INTRODUCTION

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Salmonella choleraesuis infections in swine cause a septicemic disease resulting in pneumonia and other systemic involvement, with some involvement of the intestinal tract (32, 47). In most outbreaks, mortality can be high, although morbidity is variable but usually less than 10% (47). The duration and severity of the disease in individual pigs are unpredictable, and recovered pigs have been found to be carriers and fecal shedders (47). The resulting S. choleraesuis reservoir in swine is of obvious concern due to its disease-causing potential for young pigs as well as its public health implications for humans (2).

Vaccination against S. choleraesuis is an appropriate strategy for control and prevention of this disease (47). This is particularly true because detection of carriers is difficult because of intermittent shedding of the organism (25) and because antimicrobial feed additives, which have helped to keep the disease in check, are being used with less frequency (47). The use of live-attenuated salmonellae as vaccines has been given a great deal of attention in recent years because avirulent strains of Salmonella are more effective than killed or subunit vaccines in inducing a protective immune response and attenuated strains colonize host tissues, stimulating secretory, humoral, and cellular immune responses (30).

Several attenuation strategies have been utilized to render Salmonella spp. avirulent (3, 4, 7, 10, 12). These include the use of temperature-sensitive mutants (e.g., see reference 10), auxotrophic mutants (e.g., aroA, asd, cys, or thy mutants [13, 19, 38, 43, 44]), mutants defective in purine or diaminopimelic acid biosynthesis (e.g., pur and dap mutants [5, 31, 35]), strains altered in the utilization or synthesis of carbohydrates (e.g., galE mutants [14, 20]), and mutants altered in global gene expression (e.g., cya crp or phoP mutants [7, 10, 12]). As might be expected, attempts to attenuate salmonellae by these methods have led to varying degrees of success and demonstrated differences in virulence and immunogenicity (4, 5, 7, 10, 12). For instance, aroA mutants and galE mutants of Salmonella typhimurium lacking UDP-galactose epimerase activity were avirulent and immunogenic in mice (14, 18-20). In contrast, asd, thy, and pur mutants of S. typhimurium were avirulent in mice but also were not immunogenic when mice were challenged with the virulent parent strain (10, 34). When these same mutations were tested in S. choleraesuis, all mutants were reduced in virulence, but only aroA mutants were sufficiently avirulent, and none were effective as live vaccines (33, 34).

Subsequently, Kelly et al. (23) constructed and characterized S. choleraesuis mutants defective in the cyclic AMP (cAMP)-cAMP receptor protein (CRP) global regulatory system. Preliminary studies have shown S. choleraesuis strains with cya and crp mutations to be avirulent and immunogenic in BALB/c mice (23) and pigs (45). In the present report, we extend those observations by assessing the virulence and ability of a series of cya crp derivatives, with or without additional mutations and/or the 50-kb virulence plasmid, to induce a protective immune response in pigs. In addition, these strains were compared to a commercially available vaccine attenuated by passage five times through porcine neutrophils and found to have lost its 50-kb virulence plasmid (40).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and vaccines. The S. choleraesuis strains are listed in Table 1. The highly virulent strain 3246, a swine-derived field isolate (23), was chosen as the parent strain for all subsequent genetically modified strains. They were suspended in Luria-Bertani (LB) broth (10 g of Bacto tryptone, 5 g of Bacto yeast extract, and 10 g of NaCl per liter of deionized water) containing 20% glycerol and stored frozen at 70°C. These strains were characterized for (i) type 1 pili in static broth cultures (24, 36) and motility in motility medium composed of 1.0% casein enzyme hydrolysate (Sigma, St. Louis, Mo.), 0.5% NaCl, 0.5% agar (Difco Laboratories, Detroit, Mich.), and 50 µg of triphenyltetrazolium choride per ml; (ii) the appearance of lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis when visualized by silver staining (17, 46); (iii) fermentation patterns on various carbohydrates and production of H₂S by using the API 20E system; (iv) growth rates both in minimal liquid medium (9) supplemented with DL-methionine (20 µg/ml) when required and 0.5% (wt/vol) of the desired carbohydrate and in Luria broth (27) by methods described previously (11); and (v) group C₁ O antigen and H antigen (poly a-z) as confirmed by slide agglutination with antisera (Difco Laboratories). These strains were all found to be Salmonella serotype C₁, and because the deletion in cya and crp alters their biochemical characteristics, they gave API 20E code number 4104100 (good identification as Yersinia ruckeri [1 of 5] and Hafnia alvei [1 of 213]). The reference vaccine was a commercially available vaccine (NOBL SC-54, U.S. veterinary license no. 319, serial no. 104) and was obtained from NOBL Laboratories, Inc. (Sioux Center, Iowa), through a distributor (Vetpo Distributors, Inc., Holland, Mich.). This vaccine was stored at 4°C, as per the manufacturer's recommendations. The virulent challenge strain, S. choleraesuis var. kunzendorf P92-091, was originally obtained from Lorraine J. Hoffman, Veterinary Diagnostic Laboratory, Ames, Iowa. This strain was stored at 4°C in lyophilized vials and was identified as S. choleraesuis by serotyping and API 20E.

Genetic manipulations. Transductions were performed with the bacteriophage P1L4 (8) as previously described (23). Fusaric acid selection for deletion derivatives of strains harboring Tn10 insertions was done as described by Maloy and Nunn (29).

Animals, husbandry, and housing. Male and female (n = 72) purebred and crossbred pigs, 51.5 ± 1.5 days of age and weighing 12.7 ± 2.1 kg at vaccination, were used in the present study. These animals were obtained from the Pharmacia & Upjohn swine herd and were in overall good health, free of clinical signs of enteric diseases and negative for Salmonella species by microbiological culture and serology. Groups of eight pigs were housed in separate rooms (four pens/room, two pigs/pen) in a biosafety level 2 facility. Animals were acclimated to the diet and facilities for 7 days prior to initiation of the study. Pigs were fed a nonmedicated diet (S-850), and feed and water were provided ad libitum except where noted.

Bacterial inocula. Each of the cya crp S. choleraesuis vaccine derivatives was revived from frozen stocks by inoculating 0.1 ml of thawed culture into 10 ml of LB broth in a 16- by 125-mm polystyrene tissue culture tube (FALCON). After 14 h of static incubation at 37°C, each 10-ml culture was used to inoculate separate flasks containing 90 ml of fresh LB broth in 250-ml sterile polycarbonate Erlenmeyer flasks (Corning; Corning Glass Works, Corning, N.Y.). After a 5- to 6-h static incubation at 37°C, the resulting undiluted broth culture contained approximately 2 × 10⁸ to 5 × 10⁸ CFU/ml. An inoculum volume of 10 ml containing approximately 2 × 10⁹ to 5 × 10⁹ CFU was administered to each pig. The exact inoculum sizes are given in Table 1. Nonvaccinated control animals received 10 ml of sterile water as the vaccine inoculum. The SC-54 group was vaccinated intranasally with 2.0 ml (1.0 ml per nostril), as per the manufacturer's instructions. This inoculum was expected to contain approximately 2 × 10⁹ CFU/pig.

Animal vaccination and infections. Pigs were subjected to fasting for 24 h prior to inoculation and for a minimum of 30 min postinoculation with either cya crp derivatives, the vaccine strain, or the challenge organism. All eight pigs within a room received the same inoculum size (in bacterial numbers as well as volume) and the identical strain. For delivery of bicarbonate, cya crp derivatives, vaccine strains, or sterile broth (to nonvaccinated, control pigs), animals were lightly anesthetized with ketamine HCl (20 mg/kg of body weight) and restrained by hand, and the mouth was opened with a speculum to allow gastric intubation. Five to ten minutes prior to inoculation with the cya crp S. choleraesuis vaccine strains, stomach acidity was neutralized with 25 ml of a 10% (wt/vol) sodium bicarbonate solution by gastric intubation with a 16.5-in. SILASTIC tube (1/4-in. inside diameter and 3/8-in. outside diameter). The bacterial inoculum was then delivered by gastric intubation via the same-size tubing. The SC-54 vaccine was administered to animals intranasally as per the manufacturer's recommendations.

Experimental design and protective immunity. Animals were ranked from heaviest to lightest and divided into four groups of similar weight. Pigs were paired with another pig within their weight group, and one pair from each weight group was randomly assigned to each of the nine rooms. Within each room (treatment group), animal pairs were randomly assigned to one of the four pens. There were eight animals (four pens of two animals each) per treatment group.

Monitoring and sample collection. Rectal swabs were taken daily for the first week in both the postvaccination and the postchallenge periods. Thereafter, they were taken on Monday, Wednesday, and Friday for the remainder of the study and on day 52/53 or at necropsy for each pig for determination of the presence of S. choleraesuis. Two samples (1 to 2 g) of feed were collected from two different bags of feed from each production lot used in the study. These samples were cultured for Salmonella spp. as described below. Blood samples were collected from each pig on days 1 and 25 and at necropsy to obtain serum for antibody determinations. Blood (heparinized) for the cell-mediated immune (CMI) response assays was taken from three randomly selected pigs from each group (same pigs at each time period) on days 1 and 25 and at necropsy.

Isolation of Salmonella species. Fecal samples (2 g), rectal swabs, and feed samples (2 g) were enriched in 10 ml of selenite cystine broth. Tissue samples were thawed at room temperature, a 2-g sample was minced with scissors, and the sample was homogenized for 2 min in 10 ml of selenite cystine broth with a Stomacher (Tekmar Co., Cincinnati, Ohio). The enrichments were incubated overnight (16 to 18 h) at 37°C. At the end of the incubation period, 0.1 ml of the enrichments was plated onto brilliant green agar or MacConkey agar supplemented with 100 µg of tetracycline and 100 µg of ampicillin/ml of medium (after challenge with strain P92-091 only). The agar plates were incubated at 37°C for 18 to 24 h and scored for Salmonella colonies.

cya crp

Serology and ELISA. Blood samples (~10 ml) were taken from all pigs for serology on the day of, but prior to, vaccination and challenge and on the day of necropsy. An enzyme-linked immunosorbent assay (ELISA) was used to measure antibody titers. The antigen used to coat the plates for the ELISA was S. choleraesuis UC6077, which had been killed by heating in a boiling water bath for 10 min. The nonviable bacteria were washed in saline and suspended to approximately 1.4 × 10¹¹ organisms per ml (as determined by viable count prior to boiling). A 1:40 dilution of this suspension was prepared in bicarbonate-carbonate buffer (pH 9.6), and 0.1 ml of this antigen was added to each well of a 96-well polystyrene ELISA microtiter plate (Corning Glass Works). The plates were incubated for 18 h at 4°C, and the wells were washed three times with 0.002 M imidazole-buffered saline containing 0.02% Tween 20 (wash buffer; Kirkegaard & Perry Laboratories, Gaithersburg, Md.). The unreacted sites in the wells were blocked for 1 h at 37°C with 20% goat serum (Sigma Chemical Co.) in wash buffer. The plates were then washed three times. Serum samples were diluted 1:60 in wash buffer containing 10% goat serum, and 0.1 ml of this solution was dispensed to duplicate wells. One-tenth milliliter of affinity-purified, goat anti-swine immunoglobulin G conjugated to horseradish peroxidase (Kirkegaard & Perry; diluted 1:1,000 in 10% goat serum) was added to each well. The plate was incubated at room temperature for 45 min. The wells were washed three times, and 0.1 ml of 3,3',5,5'-tetramethylbenzidine and peroxidase solution (TMB; Kirkegaard & Perry) was added to each well for 10 min. The reaction was stopped by the addition of 0.05 ml of a 1:400 dilution of hydrofluoride. The plate was read by use of an automated ELISA reader (3550 microplate reader; Bio-Rad Laboratories, Richmond, Calif.) set at 655 nm.

Lymphoproliferation (CMI) assay. Five to 10 ml (day 25) or 25 ml (day 52/53) of heparinized blood was collected by venipuncture and was mixed with an equal volume of phosphate-buffered saline (PBS; pH 6.8). The suspensions were centrifuged at 1,000 × g for 20 min at 10°C. The buffy coat was removed, suspended in 2.0 ml of PBS, and then placed in 12.0 ml of sterile H₂O for 40 s to lyse the erythrocytes. Immediately thereafter, 6.0 ml of 2.7% NaCl-phosphate buffer was added to restore isotonicity. This suspension was then centrifuged at 10°C and 121 × g for 7 min, and the supernatant was discarded. The lysis procedure was repeated. The resulting pellet was resuspended in 5.0 ml of PBS, and 2.0 ml was layered onto 3 ml of lymphocyte separation medium (Organon Teknika, Durham, N.C.). The other 3.0 ml was used for the blood cultures (see below). The cells were centrifuged at 1,000 × g for 20 min at 10°C, and the cells at the interface were harvested and washed once in PBS. These cells were then resuspended at approximately 5 × 10⁶ cells per ml in RPMI 1640 medium (Gibco, Grand Island, N.Y.) containing 10% fetal bovine serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. One-tenth milliliter of the cell suspension was dispensed into each well of a 96-well microtiter plate (Corning Glass Works). Into each column (12 columns containing eight wells labeled row A to H) were dispensed cells from a different pig. Cells in wells of rows A, B, and C were treated with 0.1 ml of S. choleraesuis UC6077 (heat inactivated as for the ELISA, resuspended to the same density, and diluted 1:1,600). Cells in wells from rows D and E (mitogen control) were treated with 0.1 ml of phytohemagglutinin (PHA) at 5 µg/ml. Cells in rows F, G, and H (negative control) were treated with 0.1 ml of medium. The plates were incubated at 37°C with 5% CO₂ for 72 h and then pulsed with 5-bromo-2'-deoxyuridine (BrdU) for 24 h (supplied as 5-bromo-2'-deoxyuridine labeling and detection kit III [Boehringer Mannheim Biochemica, Indianapolis, Ind.]).

Blood cultures. The blood samples collected on days 25 and 52/53 for the CMI response assay were cultured for Salmonella as per the procedure of Roof et al. (39). Two separate components of the blood samples were cultured: the packed erythrocyte fraction and the pellet from the lymphocyte separation medium gradient. Three-milliliter suspensions of both components were added to 5.0 ml of peptone water (Difco) and incubated overnight at 37°C. From these broths, 0.1 ml was plated onto blood agar (Trypticase soy agar supplemented with 5% sheep blood) and brilliant green agar plates, streaked for isolation, and incubated at 37°C for 18 to 24 h.

Statistical analysis. The data were analyzed for the two phases of the experiment, the postvaccination (safety) period and the postchallenge (efficacy) period. The analysis of the safety period included the data from day 5 to day 11, while the analysis of the efficacy period included day 26 to day 32. The same statistical analyses were conducted for both periods. The primary response variables were fecal consistency (diarrhea score), physical condition (morbidity score), body temperature, and average daily gain (ADG). The other variables measured, including mortality, culturing, immune response, lesion severity scores at necropsy, and histologic lesions, were secondary and supportive data but were not analyzed statistically.

	RESULTS

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Attenuation of cya crp mutants in pigs. The cya crp S. choleraesuis derivatives were administered by oral gavage at doses of approximately 10⁹ CFU/pig, preceded by 25 ml of a 10% (wt/vol) sodium bicarbonate solution, to assess their virulence in swine. The SC-54 vaccine was administered to animals intranasally as per the manufacturer's recommendations. As shown in Table 2, all cya crp S. choleraesuis derivatives clearly were avirulent. Pigs survived oral challenge with a number of organisms that represented 10 times the oral 50% lethal dose of the wild-type parent strain (data not shown). Furthermore, these animals did not show any signs of disease and remained healthy for at least 21 days after challenge. No significant clinical signs were seen for any of the parameters measured except that the strains which carried the virulence plasmids (vpl⁺; 3781 and 4186) caused a small, short-term elevation in the maximum rectal temperature ( = 40.9 and 41.2°C, respectively) compared to pretreatment temperature values (Table 2). The increase in maximum temperature was not significantly different between vpl⁺ strains but was significantly higher than that for all other groups including the other cya crp strains. The low-grade fevers for the pigs given the vpl⁺ strains returned to normal after peaking at 3 days after vaccination. No significant differences were noted in the ADG between naive animals and animals given cya crp strains or SC-54. None of the cya crp derivatives nor SC-54 was recovered by rectal swab from the pigs at any day postvaccination (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Prechallenge safetyclinical signs following vaccination with NOBL SC-54 or various vaccine candidate strains

Immunogenicity of cya crp mutants in pigs. Twenty-one days after vaccination with the cya crp derivatives or SC-54, pigs were challenged with a heterologous, virulent strain of S. choleraesuis, P92-091. Pigs challenged with P92-091 without previous exposure to the cya crp derivatives or SC-54 had a moderate to severe fever, which peaked 2 days after infection ( = 41.3°C) and persisted for at least 7 days after infection (Table 3). These pigs were depressed and had diminished appetites for 2 to 3 days. Approximately 90% of the pigs experienced watery diarrhea, which persisted for half of the monitored period (21 days). Fifty percent of these pigs also shed the challenge organism for 3 to 4 days postchallenge. The ADG was significantly lower than that of the nonvaccinated, nonchallenged pigs.

View this table: [in this window] [in a new window]   TABLE 3.   Postchallenge efficacyclinical signs after challenge with virulent S. choleraesuis P92-091e

cya crp

cya crp

cya crp

Pathology and bacteriologic examinations. In this study, only one animal died prior to termination. This animal had lesions consistent with septicemia caused by S. choleraesuis. Lesion scores for all animals at necropsy are summarized in Table 4. The challenge strain, S. choleraesuis P92-091, did not induce macroscopic lung lesions in these pigs. This was somewhat unexpected, since in preliminary studies with animals of similar age and size we had seen lung lesions in pigs suffering lethal infection with this strain (data not shown). However, two animals in the SC-54 group did have a mild interstitial pneumonia, reported to be consistent with a systemic lesion of S. choleraesuis infection. Lesion scores from the cecum and/or colon were more severe than those from the lungs. Lesion scores for pigs vaccinated with the cya crp pmi strains were more severe than scores for the nonvaccinated, challenged group. Interestingly, lesion scores for 3923-vaccinated pigs also were higher than those for the nonvaccinated group, even though pigs vaccinated with this strain were never positive by rectal swab or fecal culture for virulent S. choleraesuis. Lesions in the cecum and/or colon were consistent with S. choleraesuis pathogenesis (37).

cya crp

Serum antibody responses. The humoral immune response measured by ELISA (Table 5) revealed that groups vaccinated with all of the cya crp strains except 4814 responded with a significant increase in antibody titer by 21 days postvaccination. No significant increase in antibody response was seen with nonvaccinated pigs or pigs vaccinated with 4814 or SC-54. Four weeks after challenge (study day 52/53) with virulent S. choleraesuis, all groups except the nonvaccinated, nonchallenged group showed a significantly increased antibody response to S. choleraesuis antigen.

CMI responses. The CMI response data are summarized in Table 6. For a stimulation index (absorbance of test sample/background absorbance) to be considered positive, it generally should exceed 3.0 (6). As measured by antigen-reactive cells in the peripheral blood, there was no significant CMI response to any of the vaccine strains at 21 days postvaccination. This was unexpected, since others have reported that oral administration of a recombinant derivative of 3781 resulted in a positive delayed-type hypersensitivity (DTH) response in four of four vaccinated pigs at 28 days postvaccination (45). That these lymphocytes may not be detected unless the timing of their sampling is precise was shown by the results obtained with the positive-control pig. This pig had been hyperimmunized with acetone-killed S. choleraesuis 7 days prior to bleeding and had a high-titer antibody response as measured by ELISA (Table 5) on study day 25 and day 52/53. At day 25, when this animal had not been revaccinated for more than 2 months, a positive stimulation index was not obtained even though its ELISA titer was very high (Table 5). In anticipation of the day 52/53 bleed, the positive-control pig was immunized 7 days prior to the bleeding with acetone-killed S. choleraesuis. The stimulation index of these cells for the S. choleraesuis antigen was positive. These results suggest that the lymphocyte proliferation assay may not be as useful as a DTH assay for measuring CMI.

	DISCUSSION

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Live vaccines in general are more effective against salmonellosis than are killed vaccines (3, 11). This is thought to be because live vaccines better stimulate cellular immune responses (26), perhaps because of greater persistence (22) and/or because different antigens are expressed in vivo (42). Salmonella strains that lack the ability to synthesize adenylate cyclase and CRP have been found to be nonvirulent for mice, chickens, and swine and effective as live vaccines when given by injection or by feeding (16). In order to better characterize this attenuation strategy, we constructed a series of isogenic cya crp mutants of S. choleraesuis 3246, a strain virulent for pigs, with or without additional deletions in genes associated with virulence (cdt) or synthesis of LPS (pmi) and/or cured of the virulence plasmid (vpl). The aim was to assess the effect of additional deletions on strains that were proven to be effective live-vaccine constructs but which showed different degrees of attenuation in a rodent model (23).

The results of the present study demonstrated that all of the cya crp S. choleraesuis derivatives tested are safe for swine in that they showed essentially complete loss of virulence. This is not entirely unexpected, since many genes and operons are under the control of cAMP and CRP (11). For instance, cya and crp mutants are impaired in their abilities to transport and break down carbohydrate and amino acid catabolites (1) and are unable to synthesize functional fimbriae (41). Strains harboring a deletion in the cdt gene are unable to penetrate and persist in deeper tissues (23). Nonetheless, it was somewhat surprising to note that there was no apparent difference in virulence of, for example, 3781 and 4186. A comparison of these two strains in mice showed that 3781 was less virulent than 4186 (23). These strains are identical except that 3781 carries an additional deletion in a gene(s) adjacent to crp that is also associated with S. choleraesuis virulence and may be involved in the colonization of deep tissues (23). It may be that cya and crp derivatives of S. choleraesuis are sufficiently attenuated such that deletion of additional genes found on the virulence plasmid that are necessary for, or contribute to, virulence would result in a phenotype that is unmeasurable when the strain is given orally to swine. This can be noted from the finding that curing of the virulence plasmid from 3781 and 4186 (resulting in strains 3923 and 4497, respectively) did not further attenuate these strains (Table 2). The same was true for strains 4522 and 4814, which contain the virulence plasmid but carry an additional deletion in a gene (pmi) involved in the synthesis of LPS. In contrast, Gulig and Curtiss (15) showed that vpl strains of S. typhimurium were less invasive, and therefore more attenuated, than vpl⁺ strains.

Nonetheless, even though all of the cya crp derivatives studied were sufficiently attenuated to be of interest as vaccine candidates, they were not all protective to the same degree. It should be noted that the challenge model used did not cause excessive mortality but induced disease of sufficient severity to make possible a number of important conclusions concerning the protective efficacy of the various vaccine strains used in this study.

Pigs vaccinated once with cya crp derivatives, with or without the virulence plasmid, were well protected against challenge with ca. 10 50% lethal doses of S. choleraesuis P92-091. In contrast, similar vaccination of pigs with cya crp strains carrying an additional deletion in a gene involved in LPS biosynthesis did not protect against challenge with S. choleraesuis P92-091. This result is compatible with the hypothesis that the O antigen is the main protective immunogen in Salmonella species (34). However, we have not ruled out the possibility that the strains carrying a deletion in the pmi gene are less able to persist in pigs or are unable to induce a protective immune response for some other reason.

Maximum temperature increases in animals vaccinated with one of the four effective cya crp derivatives were significantly lower than those in animals in the nonvaccinated, challenged group and the cya crp pmi strain-vaccinated group. However, only pigs vaccinated with 3781 had mean maximum temperatures which were not significantly different from those of the nonvaccinated, nonchallenged group. The maximum temperature of pigs vaccinated with the other vpl⁺ strain, 4186, while not significantly different from that of 3781-vaccinated animals, was different from the maximum temperature of the nonvaccinated, nonchallenged group. Further statistical analysis of the area under the curve (AUC) for the temperatures after challenge with S. choleraesuis P92-091 generally supported the conclusions from analysis of the mean maximum temperature. Pigs vaccinated with strain 3781 and 4186 had temperature AUCs which were significantly lower than those of animals in the nonvaccinated, challenged group and the cya crp pmi strain-vaccinated groups but which were not significantly different from those of animals in the nonvaccinated, nonchallenged group. The apparently poorer protective activity of 4497, based solely on temperature maximum and AUC, is something of a statistical aberration (and probably not of biological significance), since pigs in this group had a lower baseline temperature before challenge (Table 3).

Of the effective cya crp strains, no shedding of virulent S. choleraesuis was noted from pigs vaccinated with 3781 or 3923, while only a few pigs which were vaccinated with 4186 and 4497 shed virulent S. choleraesuis. The former two vaccine constructs contained an additional deletion in the cdt gene. It may be that these two strains, which are less invasive (data not shown), remained more associated with the gut mucosa and caused a more localized immune response, which resulted in increased protection against gut colonization by the challenge strain. The average number of days of shedding of virulent S. choleraesuis was slightly higher for the pigs vaccinated with the pmi-deletion strains than for the nonvaccinated, challenged group (Table 3).

While the NOBL SC-54 vaccine was protective by most of the parameters measured, it was not fully protective as judged by the diarrhea scores, temperature elevation, and shedding of virulent S. choleraesuis postchallenge. The diarrhea scores, although significantly improved over those of the nonvaccinated, challenged group, were significantly higher than those for pigs given 3923 and 4186. The maximum temperature elevation for pigs given SC-54 was significantly higher than the maximum temperature of those given the effective cya crp strains and of pigs in the nonvaccinated, nonchallenged group. In fact, the maximum temperature of the SC-54 group was identical to that of the nonvaccinated, challenged group (Table 3). Moreover, the percentage of animals shedding the virulent challenge strain and the duration of shedding for pigs given SC-54 were significantly greater than those for pigs given the four effective cya crp strains. The percentage of pigs shedding after challenge was even higher in the group vaccinated with SC-54 than in the nonvaccinated, challenged group.

The lack of recovery of the vaccine strains or of S. choleraesuis P92-091 at the time of necropsy implied that these strains either do not invade tissues, do not persist in the tissues if they do invade, or are present only in low numbers and were not efficiently isolated by our culture techniques. In mice, both 3781 and 4186 were invasive and persisted in the blood, Peyer's patches, and spleen for at least 28 days postinoculation (23). Similarly, Stabel and coworkers found that a recombinant derivative of 3781 could invade and persist in the spleens and livers of pigs for 14 to 21 days (45), but they used an inoculum that was at least 10-fold larger than the vaccine inoculum used in the present study. They did not report whether they could recover the isolate by culturing rectal swabs. Since, in order to evaluate immunogenicity, we did not sample tissues until 28 days after challenge with virulent S. choleraesuis (49 days after vaccination), it is possible that we missed the window of persistence of these strains. We were able to culture S. choleraesuis P92-091 from the liver and MLN of the sole pig that died due to salmonellosis at 5 to 6 days postchallenge. Also, an incubation temperature of 37°C for cultural isolation was used in the present study. Subsequent experiments performed in order to enhance our culturing efficiency showed that a higher temperature, 39°C, provided better growth of S. choleraesuis and reduced the growth of contaminants in enrichment broths (data not shown). Thus, it may be that our culturing technique was not optimized for recovering S. choleraesuis in this study.

In an attempt to evaluate the induction of specific immune responses, anti-S. choleraesuis antibodies and antigen-reactive cells in the peripheral blood were measured. Although serum antibody titers rose significantly following vaccination with the cya crp strains (except 4814), it is unclear what role vaccination played in eliciting anti-Salmonella antibody responses, as the nonvaccinated, nonchallenged pigs had the highest ELISA value (0.305). All but two vaccine strains (3781 and 4186) induced lower responses at this time, and the two strains that induced higher responses induced only slightly higher responses. Since the nonvaccinated animals showed increases in ELISA values, this result may be nonspecific. Similarly, the highest response of all groups at day 52/53 was observed in the naive but challenged animals (ELISA value = 0.77). Thus, this suggests that challenge and not vaccination was primarily responsible for the increase in anti-Salmonella titers noted at the time of challenge.

No CMI response to any of the vaccine strains at 21 days postvaccination was noted by the lymphocyte proliferation assay. This was unexpected, since Stabel and coworkers found that oral administration of a recombinant derivative of 3781 resulted in a positive DTH response in four of four vaccinated pigs at 28 days postvaccination (45). However, as mentioned above, their vaccination dose was at least 10-fold higher than that used here, the antigen preparations used to test antigen reactivity were different, and these assays, although having certain of the cell types in common, measure very different types of responses. In the DTH reaction, macrophages activated by antigen-reactive T cells are primarily responsible for the reaction measured. In the lymphoproliferation assay used here, only antigen-specific T cells circulating in the blood are measured and the time of their presence in the blood is related to the timing of antigen stimulation (6). That these lymphocytes may not be detected unless the timing of their sampling is precise was shown by the results obtained with the positive-control pig, which had been hyperimmunized with acetone-killed S. choleraesuis and had a high-titer antibody response as measured by ELISA on day 25 and day 52/53 (Table 5). At day 25, when this animal had not been revaccinated for more than 2 months, a positive stimulation index was not obtained even though its ELISA titer was very high. However, when this pig was immunized with acetone-killed S. choleraesuis 7 days prior to the bleeding, the stimulation index then became positive. These results suggest that the lymphocyte proliferation assay may not be as useful as a DTH assay for measuring CMI response. After challenge with S. choleraesuis P92-091 at day 52/53 (the day of necropsy and 28/29 days postchallenge), several of the groups had positive stimulation indices (>3.0). The group of pigs which was most adversely affected by the challenge with S. choleraesuis P92-091, the group vaccinated with strain 4814, also had the lowest stimulation index at necropsy (day 52/53). The nonspecific response of the lymphocytes to the mitogen PHA was positive at all times for all groups.

In summary, we evaluated the safety and protective efficacy in pigs of six cya crp isogenic constructs of S. choleraesuis 3246 compared to those of a commercial vaccine which was attenuated by accidental deletion of the virulence plasmid upon serial passage through neutrophils in vitro (25, 40). Four of these strains, 3781, 3923, 4186, and 4497, were found to be as protective as or more protective than the commercially available vaccine. Moreover, the percentage of animals shedding the virulent challenge strain and the duration of shedding were significantly lower in pigs given the four effective cya crp strains than in pigs given the commercial vaccine.