肉品中肠毒素性大肠杆菌和肠沙门菌多重PCR检测方法的建立

利用肠毒索性大肠杆菌（ETEC）的LT-A、ST-1毒素基因和肠沙门菌的iroB基因设计3对引物,建立能同时检测ETEC和肠沙门菌的多重PCR方法.结果：多重PCR扩增出了预计的PCR产物,本方法的特异性为100％、灵敏度达10102 CFU/mL,用建立的多重PCR方法对30份临床可疑腹泻样品进行检测并与生化试验比较,结果两种方法检出大肠杆菌和沙门菌的阳性符合率达100％.

从患者血液培养中分离出肠沙门菌维尔肖学血清型一例

1病例患者女,13岁,学生。2005年3月18日因＂发热4d＂入院。患者入院前4d,晚饭后出现发热,体温38.8℃,自觉头痛,无明显畏寒,无咳嗽、咳痰,无胸痛、心悸、恶心、腹泻,无尿频、尿急、尿痛。用克林霉素后未见明显好转。2d后出现乏力,食欲下降,呕吐胃内容物1次,头痛加剧,体温39.7℃。入院体检：体温39.1℃,心率90次/min,呼吸17次/min.血压110/80mmHg。神志清楚,

肠沙门菌副伤寒甲血清型的萘啶酸抗性和克隆扩散

目的了解玉溪市1999年至2008年甲型副伤寒患者肠沙门菌甲型副伤寒血清型(SPA)分离株的萘啶酸抗性和克隆扩散。方法采用有对照的K-B纸片扩散技术对4060株SPA进行抗微生物药物敏感性试验;对随机选择的166个萘啶酸抗性(NAR)株和20个萘啶酸敏感(NAS)株进行SpeI消化染色体DNA脉冲场凝胶电泳(PFGE)分型、聚类分析以及氟喹诺酮最小抑菌浓度(MIC)测定。结果分离株的NAR率由1999年的12.5%、2000年82.2%、2001年93%上升到2008年的100%;NAS株在1999年占优势,但在2000年以后被NAR株替代;166个NAR株都降低了对氟喹诺酮类药物的敏感性,其MIC值比20个NAS株的高得多。186个菌株SpeI消化产物得出以SpeI01、SpeI02型占优势的9种PFGE型。结论萘啶酸筛检实验可用于检测SPA降低氟喹诺酮敏感性,SpeI01和SpeI02是玉溪流行的主要克隆,建议制定并实施氟喹诺酮药物抗性株引起爆发流行的处理方案。

Application of an indirect immunofluorescent staining method for detection of Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues

AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method （IFA） was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS： The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm.CONCLUSION： IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.

Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction

AIM： To identify and understand the regular distribution pattern for Salmonella enteritidis （S. enteritidis） in the internal organs of mice after an oral challenge over a 3 wk period. METHODS： Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction （FQ-PCR） were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS： The results showed that the spleen was positive at 12 h post inoculation （PI）, and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms.CONCLUSION： The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteriCdis infection in vivo.

Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction

AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.

Vitamin D differentially regulates Salmonella-induced intestine epithelial autophagy and interleukin-1β expression

AIM To investigate the effects of active vitamin D3 on autophagy and interleukin(IL)-1β expression in Salmonella-infected intestinal epithelial cells(IECs).METHODS Caco-2 cells, NOD2 siR NA-, Atg16L1 siR NA- or vitamin D receptor(VDR) siR NA-transfected Caco-2 cells were pretreated with 1,25-dihydroxyvitamin D3(1,25D3), and then infected by wild-type S. typhimurium strain SL1344. The conversion of LC3-I to LC3-II was detected by Western blot analysis and LC3+ autophagosome was analyzed by immunofluorescence. Caco-2 cells or VDR si RNA-transfected cells were pretreated with 1,25D3, and then infected by SL1344. Membrane protein and total RNA were analyzed by Western blot and RT-PCR for VDR and Atg16L1 protein and m RNA expression, respectively. Atg16L1 si RNA-transfected Caco-2 cells were pretreated by 1,25D3 and then infected with SL1344. Total RNA was analyzed by RT-PCR for IL-1β mR NA expression.RESULTS The active form of vitamin D, 1,25D3, showed enhanced VDR-mediated Atg16L1 mR NA expression, membranous Atg16L1 protein expression leading to autophagic LC3 II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells which was counteracted by Atg16L1 and VDR si RNA, but Atg16L1 mediated suppression of IL-1β expression. Thus, active vitamin D may enhance autophagy but suppress inflammatory IL-1β expression in Salmonella-infected IECs.CONCLUSION Active vitamin D might enhance autophagic clearance of Salmonella infection, while modulation of inflammatory responses prevents the host from detrimental effects of overwhelming inflammation.

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylorihpaA

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Effect of PLA/ZnO Packaging and Gamma Radiation on the Content of Listeria innocua, Escherichia coil and Salmonella enterica on Ham during Storage at 4℃

The effect of ZnO and low y-radiation on PLA based-films was investigated to be used for food packaging application. Ham slices were inoculated with E. coli, L. innocua and S. enterica and then covered with PLA and PLA/5% ZnO films. The samples were irradiated with a y-radiation dose of 0.3 kGy at dose rate of 13.5 kGy/h. Microbiological analysis was performed at 0, 1 and 5 days on samples stored at 4 ℃. Results showed that no consistent reduction of bacteria was obtained, even at the fifth day of storage, when the ham was covered with PLA/5% ZnO film and no γ-radiation was performed. The use of γ-radiation results necessary to reduce the bacteria growth. In fact E. coli and S. enterica were not detected after 5 days of storage; whereas in the case of test with L. innocua a reduction of 1.3 log CFU/g was observed after 5 days of storage. The antibacterial results indicate that the presence of ZnO in PLA film is effective only for E. coll. The differences of the results obtained here with those reported in literature （where ZnO particles are reported to be very effective as antimicrobial material） are accounted for the different methodologies used. In conclusion considering the positive results, even if small, obtained here at least only for the E. Coli and considering that PLA/5% ZnO film shows, compared to plain PLA film, good tensile properties （especially Young＇s modulus and stress at yielding） and good permeability （to O2 and CO2） induce to consider the PLA/5% ZnO composite film usable for food packaging when long shelf life and food safety are required, considering also that it is biodegradable and compostable.

Milk Quality in Dual Purpose Cattle with Hand or Machine Milking

To evaluate and improve the milk quality in dual purpose cattle with hand or machine milking, a good management and sanitary program was implemented （GMSP） in 40 farms. Was obtained a diagnostic by an interview and in situ evaluation of management conditions and milk samples for laboratory analysis integrating the platform and microbiological counts, before and after of GMSP. The changes achieved were no significant （P 〉 0.05） for pH and density, but alcohol and foreign material were positive modified （P 〈 0.05） by GMSP in both hand and machine milking. The quantities of sediments were decreased （P 〈 0.05） in more than 50.0%. Decrease （P 〈 0.05） was observed in the counts of CFU mL1 （colony forming unitsl） in aerobic mesophilic, coliforms, Salmonella ssp. and Staphylococcus aureus with the GMSP. After GMSP Salmonella ssp. incidence was affected （P 〈 0.05） by hand （55.5%） vs. machine （61.5%） milking type; coliforms count and positive incidence in milk were affected （P 〈 0.05） in hand （81.5%） vs. machine （53.9%）. Therefore it was confirmed that the implementation of adequate sanitary and management practices, in both hand and machine milking, decreased the presence and incidence of microorganisms in milk with potential to produce disease in animals and humans.

Composting Heap Palm Tree＇s Products in Southern Tunisia

Like many fruit trees, palm trees provide large amounts of non valued products. Composting is considered as the most promising technique for the valorisation of these products. In this work, the heap composting way is evaluated. Several physicochemical （pH, humidity, salinity, density, organic matter rate, C/N ratio, heavy metals） and some microbiological parameters （total and faecal coliforms, Escherichia coli, streptococci and salmonella） are studied after compost maturation. Main results show that palm compost is characterised by a neutral pH （7.87）, low humidity （40.77%）, high organic matter （50%）, high salinity （2.06 g/L）, acceptable C/N ratio （12.2）, low density （0.43 g/cm3）, high conductivity （3.22 ms/cm）, 109.6 ppm of NH4 and 256.66 ppm of HNO3. Finally, microbiological parameters respect hygiene requirements in comparison with compost quality standards.

Acalculous cholecystitis due to Salmonella enteritidis

Acute acalculous cholecystitis (AAC) is defi ned as an acute infl ammation of the gallbladder in the absence of stones. We herein report a case of a young man who developed AAC after a Salmonella enteritidis gastrointestinal infection.

Search of Salmonella in Meat and Dairy Products

The epidemiology of Salmonella involves food, especially meat and dairy products. These have a high salmonella outbreak in the summer. The majority of Salmonella enteritis in young children occurs in the form of scattered cases. At least 25% of summer gastroenteritis in young children is caused by Salmonella. In Western Europe, S. typhimurium accounts for nearly 70% of isolates. The objective of this study was to investigate the Salmonella contaminating meat and dairy products as well as define the percentages of contamination by Salmonella from 2004 to 2006, by the Department of Hygiene of Tunis. For this study, we collected samples in various motherboard solutions, and conducted a pre-nonselective enrichment in selective enrichment in isolation and identification and ultimately biochemical confirmation. One hundred and sixty two samples, 125 samples of meat products including 68 samples of red meat （beef and sheep, beef, offal and Merguez） and 57 samples of poultry （chicken and turkey）, 37 samples of milk products which included 32 samples of cheese and five samples milk, were analysed microbiologically from 2004 to 2006, in the Health Service in Tunis. These analyses include the detection and enumeration of Salmonella. From 2004 to 2006, the rate of infections by the Salmonella, meat and dairy products were： 55.9% for red meat, 71.9% for poultry, 68.8% for cheeses, 40% for milk. The meat of poultry is contaminated with Salmonella that the cheeses, which are more than red meats, which are also more contaminated than milk. This might be due to a lack of hygiene throughout the circuit processing （slaughter, transport, processing, etc.）. The increased risk of contamination of milk products by Salmonella is proportional to the manipulation done on these products. The latter must be handled by pasteurization and sterilization.

萘啶酸抗性甲型副伤寒病原菌的克隆扩散和遗传多样性

目的认识肠沙门菌甲型副伤寒血清型（SPA）的克隆扩散和遗传多样性，建立并确定病原菌流行克隆的分型方法。方法采用有对照的K-B纸片扩散技术对分离的3980株SPA进行抗微生物药物敏感性试验；经PCR扩增和基因测序检测15个萘啶酸抗性菌株喹诺酮抗性决定区的gyrA、gyrB、gyrC和gyrE基因；采用SpeI和XbaI消化染色体DNA脉冲场凝胶电泳（PFGE）对来自7个县的121个分离株进行分型和聚类分析。结果萘啶酸敏感菌株在1999年占有优势，但2000年以后被萘啶酸抗性菌株替代；15个萘啶酸抗性菌株的PCR扩增和基因测序显示抗性机制是由gyrA基因的单点突变引起；121个菌株SpeI和XbaI消化产物分别得出以SpeI01、SpeI02或XbaI01型占优势的5种和4种PFGE型，SpeI01和SpeI02分别占37．2％和57．9％，或XbaI01占95．0％。结论在研究期间SPA分离株萘啶酸抗性率上升；PFGE型的SpeI01和SpeI02或XbaI01是玉溪流行的主要克隆；采用SpeI和XbaI的PFGE是鉴别SPA的一项有用技术。

Evaluation of Total and Faecal Coliforms and Salmonella spp. in Irradiated Mussels from Brazil

Ilha Grande Bay is one of the biggest producers of bivalves of Rio de Janeiro State. Statistics reports of foodborne diseases are quite low in Brazil, however, this fact is a matter of Public Health. In their majority concerning consumption of bivalves meat, the availability of safe products requires the use of technology as food irradiation. The objective of this paper was to evaluate the presence of bacteria resulting from the environmental contamination and epidemiological importance, Salmonella spp., total and faecal coliforms of mussel （Perna perna） from that region and the use of irradiation on the product in natura. Fifteen indicative samples of mussel were collected from five gr owing points in Ilha Grande Bay. A sample of each point was irradiated with doses of 1.0 and 1.5 kGy. The bacteriological analysis followed the instructions of the Brazilian legislation. The samples presented irregularities in relation to Salmonella spp. and faecal coliforms, the latter for the control group. The control group was noticed as not appropriate for consumption. The dose of 1.0 kGy was effective for the reduction of faecal coliforms, but ineffective for the extinction of Salmonella spp.

Effect of Earthworms on Removal of Pathogens during Stabilization of Industrial Organics

To investigate feasibility of pathogen free industrial organics with higher agronomic value, the industrial organic wastes were subjected to vermistabilization. The body of earthworm work as ＂biofilter＂ and they can ＂purify＂ and also ＂disinfect＂ and ＂detoxify＂ municipal and industrial organics. The microbiomics of gut and cast of earthworm （Eisenia foetida Savigny） and their association with vermistabilization was studied to determine the microbial quantification in reactors （industrial organics）. Worm were reared in five reactors viz. Sewage sludge （SS）, Paper mill industry sludge （PS）, Vegetable processing industry （VP）, Tannery waste （TW） and Meat process industrial sludge （MP） for ninety days. The microbial load （Salmonella, Shigella, Escherichia, Mycobacterium, Streptococcus） in gut and cast, biomass, recovery of cast in different reactors were determined, periodically. The microbiomics of worms gut revealed the removal of Salmonella （12-18 × 10^8±0.02 to 0-4 × 10^3 ＋ 0.05 CFU/g）, Shigella （14-23 × 10^8 ± 0.04 to 0-4 × 10^3 ± 0.05 CFU/g）, Escherichia （4-16 × 10^8 ± 0.02 to 0-4 × 10^2 ± 0.05 CFU/g）, Mycobacterium （3-16 × 10^8 ± 0.02 to 0-3 × 10^2 ± 0.05 CFU/g）, Streptococcus （6-16 × 10^8 ± 0.02 to 0-4 × 10^3 ± 0.05 CFU/g） during stabilization of industrial organics. Similarly, reduction in pathogens Salmonella （12-19 × 10^8 ± 0.02 to 0-8 ×10^3 ± 0.05 CFU/g）, Shigella （7-20× 10^8 ± 0.04 to 0-2 ×10^3 ± 0.05 CFU/g）, Escherichia （2-20 × 10^8 ± 0.02 to 0.0-2 × 10^3 ± 0.05）, Mycobacterium （1-8 × 10^8 ±0.05 to 0.0-5 × 10^2 ± 0.05 CFU/g）, Streptococcus （8-18 × 10^8 ± 0.02 to 0-7 × 10^3 ± 0.05 CFU/g） in castings of industrial organics indicates the selective nature of feeding of worm. This amply demonstrates that these pathogens have been eliminated as they entered in food chain of worms. However, it may not be possible to remove pathogens completely, but at least worms change the ＂microbial make-up＂ of industrial organics to make it harmless to the soil and enable its use as a nutritive organic fertilizer.