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Salmonella fimbriae: Novel antigens in the detection and control of salmonella infections
Br. vet.J. (1995). 151, 643
REVIEW SALMONEI,I.A FIMBRIAE: NOVEL ANTIGENS IN THE DETECTION AND CONTROL OF SALMONEIJA INFECTIONS
c.j. THORNS Bacteriology DepaT~ment, Central Vetefina, 3, Laboratory, New Haw, Addlestone, SuT~'ey KT15 3NB, UK
SUMMARY Fimbriae are thin, proteinaceous surface organelles produced by members of the Enterobacteriaceae, including most salmonellas. A number of fimbrial antigens expressed by strains of Salmonella enteritidis and S. typhimurium have now been described and characterized. However, their functions are still poorly understood, although some evidence indicates they have a role in bacterial survival in the host or external environment. Diagnostic tests based on the detection of fimbriae or specific antibodies against them have recently been developed and applied successfully to the rapid and specific identification of S. enteritidis infections. The role of salmonella fimbriae in future generations of live vaccines either as protective antigens or as the carriers of heterologous antigens is also discussed. Kr~a,ORDS:Salmonella; fimbriae; pathogenesis; diagnosis; control.
INTRODUCTION Salmonella infections in farm animals continue to be a major problem worldwide. They cause substantial economic loss both directly through mortality and poor growth after clinical disease, and indirectly from animal carriage leading to cases of human salmonella infections. In recent years, SalmoneUa enteritidis has become the dominant serotype isolated from cases of human food poisoning in many countries including the United Kingdom and the United States. This increase is generally thought to be associated, in part, with an increase in eggs and other poultry products contaminated with this serotype (O'Brien, 1988; St Louis et al., 1988). National and international control policies have been set up with the aim of reducing the prevalen.ce of salmonellas in poultry and other farm animals in order to reduce outbreaks of human food-borne infections. An important part of these 0007-1935/95/060643-16/$12.00/0
© 1995 Bailli~re Tindall
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policies is tim requirement for rapid, specific and inexpensive methods of detection, and more effective procedures for reducing the infection by, and carriage of, sahnonellas in farm animals. Fimbriae are proteinaceous surface organelles expressed by many bacteria, of which those of Eschedchia coli have been studied most (Parry & Rooke, 1985). In particular, the fimbriae of enterotoxigenic E. coli such as F4 and F5 (K88 and K99), which are responsible for the attachment of the organism to the villous epithelium of the small intestine, are now used widely in diagnostic tests and as active components in subunit vaccines (Morgan et aL, 1978; Walker & Foster, 1983; Thorns el al., 1989a, b). The expression of fimbriae by certain strains of salmonella was first described over 35 ),ears ago (Duguid & Gillies, 1958) but only recently has their potential as diagnostic and protective antigens been considered. This rexdew describes the different types of fimbriae known to be expressed by Salmonella, their possible role in the disease process and recent advances in the application of fimbriae to the diagnosis and control of salmonella infections.
FIMBRIAE O F S A L M O N E L L A
Duguid and Gillies (1958) first noticed an association between those salmonella strains that expressed fimbriae as determined by electron microscopy and the ability of the hacteria to agglutinate strongly with certain types of red blood cells; a relationship previously demonstrated for E. coli and Shigella spp. (Duguid et aL, 1955; Duguid & Gillies, 1957). At that time, studies on the interaction between el-ythrocytes and bacteria expressing fimbriae resulted in the discove D, that certain substances, in particular mannose-containing carbohydrates, affected the haemagglutination reaction (Collier el al., 1955). In a follow-up study with over 1400 salmonella strains, Duguid el al. (1966) classified sahnonella fimbriae on the basis of their morpholog3, and ability to mediate erythrocyte agglutination in the presence or absence of D-mannose. This classification still forms the basis for differentiating sahnonella fimbriae and is summarized in Table I.
Table I Classification o f salmonella fimbriae Fimbda
Structure
Diameter
Type 1
rigid
7 nm
Type 2
rigid
7 nm
Non-haelnagglutinating
Type 3
flexible
3-5 mn
Type 4 ND
flexible flexible
3 nm 3 nm
Mannose resistant, with tanned red cells Mannose resistant Non-haelnagglutinating
ND, No designation.
Haemag,glutination
Mannose sensitive
Cell tvceplor
Distribution
Mannoside glycoproteins Unknown
Ubiquitous
Unknown Unknown Unknown
S. pullorun~ S. dublin S. gaUinarum S. parah,phi B S. enteritidis S. typhimudum S. typhimumtm S. enteritidis S. dublin
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Type 1 fimbriae of salmonella are rigid structures, 7 nm in diameter and up to 100 nm in length. As many as 300 type 1 fimbriae are expressed per cell [Fig. l(a)], although only about 10% of cultured bacteria appear to express them at any one time (unpublished observations). They consist of a number of identical protein subunits non-covalently linked around a hollow core, which gives a channelled appearance when viewed by electron microscopy (Muller et al., 1991). Protein subunits of between 20-22 kDa relative molecular weight (M,.) have been described for type 1 fimbriae of S. typhimurium and S. enteritidis (Korhonen et al., 1980; Muller et al., 1991). Type 1 fimbriae mediate mannose-sensitive (MS) haemagglutination (Duguid et al., 1966) and lectin-based binding to mannoside glycoprotein receptors situated on a variety of epithelial cells (Kukkonnen et al., 1993). Most salmonellas express type 1 fimbriae which show considerable conservation across the genus at both molecular and structural levels and which is confirmed by the extensive cross-reactivity seen with polyclonal and monoclonal antibodies raised against type 1 (SEF21) fimbriae of S. enteritidis (Muller et aL, 1991; Sojka et al., submitted for publication). Type 1 fimbriae of bacteria in other genera of Enterobacteriaceae such as Klebsiella also show a certain genetic homology with salmonella type 1 fimbriae where only minor DNA sequence differences have been detected in the encoding genes (Purcell et al., 1987). Type 2 fimbriae are morphologically similar to type 1 but lack the ability to agglutinate erythrocytes. They were first observed on strains of S. gaUinantm and S. pullo~Tzm (Duguid & Gillies, 1958) and have since been described on certain S. paratyphi B and S. d'ubli~ strains (Duguid et aL, 1966). There is a close antigenic relationship between types 1 and 2 fimbriae which may indicate that the type 2 fimbriae are non-agglutinating variants of type 1 (Clegg & Gerlach, 1987). This theory is plausible since the haemagglutinating property can be removed from E. coli type 1 fimbriae by the construction of recombinant plasmids encoding nonhaemagglutinating fimbriae, which suggests that there are separate genes for the structural and haemagglutinating components of type 1 fimbriae of E. coli (Minion et al., 1986). Type 3 fimbriae are thinner, more flexible structures with a diameter of between 3-5 nm, when viewed by electron microscopy, and which mediate the agglutination of tannic acid-treated erythrocytes in the presence of 0t-D-mannose (Duguid et al., 1966). Like type 1 fimbriae there is a strong antigenic crossreactivity between fimbriae expressed by Salmonella, Klebsiella and Yersinia spp. (Adegbola et al., 1983; Old & Adegbola, 1985). Type 4 fimbriae were originally defined by Duguid et al. (1966) as thin flexible fimbriae 4 nm in diameter, with the ability to mediate agglutination of fresh erythrocytes in the presence of mannose [mannose resistant (MR)] and until recently had not been described on Salmonella. However, a thin flexible fimbria 3 nm in diameter that mediates MR haemagglutination of pigeon erythrocytes has recently been described on a strain of S. typhimudum isolated from pigeons (Grund & Weber, 1988; Grund & Seiler, 1993). More recently, two other salmonella fimbriae have been identified and extensively characterized, but which do not fit into the existing classification because of their size and inability to haemagglutinate erythrocytes. SEF14 fimbriae were first described on strains of S. ente)~tidis (Thorns et al., 1990; Muller et al., 1991). The
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Fig. 1. (a)
Fig. 1. (b)
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Fig. 1. Salmonella enleritidis negatively stained with phosphottmgstic acid showing distinct surface fimbrial o,'gaqelles. (a) Rigid type 1 fimbriae with a channelled structure radiating fro,n the cell surface (a,rows). (b) Fine and flexible SEFI4 fimbriae expressed on the cell su,'[ace and a detached flagellum (arrow). (c) Co-expression of type 1 fimbriae (arrows) and SEF14 fimhriae (sel) on the same organism. Bars=100 nm. structure comprises thin, filamentous organelles, <3 nm in diameter [Fig. l(b)] composed of repeating protein subunits 14.3 kDa (M,) in size. SEF14 is uniquely specific to certain serotypes in serogroup D, including all strains of S. enteritidis so far examined and a proportion of S. dublin strains (Thorns et al., 1992). The structural gene encoding SEF14 has been cloned and sequenced (sefA) and shown to be limited in distribution to serotypes belonging to group D. Interestingly, S. gallina~tm, S. pullontm and S. typhi all possess the entire sefA gene but do not express the SEF14 fimbriae (Turcotte & Woodward, 1993). S. enteritidis organisms are able to express type 1 (SEF21) and SEF14 fimbriae simultaneously [Fig. 1 (c) ]. SEF17 fimbriae were also first described on a strain of S. enteritidis (Collinson et al., 1991). They have a similar morphology to SEF14 fimbriae but are more tightly coiled (when viewed by electron microscopy) and comprise protein subunits of 17 kDa (M,.) which contain a receptor for the tissue-matrix protein fibronectin (Collinson el al., 1993). The structural gene encoding SEF17 has been sequenced and termed agfA (Collinson et al., 1991), and appears to be widely distributed throughout the SalmoneUa genus (Doran et al., 1993). However, in vitro expression of SEF17 fimbriae is restricted to certain strains which spontaneously aggregate in a sttspension prepared from organisms cultured on solid media, and this phenotypic characteristic is termed auto-aggregative (Collinson et al., 1991). Recent studies have demonstrated a high degree of homology between SEF17 and the
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thin, coiled, fibrillar su'ucture called curli which is expressed by entero-aggregative E. coli associated with infantile dim'rhoea in humans (Collinson et al., 1992; Arnqvist et al., 1992). These two fimbriae have been classifed into a group of timbriae termed GVVPQ by Collinson et al. (1992) in recognition of common fimbrin N-terminal amino acid sequences. Other studies on fimbriae expressed by strains of S. typhimu~um and S. enteritidis describe fimbrial antigens that have morphological, biochemical and antigenic similarities with SEF17 (Rohde et al., 1975; Willems et al., 1993; S. Grund, personal communication) and sequence studies need to be carried out to ascertain if these fimbriae are SEF17 or other members of the GVVPQ family.
MOLECULAR MECHANISMS ASSOCIATED WITH FIMBRIAL EXPRESSION
The genes that encode for the expression of the SEF14 fimbriae of S. entelJtidis have been characterized more fully than any other salmonella fimbriae. The s(ABCD genes make part of a complex sefoperon responsible for SEF14 assembly and expression by S. enteritidis (Clouthier et al., 1993, 1994). The sefA gene encodes the SEF14 fimbrial subunit, whereas the sejB gene encodes subunit transport proteins homologous to E. coli and Klebsiella pneumoniae fimbrial periplasmic chaperone proteins. The seJC gene encodes the fimbrial usher proteins located in the outer membrane that are thought to be responsible for the organization and assembly of subunits into the polymeric fimbrial organelle. The seJD gene has recently been located on the sefoperon and appears to encode a distinct fimbrial structure from SEF14 which has been given the tri~dal name SEF18 (Clouthier et al., 1994). This is the first example in the Enterobacte~Jaceae of a gene cluster that encodes two distinct cell surface structures, namely SEF14 and SEF18. Recently it has been reported that a region on the 90 kb virulence plasmid of S. typhimu~Jum comprises a cluster of genes associated with the expression of a novel type of fimbria. This gene cluster has been termed the pef (plasmid-encoded fimbriae) locus and contains genes that appear to encode proteins analagous to the chaperone and usher proteins described in the biosynthesis of SEF14 and other bacterial fimbriae (Friedrich et al., 1993). This may prove to be the first description of a fimbria expressed by salmonella to be encoded by genes located on plasmid DNA.
VIRULENCE-ASSOCIATED PROPERTIES OF S A L M O N E I J A FIMBRIAE
In vitro studies It has been generally assumed for many years that salmonella fimbriae, like those expressed by enterotoxigenic and uropathogenic strains of E. coli, are involved in some way in the key, initial stages of infection; namely attachment and colonization to intestinal and urinary tract epithelial cells. Consequently, most previous in vitro studies have been directed towards confirming this hypothesis. Table II describes some of the various properties ascribed to salmonella fimbriae that might contribute to the virulence of the organism.
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Table II Properties of salmonella fimbriae Property
Fimbria
Organism
Binding or increased binding to undamaged intestinal epithelial cells Binding to tissue matrix proteins, e.g. laminin fibronectin and collagen
Type 1
S. enteritidis S. typhimurium S. typhimurium, S. entet~tidis S. typhimu~qum S. ente~tidis S. typhimurium S. typhimmqum S. enteritidis S. enteritidis
Host tissue tropism, e.g. liver Lectinophagocytosis Attto-aggregation
Type 1 Type 3 SEF17 Type 1 Type 1 SEF17? SEF17
Many studies p u r p o r t to have demonstrated that fimbriae mediate either the attachment or invasion of salmonella to a variety of undifferentiated and differentiated mammalian and avian tissue culture cell lines or isolated enterocytes (Tavendale et al., 1983; Lindquist el al., 1987; Ernst et al., 1990; Craven et al., 1992; Isaacson & Kinsel, 1992). Conversely, a n u m b e r of other studies have failed to detect any measurable effect (Finlay et al., 1988; Barrow & Lovell, 1989; Barrow et al., 1992). These anomalies are explainable because some experiments have concentrated only on type 1 fimbriae, although it is now well established that coexpression of two or even three different fimbriae can occur on the same bacterium (Thorns et aL, 1990; Grund & Seiler, 1993). Moreover, methods commonly used to determine the expression of type 1 fimbriae are either indirect (MS haemagglutination) or insensitive (electron microscopy). In addition, the increased attachment of the bacterium to epithelial cells in tissue or organ culture models has not been correlated with the in situ expression of fimbriae. Some recent studies have r e por t ed the binding of defined sahnonella fimbriae to tissue-matrix proteins. Kukkonen et al. (1993) showed that type 1 fimbriae of S. enteritidis and S. t~phimurium bind to oligomannoside chains of the laminin network in basement membranes. Type 3 fimbriae of S. typhimurium bind to hum an type V collagen (Tarkkanen et al., 1990), whereas the SEF17 fimbriae of a strain of S. ente~{tidis mediates binding to fibronectin (Collinson et al., 1993). Together, these findings provide evidence for a possible mechanism for salmonella to colonize the intestinal epithelium, particularly if the tissue has been damaged to expose basement m e m b r a n e components. As more fimbriae are defined at the molecular and structural levels and specific reagents are p r o d u c e d to detect them, future in vitro studies should provide more convincing evidence for the role of fimbriae, in the initial stages of the infective process. SEF17 fimbriae are responsible for the auto-aggregation and distinctive colonial morphology of one strain of S. enteritidis (Collinson et al., 1991). These workers speculated that the characteristic may be important in the survival of the bacteria as they e n c o u n t e r e d stomach acids, surfactants and o t h e r biocides present in the digestive tract. In addition, clumps of bacteria may ensure a sufficient and viable inoculum capable of evading phagocytosis and ot her host defences. However, expression of SEF17 has only been r e por t ed on a small n u m b e r of S. enteTJtidis
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sta'ains and evidence indicates that SEF17 is rarely expressed by cultured organisms ( T h o r n s & Dibb-Fuller, u n p u b l i s h e d data). It remains to be seen if this fimbria is expressed in vivo by the majority o f S. enteritidis strains. In vivo animal model studies Surprisingly few investigations have b e e n u n d e r t a k e n using animal models to study the role o f salmonella fimbriae in the infective process. Darekar and Duguid (1972) first described a difference in infectivity between fimbriated (tim ÷) and non-fimbriated (tim-) S. t~,phimm~um organisms using the LAC Grey mouse strain. These preliminary results were e x t e n d e d in the classic studies o f Duguid el. al. (1976) who used a tim- strain o f S. t~,phimurium isolated originally from a pig and a tim + s p o n t a n e o u s variant derived fi'om it. T h e y f o u n d that the tim ÷ variant caused m o r e infections (+26%) and deaths (+40%) than the tim- strain in orally inoculated mice. T h e y also c o m p a r e d faecal excretion at intervals up to 120 days o f mice surviving oral or conjunctival challenge. S. typhimurium was isolated m o r e frequently from animals challenged with the tim + strain (906 isolations from 384 animals infected out o f 877 challenged) than from those c h a l l e n g e d with the tim_ strain (614 isolations from 341 animals out o f 877 challenged). In this study the greatest difference in faecal e x c r e t i o n between tim ÷ and tim- strains was between groups o f mice infected for at least 13 weeks. This suggests that the increased duration o f faecal excretion o f S. t~,phimurium in this animal m o d e l is related, in part, to the ability to express type 1 fimbriae. In a series o f investigations, the virulence o f tim + and tim- S. typhimurium strains were studied by the oral infection o f the gpc m o u s e strain (Tanaka et at, 1977, 1981; T a n a k a & Katsube, 1978). T h e tim ÷ strain o f S. typhimurium was isolated originally from the faeces o f a dog that was persistently e x c r e t i n g the bacterium. T a n a k a and colleagues also d e m o n s t r a t e d an association between tim + organisms and increased virulence and showed that tim + bacteria localized in the terminal ileum and c a e c u m o f orally-infected mice, whereas tim- descendants were not associated ~fith colonization o f any sites o f the digestive tract. In all the above studies, the authors were probably c o m p a r i n g type 1 fimbrial m u t a n t strains as they used MS h a e m a g g l u t i n a t i o n to d e t e c t their presence. In addition the variants were ill-defined since they arose spontaneously f r o m the p a r e n t strains, t h e r e f o r e mutations in o t h e r regulatory, accessory or structural genes, e n c o d i n g virulence-associated antigens c a n n o t be ruled out as c o n t r i b u t i n g to the observed effects. In vivo expression o f the fimbriae was also n o t established in these studies. T h e s e reservations cast considerable d o u b t on the validity o f the conclusions. However, the overall hypothesis that type 1 fimbriae e n h a n c e faecal dissemination is backed up by a r e c e n t study in which n e o n a t a l rats were infected with r e c o m b i n a n t and m u t a n t E. coli type 1 tim + a n d tim- strains (Bloch el aL, 1992). T h e s e authors d e m o n s t r a t e d that the ability to express type 1 fimbriae was related to increased communicability o f E. coli between infected a n d n o n - i n f e c t e d litter mates and did not necessarily require the direct association o f the fimbriae with the intestinal mucosa. In the m u r i n e typhoid m o d e l , n o n - r e v e r t a n t type 1 fimbriated strains o f S. typhimurium were cleared faster f r o m the b l o o d o f i n b r e d mice than non-fimbriated organisms. Moreover, the endothelial and K u p p f e r cells o f the liver a n d phago-
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cytic cells of the spleen and kidneys were seen selectively to trap fimbriated bacteria (Leunk & Moon, 1982; Lockman & Curtiss, 1992). It has been reported that the attachment, internalization and survival in phagocytes is mediated by type 1 fimbriae (lectinophagocytosis) of E. coli and S. typhimurium (Keith et al., 1990). These findings can be interpreted as evidence that expression of t)q~e 1 fimbriae decreases the virulence of salmonella in this model by reducing bacteraemia and increasing the rate of salmonella clearance. However, S. enteritidis, S. dublin and S. typhi are able to establish persistent carriage by colonizing specific niches in the host, and selective trapping may aid this process. For example, sahnonella that are genotypically tim + can exhibit fimbrial phase variation between phenotypically fimbriated (tim +) and non-fimbriated states (tim-) states, which is influenced, in part, by the ambient environment (Eisenstein, 1987). It is possible that the differential expression of type 1 and other fimbriae, results from the microenviromnental conditions encountered by facultative intracellular pathogens such as S. typhimuHum and S. ente~tidis. In one study, tim- S. typhimurium predominated in the blood, whereas tim + organisms were most commonly isolated from the spleen and liver of mice after oral infection with a mixed population of tim- and tim + bacteria (Lockman & Curtiss, 1992). In our laboratory we have compared the survival of defined SEF14 fimbrial deletion mutants of S. ente~tidis with the parent strains in 6-week-old BALB/c mice and 3-day-old chicks. In both models, there was a small but significant increase in the duration of faecal excretion after oral infection of the parent strain of S. enteritidis (SEF14 +) compared with infection with the mutant strains (SEF14-). Parent and mutant strains both expressed type 1 fimbriae when grown under appropriate conditions (Thorns & Turcotte, unpublished data). In contrast, when 20-week-old hens were orally infected with the two strains, no differences were observed in colonization of the intestine or invasion and dissemination to various organs of the body (Thorns, unpublished observations). In a recent study, passive immunization against experimental S. enteritidis infection in CD1 mice by the oral administration of hen egg-yolk antibodies specific for SEF14 fimbriae increased the survival rate from 32% of control animals to 78% of the SEF14 antibodytreated group. A reduction in the localization of S. ente~tidis along villous margins of the small intestine was noticed in the treated group (Peralta et al., 1994). Type 3 fimbriae of S. enteritidis have also been shown to contribute to the virulence of the organism in orally-infected mice (Aslanzadeh & Paulissen, 1992). These findings are the first indication that fimbriae other than type 1 may increase gut colonization and faecal dissemination of salmonellas. Based on existing evidence it is reasonable to suggest, therefore, that pathogens like S. typhimuHum rarely express type 1 or other fimbriae in extracellular environments like blood, but switch on the expression of the fimbriae when in close association with epithelial and other cell types, thereby permitting the bacterium to adapt to and take advantage of the different niches encountered in an infected host. DIAGNOSTIC TESTS BASED O N SALMONELLA HMBRIAE
The first diagnostic tests based on the detection of fimbrial antigens were developed for the detection of enterotoxigenic E. coli (Sojka, 1971; Orskov et al., 1975;
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Guinee el al., 1976). The impetus for their development and use was the discover?, that fimbriae such as F4 and F5 are essential for the E. coli to cause disease, and so their detection on isolates froln clinical cases of diarrhoea is of diagnostic significance and a variety of tests that incorporate monoclonal antibodies are now commonly used-to detect rapidly fimbriae expressed by enterotoxigenic E. colt: (Thorns et al., 1989a, b). Although much effort has gone into the characterization of fimbriae expressed by SalmoneUa, their application to the detection of specific sahnonella infections has, to date, not been fully exploited. The two main reasons for this have been the concentration of research towards characterization of type 1 fimbriae and a lack of understanding of the role of these antigens in the lifecycle of the bacterimn, which means that their detection is of unknown clinical significance. Detection 0 f S a l m o n e l l a g e n u s Genus-specific tests based mainly on the detection of surface antigens such as lipopolysaccharide, flagellin and outer membrane proteins, have been used with some success in enzyme-linked immunosorbent assays (ELISAs) and agglutination tests (Clark et al., 1989; Kerr et aL, 1992; Manafi & Sommer, 1992; Feldsine et al., 1992; Wyatt et al., 1993). Recently a DNA-based test which targets the ag/A structural gene of SEF17 filnbriae has been developed and shown to react strongly with 603 of 604 sahnonella isolates and only ver?, weakly to 31 of 266 other members of the family Enterobacteriaceae (Doran et al., 1993). This is the first instance in which a fimbrial gene probe has been used as a genus-specific diagnostic tool and may offer quicker and more sensitive approaches than existing culture methods. Specific detection o f S a h n o n e l l a enteritidis The rapid spread of S. ente~tidis through the poultr?, population and the subsequent increase in human food poisoning cases caused by this serotype, highlights the need for more rapid, specific tests to identify S. entedtidis infections in animal and hunaan populations in order to apply prescribed control strategies. Recently, a simple, latex agglutination test has been developed for the specific identification of S. ente~tidis. It is based on the detection of the SEF14 fimbrial antigen and to ensure the differentiation of S. enteritidis from other closely related serogroup D salmonellas, a second latex reagent is used that detects a specific epitope on the flagellum of S. dublin (McLaren et al., 1992; Thorns et al., 1992, 1994). Extensive evaluation by laboratories worldwide has demonstrated that this is an accurate, presumptive test for S. enteritidis that can be carried out in routine laboratories that would normally have to send all their Salmonella isolates to specialized laboratories for serotyping. The increase in bacteriological monitoring of pouhr?, flocks for salmonella has required an evaluation of alternative screening procedures in order to reduce the time and costs involved. The most likely alternative is serological monitoring, especially as more specific ELISA-based assays become available (Barrow, 1994). A recent development is the application of SEF14 fimbriae to the specific serodiagnosis of chicken flocks infected with S. enteritidis. Results indicate that birds infected with S. ente~{tidis readily seroconvert within 10 days of infection and the IgG response persists for at least 4 weeks thereafter (Thorns el al., 1993). The pro-
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duction of SEF14 antibodies following infection is the first demonstration of a specific anti-fimbrial response to salmonella infection. Preliminary data indicate that the potential advantages of the detection of SEF14 antibodies are high specificity and sensitivity. In addition, it is possible that future generations of tests could distinguish vaccinal responses from field infection (see below).
APPLICATION OF SALMONELLA FIMBRIAE IN VACCINE DEVELOPMENT
The pathogenesis of sahnonella infections is recognized as being multifactorial so it is unlikely that a simple, defined, fimbrial subunit vaccine will elicit a sufficient protective response against challenge by salmonellas. However, Aslanzadeh and Paulissen (1990) reported that specific antibodies to type 1 and type 3 fimbriae conferred partial passive protection to mice orally-infected with S. ente~tidis. In their study, pretreatment with anti-fimbrial antibody increased survival of mice by over 60%. These results may indicate the importance of synergism between the two fimbriae in the disease process and further work needs to be carried out to confirm this finding. In particular it would be of interest to determine the combined protective effect of specific antibodies to SEF21, SEF17 and SEF14 fimbriae of S. entel~tidis in a suitable animal model. Future directions are likely to involve the use of fimbriae as carriers of protective, heterologous antigenic determinants in novel multivalent live vaccines (Woodward et al., 1993). The successful incorporation of peptides from foot-andmouth disease virus and human immunodeficiency virus type 1 into E. coli F4 timbriae has been reported, and specific antibodies to the heterologous peptides were produced in mice after inoculation with the purified fimbriae (Bakker et al., 1990). The use of sahnonella fimbriae as carriers of foreign epitopes is currently being pursued in our laboratory. Epitopes of the MPB70 antigen of Mycobacte~um bovis (Harboe & Nagai, 1984) have been successfully incorporated into SEF14 timbriae produced by a strain of S. ente~tidis (Woodward et al., 1993). The use of vaccines to reduce excretion and vertical transmission of salmonellas is likely to increase in the future. It is therefore important to develop vaccines in which the vaccinal antibody responses can be differentiated from those resulting from natural infections so as not to compromise serological monitoring. From initial observations in our laboratory and elsewhere, it seems very unlikely that SEF14 fimbriae are prerequisites for virulence, therefore one strategy being adopted in our laboratory is the construction of sefA gene deletion mutants from live vaccine strains (Cooper et al., 1990, 1992) with the aim of being able to distinguish vaccinal antibody responses devoid of SEF14 antibodies as opposed to SEF14 seroconversion seen in field infections (Thorns et aL, 1993).
CONCLUSIONS
Most salmonellas are able to express one or more types of fimbriae at some time during their lifecycle. The environmental and molecular signals that determine fimbrial expression are still poorly understood, but it is now recognized that their expression in specific niches in the infected host or in the outside environment
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play an important part in bacterial smMval. Duguid et al. 's (1976) original notion, that fimbriae se~we a fnnction that is marginally advantageous to the organism, has not yet been confirmed or expanded upon due to the paucity of experimental evidence mad the ad hoc nature of the research over the past 25 years. Nevertheless modern molecular and immunological techniques, linked with in vivo studies, will undoubtedly help to unravel the complex individual and interdependent functions of sahnonella fimbriae in the near future. Current evidence indicates that many different fimbriae mediate a variety of functions that are important for the maintenance and SUl-vival of the organisms in the host and its environment. These functions might include: (i) initiation a n d / o r stabilization of the organism to epithelial cells; (ii) colonization and microcolony formation of tissues via site-specific binding to receptors on tissue matrix proteins; (iii) maintenance of persistent infections in the host by mediating selective bacterial trapping by phagocytic cells, and subsequent SUlarival in specific niches of the infected animal or its environment; (ix,) evasion of the host's specific immunological defences by: (a) presentation of self epitopes on the fimbriae and (b) production of harmless immune responses to fimbriae which are then rapidly shed or their expression switched off from the surface of the organism. Recent advances in the characterization of sahnonella filnbriae have already resulted in flaeir application to novel diagnostic tests and demonstrated their potential in the future development of vaccines. Only when the fimbrial functions have been fnlly characterized will their potential for the detection and control of sahnonella infections be frilly exploited.
ACKNOWLEDGEMENTS
I would like to thank Diane Newell, Martin Woodward and Clifford Wray for their helpful discussions and critical evaluation of the manuscript.
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TI-IORNS, C.J., Sql~a, M. G., McLxp,EN, I. M. & DIBB-Fut.LER, M. (1992). Characterisation of monoclonal antibodies against a fimbrial structure of Sabnonella enteritidis and certain odaer serogroup D salmonellae and their application as serotyping reagents. Research in VeteHna O, Science 53, 300-8. THORNS, C.J., NtcHOt--xS,R. A.J., BELl., M. M. & CHISNALL,S. C. (1993). Preliminary results on dae use of SEF14 fimbrial antigen in an ELISA for the serodiagnosis of SalmoneUa enteritidis infection in poultry, Brussels, 7-9 June 1993. Directorate General for Agriculture, Commission of the European Comnatmities, Brussels. THORNS, C.J., MOLTEN, I. M. & Sqltc.\, M. G. (1994). The use of latex particle agglutilaation to specifically detect Salmonella enteritidis. International Journal of Food Microbiology, 21, 47-53. TuRcorrE, C. & WOODWARD,M.J. (1993). Cloning, DNA nucleotide sequence and distribution of the gene encoding SEF14, a filnbrial antigen of Sahnonella enteritidis. Journal of General Microbiolog?, 139, 1477-85. WALKER,P. D. & FOSTE,, W. (1983). Bacterial vaccines. Biologist 30, 67-74. Wit.cE..x.ts, P., DE GP,EX~, H., HE~NALSTEENS,J-P., DEWrRrE, I. & LINTt:.R~XIANS,P. (1993). Characterisation of the virulence factors from Salmonella typhimurium and Sabnonella enteHtidis as a basis for the development of a new generation of diagnostics and vaccines. In Proceedings FLAIR No. 6: Prevention and Control of Potentialll, Pathogenic Microolganisms in Poult O, and Poult O, Meat Processing, eds A. Pusztai, M. H. H'inton & R. W. A. W. Mulder, pp. 79-83. Spelderholt: COVP-DLO. WOODWARO, M. J., THom,4s, C. J. & TU~coTrE, C. (1993). Fimbriae of Salmonella enteritidi.s:. Molecular analysis of SEF14 and vaccine development potential. In Proceedings ofa NATO Advanced Studies h~stitute on Biolog), of Sahnonella, Portorosa, Italy, May 1992, eds F. Cabello et al. pp. 79-82. New York: Plenum Press. W~\rr, G. M., L.XN¢;H:.V,M. N., LEE, H. A. & MOR¢:AN,M. R. A. (1993). Further studies on the feasibility of one-day Sahnonella detection by enzyme-linked ilnmunosorbent assay. Applied and Environmental Microbiology, 59, 1383-90.
(Acceptedfor publication 16 September 1994)
REVIEW SALMONEI,I.A FIMBRIAE: NOVEL ANTIGENS IN THE DETECTION AND CONTROL OF SALMONEIJA INFECTIONS
c.j. THORNS Bacteriology DepaT~ment, Central Vetefina, 3, Laboratory, New Haw, Addlestone, SuT~'ey KT15 3NB, UK
SUMMARY Fimbriae are thin, proteinaceous surface organelles produced by members of the Enterobacteriaceae, including most salmonellas. A number of fimbrial antigens expressed by strains of Salmonella enteritidis and S. typhimurium have now been described and characterized. However, their functions are still poorly understood, although some evidence indicates they have a role in bacterial survival in the host or external environment. Diagnostic tests based on the detection of fimbriae or specific antibodies against them have recently been developed and applied successfully to the rapid and specific identification of S. enteritidis infections. The role of salmonella fimbriae in future generations of live vaccines either as protective antigens or as the carriers of heterologous antigens is also discussed. Kr~a,ORDS:Salmonella; fimbriae; pathogenesis; diagnosis; control.
INTRODUCTION Salmonella infections in farm animals continue to be a major problem worldwide. They cause substantial economic loss both directly through mortality and poor growth after clinical disease, and indirectly from animal carriage leading to cases of human salmonella infections. In recent years, SalmoneUa enteritidis has become the dominant serotype isolated from cases of human food poisoning in many countries including the United Kingdom and the United States. This increase is generally thought to be associated, in part, with an increase in eggs and other poultry products contaminated with this serotype (O'Brien, 1988; St Louis et al., 1988). National and international control policies have been set up with the aim of reducing the prevalen.ce of salmonellas in poultry and other farm animals in order to reduce outbreaks of human food-borne infections. An important part of these 0007-1935/95/060643-16/$12.00/0
© 1995 Bailli~re Tindall
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policies is tim requirement for rapid, specific and inexpensive methods of detection, and more effective procedures for reducing the infection by, and carriage of, sahnonellas in farm animals. Fimbriae are proteinaceous surface organelles expressed by many bacteria, of which those of Eschedchia coli have been studied most (Parry & Rooke, 1985). In particular, the fimbriae of enterotoxigenic E. coli such as F4 and F5 (K88 and K99), which are responsible for the attachment of the organism to the villous epithelium of the small intestine, are now used widely in diagnostic tests and as active components in subunit vaccines (Morgan et aL, 1978; Walker & Foster, 1983; Thorns el al., 1989a, b). The expression of fimbriae by certain strains of salmonella was first described over 35 ),ears ago (Duguid & Gillies, 1958) but only recently has their potential as diagnostic and protective antigens been considered. This rexdew describes the different types of fimbriae known to be expressed by Salmonella, their possible role in the disease process and recent advances in the application of fimbriae to the diagnosis and control of salmonella infections.
FIMBRIAE O F S A L M O N E L L A
Duguid and Gillies (1958) first noticed an association between those salmonella strains that expressed fimbriae as determined by electron microscopy and the ability of the hacteria to agglutinate strongly with certain types of red blood cells; a relationship previously demonstrated for E. coli and Shigella spp. (Duguid et aL, 1955; Duguid & Gillies, 1957). At that time, studies on the interaction between el-ythrocytes and bacteria expressing fimbriae resulted in the discove D, that certain substances, in particular mannose-containing carbohydrates, affected the haemagglutination reaction (Collier el al., 1955). In a follow-up study with over 1400 salmonella strains, Duguid el al. (1966) classified sahnonella fimbriae on the basis of their morpholog3, and ability to mediate erythrocyte agglutination in the presence or absence of D-mannose. This classification still forms the basis for differentiating sahnonella fimbriae and is summarized in Table I.
Table I Classification o f salmonella fimbriae Fimbda
Structure
Diameter
Type 1
rigid
7 nm
Type 2
rigid
7 nm
Non-haelnagglutinating
Type 3
flexible
3-5 mn
Type 4 ND
flexible flexible
3 nm 3 nm
Mannose resistant, with tanned red cells Mannose resistant Non-haelnagglutinating
ND, No designation.
Haemag,glutination
Mannose sensitive
Cell tvceplor
Distribution
Mannoside glycoproteins Unknown
Ubiquitous
Unknown Unknown Unknown
S. pullorun~ S. dublin S. gaUinarum S. parah,phi B S. enteritidis S. typhimudum S. typhimumtm S. enteritidis S. dublin
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Type 1 fimbriae of salmonella are rigid structures, 7 nm in diameter and up to 100 nm in length. As many as 300 type 1 fimbriae are expressed per cell [Fig. l(a)], although only about 10% of cultured bacteria appear to express them at any one time (unpublished observations). They consist of a number of identical protein subunits non-covalently linked around a hollow core, which gives a channelled appearance when viewed by electron microscopy (Muller et al., 1991). Protein subunits of between 20-22 kDa relative molecular weight (M,.) have been described for type 1 fimbriae of S. typhimurium and S. enteritidis (Korhonen et al., 1980; Muller et al., 1991). Type 1 fimbriae mediate mannose-sensitive (MS) haemagglutination (Duguid et al., 1966) and lectin-based binding to mannoside glycoprotein receptors situated on a variety of epithelial cells (Kukkonnen et al., 1993). Most salmonellas express type 1 fimbriae which show considerable conservation across the genus at both molecular and structural levels and which is confirmed by the extensive cross-reactivity seen with polyclonal and monoclonal antibodies raised against type 1 (SEF21) fimbriae of S. enteritidis (Muller et aL, 1991; Sojka et al., submitted for publication). Type 1 fimbriae of bacteria in other genera of Enterobacteriaceae such as Klebsiella also show a certain genetic homology with salmonella type 1 fimbriae where only minor DNA sequence differences have been detected in the encoding genes (Purcell et al., 1987). Type 2 fimbriae are morphologically similar to type 1 but lack the ability to agglutinate erythrocytes. They were first observed on strains of S. gaUinantm and S. pullo~Tzm (Duguid & Gillies, 1958) and have since been described on certain S. paratyphi B and S. d'ubli~ strains (Duguid et aL, 1966). There is a close antigenic relationship between types 1 and 2 fimbriae which may indicate that the type 2 fimbriae are non-agglutinating variants of type 1 (Clegg & Gerlach, 1987). This theory is plausible since the haemagglutinating property can be removed from E. coli type 1 fimbriae by the construction of recombinant plasmids encoding nonhaemagglutinating fimbriae, which suggests that there are separate genes for the structural and haemagglutinating components of type 1 fimbriae of E. coli (Minion et al., 1986). Type 3 fimbriae are thinner, more flexible structures with a diameter of between 3-5 nm, when viewed by electron microscopy, and which mediate the agglutination of tannic acid-treated erythrocytes in the presence of 0t-D-mannose (Duguid et al., 1966). Like type 1 fimbriae there is a strong antigenic crossreactivity between fimbriae expressed by Salmonella, Klebsiella and Yersinia spp. (Adegbola et al., 1983; Old & Adegbola, 1985). Type 4 fimbriae were originally defined by Duguid et al. (1966) as thin flexible fimbriae 4 nm in diameter, with the ability to mediate agglutination of fresh erythrocytes in the presence of mannose [mannose resistant (MR)] and until recently had not been described on Salmonella. However, a thin flexible fimbria 3 nm in diameter that mediates MR haemagglutination of pigeon erythrocytes has recently been described on a strain of S. typhimudum isolated from pigeons (Grund & Weber, 1988; Grund & Seiler, 1993). More recently, two other salmonella fimbriae have been identified and extensively characterized, but which do not fit into the existing classification because of their size and inability to haemagglutinate erythrocytes. SEF14 fimbriae were first described on strains of S. ente)~tidis (Thorns et al., 1990; Muller et al., 1991). The
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Fig. 1. (a)
Fig. 1. (b)
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Fig. 1. Salmonella enleritidis negatively stained with phosphottmgstic acid showing distinct surface fimbrial o,'gaqelles. (a) Rigid type 1 fimbriae with a channelled structure radiating fro,n the cell surface (a,rows). (b) Fine and flexible SEFI4 fimbriae expressed on the cell su,'[ace and a detached flagellum (arrow). (c) Co-expression of type 1 fimbriae (arrows) and SEF14 fimhriae (sel) on the same organism. Bars=100 nm. structure comprises thin, filamentous organelles, <3 nm in diameter [Fig. l(b)] composed of repeating protein subunits 14.3 kDa (M,) in size. SEF14 is uniquely specific to certain serotypes in serogroup D, including all strains of S. enteritidis so far examined and a proportion of S. dublin strains (Thorns et al., 1992). The structural gene encoding SEF14 has been cloned and sequenced (sefA) and shown to be limited in distribution to serotypes belonging to group D. Interestingly, S. gallina~tm, S. pullontm and S. typhi all possess the entire sefA gene but do not express the SEF14 fimbriae (Turcotte & Woodward, 1993). S. enteritidis organisms are able to express type 1 (SEF21) and SEF14 fimbriae simultaneously [Fig. 1 (c) ]. SEF17 fimbriae were also first described on a strain of S. enteritidis (Collinson et al., 1991). They have a similar morphology to SEF14 fimbriae but are more tightly coiled (when viewed by electron microscopy) and comprise protein subunits of 17 kDa (M,.) which contain a receptor for the tissue-matrix protein fibronectin (Collinson el al., 1993). The structural gene encoding SEF17 has been sequenced and termed agfA (Collinson et al., 1991), and appears to be widely distributed throughout the SalmoneUa genus (Doran et al., 1993). However, in vitro expression of SEF17 fimbriae is restricted to certain strains which spontaneously aggregate in a sttspension prepared from organisms cultured on solid media, and this phenotypic characteristic is termed auto-aggregative (Collinson et al., 1991). Recent studies have demonstrated a high degree of homology between SEF17 and the
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thin, coiled, fibrillar su'ucture called curli which is expressed by entero-aggregative E. coli associated with infantile dim'rhoea in humans (Collinson et al., 1992; Arnqvist et al., 1992). These two fimbriae have been classifed into a group of timbriae termed GVVPQ by Collinson et al. (1992) in recognition of common fimbrin N-terminal amino acid sequences. Other studies on fimbriae expressed by strains of S. typhimu~um and S. enteritidis describe fimbrial antigens that have morphological, biochemical and antigenic similarities with SEF17 (Rohde et al., 1975; Willems et al., 1993; S. Grund, personal communication) and sequence studies need to be carried out to ascertain if these fimbriae are SEF17 or other members of the GVVPQ family.
MOLECULAR MECHANISMS ASSOCIATED WITH FIMBRIAL EXPRESSION
The genes that encode for the expression of the SEF14 fimbriae of S. entelJtidis have been characterized more fully than any other salmonella fimbriae. The s(ABCD genes make part of a complex sefoperon responsible for SEF14 assembly and expression by S. enteritidis (Clouthier et al., 1993, 1994). The sefA gene encodes the SEF14 fimbrial subunit, whereas the sejB gene encodes subunit transport proteins homologous to E. coli and Klebsiella pneumoniae fimbrial periplasmic chaperone proteins. The seJC gene encodes the fimbrial usher proteins located in the outer membrane that are thought to be responsible for the organization and assembly of subunits into the polymeric fimbrial organelle. The seJD gene has recently been located on the sefoperon and appears to encode a distinct fimbrial structure from SEF14 which has been given the tri~dal name SEF18 (Clouthier et al., 1994). This is the first example in the Enterobacte~Jaceae of a gene cluster that encodes two distinct cell surface structures, namely SEF14 and SEF18. Recently it has been reported that a region on the 90 kb virulence plasmid of S. typhimu~Jum comprises a cluster of genes associated with the expression of a novel type of fimbria. This gene cluster has been termed the pef (plasmid-encoded fimbriae) locus and contains genes that appear to encode proteins analagous to the chaperone and usher proteins described in the biosynthesis of SEF14 and other bacterial fimbriae (Friedrich et al., 1993). This may prove to be the first description of a fimbria expressed by salmonella to be encoded by genes located on plasmid DNA.
VIRULENCE-ASSOCIATED PROPERTIES OF S A L M O N E I J A FIMBRIAE
In vitro studies It has been generally assumed for many years that salmonella fimbriae, like those expressed by enterotoxigenic and uropathogenic strains of E. coli, are involved in some way in the key, initial stages of infection; namely attachment and colonization to intestinal and urinary tract epithelial cells. Consequently, most previous in vitro studies have been directed towards confirming this hypothesis. Table II describes some of the various properties ascribed to salmonella fimbriae that might contribute to the virulence of the organism.
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Table II Properties of salmonella fimbriae Property
Fimbria
Organism
Binding or increased binding to undamaged intestinal epithelial cells Binding to tissue matrix proteins, e.g. laminin fibronectin and collagen
Type 1
S. enteritidis S. typhimurium S. typhimurium, S. entet~tidis S. typhimu~qum S. ente~tidis S. typhimurium S. typhimmqum S. enteritidis S. enteritidis
Host tissue tropism, e.g. liver Lectinophagocytosis Attto-aggregation
Type 1 Type 3 SEF17 Type 1 Type 1 SEF17? SEF17
Many studies p u r p o r t to have demonstrated that fimbriae mediate either the attachment or invasion of salmonella to a variety of undifferentiated and differentiated mammalian and avian tissue culture cell lines or isolated enterocytes (Tavendale et al., 1983; Lindquist el al., 1987; Ernst et al., 1990; Craven et al., 1992; Isaacson & Kinsel, 1992). Conversely, a n u m b e r of other studies have failed to detect any measurable effect (Finlay et al., 1988; Barrow & Lovell, 1989; Barrow et al., 1992). These anomalies are explainable because some experiments have concentrated only on type 1 fimbriae, although it is now well established that coexpression of two or even three different fimbriae can occur on the same bacterium (Thorns et aL, 1990; Grund & Seiler, 1993). Moreover, methods commonly used to determine the expression of type 1 fimbriae are either indirect (MS haemagglutination) or insensitive (electron microscopy). In addition, the increased attachment of the bacterium to epithelial cells in tissue or organ culture models has not been correlated with the in situ expression of fimbriae. Some recent studies have r e por t ed the binding of defined sahnonella fimbriae to tissue-matrix proteins. Kukkonen et al. (1993) showed that type 1 fimbriae of S. enteritidis and S. t~phimurium bind to oligomannoside chains of the laminin network in basement membranes. Type 3 fimbriae of S. typhimurium bind to hum an type V collagen (Tarkkanen et al., 1990), whereas the SEF17 fimbriae of a strain of S. ente~{tidis mediates binding to fibronectin (Collinson et al., 1993). Together, these findings provide evidence for a possible mechanism for salmonella to colonize the intestinal epithelium, particularly if the tissue has been damaged to expose basement m e m b r a n e components. As more fimbriae are defined at the molecular and structural levels and specific reagents are p r o d u c e d to detect them, future in vitro studies should provide more convincing evidence for the role of fimbriae, in the initial stages of the infective process. SEF17 fimbriae are responsible for the auto-aggregation and distinctive colonial morphology of one strain of S. enteritidis (Collinson et al., 1991). These workers speculated that the characteristic may be important in the survival of the bacteria as they e n c o u n t e r e d stomach acids, surfactants and o t h e r biocides present in the digestive tract. In addition, clumps of bacteria may ensure a sufficient and viable inoculum capable of evading phagocytosis and ot her host defences. However, expression of SEF17 has only been r e por t ed on a small n u m b e r of S. enteTJtidis
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sta'ains and evidence indicates that SEF17 is rarely expressed by cultured organisms ( T h o r n s & Dibb-Fuller, u n p u b l i s h e d data). It remains to be seen if this fimbria is expressed in vivo by the majority o f S. enteritidis strains. In vivo animal model studies Surprisingly few investigations have b e e n u n d e r t a k e n using animal models to study the role o f salmonella fimbriae in the infective process. Darekar and Duguid (1972) first described a difference in infectivity between fimbriated (tim ÷) and non-fimbriated (tim-) S. t~,phimm~um organisms using the LAC Grey mouse strain. These preliminary results were e x t e n d e d in the classic studies o f Duguid el. al. (1976) who used a tim- strain o f S. t~,phimurium isolated originally from a pig and a tim + s p o n t a n e o u s variant derived fi'om it. T h e y f o u n d that the tim ÷ variant caused m o r e infections (+26%) and deaths (+40%) than the tim- strain in orally inoculated mice. T h e y also c o m p a r e d faecal excretion at intervals up to 120 days o f mice surviving oral or conjunctival challenge. S. typhimurium was isolated m o r e frequently from animals challenged with the tim + strain (906 isolations from 384 animals infected out o f 877 challenged) than from those c h a l l e n g e d with the tim_ strain (614 isolations from 341 animals out o f 877 challenged). In this study the greatest difference in faecal e x c r e t i o n between tim ÷ and tim- strains was between groups o f mice infected for at least 13 weeks. This suggests that the increased duration o f faecal excretion o f S. t~,phimurium in this animal m o d e l is related, in part, to the ability to express type 1 fimbriae. In a series o f investigations, the virulence o f tim + and tim- S. typhimurium strains were studied by the oral infection o f the gpc m o u s e strain (Tanaka et at, 1977, 1981; T a n a k a & Katsube, 1978). T h e tim ÷ strain o f S. typhimurium was isolated originally from the faeces o f a dog that was persistently e x c r e t i n g the bacterium. T a n a k a and colleagues also d e m o n s t r a t e d an association between tim + organisms and increased virulence and showed that tim + bacteria localized in the terminal ileum and c a e c u m o f orally-infected mice, whereas tim- descendants were not associated ~fith colonization o f any sites o f the digestive tract. In all the above studies, the authors were probably c o m p a r i n g type 1 fimbrial m u t a n t strains as they used MS h a e m a g g l u t i n a t i o n to d e t e c t their presence. In addition the variants were ill-defined since they arose spontaneously f r o m the p a r e n t strains, t h e r e f o r e mutations in o t h e r regulatory, accessory or structural genes, e n c o d i n g virulence-associated antigens c a n n o t be ruled out as c o n t r i b u t i n g to the observed effects. In vivo expression o f the fimbriae was also n o t established in these studies. T h e s e reservations cast considerable d o u b t on the validity o f the conclusions. However, the overall hypothesis that type 1 fimbriae e n h a n c e faecal dissemination is backed up by a r e c e n t study in which n e o n a t a l rats were infected with r e c o m b i n a n t and m u t a n t E. coli type 1 tim + a n d tim- strains (Bloch el aL, 1992). T h e s e authors d e m o n s t r a t e d that the ability to express type 1 fimbriae was related to increased communicability o f E. coli between infected a n d n o n - i n f e c t e d litter mates and did not necessarily require the direct association o f the fimbriae with the intestinal mucosa. In the m u r i n e typhoid m o d e l , n o n - r e v e r t a n t type 1 fimbriated strains o f S. typhimurium were cleared faster f r o m the b l o o d o f i n b r e d mice than non-fimbriated organisms. Moreover, the endothelial and K u p p f e r cells o f the liver a n d phago-
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cytic cells of the spleen and kidneys were seen selectively to trap fimbriated bacteria (Leunk & Moon, 1982; Lockman & Curtiss, 1992). It has been reported that the attachment, internalization and survival in phagocytes is mediated by type 1 fimbriae (lectinophagocytosis) of E. coli and S. typhimurium (Keith et al., 1990). These findings can be interpreted as evidence that expression of t)q~e 1 fimbriae decreases the virulence of salmonella in this model by reducing bacteraemia and increasing the rate of salmonella clearance. However, S. enteritidis, S. dublin and S. typhi are able to establish persistent carriage by colonizing specific niches in the host, and selective trapping may aid this process. For example, sahnonella that are genotypically tim + can exhibit fimbrial phase variation between phenotypically fimbriated (tim +) and non-fimbriated states (tim-) states, which is influenced, in part, by the ambient environment (Eisenstein, 1987). It is possible that the differential expression of type 1 and other fimbriae, results from the microenviromnental conditions encountered by facultative intracellular pathogens such as S. typhimuHum and S. ente~tidis. In one study, tim- S. typhimurium predominated in the blood, whereas tim + organisms were most commonly isolated from the spleen and liver of mice after oral infection with a mixed population of tim- and tim + bacteria (Lockman & Curtiss, 1992). In our laboratory we have compared the survival of defined SEF14 fimbrial deletion mutants of S. ente~tidis with the parent strains in 6-week-old BALB/c mice and 3-day-old chicks. In both models, there was a small but significant increase in the duration of faecal excretion after oral infection of the parent strain of S. enteritidis (SEF14 +) compared with infection with the mutant strains (SEF14-). Parent and mutant strains both expressed type 1 fimbriae when grown under appropriate conditions (Thorns & Turcotte, unpublished data). In contrast, when 20-week-old hens were orally infected with the two strains, no differences were observed in colonization of the intestine or invasion and dissemination to various organs of the body (Thorns, unpublished observations). In a recent study, passive immunization against experimental S. enteritidis infection in CD1 mice by the oral administration of hen egg-yolk antibodies specific for SEF14 fimbriae increased the survival rate from 32% of control animals to 78% of the SEF14 antibodytreated group. A reduction in the localization of S. ente~tidis along villous margins of the small intestine was noticed in the treated group (Peralta et al., 1994). Type 3 fimbriae of S. enteritidis have also been shown to contribute to the virulence of the organism in orally-infected mice (Aslanzadeh & Paulissen, 1992). These findings are the first indication that fimbriae other than type 1 may increase gut colonization and faecal dissemination of salmonellas. Based on existing evidence it is reasonable to suggest, therefore, that pathogens like S. typhimuHum rarely express type 1 or other fimbriae in extracellular environments like blood, but switch on the expression of the fimbriae when in close association with epithelial and other cell types, thereby permitting the bacterium to adapt to and take advantage of the different niches encountered in an infected host. DIAGNOSTIC TESTS BASED O N SALMONELLA HMBRIAE
The first diagnostic tests based on the detection of fimbrial antigens were developed for the detection of enterotoxigenic E. coli (Sojka, 1971; Orskov et al., 1975;
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Guinee el al., 1976). The impetus for their development and use was the discover?, that fimbriae such as F4 and F5 are essential for the E. coli to cause disease, and so their detection on isolates froln clinical cases of diarrhoea is of diagnostic significance and a variety of tests that incorporate monoclonal antibodies are now commonly used-to detect rapidly fimbriae expressed by enterotoxigenic E. colt: (Thorns et al., 1989a, b). Although much effort has gone into the characterization of fimbriae expressed by SalmoneUa, their application to the detection of specific sahnonella infections has, to date, not been fully exploited. The two main reasons for this have been the concentration of research towards characterization of type 1 fimbriae and a lack of understanding of the role of these antigens in the lifecycle of the bacterimn, which means that their detection is of unknown clinical significance. Detection 0 f S a l m o n e l l a g e n u s Genus-specific tests based mainly on the detection of surface antigens such as lipopolysaccharide, flagellin and outer membrane proteins, have been used with some success in enzyme-linked immunosorbent assays (ELISAs) and agglutination tests (Clark et al., 1989; Kerr et aL, 1992; Manafi & Sommer, 1992; Feldsine et al., 1992; Wyatt et al., 1993). Recently a DNA-based test which targets the ag/A structural gene of SEF17 filnbriae has been developed and shown to react strongly with 603 of 604 sahnonella isolates and only ver?, weakly to 31 of 266 other members of the family Enterobacteriaceae (Doran et al., 1993). This is the first instance in which a fimbrial gene probe has been used as a genus-specific diagnostic tool and may offer quicker and more sensitive approaches than existing culture methods. Specific detection o f S a h n o n e l l a enteritidis The rapid spread of S. ente~tidis through the poultr?, population and the subsequent increase in human food poisoning cases caused by this serotype, highlights the need for more rapid, specific tests to identify S. entedtidis infections in animal and hunaan populations in order to apply prescribed control strategies. Recently, a simple, latex agglutination test has been developed for the specific identification of S. ente~tidis. It is based on the detection of the SEF14 fimbrial antigen and to ensure the differentiation of S. enteritidis from other closely related serogroup D salmonellas, a second latex reagent is used that detects a specific epitope on the flagellum of S. dublin (McLaren et al., 1992; Thorns et al., 1992, 1994). Extensive evaluation by laboratories worldwide has demonstrated that this is an accurate, presumptive test for S. enteritidis that can be carried out in routine laboratories that would normally have to send all their Salmonella isolates to specialized laboratories for serotyping. The increase in bacteriological monitoring of pouhr?, flocks for salmonella has required an evaluation of alternative screening procedures in order to reduce the time and costs involved. The most likely alternative is serological monitoring, especially as more specific ELISA-based assays become available (Barrow, 1994). A recent development is the application of SEF14 fimbriae to the specific serodiagnosis of chicken flocks infected with S. enteritidis. Results indicate that birds infected with S. ente~{tidis readily seroconvert within 10 days of infection and the IgG response persists for at least 4 weeks thereafter (Thorns el al., 1993). The pro-
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duction of SEF14 antibodies following infection is the first demonstration of a specific anti-fimbrial response to salmonella infection. Preliminary data indicate that the potential advantages of the detection of SEF14 antibodies are high specificity and sensitivity. In addition, it is possible that future generations of tests could distinguish vaccinal responses from field infection (see below).
APPLICATION OF SALMONELLA FIMBRIAE IN VACCINE DEVELOPMENT
The pathogenesis of sahnonella infections is recognized as being multifactorial so it is unlikely that a simple, defined, fimbrial subunit vaccine will elicit a sufficient protective response against challenge by salmonellas. However, Aslanzadeh and Paulissen (1990) reported that specific antibodies to type 1 and type 3 fimbriae conferred partial passive protection to mice orally-infected with S. ente~tidis. In their study, pretreatment with anti-fimbrial antibody increased survival of mice by over 60%. These results may indicate the importance of synergism between the two fimbriae in the disease process and further work needs to be carried out to confirm this finding. In particular it would be of interest to determine the combined protective effect of specific antibodies to SEF21, SEF17 and SEF14 fimbriae of S. entel~tidis in a suitable animal model. Future directions are likely to involve the use of fimbriae as carriers of protective, heterologous antigenic determinants in novel multivalent live vaccines (Woodward et al., 1993). The successful incorporation of peptides from foot-andmouth disease virus and human immunodeficiency virus type 1 into E. coli F4 timbriae has been reported, and specific antibodies to the heterologous peptides were produced in mice after inoculation with the purified fimbriae (Bakker et al., 1990). The use of sahnonella fimbriae as carriers of foreign epitopes is currently being pursued in our laboratory. Epitopes of the MPB70 antigen of Mycobacte~um bovis (Harboe & Nagai, 1984) have been successfully incorporated into SEF14 timbriae produced by a strain of S. ente~tidis (Woodward et al., 1993). The use of vaccines to reduce excretion and vertical transmission of salmonellas is likely to increase in the future. It is therefore important to develop vaccines in which the vaccinal antibody responses can be differentiated from those resulting from natural infections so as not to compromise serological monitoring. From initial observations in our laboratory and elsewhere, it seems very unlikely that SEF14 fimbriae are prerequisites for virulence, therefore one strategy being adopted in our laboratory is the construction of sefA gene deletion mutants from live vaccine strains (Cooper et al., 1990, 1992) with the aim of being able to distinguish vaccinal antibody responses devoid of SEF14 antibodies as opposed to SEF14 seroconversion seen in field infections (Thorns et aL, 1993).
CONCLUSIONS
Most salmonellas are able to express one or more types of fimbriae at some time during their lifecycle. The environmental and molecular signals that determine fimbrial expression are still poorly understood, but it is now recognized that their expression in specific niches in the infected host or in the outside environment
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play an important part in bacterial smMval. Duguid et al. 's (1976) original notion, that fimbriae se~we a fnnction that is marginally advantageous to the organism, has not yet been confirmed or expanded upon due to the paucity of experimental evidence mad the ad hoc nature of the research over the past 25 years. Nevertheless modern molecular and immunological techniques, linked with in vivo studies, will undoubtedly help to unravel the complex individual and interdependent functions of sahnonella fimbriae in the near future. Current evidence indicates that many different fimbriae mediate a variety of functions that are important for the maintenance and SUl-vival of the organisms in the host and its environment. These functions might include: (i) initiation a n d / o r stabilization of the organism to epithelial cells; (ii) colonization and microcolony formation of tissues via site-specific binding to receptors on tissue matrix proteins; (iii) maintenance of persistent infections in the host by mediating selective bacterial trapping by phagocytic cells, and subsequent SUlarival in specific niches of the infected animal or its environment; (ix,) evasion of the host's specific immunological defences by: (a) presentation of self epitopes on the fimbriae and (b) production of harmless immune responses to fimbriae which are then rapidly shed or their expression switched off from the surface of the organism. Recent advances in the characterization of sahnonella filnbriae have already resulted in flaeir application to novel diagnostic tests and demonstrated their potential in the future development of vaccines. Only when the fimbrial functions have been fnlly characterized will their potential for the detection and control of sahnonella infections be frilly exploited.
ACKNOWLEDGEMENTS
I would like to thank Diane Newell, Martin Woodward and Clifford Wray for their helpful discussions and critical evaluation of the manuscript.
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type 3-like fimbriae) of Salmonella strains. FEMS Microbiological Letters 19, 233-8. ARNQVIST,A., OLSEN,A., PFEIFER,j., RUSSELL, D. G. & NORMARK,S. (1992). The Crl protein activates cl3,ptic genes for curli formation mad fibronectin binding in Escherichia coli HB 101. Molecular Microbiolo~, 6, 2443-52. ASL-~NT_,\DEI-I,J. 8¢ PAUI,ISSEN,L.J. (1990). Adherence and pathogenesis of Salmonella enteritidis in mice. Microbiology and hnmunology 34, 885-93. ASL~NZADIm,J. & PAVLISSEN,L.J. (1992). Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella ente~itidis in mice. Microbiolo~, and hnmunolog), 36, 351-9. BAKKER,D., VANZI.IDERFELD, F. G., VANDI.:RVEEN,S., OUDE(;A,B. 8c DE GP~-~-.xv,F. K. (1990). 1(88 fimbriae as carriers of heterologous antigenic determinants. Microbial Pathogenesis 8, 343-52.
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(Acceptedfor publication 16 September 1994)