饲喂乳杆菌雏鸡对鸡白痢菌感染的影响

雏鸡饲喂乳杆菌培养物后，人工感染鸡白痢沙门氏菌，观察雏鸡盲肠主要正常菌群定植及雏鸡抵抗力情况。研究结果表明：人工感染鸡白痢沙门氏菌后，饲喂乳杆菌组同未饲喂乳杆菌组相比，雏鸡盲肠内正常菌群有一定变化，大肠杆菌数明显降低（Ｐ＜００１），双歧杆菌和乳杆菌数明显增加（Ｐ＜００１），而且死亡率降低２０％。     

雏鸡沙门氏菌病的病原分离与鉴定

邯郸地区某县养殖场于2008年2月引进一批海兰白雏鸡共6500只,4日龄时部分雏鸡开始发病,6日龄时开始死亡,7d内共死亡1248只,死亡率达19.2%。剖检不同日龄死雏21只,活病雏13只,死雏呈现严重败血症病变,活雏心肌、肝、肺、肌胃等脏器出现黄白色或大小不等的灰白色坏死灶。取其卵黄、肝、脾、心血等病料进行病原分离培养,从获得的153个菌落中挑选可疑菌落27个,经细菌学鉴定,确认17株为鸡白痢沙门氏菌,抗原式为1,9,12∶-∶-,10株为德尔卑沙门氏菌(S.derby),抗原式为1,4,12∶f,g∶-。     

Dot-ELISA检查香肠中沙门氏菌的研究

以硝酸纤维膜为固相载体,利用沙门氏菌多价血清和辣根过氧化物酶(HRP)标记制备的羊抗兔IgG,成功地建立对香肠的Dot-ELISA检查法,同时,采用常规分离培养鉴定技术作对照试验。结果显示,在86份香肠中,用Dot-ELISA检出沙门氏菌阳性为36份,阳性率为41.86%;而常规分离培养鉴定技术检出沙门氏菌阳性为34份,阳性率为39.53%,两种方法的阳性符合率为86.17%,经统计分析,t=0.311 1,p>0.05,两种方法差异不显著。     

Epidemiological Cut-off Values (ECOFFs) of Salmonella from Chicken Intestinal Tract for Three Animal-specific Antibiotics

In order to establish epidemiological cut-off values (ECOFFs) of Salmonella for three animal-specific antibiotics (florfenicol,apramycin and danofloxacin) in Guangdong province.A total of 166 strains of Salmonella isolates from chicken intestinal tract from veterinary clinics and farms in Guangdong province in 2017 were recruited in this study,the minimum inhibitory concentrations (MIC) of florfenicol,apramycin and danofloxacin were determined in triplicate for each bacterial strain using the agar dilution method on Mueller-Hinton agar plates according to the CLSI reference method (CLSI-M100-S26).ECOFFs were calculated for the MIC data sets by application of nonlinear regression analysis,NRI(normalized resistance interpretation) and ECOFFinder software.MIC distribution of florfenicol,apramycin and danofloxacin against Salmonella from 2 to >512,4 to >512 and 0.015 to 64 μg/mL,respectively.MIC₅₀ and MIC₉₀ for apramycin,florfenicol and danofloxacin were 256 and >512,16 and >512,0.5 and 16 μg/mL,respectively.MIC distribution of apramycin and florfenicol presented obvious unimodal shape,while that of danofloxacin was a discontinuous multi-peak.The ECOFFs of florfenicol,apramycin and danofloxacin were recommended to be 16,16 and 0.125 μg/mL,respectively.In present study,the ECOFFs of Salmonella from chicken intestinal tract for apramycin,florfenicol and danofloxacin were determined,which provided scientific basis for resistance surveillance.     

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。     

鸭源沙门菌的分离鉴定、耐药性及致病性分析

【目的】为防控鸭源沙门菌感染,本试验针对四川德阳地区鸭源沙门菌的感染情况开展研究。【方法】从该地区3个种鸭场和1个鸭孵化场共采集不同类型样本222份,按国标GB 4789.4－2016方法对沙门菌进行分离鉴定,进一步通过K-B法检测分离菌对14种抗菌药物的敏感性,并对鉴定出的稀有血清型沙门菌进行致病性研究。【结果】疑似沙门菌分离株在BS琼脂上呈黑色、灰色或棕褐色、有金属光泽菌落,在XLD琼脂上呈无色透明、或中心黑色或几乎全黑的菌落。将疑似沙门菌株接种三糖铁琼脂斜面进行生化鉴定,疑似沙门菌分离株均能使三糖铁斜面变为红色,底部变为黄色带黑色,符合沙门菌生化特性。通过PCR对沙门菌特异性基因invA进行扩增,经琼脂糖凝胶电泳后可在500 bp左右观察到目的条带,确认共分离到17株沙门菌,总分离率为7.7%（17/222）,包括鼠伤寒沙门菌、哈托沙门菌和波恩沙门菌3种血清型,其比例分别为47.1%（8/17）、29.4%（5/17）和23.5%（4/17）,优势血清型为鼠伤寒沙门菌。分离菌对β-内酰胺类和氨基糖苷类抗菌药耐药率最高,对氨苄西林和链霉素的耐药率分别为82.4%（14/17）和88.2%（15/17）,对喹诺酮类抗菌药最敏感,其中对萘啶酸100%（17/17）敏感。分离菌存在严重的多重耐药现象,其中耐10种以上（包括10种）抗菌药的6株,占比35.2%,且10种抗菌药分别来自至少6类不同种类抗菌药;耐8种抗菌药物的2株,占比11.8%;耐5~7种抗菌药的6株,占比35.3%;耐2~3种抗菌药物的共3株,占比17.6%。通过寇氏改良法测得稀有血清型菌株H4、B2的LD₅₀分别为3.98×10⁶和1.58×10⁶ CFU,致病性试验结果表明,哈托和波恩沙门菌均能引起小鼠急性死亡,脏器充血、出血,细胞变性等。【结论】本研究成功分离到17株鸭源沙门菌,从鸭中分离到哈托沙门菌在国内外属首次。分离菌具有严重的耐药性和多重耐药现象,2株稀有血清型沙门菌对小鼠的致病力均较强,且致病作用相似。本研究结果可为鸭场沙门菌病的防治提供参考。     

甘草多糖对鼠伤寒沙门菌诱导的巨噬细胞氧化-抗氧化平衡紊乱的调解

为了探讨甘草多糖对鼠伤寒沙门菌诱发巨噬细胞氧化-抗氧化平衡紊乱的调节作用。将0、1、20和100 mg/L甘草多糖4个质量浓度组与小鼠腹腔巨噬细胞共孵育,同时每组按1∶1加入鼠伤寒沙门菌SL1344;利用ELISA试剂盒检测各组丙二醛(malondialdehyde,MDA),活性氧(reactive oxygen species,ROS),一氧化氮(nitricoxide,NO),诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS),谷胱甘肽过氧化物酶(glutathione peroxidase,GSHPX),超氧化物歧化酶(superoxide dismutase,SOD)和总抗氧化能力(total antioxidant capacity,T-AOC)变化情况;利用荧光显微镜和荧光酶标仪观察甘草多糖对鼠伤寒沙门菌引起巨噬细胞产生ROS的影响;利用乳酸脱氢酶(lactate dehydrogenase,LDH)细胞毒性试剂盒检测巨噬细胞上清LDH水平。结果表明,鼠伤寒沙门菌可诱导小鼠腹腔巨噬细胞发生氧化损伤,导致NO(4.65±1.07)μmol/L、iNOS(15.46±0.92)U/mL、MDA(3.46±0.39)nmol/L和ROS(3.69±0.31)U/mL氧化水平以及LDH水平显著升高,而GSH-PX(15.58±1.35)nmol/L、SOD(13.32±0.92)NU/mL和T-AOC(8.36±0.60)U/mL抗氧化水平显著降低。荧光显微镜和荧光酶标仪检测显示,鼠伤寒沙门菌感染小鼠腹腔巨噬细胞ROS水平显著升高。甘草多糖呈剂量依赖明显降低细胞氧化水平和增强抗氧化水平。甘草多糖作为重要的免疫调节剂,能够调节鼠伤寒沙门菌引起的巨噬细胞氧化损伤,维持细胞氧化-抗氧化平衡。     

Factors affecting costs for on-farm control of salmonella in Swedish dairy herds

Background

The Swedish control program for salmonella includes restrictions and on-farm control measures when salmonella is detected in a herd. Required control measures are subsidised by the government. This provides an opportunity to study costs for on-farm salmonella control. The aim of this study was to describe the costs for on-farm salmonella control in Swedish cattle herds and to investigate the effects of herd factors on these costs in dairy herds.

Results

During the 15 years studied there had been a total of 124 restriction periods in 118 cattle herds; 89 dairy herds, 28 specialised fattening herds and three suckler herds. The average costs per herd for on-farm salmonella control was 4.60 million SEK with a median of 1.06 million SEK corresponding to approximately 490 000 and 110 000 EUR. The range was 0.01 to 41 million SEK corresponding to 1080 EUR to 4.44 million EUR per farm. The costs cover measures required in herd-specific control plans, generally measures improving herd hygiene. A mixed linear model was used to investigate associations between herd factors and costs for on-farm salmonella control in dairy herds. Herd size and length of the restriction period were both significantly associated with costs for on-farm control of salmonella with larger herds and longer periods of restrictions leading to higher costs. Serotype detected and administrative changes in the Swedish Board of Agriculture aiming at reducing costs were not associated with costs for on-farm salmonella control.

Conclusions

On-farm control of salmonella in Swedish cattle herds incurred high costs but the costs also varied largely between herds. Larger herds and longer restriction periods increased the costs for on-farm control of salmonella in Swedish dairy herds. This causes concern for future costs for the Swedish salmonella control program as herd sizes are increasing.     

Isolation,Identification and Analysis of Serotype and Antibiotic Susceptibility of Pathogenic Salmonella from Chickens

To investigate the situations of predominant strain and antibiotic resistance of pathogenic Salmonella from chickens in Guangxi Zhuang Autonomous region, Salmonella was pre-enriched and isolated from tissues of clinical suspected chickens, and the Salmonella isolates were identified by biochemical test using ID 32E System for the identification of Enterobacteriaceae of VITEK System ATB Expression, serotypes were determined by slid agglutination test, antibiotic susceptibility was analyzed by ATB VET Susceptibility Test Strip of Enterobacteriaceae antibiotics.The results showed that 34 Salmonella strains were obtained from 310 clinical samples, of which 1 strain belonged to A serogroup, 1 to C2 serogroup, 15 to B serogroup, 14 to D serogroup and 3 to untyped serogroup, and S.typhimurium of B serogroup and S.pullorum of D serogroup were the predominant serotypes.All Salmonella isolates were sensitive to meropenem, imipenem, spectinomycine and apramycine, and the resistance rates to chloramphenicol, kanamycine and gentamicine were less than 10%, and the resistance rates to amoxicillin, streptomycine, flumequine, oxolinique, sulfamethizol, tetracycline and nitrofurantoine ranged between 50% and 90%, while the resistance rates to penicilline, oxacilline, fusidique, rifampicine and metronidazole reached to 100%.The results indicated that the serotype distribution of pathogenic Salmonella from chickens exhibited regional characteristics, and S.typhimurium and S.pullorum were the predominant serotypes in Guangxi Zhuang Autonomous region, and antibiotic resistance of Salmonella isolates was very serious which should be highly concerned.     

Comparative Phenotypic and Genotypic Analyses of Salmonella Rissen that Originated from Food Animals in Thailand and United States

Salmonella enterica serovar Rissen has been recognized as one of the most common serovar among humans and pork production systems in different parts of the world, especially Asia. In the United States, this serovar caused outbreaks but its epidemiologic significance remains unknown. The objectives of this study were to compare the phenotypic (antimicrobial susceptibility) and genotypic attributes of Salmonella Rissen isolated in Thailand (Thai) and the United States (US). All the Thai isolates (n = 30) were recovered from swine faecal samples. The US isolates (n = 35) were recovered from swine faecal samples (n = 29), cattle (n = 2), chicken (n = 2), dog (n = 1) and a ready‐to‐eat product (n = 1). The antimicrobial susceptibility of isolates was determined using the Kirby‐Bauer disk diffusion method with a panel of 12 antimicrobials. Pulse‐field gel electrophoresis (PFGE) was used to determine the genotypic diversity of isolates. All Thai isolates showed multidrug resistance (MDR) with the most frequent antibiotic resistance shown against ampicillin (100%), sulfisoxazole (96.7%), tetracycline (93.3%), streptomycin (90%) and chloramphenicol (30%). About half of the isolates of USA origin were pan‐susceptible and roughly 30% were resistant to only tetracycline (R‐type: Te). Salmonella Rissen isolated from Thailand and the USA in this study were found to be clonally unrelated. Genotypic analyses indicated that isolates were clustered primarily based on the geographic origin implying the limited clonality among the strains. Clonal relatedness among different host species within the same geography (USA) was found. We found genotypic similarity in Thai and US isolates in few instances but with no epidemiological link. Further studies to assess propensity for increased inter‐regional transmission and dissemination is warranted.