Immunity to Enteric Infection in Mice

Transcription

1 INFECrlON AND IMMuNrrY, Mar. 1970, p Vol. 1, No American Society for Microbiology Printed in U.S.A. Immunity to Enteric Infection in Mice FRANK M. COLLINS Trudeau Institute, Inc., Saranac Lake, New York 1983 Received for publication 13 October 1969 Specific pathogen-free CD-1 mice infected orally with sublethal doses (104 to 106 viable organisms) of Salmonella enteritidis rapidly developed extensive bacterial populations in the liver, spleen, and mesenteric lymph nodes. Although the pathogen did not multiply extensively in the gut, the infection persisted in the intestine at between 104 and 105 viable organisms throughout the experiment. S. gailinarum was less invasive than S. enteritidis when given by mouth; S. pullorwn failed to survive in the intestine or to invade the tissues of orally infected mice. Vaccination with a sublethal dose of living S. enteritidis, either orally or intravenously, completely prevented the establishment of liver and spleen populations of a drugresistant, virulent strain of S. enteritidis. Vaccination with an ethyl alcohol-killed vaccine given by various routes delayed the spread of the orally introduced challenge population to the liver and spleen by 1 to days but was unable to prevent the subsequent growth of the pathogen in vivo, although the vaccinated mice survived the infection. The importance of these findings in relation to vaccination against typhoid fever in man is discussed. Studies of anti-salmonella immunity, both in this laboratory (4, 7, 10) and elsewhere (16,, 3, 9), have established that, once phagocytosis is complete, the expression of acquired resistance to S. enteritidis depends upon cellular rather than humoral factors (6). This conclusion is consistent with the observation that both delayed hypersensitivity to one or more Salmonella antigens and the presence of an effective antibacterial immunity develop in mice vaccinated with living, but not with dead, organisms (8). These studies are subject to the criticism, however, that the challenge infection was always presented parenterally, so that the resulting host-parasite interactions may not be relevant to the natural infection (15). Studies with the oral route of challenge of a number of animal species support the contention that both living and killed vaccines have protective value in terms of host survival against Salmonella infections (5, 8). In the mouse, the degree of protection afforded by a given vaccinating regimen is difficult to assess largely because of the size of the oral challenge necessary to bring about the death of the normal controls (3). Earlier studies with Salmonella-infected mice (6, 10) indicated that assessment of anti-salmonella immunity could be made by comparing the fate of the infecting population of salmonellae in the livers and spleens of vaccinated and normal mice. The results of such studies gave a better picture of the host-parasite relationships involved 43 in the immune reaction than when it was merely observed whether a higher proportion of the immunized mice somehow survived a lethal challenge, as compared with the untreated controls (, 17). The use of daily serial enumeration of the intestinal, liver, spleen, and mesenteric node populations in orally infected mice preimmunized with living or ethyl alcohol-killed vaccines gave results which were consistent with those obtained earlier for subcutaneously and intravenously infected animals (6). MATERIALS AND METHODS Organisms. S. enteritidis 5694 and S. enteritidis 5694 SMR (resistant to 10,ug of streptomycin per ml) were described earlier (4). Animals. Specific pathogen-free CD-1 mice (Charles River Farms, Inc.) were maintained as described previously (9). Five-week-old females (18 to 4 g) were used throughout. The mice were maintained under isocaps (Carworth-Lab Cages, New City, Rockland County, N.Y.). Vaccines. Sixty mice were vaccinated orally with 106 viable S. enteritidis 5694 delivered in 0. ml of saline by a gauge 19 gavage needle (group 1). Eighty mice were vaccinated intravenously with 0.5 LD5o (approximately 10' viable organisms) of S. enteritidis (group ). The surviving animals were challenged by mouth 14 days later with 106 viable streptomycinresistant S. enteritidis delivered in 0. ml of saline by gavage tube. An ethyl alcohol-inactivated S. enteritidis vaccine was prepared and administered as described previously (7, 8). Sterility tests were carried out on the

2 44 COLLINS killed vaccine and on homogenates of the livers and spleens of the vaccinated mice before challenge. Forty mice were vaccinated with 3 weekly doses of 106 ethyl alcohol-killed S. enteritidis for 3 weeks (group 3). Injections were made by the intravenous, intraperitoneal, and subcutaneous routes to involve as much of the reticuloendothelial system as possible in the immune response (6). A second group of 40 mice were injected with 107 ethyl alcohol-killed organisms given as two intramuscular injections 14 days apart (group 4). All four groups of vaccinated mice (together with a group of normal controls of the same age) were infected by mouth with approximately 106 virulent S. enteritidis SMR, and the fate of the challenge population was followed for 8 to 10 days. The viability of the living vaccines and the challenge populations was checked by plating suitable 10-fold saline dilutions on nutrient agar plates. Enumeration of the in vivo population. The number of bacteria in the blood, liver, spleen, and mesenteric lymph node was estimated daily on Tryptone Soy Agar plates (Difco) by using five randomly selected mice as described previously (5, 7). Gut counts were made by dissecting out the large and small intestines and the cecum (leaving the stomach in situ) and freeing them carefully from the mesentery and the mesenteric node. The gut was macerated for min in 10 ml of saline by using a high-speed blendor (model 45; The VIRTIS Co., Inc., Gardiner, N.Y.). The homogenate was diluted suitably in saline and plated on MacConkey and DCA agar plates immediately. When in doubt, the lactose-negative colonies were checked with group D antiserum by slide agglutination (4). The relative counting error for the gut counts is shown in Table 1. Drug treatment. Normal 4-week-old, specific pathogen-free mice were given sterile water containing 50 pug of streptomycin, 00 plg of neomycin, and 00 pug of succinyl sulfathiazole per ml to drink ad lib. for 7 days. They were then returned to sterile drinking water and 1 to 5 days later were challenged by mouth with increasing doses of S. enteritidis. RESULTS Growth of an oral challenge of S. enteritidis in drug-treated and normal mice. Normal specific pathogen-free mice were infected by mouth with increasing doses of S. enteritidis, and the fate of the organism in the gut and the rate of spread of the pathogen to the mesenteric node, the liver, and the spleen were followed over a period of 10 days (Fig. 1). Increasing the challenge inoculum from X 104 to 4 X 107 viable organisms had relatively little influence on the overall outcome of the infection. It did, however, markedly influence the rate of spread of the infection in vivo, with the result that organisms could be detected in the liver within 3 hr and in the spleen after 4 hr when the mice received the largest dose of organisms, but could not be found in these tissues until the 4th day in animals receiving the smallest dose (Fig. 1). Once the infection reached 4 u co Lu -j Cie co z 0 0 INFEC. IMMUN TIME IN DAYS FIG. 1. Growth curves obtained in normal CD-I mice infected orally with increasing doses ofsalmonella enteritidis. The size of the inoculum is indicated by the arrows. Abbreviations: Gut, viable bacteria in the gut wall and contents; Sp, spleen; Lr, liver; Mes, mesenteric lymph node; Bl, blood. the liver and spleen, however, the organisms grew in these organs at the same rate regardless of the size of the infecting dose. In all cases, the bacterial population reached a maximum in the spleen and liver at about the 8th day, after which the bacterial population tended to decline. The behavior of the bacterial population within the gut itself was of particular interest. Regardless of the size of the inital infecting population, the number of organisms in the intestine stabilized at levels ranging from 104 to 105 viable bacteria (Fig. 1). Independent determination of the bacterial populations present in the gut wall and in the gut contents showed that most of the organisms were associated with the wall (Table 1). It is uncertain, however, whether

3 VOL. 1, 1970 ENTERIC INFECTION IN MICE 45 TABLE 1. Organ Growth of Salmonella enteritidis in specific pathogen-free CD-I mice after oral infection with 4 X 107 living organisms Time (hr) Blood Oa St 8.0 XlOsb X 10 Spleen X X d0.8 X X 105 Liver X X X X d 6.0 X 104 Mesenteric X X X 104 node Duodenum Lumen X t1.3 X 103 c X X 103 Contents 0 X X X 10 Small intestine Lumen.0 d1.8 X X X X 103 Contents 0 X X X 10 Cecum X i 3.7 X X I 1.1 X 103 Large intestine Lumen X X i.4 X X 10 Contents Total gut X i1.0 X X X X 103 a Less than 50 viable bacteria. b Average for five determinations c Not determined. standard deviation. the organisms were within the gut wall itself or merely located on its surface. A further point of interest concerns the increase in bacterial numbers within the gut at about the 8th day of the infection, at a time corresponding to the maximum bacterial populations in the liver and spleen. It seemed possible that this increase in the gut population could be related in some way to the large numbers of bacteria found in the liver and spleen. To obtain more information on this point, animals were infected intravenously with a standard inoculum of 104 viable organisms ( to 5 LD5o). The populations of bacteria found in the blood, the gut, the liver, and the spleen were determined, along with the bacterial contents of the inguinal and mesenteric lymph nodes. The challenge infection was substantially cleared from the circulation by 4 hr but then reappeared again in large numbers from day onwards (Fig. ). Serial enumeration studies revealed that the gut was initially free from salmonellae, but that from day onwards the intestinal population increased in parallel with that of the liver and the spleen, the blood, and the inguinal and mesenteric lymph nodes. It would seem therefore that the increasing numbers of bacteria present in the mesenterc and inguinal lymph nodes and in the gut are due to the reseeding of these organs by blood-borne bacteria. In the light of these findings, it seems likely that most of the organisms found in the gut at the height of an orally induced infection are derived from the systemic population and are not the descendants of the original intestinal infection. Despite the ability of a small oral challenge by S. enteritidis to bring about a widespread systemic infection, the slow rate of evolution of the disease allowed sufficient time for the development of a high level of acquired resistance to the challenge infection by the host. As a result, the host has a better chance of controlling the infection before it can reach lethal proportions, so that the LD50 by the oral route is extremely high (Table ). Claims have been made for increased susceptibility of mice exposed to oral infection after treatment with streptomycin (1) and other drugs (6). An attempt was made to use this finding in the present studies, together with an examination of the effects of starvation (1) and the stabilization of the ph of the challenge suspension by use of a strong buffering agent on the infectivity of the oral challenge. The LD5o determinations recorded in Table reveal that it was not possible to increase greatly the susceptibility of CD-1 mice to oral S. enteritidis challenge by these means. On the other hand, the growth curves shown in Fig. 3 make it clear that the oral challenge, presented 4 hr after cessation of the drug treatment, rapidly spread to the liver and spleen. The increasing bacteremia detected from

4 46 COLLINS the 4th day was probably responsible for the subsequent increase in the gut population. As observed earlier (1, 6), the drug-induced changes in the intestinal population were only transitory, since oral challenge 5 days after cessation of drug treatment showed no such effects (Fig. 3). Starving the mice overnight actually increased their resistance to oral infection. Suspension of the organisms in bicarbonate buffer (ph 8.5) provided no advantage over saline, and the drugtreated mice appeared to be at least as resistant to infection by the oral route as they were to 8 Intravenous challenge +Gut < Go Lr Normal mice S Sp < 6- >4" U. z 0 0 z 41 A,' A Mes v Ing 4B1 TIME IN DAYS. FIG.. Growth ofsalmonella enteritidis in the liver (Lr), spleen (Sp), mesenteric lymph node (Mes), inguinal lymph node (Ing), blood (BO), and intestine (Gut) after intravenous infection. The inoculwn size is represented by the arrowhead. INFEC. IMMUN. subcutaneous infection (Table ). The change in the level of susceptibility to oral challenge after drug treatment was not sufficient to warrant its further application to the present study. 8 - Mice given drugs by mouth for 7 days. _ Challenged by mouth 5 days later. 6 ux, m 0 08 co Challenged day later wo{ 4- I I f,~~~~~i Lr SP *MN TIME IN DAYS FiG. 3. Growth of an oral challenge dose of Salmonella enteritidis I day (bottom) and 5 days (top) after cessation of an oral drug treatment regimen in CD-I mice. TABLE. LD6 for Salmonella enteritidis in specific pathogen-free CD-I mice challenged by various routes Mice Pretreatment Challenge route" Diluent LD60 Normal Fed normally IV Saline 3. X 10' Normal Fed normally IV Bicarbonate 6.3 X 108 Drug-treated Fed normally IV Saline 3. X 103 Drug-treated Fed normally IV Bicarbonate 5.0 X 103 Normal Fed normally IP Saline 10 Normal Fed normally SC Saline.5 X 105 Normal Fed normally BM Saline 1.8 X 106 Normal Fed normally BM Bicarbonate 8.0 X 106 Normal Starved overnight BM Saline 1.6 X 107 Normal Starved overnight BM Bicarbonate 3. X 107 Drug-treated Fed normally BM Saline 3.0 X 10' Drug-treated Fed normally BM Bicarbonate 4.0 X 106 Abbreviations: IV, intravenous; IP, intraperitoneal; SC, subcutaneous; BM, by mouth.

5 VOL. 1, 1970 ENTERIC INFECTION IN MICE 47 Fate of S. gailinarum and S. pullorum in orally infected mice. When normal mice were infected by mouth with 3 X 107 S. gallinarum, the organisms survived in the gut for at least 10 days (Fig. 4). A small number of organisms reached the liver and spleen by day 8 but then were rapidly eliminated. S. pullorum failed to persist in vivo, even when mice were orally infected with nearly 108 viable organisms (Fig. 4). The behavior of both strains was consistent with that reported earlier for other routes of infection (7, 8). Fate of orally introduced S. enteritidis in vaccinated mice. Having established the normal growth patterns of S. enteritidis in orally infected mice, the effect of living (groups 1 and ) and dead vaccines (groups 3 and 4) on the course of an orally introduced challenge was examined. The growth curves shown in Fig. 5 indicate that living vaccines completely prevented the invasion of the tissues from the gut by a virulent, streptomycin-resistant mutant strain of S. enteritidis. The challenge organism could not be detected at any time in the liver or spleen despite its contin- co _ Oral challenge S. pullorum in normal mice u. Oral challenge S. gallinarum in normal mice Z 6 O 5 O TIME IN DAYS FiG. 4. Growth of Salmonella pullorum (top) and S. gallinarum (bottom) in normal CD-I mice after oral challenge. No organisms resembling S. pullorum could be detected in the liver, spleen, blood, or mesenteric lymph node at any time during the experiment. u a] co z Living oral vaccine S enteritidis Oral challenge S entertidis SMR Living intrcavenous vcaccine _ Oral challenge S.enteritidis SMR Gut [ ''n+ G ut 0 I!L-4---,----U' Sp *-, & Mes TIME IN DAYS FIG. 5. Effect of vaccination with living Salmonella enteritidis by the oral (group 1) route (top) or the intravenous (Group ) route (bottom) on the growth of an oral challenge population of a virulent streptomycinresistant strain of Salmonella enteritidis. The broken lines in the lower section represent the growth of a similar challenge in normal mice. Symbols: *, liver; *, spleen; A, mesenteric lymph node; +, gut. ued presence in the gut for at least 8 days. However, the intestinal population in these mice declined steadily throughout the experiment, presumably because of the absence of reseeding from the liver and spleen (Fig. 5). Mice which received the killed vaccine were able to delay, but not to prevent, the spread of the pathogen from the gut to the liver and spleen (Fig. 6); however, once within the tissues, there was no discernible effect of the killed vaccine on the growth of the organism. DISCUSSION One of the major limitations of the use of the mouse as an experimental model for the study of immunity to typhoid fever has been the difficulty of obtaining a realistic infection by mouth (3, 15). It has been known for many years that the introduction of enormous numbers of virulent salmonellae by the oral route will establish a progressive lethal murine infection (1, 3, 1), but the very size of the infective dose required ex-

6 48 COLLINS z O x doses M Normal %%~~~~~~~~~~~~~~~% A 0/ TIME IN DAYS~ FIG. 6. Effect of preimmunization with (group 3; top) or two intramuscular doses middle) ofan ethyl akcohol-killed vaccine on of an oral challenge of Salmonella enter, INFEC. IMMUN. mensal flora of the gut (7). However, both drug-.l r treated and germ-free mice constitute highly GGut artificial experimental models, and any protec- I 'es tion data obtained with them may hold little relevance for the natural infection. The explosive growth of the pathogen in the intestinal contents of such mice is somewhat reminiscent of that QBl observed in the peritoneal cavity of mice challenged intraperitoneally (5, 9). As has been explained elsewhere (, 6), the rapid and exten-, sive extracellular growth of unopsonized S. enteritidis within the peritoneal cavity makes this \ model of limited value in studying the mechanism of anti-salmonella immunity. In the germ-free or drug-treated animal, it is presumably the massive overgrowth by the pathogen within the intestine that renders the host so susceptible to oral challenge. Since drugs had little effect on the susceptibility of the mice used in the present experiments, it must be assumed that the doses of streptomycin, neomycin, and succinyl sulfathiazole were inadequate or that specific-pathogen-free \> CD-1 mice are already maximally susceptible to oral infection because their gut flora is quite different from that of conventional mice (Collins, unpublished data). At present, most workers believe that pathogenic salmonellae invade the tissues of the susceptible host by crossing the lumen of the small intestine to enter the mesenteric lymphatics and so gain entry to the blood by way of the intervening lymph nodes (3, 15, 4). However, there 8 10 is also evidence that the organisms may enter the host through the tonsils (1, 14). Gerichter (15) nine doses reported that 64% of massively infected mice (group 4; (5 X 109 S. typhi) were bacteremic within minthe growth utes of delivering the oral challenge. These *itidis. The organisms presumably gained direct entry into growth of the challenge dose in unvaccinatred mice is the blood through mucosal capillaries (15). It is shown in the bottom section. not certain whether this route of entry was important in the present study, because very criticism small numbers of blood-borne organisms are poses such protection experiments to the that the experimental model no longe,r bears a difficult to detect. However, the growth curves meaningful relationship to the natural infection shown in Fig. 1 indicate that infection occurred in man (14). simultaneously in the liver and spleen. More- by the over, significant numbers of organisms were This objection was largely overcomie often experiments of Savage and Dubos (6) and detected in the liver before any organisms could Miller and Bohnhoff (1), who showedithat oral be cultured from the mesenteric lymph node. treatment with antibacterial agents ailters the This is an entirely different picture from that normal gut flora transiently, rendering t'he mouse observed in mice infected subcutaneously, in more susceptible to experimental infecition (13), which the draining lymph node was invariably The same treatment increases suscepttibility to heavily infected several days before organisms oral infection with salmonellae, pr'esumably appeared in the liver and spleen (5, 6). Clearly, because the normal intestinal flora tends to an- more quantitative information is still required, tagonize the growth and survival of S. enteritidis but, if it is assumed that a proportion of the chalmice are lenge dose does gain entry to the liver and spleen in the gut (3, 15). Similarly, germ-free exquisitely sensitive to oral infection, even by via the blood without primarily involving the organisms which are normally part of the com- mesenteric lymph node or the tonsils, then mice

7 VOL. 1, 1970 ENTERIC INFECTION IN MICE 49 infected intravenously are a suitable model for studying the mechanism of acquired resistance to the natural Salmonella infection (6). Serial enumeration of spleen and liver populations made it possible to study the behavior in vivo of small oral doses of S. enteritidis and thus to evaluate different vaccinating regimens without having to resort to the use of a massive oral challenge to kill a significant proportion of the control mice. The results obtained in this study are strikingly similar to those reported previously, when the effects of living and killed vacines were compared in animals challenged subcutaneously (6). The living vaccine was found to prevent the progression of the pathogen beyond the regional lymph node with the result that systemic infection could not develop. The killed vaccine merely delayed the spread of the subcutaneous infection and had no effect on the subsequent growth of the organism in the liver and spleen. The present study indicates that the oral route of challenge is no exception to this general pattern. Mice hyperimmunized with a killed vaccine only acquire the ability to terminate the infection when the host responds to the challenge infection itself. Thus, these results reinforce the contention that, irrespective of the route of infection, there is a real difference in the quality of the host's response to living and to dead bacterial vaccines (8). This view of acquired resistance to Salmonella infections raises questions regarding the proper approach to immunization against typhoid fever (14, 15). If the findings of the present study are applicable to human typhoid, it follows that vaccination with dead organisms, (11, 0) or extracts thereof (16) is unlikely to produce a level of protection sufficient to prevent systemic invasion after alimentary infection (15). Considerable controversy has developed over the relative importance of the Vi antigen content of the vaccine strain and the influence of the inactivating procedure on the protective value of the vaccine for man (14, 16, 4). From the present studies, one would expect that the vaccine best able to stimulate the production of those humoral antibodies which can delay the spread of the pathogen from the gut to the tissues would have higher "protective" value by bringing about an increased incidence of attenuated or subclinical infections. This prediction is consistent with the statistical evidence of recent field trials which showed a substantial reduction in the incidence of clinical infections in vaccinated subjects, without affording complete protection against the disease (11, 16, 0). It would seem that dead vaccines can never provide total protection against clinical infection with S. typhi, since they are unable to induce the cellular immunity which can eliminate the infectious agent at, or near, its portal of entry. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. I thank Oliver Duprey and William Woodruff for excellent technical assistance throughout this study. LITERATURE CITED 1. Abrams, G. D., and J. E. Bishop Effect of the normal microbial flora on the resistance of the small intestine to infection. J. Bacteriol. 9: Blanden, R. V., G. B. Mackaness, and F. M. Collins Mechanisms of acquired resistance in mouse typhoid. J. Exp. Med. 14: Bohnhoff, M., C. P. Miller, and W. R. Martin Resistance of the mouse's intestinal tract to experimental Salmonella infection. 1. Factors which interfere with the initiation of infection by oral inoculation. J. Exp. 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