Pathogenicity of Salmonella enteritidis in poultry

International Journal of Food .~licrobiolo~', 21 (1994) 89-105

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e2 1994 Elsevier Science B.V. All rights reserved 0168-1605/94/$[)7.00 FOOD 00672

Pathogenicity of Salmonella enteritidis in poultry Shoko Suzuki * National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokura 1-15-1, Kokubunji-shi, Tok3'o 185, Japan

Salmonella enteritidis is a common pathogen of all species of mammals and fowls. The recent increase in the number of outbreaks of food poisoning due to S. enteritidis in man was epidemiologically analysed, and it was considered that contaminated eggs or egg products were the major source of this infection. To assist in prevention and eradication of human food poisoning many investigators have studied the pathogenicity of S. enteritidis in poultry. Gross pathological observations after natural and experimental infections with S. enteritidis in poultry revealed that this organism may cause systemic infection in chicks and laying hens accompanied by prolonged faecal shedding. Some variations in the mortality rates, clinical symptoms, faecal shedding and frequency of production of contaminated eggs were observed in the chicks and hens experimentally infected with S. enteritidis isolates, Choice of bacterial strain, phage type, age of bird and inoculum size may affect the outcome of an infection. Moreover, isolation of the organisms from the ovaries, oviducts and egg contents indicates the possibility of transovarian infection of S. enteritidis in chickens. Some virulence factors associated with S. enteritidis are also reviewed in the present paper. Key words: Salmonella enteritidis; Pathogenicity; Chicken; Virulence factor

Introduction Salmonella enteritidis is a common pathogen of many species of mammals and fowls. Prior to the 1980s, S. enteritidis was rarely isolated from poultry (Faddoul and Fellows, 1966; Snoeyenbos et al., 1969), and most isolates may have been derived from contaminated feed (Williams, 1981). Recently, however, the incidence of S. enteritidis infection in poultry flocks has been increasing in Britain, the United States and other countries (Dreesen et al., 1992; Hopper and Mawer, 1988; Khakhria et al., 1991). Concurrently, a dramatic increase in the number of outbreaks of food poisoning due to S. enteritidis in man has been reported (Duguid and North, 1991; Stevens et al., 1989; Rodrigue et al., 1990). Epidemiological studies have attributed the outbreaks of S. enteritidis food poisoning to the consumption of contaminated eggs or egg products (Hedberg et al., 1993; St. Louis et al., 1988). Thus, S. enteritidis has become the most serious pathogen for man * Tel. 0423-21-1841; Fax 0423-21-1769.

SSDI 0168- 1 6 0 5 ( 9 3 ) E 0 0 8 8 - 9

90 and the poultD, industo ~. It is thus important to analyse the behavior of the organism in chickens for the prevention and elimination of food poisoning. Most Salmonella infections in poultry arise from the ingestion of these organisms. Ingested organisms proceed through the alimentary, tract, where they may interact with the mucosal surface at the Peyer's patches, and may adhere to and penetrate into the intestinal epithelial cells. After proceeding through the intestinal wall and into deeper tissues, some Salmonella can invade, survive and multiply in the reticuloendothelial system, and disseminate to other tissues, causing serious systemic diseases (Barrow et al., 1987). Recently, some reports concerning the virulence factors associated with Salmonella have been published (D'Aoust, 1991; Finlay and Falkow, 1988). Such virulence factors are necessary for bacteria to invade, colonize, survive and multiply in the cells, and to overcome the host defence systems. Based on current investigations carried out by many researchers, the present paper reviews the pathogenicity of S. erzteritidis in poultry and some virulence factors of this serotype.

Clinical symptoms in chickens It is generally recognized that non-typhoid types of Salmonella such as S. enteritidis and S. ~'phimurium responsible for food poisoning cause both overt and symptomless intestinal infections in a wide range of domestic and wild animals and birds. In general, acute outbreaks of S. enteritidis infection occur in young birds and in birds under extreme stress conditions, and seldom occur in semimature and adults under natural conditions. Also, in the recent cases of natural infection with S. enteritidis, a high mortality with rates sometimes up to 20% has been observed in chicks less than 2 weeks of age, and some chicks of the affected flocks have showed signs of stunting (Lister, 1988; O'Brien, 1988). The response to S. enteritidis infection in hens is different from that in younger birds. The infection seldom causes mortality in birds more than 1 month old. The resistance to salmonellosis in chicks generally increases in proportion to the age of chickens possibly due to the development of normal flora in the intestine and of the immune system with aging (Corrier et al., 1991; Smith and Tucker, 1980; Ziprin et al., 1989). None of the chickens from the broiler breeder and layer flocks naturally infected with S. enteritidis showed obvious clinical abnormalities, increase in mortality or decrease of egg production (Cooper et al., 1989; H o p p e r and Mawer, 1988; Humphrey, 1990a), although the body condition of some of them was rather poor (Lister, 1988). No signs of illness were also observed in 52-week and 18-week-old laying hens with experimental oral administration of S. enteritidis phage type 4 (Humphrey et al., 1989a). Most birds were in full lay, without gross pathological abnormalities, although some hens were found to be carrying S. enteritidis in their caeca (Hopper and Mawer, 1988; Lister, 1988). These symptomless carriers may spread the infection in the flock due to the contamination of their intestinal contents with S. enteritidis.

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On the other hand, variations in the mortality rates were observed in experimental infections in chicks. Gast and Beard (1992) suggested the presence of significant differences in the mortality rates (14.5-89.5%) in day-old chickens orally inoculated with eight S. enteritidis isolates. Barrow (1991) also reported the presence of variations in mortality ranging from 96% (phage type 4) to 20% (phage type 13a) in four phage types of S. enteritidis (phage types 4, 6, 8 and 13a). In contrast, none of the nalidixic acid-resistant mutants of S. enteritidis phage type 4 isolates were lethal to the chicks (Hinton et al., 1989). Although most of the infected hens failed to show clinical signs, as described above, some experimental inoculations of S. enteritidis strains caused depression, anorexia, diarrhea, reduced egg production a n d / o r mortality in laying hens (Gast and Beard, 1992; Humphrey et al., 1991a,b; Shivaprasad et al., 1990; Timoney et al., 1989). As indicated in the mortality among chicks, there were significant differences in the total egg production among hens after infection with S. enteritidis strains of different origin or phage types (Gast and Beard, 1992; Shivaprasad et al., 1990), as well as in the virulence for laying hens between the isolates from man or eggs and from the ovary of a hen (Shivaprasad et al., 1990). These data suggest that in addition to the age of the birds, the selection of bacterial strains and phage types may be an important factor affecting the outcome of the S. enteritidis infection. However these factors are not essential for the pathogenesis of the organisms because the strains used were isolated from diseased humans and chickens. Further studies may enable to define the role of these factors in the S. enteritidis infection in poultry.

Pathological changes in chickens In the infection of chicks with S. enteritidis, pericarditis, necrotic foci in the liver a n d / o r an indurated yolk sac remnant, which was always detected through the abdominal wall by palpation, were observed (Barrow, 1991; O'Brien, 1988). Postmortem findings of the infected hens revealed the presence of deformed, shrunken, discoloured and or congested ovaries and ovules, shrunken or extended and malformed follicles with a fluid-filled cyst attached, soft-shelled eggs a n d / o r egg peritonitis (Cooper et al., 1989; Hopper and Mawer, 1988; Lister, 1988), suggesting the transovarian infection of S. enteritidis. Although Humphrey et al. (1989a, 1991a,b) reported that no macroscopic or microscopic lesions were present in the visceral organs of most infected hens at necropsy, several birds showed peritonitis and numerous small pale foci in the liver and kidneys suggesting that S. enteritidis may cause systemic infection in chicks and hens.

Recovery of organisms from chickens As described in recent reports, S. enteritidis was recovered from the heart, liver, spleen, caeca, yolk sac, ovary, oviduct, peritoneum, egg a n d / o r faeces of infected birds. The recovery of organisms from the infected chicks and hens lasted for a

~2 long period of time. In some studies, the organisms had been detected in the heart, yolk sac, caeca a n d / o r yolk of naturally infected broiler chicks up to the time of slaughter (Lister, 1988; O'Brien, 1988). As in the case of naturally infected birds, the organisms persisted in the viscera of hens with experimental oral administration from 70 days (Humphrey et al., 1989a) to 22 weeks after the infection (Gast and Beard, 1990b). Prolonged faecal shedding from chickens exposed by contact was also observed (Barrow, 1991; Gast and Beard, 1990b; Nakamura et al.. 1993b). Chicks which were infected by contact with S. enteritidis phage type 4 within 24 h of hatching, yielded the organisms in the viscera and remnant yolk and intermittently shed organisms at least until 28 weeks after exposure (Nakamura et al., 1993b). Faecal shedding from the experimentally infected hens was also intermittent and lasted until 6 weeks ~fter inoculation (Shivaprasad et al., 1990). As described above, significant differences in the frequency of production of contaminated eggs (0-8.1%) were observed among the different sources or phage types of strains used (Gast and Beard, 1992; Shivaprasad et al., 1990). Barrow (1991) showed that faecal shedding in hens infected with strains of phage types 4 or 13a lasted longer than that with strains of phage types 6 or 8. In another experiment, S. enteritidis phage type 4 was isolated from the viscera and egg content in the birds infected at 52 weeks of age, but not in the younger birds, and prolonged faecal excretion was also observed in the former birds (Humphrey et al., 1991b). Though it remains to be determined why the older birds were more affected than the younger birds, stress or some unknown factors may be involved in the outcome of infection. The dose of the pathogen administered was closely related to the duration of faecal shedding, but not to the ability to cause systemic infection in birds or contamination of egg contents (Humphrey et al., 1989a, 1991a). Ingested bacteria must first overcome the defence mechanisms of the alimentary canal, including the bactericidal action of the acid in the stomach (Giannella et al., 1972), rapid removal by peristaltism in the small intestine (Dixon, 1960) and the protective effect of the normal intestinal flora (Barnes and Impey, 1980). Though large doses of bacteria are required to overcome these defence mechanisms, even a small number of bacteria may be able to cross the intestine, proliferate and cause systemic infection when bacteria avoid these barriers.

Infection of S. enteritidis in eggs

Contamination of egg contents with S. enteritidis may occur by penetration of the organism through the shell or by transovarian infection with the organism. Previous studies demonstrated that the infection of S. typhimuriurn occurred through the egg shell (Williams et al., 1968). Faecal shedding of S. enteritidis from the infected chickens for long periods of time as described above, increases the opportunity of egg soiling with infected droppings. S. enteritidis phage type 4 is more heat-resistant than some other egg-associated Sabnonella (Humphrey et al., 1990b). Therefore, it is possible that S. enteritidis penetrates into the egg through the shell when the egg is soiled with faeces containing the organisms. However,

t13 Gast and Beard (1990a) suggested that this mode of infection with S. enteritidis was not essential to cause the contamination of egg contents, as the organisms were more frequently recovered from the yolk or albumen of eggs laid by infected hens than from eggshells. S. enteritidis aggressively penetrated into the tissues of the adult hen causing a systemic infection in many visceral organs including peritoneum, ovules and oviduct, and the organisms were predominantly isolated in the yolk or albumen of about 10% of the eggs during the first 2 weeks following experimental oral inoculation, suggesting the possibility of transovarian infection with S. enteritidis (Timoney et al., 1989). The ability of transovarian infection was also demonstrated with the isolation of organisms from the yolk a n d / o r albumen of eggs laid by hens infected with S. enteritidis (Gast and Beard, 1990a; Humphrey et al., 1991a.b; Nakamura et al., 1993a; Shivaprasad et al., 1990). The gross pathological changes observed in the ovary and oviduct of laying hens also indicated the possibility of transovarian transmission of S. enteritidis in the birds (Cooper et al., 1989). The yolk or albumen is presumably contaminated when the egg laid by an infected hens moves from the ovary through the oviduct before being covered by the shell. Furthermore, the fact that the albumen was more frequently contaminated than the yolk suggested that some eggs became infected in the peritoneum or oviduct (Shivaprasad et al., 1990; Timoney et al., 1989). The infection of the oviduct may occur through haematogeneous spread after intravenous administration of S. enteritidis (Barrow and Lovell, 1991). While the frequency of contamination of the egg contents showed considerable variations, in general, the proportion of eggs naturally or artificially infected with S. enteritidis was estimated as approximately 1% or less (Humphrey et al., 1989b). The difference in the proportion of eggs containing Salmonella may depend on the size of the flocks examined because a large and small proportion was observed in small and large flocks, respectively (Duguid and North, 1991). Also Hopper and Mawer (1988) suggested that the low frequency of infected eggs was due to the variations in the number of affected ovules in birds with S. enteritidis infection and that normal uninfected ovules may also coexist in the same ovary. Organisms were isolated in small numbers (most probable number less than 100/100 ml) from eggs laid by hens infected with S. enteritidis (Hopper and Mawer, 1988; Humphrey et al., 1989b). The complex system of membrane barriers, anti-bacterial components in the albumen and the large content of antibodies in the yolk may account for the presence of only small numbers of bacteria in the egg (Board and Fuller, 1974; Dadrast et al., 1990; Tranter and Board, 1982).

Stress factors involved in infection in chickens

As some birds in the flocks may remain indefinitely infected and shed the organism, the spread of infection may occur within the flocks when the birds are exposed to stresses. In poultry breeding, stresses including vaccinations, transfer of birds, rearing at a high density and induced molting may occur. Recent trial

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showed that the bird density did not appear to affect the S. typhimurium contamination rates of prechilled carcasses (Waldroup et al., 1992). Fasting conditions for induced molting in hens orally infected with S. enteritidis increased the incidence of inflammation of the epithelium and lamina propria of the colon and caeca, promoted the shedding of the organisms, and enhanced the susceptibility of the hens to the organism (Holt, 1993; Holt and Porter, 1992a,b). In another study, starvation prolonged the survival of S. enteritidis in the crop and decreased the speed of spread of the bacterium from the upper to the lower gut (Humphrey et al., 1993). These conditions may be conducive to the adhesion to and colonization of the intestinal epithelium.

Non-oral infection

Recent studies indicate that Salmonella present on the feathers of inoculated chicks can be easily and quickly disseminated to the pen mates (Bailey et al., 1990). This observation suggests that airborne droplets or dust particles contaminated with organisms may contribute to the spread of the infection through non-oral routes, such as the conjunctiva and respiratory tract. The conjunctival route has been found to be more effective than the oral route for infection of guinea-pigs with S. enteritidis (Moore, 1957). As in the case of the contact exposure with S. enteritidis, airborne or conjunctival infection caused systemic infection and faecal shedding in laying hens, suggesting that the spread of infection by airborne droplets or dust particles containing S. enteritidis may occur in a poultry house under natural conditions (Baskerville et al., 1992; Humphrey et al., 1992).

Virulence factors of S. enteritidis

Although the microbial factors that contribute to the virulence in S. enteritidis have not yet been fully elucidated, investigations on the virulence characteristics of S. enteritidis may contribute to a better understanding of the pathogenesis of avian infection with strains of this serotype. En terotoxin Enterotoxins have been identified in a number of genera of enteric bacilli. Several studies on the pathogenesis of Salmonella infections have suggested the potential involvement of exotoxins. Investigations of 68 Salmonella isolates, representing 39 serotypes including S. enteritidis, for the production of enterotoxin showed that most of the isolates produced a heat-labile enterotoxin (Jiwa, 1981). Koupal and Deibel (1975) described a cell wall-associated enterotoxic factor in S. enteritidis that caused intestinal fluid loss when administered orally to infant mice. This profuse toss of intestinal fluids was attributed to the enterotoxin-mediated activation of adenylate cyclase localized in the cytoplasmic membrane of host epithelial cells and to the attendant high concentrations of cyclic AMP in the cytoplasm (Peterson et at., 1983).

95 Enterotoxin of Salmonella, which is a heat-labile protein with a molecular weight of 110000 and an isoelectric point in the pH range from 4.3 to 4.8 (Houston et al., 1981), shows a subunit composition (Chopra et al., 1987b; Finkelstein et al., 1983) and appears to be closely related to the cholera toxin (CT) of Vibrio cholerae and heat-labile toxin (LT) of Eseherichia coli functionally, immunologically and at the genetic level (Jiwa, 1981; Jiwa and Mansson, 1983; Sandefur and Peterson, 1977). Like CT and LT the enterotoxin causes cytotonic or cytotoxic effects in CHO, Y1 or Vero cells, secretes fluids in the ligated loop model with adult rabbits, promotes the increase of vascular permeability of rabbit skin. Furthermore, the toxin also binds to GM~ gangliosides and increases the cyclic AMP levels by affecting the adenylate cyclase enzyme and each of these activities is neutralized by antisera to CT. DNA hybridization with the probes derived from the CT gene (ctxAB) demonstrated that a 6.3-kilobase enterotoxin (stx) gene of S. typhimurium was located on chromosomal DNA and that there was some degree of homology between its gene sequences and those of CT (Chopra et al., 1987a,b). The amount of toxin produced is very low (nanogram levels), although the toxin can be detected within hours in cell sonic extracts, and its production is influenced by the characteristics of the growth medium, aerobicity, pH, temperature and period of incubation (Jiwa, 1981; Peterson et al., 1981).

Cytotoxin In addition to the enterotoxin, cytotoxin production has also been demonstrated in most Salmonella strains including S. enteritidis (Ashkenazi et al., 1988; Baloda et al., 1983; Ketyi et al., 1979; Koo and Peterson, 1982; O'Brien et al., 1987). Salmonella cytotoxin is a heat-labile protein with a molecular weight of 5600078000 which is a component of the bacterial outer membrane (Ashkenazi et al., 1988; Reitmeyer et al., 1986) and causes cytotoxic effects on human AV-3, Vero, Hela and CHO cells (Ashkenazi et al., 1988; Ketyi et al., 1979; Koo and Peterson, 1982; Reitmeyer et al., 1986). Neutralization studies by Ketyi et al. (1979) showed that the cytotoxic activity of the Salmonella cytotoxin was neutralized by anti serum to the Shiga toxin of Shigella dysenteriae 1. In contrast, Ashkenazi et al. (1988) demonstrated that the cytotoxins produced by S. enteritidis, S. choleraesuis and S. typhi were genetically and immunologically distinct from the Shiga toxin and from Shiga-like toxins type I and type II of E. coli. Therefore, some toxins may be related to the Shiga toxin unlike others. In Salmonella gastroenteritis extensive damage of the intestinal mucosal surface is observed. The inhibition of protein synthesis in intestinal epithelial ceils by the Salmonella cytotoxin (Koo and Peterson, 1982; Koo et al., 1984) probably accounts for the morphological alterations of the intestinal mucosa observed in experimental infection. Thus, the Salmonella cytotoxin may play a role in local damage of the intestinal mucosa that results in enteric symptoms and inflammatory diarrhea. Lipopolysaccharide Lipopolysaccharide (LPS) of the bacterial cell wall which is also an important factor in the virulence of Salmonella consists of three components: lipid A, inner

96 core and O-side chain oligosaccharides. Lipid A is the endotoxic principle of LPS and activates macrophages. Several factors released from activated macrophages exert biological effects associated with fever and shock. The length and the structure of the O-side chain of LPS molecules may affect the Salmonella virulence in mice (Nakano and Saito, 1969; Valtonen et al., 1975). S. enteritidis phage type 7 which was spontaneously derived from S. enteritidis phage type 4 following the loss of the ability to express long-chain LPS was avirulent in mice, while S. enteritidis phage type 4 was virulent (Chart et al., 1989a). Full expression of long-chain LPS is also necessary for the virulence of S. enteritidis for mice, whereas it was shown that partial expression did not to confer virulence (Chart et al., 1993). O-side chain of LPS molecules is associated with the resistance of Salmonella to the lytic action of the complement cascade. Smooth phenotype strains are more resistant to lysis than isogenic rough variants because the O antigen polysaccharide through steric hindrance prevents the C5b-9 complex from the classical or alternative complement pathways from reaching the hydrophobic domain of the outer membrane (Grossman et al., 1987). Moreover, the ability of a Salmonella strain to colonize the mouse large intestine decreases when its LPS structure becomes more defective (Nevola et al., 1985). Resistance of Salmonella to macrophage phagocytosis is also affected by the composition of O-chains. Differences in the O-side chain structure of LPS affect the virulence in mice, the extent of activation of C3 and the subsequent rate of phagocytosis of Salmonella strains by murine macrophage-like cell line (Liang-Takasaki et al., 1982, 1983). Therefore, the virulence of a strain may be influenced by the ability of the polysaccharide structure of its LPS to activate the alternative pathway of complement, resulting in subsequent phagocytosis (Liang-Takasaki et al., 1983). Adherence to host cells Adherence of many pathogenic organisms to the host cells has been associated with the presence of fimbriae and mannose-resistant hemagglutinin (MRHA). Fimbriae are classified according to their morphology and ability to agglutinate erythrocytes (Duguid et al., 1966). Type 1 fimbriae which are surface organelles found in most of the Gram-negative bacteria hemagglutinate erythrocytes in the absence of D-mannose. Type 3 fimbriae are smaller, more flexible structures that mediate the agglutination of the tannic acid-treated erythrocytes in the presence of D-mannose. S. enteritidis is known to display type 1 a n d / o r type 3 fimbriae (Duguid et al., 1966, 1979; Aslanzadeh and Paulissen, 1990). Both type 1 and 3 fimbriae contribute to the adherence of S. enteritidis to the human buccal and mouse small intestine epithelial cells (Aslanzadeh and Paulissen, 1990, 1992). Type 1 fimbriae of S. enteritidis may directly bind to mannose residues present on the surface of the epithelial cells since o-mannose inhibits the binding. Type 3 fimbriae may also contribute to the adherence of the organism to the cells by the reduction of the surface hydrophobicity and enhancement of the initial contact between the bacteria and epithelial cells. Recently, Tarkkanen et al. (1990) have demonstrated that type 3 fimbriae of S. typhimurium and S. enteritidis are involved in the binding of

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these strains to type V collagen. Based on morphological, biochemical and serological studies, three fimbrial antigens, SEF21, SEFI7 and SEF14 (SEFA). have been identified in S. enteritidis (Collinson et al., 1991; Feutrier et al., 1986; Mi.iller et al., 1991; Thorns et al., 1990). The SEF21 fimbriae which are mannose-sensitive type 1 fimbriae are morphologically, biochemically and serologically distinct from the SEF17 and SEF14 fimbriae (Mi.iller et al., 1991). The SEF21 fimbrial subunit, with a molecular weight of 21000, shares a significant N-terminal amino acid sequence homology with the structural subunit of type 1 fimbriae of S. typhimurh~m, and the antiserum to the SEF21 subunit reacted with type 1 fimbriae of Salmonella spp. but not with E. coli type 1 fimbriae or with other fimbriae produced by S. enteritidis. Thin, aggregative SEF17 fimbriae from S. enteritidis are composed of fimbrial subunits, each with a molecular weight of 17000 (Collinson et aI., 1991). The SEF17 fimbrial subunit shares N-terminal amino acid sequence homology with the structural subunit of curli of E. coli. A recent study by Collinson et al. (1993) demonstrated that the SEF17 fimbriae mediated the binding of S. enteritidis to fibronectin and were responsible for the high autoaggregation and the ability to bind Congo red. The SEF14 fimbriae have been previously reported as type 1 fimbriae by Feutrier et al. (1986) and as a fimbrialike structure SEFA by Thorns et al. (1990). N-terminal amino acid sequence of the fimbrial subunit, with a molecular weight of apparently 14000, shows some degree of homology with that of type 1 fimbriae of S. typhimurium and E. coli (Feutrier et al., 1986). The fimbria structural gene and other two genes involved in fimbria production and processing and in fimbriation have been located in the chromosomal DNA of S. enteritidis (Miiller et al., 1989). Subsequent study by Turcotte and Woodward (1993) revealed that this fimbria structural gene, designated as sefA, encoded SEF14 and was detected in the strains belonging to Salmonella group D. The nucleotide sequence of the sefA gene did not share any homolo~ with the Salmonella fimA gene encoding type 1 fimbriae.

Invasion The ability of the bacterium to invade the host cells is one of the virulence factors of the organism. S. enteritidis is a very invasive pathogen causing systemic infection in chickens as described above. Recent studies revealed a gradation (35-83%) in the ability of the strains isolated in 1978, 1984 or 1988 to invade the liver of chicken (Hinton et ai., 1990). It is suggested that the recent isolates may become more virulent to chickens in the past 10 years. Phage type 4 strains were slightly more invasive than types 6, 8 and 13a, although all the strains were highly invasive in the Vero cells (Barrow, 1991). This observation may account for the variations in the mortality of chicks, total egg production, contaminated egg production and faecal shedding in hens. Intracellular survival and multiplication After penetration into a ceil, Salmonella require nutrients present in the cell to survive and multiple. Some auxotrophic mutants of Salmonella were found to be

98 avirulent in mice. Galactose epimerase (galE) mutation and aromatic (aroA) and purin (pur) mutation of S. typhimurium are attenuated and showed a decreased virulence in mice (Germanier and Fiirer, 1971; O'Callaghan et al., 1988), while the S. enteritidis aroA mutant was also avirulent in chickens (Cooper et al., 1990). S. typhimurium strains lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp) were found to be avirulent in mice and chickens (Curtiss and Kelly, 1987). The ability of Salmonella to survive and persist within macrophages has been correlated with the virulence in mice (Buchmeier and Heffron, 1989; Fields et al., 1986). The PhoP/PhoQ two-component regulatory system promotes Salmonella survival within macrophages, defensin resistance, acid resistance and virulence in mice (Miller, 1991). One of the phoP activated gene (pag) loci, pagC, encodes an outer membrane protein that is involved in the survival within macrophages. Insertion of TnphoA downstream of the signal sequence-encoding region of pagC of S. enteritidis resulted in a macrophage survival defect and avirulence in mice. Another insertion mutant in which TnphoA was inserted into the signal sequenceencoding region of pagC showed invasion- and macrophage survival-defective phenotypes and was less virulent than the former strain (Miller et al., 1992).

Virulence plasmid S. enteritidis contains the serotype-specific 36 megadalton (MDa) (54 kilobase) plasmid which is responsible for the virulence in mice (Hovi et al., 1988; Nakamura et al., 1985; Suzuki et al., 1989), as other non-typhoidal Salmonella which carry the serotype-specific virulence plasmid (Gulig, 1990). During the investigations on the role of these plasmids in the manifestation of virulence, it was revealed that the virulence-associated plasmids of S. typhimurium, S. dublin and S. enteritidis were primarily responsible for spreading the infection beyond the small intestine to mesenteric lymph nodes, spleen and liver of mice (Gulig and Curtiss, 1987; Heffernan et al., 1987; Suzuki et al., 1992). Moreover, the S. enteritidis virulence plasmid has not affected the structure and components of lipopolysaccharide, acquisition of iron or the serum resistance of the organisms (Hovi et al., 1988; Kawahara et al., 1989; Suzuki et al., 1992). In a recent study, most of the S. enteritidis isolates from avian sources carried a 36 MDa plasmid (Chart et al., 1989b; Dorn et al., 1992; Poppe and Gyles, 1987; Shivaprasad et al., 1990; Singer et al., 1992). The 36 MDa plasmid of the strains isolated from chickens may also contribute to the virulence in mice, but not to the serum resistance, the expression of smooth LPS and outer membrane proteins, the production of toxins and a haemolysin, the invasion of HEp-2 cells, or the aerobactin-mediated iron uptake system (Chart et al., 1989b; Poppe and Gyles, 1987). Montenegro et al. (1991) indicated that virulence plasmids were detected in nearly 100% of non-typhoidal Salmonella strains isolated from animal organs and human blood and in 48-87% of the strains of faecal, food or environmental origin, suggesting the involvement of virulence plasmids in systemic infection in humans and livestock. Although the 36 MDa plasmid-carrying strain isolated from chicken

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or man was more virulent in mice than its plasmid-free derivative, there were no significant differences in the mortality in newly hatched chickens and in the ability to colonise the caeca and to invade viscera of hens (Halavatkar and Barrow, 1993; Hinton et al., 1990). Moreover, the current isolates, which showed an enhanced virulence to chickens compared with the strains isolated before 1979, were as virulent in mice as the formerly isolated strains, although all the strains carried the 36 MDa plasmid (Chart et al., 1989b). However, the current isolates were more invasive in the liver of hens than the formerly isolated strains (Hinton et al., 1990). Therefore, the enhanced virulence to chickens of the current isolates is unlikely to be associated with the presence of the 36 MDa plasmid. Shivaprasad et al. (1990) also reported that the difference in the behavior of S. enteritidis isolate in the hen seems to be unrelated to the phage type or plasmid presence. The contribution of the 36 MDa plasmid to the full expression of bacterial virulence may depend on the susceptibility of the host or the presence of functional chromosomal genes in the strain to complement the virulence because the introduction of the plasmid to a naturally plasmid-lacking strain did not increase the virulence in mice (Halavatkar and Barrow, 1993). Recently, the virulence regions of the S. typhimurium, S. dublin and S. choleraesuis plasmids have been located in the 6.4 kb SalI-EcoRI fragment of the plasmid (Gulig, 1990; Kawahara et al., 1990; Krause et al., 1991; Lax et al., 1990). Based on DNA hybridization studies and introduction of virulence plasmids into the different serotypes of Salmonella, it was predicted that the virulence plasmids of S. typhimurium, S. dublin and S. enteritidis shared the homologous region contributing to the virulence (Beninger et al., 1988; Hovi et al., 1988; Jones and Osborne, 1991; Korpela et al., 1989; Montenegro et ai., 1991; Popoff et al., 1984; Poppe et al., 1989; Roudier et al., 1990; Williamson et al., 1988; Woodward et al., 1989). Furthermore, DNA sequence analysis of these virulence regions indicated that the virulence regions of the S. typhimurium, S. dublin and S. choleraesuis plasmids are almost identical with each other, and contain at least four Salmonella plasmid virulence genes (spvR, spvA, spvB and spvC) which express proteins 28-70 kDa in size (Gulig, 1990; Kawahara et al., 1990; Krause et al., 1991; Lax et al., 1990). In a recent study, the 6.4 kb SalI-EcoRI virulence region of S. enteritidis 36 MDa plasmid was found to be almost identical with the conserved virulence region of the S. typhimurium, S. dublin and S. choleraesuis plasmids by DNA sequence analysis (Suzuki et al., unpublished data). The SpvR protein is a transcriptional activator for the downstream spvABC genes (Caldwell and Gulig, 1991; Coynault et al., 1992; Fang et al., 1991; Krause et al., 1992; Matsui et al., 1991; Taira et al., 1991). The expression of the spy genes is induced by starvation (Coynault et al., 1992; Fang et al., 1991; Krause et al., 1992) and the katF (rpoS) gene mediates the transcription of the spy genes during bacterial starvation (Fang et al., 1992; Norel et al., 1992). This starving condition resembles the environment in the macrophage. In fact, it was demonstrated that virulence plasmid is associated with the growth within murine liver and spleen macrophages. Though the exact mechanism by which the virulence plasmid is involved in

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systemic infection remains to be elucidated, these genetical findings may cont r i b u t e t o a b e t t e r u n d e r s t a n d i n g o f t h e p a t h o g e n e s i s o f S. enteritidis.

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