Supplemental N-acyl homoserine lactonase alleviates intestinal disruption and improves gut microbiota in broilers challenged by Salmonella Typhimurium

Background Salmonella Typhimurium challenge causes a huge detriment to chicken production.N-acyl homoserine lactonase(AHLase),a quorum quenching enzyme,potentially inhibits the growth and virulence of Gram-negative bacteria.However,it is unknown whether AHLase can protect chickens against S.Typhimurium challenge.This study aimed to evaluate the effects of AHLase on growth performance and intestinal health in broilers challenged by S.Typhimurium.A total of 240 one-day-old female crossbred broilers(817C)were randomly divided into 5 groups(6 replicates/group):negative control(NC),positive control(PC),and PC group supplemented with 5,10 or 20 U/g AHLase.All birds except those in NC were challenged with S.Typhimurium from 7 to 9 days of age.All parameters related to growth and intestinal health were determined on d 10 and 14.Results The reductions(P<0.05)in body weight(BW)and average daily gain(ADG)in challenged birds were alleviated by AHLase addition especially at 10 U/g.Thus,samples from NC,PC and PC plus 10 U/g AHLase group were selected for further analysis.S.Typhimurium challenge impaired(P<0.05)intestinal morphology,elevated(P<0.05)ileal inflammatory cytokines(IL-1βand IL-8)expression,and increased(P<0.05)serum diamine oxidase(DAO)activity on d 10.However,AHLase addition normalized these changes.Gut microbiota analysis on d 10 showed that AHLase reversed the reductions(P<0.05)in several beneficial bacteria(e.g.Bacilli,Bacillales and Lactobacillales),along with increases(P<0.05)in certain harmful bacteria(e.g.Proteobacteria,Gammaproteobacteria,Enterobacteriaceae and Escherichia/Shigel a)in PC group.Furthermore,AHLase-induced increased beneficial bacteria and decreased harmful bacteria were basically negatively correlated(P<0.05)with the reductions of ileal IL-1βand IL-8 expression and serum DAO activity,but positively correlated(P<0.05)with the increased BW and ADG.Functional prediction revealed that AHLase abolished S.Typhimurium-induced upregulations(P<0.05)of certain pathogenicity-related pathways such as lipopolysaccharide biosynthesis,shigellosis,bacterial invasion of epithelial cells and pathogenic Escherichia coli infection of gut microbiota.Conclusions Supplemental AHLase attenuated S.Typhimurium-induced growth retardation and intestinal disruption in broilers,which could be associated with the observed recovery of gut microbiota dysbiosis.

Dietary supplementation of bilberry anthocyanin on growth performance,intestinal mucosal barrier and cecal microbes of chickens challenged with Salmonella Typhimurium

Background Anthocyanins(AC)showed positive effects on improving the intestinal health and alleviating intestinal pathogen infections,therefore,an experiment was conducted to explore the protective effects of supplemented AC on Salmonella-infected chickens.Methods A total of 240 hatchling chickens were randomly allocated to 4 treatments,each with 6 replicates.Birds were fed a basal diet supplemented with 0(CON,and ST),100(ACL)and 400(ACH)mg/kg of AC for d 60,and orally challenged with PBS(CON)or 10^(9) CFU/bird(ST,ACL,ACH)Salmonella Typhimurium at d 14 and 16.Results(1)Compared with birds in ST,AC supplementation increased the body weight(BW)at d 18 and the average daily gain(ADG)from d 1 to 18 of the Salmonella-infected chickens(P<0.05);(2)AC decreased the number of Salmonella cells in the liver and spleen,the contents of NO in plasma and inflammatory cytokines in ileal mucosa of Salmonella-infected chickens(P<0.05);(3)Salmonella infection decreased the ileal villi height,villi height to crypt depth(V/C),and the expression of zonulaoccludins-1(ZO-1),claudin-1,occludin,and mucin 2(MUC2)in ileal mucosa.AC supplementation relieved these adverse effects,and decreased ileal crypt depth(P<0.05);(4)In cecal microbiota of Salmonella-infected chickens,AC increased(P<0.05)the alpha-diversity(Chao1,Pd,Shannon and Sobs indexes)and the relative abundance of Firmicutes,and decreased(P<0.05)the relative abundance of Proteobacteria and Bacteroidota and the enrichment of drug antimicrobial resistance,infectious bacterial disease,and immune disease pathways.Conclusions Dietary AC protected chicken against Salmonella infection via inhibiting the Salmonella colonization in liver and spleen,suppressing secretion of inflammatory cytokines,up-regulating the expression of ileal barrier-related genes,and ameliorating the composition and function of cecal microbes.Under conditions here used,100 mg/kg bilberry anthocyanin was recommended.

Pretreatment with probiotics Enterococcus faecium NCIMB 11181 attenuated Salmonella Typhimurium-induced gut injury through modulating intestinal microbiome and immune responses with barrier function in broiler chickens

Background:Preventing Salmonella infection and colonization in young birds is key to improving poultry gut health and reducing Salmonella contamination of poultry products and decreasing salmonellosis for human consumption(poultry meat and eggs).Probiotics can improve poultry health.The present study was conducted to investigate the impact of a probiotics,Enterococcus faecium NCIMB 11181(E.faecium NCIMB 11181)on the intestinal mucosal immune responses,microbiome and barrier function in the presence or absence of Salmonella Typhimurium(S.Typh-imurium,ST)infection.Methods:Two hundred and forty 1-day-old Salmonella-free male broiler chickens(Arbor Acres AA+)were randomly allocated to four groups with 6 replicate cages of 10 birds each.The four experimental groups were follows:(1)nega-tive control(NC),(2)S.Typhimurium,challenged positive control(PC),(3)the E.faecium NCIMB 11181-treated group(EF),(4)the E.faecium NCIMB 11181-treated and S.Typhimurium-challenged group(PEF).Results:Results indicated that,although continuous feeding E.faecium NCIMB 11181 did not obviously alleviate growth depression caused by S.Typhimurium challenge(P>0.05),E.faecium NCIMB 11181 addition significantly blocked Salmonella intestinal colonization and translocation(P<0.05).Moreover,supplemental E.faecium NCIMB 11181 to the infected chickens remarkably attenuated gut morphological structure damage and intestinal cell apoptosis induced by S.Typhimurium infection,as evidenced by increasing gut villous height and reducing intes-tinal TUNEL-positive cell numbers(P<0.05).Also,E.faecium NCIMB 11181 administration notably promoting the production of anti-Salmonella antibodies in intestinal mucosa and serum of the infected birds(P<0.05).Addition-ally,16S rRNA sequencing analysis revealed that E.faecium NCIMB 11181 supplementation ameliorated S.Typhimu-rium infection-induced gut microbial dysbiosis by enriching Lachnospiracease and Alistipes levels,and suppressing Barnesiella abundance.Predicted function analysis indicated that the functional genes of cecal microbiome involved in C5-branched dibasic acid metabolism;valine,leucine and isoleucine biosynthesis;glycerolipid metabolism and lysine biosynthesis were enriched in the infected chickens given E.faecium NCIMB 11181.While alanine,asparate and glutamate metabolism;MAPK signal pathway-yeast;ubiquine and other terpenoid-quinore biosynthesis,protein processing in endoplasmic reticulum;as well as glutathione metabolism were suppressed by E.faecium NCIMB 11181 addition.Conclusion:Collectively,our data suggested that dietary E.faecium NCIBM 11181 supplementation could ameliorate S.Typhimurium infection-induced gut injury in broiler chickens.Our findings also suggest that E.faecium NCIMB 11181 may serve as an effective non-antibiotic feed additive for improving gut health and controlling Salmonella infection in broiler chickens.

Exopolysaccharides of Lactobacillus rhamnosus GG ameliorate Salmonella typhimurium-induced intestinal inflammation via the TLR4/NF-κB/MAPK pathway

Background Salmonella typhimurium(S.T),as an important foodborne bacterial pathogen,can cause diarrhea and gastroenteritis in humans and animals.Numerous studies have confirmed that exopolysaccharides(EPSs)have various biological functions,but the mechanism through which EPSs improve the immunity of animals against the invasion of pathogenic bacteria is unclear.Here,we explored the protective effect of EPSs of Lactobacillus rhamnosus GG(LGG)on the S.T-infected intestine.Methods Mice received adequate food and drinking water for one week before the start of the experiment.After 7 d of prefeeding,2×108 CFU/mL S.T solution and an equivalent volume of saline(control group)were given orally for 1 d.On the fourth day,the mice were treated with 0.5 mg/mL EPSs,1.0 mg/mL EPSs,2.0 mg/mL EPSs,or 2.0 mg/mL penicillin for 7 d.Finally,the body and relative organ weight,histological staining,and the levels of antioxidant enzyme activity and inflammatory cytokines were determined.Results The S.T-infected mice exhibited symptoms of decreased appetite,somnolence,diarrhea and flagging spirit.Treatment with EPSs and penicillin improved the weight loss of the mice,and the high dose of EPSs showed the best therapeutic effect.EPSs significantly ameliorated S.T-induced ileal injury in mice.High-dose EPSs were more effective than penicillin for alleviating ileal oxidative damage induced by S.T.The mRNA levels of inflammatory cytokines in the ileum of mice showed that the regulatory effects of EPSs on inflammatory cytokines were better than those of penicillin.EPSs could inhibit the expression and activation of key proteins of the TLR4/NF-κB/MAPK pathway and thereby suppress the level of S.T-induced ileal inflammation.Conclusions EPSs attenuate S.T-induced immune responses by inhibiting the expression of key proteins in the TLR4/NF-κB/MAPK signaling pathway.Moreover,EPSs could promote bacterial aggregation into clusters,which may be a potential strategy for reducing the bacterial invasion of intestinal epithelial cells.

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.

Oral Immunization of Mice With Vaccine of Attenuated Salmonella typhimurium Expressing Helicobacter pylori Urease B Subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Salmonella typhimurium myopericarditis: A case report and review of literature

BACKGROUND Non-typhoidal salmonella(NTS)is a rare,but well-established cause of myopericarditis.Presenting symptoms may be varied,however often revolve around the dual presentation of both myopericarditis and infectious diarrhoea.Given the rarity of NTS related myopericarditis,we conducted a systematic review of the literature,identifying 41 previously reported cases.CASE SUMMARY We present the case of an otherwise healthy 39-year old male,presenting with chest pain in the setting of documented Salmonella typhimurium infection.After further investigation with echocardiogram and laboratory blood tests,a diagnosis of NTS associated myopericarditis was made,and the patient received antibiotic treatment with an excellent clinical outcome.Overall,myopericarditis is rare in NTS.Although treatment for myopericarditis has not been well established,there are guidelines for the treatment of NTS infection.In our review,we found that the majority of NTS cases has been pericarditis(27/42,64.3%),with an average age of 48.3 years,and 71.4%being male.The average mortality across all cases was 31%.CONCLUSION Myopericarditis is a rare,but potentially serious complication of NTS infection,associated with an increased morbidity and mortality.

Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection,intestinal permeability,and cecal microbiota composition in broiler chickens

Background:Interest in the use of natural feed additives as an alternative to antimicrobials in the poultry industry has increased in recent years because of the risk of bacterial resistance.One of the most studied groups are polyphenolic compounds,given their advantages over other types of additives and their easy potentiation of effects when complexes are formed with metal ions.Therefore,the objective of the present study was to evaluate the impact of dietary supplementation of copper acetate(CA),curcumin(CR),and their combination(CA-CR)against Salmonella Typhimurium colonization,intestinal permeability,and cecal microbiota composition in broiler chickens through a laboratory Salmonella infection model.S.Typhimurium recovery was determined on day 10 post-challenge by isolating Salmonella in homogenates of the right cecal tonsil(12 chickens per group)on Xylose Lysine Tergitol-4(XLT-4)with novobiocin and nalidixic acid.Intestinal integrity was indirectly determined by the fluorometric measurement of fluorescein isothiocyanate dextran(FITC-d)in serum samples from blood obtained on d 10 post-S.Typhimurium challenge.Finally,microbiota analysis was performed using the content of the left caecal tonsil of 5 chickens per group by sequencing V4 region of 16S rRNA gene.Results:The results showed that in two independent studies,all experimental treatments were able to significantly reduce the S.Typhimurium colonization in cecal tonsils(CT,P<0.0001)compared to the positive control(PC)group.However,only CA-CR was the most effective treatment in reducing S.Typhimurium counts in both independent studies.Furthermore,the serum fluorescein isothiocyanate dextran(FITC-d)concentration in chickens treated with CR was significantly lower when compared to PC(P=0.0084),which is related to a decrease in intestinal permeability and therefore intestinal integrity.The effect of dietary treatments in reducing Salmonella was further supported by the analysis of 16S rRNA gene sequences using Linear discriminant analysis effect size(LEfSe)since Salmonella was significantly enriched in PC group(LDA score>2.0 and P<0.05)compared to other groups.In addition,Coprobacillus,Eubacterium,and Clostridium were significantly higher in the PC group compared to other treatment groups.On the contrary,Fecalibacterium and Enterococcus in CR,unknown genus of Erysipelotrichaceae at CA-CR,and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively.Conclusions:CR treatment was the most effective treatment to reduce S.Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Salmonella Typhimurium infection disrupts but continuous feeding of Bacillus based probiotic restores gut microbiota in infected hens

Background:The gut microbiota plays an important role in the colonisation resistance and invasion of pathogens.Salmonella Typhimurium has the potential to establish a niche by displacing the microbiota in the chicken gut causing continuous faecal shedding that can result in contaminated eggs or egg products.In the current study,we investigated the dynamics of gut microbiota in laying chickens during Salmonella Typhimurium infection.The optimisation of the use of an infeed probiotic supplement for restoration of gut microbial balance and reduction of Salmonella Typhimurium load was also investigated.Results:Salmonella infection caused dysbiosis by decreasing(FDR<0.05)the abundance of microbial genera,such as Blautia,Enorma,Faecalibacterium,Shuttleworthia,Sellimonas,Intestinimonas and Subdoligranulum and increasing the abundance of genera such as Butyricicoccus,Erysipelatoclostridium,Oscillibacter and Flavonifractor.The higher Salmonella Typhimurium load resulted in lower(P<0.05)abundance of genera such as Lactobacillus,Alistipes,Bifidobacterium,Butyricimonas,Faecalibacterium and Romboutsia suggesting Salmonella driven gut microbiota dysbiosis.Higher Salmonella load led to increased abundance of genera such as Caproiciproducens,Acetanaerobacterium,Akkermansia,Erysipelatoclostridium,Eisenbergiella,EscherichiaShigella and Flavonifractor suggesting a positive interaction of these genera with Salmonella in the displaced gut microbiota.Probiotic supplementation improved the gut microbiota by balancing the abundance of most of the genera displaced by the Salmonella challenge with clearer effects observed with continuous supplementation of the probiotic.The levels of acetate and butyrate in the faeces were not affected(P>0.05)by Salmonella challenge and the butyrate level was increased by the continuous feeding of the probiotic.Probiotic supplementation in Salmonella challenged chickens resulted in higher level of propionate.Continuous probiotic supplementation decreased(P<0.05)the overall mean load of Salmonella in faeces and had a significant effect on Salmonella load reduction in internal organs.Conclusions:Salmonella challenge negatively impacts the diversity and abundance of many gut microbial genera involved in important functions such as organic acid and vitamin production.Strategic feeding of a Bacillus based probiotic helps in restoring many of the microbial genera displaced by Salmonella Typhimurium challenge.

Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPI1-mediated gene expression and infectivity

Background： Salmonella enter/ca serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands （SPI）, producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell infection： pyruvate formate lyase I （pf/B） and b/functional acetaldehyde-CoA/alcohol dehydrogenase （adhE）. We were therefore interested in examining the role these genes may play in the transmission of Salmonella to humans. Methods： Mutant strains of Salmonella with single gene deletions for pflB and adhE were created. Invasion and growth in human HCT-8 intestinal epithelial cells and THP-1 macrophages was examined. Quantitative PCR was performed on 19 SPI-1 genes. Results： In HCT-8 cells, both mutant strains had significantly higher intracellular counts than the wild-type from 4 to 48 h post-infection. Various SPI-1 genes in the mutants were up-regulated over the wild-type as early as 1 h and lasting until 24 h post-infection. In THP-1 cells, no significant difference in internal Salmonella counts was observed; however, SPI-1 genes were largely down-regulated in the mutants during the time-course of infection. We also found five SPI-1 genes - hilA, hiIC hill）, sicP and rtsA - which were up-regulated in at least one of the mutant strains in log-phase broth cultures alone. We have therefore identified a set of SPI-1 virulence genes whose regulation is effected by the central metabolism of Salmonella.

Mutagenic activity in Salmonella typhimurium mutants of the dust organic extract and its five fractions derived from an aluminum electrolytic plant

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Understanding the effects of intramuscular injection and feed withdrawal on Salmonella Typhimurium shedding and gut microbiota in pullets

Background:Gut microbiota plays a key role in health,immunity,digestion,and production in layers.Factors such as environment,diet,diseases,stress,and flock management significantly affect gut microbiota;however,it is not known how potential stressors such as intramuscular injections or feed withdrawal alter the composition of gut microbiota that result in increased the shedding level of foodborne pathogens.In the current study,the effects of intramuscular corticosterone injection and feed withdrawal were evaluated to understand their role in Salmonella Typhimurium shedding and changes in the composition of gut microbiota in layers.Results:Salmonella shedding was observed for 8 weeks post-infection.There was a significant increase in Salmonella Typhimurium count after intramuscular injection and feed withdrawal.The Salmonella infected and the negative control groups showed significant differences in the abundance of different genera in gut microbiota at week 1 and up to week 7 post infection.The infected group showed a significant reduction in alpha diversity of gut microbiota.Firmicutes reduced significantly(P<0.05)after intramuscular injection,while the feed withdrawal groups did not cause any significant changes in Proteobacteria-Firmicutes ratio.Furthermore,intramuscular injection resulted in a significant change in alpha diversity of gut microbiota.Conclusions:Exposure of chicks to relatively low dose of Salmonella Typhimurium can lead to persistent shedding in pullets.The Salmonella Typhimurium infection disrupted the gut microbiota composition immediately after infection.The potential stress of intramuscular injection and feed withdrawal significantly increased the Salmonella Typhimurium count in faeces.The intramuscular injection also resulted in a significant alteration of the Proteobacteria-Firmicutes ratio,which could increase the risk of dysbiosis.

Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

Rapid,sensitive,point-of-care detection of pathogenic bacteria is important for food safety.In this study,we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip(QBs-labelled LFIAS)combined with strand displacement loop-mediated isothermal amplification(SD-LAMP)for quantitative Salmonella Typhimurium(ST)detection.Quantum dot nanobeads(QBs)served as fluorescence reporters,providing good detection efficiency.The customizable strand displacement(SD)probe was used in LAMP to improve the specificity of the method and prevent by-product capture.Detection was based on a sandwich immunoassay.A fluorescence strip reader measured the fluorescence intensity(FI)of the test(T)line and control(C)line.The linear detection range of the strip was 10^(2)–10^(8) colony forming units(CFU)·mL^(-1).The visual limit of detection was 10^(3) CFU·mL^(-1),indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips.ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction(PCR)and SD-LAMP.We detected ST in foods with an acceptable recovery of 85%–110%.The method is rapid,simple,almost equipment-free,and suitable for bacterial detection in foods and for clinical diagnosis.

Anti-angiogenesis Effect on Glioma of Attenuated Salmonella Typhimurium Vaccine Strain with flk-1 Gene

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial Gl261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.

Plant Extracts and Essential Oils at Concentrations Acceptable to a Sensory Panel Inactivate Salmonella Typhimurium DT104 in Ground Pork

A potential method to inhibit pathogenic bacterial growth in meat is through the introduction of plant-derived antimicrobials. Because these antimicrobials may also adversely affect the sensory characteristics of the meat, the objectives of this study were 1) to define the appropriate concentrations of olive extract, apple extract, oregano oil, and cinnamon oil added to ground pork that are acceptable to a sensory panel, and 2) to determine their antimicrobial activities against Salmonella Typhimurium DT104 in inoculated ground pork. Plant extracts were evaluated against two initial inoculum levels (6 and 4 log CFU/g of pork) of Salmonella. Sensory tests showed that acceptable concentrations of oregano and cinnamon oils were 0.5% and of olive and apple extracts were 3%, respectively. Ground pork samples were inoculated with Salmonella, treated with antimicrobials at various concentrations (0.1% - 0.5% cinnamon and oregano essential oils and 3% - 5% olive and apple extracts), and stored at 4°C for 7 days. Survivors were enumerated at days 0, 3, 5, and 7. Cinnamon oil at 0.5% and olive extract at 3% induced a 1.0 and a 0.9 log CFU/g (from 6-log CFU/g initial inoculum) reduction, respectively, at day 7. At 3%, olive extract showed a 1.06 log CFU/g maximum reduction of Salmonella from a 4-log CFU/g initial inoculum. Pork samples containing oregano oil and apple extract did not show a significant reduction compared to the control without the antimicrobials. The results indicate that cinnamon oil and olive extract can potentially be applied at consumer-acceptable concentrations against low levels of S. Typhimurium DT104 in ground pork.

Culturability and Viability of Salmonella Typhimurium during Photo-Fenton Process at pH 5.5 under Solar Simulated Irradiation

Culturability and viability techniques such as plate count on solid agar (PC), Most Probable Number (MPN) and Direct Viable Count-Fluorescence in Situ Hybridation (DVC FISH) were used to study the inactivation of Salmonella typhimurium by photo-Fenton process at pH 5.5. In the presence of only simulated solar irradiation (500 W·m-2), S. typhimurim showed that both culturability measured by MPN and viability (measured by DVC FISH) underwent just a slight decreasing of 2 and 1 log respectively after 240 min of light exposition while culturability measured by PC did not show any change. Results after 48 h of dark conditions did not reveal re-growth. However, when experiment was carried out in the presence of 2 mg L-1 of Fe3+ and 20 mg L-1 of H2O2 and pH 5.5, culturability was strongly affected after 240 min of simulated solar irradiation;nevertheless, viability was only slightly altered (~1 log). During dark period of 48 h changes on culturability and viability were not observed. On the other hand, it was also found that sugar metabolism was affected rather the amino-acids in S. typhimurium cells irradiated at different times upon photo-Fenton conditions. These findings might suggest for the first time that photo-Fenton process at pH 5.5 could induce viable but nonculturable state (VBNC) on waterborne S. typhimurium and that probably sugar metabolism damage could activate the VBNC state.

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicity of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 4 wk post-challenge. RESULTS: The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistentwith the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P < 0.01). CONCLUSION: Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.

Detection of Salmonella typhimurium in retail chicken meat and chicken giblets

Objective:To detect Salmonella typhimurium(S.typhimurium),one of the most frequently isolated serovars from food borne outbreaks throughout the world,in retail raw chicken meat and giblets.Methods:One hundred samples of retail raw chicken meat and giblets(Liver,heart and gizzard)which were collected from Assiut city markets for detection of the organism and by using Duplex PCR amplification of DNA using rfbj andfliC genes.Results:S.typhimurium was detected at rate of 44%,40%and 48%in chicken meat,liver and heart,respectively,but not detected in gizzard.Conclusions:The results showed high incidence of 5.typhimurium in the examined samples and greater emphasis should be applied on prevention and control of contamination during processing for reducing food-borne risks to consumers.

Systemic immune responses to oral administration of recombinant attenuated Salmonella typhimurium expressing Helicobacter pylori urease in mice

AIM: To evaluate whether attenuated Salmonellatyphimurium producing Helicobacter pylori ( H pylori) urease subunit B (UreB) could induce systemic immune responses against H pylori infection.METHODS: Attenuated S. typhimurium SL3261 was used as a live carrier of plasmid pTC01-UreB, which encodes recombinant H pylori UreB protein. Balb/c mice were given oral immunization with two doses of SL3261/pTC01-UreBat a 3-wk interval. Twelve weeks after oral immunization of mice, serum IgG antibodies were evaluated by ELISA assay. Gamma interferon (IFN-γ) and interleukin 10 (IL-10)in the supernatant of spleen cell culture were also assessed by ELISA.RESULTS: After oral immunization of mice, serum specific IgG antibodies against UreB in vaccine group were much higher than that in PBS and native Salmonella SL3261control groups (A450, 0.373±0.100 vs 0.053±0.022, 0.142±0.039, respectively, P＜0.01). Moreover, IFN-γ in vaccine group was on average 167.53±29.93 pg/mL, which showed a significant increase vs that of PBS control group (35.68±3.55 pg/mL, P＜0.01). There was also a tremendous increase of IL-L0 in vaccine group compared to PBS and SL3261 control groups (275.13±27.65 pg/mL vs 56.00±7.15 pg/mL, 68.02±15.03 pg/mL, respectively, P＜0.01). In addition, no obvious side effects in mice and no change in gastric inflammation were observed. CONCLUSION: The multiple oral immunizations with the attenuated S. typhimurium expressing H pylori UreB could induce significant systemic immune responses, suggesting it may be used as oral vaccine against H pylori infection.