Institute for Animal Health, Compton, Newbury RG20 7NN, UK1
Author for correspondence: Timothy S. Wallis. Tel: +44 1635 577230. Fax: +44 1635 577263. e-mail: timothy.wallis{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: Salmonella serotypes, porcine virulence, macrophages, apoptosis, cytokine bioassays
Abbreviations: LDH, lactate dehydrogenase
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INTRODUCTION |
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The factors influencing Salmonella serotype host-specificity remain poorly defined, although there is some evidence that bacterial survival within macrophages is important. S. typhimurium can persist in significantly higher numbers than S. typhi in primary murine macrophages in vitro (Lissner et al., 1985 ; Vladoianu et al., 1990
; Alpuche-Aranda et al., 1995
; Ishibashi & Arai, 1996
; Schwan & Kopecko, 1997
), which correlates with the virulence of these serotypes for mice. Similarly, S. typhi persists better in human compared to murine macrophages in vitro, which again correlates with virulence. However, the relative persistence of S. typhi and S. typhimurium in human macrophages varies considerably between different studies (Vladoianu et al., 1990
; Alpuche-Aranda et al., 1995
; Ishibashi & Arai, 1996
). This variation may be explained by unquantified differences in the magnitude of macrophage lysis, which can have a major effect on the interpretation of results from bacterial persistence studies (Guilloteau et al., 1996
; Sizemore et al., 1997
).
This study has used Salmonella serotypes of defined virulence in pigs to characterize the interaction of Salmonella serotypes with porcine macrophages. Bacterial persistence, the magnitude and nature of Salmonella-induced macrophage damage, and the production of pro-inflammatory cytokines were assessed and correlated with virulence.
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METHODS |
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Virulence of Salmonella serotypes following inoculation of pigs.
Camborough pigs (67 weeks old) were supplied by the Institute for Animal Health farm. Pigs were weaned at 2 weeks and given a 3 d course of enrofloxacin (Baytril; Bayer), to compensate for their lowered resistance to bacterial infections during weaning and transport to the disease-secure animal unit. Thereafter, pigs were fed on a diet of antibiotic-free, irradiated pellets. None of the pigs excreted salmonellas at 0, 2 or 4 weeks before infection as determined by enrichment of faecal swabs in Rappaport broth (at 37 °C for 18 h) and selenite brilliant green broth (at 42 °C for 18 h) followed by overnight incubation at 37 °C on modified brilliant green agar (Difco).
Bacterial cultures of S. typhimurium ST4/74, S. dublin SD3246 or S. choleraesuis SCS14/74 were prepared by inoculating Bacto-Tryptose broth (Jones, 1975 ) with several bacterial colonies and incubating statically at 37 °C for 18 h. For oral inoculation, approximately 3x108 c.f.u. were suspended in 10 ml ant-acid [5% Mg(SiO3)3, 5% NaHCO3 and 5% MgCO3 in H2O] and administered directly into the stomach of each pig immediately before the morning feed using a 10FG catheter (Arnolds Veterinary Products). For intravenous inoculation, approximately 1x106 c.f.u. in a volume of 1 ml were injected into the superior vena cava. Rectal temperatures were recorded and animals were monitored for clinical signs of disease twice daily. All animals were humanely killed at 7 d after inoculation. The numbers of bacteria in the lung, spleen, liver, ileum, caecum and colon and in the lymph nodes associated with these sites were determined by viable counts of one sample per site as described by Watson et al. (1998)
. The limit of accurate quantification was 2·0 log10c.f.u. (g tissue)-1 and any samples containing fewer bacteria were enriched in Rappaport and selenite brilliant green broth as described above. Samples that were positive on enrichment were given a value of 2·0 log10c.f.u. g-1 and those that were negative were given a value of 0 c.f.u. g-1.
Salmonella infection of porcine alveolar macrophages and preparation of conditioned supernatants for cytokine bioassay.
Alveolar macrophages were isolated from healthy Camborough pigs by bronchoalveolar lavage as described previously for cattle (Guilloteau et al., 1996 ). Viability of cells, of which 9099% are alveolar macrophages (Ganter & Hensel, 1997
), was >99% as assessed by trypan blue exclusion. Cells were allowed to adhere to 24-well tissue culture plates at 5x105 cells ml-1 in Iscoves modified Dulbeccos medium containing 10% foetal calf serum (FCS), 100 units penicillin ml-1, 100 µg streptomycin ml-1 and 100 µg gentamicin ml-1 and incubated overnight at 37 °C in 5% CO2 and 95% humidity. Two hours before infection, non-adherent cells were washed off and the culture medium was changed to Dulbeccos modified Eagles medium Hams nutrient mix F-12 without phenol red (DME/F12) containing 5% FCS. Bacterial cultures were prepared as described above and diluted in DME/F12 medium to give a ratio of infection of five bacteria per macrophage. Viable and heat-inactivated bacteria were added to the macrophage monolayers and the monolayers were incubated for 1, 3, 24 or 48 h. Overgrowth of extracellular bacteria in monolayers incubated for 24 or 48 h was prevented by washing the monolayers 1 h after infection and adding DME/F12 containing 5% FCS and 100 µg gentamicin ml-1, followed by washing the monolayers after a further hour and adding DME/F12 containing 5% FCS and 10 µg gentamicin ml-1. Gentamicin was not added to the monolayers incubated for 1 or 3 h, because changing the media would have prevented the measurement of cytokines released immediately after infection. After the appropriate incubation time, the supernatants, termed conditioned macrophage supernatants, were collected and centrifuged (13500 g, 2 min, 4 °C) to reduce the amount of cell debris and bacteria. An aliquot (50 µl) was used to quantify macrophage damage by measuring the amount of lactate dehydrogenase (LDH) released by the monolayers using the cytotoxic 96 kit (Promega). Results obtained using this method correlate with the severity of macrophage damage by microscopic assessment (Guilloteau et al., 1996
). The results were expressed as the percentage of LDH released by infected monolayers compared to the LDH released by detergent treatment of control monolayers. The remaining conditioned macrophage supernatants were stored at -70 °C in aliquots. The number of macrophage-associated bacteria was determined by washing monolayers twice with DME/F12 without supplements and adding 1 ml PBS containing 0·1% sodium deoxycholate. Serial dilutions of the lysates were plated onto MacConkey agar plates and incubated overnight at 37 °C.
Bioassay for IL-1 activity.
Cytokine release was quantified using biological assays, which have the following characteristics: they measure cytokine release rather than expression; they are very sensitive; and they are unlikely to be affected by unrecognized inhibitors often present in serum, when using monocultures of cells. In addition, ELISAs for porcine cytokines are not readily available. IL-1 activity was measured using the A375 cell line. The growth of A375 cells is inhibited in the presence of human and porcine IL-1 (Nakai et al., 1988 ; Cendan et al., 1994
). The cells were maintained in Eagles minimal essential medium with Earles salts containing 10% FCS, 100 units penicillin ml-1, 100 µg streptomycin ml-1 and 100 µg gentamicin ml-1. Aliquots of the conditioned macrophage supernatants were thawed and serially diluted 1 in 2 in the above media to give a final volume of 50 µl per well, in 96-well plates. Medium alone was used as a negative control. Fifty microlitres of A375 at 1x105 cells ml-1 in Eagles minimal essential medium with supplements was added to each well and incubated for 96 h at 37 °C in 5% CO2 and 95% humidity. Following incubation, the culture medium was removed and the wells were washed once with PBS. The remaining cells were stained with freshly prepared crystal violet stain (0·1% crystal violet, 1% acetic acid, 5% formaldehyde, 4·25 g NaCl l-1, 2·25 g NaH2PO4 anhydrous l-1, 2·75 g Na2HPO4 anhydrous l-1) for 2 h at room temperature and then the excess stain was removed by washing with PBS. The retained crystal violet was dissolved in 100% methanol and quantified by spectrophotometry (OD595). The percentage growth inhibition was calculated using the formula: [(OD595 negative control - OD595 test sample)/OD595 negative control]x100. The range of detection of the assay was 0·1 units per well (resulting in 7% growth inhibition) to >10 units per well (resulting in 75% growth inhibition) as estimated from a dose response curve to recombinant human IL-1
(Genzyme Diagnostics) which was determined in parallel with each assay.
Bioassay for IL-6 activity.
IL-6 activity was measured using the 7TD1 cell line (kindly supplied by P. Kaiser, IAH, Compton, UK), whose growth is dependent on the presence of IL-6 (Van Snick et al., 1986 ). The suitability of this cell line in a bioassay for porcine IL-6 has been reported previously (Frank et al., 1996
). The cells were maintained in RPMI 1640 containing 10% FCS, 100 units penicillin ml-1, 100 µg streptomycin ml-1, 100 µg gentamicin ml-1, 0·05 mM 2-mercaptoethanol. Recombinant murine IL-6 (R&D Systems) was added at a concentration of 4 ng ml-1 for routine growth of the cells. For use in the bioassay, cells were washed three times to remove IL-6 and were resuspended in RPMI 1640 with supplements, but without IL-6. Aliquots of the conditioned macrophage supernatants were thawed and serially diluted 1 in 5 in RPMI 1640 with supplements to give a final volume of 50 µl per well, in 96-well plates. Fifty microlitres of 7TD1 cell suspension at 1x105 cells ml-1 in RPMI with supplements was added to each well. The assay plates were incubated for 72 h at 37 °C in an atmosphere of 5% CO2 and 95% humidity. Growth of the 7TD1 cells was estimated by measuring DNA synthesis. An aliquot of [3H]thymidine containing 0·015 MBq radioactivity was added to each well 18 h before the end of the incubation period. The cells were harvested onto glass fibre filters and incorporation of [3H]thymidine into the cells was measured using a betaplate liquid scintillation counter. The range of detection was 0·001 pg ml-1 (5000 c.p.m. incorporated tritiated thymidine) to greater than 8 pg ml-1 (45000 c.p.m.) as estimated from a dose response curve to murine IL-6 determined in parallel to each assay.
Bioassay for TNF- activity.
TNF- activity was measured using the WEHI 164 clone 13 cell line (kindly supplied by G. Entrican, Moredun Research Institute, Edinburgh, UK). This cell line is sensitive to the cytotoxic activity of human TNF-
(Espevik & Nissen-Meyer, 1986
) and is suitable as a bioassay for porcine TNF-
(Kim et al., 1991
). Cells were maintained in Dulbeccos modified essential medium containing 10% FCS, 100 units penicillin ml-1, 100 µg streptomycin ml-1 and 100 µg gentamicin ml-1. Semi-confluent monolayers were prepared by adding 3x104 WEHI cells into wells of a 96-well plate and incubating overnight at 37 °C in an atmosphere of 5% CO2 and 95% humidity. The medium overlying the monolayers was replaced with 50 µl fresh medium. Aliquots of the conditioned macrophage supernatants were thawed and serially diluted 1 in 2 into the medium overlying the monolayers to give a final volume of 50 µl. Medium alone was used as a negative control. Finally, 50 µl media containing 0·5 µg actinomycin D-mannitol ml-1 (Sigma) was added to each well. The monolayers were incubated for 18 h as above. Following incubation, the remaining cells were stained with crystal violet stain as described for the IL-1 bioassay. The percentage cytotoxicity was calculated according to the formula: [(OD595 negative control - OD595 test sample)/OD595 negative control]x100. The range of detection was 0·001 units per well (3% cytotoxicity) to greater than 0·1 units per well (85% cytotoxicity), as estimated from a dose response curve to recombinant human TNF-
(Genzyme Diagnostics) determined in parallel to each assay.
Characterization of DNA from macrophages.
Porcine alveolar macrophages were isolated and maintained in 24-well plates as described above. Bacterial cultures were prepared as described above and, where required, were opsonized in 10% autologous serum for 40 min on a rolling platform. The monolayers were infected and subsequently incubated as described above: the overgrowth of bacteria in the culture medium of infected monolayers incubated for 24 and 48 h was prevented with the use of gentamicin. Actinomycin D-mannitol was added to three control monolayers to give a final concentration of 1 µg ml-1. DNA was extracted from macrophages by the method of Zychlinsky et al. (1992) with minor modifications as follows. Monolayers were washed twice with PBS and 200 µl lysis buffer (10 mM Tris; 5 mM EDTA; 0·5% SDS) was added and incubated at room temperature for 15 min. The lysate was pooled from triplicate wells, 1 µl 20 mg proteinase K ml-1 was added and the mixture was incubated for 2 h at 56 °C. An equal volume of Tris-saturated phenol/chloroform (1/1, v/v) was added and mixed gently for 5 min. The mixture was centrifuged (13500 g, room temperature, 15 min) and the upper aqueous layer was recovered. DNA was precipitated overnight at -20 °C by the addition of 1/10 volume 3 M sodium acetate and 2 vols ethanol and collected by centrifugation (13500 g, room temperature, 20 min). The DNA was washed once in 70% ethanol, air-dried and dissolved in 30 µl TE buffer containing 10 µg RNase ml-1. Samples (10 µl) were separated through a 1·5% agarose gel and examined on a UV transilluminator.
Electron microscopy of macrophages.
Porcine alveolar macrophages were isolated and maintained (at 30 ml of 1x105 cells ml-1 in 250 ml flasks) as described above. Monolayers were infected with bacteria with or without opsonization as described above. Actinomycin D-mannitol was added to control monolayers to give a final concentration of 1 µg ml-1. At 24 h after infection, the monolayers were washed once with 0·1 M cacodylate-buffered 2·5% glutaraldehyde and incubated overnight. The fixed macrophages were washed twice in 0·1 M phosphate buffer for 30 min and then post-fixed in 1% 0·1 M phosphate-buffered osmium tetroxide for 90 min. The macrophages were washed five times in double-distilled water and resuspended in 1% aqueous uranyl acetate for 1 h in the dark. After a further wash in water, the macrophages were embedded in 0·5 ml 1% agar. The solidified agar blocks were cut into 2 mm3 cubes, dehydrated through a methanol gradient and incubated for 2x15 min in propylene oxide. The blocks were embedded in araldite resin and incubated at 60 °C for 48 h to allow polymerization to occur. Blocks were sectioned at a thickness of approximately 90 nm and the sections were collected on 3-mm-diameter nickel Athene grids and stained with lead citrate for 10 min. The sections were examined using a Philips EM 300 transmission electron microscope at an accelerating voltage of 80 kV.
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RESULTS |
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S. typhimurium and S. dublin persist within, and induce damage to, porcine alveolar macrophages at significantly greater magnitude than S. choleraesuis
The relative recovery of S. typhimurium, S. dublin and S. choleraesuis was assessed up to 48 h after infection of porcine alveolar macrophages. Gentamicin was added after 1 h of infection to macrophages incubated for 24 or 48 h to prevent overgrowth of extracellular bacteria. Non-adherent bacteria and gentamicin were removed by washing the macrophages immediately before lysing the macrophages for bacterial recovery. Therefore, the bacterial recovery at 1 and 3 h is obtained from adherent and intracellular bacteria, and at 24 and 48 h from only intracellular bacteria. The number of bacteria associated with the macrophages at 1 h after infection is significantly greater for S. typhimurium compared to either S. dublin or S. choleraesuis (P <0·05) (Table 1). Between 1 and 3 h, the recovery of S. typhimurium and S. dublin decreased slightly (0·10·3 log10c.f.u. ml-1) whereas the recovery of S. choleraesuis decreased by almost 1·0 log10c.f.u. ml-1, and it was recovered in significantly lower numbers than either S. typhimurium or S. dublin (P<0·05). Between 24 and 48 h, the recovery of S. typhimurium and S. dublin remained constant, whereas the recovery of S. choleraesuis continued to decrease. At 48 h after infection, S. choleraesuis was recovered in significantly lower numbers than either S. typhimurium or S. dublin (P <0·02).
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DISCUSSION |
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In the present study, the relative virulence of S. choleraesuis, S. typhimurium and S. dublin for pigs was determined. Pigs can be experimentally infected with S. typhimurium and S. choleraesuis by both oral and intranasal inoculation (Reed et al., 1986 ; Fedorka-Cray et al., 1995
; Gray et al., 1995
), and infection via other routes, independent of the gastro-intestinal tract, may also be possible. The relative importance of different routes of natural infection in pigs is not known, and therefore the virulence of the three serotypes was assessed following both oral and intravenous inoculation. S. choleraesuis induced severe symptoms and infected systemic tissues in high numbers, S. typhimurium induced an acute, self-limiting enteritis and S. dublin was of low virulence. All three strains used to infect pigs are highly virulent for mice (Baird et al., 1985
; Plested, 1995
; Guilloteau et al., 1996
; A. V. Gautier & T. S. Wallis, unpublished data). These results confirm the association of S. choleraesuis with systemic disease in pigs, and the general lack of association of S. dublin with porcine salmonellosis. They also suggest that the mechanism of serotype-host specificity in pigs does not depend on intestinal colonization and translocation, since S. choleraesuis was the most virulent serotype by both routes of inoculation. This supports the recent observation that serotype-host specificity of S. dublin and S. choleraesuis does not correlate with intestinal invasion of bovine or porcine intestinal mucosa (Bolton et al., 1999
).
For many years it has been widely accepted that a key stage in the pathogenesis of Salmonella is its persistence within cells of the reticuloendothelial system. For example, susceptibility of mice to salmonellosis correlates with net growth of S. typhimurium within isolated macrophages (reviewed by Dunlap et al., 1994 ) and Salmonella mutants that are unable to survive in murine macrophages have reduced virulence in mice (Fields et al., 1986
; Libby et al., 1994
; Hensel et al., 1998
). However, several independent groups have recently reported that Salmonella is highly cytolytic for macrophages (Guilloteau et al., 1996
; Chen et al., 1996
; Lindgren et al., 1996
), suggesting that the contribution of macrophage infection to pathogenesis is more complex than previously believed. We have therefore investigated several parameters of the interaction of our strains of defined porcine virulence with primary porcine alveolar macrophages. This macrophage population has been relatively well characterized and is phagocytic, bactericidal, produces pro-inflammatory mediators, expresses several surface receptors typical of other macrophages and is easy to isolate in high numbers and purity (reviewed by Van Reeth & Adair, 1997
). In addition, in pigs the lungs are an important site for clearance of bacteria and particulates from blood (Winkler, 1988
), and pneumonia is a common feature of S. choleraesuis infection of pigs and therefore it is probable that S. choleraesuis interacts with alveolar macrophages during pathogenesis. However, porcine alveolar macrophages may differ in some of their properties to tissue macrophages in other systemic organs, and therefore may not necessarily accurately model the interaction of Salmonella with macrophages in the porcine liver and spleen. Macrophage-derived cell lines were not used because they are dramatically more permissive for uptake and/or intracellular replication of Salmonella serotypes compared to primary macrophages (Buchmeier & Heffron, 1989
; Vladoianu et al., 1990
) and would appear to be inherently unsuitable for such studies. For example, in murine macrophage-like cells, some studies find no difference between S. typhimurium and S. typhi (Vladoianu et al., 1990
; Hirose et al., 1997
), and others report differences in either uptake, but not persistence (Pascopella et al., 1995
), or persistence, but not uptake (Schwan & Kopecko, 1997
). In the present study, the majority of macrophage experiments were performed using non-opsonized bacteria, since preliminary experiments showed that opsonization was not a prerequisite for efficient phagocytosis and did not influence subsequent persistence, as has sometimes been reported for other serotypehost combinations (Ishibashi & Arai, 1996
).
The relative persistence of S. typhimurium, S. dublin and S. choleraesuis in porcine alveolar macrophages did not directly correlate with their virulence in pigs. S. typhimurium and S. dublin, but not S. choleraesuis, induced damage to the macrophage plasma membrane. This may allow gentamicin to enter the macrophage and kill intracellular bacteria, and therefore the difference in recovery of S. typhimurium and S. dublin compared to S. choleraesuis is probably greater than that measured by viable counts. These persistence data appear to be in contrast to those obtained with murine macrophages, from which it was concluded that the persistence of S. typhimurium and S. typhi directly correlated with their host specificity (Lissner et al., 1985 ; Vladoianu et al., 1990
; Alpuche-Aranda et al., 1995
; Ishibashi & Arai, 1996
; Schwan & Kopecko, 1997
). However, S. typhimurium can persist relatively well not only in murine macrophages, but also in human-monocyte-derived macrophages, chicken peritoneal exudate cells and porcine alveolar macrophages (Vladoianu et al., 1990
; Alpuche-Aranda et al., 1995
; Ishibashi & Arai, 1996
; M. S. Chadfield & J. E. Olsen, personal communication; this study). Taking these results together, there is a direct correlation between S. typhimurium persistence within macrophages and its ability to cause disease in a wide range of animals.
Macrophages are important in the regulation of the hosts immune response to infection through the release of pro-inflammatory cytokines and other mediators of inflammation. This contributes to the mobilization of host defences and recruitment of other immune cells required for control of an infection, but may also exacerbate some aspects of disease (reviewed by Murtaugh et al., 1996 ). The release of three pro-inflammatory cytokines by macrophages infected with different serotypes was quantified to determine whether this correlates with the different disease syndromes. Infection of porcine macrophages with all three serotypes resulted in a rapid and sustained release of IL-1 and TNF-
activity and a more gradual increase in the release of IL-6 activity. This is despite differences in the numbers of associated bacteria and the magnitude of macrophage damage. Therefore, the ability of different Salmonella serotypes to induce the release of pro-inflammatory cytokines from macrophages in vitro does not correlate with serotype-host specificity. Heat-inactivated bacteria were included in the cytokine assays to determine if any differences between the Salmonella serotypes were related to differences in surface antigens such as LPS. Although it turned out that this control was not necessary, it did reveal a difference in cytokine release induced by viable and killed bacteria. This supports a previous report showing differences in IL-12 induction in murine macrophages by viable and killed S. dublin (Chong et al., 1996
) and is relevant to the design of vaccines for salmonellosis.
Salmonella-induced macrophage damage has been reported to occur in vivo in both mice and pigs (Baskerville et al., 1972 ; Richter-Dahlfors et al., 1997
). However, in the present study there was no direct correlation between Salmonella-induced macrophage damage in vitro and virulence of the same strains in pigs. Furthermore, macrophage damage induced by S. typhimurium and S. dublin did not have any of the typical characteristics of apoptosis (reviewed by Allen et al., 1997
; McConkey, 1998
), which is in contrast to the conclusions of several other studies on Salmonella-induced macrophage damage (Chen et al., 1996
; Monack et al., 1996
; Hersh et al., 1999
). This is unlikely to be due to the use of different cell types or bacterial strains, since we have previously shown that Salmonella can damage macrophages by a non-apoptotic mechanism using both an immortalized macrophage-like cell line and alveolar macrophages isolated from cattle following infection with several commonly studied S. typhimurium strains (Watson et al., 2000
). In addition, opsonization of the bacteria did not alter the mechanism of macrophage damage. The induction of macrophage damage by a non-apoptotic mechanism is consistent with several features of Salmonella pathogenesis. For example, Salmonella infection of the intestines is associated with a large inflammatory response whereas apoptosis is generally believed to limit inflammation (reviewed by Savill, 1997
). In addition, there seems little benefit to an intracellular pathogen in inducing apoptosis in its host cell, since the pathogen may then become trapped within the apoptotic cell. This is analogous to many viral infections, in which apoptosis of the host cell is blocked to facilitate viral replication and spread (reviewed by Tschopp et al., 1998
). Conversely, the induction of apoptosis by predominately extracellular pathogens, such as Yersinia spp., may prevent phagocytosis and subsequent bacterial killing (Monack et al., 1997
; Ruckdeschel et al., 1997
).
This study reports on several in vitro assays which model the interaction of Salmonella and macrophages during pathogenesis with respect to host specificity. Although persistence within macrophages correlated with the broad host specificity of S. typhimurium, there were no direct correlations between persistence within macrophages, induction of cytokine release by macrophages or the severity or nature of macrophage damage with Salmonella serotype specificity for pigs. There is relatively little known about the interaction of Salmonella and macrophages in vivo and it is therefore possible that such in vitro assays do not accurately model the interaction in vivo. Further work will address this problem by characterizing the interaction between Salmonella and host cells in intestinal and systemic tissues following experimental infection of natural host animal species.
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ACKNOWLEDGEMENTS |
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Received 10 January 2000;
revised 23 March 2000;
accepted 27 March 2000.