Molecular Microbiology and Cell Biology Group, School of Biological Sciences1 and Department of Physiology, The Medical School2, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Institute for Animal Health, Compton, Newbury RG20 7NN, UK3
Author for correspondence: John Stephen. Tel: +44 121 414 6550. Fax: +44 121 414 6557. e-mail: J.Stephen{at}bham.ac.uk
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ABSTRACT |
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Keywords: invasion, intestine, M cells, host restriction, histotoxicity
Abbreviations: AE, absorptive epithelium; FAE, follicle-associated epithelium; PMN, polymorphonuclear leucocyte; TEM, transmission electron microscopy
a Present address: Dept of Microbiology & Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada T2N 4N1.
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INTRODUCTION |
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The second objective was to ascertain the portal of entry into porcine gut epithelium. Many studies of Salmonella/intestinal interactions are carried out in mice with strains of S. typhimurium, which have given rise to the perception that the portal of entry into the host for Salmonellae is via Peyers patches, in particular M cells (Jones et al., 1994 ;Clark et al., 1994
, 1996
; Penheiter et al., 1997
). However, we have shown that S. typhimurium (Wallis et al., 1986
; Worton et al., 1989
), and S. dublin and S. choleraesuis (Bolton et al., 1999
), enter rabbit enterocytes directly. Two recent studies in calves (Frost et al., 1997
; Watson et al., 1995
) demonstrated that both follicle-associated epithelium (FAE), which primarily consists of both enterocytes and M cells, and absorptive epithelium (AE), which lacks M cells, were rapidly invaded. An examination, by light and electron microscopy, of bovine ileal mucosa from loops infused with S. typhimurium showed that within 5 min, organisms were interacting with and destroying M cells lining domed villi associated with FAE; and by 1 h, bacteria were seen in macrophages in the lamina propria. However, in absorptive villi, bacteria were also seen adhering to enterocytes by 15 min; the process of bacterial internalization, exfoliation of bacteria-laden enterocytes and stunting of villi had begun by 30 min, and by 2 h these changes were severe (Frost et al., 1997
). In a quantitative in vivo study no differences were found in the numbers of tissue-associated S. typhimurium and S. dublin organisms recovered from bovine ileal loops, with or without Peyers patches, 3 h after inoculation (Watson et al., 1995
). Since the nature of the Salmonella/host interaction is both serotype- and host-dependent, we have now examined, by electron microscopy, porcine tissues challenged with S. dublin and S. choleraesuis to determine if entry into M cells is a sine qua non for Salmonella invasion of ileal epithelia in pigs.
The third objective was to determine if the differential histotoxicity shown by S. dublin but not S. choleraesuis for rabbit tissue (Bolton et al., 1999 ; Lodge et al., 1999
) was expressed in porcine and bovine tissue.
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METHODS |
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Bovine/porcine ileal loop invasion assays.
The surgical techniques were carried out as described previously (Watson et al., 1995 ). Animals were terminally anaesthetized with sodium pentabarbitone. Commencing at the distal ileum, a maximum of 14 sequential loops (each 9 cm in length and containing both AE and FAE) were ligated, separated by 1 cm loops. Approximately 109 organisms, suspended in Hartley Digest Broth (HDB) medium (Oxoid), were injected into appropriate loops, with alternating strains in sequential loops. In a typical experiment, three loops were infected with S. choleraesuis, three loops were infected with S. dublin, one loop was infected with the invasive wild-type S. typhimurium strain 4/74, one with its less invasive isogenic invH mutant, and one with HDB. The ligated loops were replaced inside the abdomen and the wound repaired. Following 2 h incubation, 4·5 ml of 300 µg gentamicin ml-1 in mucosal medium (MM; Amin et al., 1994
) was injected into each loop. After a further 1 h incubation, designated loops were fixed for electron microscopy, or biopsies were taken for quantitative bacterial viable counts as follows.
Loops were placed immediately into ice-cold MM and washed in the same medium. Three biopsies of AE and three biopsies of FAE were taken from each loop with a 12 mm cork borer and homogenized separately for 30 s in 3 ml of 1% (v/v) Triton X-100 (Sigma) in phosphate-buffered saline (NaCl, 8·0 g; KCl, 0·2 g; Na2HPO4, 1·43 g; and KH2PO4, 0·2 g dissolved and made up to 1 litre with distilled water; pH adjusted to 7·3 with HCl). Serial dilutions were prepared for each sample and plated in triplicate on to both MacConkey and Brilliant green agar and incubated overnight at 37 °C. Brilliant green agar was used to suppress growth of indigenous Escherichia coli. Counts were made of the number of viable, i.e. gentamicin-resistant, organisms recovered per sample. The numbers of viable gentamicin-resistant bacteria recovered were used as a measure of invasiveness.
Sample tissues became available for electron microscopic analyses only, from one pig in which exposure to challenge was 90 min. In this experiment, two additional strains were used: S. dublin 2229 and S. choleraesuis 14/74.
The effectiveness of gentamicin killing.
Loops containing either AE or FAE were challenged with approximately 109 adherent non-invasive E. coli B44; control loops were injected with MM. After 1 h incubation, 4·5 ml gentamicin (300 µg ml-1 in MM) was injected into loops; 4·5 ml MM was injected as control. By this means it was established that >99% of the organisms were killed.
Preparation of inocula for invasion assays.
Organisms were grown as described by Amin et al. (1994 ) but were held on ice overnight at 4 °C in fresh HDB medium to suppress histotoxic activity of S. dublin (Bolton et al., 1999
). Approximately 109 stored organisms were suspended in fresh HDB medium and added to designated 9 cm loops.
For the one 90 min experiment in pigs, organisms were grown and used fresh without overnight storage at 4 °C; in our recent work (Bolton et al., 1999 ), S. dublin 3246 organisms grown at 37 °C and used without storage at 4 °C were highly histotoxic to rabbit tissues.
Scanning electron microscopy.
Biopsies from control and test loops were taken for analysis after 3 h or after 90 min bacterial challenge. Tissues were fixed in 2·5% glutaraldehyde in phosphate buffer for 24 h. Tissues were then dehydrated through a graded acetone series: 50% (v/v H2O), 70%, 90%, 95%, 100%, 100% dried (two 15 min washes for each acetone concentration). The tissue then underwent critical-point drying with liquid CO2 in a Polaron E3000 series II critical-point drier. Pieces of tissue were then mounted on copper stubs with Araldite Rapid (Ciba-Geigy) and sputter coated with Pt in an EMSCOPE SC500 low-voltage sputter coater operated at 100150 V with 1020 mA current. Tissues were examined in a JEOL 120 CX II electron microscope with a TEMSCAN attachment. Images were obtained via a secondary electron detector at 40 kV.
Transmission electron microscopy (TEM).
Biopsies from control and test loops were taken for analysis after 3 h or after 90 min bacterial challenge. Tissues were fixed in 2·5% glutaraldehyde for 1 h and post-fixed in 1% OsO4 for 1 h; both the glutaraldehyde and OsO4 solutions were made up in phosphate buffer (pH 7·2; adjusted to 350 mM with sucrose). Tissues were then dehydrated serially in 70%, 90% and 100% ethanol and embedded in Epon-Araldite (Mollenhauer et al., 1964 ). Sections (approx. 2·5 µm thick) were stained with 1% toluidine blue and observed by light microscopy. Ultra-thin sections (approx. 90 nm thick) were cut, mounted on Formvar-coated copper grids and stained with methanolic uranyl acetate and Reynolds lead citrate and examined at an accelerating voltage of 60 kV in a JEOL 100 CX II electron microscope.
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RESULTS |
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In three pigs (1, 2, 3; Fig. 1a, b), there were clear differences in the recoveries of the non-invasive mutant S. typhimurium 4/74 invH and its invasive parent S. typhimurium 4/74 (used as negative and positive controls respectively), demonstrating that this bioassay in pigs could differentiate between strains which differed in their ability to invade either AE or FAE.
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The recoveries of S. dublin 3246 and S. choleraesuis A50 from porcine AE were comparable to each other; this was also true for FAE (Fig. 1a, b).
In all three pigs, wild-type S. typhimurium 4/74 was more invasive than either S. dublin 3246 or S. choleraesuis A50 (Fig. 1a, b).
Recoveries of S. dublin and S. choleraesuis from bovine ileal loop mucosae
A similar picture was obtained to that described above for pigs. In two calves (1, 2; Fig. 2a, b), there were clear differences in the recoveries of S. typhimurium 4/74 invH and S. typhimurium 4/74, demonstrating that this bioassay in calves could differentiate between strains which differed in their ability to invade either AE or FAE.
In all five calves (1, 2, 3, 4, 5; Fig. 2a, b), the recoveries of S. dublin 3246 from bovine AE and FAE were comparable to each other; this was also true for S. choleraesuis A50.
The recoveries of S. dublin 3246 and S. choleraesuis A50 from bovine AE were comparable to each other; this was also true for FAE (Fig. 2a, b).
The susceptibility of bovine ileal mucosae to invasion (Fig. 2a, b) seemed to be greater than that of corresponding porcine mucosae (Fig. 1a, b
).
Electron microscopic analyses of porcine ileal mucosae after challenge in vivo with S. choleraesuis and S. dublin
Unless otherwise stated, the following data were derived from one 3 h experiment in one animal but are typical of what was observed in comparable tissues from other animals. Porcine ileal AE appeared normal after exposure to HDB medium (Fig. 3a). S. choleraesuis A50 caused AE villus tips to become more wrinkled, showing classic signs of incipient accelerated cell shedding in response to bacterial invasion (Fig. 3b
). Exposure to S. dublin 3246 caused greater disruption of AE architecture than did exposure to S. choleraesuis A50: villi were much less well-defined, shorter and overlaid with debris (Fig. 3c
). The extent of the tissue damage caused by S. dublin 3246 was far greater than could be accounted for by the observed presence of organisms. S. choleraesuis A50 organisms were observed in otherwise intact-looking cells, nearly always singly or in small numbers, inside vacuoles of varying size. For example, Fig. 3(d)
(main panel) shows two organisms without a discernible surrounding membrane. The inset in Fig. 3(d)
(tissue taken after 90 min in another experiment) shows a typical newly formed membrane-bounded vacuole containing an internalized organism; Fig. 4(f)
shows an organism inside a much smaller vacuole.
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One of the objectives of this work was to determine the cellular route of entry of Salmonella into porcine gut epithelia. A special search was therefore made for evidence implicating M cells in invasion, particularly of FAE. Under the conditions of our experiments, it was exceedingly difficult to find structures which could be unequivocally identified as M cells in S. dublin-challenged epithelia: in both the 3 h experiments with stored organisms and the 90 min experiment with freshly grown organisms, major tissue destruction was induced by this serotype. What is abundantly clear, however, is that, while S. dublin organisms may enter porcine gut via M cells, they also enter directly into cells with brush borders. Such entry is neither tissue nor animal specific: S. dublin 3246 could be seen entering microvillus-bearing cells in FAE (Fig. 4a), and enterocytes in AE (see Fig. 3e
); these tissues were from two different pigs. Entry into enterocytes was not strain specific: from the 90 min experiment S. dublin 2229 could be seen entering directly into enterocytes (Fig. 4b, c
).
In contrast, it was possible to be more definite about S. choleraesuis FAE interactions since in both pigs and calves S. choleraesuis did not cause the same kind of structural damage as S. dublin. S. choleraesuis A50 could be seen entering M cells (Fig. 4d, e), and concurrently, directly into enterocytes (Fig. 4e, f
). The enterocyte containing the S. choleraesuis A50 organism in Fig. 4(f)
was four cells removed from the M cell in Fig. 4(d)
. From the 90 min experiment, a similar picture of concurrent entry of S. choleraesuis 14/74 into both M cells and enterocytes was obtained (not shown).
Electron microscopic analyses of bovine ileal mucosae after challenge in vivo with S. choleraesuis and S. dublin
A less detailed study of bovine tissues was carried out since the early stages of epithelial invasion had already been thoroughly addressed (Watson et al., 1995 ; Frost et al., 1997
). The picture obtained with bovine gut essentially paralleled that obtained with porcine gut. The following data were derived from one 3 h experiment in one animal but are typical of what was observed in comparable tissues from other animals.
After 3 h exposure to HDB medium, control AE villi were well preserved (Fig. 5a). Exposure to S. choleraesuis A50 induced wrinkling of villi, a classic sign of incipient accelerated cell shedding in response to bacterial invasion (Fig. 5b
). Organisms were observed inside otherwise intact goblet cells (Fig. 5g
) and enterocytes (Fig. 5h
). In contrast, villi of AE in loops challenged with S. dublin 3246 were shorter, and some were frankly damaged and overlaid with cellular debris (Fig. 5c
).
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DISCUSSION |
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Wild-type virulent S. typhimurium 4/74 was recovered (from both porcine and bovine hosts) in approximately 10-fold higher numbers than the attenuated S. typhimurium 4/74 strain, indicating that the system could discriminate between highly invasive and non-invasive strains. In the study reported here, S. dublin was recovered from both bovine and porcine ileal mucosae in numbers comparable to S. choleraesuis despite inducing more mucosal damage to bovine and porcine ileal mucosae than S. choleraesuis. Therefore, in these experiments, we found no evidence for the idea that the host-restriction exhibited by these serotypes for cattle and pigs is explicable solely in terms of major differences in their respective invasiveness for terminal ileum. However, a double rider must be added. First, it is obvious that the magnitude of mucosal damage must be taken into account when interpreting the biological significance of the data. Second, the dose response around an inoculum size of 109 for invasiveness in calves was flat, indicating probable saturation of Salmonella binding sites; we do not know the minimum inoculum sizes which would result in effective invasion of either bovine or porcine ileal epithelia. It would be extremely useful to be able to estimate the numbers of organisms surviving passage through the alimentary tract to distal ileum after oral administration of infectious doses of S. dublin and S. choleraesuis in pigs and calves. Such information could result in more meaningful comparative studies being carried out in terms of the size and phenotype of challenge inocula on the invasiveness of host-restricted Salmonella serotypes in pigs and calves. The results of such experiments would either confirm that differential invasiveness of the gut accounts for host restriction or require that explanations for host restriction in systemic Salmonella infections be sought at stages of the complex host/pathogen interaction other than invasiveness for gut epithelia. They would also provide data on the survivability of such strains through the alimentary tract, an unglamorous area that has been completely ignored in this context.
Previously we had shown a correlation between invasiveness for distal rabbit ileum and virulence in human strains of S. typhimurium (Amin et al., 1994 ; Bolton et al., 1999
), which, taken together with this work involving host-restricted strains, suggests the following important points. While all virulent strains of Salmonella are invasive, the converse is apparently not true: invasiveness per se is not a general predictor of Salmonella virulence since the S. dublin strains used in this work did not cause clinical disease in pigs when administered orally in doses which routinely infected calves (T. S. Wallis, unpublished observations). We have also shown that Salmonella-induced enteritis in rabbits is not due solely to the intestinal invasiveness of salmonellae. Invasive and enteropathogenic strains of S. typhimurium never induced fluid secretion in rabbit ileal loops in the absence of a PMN influx, although a PMN influx per se was not responsible for fluid secretion (Wallis et al., 1989
). More recently a pathogenicity island (SPI-5) has been identified in S. dublin 2229 and shown to be conserved in a range of different serotypes (Wood et al., 1998
). Genes encoded by SPI-5 are involved in S. dublin-induced enteropathogenesis in calves, but not in systemic pathogenesis in mice. Mutation of the SPI-5-associated gene sopB, which encodes a secreted effector protein SopB (an inositol phosphate phosphatase; Norris et al., 1998
), significantly reduced (but did not abolish) fluid secretion and PMN influxes, without affecting the invasive phenotype in vivo (Galyov et al., 1997
). The enteropathogenicities for calves of S. typhimurium and S. dublin strains of equal invasiveness in vivo have also been compared. Strains of S. typhimurium were more enteropathogenic than those of S. dublin (Watson et al., 1998
), suggesting that the virulence mechanisms of these serotypes may differ. The elucidation of these differences may hold the key to defining host restriction in terms of clinical disease causation rather than in susceptibility to initial infection. Clearly much remains to be unravelled.
There were clear differences in the reaction of both porcine and bovine tissues to the two Salmonella serotypes: S. dublin was clearly more histotoxic than S. choleraesuis to both porcine and bovine tissues. We believe the damage to be toxin-mediated, since damage was much more extensive than might have been expected if it were linked to the direct physical presence of bacteria: it was relatively easy to find examples of damage in areas where it was difficult to find attaching/invading bacteria. If, as would seem highly likely, the damage reported here is mediated by the toxin previously described (Lodge et al., 1999 ; Bolton et al., 1999
), it would follow that the toxin shows no specificity for the tissues of rabbit (Bolton et al., 1999
), calf and pig (this work). These experiments suggest that (in the context of gut invasion) there are at least two different biotypes of Salmonella, which cross serotypic boundaries one which causes rapid initial histotoxic damage to mucosal epithelia, the other which does not. Histotoxic strains will open up additional cellular routes for invasion of gut mucosae by virtue of early damage to gut enterocytes, and denudation of epithelia of enterocytes with subsequent exposure of subepithelial cells and tissues. In addition, the invasiveness of histotoxic strains like S. dublin 3246 will always be underestimated. However, in the context of this work, allowance for this possibility does not provide an explanation of host-restriction of S. dublin in terms of invasiveness: this serotype was both invasive and histotoxic in pigs, calves and rabbits.
The bulk of the work reported here was carried out over 3 h with organisms grown at 37 °C and stored on ice at 4 °C overnight. This protocol was designed to give a truer measure of the relative intrinsic invasiveness of the strains of S. dublin and S. choleraesuis by hopefully suppressing the expression of histotoxicity of the former, as was the case for rabbit ileal tissue in vitro (Bolton et al., 1999 ). Unfortunately it was not possible to carry out a time-based study to measure the rate of initial invasion. As alluded to above, our estimates of S. dublin invasiveness were clearly underestimates since by 3 h there was evidence of S. dublin-induced tissue damage in porcine AE and FAE and particularly in bovine FAE. The limited data from the 90 min pig experiment, generated with organisms which were grown and used fresh without overnight storage at 4 °C, demonstrates that the expression of overt histotoxicity by S. dublin occurs early when an appropriate phenotype is used to inoculate the loops. This observation lends additional weight to the view that definitive experiments on the biological significance of differential invasiveness of these Salmonella serotypes for gut will require the use of appropriate in vivo phenotypes. Tissue destruction by S. dublin is thus not only extensive but rapid, from which it follows that organisms present in gentamicin-treated tissues must be intrinsically highly invasive as well as toxigenic. In fact, S. dublin organisms were found in deeper tissues and in some cases even near to vascular elements.
Our approach enabled us to compare the relative invasiveness of these S. dublin and S. choleraesuis strains for bovine and porcine ileal mucosae with or without Peyers patch tissue in a competitive situation in the same loop. At three hours post-infection both serotypes were recovered in comparable numbers from AE and FAE, confirming and extending previous observations (Frost et al., 1997 ; Watson et al., 1995
) implicating both AE and FAE in the early stages of the infection process. Clearly, in natural Salmonella/host interactions salmonellae are able to penetrate the apical membranes of M cells in FAE and also enterocytes in both FAE and AE. This point needs to be emphasized since, as stated in the Introduction, there is now a growing acceptance, based largely on studies in murine ligated loops, that M cells are the main primary route of Salmonella entry into the gut in vivo. M cells then die, creating a gap in the epithelium through which bacteria can penetrate and then infect adjacent enterocytes via basolateral membranes, resulting in exfoliation of enterocytes, denudation of FAE epithelium and exposure of the basement membrane to luminal bacteria (Jones et al., 1994
). It is interesting to note that the damage seen in murine ligated loops 2 h after infection with very large doses of the mouse-virulent strain S. typhimurium SL 1344 (200 µl culture containing approx. 108 c.f.u. into a 45 cm murine ileal loop; Jones et al., 1994
) bears some similarity to that caused by histotoxic S. dublin in pigs, calves (this work) and rabbits (Bolton et al., 1999
; Lodge et al., 1999
). It is possible that the disruption of murine epithelia reported by Jones et al. (1994
) was toxin mediated as distinct from being a secondary undefined consequence of M cell destruction. The M cell scenario may hold for S. typhimurium in mice but not necessarily in calves, where both M cells and enterocytes were infected very early in infection (Frost et al., 1997
; Watson et al., 1995
). Infection of M cells in bovine FAE preceded the initiation of independent infection of enterocytes in AE by only 10 min. It is also manifestly not true that M cells are the major route of invasion in rabbits, in which we have used large numbers of ligated ileal loops (Wallis et al., 1986
) each one deliberately constructed in segments of gut free of Peyers patches, something which is very easy to do by virtue of the physical distribution of Peyers patches in rabbit small intestine. It was logistically not possible for us to conduct a series of time-course observations with pigs similar to what had already been done with calves. Hence we cannot make definitive statements about the detailed kinetics of infection of different cell types. However, the point we make from this work is that one can demonstrate concurrent internalization of non-histotoxic S. choleraesuis by enterocytes physically removed from active foci of M cell penetration in porcine ileal epithelia. The biological significance of interactions between S. typhimurium and M cells may well depend on the precise Salmonella serotype (or strain)/host combination.
As with S. typhimurium TML (Worton et al., 1989 ), S. dublin was nearly always seen entering the gut in micro-colonies; in sharp contrast, S. choleraesuis was observed nearly always entering as single organisms or in groups of two or three. The entry by S. typhimurium and S. dublin as microcolonies is not due to clumping, since it is not difficult to prepare single cell suspensions for use in challenging gut either in vivo or in vitro. It is a highly reproducible but an as yet unexplained phenomenon which might be significant in initiating infection and an example of bacterial co-operative activity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bolton, A. J., Martin, G. D., Osborne, M. P., Wallis, T. S. & Stephen, J. (1999). Invasiveness of Salmonella typhimurium, Salmonella choleraesuis and Salmonella dublin for rabbit terminal ileum in vitro. J Med Microbiol 48, 800-810.
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Watson, P. R., Paulin, S. M., Bland, A. P., Jones, P. W. & Wallis, T. S. (1995). Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene. Infect Immun 63, 2743-2754.[Abstract]
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Received 13 January 1999;
revised 7 May 1999;
accepted 9 June 1999.