic agonist phentolamine (64). Understanding the mechanisms by which Salmonella uses stress hormones to modulate virulence gene expression will hopefully provide further insight into Salmonella pathogenesis.
Molecular Basis of Pathogenesis of Salmonellosis in Pigs.
The roles of genes attributed to virulence in other animal species have been little studied in pigs. Not surprisingly SPI-1 genes also influence the pathogenicity of salmonellosis in pigs. Recently it was shown that mutation of hilA, a regulator of TTSS-1, influenced the virulence of serovar Choleraesuis in pigs after oral but not intraperitoneal challenge (98). This implicates a role for TTSS-1 in the enteric but not the systemic phase of infection, reflecting that which is seen with serovar Typhimurium infections of mice.
Like other serovars, serovar Choleraesuis carries a large virulence plasmid, which influences systemic pathogenesis in pigs (40). The complete nucleotide sequence of pKDSC50, a plasmid from serovar Choleraesuis strainRF-1, has been determined, and 48 open readingframes (ORFs) are predicted to be encoded by the 49-kbp molecule (71). Analysis of the genetic organization of pKDSC50 suggeststhat the plasmid is composed of several virulence-associated genes,which include the spvRABCD genes, plasmid replication and maintenancegenes. A second virulence-associatedregion including the pef operon andrck (resistance to complement killing) gene, which has been identifiedon the virulence plasmid of serovar Typhimurium, wasabsent. Comparative analysis of the nucleotide sequencesof the 50-kb virulence plasmid of serovar Choleraesuis and the94-kb virulence plasmid of serovar Typhimurium revealed high levels of homology, suggesting at least some degree of commonancestry.
The prevalence of Salmonella serovars in domestic fowl varies in different countries and with time. Certain serovars have been known to emerge within a country or region for a period and then disappear with no obvious cause or intervention measure. In the 1950s serovar Agona was prevalent in the United Kingdom poultry industries, yet disappeared without any obvious intervention. Historically, serovar Typhimurium has been among the most prevalent serovars isolated from poultry. In the United Kingdom between 1968 and 1973, serovar Typhimurium accounted for more than 40% of all Salmonella isolations associated with poultry, followed by serovar Enteritidis (6%), serovar Pullorum (4%), and serovar Gallinarum (3%) (154). During this time serovar Hadar became established in the United Kingdom poultry industry, initially in turkeys and then also in chickens. This also gradually disappeared without apparent reasons. During the 1980s, serovar Enteritidis phage type 4 (PT4) emerged as the predominant serovar, exceeding the isolation rates of serovar Typhimurium. The reasons for this epidemic remain unclear, but the introduction of intensive screening and control measures, which included an active immunization program in the United Kingdom poultry industries, has been attributed to the recent decline of PT4 during the past few years. One of the interesting aspects of this period is that unrelated phage types of serovar Enteritidis, such as 1,6,8 and 13a, also became established as major causes of infection in poultry and humans in the western hemisphere and other areas of the world, such that serovar Enteritidis was effectively the cause of a pandemic (136). The reason for this has never been understood, although it is likely that a combination of factors in the poultry industry involving breed genetics, feed production, and consolidation of changes in company infrastructures probably contributed in a major way. Other hypotheses have been suggested. One proposes that the pandemic was a consequence of the diminution of an exclusion effect due to the gradual control of the serologically related serovar Gallinarum-Pullorum (131), although, given the timescale over which this has happened in most countries, this hypothesis seems highly unlikely.
In recent years, large numbers of Salmonella isolations have continued to be reported in the United Kingdom poultry industries. After serovar Typhimurium and serovar Enteritidis, the most commonly isolated serovars are Livingstone, Seftenberg, Kedougou, and Montevideo (7). However, the isolation of these serovars together with the isolation of other rarer serovars is probably a reflection of the surveillance activity and is not due to the investigation of clinical disease.
The high prevalence of serovar Enteritidis, serovar Typhimurium, and other serovars in poultry is reflected in many other parts of the world. There are some exceptions; for example, serovar Enteritidis is virtually absent from Australasia. This is likely to be a result of strict controls on the importation of poultry. However, these countries have their own epidemiological peculiarities, such as the high incidence of serovar Sofia in poultry. However, this is not reflected by a high incidence of serovar Sofia infections in humans. Other countries have a relatively higher incidence of some group C serovars, such as Hadar and Infantis. However, these serovars are largely restricted to the alimentary tract of poultry and are responsible for outbreaks of gastroenteritis in humans, with exceedingly rare systemic involvement.
A traditional association has existed between serovar Enteritidis infection in poultry and human infection arising from consumption of eggs. Despite this strong correlation the apparent tropism of this organism for the reproductive tract of poultry has not been fully elucidated. The greatest reservoir of Salmonella for humans, however, is in the broiler industry, such that the consumption of poultry meat, rather than eggs, is likely to result in most human infection.
Poultry-specific serovars Gallinarum and Pullorum have largely been eradicated from the poultry industries of Europe and North America. However, in regions of the world with less developed industries and, in particular, in facilities with poor biosecurity, these serovars still represent major threats to bird health, welfare, and the associated effects on local economy. Although chickens are the natural hosts of serovars Gallinarum and Pullorum, natural outbreaks caused by these serovars in turkeys, guinea fowl, and other avian species have been described (123, 152). In addition, the increase in the popularity of free-range rearing in northern Europe has resulted in an increase in incidence of serovar Pullorum infection. These serovars are rarely isolated from mammalian species (33), although where clinical disease is extensive in poultry, transmission into the human food chain must occur, and it is thought that this has resulted in human infections, largely gastrointestinal in nature. The isolation of serovars Gallinarum and Pullorum from other animals such as cattle has been reported, but this may have been the result of confusion with nonmotile serovar Dublin strains.
The sources of infection in poultry are many, although the major sources are poultry themselves through introduction and, in serovar Gallinarum, serovar Pullorum, and serovar Enteritidis, through vertical transmission, feed, and the environment. Asymptomatic shedding of Salmonella from the intestines leads to the contamination of eggs, which can also contribute to vertical transmission, in addition to the result of contamination of the egg contents from infected ovaries or oviduct. Immediately after hatching, oral ingestion by the chicks results in very high numbers of Salmonella in the gut and extensive shedding in the feces. This leads to rapid horizontal spread around the hatchery and in the first few weeks of life of the bird.
The capacity of Salmonella to cause disease in poultry is closely related to the infecting serovar and the age and genetic background of the bird.
Infections Caused by Serovar Gallinarum and Serovar Pullorum.
Fowl typhoid (FT) and pullorum disease (PD) are systemic diseases that occur primarily in chickens and turkeys but are also important in game birds. Fowl typhoid is caused by serovar Gallinarum; pullorum disease is caused by the serologically identical serovar Pullorum. Both of these serovars are highly host specific and are rarely associated with systemic disease in nonavian species. FT is widely considered to be a disease of adult birds and PD is considered to be a disease of chicks and poults; however, serovar Gallinarum is able to infect both young and older birds.
The clinical signs and pathology of these infections have been described in detail previously (123, 152). The course of serovar Gallinarum infection in a susceptible bird is of a severe systemic form. Horizontal transmission of serovar Gallinarum is largely fecal-oral; the organism colonizes the gut very poorly and soon disappears from the feces, but it is taken up, primarily it is thought, by the cecal tonsil and Peyer’s patch, as indicated by higher bacterial counts soon after oral infection (10). Serovar Gallinarum bacteria then are soon found in small numbers in organs rich in the monocyte-macrophage cell lineage, mainly spleen and bone marrow, where it is thought that they multiply. After bacterial counts have increased considerably at these sites a bacteremia occurs and the organisms are then found in localized areas in the intestinal wall, possibly in aggregates of lymphoid cells. From these sites they are shed into the intestine in large numbers. In commercial flocks transmission also occurs as a result of cannibalism, as indicated by the ability to reduce the incidence of disease by removal of dead birds (A. Berchieri, personal communication). Pathological signs in acute cases include hepatomegaly and bronzing of the liver.
Surviving birds show large areas of necrosis in the myocardium, from which, unlike serovar Enteritidis, it is not possible to isolate bacteria. Older literature suggests that vertical transmission is an important mode of spread. However, it is very difficult to reproduce this experimentally (22). Whether this is a real result or an indication that host genetics also play a role in determining the course of infection is unclear. Certainly, with an organism that is able to produce mortality in excess of 80% in a susceptible host, horizontal transmission may be sufficient for transmission, under existing commercial conditions.
Serovar Pullorum is regarded by many as a biotype of serovar Gallinarum, although multilocus sequencing indicates both have a common ancestor (96), and more recent work on the glycogen biosynthesis genes indicates that the sequence of glgC, encoding ADP glucose pyrophosphorylase, more closely resembles that of serovar Typhimurium than of serovar Gallinarum (P. A. Barrow and M. A. Lovell, unpublished results). However, the organism is less virulent in older birds than serovar Gallinarum. High mortality occurs only when birds are infected within a few days of hatching. After this time no disease occurs and, like serovar Gallinarum in older birds, it colonizes the intestine very poorly. However, in the absence of a mature gut flora, infection soon after hatching results in massive bacterial replication within the intestines. How the organism becomes systemically disseminated is uncertain, but bacterial multiplication is thought to take place in the spleen and liver with an ensuing septicemia. Mortality is strain and host background dependent (15, 31). Birds may also be infected as a result of vertical transmission and, on hatching, infected eggs lead to moribund and dead chicks in the incubator. Birds may huddle together, exhibit ruffled feathers, and manifest depression, anorexia, and diarrhea. The highest mortality occurs in birds at 2 to 3 weeks of age. In surviving birds the organism persists in the spleen and, despite high levels of circulating antibody, is not eliminated until the birds (hens only) become sexually mature at 16 to 20 weeks, when the organism multiplies in the tissues again and spreads to other sites, including the reproductive tract and, in particular, the ovaries, from which eggs may become infected (175). The tropism of serovar Pullorum for the reproductive tract is much greater than for the ubiquitous serovars. Serovar Pullorum readily infects the reproductive tract and developing eggs after oral infection, with particularly high numbers in the oviduct at the point of lay (175). The molecular basis for this tissue tropism remains unknown.
In both organisms host genetic background is an important component defining the course of infection. Lines of bird showing the SAL1-resistant phenotype are almost completely resistant to infection, and the difference in LD50 after parenteral inoculation with serovar Gallinarum may be as great at 10-million-fold (31) (see below).
Infections Caused by Other Salmonella Serovars.
The capacity of serovars other than Gallinarum and Pullorum to cause disease is relatively poorly understood. Serovar Typhimurium, serovar Enteritidis, and some strains of other serovars are capable of producing clinical salmonellosis in very young birds, probably by a mechanism common to that of serovar Pullorum (14, 43, 66). Several experimental infection studies have shown that strains of serovar Enteritidis PT4 appear to be more virulent in chicks than other phage types of this serovar (44, 125, 126) and that the virulence of PT4 strains can approach that of serovar Pullorum strains in chicks (44, 59). Many serovars derived from poultry products have been associated with food poisoning in humans. However, the potential for such serovars to infect poultry has been studied little in controlled experiments. A chick isolate of serovar Kedougou was shown to colonize the gut but not invade the mucosa of experimentally infected day-old chicks (30). Similarly strains of serovars Heidelberg, Seftenberg, Infantis, Montevideo, and Menston all efficiently colonized the intestines of young birds but were less invasive than a strain of serovar Typhimurium (18). More recently the virulence of several different serovars of Salmonella was assessed in day-old specific-pathogen-free chicks. Not surprisingly, the host-specific serovar Pullorum proved to be the most virulent, followed by the ubiquitous serovars Typhimurium and Enteritidis. Three of four strains of Heidelberg caused low levels of mortality, whereas birds infected with isolates of serovars Montevideo, Hadar, and Kentucky all survived. However, these latter serovars all colonized the intestines efficiently and caused a reduction in body weight (137), indicating that subclinical Salmonella infections can still be detrimental to bird health, welfare, and productivity. The reasons why such serovars are apparently much less virulent in chicks yet retain the capacity to cause food poisoning in humans are not understood.
The potential for Salmonella to invade the intestines of poultry has been assessed in several studies. The passage of serovar Enteritidis and serovar Thompson across the cecal mucosa of freshly hatched chicks was visualized by electron microscopy (124). The uptake of Salmonella by macrophages was observed in the cecal lumen; the macrophages then became abnormal in appearance and were often ruptured, releasing organisms back into the lumen. Epithelial cell death was related to large numbers of bacteria. Bacteria were never observed in large numbers below the basement membrane, and there was no significant pathology in the lamina propria tissue. Wandering cells, identified as macrophages, appeared to contain bacteria and were observed spanning the epithelial and lamina propria regions through breaks in the basement membrane. It is suggested that the passage of bacteria from the epithelium to the lamina propria is primarily the result of capture and transport within host macrophages.
The invasiveness of different serovars of Salmonella was directly compared in two studies in chickens. In ligated jejunal loops, zoonotic serovars of Salmonella were more invasive than serovars not normally considered to be horizontally transmitted (1). Invasiveness of different serovars was assessed more comprehensively after oral inoculation of 1-week-old chicks in addition to inoculation onto cecal tonsils and into ligated jejunal loops. Serovar Typhimurium was found to be more invasive than serovar Gallinarum at all sites tested, demonstrating that primary invasion does not correlate with systemic pathogenesis in chickens (35). Infection with serovar Typhimurium is typified by acute enteropathogenic responses characterized by expression of CXC chemokines and an influx of PMNs (183) followed by massive multiplication in spleen and liver with septicemia.
Mortality rates vary enormously, from less than 10% to more than 80% in severe outbreaks, depending on bacterial strain and host genetic background. Death probably results from a combination of toxicity and dehydration due to diarrhea and malaise. Resistance to infection develops rapidly during the first 72 h of life and has been attributed to maturation of macrophages and increasing heterophil bactericidal activity since the resistance is apparent in orally and parenterally infected birds. However, the gradual development of a commensal flora in the gut also leads to increased resistance by a competitive exclusion mechanism (10).
Strains of serovar Enteritidis, in addition to being highly virulent for young chicks, can cause asymptomatic and chronic infections in older birds, including commercial layers and broiler breeders (75, 80, 99), and infection soon after hatching can result in infection persisting until birds come into lay with the consequential vertical transmission (23). The extent to which egg contamination is a result of systemic bacteria reaching ovules, or a result of contamination, either in the oviduct or cloacae after egg formation, and the extent to which this is a characteristic unique to serovar Enteritidis, remains relatively unclear. A study of orally infected adult birds showed that serovar Enteritidis and serovar Typhimurium strains are similarly able to colonize both the reproductive tract and any eggs that are forming in the oviduct before oviposition (92). Unfortunately serovar Enteritidis PT4 strains were not included in the study. More recently, in a comparison of six serovars injected intravenously into adult birds, serovar Enteritidis PT4 was shown to colonize the reproductive organs of mature laying hens most efficiently (118), which may well explain the association of this serovar with infected eggs. Very rarely are serovars other than serovar Enteritidis found associated with table eggs (140, 144, 180). Serovar Enteritidis may be isolated from the reproductive tract of naturally infected (78, 82) and experimentally infected (58, 82, 159) birds. A number of studies indicate that the inner shell and the shell membranes are frequently the main site of infection (26, 84, 118) and egg infection may continue after fecal excretion ceases (83), suggesting that the lower reproductive tract (isthmus and uterus) are the main sites of colonization.
Earlier work suggested that most serovar Enteritidis egg infections arise from fecal contamination (17). In contrast, serovar Enteritidis colonization of the preovulatory follicles and ability to attach to ovarian granulosa cells was demonstrated, suggesting infection of eggs may occur as a result of systemic infection (158). More recent work suggests that serovar Enteritidis may be able to colonize the reproductive tract by virtue of its ability to bind to secretions within the isthmus by using type 1 fimbriae (42). However, all this latter work was carried out by using in vitro assays and needs to be confirmed in vivo.
Host genetic background also has a profound effect on pathogenesis in acute serovar Typhimurium infection of chicks and serovar Gallinarum infections of adult birds (31, 32) and bacterial carriage in persistently infected chickens carrying serovar Pullorum (22). Resistance is dominant, not sex-linked and not obviously associated in with MHC (39). The major phenotype is linked with the avian SAL1 gene, on chicken chromosome 5. The pattern of heredity is consistent with a single dominant gene on chicken chromosome 5 (106), expression of which is manifested by reduced bacterial counts in the spleen in vivo and by different rates of bacterial killing by peripheral blood monocyte-derived macrophages and levels of Salmonella-induced oxidative burst in vitro (176). The SAL1 gene has little effect on the intestinal carriage, which is regulated by other chicken genes (12, 46). NRAMP1 (termed Slc11a1 on chicken chromosome 7) and TLR4, which have a major effect on murine resistance to salmonellosis in inbred mice, have only relatively minor effects on resistance to salmonellosis in chickens (81, 95).
Our understanding of the virulence mechanisms of Salmonella for poultry is restricted by the limited number of studies that have been carried out comparing the behavior of wild-type strains of defined virulence with isogenic strains carrying defined mutations. The subject is further complicated by the differing virulence of different serovars for poultry, together with the profound effects that bird age, intestinal flora, and genetic resistance have on Salmonella pathogenesis. However, what is clear is that different serovars rely to varying degrees on different virulence gene clusters to infect poultry. This is best illustrated by Salmonella pathogenicity islands 1 and 2, which encode type III secretion systems TTSS-1 and TTSS-2, respectively. TTSS-1 and TTSS-2 influence different stages of Salmonella pathogenesis. Different Salmonella serovars induce different forms of disease in poultry and, not surprisingly, they show a different reliance on these virulence factors during infection. Serovar Gallinarum was shown to require a functional TTSS-2 for infection of 3-week-old birds via the oral route, but surprisingly, disruption of TTSS-1 did not influence virulence (89). The TTSS-1 mutant (insertional inactivation of spaS) was unable to invade avian epithelial cells in vitro, but it persisted within chicken macrophages as well as the wild type. In contrast, the TTSS-2 mutant (insertional inactivation of ssaU) was fully invasive but less able to persist within chicken macrophages. These observations confirm a key role for TTSS-2 in influencing the systemic pathogenesis of serovar Gallinarum and suggest that serovar Gallinarum penetrates the intestinal mucosa by a mechanism independent of TTSS-1. A similar role for these virulence factors was observed for serovar Pullorum in one-week-old birds (177). Again a functional TTSS-2 was shown to be key for systemic pathogenesis and for persistent infection, which would normally lead to the carrier state. However, in contrast to serovar Gallinarum, disruption of TTSS-1 was mildly attenuating.
In poultry a role for large-molecular-weight plasmids in virulence has been found for some but not all serovars. Serovar Gallinarum carries an 85-kb plasmid that influences the virulence of serovar Gallinarum in day-old and 2-week-old birds following oral and intravenous routes of infection (14). Failure to recover organisms from the livers and spleens of birds after oral infection was interpreted as suggestive of a role for plasmid genes in intestinal invasion. But clearly, for bacteria to be recoverable from such organs, they would first need to adhere to intestinal epithelium, invade mucosa, replicate within tissues, and resist innate host defense mechanisms. Therefore plasmid virulence genes could be influencing any of these processes. Further work showed that a serovar Gallinarum Tn3 mutant was less able to colonize and or invade intestinal mucosa following oral infection of chickens. The transposon was found to have inserted near an ORF with no homologies in the data banks. This ORF was adjacent to two additional ORFs with a high degree of homology of Escherichia coli genes encoding the minor structural subunits (FaeH and FaeI) of the K88 fimbria. A similar region of homology was found by DNA-DNA hybridization on the virulence plasmids of serovar Pullorum, serovar Dublin, and other serovar Gallinarum strains but not in the plasmids of serovar Typhimurium, serovar Enteritidis, or serovar Choleraesuis (139).
An 85-kb plasmid was also shown to influence the virulence of serovar Pullorum for newly hatched chicks (15). The plasmids from these two serovars are functionally homologous since, although plasmid curing results in total loss of virulence, this can be completely restored by introducing the plasmid from the other serovar (16). However, other aspects of virulence, such as the greater virulence of serovar Gallinarum for older birds and the specificity for poultry, was not associated with the plasmid. In contrast a 54-kb plasmid of serovar Enteritidis, required for virulence in mice, did not influence the pathogenesis of serovar Enteritidis PT4 for young chicks or adult laying birds by any route of infection (66) and this was also found for the 85-kb plasmid of serovar Typhimurium in virulence for newly hatched chickens (P. A. Barrow, unpublished results). An explanation for this result is that serovars Gallinarum and Pullorum are true systemic pathogens that require a full repertoire of virulence determinants to overcome the avian immune system. In contrast, serovars Enteritidis and Typhimurium appear to be opportunists exploiting the immunodeficiencies of young birds or immunocompromised older birds.
Both serovar Gallinarum and serovar Pullorum also possess a much smaller plasmid containing two to three ORFs that does not contribute to virulence (19).
Pigeons are susceptible to infection by Salmonella. Clinical signs vary enormously. At postmortem intestinal inflammatory responses are prevalent in "squabs" (young birds), whereas systemic disease is evident in adult birds as indicated by pneumonia, brain abscesses, and congestion of the liver (50). Phage types 2 and 99 of serovar Typhimurium variant Copenhagen (O5-negative) are almost exclusively associated with infections of pigeons. A certain degree of host "adaptation" of pigeon strains of serovar Typhimurium variant Copenhagen has therefore been postulated (130). A recent study documented the host specificity of pigeon-derived serovar Typhimurium variant Copenhagen strains by determining the bacterial characteristics that were associated with any adaptation of these strainsfor pigeons (121). Pulsed-field gel electrophoresis (PFGE) patterns of 38 pigeon strains were compared with those obtained from 89 porcine, poultry, and human strains of variant Copenhagen. The pigeon strains were very closely related, whereas the strains from the other host species were relatively unrelated. Pigeon-derived strains were much more cytotoxic for pigeon macrophages than were the porcine strains. After experimental infection of pigeons with a pigeon strain, clinical symptoms, fecal shedding, and colonization of internal organs were more pronounced than those after infection with a porcine strain.Together the data suggest that the DT 99 strains are adapted to pigeons. The molecular basis of the specificity of this serovar for pigeons remains unknown.
There are two possible explanations to account for the apparent host specificity of certain Salmonella serovars. Environmental factors may increase exposure of particular animal species to certain serovars. For example, ruminants may be more exposed to serovar Dublin than serovar Choleraesuis and vice versa for pigs. Alternatively, there are genetic differences between these serovars, which may allow them to survive and/or grow in specific niches only found within ruminants or pigs. However, these two explanations are not mutually exclusive and the host species genetic background may also play a role.
The role of environmental factors in influencing Salmonella host specificity can be investigated by comparing the virulence of different serovars in different animal species in controlled experimental infection studies. In a coordinated study using the same strains, the virulence of serovars Typhimurium, Dublin, Choleraesuis, Gallinarum, and Abortusovis were compared in orally infected cattle, pigs, sheep, poultry, and mice. In calves, serovars Typhimurium, Dublin, and Choleraesuis were all highly virulent, causing severe diarrheal disease. In contrast, serovars Abortusovis and Gallinarum were avirulent (122, 169, 170). In pigs, serovar Typhimurium caused self-limiting diarrhea and serovar Choleraesuis caused severe systemic disease, whereas serovars Dublin, Gallinarum, and Abortusovis were avirulent (173) (P. A. Wallis and Paulin, unpublished observations). In sheep, only serovars Dublin, Abortusovis, and Gallinarum were compared and none of these serovars caused diarrheal disease. Only serovar Dublin caused a prolonged pyrexial response although both Dublin and Abortusovis were recoverable in significant numbers from systemic tissues (164). In 1-week-old poultry only serovar Gallinarum was virulent (J. Olsen, personal communication). In mice serovars Typhimurium and Dublin were highly virulent, and serovars Choleraesuis and Abortusovis were moderately virulent (51, 163); serovars Abortusovis and Gallinarum were of low virulence (24). A more restricted study using mice, chickens, and other laboratory animals and concentrating on the capacity to produce systemic disease in adult animals similarly showed that serovar Gallinarum produced systemic disease only in chickens (13), serovar Dublin and serovar Enteritidis produced clinical disease only in mice, whereas serovar Choleraesuis produced disease in mice and other laboratory mammals.
These studies illustrate that representative strains of these serovars do exhibit clear differences in virulence for different host species and as such show host specificity. Serovars Abortusovis and Gallinarum were virulent only for sheep and poultry, respectively, and these are examples of host-specific serovars. Serovars Dublin and Choleraesuis were virulent for several distinct host species but not for all and therefore represent host-restricted serovars. Finally, serovar Typhimurium has a broad host range typical of the ubiquitous serovars. These strains represent a unique collection and a valuable resource for further probing factors that influence host specificity. In terms of the ability to produce systemic infections in adult animals, it has also been suggested that the major division between serotypes was between those able to produce disease in birds (serovar Gallinarum) as opposed to in mammals (serovars Dublin, Choleraesuis, and Typhi) with additional characteristics, such as the wider host range exhibited by serovars Choleraesuis and Dublin compared with the highly host-specific serovars such as serovars Typhi and Sendai. A number of questions can thus be posed relating to explaining the infection phenotype of these strains. These include (i) whether host specificity is exhibited among strains able to produce gastroenteritis, such as serovar Typhimurium and serovar Enteritidis; (ii) whether early interactions between Salmonella and mucosal surfaces determine the ability of a strain to produce systemic disease; and (iii) whether the ability to survive and multiply systemically explains the capacity to produce systemic infections
The increasing availability of bacterial genomic sequence information is providing valuable insight into the potential of different serovars of Salmonella to cause disease. Genome sequencing has enabled the characterization of the repertoire of virulence genes carried by different serovars, which can further our understanding as to how and why different serovars are associated with different types of infection and host specificity.
The first two serovars to be sequenced were the human-specific serovar Typhi (120) and the broad-host-range serovar Typhimurium (107). The information engendered by a comparison of these two serovars alone can provide insight into the roles of different genetic loci of serovar Typhimurium genes that are associated with gastroenteritis and those of serovar Typhi that are associated with systemic infections in humans. More recently the complete genome sequencing of other serovars has been initiated (www.sanger.ac.uk/Projects/Salmonella). These include serovar Typhimurium SL1344 and DT104, serovar Enteritidis, serovar Gallinarum, and S. bongori. The inclusion of S. bongori will enable information to be generated that will increase our understanding of the evolution of Salmonella and the acquisition of virulence determinants, since S. bongori is widely considered to be avirulent. Preliminary analysis of the S. bongori genome has demonstrated that this species lacks most of the genes normally found on the key virulence locus Salmonella pathogenicity island 2 (SPI-2), illustrating the power of genomic comparisons for providing insight into the molecular basis of pathogenesis. The inclusion of serovar Enteritidis and serovar Gallinarum in the sequencing program is valuable, because these serovars are antigenically related and this, together with other evidence, points to these serovars having a common ancestor (96). A comparison of the genomic sequences of these two serovars will add to our understanding of host specificity because serovar Enteritidis has a broad host range and serovar Gallinarum specifically infects birds. Sequencing other closely related serogroup D serovars of great economic significance, serovars Dublin and Pullorum, has also been started and this will add greatly to a more complete understanding of the infection biology of these different virulent pathotypes. Further proposals are also under way to sequence additional strains of serovar Dublin, serovar Pullorum, and serovar Choleraesuis by using strains of defined virulence and host specificity (172, 173, 175). It has also been recommended that this collection of sequenced strains should be extended to include isolates of serovars Paratyphi B and C, which bear more than superficial resemblance to serovars Typhimurium and Choleraesuis but are serious human pathogens, and also serovar Typhimurium DT2, which produces a severe systemic disease in adult pigeons (see below).
Some conclusions of relevance to pathogenesis and host specificity can be drawn from a superficial comparison of the serovar Typhi and serovar Typhimurium genome sequences (107, 120). The 4.81-Mbp genome of serovar Typhi is predicted to contain 204 pseudogenes. In contrast, the 4.86-Mbp chromosome of serovar Typhimurium is predicted to contain only 39 pseudogenes. In serovar Typhi, 75 of the 204 pseudogenes are predicted to be involved in housekeeping functions (including cobalamin biosynthesis genes cbiC, -J, -K, and –M, the proline transporter proV, and dmsA and –B, involved in utilization of dimethyl sulfoxide anaerobically as an electron acceptor in respiration); thus, apparently these genes are redundant during serovar Typhi infections of humans. Forty-six of the pseudogenes are thought to be in virulence genes. Seven of the 12 chaperone-usher fimbrial operons of serovar Typhi carry pseudogenes. Such fimbriae are likely to promote adhesion of Salmonella to target host cells. Consistent with this, narrow host range serovars such as serovar Typhi are less likely to require a broad repertoire of fimbrial adhesions. Many of the pseudogenes of serovar Typhi lie within or associated with Salmonella pathogenicity islands. These include sopE2 and sopA of SPI-1, slrP and sseJ of SPI-2, and cigR, marT, and misL of SPI-3. SPI-1 and SPI-2 encode type three secretion systems that deliver effector proteins into target host cells and influence, respectively, Salmonella-induced intestinal inflammatory responses and net growth in vivo. The implications of losing virulence factors that promote host inflammatory responses and bacterial replication are discussed elsewhere in this chapter.
The prevalence and polymorphisms of genes encoding TTSS-1-dependent translocated effector proteins were assessed in clinical isolates of S. enterica to determine whether the repertoire of effector proteins correlates with host specificity or emergence of epidemic strains. As assessed by PCR and Southern blotting sopB, sopD, sopE2, sipA, sipB, sipC, and sptP were highly conserved, whereas avrA and sopE were present in only some isolates, suggesting that these latter effectors may play a role in the emergence of epidemic strains (128). Consistent with these observations it was recently reported that sopE was prevalent (although not exclusively) in epidemic strains (79). However, of key importance in this type of analysis is the need to assess the repertoire of effector protein expression rather than carriage. This issue was addressed, in part, in a study comparing the repertoire of secreted effector proteins in serovar Paratyphi B to distinguish between strains associated with systemic and enteric disease. All serovar Paratyphi B strains from systemic infections were found to be somewhat genetically related with respect to the pattern of their virulence gene carriage, including sopB, sopD, sopE1, avrA, and sptP as assessed by PCR and Southern blot analysis, as well as other molecular properties (multilocus enzyme electrophoresis type, PFGE type, ribotype, and IS200 type). Such strains were classified as members of the systemic pathovar (SPV). All SPV strains lacked avrA yet possessed a novel sopE1-carrying bacteriophage. Western blotting analysis showed SPV strains expressed high levels of SopE1 and normal levels of SopB, but they failed to express SopD. In contrast, strains from enteric infections (classified as belonging to the enteric pathovar), possessed various combinations of the respective virulence genes and displayed a range of PFGE patterns and ribotypes. The authors propose that a PCR technique for testing for the presence of the genes sopE1 and avrA may be used as a diagnostic tool for distinguishing enteric and systemic pathovars of serovar Paratyphi B that would enable the potential risk to public health of new field isolates to be assessed.
Auxotrophy is more frequent among host-specific serovars. However, there is no clear correlation between auxotrophy and host specificity. The occurrence of auxotrophy in serovars that do not normally colonize the intestine suggests that metabolic flexibility may be essential for efficient intestinal colonization when in competition with the commensal intestinal flora. However, the physiological complexity and metabolic redundancy of the Enterobacteriaceae suggest that this may be difficult to determine.
Recent evidence suggests that genome plasticity in Salmonella may play an important role in host specificity and adaptation to host-specific niches. It was reported that field isolates of host-specific Salmonella serovars Typhi, Paratyphi C, Gallinarum, and Pullorum frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons (101, 102, 104), while broad-host-range serovars consistently have a conserved chromosomal arrangement (100, 103). Such chromosomal rearrangements appear to be a consequence of lifestyle differences between broad-host-range and host-specific serovars, because no intrinsic differences in recombination frequency at rrn operons could be detected in vitro in strains of serovar Typhi and serovar Typhimurium (73). Thus, what is driving chromosomal rearrangements in these host-specific serovars? The ability to regulate the expression of a large genome with a complex repertoire of metabolic and virulence gene is essential for a pathogen that has to survive both within a range of animal species and while free in the environment. The greater the range of environments in which the pathogen may survive, the less likely there is to be less selection pressure on the genome of the organism. Host-specific Salmonella serovars will by definition have fewer niches in which to persist compared with broad-host-range serovars. Thus selection pressure on the genomes of host-specific serovars is likely to be greater than broad-host-range serovars. Reorganization of the sequence of genes within a bacterial chromosome through large-scale recombination around the rrn operons, bringing different sets of genes closer to the origin of replication of the chromosome, will influence gene dosage and therefore potentially expression and provide additional opportunities to respond to a restricted and hostile environment. Having to persist within the hostile environment of host tissues, while being targeted by a sophisticated immunological response, may be the selective pressures that drive genomic plasticity in the host-specific but not broad-host-range serovars.
The host and bacterial factors that determine whether Salmonella serovars remain restricted to the gastrointestinal tract or penetrate beyond the mucosa and cause systemic disease remain largely undefined. Salmonella must first survive passage through the stomach and then initial adhesion of bacteria to epithelial cells if an invasive infection is to be established. Much effort has been spent in assessing the repertoire of fimbrial and nonfimbrial adhesins of Salmonella, because bacterial adhesins are frequently highly host specific and therefore an obvious factor that potentially influences host range. The repertoire of fimbrial operons encoded by the sequenced strain of serovar Typhi CT18 has been compared with other clinical isolates of serovar Typhi and other serovars of Salmonella by DNA hybridizations. The serovar Typhi CT18 genome contains a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. Hybridization analysis showed that all 14 putative fimbrial operons of serovar Typhi were also present in a number of nontyphoidal Salmonella serovars. Thus, a simple correlation between host range and the presence of a single fimbrial operon is unlikely. However, the serovar Typhi genome differed from that of all other serovars investigated in that it contained a unique combination of putative fimbrial operons (160). These observations do not rule out a role for adhesins in influencing host range and pathogenesis, although it would seem likely that a component of the host-specific phenotype would be expressed systemically. DNA hybridization analysis provides no information about gene expression. Clearly, it is important to look at the repertoire of fimbrial expression by different serovars during infections of different host species. Such an analysis may provide a greater insight into how different serovars use adhesins during interactions with different hosts and cell types during pathogenesis.
If host specificity is determined by Salmonella interactions with host species in the intestines, it should be possible to overcome any intestinal barriers to infection by parenteral inoculation. Strains of serovar Dublin that were avirulent for pigs after oral infection were also shown to be avirulent following intravenous challenge (173), suggesting that the porcine resistance to serovar Dublin infections is not determined by intestinal factors. Studies assessing early host-pathogen interactions in the gut by using strains of defined virulence have found no correlation between host specificity and the magnitude of intestinal invasion in cattle (172), sheep (164, 172), pigs (28), mice (13), or poultry (35), suggesting that the major site of expression of host specificity and the capacity to produce systemic disease are expressed systemically.
The predominant pathology associated with broad-host-range serovars like Typhimurium is the characteristic inflammatory response induced within intestinal mucosa. Enteropathogenic Salmonella serovars have acquired, through horizontal gene transfer, the apparatus and effector proteins necessary to elicit such responses. Epithelial cells play a key role in orchestrating the intestinal inflammatory responses to intestinal pathogens. The interaction of Salmonella with epithelial cells in vitro leads to the basolateral release of chemokines and apical secretion of pathogen-elicited epithelial chemoattractant (94). These substances are, at least in part, responsible for directing the recruitment and trafficking of PMNs across intestinal epithelial cells, and a functional TTSS-1 is required for the induction of PMN transmigration in vitro (108). Salmonella-induced PMN influxes in vivo are also TTSS-1 dependent (57, 161, 170). Recently, serovar Typhimurium was shown to induce the up-regulation of CXC chemokines in bovine (143) and chick (183) intestinal mucosa in vivo, which is likely to be responsible for the PMN influx observed. Thus through the activity of TTSS-1, serovar Typhimurium actively induce an acute inflammatory cell influx, which presumably confers some evolutionary advantage. However, PMNs that have migrated across model intestinal epithelia appear to have an enhanced ability to kill serovar Typhimurium (113), which can be used prophylactically (54). Thus, the strategy of evoking an inflammatory response does not come without risks.
TTSS-1 has little impact on the systemic virulence of serovar Typhimurium, as assessed in mice (56), and on serovar Gallinarum infections in chickens (89). Thus, it is likely that the primary functions of effector proteins secreted by TTSS-1 are to enable the pathogen to induce diarrheal disease in host animals. This ability greatly enhances Salmonella dissemination into the external environment and significantly increases the likelihood of transmission via the fecal-oral route. Therefore, the strategy of inducing of acute intestinal inflammatory responses by broad-host-range serovars such as serovar Typhimurium may restrict Salmonella infections to the gut but facilitate rapid host-to-host transmission through the induction of diarrhea.
There is increasing evidence that the host-specific serovars are less capable of inducing intestinal inflammatory responses. For example, strains of serovar Dublin are reproducibly less enteropathogenic than strains of serovar Typhimurium in bovine ligated ileal loops (170). Similarly, strains of serovar Choleraesuis are less enteropathogenic than strains of serovar Typhimurium in porcine ligated ileal loops (Wallis and Paulin, unpublished data), and serovar Gallinarum is less enteropathogenic than serovar Typhimurium in young birds (P. Wigley, personal communication). In a study using chicken nonpolarized primary kidney cells, cytokine responses induced by the serovars Enteritidis, Typhimurium, and Gallinarum were measured by quantitative RT-PCR and bioassays. Invasion of cells by serovar Typhimurium and serovar Enteritidis caused an 8- to 10-fold increase in production of the proinflammatory cytokine IL-6, while invasion by serovar Gallinarum caused no such increase (91). Serovar Gallinarum also induced lower levels of IL-1β than these two other serovars. In addition to other factors, this effect may be the direct result of the presence/absence of flagellin, which is a potent stimulator of proinflammatory signals in nonpolarized cell cultures (60) and, unlike serovar Typhimurium and the very closely related serovar Enteritidis, serovar Gallinarum is nonflagellate. Thus, these potentially important observations need to be confirmed in a polarized cell culture model or preferably in vivo and the effect of flagella explored.
Avoidance of intestinal inflammatory responses could facilitate immune-evasion and systemic spread through tissues. This may be achieved either passively by loss of effector proteins involved in eliciting proinflammatory responses or actively through the evolutionary acquisition of effector proteins involved in immunosuppression. Consistent with the model of systemic salmonellosis occurring through a "passive" strategy of stealth is the observation that serovar Typhi has lost the ability to express some proinflammatory TTSS-1-dependent effector proteins, including SopA, SopE2, and SopD2, by gene decay (120). Evidence for an "active" strategy of stealth comes from the observation that the TTSS-1-dependent effector AvrA inhibits activation of the key proinflammatory NF-κβ transcription factor (37). However, AvrA is expressed (at least in vitro) by serovar Typhimurium, which is arguably one of the most proinflammatory pathogens known to humans and not by serovar Typhi. These observations question a role for this protein in suppression of inflammatory responses. Further doubt is engendered by the observation that a serovar Dublin avrA mutant was no more enteropathogenic than the wild-type parental strain (145). Furthermore, serovar Paratyphi B strains associated with systemic infections lack avrA (129), ruling out a role for this protein in facilitating systemic spread. Thus, the role of AvrA in pathogenesis remains unclear and the regulation of expression of this protein in vivo awaits clarification. Further genomic analysis of more strains of different serovars to assess the repertoire of TTSS-dependent secreted effector proteins, together with analysis of gene expression in vivo, will provide more insight into the role of inflammation and the control of the spread of Salmonella to systemic sites.
The host-specific serovars are typically associated with systemic infections accompanied by higher levels of mortality in adult animals. In evolutionary terms killing one’s host is arguably not an ideal strategy for successful proliferation. A common feature of some of the host-specific Salmonella serovars is infection of reproductive tissues, resulting in infection of eggs in birds and induction of abortion in mammalian species. This is particularly the case for serovar Pullorum in poultry, serovar Dublin in cattle, serovar Abortusovis in sheep, and serovar Abortusequi in horses. Salmonella replicate to high numbers in fetal tissues, which leads to abortion and subsequent dissemination of high numbers of bacteria into the environment, facilitating further infection of other animals in the near vicinity. Thus, systemic infections have the potential to promote animal-to-animal spread of Salmonella by a mechanism that is quite distinct from diarrheal disease. There is some argument suggesting that these serovars are better and thus further adapted to the host, facilitating transmission with reduced injury to the adult.
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