Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.     

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.     

Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 10⁸ colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 10⁶ CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine’s value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.     

Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis ∆crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis ∆crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis ∆crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis ∆crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis ∆crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis.     

Induction of seroconversion and persistence of Salmonella Typhimurium in pigs are strain dependent

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Salmonella Typhimurium pathogenesis is host species specific. In addition, differences in in vitro behaviour of Salmonella Typhimurium strains have also been described, which may be reflected by a different course of infection within a host species. We compared the course of a Salmonella Typhimurium infection in pigs, using two Salmonella Typhimurium strains that were able to interfere with MHC II expression on porcine macrophages to a different extent in vitro. After experimental inoculation, blood and faecal samples from all pigs were collected at regular time points. At 40 days post inoculation (pi), animals were euthanized and tissue samples were bacteriologically analysed. The proportion of serologically positive piglets at 33 days pi was significantly higher in pigs that were inoculated with the strain that did not downregulate MHC II expression in vitro. Furthermore, this strain was less frequently shed and isolated in lower numbers from tonsils and ileocaecal lymph nodes than the strain that was able to markedly downregulate MHC II expression in vitro. We thus found that the delayed onset of seroconversion after oral inoculation of piglets with a particular Salmonella Typhimurium strain coincided with higher faecal shedding and increased persistence. Strain specific differences in Salmonella pathogenesis might thus have repercussions on the serological detection of Salmonella Typhimurium infections in pigs.     

6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A ΔqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the ΔqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.     

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

Effects of feeding Salmonella enterica serovar Typhimurium or serovar Choleraesuis on growth performance and circulating insulin-like growth factor-I, tumor necrosis factor-alpha, and interleukin-1beta in weaned pigs

The most common Salmonella serovars causing clinical disease in pigs are Salmonella enterica serovars Typhimurium (Typhimurium) and Choleraesuis. Given that the swine host-adapted serovar Choleraesuis has been reported to cause systemic disease, a different disease outcome from that of Typhimurium, our working hypothesis was that this serovar would likely engage systemic immune-inflammatory mechanisms, resulting in elevated systemic cytokine secretion. Forty-eight weaned pigs were blocked by BW and sex, and randomly allotted to 1 of 3 treatments in a 14-d study. Each treatment had 8 replicates (pens), with 2 pigs/pen. The treatments consisted of a negative control and pigs repeatedly fed 10(8) cfu of Typhimurium or Choleraesuis. On d 0, the pigs were fed Choleraesuis or Typhimurium in dough balls, and the bacteria were refed twice weekly throughout the experiment. Control pigs received dough balls without bacteria. All pigs were housed in temperature-controlled rooms under constant lighting and were fed a standard corn-soybean meal-based nursery diet. Pig BW and feed disappearance were used to determine ADG, ADFI, and G:F. Rectal temperatures were obtained daily from 1 pig/pen beginning 2 d before the first bacterial feeding through d 7 using rapid-response digital thermometers. Serum was collected on d 0, 7, and 14 from a single pig/pen for analysis of IGF-I, tumor necrosis factor-alpha , and IL-1beta. There was no change in the rectal temperature of the control or the Typhimurium-challenged pigs (compared with d 0) or when comparing Typhimurium-challenged pigs with control animals. In contrast, pigs fed Choleraesuis had increased rectal temperatures beginning on d 2 and continuing through d 7 (P < 0.05), with the greatest elevation on d 3 (P < 0.001) compared with the control pigs. Average daily gain and ADFI of pigs challenged with Typhimurium did not differ from those of the control animals. Pigs fed Choleraesuis had a 25% reduction in ADG (P < 0.0001) and ADFI (P < 0.002) compared with the control pigs. On d 7, pigs fed Choleraesuis had reduced serum IGF-I compared with control (P < 0.01) or Typhimurium-challenged pigs (P = 0.01). Bacterial feeding did not affect serum tumor necrosis factor-alpha or IL-1beta compared with control pigs at any time throughout the experiment. We conclude that repeated exposure of weaned pigs to Choleraesuis reduced growth performance in the absence of changes in systemic inflammatory cytokines.     

Porcine circovirus type 2 (PCV2) vaccination reduces PCV2 in a PCV2 and Salmonella enterica serovar Choleraesuis coinfection model

We previously reported that prior porcine circovirus type 2 (PCV2) infection potentiates the severity of clinical signs, lung lesions, and fecal shedding and tissue dissemination of Salmonella enterica serovar Choleraesuis in infected pigs. Here, we evaluated whether PCV2 vaccination is effective in reducing fecal shedding and tissue dissemination of S. Choleraesuis and improving clinical signs associated with PCV2 and S. Choleraesuis infection in 15 Cesarean-derived, colostrum-deprived pigs randomly assigned to 3 groups (n = 5/group). The vaccinated and co-infected (VAC-COINF) group received 2 ml of a commercial PCV2 vaccine at age 3 weeks. The VAC-COINF and co-infected (COINF) groups were inoculated intranasally with PCV2 and S. Choleraesuis at 5 and 7 weeks of age, respectively. The CONTROL group pigs received a similar volume of PBS for sham-vaccination and sham-inoculation. PCV2 vaccination clearly reduced PCV2 DNA load in the serum and postmortem tissue samples and decreased PCV2 antigen levels in tissue samples of the VAC-COINF group. After S. Choleraesuis infection, the incidence of several clinical signs increased in the VAC-COINF group compared to that in the COINF group. The microscopic lung lesions and weight gain, fecal shedding and tissue dissemination of S. Choleraesuis except in the spleen were not significantly different in the VAC-COINF and COINF groups. Thus, PCV2 vaccination reduced PCV2 in the S. Choleraesuis and PCV2 coinfection model and the effects on S. Choleraesuis were minimal.     

On-farm risk factors for Salmonella Enteritidis contamination

Forty layer farms from 2 states participated in a study to examine the risk factors and incidence of Salmonella Enteritidis from multiple samples, including environmental drag swabs from the bird areas, feed, water, flies, rodents, live rodent traps, and environmental swabs from areas occupied by other livestock. Twenty-four of these farms had between 3,000 and 31,000 bird flocks (medium-sized flocks) and 16 had less than 3,000 birds (small-sized flocks). All were housed in cage-free production systems. Twenty-two farms included outside pasture areas for the birds. Most of the participants had just come under the FDA Egg Rule and had not yet tested their flocks (flocks under 3,000 birds are exempt) for Salmonella Enteritidis. Many, however, obtained their pullets from commercial Salmonella Enteritidis-clean breeder sources hatched in National Poultry Improvement Plan hatcheries. Vaccination against Salmonella Enteritidis was performed on 21 of the 40 farms (combination of live and killed vaccines). Salmonella Enteritidis was detected on 7 out of the 40 farms, primarily in rodents, their feces, or from swabs taken inside live traps. Of these 7 Salmonella Enteritidis-positive farms, 3 farms that had vaccinated their pullets with live Salmonella Typhimurium vaccine and killed-Salmonella Enteritidis vaccine; no Salmonella Enteritidis was isolated from the environmental drag swabs taken from the bird area or from the eggs on these farms. However, on the farms that had not vaccinated for Salmonella Enteritidis, the organism was isolated from 4 environmental drag swabs and 3 egg pools. The last 4 farms had flocks under 3,000 birds. No Salmonella Enteritidis was isolated from any of the samples of feed, flies, water, or swabs taken from other livestock areas. Based on the initial findings in this study, we suggest the 2 most important risk factors for Salmonella Enteritidis contamination inside the bird area and in the eggs in these small- and medium-sized flocks are the presence of infected rodents and the absence of an Salmonella Enteritidis vaccination program.     

Assessment of attenuated Salmonella vaccine strains in controlling experimental Salmonella Typhimurium infection in chickens

Salmonella hold considerable promise as vaccine delivery vectors for heterologous antigens in chickens. Such vaccines have the potential additional benefit of also controlling Salmonella infection in immunized birds. As a way of selecting attenuated strains with optimal immunogenic potential as antigen delivery vectors, this study screened 20 novel Salmonella Typhimurium vaccine strains, differing in mutations associated with delayed antigen synthesis and delayed attenuation, for their efficacy in controlling colonization by virulent Salmonella Typhimurium, as well as for their persistence in the intestine and the spleen. Marked differences were observed between strains in these characteristics, which provide the basis for selection for further study as vaccine vectors.     

Efficacy of a novel virulence gene-deleted Salmonella Typhimurium vaccine for protection against Salmonella infections in growing piglets

We have previously developed a novel attenuated Salmonella Typhimurium (S. Typhimurium) ΔcpxR Δlon vaccine. This study was carried out to examine whether this vaccine could effectively protect growing piglets against Salmonella infection. Attenuated S. Typhimurium secreting the B subunit of Escherichia coli heat-labile enterotoxin was also used as a mucosal adjuvant. Pregnant sows in groups A and B were primed and boosted with the vaccine and mucosal adjuvant, whereas sows in groups C, D and E received PBS. Piglets in groups A and C were intramuscularly primed with formalin-inactivated vaccine and orally boosted with live vaccine, while piglets in groups B, D and E received PBS. Piglets in groups A, B, C, and D were challenged with a wild type virulent S. Typhimurium at the 11th weeks of age. Colostrum sIgA and IgG titers in vaccinated groups A and B sows were approximately 50 and 40 times higher than those of non-vaccinated groups C, D and E sows (P < 0.001). Serum IgG titers of group A piglets were also significantly higher than those of groups D and E piglets during the study (P < 0.001). Furthermore, no clinical signs were observed in group A piglets during the entire experimental period after the challenge, while diarrhea was observed in many of the piglets in groups B, C, and D. No Salmonella was isolated from fecal samples of the groups A and C piglets on day 14 after challenge, whereas the challenge strain was isolated from several piglets in groups B and D. These results indicate that vaccination of the piglets with the vaccine and mucosal adjuvant in addition to vaccination of their sows induced effective protection against Salmonella infections in the growing piglets.     

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.     

Serotype identification and antimicrobial resistance profiles of <Emphasis Type="Italic">Salmonella</Emphasis> spp. isolated from chicken carcasses

In this study, 32 Salmonella strains isolated from 400 chicken carcasses were serotyped, and antibiotic resistance profiles were detected against 12 selected antimicrobial agents using disc diffusion method. Thirty-two isolates were identified as follows; 22 (68.7%) Salmonella Enteritidis, five (15.6%) Salmonella Virchow, three (9.3%) Salmonella Typhimurium and two (6.2%) Salmonella Hadar. In all Salmonella isolates, antibiotic resistance were detected. Out of 32 Salmonella strains, 22 (68.75%) displayed multi-drug resistance. Thirty-two (100.0%) of the isolates were found to be resistant to penicillin G, 20 (62.5%) to nalidixic acid, four (12.5%) to cephalothin, two (6.2%) to streptomycin and two (6.2%) to tetracycline. Fifteen (68.1%) Salmonella Enteritidis, one (33.3%) Salmonella Typhimurium, two (100.0%) Salmonella Hadar and two (40.0%) Salmonella Virchow were shown to be resistant to nalidixic acid. Cephalothin resistance was detected in 9.0%, 33.3%, and 20.0% for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Virchow, respectively. The results indicate that Salmonella recovered from chicken carcasses were resistant to multiple antimicrobials and that resistance among these isolates varies by serotype. Also, this emerged as a significant public health problem.     

A vaccine candidate for post-weaning diarrhea in swine constructed with a live attenuated Salmonella delivering Escherichia coli K88ab,K88ac,FedA, and FedF fimbrial antigens and its immune responses in a murine model

In order to construct a novel vaccine candidate for preventing post-weaning diarrhea in swine, the individual genes for Escherichia coli K88ab, K88ac, FedA, and FedF fimbriae were inserted into a secretion plasmid pBP244 containing asd, lepB, secA, and secB. These were transformed into Salmonella Typhimurium Δlon ΔcpxR Δasd. Secretion of the individual recombinant fimbrial antigens was confirmed by immunoblot analysis. Groups 1 and 2 mice received a single oral dose of the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In groups 3 and 4, mice were primed and boosted with the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In general, all immunized mice had significantly increased serum immunoglobulin (Ig)G (P < 0.05) and intestinal secretory IgA against the individual fimbrial antigens compared with those mice in the control group. In the IgG2a and IgG1 titer assay, only IgG2a titer was increased in group 1, while both IgG2a and IgG1 titers were increased in group 3. Furthermore, the vaccine strains were not detected in the excreted feces of any immunized mice. Thus, the vaccine candidate can be highly immunogenic and be safe to the environment.     

Salmonella Incidence in Broilers from Breeders Vaccinated with Live and Killed Salmonella

Salmonella reduction in broilers from commercial broiler breeders vaccinated with live and killed Salmonella vaccines was evaluated. Broiler breeders were vaccinated with Poulvac ST live Salmonella Typhimurium vaccine at 1 d of age, and this was repeated at 2 and 6 wk of age. The breeders were then administered a killed autogenous oil emulsion adjuvanted vaccine containing Salmonella Kentucky, Salmonella Heidelberg, and Salmonella Hadar, at 10 and 18 wk of age. From the ages of 36 to 52 wk, eggs from the breeder flock were hatched, and progeny were challenged in 4 separate experiments at 1 d of age by oral gavage with 1 × 10⁶ cfu/chick by either Salmonella Kentucky, Salmonella Heidelberg, Salmonella Hadar, or Salmonella Enteritidis, each containing resistance to naladixic acid at 32 μg/mL. At 17 to 21 d of age, the broilers were euthanized, and 1 side of the cecum was cultured for Salmonella, and the other side of the cecum was used for enumeration on positive samples. Recovered Salmonella was confirmed to be the challenge strain by O-antisera grouping. This study indicated a difference in Salmonella incidence and enumeration between the vaccinated and nonvaccinated breeder groups for certain species. When challenged with serotypes Salmonella Kentucky, Salmonella Hadar, and Salmonella Heidelberg, protection was noted with a reduction of 28, 17, and 11%, respectively, when compared with the control groups. However, protection was not seen when challenged with S. enteritidis. Under the conditions of this study, live and killed vaccination of commercial broiler breeders with Salmonella contributes protection to progeny when challenged at 1 d of age.     

Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.     

Characterization of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from pigs presenting with diarrhea in Korea

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.     

Evaluation of protection conferred by a Salmonella Typhimurium inactivated vaccine in Salmonella-infected finishing pig farms

The efficacy of an inactivated S. Typhimurium vaccine administered to pigs at the beginning of the fattening period was evaluated in four clinical trials (trials A, B, C and D). Faecal shedding and the systemic antibody response during fattening, as well as, the cecal contents and mesenteric lymph nodes collected after slaughtering were used to assess the outcome. Salmonella shedders prevalence in the control groups was six times higher than in the treated groups in trials A and D, both herds infected by S. Typhimurium. The risk of positive pens was also four or five times higher for the pens housing control pigs in trials A and C. Lower prevalence of Salmonella was observed in the slaughter samples from the vaccinated pigs in trial D and in the cecal content samples in trial A, when just the S. Typhimurium results were compared. The results suggest the effective homologous protection of the vaccinated pigs; however, the high humoral response elicited in the vaccinated pigs complicates their use in farms under serological surveillance programmes.     

A clinical field trial to evaluate the efficacy of vaccination in controlling Salmonella infection and the association of Salmonella-shedding and weight gain in pigs

A clinical field trial was performed to determine the effectiveness of an autogenous Salmonella Typhimurium bacterin compared with a commercial live S. Choleraesuis vaccine in pigs. The association between Salmonella shedding and weight gain was also investigated. Nine cohorts of weaned pigs, (330 to 350 pigs per cohort), were randomly assigned to 1 of 3 treatment groups (injection with S. Typhimurium bacterin, vaccination via water with S. Choleraesuis vaccine, or a control group receiving no vaccine). In each cohort, the average daily gain was calculated for a selected pen throughout the production stage. Pen (pooled) fecal samples were collected bi-weekly and cultured. The odds of Salmonella shedding in both vaccinated groups was higher than in the control group (P < 0.05). The prevalence of Salmonella shedding declined overall as pigs aged (P = 0.04). However, the control pigs showed the smallest decrease in Salmonella shedding over the entire production stage, while prevalence of Salmonella shedding in the vaccinated groups decreased twice as much as the control group over the entire production stage. Salmonella Typhimurium var. Copenhagen DT104, S. Cerro, and S. Agona, which had been isolated on the study farm previously, were recovered from pigs in this study. Shedding of S. Typhimurium var. Copenhagen decreased over time in both vaccine treatment groups. On the other hand, S. Cerro shedding rate was lower in the control pigs compared with vaccinated pigs and S. Agona could be recovered only from the samples collected from S. Choleraesuis vaccinated pigs. The pigs from pens with a higher Salmonella recovery rate experienced slower growth compared with pigs from pens where Salmonella was not isolated. This latter finding indicates that there might be an economic incentive for producers to try to control endemic salmonellosis if effective programs could be developed.