The Salmonella dublin Virulence Plasmid Does Not Modulate Early T ...

INFECTION AND IMMUNITY, Jan. 1996, p. 222–229 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 1

The Salmonella dublin Virulence Plasmid Does Not Modulate Early T-Cell Responses in Mice LAURENCE A. GUILLOTEAU,1† ALISTAIR J. LAX,2 SHEILA MACINTYRE,1 2 AND TIMOTHY S. WALLIS * School of Animal and Microbial Sciences, University of Reading, Reading,1 and Institute for Animal Health, Compton, near Newbury,2 Berkshire, United Kingdom Received 10 March 1995/Returned for modification 9 May 1995/Accepted 1 November 1995

The virulence plasmid in Salmonella dublin mediates systemic infection in mice and cattle. The role of gd T cells or hepatic extrathymic T cells has recently been reported to be important in the control of the early stage of Salmonella choleraesuis infections of mice. Here, we report on T-cell responses in conventional mice after challenge with a virulent strain of S. dublin carrying a virulence plasmid or with a strain cured of the plasmid. Over a period of 4 days postinfection, when both strains could be compared, similar changes in ab and gd T-cell subsets in peritoneal cavities, livers, and spleens were recorded, demonstrating no clear role of the virulence plasmid in modulation of early T-cell responses. To investigate further the role of the virulence plasmid in pathogenesis, the growth of the plasmid-cured strain was assessed in SCID, SCID bg, and irradiated mice. During the first 6 days after infection, there was no statistically significant difference in the net growth of Salmonella cells in the livers and spleens of SCID and SCID bg mice compared with conventional BALB/c mice. This observation excludes a key role for a T- or B-cell-mediated immune response in controlling the initial growth of the plasmid-cured S. dublin strain. Thereafter, the immunocompromised mice were no longer able to control infection, although SCID mice were more efficient at controlling net bacterial multiplication than SCID bg mice, potentially implicating NK cells in the control of infection in SCID mice. The early control of net bacterial multiplication in the spleens and livers of BALB/c mice was ablated by whole-body X-irradiation. Both wild-type and plasmid-cured strains multiplied significantly more rapidly in irradiated than in conventional BALB/c mice. However, the numbers of wild-type bacteria still increased more rapidly than the numbers of the cured strain. These results are consistent with a role of the S. dublin virulence plasmid in promoting in vivo growth of Salmonella cells. are generally serotype specific, and a common 8-kb region is required for expression of virulence (23, 44). Five open reading frames are believed to form a gene system, which is now designated spv (Salmonella plasmid virulence) (15). The role of virulence plasmids in pathogenesis is not known. The virulence plasmid has been shown to mediate systemic infection in mice (13, 14, 25, 32). Similarly, the virulence plasmid is necessary for systemic infection of calves infected with S. dublin but does not mediate intestinal invasion, enteritis, or the extracellular growth rate (43). One report suggested that the virulence plasmid may exert its effect on the growth rates of organisms by promoting intracellular growth of Salmonella cells in the host (16). In vitro studies have not shown any relationship between the carriage of the virulence plasmid and the survival of organisms in macrophages (12, 34). In vivo, virulence plasmid carriage is related to a strong inflammatory response as displayed by a marked splenomegaly and hepatic abscess formation (18), whereas a cured strain produced a radiosensitive mononuclear response in the liver (17). Although the role of the virulence plasmid in vivo appears to be related to interference with the function of the phagocyte system, there is no direct evidence for this. It has been suggested recently that expansion of gd T cells and hepatic extrathymic ab T cells may be involved in controlling growth of plasmidcured but not wild-type strains (7, 27). This observation potentially implicates the virulence plasmid in immunomodulation of the host response. Immunocompromised mice provide a model to study cellular components of the nonspecific immune response. Severe combined immunodeficient mice (SCID) lack functional T and B lymphocytes (4). SCID bg mice (6) have, in addition, a

Salmonellosis is a disease of humans and domestic animals ranging in severity from enteric fever to mild food poisoning. The relative importance of gastroenteritis and/or systemic infection varies in different Salmonella-host combinations. Salmonella dublin is host specific for cattle, since it causes both enteric and systemic disease but can also infect humans. The pathogenicity of S. dublin has been investigated mainly for mice (17, 25), but also for cattle (22, 43). Studies with Salmonella typhimurium have demonstrated that during the first days of infection, bacterial growth seems to be controlled by a T-cell-independent mechanism (20, 24, 26, 30). This innate resistance is dependent on the host genetic background, in part by regulating the growth rate of Salmonella cells in macrophages (31) or the responsiveness to lipopolysaccharide (5). Virulent strains appear to overwhelm this early innate-resistance mechanism. If the infection is controlled by these early events, bacterial numbers reach a plateau. This plateau is normally followed by the clearance of the organisms from the mononuclear phagocyte system, a phase requiring the presence of functional T cells (20, 29, 30). Several molecular studies have sought to identify the virulence genes involved in the systemic phase of infection. Some of these are located on a large plasmid (3, 14). These plasmids

* Corresponding author. Mailing address: Institute for Animal Health, Compton Nr Newbury, Berkshire RG20 7NN, United Kingdom. Phone: (44) 1635 578411. Fax: (44) 1635 577263. Electronic mail address: [email protected]. † Present address: Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. 222

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decreased natural killer (NK) cell activity (35) and phagocytic dysfunction in neutrophils (38). The present paper reports the kinetics of T-cell responses induced by a wild-type strain of S. dublin and its plasmid-cured derivative strain in immunocompetent mice. In addition, SCID and SCID bg mice were infected with the plasmid-cured strain to investigate the role of lymphocytes in controlling the in vivo growth of a plasmidcured S. dublin strain. Effector cells were assessed for their radiosensitivity. MATERIALS AND METHODS Bacterial strains. The strains of S. dublin used in this study are SD2229, a wild-type isolate from an outbreak of bovine salmonellosis, and SDM173c, a plasmid-cured derivative of SD2229 (25). After overnight culture in LuriaBertani broth, bacteria were subcultured and grown in Luria-Bertani broth at 378C for 6 h with shaking to stationary phase (optical density at 550 nm of 1.6 5 109 bacteria z ml21). Bacteria were enumerated by serial dilution and plating on MacConkey or Luria-Bertani agar. Mice. All mice used were female, aged 7 to 8 weeks. BALB/c mice were bred in our animal facilities. CB-17/IcrCru-scid scid/scid (SCID) mice were purchased from Charles River (UK Limited). scid/scid bg/bg (SCID bg) mice (6) were bred in microisolator cages and fed sterile food and water. Experimental infection and samples. Mice were injected intraperitoneally (i.p.) with 102 CFU of strain SD2229, with 103 CFU of strain SDM173c, or phosphate-buffered saline (PBS). A dose of 102 CFU for strain SD2229 was chosen, since this represents the lowest dose which could produce systemic infection with highly reproducible bacterial recoveries from livers and spleens. At least three mice were killed at the indicated times after challenge, and either (i) spleens and livers were taken for bacterial counts by homogenizing the organs in physiological saline and plating on MacConkey agar or (ii) leukocyte cell suspensions from peritoneal cavity exudates, spleens, and livers were prepared for phenotypic analysis. Cell preparation. Peritoneal exudate cells (PEC) were obtained by washing the peritoneal cavity with 3 ml of IMDM (Iscove’s modified Dulbecco’s modified Eagle’s medium; Gibco, Life Technologies Ltd., Paisley, United Kingdom) containing heparin (5 U z ml21). PEC were centrifuged at 600 3 g for 10 min and were suspended in PBS containing 0.2% (wt/vol) bovine serum albumin and 0.01% (wt/vol) NaN3 (PBS-B). Liver cells were prepared as described elsewhere (7), with slight modifications. Briefly, the livers were perfused with sterile IMDM enriched with heparin to eliminate blood. The livers were pressed through nylon gauze and suspended in IMDM. After centrifugation at 600 3 g, the cell pellet was resuspended in 15 ml of the IMDM, and the cell suspension was incubated at 48C for 30 min to allow large aggregates to settle. The supernatant was centrifuged at 600 3 g for 10 min, and the pellet was resuspended in 8 ml of 44% Percoll (Pharmacia, Milton Keynes, United Kingdom) and layered on 5 ml of 67.5% Percoll. The gradient was centrifuged at 1,100 3 g for 20 min at 208C. Lymphocytes at the interphase were harvested, washed twice in IMDM, and suspended in PBS-B. Spleen cells were obtained by the conventional method of pressing through nylon gauze, erythrocytes were lysed by the addition of 0.155 M NH4Cl to the cell pellet, and the cells were washed. MAbs and flow cytometry (FCM) analysis. Fluorescein isothiocyanate (FITC)conjugated anti-T-cell receptor (TCR) ab monoclonal antibody (MAb) H57-597 and FITC-conjugated anti-TCR gd MAb GL3, both of which were produced in hamsters, were obtained from Pharmingen (AMS Biotechnology Ltd., Witney, United Kingdom) and were used at a working dilution of 1:200. Phycoerythrin (PE)-conjugated anti-CD8 (53 to 6.7) (working dilution, 1:20) and FITC antiMac-1 (complement receptor type 3 [CD11b/CD18]) (M1/70) (working dilution, 1:20) MAbs were from Boehringer Mannheim (Lewes, United Kingdom). PEconjugated anti-CD4 MAb GK1.5 (working dilution, 1:25) was obtained from Becton Dickinson, Ltd. (Conley, United Kingdom). Anti-F4/80 MAb (working dilution, 1:10) was obtained from Serotec (Kidlington, United Kingdom). With the exception of MAbs H57-597 and GL3, these MAbs were produced in rats (immunoglobulin G2b [IgG2b] isotype). An FITC rabbit anti-rat Ig [F(ab9)2; STAR 49] was used as the secondary antibody (working dilution, 1:50) for unconjugated F4/80 MAb and was obtained from Nordic Immunology, Ltd. (Maidenhead, United Kingdom). All MAbs were diluted in PBS-B alone or with 10% mouse serum for FITC anti-rat Ig. Cells (106) were incubated at 48C for 20 min with 50 ml of MAb at an appropriate concentration, washed twice with PBS-B, and fixed with 0.5% paraformaldehyde in PBS. Cells were singly stained with FITC anti-TCR gd MAb or doubly stained with (i) PE anti-CD4 MAb or PE anti-CD8 MAb and then with (ii) FITC anti-TCR ab MAb. Cells were analyzed on a FACScan (Becton Dickinson, Ltd.) by using forward- and side-scatter gating to select lymphocyte populations. The proportion of singly or doubly stained cells in total gated lymphocytes was expressed as a histogram or as a dot plot, respectively. Mac-1 (39) and F4/80 (2) markers were used to analyze by FCM the effects of X-irradiation on newly recruited phagocytes and on mature macrophages, re-

FIG. 1. Infection kinetics of S. dublin in the spleens (h) and the livers (E) of BALB/c mice. Mice were injected i.p. with 102 CFU of wild-type strain SD2229 (closed symbols) or with 103 CFU of plasmid-cured strain SDM173c (open symbols). Data are the means 6 SE for three mice at each time point.

spectively (9). Cells were stained by using FITC anti-Mac-1 MAb or anti-F4/80 MAb and then by FITC anti-rat Ig. An irrelevant FITC rat IgG2b isotypic control (working dilution, 1:20) was obtained from V. H. Bio Ltd. (Newcastle-uponTyne, United Kingdom) and was used as a negative control. Cells were gated on the mononuclear population present, and the proportion of positive cells was expressed as a histogram. Binding of PBS-B or secondary antibody was used to determine nonspecific immunofluorescence for each set of staining (less than 1%) and was deduced from the proportion of positive cells. X-irradiation. BALB/c mice received 6 Gy of whole-body X-irradiation 2 days before the challenge; irradiation was performed at the MRC Radiobiology Unit (Harwell, United Kingdom). The effects of this treatment on the macrophage population in the peritoneal cavities, spleens, and livers were monitored by FCM analysis. Murine Ig assay. We examined pooled sera from each group, at day 16 after challenge to assess the presence of IgM and IgG in serum specific to S. dublin; an enzyme-linked immunosorbent assay (ELISA) was done with S. dublin SD2229 sonicate as the coating antigen. Statistics. The results were expressed as the means 6 standard errors of the means (SE) of values and were analyzed by one-way analysis of variance and then by an unpaired Student-Newman-Keuls multiple-comparison test to calculate significant differences (P , 0.05) between two groups or by a Student’s t test (InStat for MacIntosh GraphPad).

RESULTS Kinetics of T cells in the peritoneal cavity, spleen, and liver during S. dublin infection. The role of the virulence plasmid on bacterial infection kinetics was assessed with wild-type and plasmid-cured strains of S. dublin in BALB/c mice. The number of bacteria in both spleens and livers increased steadily in mice infected with the wild-type SD2229, resulting in the death of mice within 6 days. In contrast, although the cured-strain SDM173c was disseminated to these organs, the net number of bacteria did not increase after 6 days and death did not follow (Fig. 1). To elucidate the relationship between the T-cell response and the virulence of S. dublin, the leukocyte subset kinetics in the peritoneal cavity, spleen, and liver were analyzed following i.p. injection of either strain SD2229 or SDM173c or PBS. The total number of lymphocytes per organ was calculated from the total number of cells in PEC, spleens, or livers per mouse, and the proportion of lymphocytes was determined by FCM. Over a period of 4 days postinfection, when both strains could be compared, the numbers of lymphocytes in PEC, livers, and spleens were similar between groups, except on day 4 for spleens. The number of splenic lymphocytes in mice infected with SD2229 was about double that for mice infected with SDM173c or injected with PBS (Fig. 2). The proportion of different leukocyte subsets was quantified by FCM on PEC, spleens, and livers. Representative histograms and scatter plots

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FIG. 2. Lymphocyte kinetics in the peritoneal cavities (a), livers (b), and spleens (c) after an i.p. challenge with S. dublin. Mice were injected i.p. with 102 CFU of strain SD2229 (F), with 103 CFU of strain SDM173c (h), or with PBS (Ç). Values were calculated on the basis of the number of total cells per organ per mouse and the proportion of lymphocytes in these organs.

of singly and doubly stained cells indicating the specificity of staining are shown in Fig. 3. In comparison with the noninfected group, a significant (P , 0.05) and similar expansion of the proportion of TCRgd1 cells was observed in the peritoneal cavities on day 4 with both strains of S. dublin as well as on days 6 and 10 for strain SDM173c (Fig. 4a); all mice infected with strain SD2229 were killed by 6 days (Fig. 1). A similar pattern of TCRgd1 cell expansion was observed in the livers and spleens, although it was not significantly different from that for the noninfected group (Fig. 4b and c). A significant increase in the proportion of CD41 TCRab1 cells was observed in the peritoneal cavities on days 4 and 10 after the challenge only from mice infected with SDM173c (Fig. 5a), but there was not a significant difference between mice infected with SDM173c or SD2229. The proportion in CD41 TCRab1 cells in the livers and spleens did not change significantly (Fig. 5b and c). There was no change in the proportion of CD81 TCRab1 cells in these organs following challenge with either strain (data not shown). Resistance of immunocompromised mice to infection by a plasmid-cured strain of S. dublin. We investigated the infection kinetics of plasmid-cured S. dublin in the absence of func-

INFECT. IMMUN.

tional lymphocytes using both SCID and SCID bg mice. When immunocompetent mice (BALB/c) were challenged with strain SDM173c, the number of Salmonella cells per spleen increased to approximately 104 CFU 4 days after challenge, and there was no net change over 25 days (Fig. 6a). In the livers, the number of viable Salmonella cells remained low (102 CFU) for the duration of the experiment (25 days) and started to decrease after 6 days infection (Fig. 6b). In immunocompromised mice, net growth of strain SDM173c was greater than that in BALB/c mice. In the initial stages of infection, there were no statistically significant differences in the recovery of SDM173c from BALB/c, SCID, and SCID bg mice (Fig. 6). Thereafter, the immunocompromised mice, unlike BALB/c mice, were no longer able to control net multiplication of the cured strain. From day 6 onward, the number of Salmonella cells in both spleens and livers continually increased, and SCID bg mice started to succumb to the disease 19 days after challenge (Fig. 6). Over this period, SCID mice were more efficient at controlling multiplication of SDM173c than SCID bg mice, particularly in spleens, in which a significant increase in numbers was not observed until 15 days after infection. However, even with SCID bg mice, the net increase in bacterial numbers was slower and the length of survival of mice was longer compared with the infection kinetics of the wild-type strain SD2229 in BALB/c mice (Fig. 1). At postmortem examination, a strong splenomegaly in SCID mice (4.8 times higher relative weight at day 16 versus day 2) and SCID bg mice (5.5 times higher relative weight at day 16 versus day 2) was observed, compared with BALB/c mice (1.6 times higher relative weight at day 16 versus day 2) (Table 1). Livers from SCID and SCID bg mice showed focal inflammatory lesions, giving the organ a mottled appearance. Anti-S. dublin Ig could not be detected by ELISA in serum from SCID or SCID bg mice (data not shown), whereas infection of conventional mice with this strain results in potent serum antibody responses (42). Radiosensitivity of effector cells in the early control of S. dublin. We analyzed the effect of whole-body X-irradiation on the infection kinetics of S. dublin in the spleens and the livers of BALB/c mice. FCM analysis was performed on cells from X-irradiated mice injected with PBS to monitor the effects on macrophage populations. The numbers of Mac-11 cells in the peritoneal cavities, spleens, and livers were maximally depleted ($97%) at 6, 4, and 8 days, respectively after X-irradiation (Fig. 7a). The number of F4/801 cells dropped 90% in spleen and PEC by 6 days after treatment (Fig. 7b). A similar pattern of depletion was detected in infected mice, 4 days after infection, i.e., 6 days after irradiation (data not shown). Under these conditions, a significant increase in the numbers of both strains was observed in spleens and livers from X-irradiated mice compared with those for nontreated mice (P , 0.05) (Fig. 8). These results demonstrated the primary role of radiosensitive cells in the control of infection with both of these strains. DISCUSSION The use of athymic mice or T-cell depletion in immunocompetent mice has provided indirect evidence that the early suppression of Salmonella growth by the host is independent of T cells (20, 24, 26, 30). However, it was reported that the gd1 T-cell subset was expanded in mice infected with plasmid-free but not virulent strains of Salmonella choleraesuis. It was further suggested that this T-cell subset was protective in the early stage of Salmonella infection (7), implicating a possible role for the virulence plasmid in immunomodulation of the host T-cell response. To investigate this hypothesis further, we have stud-

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FIG. 3. Scatter plots depicting the proportion of TCRgd1 cells (a) and CD41 TCRab1 cells (b) in the peritoneal cavities, spleens, and livers on day 4 after an i.p. challenge with S. dublin. Mice were injected i.p. with 102 CFU of strain SD2229 or 103 CFU of strain SDM173c or were noninfected. Cells were singly stained with FITC-conjugated anti-TCRgd MAb or doubly stained with anti-CD4 MAb and finally with anti-TCRab MAb.

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FIG. 4. Kinetics of TCRgd1 cells in the peritoneal cavities (a), livers (b), and spleens (c) after challenge with S. dublin. Mice were injected i.p. with 102 CFU of strain SD2229 (t) or 103 CFU of strain SDM173c (u) or were noninfected (1). Data are the percentages of total gated lymphocytes and represent the means 6 SE from two separate experiments; three mice were used at each time point per experiment. †, mice infected with strain SD2229 that were dead by day 5.

ied the ability of wild-type and plasmid-cured strains of S. dublin to initiate T-cell responses in mice. Up to day 4, when the immunomodulating activities of the strains could be compared, both strains induced similar changes in the proportion of lymphocyte populations at all sites examined. This result demonstrates that, at least during the early phase of infection, there is no correlation between the expansion of the proportion of gd T cells in the inflamed sites and the presence of the virulence plasmid in S. dublin in contrast to that reported in S. choleraesuis infections (7). On the contrary, mice infected with the virulent strain showed an increase in the number of lymphocytes, including gd T cells. These conflicting results may reflect the inherent differences in the virulence of the S. dublin and S. choleraesuis strains tested. The time to death following i.p. challenge with 50 CFU of wild-type S. choleraesuis was greater than 10 days. Following i.p. challenge with 100 CFU of wild-type S. dublin, all mice were dead by day 6. Lower doses of S. dublin SD2229 (as low as 10 CFU) show similar kinetics to death (22). This difference in virulence was also reflected in the infection kinetics of the plasmid-cured S. choleraesuis and S. dublin strains. However, clearly the difference in the virulence of the wild-type and plasmid-cured strains of S. dublin used here cannot be explained by plasmid-mediated immunomodulation. Both strains evoked similar changes in the proportion of lymphocyte subsets at all sites tested when the net growth rate of the wild-type strain was considerably greater than that of the plasmid-cured strain. This shows that the previously reported plasmid-mediated difference in gd T-cell expansion is not necessarily true for all Salmonella serotypes. Although the virulence plasmid did not clearly influence quan-

titatively the lymphocyte kinetics during the early phase of infection, we could not exclude differences in biological functions such as cytokine production (36, 37). However, there is evidence for gd T cells in controlling Salmonella infections. In vitro, the relative virulence of the Salmonella strain (7) and the genetic resistance of the host (8) influence the ability of macrophages to express stress proteins, which in turn induce the expansion of gd T cells. Further direct evidence was reported recently. Mice were depleted of gd T cells from the intraepithelial lymphocyte population and undefined lymph nodes. The 50% lethal dose in such mice following oral challenge with Salmonella enteritidis was decreased 100-fold (28). We have shown that SCID and SCID bg mice were as effective as conventional mice in controlling the early phase of plasmid-cured S. dublin infections. Immunocompromised mice developed a strong splenomegaly and hepatic inflammatory foci, which are reactions mainly mediated by accumulation of phagocytic cells (11). The deficiency in NK activity (35) and abnormalities in neutrophil function (38) attributed to the beige mutation could explain why SCID mice have better control of Salmonella growth in the later stage of infection than SCID bg mice. NK cells of SCID mice produce gamma interferon, the main cytokine involved in macrophage activation, by a T-cell-independent mechanism (33). These results suggest a protective role of NK cells in Salmonella infection, in contrast to previous studies (10). The dysfunction of neutrophils in SCID bg mice may also influence the susceptibility of these mice to salmonellosis. A correlation between host susceptibility and granulocyte bactericidal activity has been demonstrated

FIG. 5. Kinetics of CD41 TCRab1 cells in the peritoneal cavities (a), livers (b), and spleens (c) after a challenge with S. dublin. Mice were injected i.p. with 102 CFU of strain SD2229 (t) or with 103 CFU of strain SDM173c (u) or were noninfected (1). Data are percentages of total gated lymphocytes and represent the means 6 SE from two separate experiments; three mice were used at each time point per experiment.


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FIG. 7. Kinetics of Mac-11 cells (a) and F4/801 cells (b) in the peritoneal cavities (u), spleens (s), and livers (■) from whole-body X-irradiated mice. Data are the absolute numbers of positive cells per organ, obtained from three mice at each time point. ND, not done.

FIG. 6. Infection kinetics of S. dublin SDM173c in the spleens (a) and livers (b) of BALB/c (F), SCID (h), and SCID bg (Ç) mice. Mice were injected i.p. with 103 CFU. Data are the means 6 SE for four mice at each time point. **, significantly different (P , 0.001) compared with BALB/c mice, determined by a Student-Newman-Keuls test. †, SCID bg mice infected with SDM173c that were dead by day 19.

elsewhere (41), but probably only at the early stage of infection which was not apparent here. The numbers of plasmid-cured S. dublin steadily increased in the immunocompromised mice over 2 to 3 weeks, demonstrat-

TABLE 1. Spleen weights from SCID, SCID bg, and BALB/c mice infected i.p. with S. dublin SDM173ca Spleen wt (g) Day

2 4 6 11 16 24

SCID

SCID bg

BALB/c

0.05 6 0.01 0.05 6 0.01 0.09 6 0.02 0.14 6 0.01 0.24 6 0.07b 0.38 6 0.09c

0.06 6 0.01 0.06 6 0.00 0.08 6 0.02 0.24 6 0.04b 0.33 6 0.06c †

0.10 6 0.01 0.09 6 0.01 0.13 6 0.01 0.16 6 0.01 0.16 6 0.01 0.16 6 0.01

a Mice were injected with 103 CFU. Spleen weights are the means 6 SE for four mice at each time point. †, SCID bg mice infected with SDM173c were dead at this time. b Significantly different (P , 0.05) compared with day 2, as determined by a Student-Newman-Keuls test. c Significantly different (P , 0.001) compared with day 2, as determined by a Student-Newman-Keuls test.

ing that a specific immune response is necessary to control the plateau phase of plasmid-cured S. dublin infection (29), as seen with wild-type Salmonella strains in immunocompetent genetically resistant mice (20). All of this evidence supports an essential role of the nonspecific immune system in the control of the first stage of plasmid-cured S. dublin infection. We have confirmed the radiosensitivity of effector cells in the control of the early stage of infection (1, 20, 26), in which bone marrow cells are crucial (19). FCM analysis on cells from X-irradiated mice confirmed the susceptibility of newly recruited phagocytes (Mac-11) and mature macrophages (F4/801) to this treatment and thus the prevention of inflammatory cell recruitment in response to Salmonella infection. Whole-body X-irradiation resulted in net growth of plasmid-cured S. dublin in spleen and livers comparable to that of the wild-type strain in unirradiated BALB/c mice (this work). These observations are different from results previously reported with S. dublin in irradiated BALB/c mice (17), which showed that plasmid-cured strains had an increased growth rate in irradiated mice only 7 days postinfection. This may relate to differences in the virulence of strain SD2229 compared with the Lane strain used in the latter study. Our results clearly demonstrate that plasmid-cured salmonellae are capable of growing rapidly in X-irradiated mice, in contrast to the behavior of other attenuated strains (21, 40). This suggests that the recruitment of inflammatory cells at the site of infection is necessary to control the net growth of plasmid-cured salmonellae. Significantly, the net growth rate of the wild-type S. dublin strain was still greater than that of the plasmid-cured strain in X-irradiated mice. Clearly, X-irradia-

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8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18.

19. 20. FIG. 8. Infection kinetics of S. dublin SD2229 (closed symbols) and SDM173c (open symbols) in the spleens (a) and livers (b) in conventional (circles) and X-irradiated (squares) BALB/c mice. Viable bacteria were enumerated as CFU in spleens and livers. Each point represents the means (m) of results from three mice. p, significantly different (P , 0.01) versus untreated infected mice, determined by a Student-Newman-Keuls test.

21. 22. 23.

tion-resistant factors also control the net growth rate of the plasmid-cured strain. These data are consistent with the proposal that the virulence plasmid in some way promotes the growth rate of Salmonella cells (16) but do not exclude the alternative possibility that plasmid-encoded factors modulate some component of the innate defense mechanisms.

24.

ACKNOWLEDGMENTS

27.

This work was supported by a BBSRC Link grant. We thank P. Sopp and A. Gautier for FCM analysis, R. Barclay for help in initiating this work, D. Buzoni Gatel for her helpful suggestions, and M. Gardiner, P. Plank, and P. Prior for animal husbandry.

25.

26.

28.

29. 1.

2. 3. 4. 5. 6. 7.

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