FliC蛋白R91S突变对肠炎沙门菌鞭毛形态和小鼠体内定植的影响

沙门菌血清D群3个血清型FliC蛋白氨基酸序列比对分析表明肠炎沙门菌与鸡伤寒沙门菌完全相同，二者与鸡白痢沙门菌存在第91位氨基酸位点差异。本研究旨在探究肠炎沙门菌FliC蛋白第91位精氨酸突变对鞭毛形态、细菌运动性和小鼠体内定植能力的影响。运用λ-Red同源重组技术删除肠炎沙门菌CICC10467 fliC基因，构建系列反式回补突变株，通过体外生长特性试验和Western blot试验分析各菌株生长和FliC蛋白表达情况，运动性试验分析各菌株在半固体琼脂中的泳动能力，电子显微镜观察各菌株鞭毛形态，细胞感染试验分析各菌株的细胞黏附和入侵能力，动物感染试验分析各菌株的组织侵染能力。结果表明，fliC基因缺失株及点突变回补株与野生株的体外生长能力无显著差异（P ≥ 0.05）。fliC基因缺失后肠炎沙门菌不表达鞭毛蛋白，各点突变回补株与野生株鞭毛蛋白表达量无明显差异。FliC蛋白R91S突变导致肠炎沙门菌鞭毛形态由超螺旋形态转变为钝直、柔韧度减弱，运动性显著降低（P<0.000 1），对RAW264.7和HCT116细胞的黏附入侵能力显著下降（P<0.001），对BALB/c小鼠的器官侵染能力显著减弱（P<0.001）。综上表明，FliC蛋白第91位精氨酸对维持细菌运动性至关重要，第91位精氨酸突变能够显著改变肠炎沙门菌鞭毛形态，减弱肠炎沙门菌在小鼠体内定植能力。     

Development of a PCR system for the characterisation of Salmonella flagellin genes

Analysis of flagellin genes was carried out on strains of Salmonella Typhimurium, Salmonella Hadar, Salmonella Abortusequi, Salmonella Enteritidis and Salmonella Gallinarum serovars, using a PCR system designed in this study. The purpose of these studies was to explore the flagellin genes of biphasic and monophasic Salmonellae for future targeted genetic interventions. The PCR primers were designed for two different structural genes of flagellin (fliC, fljB), for the repressor of fliC (fljA), for the operator region of fliC, and for the invertase system responsible for phase variation in Salmonella (hin, hixL, hixR). PCR analysis revealed that all of the examined genes (fliC, fliC-operator, fljB, fljA, hin, hixL, hixR) were present in all S. Typhimurium (n = 10) and S. Hadar (n = 10) strains tested. The results obtained on S. Typhimurium and S. Hadar strains confirmed their biphasic character at DNA level. However, the S. Enteritidis (n = 46) and S. Gallinarum (n = 5) strains lacked the invertase system (hin, hixL, hixR) as well as the fljA and fljB genes, while fliC and its operator were detectable. Consequently, the S. Enteritidis strains could only express fliC gene resulting in phase H1 flagellin. The examined S. Gallinarum strains were also demonstrated to have a cryptic flagellin gene (fliC). On the other hand, PCR results on S. Abortusequi (n = 2) indicated that both flagellin genes (fliC, fljB) and the whole phase variation system were present in both strains tested but only the H2 phase gene (fljB) was expressed. The phenotype of these strains could be clarified by motility test and/or by classical flagellar serology. The findings are also substantiated by the results of serovar-specific PCR for S. Typhimurium and S. Enteritidis. In conclusion, the PCR system developed in this study proved to be suitable for characterisation of Salmonella flagellin genes and confirmed serological results regarding all S. Typhimurium, S. Hadar and S. Enteritidis strains. This system could also identify cryptic flagellar genes of S. Abortusequi and S. Gallinarum.     

Disruption of srtA gene in Streptococcus suis results in decreased interactions with endothelial cells and extracellular matrix proteins

Streptococcus suis, a major pathogen of swine, is an emerging zoonotic agent which causes meningitis and septic shock. In this study, we investigated the ability of S. suis mutant strain (SRTDeltaA) lacking the sortase A gene (srtA) to interact with host cells and extracellular matrix (ECM) proteins, as well as its virulence in a mouse infection model. We demonstrated that mutant SRTDeltaA had reduced capacity to adhere to and invade porcine brain microvascular endothelial cells compared to the wild-type strain. In addition, mutant SRTDeltaA also showed significantly less adherence to plasma fibronectin, cellular fibronectin and collagen type I. However, disruption of srtA had little effect on the virulence of S. suis in a mouse intraperitoneal model of infection. These results indicate that surface proteins anchored by sortase A are required for a normal level of bacterial binding. However, other factors may also be important for S. suis virulence and interaction with host tissues.     

Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues

Surface-layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. This study aimed to isolate and identify Slps carrying Lactobacillus species from the porcine intestine and faeces and to characterize these S-layer-expressing strains for their ability to adhere to the pig and human intestinal cells and to extracellular matrix (ECM) proteins. In total 99 strains, putatively belonging to the genus Lactobacillus, were isolated as pure cultures. SDS-PAGE and a gene probe specific for the Lactobacillus brevis ATCC 8287 S-layer protein gene (slpA) were used to screen the presence of strains possessing putative Slps. Eight of the 99 pure cultures exhibited Slps according to the SDS-PAGE analyses. In these strains the presence of genes encoding Slps was confirmed by PCR and partial sequencing. Only one isolate of the 99 strains gave a positive hybridisation signal with the L. brevis slpA probe but did not appear to produce S-layer protein. Their taxonomic identification, based on phenotyping and the 16S rRNA sequences, revealed that the eight S-layer protein-producing strains were closely related to Lactobacillus amylovorus, Lactobacillus sobrius and Lactobacillus crispatus. The strain with the slpA positive hybridisation result was identified as Lactobacillus mucosae. The SDS-extractable protein profile, the size of the putative S-layer protein and binding capability of the strains varied greatly, even among the isolates belonging to the same Lactobacillus cluster. Removal of the intact Slps from the bacterial surface by extraction with guanidine hydrochloride reduced the adhesion of some strains to fibronectin and laminin, whereas, the adhesiveness to laminin increased with some strains.     

The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in a IPEC-J2 cell model in vitro

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.     

Detection and identification of salmonellas from poultry-related samples by PCR

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.     

Role of beta1 integrins in adhesion of canine mastocytoma cells to extracellular matrix proteins

The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with alpha and beta subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin beta1 and alpha1 through alpha5 subunits and that they moderately expressed the alpha6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-beta1 and alpha5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-beta1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-alpha integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.     

鼠伤寒沙门菌鞭毛蛋白与新城疫病毒F蛋白的融合表达及免疫原性分析

应用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白fliC基因以及含有新城疫病毒F蛋白部分表位的基因片段,通过柔性肽(Gly4Ser)2编码序列将二者串联并克隆到质粒pET30a+上,获得重组原核表达质粒pET-fliC-F,将其转化入大肠埃希菌BL21(DE3)中表达。Western blot证实重组菌表达的fliC-F融合蛋白能与小鼠抗fliC和抗F蛋白两种多抗血清发生特异性反应。动物试验显示,fliC-F融合蛋白能够刺激C3H/HeJ小鼠产生针对F蛋白的特异性血清抗体,说明原核表达系统表达的fliC-F融合蛋白具有较好的免疫原性。     

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.     

Factors influencing the cell adhesion and invasion capacity of Mycoplasma gallisepticum

Background

The cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, R_high and R_low, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.

Results

Collagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain R_high, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that R_high as well as R_low are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.

Conclusion

ECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.     

Effects of nonimmunized egg yolk powder-supplemented feed on Salmonella enteritidis prevention and elimination in broilers

Chicken consumption is a newly identified risk factor in Salmonella enterica serovar Enteritidis (SE) infection in humans. SE is widely distributed in commercial chicken flocks and high levels of cecal carriage and shedding may lead to broiler meat contamination. In the present study, the preventive and eliminative effect of nonimmunized freeze-dried egg yolk powder (EYP) on SE in broilers was investigated. In the prevention trial, reduced SE counts were observed in liver (P < or = 0.05), cecal contents, and fecal shedding (P < or = 0.05) in birds fed 10% or 5% EYP. Histological examination of cecal wall and cecal tonsils at 23 days postinfection indicated a lesser degree of intestinal pathology. In the elimination trial, a significantly lower (P < or = 0.05) number of SE reached the liver and spleen, and a reduction in cecal carriage and fecal shedding was observed. The histological changes in the cecal mucosa and cecal tonsils reflected an apparent inflammation and mucosal repair and also suggested that the infection had not completely resolved, confirming SE bacterial isolations in the cecal tissue. The present study indicates that supplementing the diets of broilers with 5% nonimmunized EYP, at the early stages of the growing period, reduces preharvest Salmonella load with a minimal degree of intestinal pathology.     

Decellularization of Extracellular Matrix from Equine Skeletal Muscle

Equine represents an attractive animal model for musculoskeletal tissue diseases, exhibiting much similarity to the injuries that occur in humans. Cell therapy and tissue bioengineering have been widely used as a therapeutic alternative by regenerative medicine in musculoskeletal diseases. Thus, the aim of this study was to produce an acellular biomaterial of equine skeletal muscle and to evaluate its effectiveness in supporting the in vitro culture of equine induced pluripotency stem cells (iPSCs). Biceps femoris samples were frozen at −20°C for 4 days and incubated in 1% sodium dodecyl sulfate (SDS), 5 mM EDTA + 50 mM Tris and 1% Triton X-100; the effectiveness of the decellularization was evaluated by the absence of remnant nuclei (histological and 4′,6-diamidino-2-phenylindole [DAPI] analysis), preservation of extracellular matrix (ECM) proteins (immunofluorescence and immunohistochemistry) and organization of ECM ultrastructure (scanning electron microscopy). Decellularized samples were recellularized with iPSCs at the concentration of 50,000 cells/cm² and cultured in vitro for 9 days, and the presence of the cells in the biomaterial was evaluated by histological analysis and presence of nuclei. Decellularized biomaterial showed absence of remnant nuclei and muscle fibers, as well as the preservation of ECM architecture, vascular network and proteins, laminin, fibronectin, elastin, collagen III and IV. After cellularization, iPSC nuclei were present at 9 days after incubation, indicating the decellularized biomaterial-supported iPSC survival. It is concluded that the ECM biomaterial produced from the decellularized equine skeletal muscle has potential for iPSC adhesion, representing a promising biomaterial for regenerative medicine in the therapy of musculoskeletal diseases.     

Protein-carbohydrate interactions between Lactobacillus salivarius and pig mucins

Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.     

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.     

A restriction fragment length polymorphism-based polymerase chain reaction as an alternative to serotyping for identifying Salmonella serotypes

The phase 1 (fliC) and phase 2 (fljB) Salmonella flagella genes were analyzed by restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR) to aid in the identification of different Salmonella serotypes. Twenty-four phase 1 flagellin and eight phase 2 flagellin genes could be differentiated among each other with restriction endonucleases Sau3A and HhaI in RFLP-PCR analysis. These flagellin genes comprise the major antigenic formulas for 52 serotypes of Salmonella sp., which include the common serotypes found in poultry and other important food animal species. With the knowledge of the O antigen composition determined from conventional O serotyping, 90% of the Salmonella serotypes could be identified by this double restriction enzyme RFLP analysis of fliC and fljB genes. This RFLP-PCR flagellar typing scheme was successfully applied to the identification of serotype for 112 Salmonella isolates obtained from poultry environment. There was a significant correlation between RFLP-PCR and conventional serotyping (chi-square, P < 0.001). Overall, PCR-RFLP proved to be a fast, accurate, and economical alternative approach to serotyping Salmonella sp.     

The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice

ABSTRACT: The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SEΔwaaL) or deep-defective (SEΔgal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SEΔwaaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SEΔwaaL as non-live vaccine in the mouse model.     

Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens.

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.     

细菌长极性菌毛的研究进展

长极性菌毛(long polar fimbriae,LPF)是一种最早发现于鼠伤寒沙门菌(Salmonella typhimurium,S.typhimurium)中的CU(chaperone/usher)类型的菌毛,后来该菌毛在其他许多细菌中也得到了鉴定。事实上,LPF不仅作为一种黏附素促进病原体黏附到易感细胞靶向部位,而且参与细菌生物被膜形成和感染组织嗜性。此外,LPF还能增强细菌在宿主细胞中的定植和持续存在能力,并介导宿主的炎症反应。在多种细菌,尤其是在对公共卫生有重要影响的肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)O157:H7菌株中,已经充分阐明了LPF是必不可少的毒力因子。现综述LPF的形态特征和分布、操纵子组成和功能、表达调节机制和生物学功能等方面的研究进展,旨在为研究LPF介导的细菌发病机制提供一定的理论基础。     

A genetically engineered derivative of Salmonella Enteritidis as a novel live vaccine candidate for salmonellosis in chickens

To construct a novel live Salmonella Enteritidis (SE) vaccine candidate, SE was genetically engineered using the allelic exchange method to delete two virulence genes, lon and cpxR. The lon gene deletion is essential to impair Salmonella replication and avoid overwhelming systemic disease in the host. The cpxR gene deletion is needed to enhance the ability of bacteria to adhere and invade the host cell. Scanning electron microscopy revealed that the derivatives JOL917 (Δlon), JOL918 (ΔcpxR), and JOL919 (Δlon/ΔcpxR) had increased surface fimbrial filamentous structures. Significant elevations of extracellular polysaccharide and FimA expression were observed for the derivatives compared to the parental wild type JOL860, while biochemical properties of the derivatives were not altered. In the safety examination by inoculation of the derivatives in chickens, gross lesion scores of the liver, spleen, kidney, small intestine and caecal tonsils were moderate in the JOL917 and JOL918 groups, and significantly lower in the JOL919 group than those of the JOL860. Bacterial counts from the spleen and caeca of the JOL917 and JOL918 groups were moderate, and significantly reduced in the JOL919 group compared to the JOL860 group. In addition, only the JOL919 group showed significantly lower bacterial counts in the faecal samples than those of the JOL860 group. Significant elevations of IgG and secretory IgA levels observed in the derivative groups, while the JOL919 and JOL860 groups showed a potent lymphocyte proliferation response as compared to those of the control group. In the protection efficacy examination, JOL919 immunized group showed significantly lower depression, lower gross lesion in the liver and spleen, and lower number of the SE positive internal organs than those of the control group against a virulent wild type SE challenge.     

Impact of a mutator phenotype on motility and cell adherence in Salmonella Heidelberg

In this study, we investigated adherence and motility of the hypermutator Salmonella enterica Heidelberg B182 bovine strain related to a 12bp deletion in mutS. This mutator phenotype was associated with increased adherence to epithelial cells and with high expression of fimA as shown by real-time RT-PCR. Motility studies showed that fliC were up-regulated in the B182 strain, while fljA and fljB were down-regulated. In order to determine if mutated mutS is implicated in this genes expression, isogenic strains, derived from a WT strain, containing the 12bp deletion in mutS (Δ12bpmutS) or an inactivated mutS (ΔmutS) were generated. Δ12bpmutS and ΔmutS strains showed a spontaneous mutation rate similar to the environmental strain B182, but exhibited lower adherence capacity and fimA expression. In contrast to the fimbriae genes, in Δ12bpmutS, fliC expression was up-regulated, but fljA and fljB expression were decreased, as in the B182 strain. Only fljB expression was increased in ΔmutS mutants. Taken together, our data suggest that mutS alteration does not influence fimbriae expression but can impact flagella genes.