Phenotypic and genotypic characterization of Salmonella spp. Isolated from pigs and their farm environment in Korea

This study's objective was to determine the prevalence of Salmonella spp. in pigs and their farm environments in Korea, and to investigate the relationship between the strains based on their phenotypic and genotypic characteristics. A total of 36 Salmonella spp. were isolated in this study: 18 isolates from 492 pigs (3.7%) and 18 isolates from 418 (4.3%) farmhouse environmental samples from 16 different pig farms. Of the Salmonella strains isolated from the numerous environmental samples, the highest prevalence was observed in slurry or manure, followed by partitions, farmer's hands, floors, water/ nipples, ventilation sources, and feed, respectively. All the Salmonella isolates originating from different farms were genetically distinct. In three farms, however, identical phage types and pulse-field gel electrophoresis patterns were observed among Salmonella isolates from pig feces and environmental samples. This study suggests that environments contaminated with Salmonella could pose an infection risk to pigs on pig farms.     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress.

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

Studies on the effect of an Enterococcus faecium probiotic on T cell populations in peripheral blood and intestinal epithelium and on the susceptibility to Salmonella during a challenge infection with Salmonella Typhimurium in piglets

Although Enterococcus faecium is used as a probiotic feed supplement in animal production, feeding of the bacterium to piglets resulted in a more severe infection with Salmonella Typhimurium DT104 during a challenge experiment. To enlighten the mode of action by which E. faecium affected the piglets' health, we investigated the influence of the probiotic bacterium on the development of intestinal and circulating immune cells during a challenge experiment with S. Typhimurium DT104. To minimise varying impacts of the maternal immunity on the course of infection, only piglets were implemented that descended from Salmonella-free sows. In addition, the potency of purified blood and intraepithelial immune cells to control the growth of Salmonella was tested in vitro. In animals treated with E. faecium, a reduction of intraepithelial CD8alphabeta T cells, reduced circulating CD8alphabeta T cells and a less efficient control of intracellular Salmonella growth, mediated by peripheral blood mononuclear cells, were observed.     

鼠伤寒杆菌主要外膜蛋白作为保护性抗原的研究

采用超声破碎,ＴｒｉｔｏｎＸ─１００处理和Ｓｅｐｈａｃｒａｌ超细Ｓ─３００凝胶过滤技术提取了鼠伤寒杆菌的主要外膜蛋白（ＭＯＭＰｓ）。ＭＯＭＰｓ的脂多糖（ＬＰＳ）含量约为０．２％。经ＳＤＳ─ＰＡＧＥ图谱显示蛋白在３６─４１ＫＤ之间。ＭＯＭＰｓ能使小鼠产生典型的足垫肿胀（ＤＴＨ）及高水平的ＩＬ─２;可保护５００ＬＤ５０鼠伤寒杆菌及伤寒杆菌的攻击,其免疫保护率分别为９０％和３３．３％,用５０ｕｇＭＯＭＰｓ免疫的小鼠的Ｔ淋巴细胞经尾静脉注射给非免疫小鼠,可使后者得到被动免疫保护,其保护率为４２．９％。基于上述实验结果,本文认为鼠伤寒杆菌的ＭＯＭＰｓ是一良好的保护性抗原。     

Evaluation of efficacy of disinfectants against Salmonella from the feed industry

Aims:  To evaluate disinfectants against Salmonella under conditions relevant for the feed industry.
Materials and Results:  A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70–80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S.
Conclusions:  Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use.
Significance and Impact of the Study:  Use of the disinfectants found to be efficient against surface associated Salmonella , may assist the industry in combating Salmonella .     

Salmonella species isolated from animal feed in Iraq.

Of 700 animal feed samples, 32 (4.5%) harbored Salmonella. The highest percentage of contamination was found in sheep feed and local protein. A total of 17 Salmonella serotypes were identified. The most frequent serotypes were Salmonella meleagridis. S. bornum, S. montevideo, and S. drypool. S. bornum was isolated for the first time in Iraq and from both local feed and its ingredients. The common somatic group found was that of Salmonella group C; then came groups E, G, B, and D. Three serotypes (S. enteritidis, S. california, and S. muenchen) seemed to form a link of infection among feed, food, patients, and carriers.     

Salmonella species isolated from animal feed in Iraq.

Of 700 animal feed samples, 32 (4.5%) harbored Salmonella. The highest percentage of contamination was found in sheep feed and local protein. A total of 17 Salmonella serotypes were identified. The most frequent serotypes were Salmonella meleagridis. S. bornum, S. montevideo, and S. drypool. S. bornum was isolated for the first time in Iraq and from both local feed and its ingredients. The common somatic group found was that of Salmonella group C; then came groups E, G, B, and D. Three serotypes (S. enteritidis, S. california, and S. muenchen) seemed to form a link of infection among feed, food, patients, and carriers.     

Some properties and protective activity of specific DLE against Salmonella cholerae suis infection

From a rabbit lymphoid tissue, twice immunized with a Salmonella ch. suis vaccine, was obtained a dialysable leucocyte extract (DLE) (m.w. 10 000Da; protein content 1.14 mg/ml; content of ribose 2.7 mg/ml; A₂₆₀/A₂₈₀ ratio 2.17 and pH 6.8). By gel filtration on Sephadex G-25, six peaks were obtained and activity was found in peak IV. The activity of the extract was determined by a dermo-application test (DAT) on 10 cows. The protective effect was tested by a challenge with Salmonella ch. suis and Salmonella dublin pathogen strains on white mice intraperitoneally treated with DLE. The DAT proved to be positive in 8 of the 10 cows. When applied on white mice, it induced a high specific protective effect against Salmonella ch. suis (70%), but not against Salmonella dublin infection.     

Peptide-based synthetic recombinant vaccines with anti-viral efficacy.

Synthetic recombinant vaccines are constructs in which a synthetic oligonucleotide coding for a protective epitope is inserted into an adequate gene for expression of the epitope. We report the results obtained using recombinant flagella of Salmonella vaccine strain expressing epitopes of influenza virus or of the parasite Schistosoma mansoni. In the case of influenza virus, three conserved epitopes of the haemagglutinin and the nucleoprotein of the virus inducing B- and T-cell immune response, were expressed and the flagella were used for intranasal immunization without any adjuvant. Both humoral and cellular immune responses specific to the virus induced in mice cross-strain long-term protection against challenge infection. Aged mice were also able to resist infection. For the design of a human influenza vaccine, epitopes recognized by the HLAs prevalent in Caucasian populations were used, and the resulting vaccine was evaluated in human/mouse radiation chimaera in which human PBMC are functionally engrafted. The vaccinated mice demonstrated efficient clearance of the virus after challenge and resistance to lethal infection. In the case of the parasitic disease schistosomiasis, a 14-residue peptide denoted 9B peptide 1 was expressed in the flagella. Intranasal vaccination of mice with this construct, without the use of adjuvant, resulted in 40% protection against challenge infection.     

SPF鸡盲肠复合微生态制剂代替抗生素预防肉仔鸡沙门茵感染

目的在雏鸡盲肠尽快建立完整健康菌群,通过“竞争排除（Competitiveexclusion,CE）”抑制病原菌定植感染。方法提取SPF鸡盲肠菌群,排除沙门菌感染的可能,制成复合微生态制剂,在鸡胚孵化19d啄壳时和21d雏鸡全出壳时进行两次喷雾接种,监测鸡群沙门菌抗体和粪便中沙门菌,测定成活率、生产性能。结果进行喷雾接种后的小鸡至12日龄可以完全不使用抗生素,成活率、采食量与体重略高于使用抗生素的对照组,能提高饲料报酬,饲养12、24、35d均未检出粪便沙门菌和血液沙门菌抗体,全程无沙门菌感染。结论SPF鸡盲肠复合微生态制剂能代替抗生素预防肉仔鸡沙门菌感染。     

Roles of diet and the acid tolerance response in survival of common Salmonella serotypes in feces of finishing pigs

The persistence of Salmonella in the environment is an important factor influencing the transmission of infection in pig production. This study evaluated the effects of acid tolerance response (ATR), organic acid supplementation, and physical properties of feed on the survival of a five-strain Salmonella mixture in porcine feces held at 4 and 22°C for 88 days. Acid-adapted or non-acid-adapted nalidixic acid-resistant Salmonella strains were used to inoculate feces of pigs fed four different diets, which consisted of a nonpelleted, finely ground meal feed or a finely ground, pelleted feed that was left unsupplemented or was supplemented with K-diformate. Organic acid supplementation and physical properties of feed markedly influenced Salmonella survival, but the effects were highly dependent on storage temperature; survival was unaffected by ATR. The most pronounced effects were observed at 22°C, a temperature similar to that of finishing pig houses. The supplementation of meal diets with K-diformate significantly reduced the duration of survival (P < 0.1) and increased rates of decline (P < 0.0001) of salmonellae in feces compared to survival in feces of pigs fed unsupplemented meal. The pelleting of feed, compared to feeding meal, significantly reduced (P < 0.1) the duration of survival in feces held at 22°C. Only minor effects of feed form and acid supplementation on survivor numbers were observed at 4°C. Differences in the fecal survival of Salmonella could not be related to diet-induced changes in fecal physiochemical parameters. The predominant survival of S. enterica serovar Typhimurium DT193 and serotype 4,[5],12:i:- in porcine feces demonstrates the superior ability of these serotypes to survive in this environment. Fecal survival and transmission of Salmonella in pig herds may be reduced by dietary approaches, but effects are highly dependent on environmental temperature.     

Early antibody response in mice to either infection or immunization with Salmonella typhimurium

After immunization with either live or heat-killed Salmonella typhimurium, mice responded with an extremely rapid production of bactericidal antibody which was correlated with the appearance of immunity to a heavy challenge dose (100 ld(50)) of the virulent bacteria. Inactivation of sera with mercaptoethanol along with Sephadex fractionation indicated that the observed bactericidal activity was associated with a macroglobulin which was completely mercaptoethanol-sensitive. The unexpected finding, that a heat-killed vaccine gave excellent protection from a challenge dose which killed all unimmunized control mice, seriously challenges the theory attributing immunity against typhoid infection entirely to a cellular host factor produced only in response to a live vaccine.     

Effect of dietary selenium on the response of stressed and unstressed chickens toEscherichia coli challenge and antigen

Selenium was added to the feed of White Leghorn type chickens 1 day prior to challenge with eitherEscherichia coli or sheep erythrocyte antigen. the incidence of death or lesions was reduced from 86% to 21% at the optimal dose of selenium (0.4 mg/kg resulting in feed concentration of 0.45 mg/kg). After the chickens were stressed by chilling, selenium was ineffective againstE. coli. Dietary additions of selenium between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 to the log₂ against sheep erythrocytes (SRBC). Followng chilling, antibody titer response was reduced from 4.9 to 2.4 to the log₂. This titer reduction could be prevented with dietary additions of selenium between 0.1 and 1.2 mg/kg. The effects of a nitrofuran and selenium were additive againstE. coli challenge infection.     

Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104

Commercial laying hens were infected with Salmonella typhimurium DT104 strain 16 alternatively via the crop (10(7) cfu per bird) or by an aerosol delivered directly to the beaks using a Collison nebuliser and Henderson apparatus (2 x 10(2) or 2 x 10(4) cfu per bird). Infection by both routes caused systemic infection and prolonged contamination of faeces. Contamination rates of eggs and muscle were much higher following the aerosol challenges despite the much lower doses given by this route. The frequency of Salmonella isolation from eggs rose from 1.7% following oral challenge to 14% and 25%, for each of the aerosol challenges respectively, and the frequency of isolation from muscle rose from 0% following the oral challenge to 27% following each of the aerosol challenges.     

Deoxycholate-treated, nontoxic, whole-cell vaccine protective against experimental salmonellosis of mice

A vaccine prepared from the residue after extraction of whole cells of Salmonella typhimurium with 2% sodium deoxycholate proved to be nontoxic and highly immunogenic. The material was not lethal for mice at 6.0 mg and was essentially nontoxic in rabbit skin, whereas endotoxic activity was found in the dialyzed extract. A high dosage, above 2.0 mg, was less protective than lower doses, indicating a degree of "immunologic paralysis." Three inoculations of low doses, 0.25 mg each, induced protection against death and tissue infection in animals challenged with 2,000 ld(50) of virulent homologous S. typhimurium and against death, but not against tissue infection, after heterologous challenge with S. enteritidis. Residues of purified cell walls were as effective as residues of whole cells, indicating that the immunizing antigen(s) resided in the cell wall.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154-/-) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154-/- mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154-/- mice exhibited a defect both in the production of IL-12, IFN-gamma, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154-/- animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in > or =90% protection. The lack of protective immunity in CD154-/- mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154-/- mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.     

Cross-protection aginst Salmonella enteritidis infection in mice. I. Immunization trials.

Mice were immunized subcutaneously with live and killed vaccines, with and without complete adjuvant incorporating Salmonella typhi-murium M206, Salmonella gallinarum 9R, Salmonella pullorum Sp223 as well as homologous Salmonella enteritidis Se795. The animals were challenged 21 days post-vaccination with 100 LD50 of virulent S. enteritidis 5694 SMR subcutaneously along with unvaccinated control mice. To assess the immunity against acute and chronic infections, the percentage of absolute survivors i.e. survivors without lesions and without the challenge organism, was taken as the criterion. Live vaccines proved better than killed vaccines. Live vaccines with complete adjuvant induced a good protection. Cross-protection could be induced with the live vaccine with complete adjuvant against S. enteritidis infection in mice.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.