Characterization of Salmonella Dublin and Salmonella Typhimurium (Copenhagen) Isolates from Cattle

Brackelsberg, C.A., Nolan, L.K. and Brown, J., 1997. Characterization of Salmonella dublin and Salmonella typhimurium (Copenhagen) isolates from cattle. Veterinary Research Communications, 21 (6), 409-420Eight Salmonella typhimurium (Copenhagen) and eight Salmonella dublin isolates from cattle were compared by their antibiotic resistance patterns, by their production of colicin, aerobactin, haemolysin and capsule, by their possession of transmissible R plasmids and the spvC gene, and by their ability to invade and replicate within cultured epithelial cells. The two groups differed in their antibiotic resistance profiles, with more of the host-adapted S. dublin isolates resistant to tetracycline than were the non-host-adapted S. typhimurium (Copenhagen) group, but more of the S. typhimurium (Copenhagen) isolates resistant to the other antibiotics tested. None of the isolates produced colicin, but all produced aerobactin. One isolate in each group was encapsulated. All of the S. typhimurium (Copenhagen) and S. dublin isolates contained plasmids, and all of them contained the spvC-homologous sequences. Four of the S. typhimurium (Copenhagen) isolates were able to transfer an R plasmid to a recipient organism by conjugation. One of the five S. dublin isolates, which showed resistance to some of the antibiotics tested, was able to transfer an R plasmid by conjugation. Both groups of isolates invaded cultured epithelial cells to a similar degree after 1 h, but the S. dublin isolates reached significantly higher levels within the cells than did S. typhimurium (Copenhagen) after 9 h. This ability may, in part, explain the association of S. dublin with more severe forms of salmonellosis and prolonged carrier states. Further study of the intracellular growth of these isolates seems warranted.     

A comparison between longitudinal shedding patterns of Salmonella Typhimurium and Salmonella Dublin on dairy farms

Salmonella in cattle herds may behave as epidemic or endemic infections. An intensive longitudinal sampling study across all management groups and ages on six dairy farms in the UK was used to examine patterns of Salmonella shedding, following the prior identification of either Salmonella Dublin (SD) (three farms) or Salmonella Typhimurium (ST) (three farms) on the premises in the context of clinical salmonellosis. Individual faeces, pooled faeces and environmental samples (total 5711 samples), taken approximately every six weeks for 15-24 weeks, were cultured for Salmonella. SD was detected at low frequency (on any visit, 0.5-18.3 per cent of samples positive) and most consistently in calves. By contrast, ST was isolated at higher frequency (on any visit, 6.8-75 per cent of samples positive), and in higher numbers, up to 10(7) cfu/g faeces. Significantly more samples from calves were positive for ST than were positive for SD (50.6 per cent v 3.1 per cent; P?相似文献   

Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: Deletion of the lon gene

Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain.     

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types

The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.     

Comparison of early ileal invasion by Salmonella enterica serovars Choleraesuis and Typhimurium

The mechanisms of Salmonella serovar-host specificity are not well defined. Pig ileal loops were used to compare phenotypic differences in early cellular invasion between non-host-adapted Salmonella serovar Typhimurium (SsT) and host-adapted Salmonella serovar Choleraesuis (SsC). By 10 minutes postinoculation, both serovars invaded a small number of M cells, enterocytes, and goblet cells. Multiple SsC organisms (up to 6 per cell) simultaneously invaded M cells, whereas SsT often invaded as one to two organisms per M cell. Internalization of both serovars resulted in vacuoles containing a single bacterium. The follicle-associated epithelium (FAE) of SsC-inoculated loops responded with more filopodia and lamellipodia although exhibiting less cell swelling than SsT. Additionally, SsT showed an enhanced affinity for sites of cell extrusion compared with SsC at 60 minutes. These results suggest: 1) both SsC and SsT exhibit non-cell-specific invasion as early as 10 minutes postinoculation, 2) Salmonella serovars exhibit differences in early invasion of FAE and M cells, and 3) cells undergoing extrusion may provide a site for preferential adherence by SsT and SsC.     

A bioreactor system to study survival of Salmonella Typhimurium in pig gut content

The batch culture system included six bioreactors. Three bioreactors containing stomach slurry were maintained at pH 4.5 and 6 respectively. Bioreactors containing small intestine slurry were maintained at pH 5.6 and 7 respectively. The bioreactors were inoculated with 10 ml of viable Salmonella. The bioreactors were maintained for 6 hours. Samples of 10 ml were taken at 0 time and at 1, 2, 4 and 6 hours. The samples were analysed for the presence of Salmonella and SCFA. In the stomach samples Salmonella numbers increased at pH 6 but fell at pH 4. In the small intestine sample Salmonella numbers increased at pH 6 and 7. In terms of SCFA production, in the stomach, with samples at pH 6 there was little change in the amounts of lactate, succinate and formate to that detected at 0 time, however levels of acetate did increase slightly. In the small intestine samples levels of succinate and formate increased slightly up to 4 hours, levels of acetate increased significantly from 0 to 6 hours. In terms of the specific growth rates of the individual strains, both strains grew at pH 6 in the stomach content and to a greater extent in the small intestinal content. A bactericidal effect was observed at pH 4 in the stomach content while neither killing nor growth occurred at pH 5 either in the stomach or the small intestine content. Both strains grew well in the small intestine content at pH 7, showing generation times of up to 24 min.     

Isotype-specific antibody responses of cattle to Salmonella Dublin lipopolysaccharide and porin following Salmonella Dublin vaccination and acute and chronic infection.

Stimulation of different T-cell subsets during antigen presentation influences the antibody isotype response to an antigen. Salmonella infection and Salmonella bacterin vaccination are likely to stimulate different T-cell subtypes. The objective of this study was to determine whether there are differences in the isotype response of cattle to Salmonella antigens following Salmonella infection and Salmonella bacterin vaccination. Sera from Salmonella bacterin-vaccinated, experimentally infected, and chronically infected (carrier) adult cattle collected during previous studies was used to evaluate the IgG1, IgG2, and IgM isotype responses of cows to Salmonella serotype Dublin lipopolysaccharide (LPS) and porin. Following vaccination and experimental oral infection, IgG1 titers to LPS and porin rose more quickly and persisted longer than did IgG2 titers. In contrast to Salmonella infection, bacterin vaccination stimulated a weak response to Salmonella porin. Salmonella infection also induced a higher IgG2:IgG1 titer ratio to LPS than did bacterin vaccination. Chronic Salmonella infection induced the highest LPS and porin IgG2:IgG1 titer ratios and the highest correlation between LPS and porin titers. Response operating characteristic curves for each isotype-specific enzyme-linked immunosorbent assay (ELISA) were determined to evaluate the effect of isotype on the sensitivity and specificity of Salmonella ELISA serology for distinguishing sera of Salmonella carriers from those of vaccinated and acutely infected cows. IgG2 titers to LPS and porin provide a more specific indicator of chronic Salmonella infection status than do IgG1 titers to the same antigens with little to no loss in sensitivity.     

Outbreak of Salmonella Typhimurium associated with feeder rodents

In December 2012, an increase in human Salmonella Typhimurium cases was identified in the province of Ontario, Canada launching an outbreak investigation. The outbreak spanned 3 years (2012–2014), with 134 cases reported from five Canadian provinces. There was a substantial burden of illness among children: 45% of cases were children 12 years old or under, and 23% of cases were under 5 years old. Epidemiologic, traceback and laboratory findings linked this outbreak to feeder rodents (used to feed snakes) supplied by a network of rodent breeders in Ontario. Cases likely acquired their illness through either direct or indirect contact with feeder rodents. This investigation not only contributes to the weight of evidence on the risk that feeder rodents pose, but also underscores the importance of investigating indirect animal contact and associated risks, especially for high‐risk individuals.     

Nose-to-nose transmission of Salmonella Typhimurium between weaned pigs

Little attention has been paid to the possibility of transmission of Salmonella in intensive pig production systems through alternate methods, such as airborne or direct nose-to-nose contact. This experimental study tested the hypothesis of nose-to-nose transmission of Salmonella enterica serovars Typhimurium (Trial I) and Agona (Trial II) in weaned pigs using stainless steel/glass isolation cabinets. In each trial, cabinet 1 (control pigs) and cabinet 2 (sentinel pigs) were connected directly to the fan unit. Cabinet 3 (seeded pigs) was not directly linked to the fan, but was arranged to receive a constant unidirectional airflow from cabinet 2 (sentinel pigs) through a 10cm diameter hole, which also allowed nose-to-nose contact between pigs housed in these two cabinets. Air was taken out of the system through ducts connecting cabinets 1 and 3 to the exhauster. Therefore, direct contact among seeded and sentinel pigs was allowed but possible aerial transference of contaminated particles between those cabinets was prevented. The system was opened 21 days post-inoculation and tissue samples were collected for bacteriological analysis. The recovery of nalidixic acid-resistant Salmonella Typhimurium from sentinel pigs corroborates the hypothesis of nose-to-nose transmission of that pathogen in pigs. However, serovar-related differences might exist regarding the nose-to-nose transmissibility of Salmonella in pigs, since Salmonella Agona was not detected in sentinel pigs (Trial II).     

Prevalence of the virulence plasmid in Salmonella Typhimurium isolates from pigs

To determine the prevalence of the virulence plasmid in Salmonella Typhimurium isolates from pigs in Japan, a total of 106 porcine isolates were subjected to PCR amplification for the detection of the virulence plasmid. Out of the isolates of S. Typhimurium, 38 (35.8%) harbored the virulence plasmid. The presence of the virulence plasmid was widely observed in the isolates from systemically infected pigs (92.0%, 23/25), compared with diarrheic (18.8%, 12/64) and apparently healthy pigs (17.6%, 3/17) (P<0.01).     

Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium

Salmonella continues to be a significant human health threat, and the objective of this study was to identify microorganisms with the potential to improve porcine food-safety through their antagonism of Salmonella. Anaerobic culture supernatants of 973 bacterial isolates from the gastrointestinal tract and feces of swine were screened for their capacity to inhibit the growth of Salmonella enterica serovar Typhimurium. Growth inhibition of 1000-fold or greater was observed from 16 isolates, and 16S rRNA sequencing identified the isolates as members of the genera Mitsuokella, Escherichia/Shigella, Anaerovibrio, Selenomonas, and Streptococcus. Four isolates were identified as Mitsuokella jalaludinii, and the mechanism of Salmonella Typhimurium growth inhibition by M. jalaludinii was further investigated. M. jalaludinii stationary phase culture supernatants were observed to significantly inhibit growth, and featured the production of lactic, succinic, and acetic acids. Aerobic and anaerobic S. Typhimurium growth was restored when the pH of the culture supernatants (pH 4.6) was increased to pH 6.8. However, S. Typhimurium growth in fermentation acid-free media was the same at pH 4.6 and pH 6.8 - indicating a synergistic effect between fermentation acid production and low pH as the cause of S. Typhimurium growth inhibition. Furthermore, exposure of S. Typhimurium to M. jalaludinii culture supernatants inhibited Salmonella invasion of HEp-2 cells by 10-fold. The results identify M. jalaludinii as a possible inhibitor of Salmonella growth and invasion in swine, and thus a potential probiotic capable of improving food safety.     

Differential responses of macrophages to Salmonella enterica serovars Enteritidis and Typhimurium

Macrophages are major effectors against Salmonella infection, and also transport bacteria between host tissues and provide a protected site for intracellular bacterial replication. We hypothesized that differences in chicken macrophage responses to Salmonella enterica serovar Enteritidis (SE) and serovar Typhimurium (ST) played a role in preferential infection of eggs by SE compared with ST. To test this hypothesis, we determined bacterial phagocytosis and intracellular viability and macrophage nitric oxide (NO) production following in vitro infection with SE or ST in the presence or absence of interferon-gamma (IFN-gamma). The effects of bacterial components, lipopolysaccharide (LPS), outer membrane proteins (OMP) and flagella, on NO production were also assessed. Our results showed: (1) in the presence or absence of IFN-gamma, the percentage macrophages phagocytizing SE and ST was similar; (2) the number of intracellular viable SE was significantly reduced compared with ST in the presence or absence of IFN-gamma; (3) increased macrophage necrosis was seen in the presence of IFN-gamma and ST; (4) Salmonella infection acted synergistically with IFN-gamma in induction of nitric oxide production; and (5) in the absence of IFN-gamma, macrophages produced significantly greater NO following treatment with SE outer membrane protein or flagella compared with ST OMP or flagella, while in the presence of IFN-gamma significantly less NO was produced following treatment with SE-LPS compared with ST-LPS. These results suggest that differential responses of chicken macrophages to SE versus ST may result in increased macrophage death with ST, which could result in an increased inflammatory response as compared to SE.     

鼠伤寒沙门菌毒力基因SptP的原核表达及生物信息学分析

【目的】试验旨在获得高效表达的鼠伤寒沙门菌SptP蛋白并进行生物信息学分析,为其功能研究和互作蛋白的筛选提供理论依据。【方法】通过PCR技术扩增SptP基因,并将该序列连接至pET-32a （+）载体,构建鼠伤寒沙门菌SptP基因原核表达载体pET32a-SptP。通过热激法将重组质粒导入大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达、纯化重组蛋白,并经过SDS-PAGE和Western blotting验证;应用在线软件对SptP蛋白进行生物信息学分析。【结果】PCR成功扩增出大小为1 632 bp的SptP基因。SptP重组蛋白在大肠杆菌BL21（DE3）感受态细胞中成功诱导表达、纯化,得到分子质量为79.7 ku的蛋白。SptP蛋白分子式为C₂₆₂₅H₄₂₅₇N₇₄₅O₈₁₂S₂₅,分子质量为60 047.68 u,无跨膜结构,无信号肽存在,理论等电点为8.75,有57个潜在的磷酸化位点,主要定位于细胞核、细胞质、高尔基体、细胞骨架、分泌系统的囊泡、质膜,占比分别为43.5%、34.8%、8.7%、4.3%、4.3%和4.3%。SptP蛋白二级结构由α-螺旋、延伸链、β-转角及无规则卷曲组成,占比分别为43.65%、14.92%、4.42%和37.02%。【结论】本研究构建了表达SptP蛋白的重组质粒pET32a-SptP,获得分子质量为79.7 ku的SptP重组蛋白,阐明了SptP蛋白的基本理化性质和生物学功能,为后续SptP蛋白与宿主细胞互作的作用机制及鼠伤寒沙门菌新疫苗的制备提供理论基础和试验依据。     

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.     

Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium

Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs.     

Comparison of Salmonella enterica subspecies enterica serovar Typhimurium isolates from dairy cattle and humans using in vitro assays of virulence

Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella typhimurium) can infect and cause disease in a wide range of host species however there have been suggestions that this serovar may have genes involved with host range or specificity [Tsolis, R.M., Townsend, S.M., Miao, E.A., Miller, S.I., Ficht, T.A., Adams, L.G., Baumler, A.J., 1999. Identification of a putative S. enterica serotype Typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis. Infect. Immun. 67 (12), 6385-6393]. Our goal in this study was to determine if in vitro virulence assays would support this suggestion. Twelve human and 10 bovine isolates of S. typhimurium from a single county in California were evaluated using in vitro virulence assays of adhesion and invasion. The resulting data was combined with results from previously reported genotypic and phenotypic testing of the isolates and statistical analysis performed using multivariate general linear models. Human isolates had higher adhesion values in each of the statistical models tested (p<0.05) but no statistical differences were found in the invasion values of human and bovine source isolates. Both adhesion and invasion values differed between the two largest groups of isolates segregated on the basis of pulsed-field gel patterns. The findings suggest there may be genetically defined in vitro virulence attributes in S. typhimurium that are associated with host species.     

减毒沙门氏菌作为口服活疫苗载体的研究进展

文章就沙门氏菌相关基因的特点、减毒沙门氏菌载体激发的免疫应答、进入机体免疫系统的机制、减毒沙门氏菌的应用、载体疫苗的优越性及潜在危险性几方面综述了减毒沙门氏菌作为口服疫苗载体的研究进展,为新型菌苗的研制提供一种新的思路。