A wild-type S. Typhimurium UK-1 strain was utilized to construct potential vaccine strains. A nalidixic acid (NA) resistant S. Typhimurium UK-1 derived from the wild type was used as the challenge strain. The P_BAD-mviN vaccine strain discussed in the current study for comparative purposes was generated from UK-1 as described in our previous report (11).

Single and double deletion mutants affecting methionine metabolism were produced in S. Typhimurium strain UK-1. This strain and the plasmids for the Red recombinase system were obtained from Dr. Young Min Kwon, Department of Poultry Science, University of Arkansas (Fayetteville, AR, USA). The Red recombinase system was used for the targeted gene deletions as previously described (13). Briefly, disruption of the targeted genes was accomplished by first transforming strain UK-1 with plasmid pKD46. This plasmid confers ampicillin resistance and harbors the genes for phage lambda Red recombinase, which mediates the exchange of DNA between the gene of Salmonella to be deleted and the gene disruption construct. This plasmid also contains a temperature-sensitive origin of replication, which facilitates its removal following recombination. Gene disruption constructs were synthesized by amplifying a region of plasmid pKD4 (13) by polymerase chain reaction (PCR), from the P1 site (nucleotides 31–50 of pKD4) to the P2 site (nt. 1488–1507). This region consists of a central kanamycin (Kan) resistance gene (encoding aminoglycoside 3′-phosphotransferase), flanked by two FLP recognition target (FRT) sites. Genomic DNA sequences corresponding to upstream and downstream regions surrounding the appropriate target gene of strain UK-1 were subsequently introduced on either side of the FRT-Kan^R-FRT region by overlap extension PCR (14), using the primers indicated in Table 1.

The method for deletion of the metD transporter sequence, which is comprised of the metNIQ operon is shown in Figure 1. The resulting PCR products were gel purified, treated with restriction endonuclease Dpn1 to degrade any trace amount of template pKD4, gel purified again, and then electroporated into electroporation-competent UK-1:pKD4 cells that had been grown at 30°C in Luria-Bertani (LB) broth with 1 mM arabinose to induce the Red recombinase. After a 1 h incubation of the electroporated cells at 37°C in SOC medium, the electroporated cells were spread on LB/Kan agar plates and grown at 37°C overnight. Four tranformants were then streaked for isolation on LB/Kan, and tested on LB/ampicillin plates to confirm curing of plasmid pKD46.

The Kan resistance marker was subsequently removed from the genomic insertion sites, leaving a gene deletion, by introducing a second plasmid, pCP20, which expresses a second recombinase, the S. cerevisiae FLP recombinase and confers ampicillin resistance (15). The FLP mediates the removal of the antibiotic resistance marker by recombining the flanking FRT sites. The FLP was induced and pCP20 removed by shifting the temperature from 30 to 42°C as described previously (13). Removal of the Kan^R genomic insertion and pCP20 were confirmed by failure to grow on LB/Kan and LB/ampicillin, respectively, with appropriate growth on LB in parallel.

Whole-cell affinity measurements were conducted as described previously (16). Briefly, cultures of the ΔmetR single mutant and ΔΔmetRmetD double mutant were grown in M9 minimal medium + 10 μM L-methionine at 37°C for 16 h and then diluted to an OD₆₀₀ of 0.05 in M9 minimal medium + L-methionine at 3, 7, 10, 13, 15, and 17 μM in culture tubes containing 4 ml each of minimal medium at the L-methionine concentrations indicated, and three technical replicate culture tubes were prepared at each methionine concentration. Cultures were grown at 37°C in a shaking water bath at 220 RPM, and the OD₆₀₀ was measured every 15 min for a total of 6 h. The replicate data were then averaged and transformed for Lineweaver–Burk plots using Microsoft Excel software.

Details of the vaccination challenge trial have been described elsewhere (11). Briefly male Cobb 500 broiler chicks (Siloam Springs, AR, USA) were obtained on day of hatch and randomly assigned to four pre-sterilized Horsfall units. A University of Arkansas Institutional Animal Care and Use Committee-approved protocol was used to ensure humane treatment of the chickens. Chicks vaccinated with ΔmetRΔmetD double mutant Salmonella was one treatment group (designated Group 2) of the four treatment groups (Group 1: unvaccinated, challenged; Group 3: vaccinated with the P_BAD-mviN vaccine strain Salmonella, challenged), and Group 4: vaccinated with the wild-type parent strain UK-1, challenged. The vaccine and control inocula were grown for 16 h in LB broth at 37^oC, followed by three washes in phosphate-buffered saline (PBS) and adjustment of the cell density by dilution in PBS to 5 × 10⁸ cells/ml. Chicks were orally inoculated with 1 × 10⁸ CFU Salmonella cells via sterile gavage needle on Day 2 post-hatch and again on Day 7 post-hatch. The unvaccinated chicks received an equal volume (0.2 ml) of sterile PBS via sterile gavage needle on Days 2 and 7 post-hatch. The challenge strain, which had been passaged repeatedly through chicks to increase its virulence followed by cryopreservation, was grown for 16 h in LB + 20 μg/ml NA. The challenge strain was then passaged twice for 8 h each passage to ensure a log phase culture. The resulting cells were then diluted to 5 × 10⁸ cells/ml with PBS and orally inoculated via sterile gavage needle to chickens at 2 weeks post-hatch with 1 × 10⁸ cells (0.2 ml).

At the time of chicken necropsy reported previously (11), ceca and ilea organs were collected aseptically and transferred to sterile sample bags, subsequently removed and transferred to 10 ml tetrathionate (TT) broth for enrichment. The TT broth was incubated for 24 h at 37°C, followed by streaking of a 10 μl loopful of the TT broth for isolation on Brilliant Green (BG) agar supplemented with 20 μg/ml NA and another 10 μl loopful on BG agar supplemented with 50 μg/ml Kan + 1 mM arabinose. Agar plates were incubated for 24 h at 37°C, examined for Salmonella colony appearance to enumerate the NA—resistant S. Typhimurium challenge strain per gram cecal content, and live vaccine strains (Kan—resistant Salmonella per gram of cecal content). A 0.1 g portion of cecal content was aseptically added to a sterile microtube, weighed, and then combined with nine volumes of sterile PBS to obtain a 1:10 dilution, followed by vortexing. After serial dilution in sterile PBS, mixed cecal contents were spread-plated aseptically onto selective media (BG + 20 μg/ml NA) for enumeration of the respective treatment groups. Since S. Typhimurium is naturally resistant to novobiocin (NO), the wild-type control NA-sensitive strain was distinguished from the NA-resistant challenge strain by direct plating of cecal contents from the wild-type-inoculated chickens on both BG + NA and BG + NO plates, and subtracting the number of colonies on BG + NA (challenge strain) from the total colonies on BG + NO (challenge strain + wild-type control strain).

As described previously (11), indirect ELISA reactions were performed by placing 1 μg of Salmonella protein from sonicated UK-1 cells in each well of 96-well microlon medium binding microtiter plates (Grainer, Frickenhausen, Germany) after diluting to 100 μl in carbonate buffer, pH 9.6. After allowing proteins to bind for 2 h 37°C, the plates were allowed to air-dry overnight at 23°C then blocked with Superblock (Thermo Scientific, Rockford, IL, USA) 2 h, 37°C. Chicken sera from unvaccinated and vaccinated chickens from all treatment groups were serially diluted in Superblock and the plates incubated at 37°C, 2 h followed by washing four times with ELISA plate wash buffer (50 mM Tris pH 8, 140 mM NaCl, 0.05% Tween-20). Anti-chicken IgG-HRP conjugate was diluted 1:20,000 with Superblock and plates incubated 37°C, 1 h, followed by washing four times in wash buffer. The TMB substrate (KPL, Gaithersburg, MD, USA) was incubated 10 min in each well, stopped with 1 N HCl and absorbance measured in a Tecan Infinite M200 plate reader at 450 nm.

Wild-type parent strain and mutants ΔmetR and ΔmetRΔmetD were grown in M9 minimal medium supplemented with 10 μM L- or D-methionine as indicated for 16 h and subsequently washed three times in PBS. Five grams of chicken feed were blended with 100 ml dionized water at high speed for 3 min. The same was done for 5 g of chicken feces, separately. These blends were then filtered through cheese cloth and autoclaved. These were subsequently aliquoted into sterile culture tubes and the washed Salmonella was added to a final density of 1 × 10⁶ cells/ml. The cultures were incubated at 37°C, 200 RPM and growth of the cultures was monitored by spread-plating and colony counting of appropriate dilutions on LB medium at 0, 2, 5, 24, 48, 96, 264, and 504 h.

The enumeration of the challenge (NA-resistant marker) strain in ceca of unvaccinated and vaccinated chickens was compared by a two-tailed Student’s t-test, using the Microsoft Excel program.