空肠弯曲菌共同抗原单克隆抗体的研制及其在细菌检验中应用——Ⅲ.在细菌快速检验中的应用

应用抗空肠弯曲菌共同抗原的单克隆抗体(4A7),建立了适用于空肠弯曲菌快速检验的间接-ELISA和BA-ELISA。两种方法的特异性检查表明,对受试的32种其它细菌,除与幽门弯曲菌发生交叉反应外,其余均为阴性反应。对空弯菌纯培养物检测的敏感性,BA-ELISA为3.2×104个菌/mL,间接-ELISA为1.6×105个菌/mL;对细菌动态培养物检测,BA-ELISA可检出接种约5个菌/mL、间接-ELISA可检出接种约10个菌/mL的24 h增菌培养物。应用BA-ELISA对人(140份)、猪(140份)、禽类(364份)及特种经济动物(265份)标本进行检测,阳性率依次为121%、52%、58.2%和32.8%;应用间接-ELISA对人(140份)、禽类(222份)及特种经济动物(265份)标本进行检测,阳性率依次为11.4%、61.7%、31.3%。两种方法的结果均与常规培养法相差不显著,可在27h内报告结果。     

应用McAb·BA-ELISA快速检测动物源空肠弯杆菌的研究

应用抗空肠弯杆菌共同抗原的单克隆抗体,建立了快速检测动物源空肠弯杆菌的 BA-ELISA 程序。此程序可检出每 ml 含5个空肠弯杆菌24小时增菌培养物或每ml 含3.2×10~4个空肠弯杆菌纯培养物,不与金黄色葡萄球菌等16种对照菌及混合菌液发生交叉反应。用其对雉鸡等10种动物456份肛拭粪便标本进行检测,总阳性率为45.5%,与常规分离鉴定法的阳性符合率为99.1%.敏感性为99.2%.特异性为100%。统计学分析表明,所建立的 BA-ELISA 检测程序与常规分离鉴定法间具有极显著的一致性(p<0.01),能使检测时间缩短4～5天.     

应用Dot—ELISA法检测鱼粉中的沙门氏菌

应用Dot—ELISA法检测鱼粉85份,对沙门氏菌的最低检出量为400个/m1,沙门氏菌阳性检出率75.29%(64/85),常规分离培养阳性检出率为62.35%(53/85),两者符合率为78.13%,差异不显著(P>0.05)。     

应用斑点酶联免疫吸附试验检测蛋白料精中的沙门氏菌

选用硝酸纤维素(NC)膜作固相载体,建立检测沙门氏菌的斑点酶联免疫吸附试验诊断方法,检测蛋白料精50份,对沙门氏菌的最低检出量为400个/mL,沙门氏菌阳性检出率66%(33/50),常规分离培养阳性检出率为60%(30/50),两者阳性符合率为84.85%,差异不显著(P>0.05)。试验表明,该方法简便、特异、快速、结果直观,便于在基层推广使用。     

Dot-ELISA检测猪胴体中沙门氏菌的研究

应用Dot-ELISA法对西宁某猪屠宰点 85份猪胴体进行了沙门氏菌的检测 ,同时采用常规分离培养鉴定技术作对照实验。结果在 85份肉样中 ,Dot-ELISA检出沙门氏菌阳性 6 5份 ,阳性率为 76 .4 7% (6 5 /85 ) ;而常规分离培养鉴定技术检出沙门氏菌阳性 6 7份 ,阳性率为 78.82 % (6 7/85 ) ,此两种方法的阳性符合率为 86 .5 7%。经统计分析 ,两种方法差异不显著 (P >0 .0 5 )。     

应用Dot-ELISA检测羊胴体中沙门氏菌的研究

应用Dot ELISA法对西宁某屠宰点 1 0 1份羊胴体淋巴结进行了沙门氏菌的检测 ,同时采用常规分离培养鉴定技术作为对照。结果显示在1 0 1份羊胴体中 ,Dot ELISA检出沙门氏菌阳性 56份 ,阳性率为 55 44% (56/ 1 0 1 ) ;而常规分离培养鉴定技术检出沙门氏菌阳性 48份 ,阳性率为 47 52 % (48/ 1 0 1 )。 2种方法的阳性符合率为 85 71 % ,差异不显著 (P >0 0 5)。     

乳胶凝集试验检测牛羊淋巴结中的沙门氏菌

采用沙门氏菌多价血清致敏乳胶并制成乳胶抗体,建立沙门氏菌乳胶凝集试验检测方法(LAT),分别用LAT法和常规分离培养检测法对38份牛淋巴结、101份羊淋巴结进行检测.结果:乳胶凝集试验牛淋巴结阳性21份,羊淋巴结阳性44份;常规分离培养检测法牛淋巴结阳性22份,羊淋巴结阳性48份,两种方法的阳性符合率均超过80%.试验表明乳胶凝集试验操作简便、快速、敏感性高、特异性强且可用于现场检测,是一种适合基层单位检测沙门氏菌的可靠方法.     

乳胶凝集试验检测牛、羊淋巴结中的沙门氏菌

用沙门氏菌多价血清致敏乳胶并制成乳胶抗体,建立沙门氏菌乳胶凝集试验检测方法（LAT）。用该方法和常规分离培养检测法检测38份牛淋巴结、101份羊淋巴结,结果乳胶凝集试验牛淋巴结阳性21份,羊淋巴结阳性44份;常规分离培养检测法牛淋巴结阳性22份,羊淋巴结阳性48份,两种方法的阳性符合率牛淋巴结为81.8%,羊淋巴结为81.3%。试验表明乳胶凝集试验操作简便、快速、敏感性高、特异性强且可用于现场检测,是一种适合基层单位用来检测沙门氏菌的可靠方法。     

动物性饲料中沙门氏菌快速检验的研究——协同凝集试验及HRP-SPA酶染色法的应用

本研究确定了适用于动物性饲料中沙门氏菌检验的增菌程序、协同凝集试验(COAG)及HRP—SPA染色法(酶染法)的最适实验条件。以这种条件进行增菌,并以COAG及酶染法进行检查,具有快度、敏感、特异性强的优点。应用COAG、酶染法及沙门氏菌常规分离鉴定法,对236份动物性饲料标本进行了检测。结果两种快速法与常规法有较好的符合率,酶染法在检出阳性标本上优于常规法.且可在16～18h内报告结果,比常规法的时间明显缩短。因此认为,COAG和酶染法适用于动物性饲料中沙门氏菌的快速检测。动物性饲料的沙门氏菌污染,依饲料的种类不同而异,其中鱼粉为31.5％、血粉25％骨粉10.3％,平均为17.4％,而且存在着多种血清型的沙门氏菌,其中以鼠伤寒沙门氏菌占优势。因此认为动物性饲料中的沙门氏菌污染在人畜沙门氏菌的流行病学上具有重要的作用。     

间接ELISA对鸡猪空肠弯曲菌快速检验的实验研究

以方阵滴定法确定了空肠弯曲菌抗血清、酶结合物的最佳工作浓度和孵育时间,从而立了快速检测空肠弯曲菌的间接ELISA。该试验可检出含8～10个空肠弯曲菌经24h培养的标本(含30万个菌/ml的肉汤培养物);不与沙门氏菌等15种对照菌及其混合液发生交叉反应。以鸡源和猪源空肠弯曲菌抗血清分别作为ELISA的第一抗体,对144份鸡泄殖腔粪便标本和116份猪直肠粪便标本的选择性增菌肉汤培养物进行了检测。其阳性率分别为83.3％和79.3％,可于28h内报告结果;常规法对上述标本的阳性检出率分别为85.4％和81％,但程序繁琐,需5～7d方能获得最终检查结果。两种方法对鸡、猪粪便标本检测结果的阳性符合率,分别为97.5％与97.9％。     

EMA与PCR结合检测沙门氏菌方法的研究

为了建立一种快速区分样品中沙门氏菌的死细菌与活细菌的检测方法,将叠氮溴化乙锭（EMA）与PCR技术相结合。以沙门氏茵的invA为靶基因,以沙门氏菌的纯培养细胞做模板进行扩增,并进行灵敏度、特异性、曝光时间及EMA浓度试验。结果显示,灵敏度为14CFU／mL,最佳曝光时间为2min,当EMA的浓度小于18μg／mL时．EMA对活菌靶基因的扩增没有明显的抑制,而终浓度为1μg／mL的EMA,能有效抑制lxlosCFU／mL沙门氏菌死菌的扩增。EMA—PCR能有效降低沙门氏菌检测过程中的假阳性。     

Salmonella serotypes encountered in animal feed additives in Lebanon.

Animal feed-additive samples (n = 300) were examined for the presence of salmonellae, using the selenite-F broth-enrichment method followed by subculturing on Salmonella-Shigella and brilliant green agar with sulfadiazine selective agar plates. Samples consisted of a variety of feed additives: 119 bone meal samples, 77 meat meal samples, 40 fish meal samples, and 64 miscellaneous meal samples. Results of examination found 49 (41.2%) of the bone meal samples, 6 (7.8%) of the meat meal samples and 2 (5%) of the fish meal samples contained salmonellae. Of 57 isolates representing 24 serotypes, 4 most frequently isolated serotypes were Salmonella meleagridis (35.1%), Salmonella tennessee (7%), Salmonella chester (5.2%), and Salmonella senftenberg (5.2%). This study shows a high Salmonella-contamination rate of bone meal compared with meat meal and fish meal samples. Of 12 known positive bone meal samples that were examined, 100% of 25-g samples, compared with 70% to 100% of 2.5-g samples and 30% to 90% of 0.25-g samples and 30% to 90% of 0.25-g samples, were positive for salmonellae.     

Effect of Temperature and Dissolved Oxygen Concentration on Edwardsiella ictaluri in Experimentally Infected Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were experimentally infected with Edwardsiella ictaluri by immersion exposure. After clinical disease ran its course for 52 d, the surviving fish were exposed to one of the following environmental regimes in troughs: 25°C with aeration, 25°C with no aeration, or variable temperature (18–23°C) with no aeration. After 29 d of exposure to the environmental regimes, various organs and tissues of the fish were assayed to determine the effects of these conditions on E. ictaluri concentrations (colony-forming units/mL of tissue sample). The concentrations of this pathogen were significantly (P < 0.05) higher in all tissues (trunk kidney, liver, head kidney, blood, spleen, gallbladder, muscle, brain, and gonad) 52 d postinfection than 29 d after exposure to any of the environmental regimes (81 d postinfection). Fish exposed to a near-normal concentration of dissolved oxygen (6.4 mg/L) and a constant temperature of 25°C had E. ictaluri concentrations that were significantly (P < 0.01) lower than those offish exposed to a low oxygen concentration (2.6 or 1.8 mg/L) and either a constant or a variable temperature.     

LUX^TM荧光PCR快速检测沙门氏菌的研究

本研究针对沙门氏菌invA基因的保守序列,设计特异的LUX^TM荧光标记引物,通过优化反应奈件和参数,建立可快速检测沙门氏菌的LUX^TM荧光PCR检测方法。结果显示,该方法高度敏感,其对纯菌的检测低限达到10^2cfu／mL,经6h增菌培养后检测,对样品液的检测低限达到1cfu／mL;特异性强,测试的全部13株沙门氏菌标准和参考菌株均呈阳性反应,测试的全部27株非目标菌均呈阴性反应;重复性好,定量检测批内和批间的变异系数均小于2％。应用本方法检测食品样品240份,结果检出阳性4份,与TaqMan荧光PCR和SN标准检测结果完全一致。本方法可在8h内完成对样品中沙门氏菌的检测,其检测的快速性、敏感性和特异性与TaqMan荧光PCR技术相当,且检测成本较低。     

Use of an amplified ELISA technique for detection of a house dust mite allergen (Der f 1) in skin and coat dust samples from dogs

OBJECTIVE: To use an amplified ELISA technique to document the presence and quantify the concentration of the house dust mite allergen, Der f 1, in skin and coat dust samples collected from dogs. ANIMALS: 29 pet dogs of various breeds. PROCEDURE: Dogs were weighed, and body surface area in square meters was determined. Skin and coat dust samples were obtained by vacuuming dogs. Collected dust was analyzed by use of standard and amplified ELISA techniques. RESULTS: By use of the standard ELISA technique, Der f 1 was detected in skin and coat dust samples from 6 of 29 (21%) dogs. Mean concentration of Der f 1 in the 6 samples with positive assay results was 16.16 ng/mL (range, 5.61 to 31.24 ng/mL). Samples with negative assay results were retested for dust mite allergen by use of an amplified ELISA technique; an additional 14 dogs had positive assay results. Mean concentration of allergen was 0.36 ng/mL (range, 0.19 to 2.20 ng/mL). Combining both techniques, 20 of 29 (69%) dogs had positive assay results for Der f 1. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that house dust mite allergens are present on the skin and in the coat of dogs, and this source of allergen may act as a reservoir for allergen exposure in hypersensitive dogs. Use of an amplified ELISA technique to determine environmental concentrations of house dust mite allergens in homes and on dogs will help to identify the relationship between immunologic findings and environmental exposures in dogs with atopic dermatitis.     

Plasma/muscle ratios of sulfadimethoxine residues in channel catfish (Ictalurus punctatus)

Channel catfish ( n = 84) maintained at a water temperature of 27°C were used in a feeding study to determine the plasma to muscle concentration ratios of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine residues. Sulfadimethoxine medicated feed was provided free choice at 42 mg SDM/kg body weight once daily for 5 days and the plasma and muscle concentrations of SDM were determined at selected withdrawal times (6, 12, 24, 48, 72, and 96 hours) following the last dose. Considerable variation in total SDM tissue concentration among fish within a sampling period was observed. For fish ( n = 12) at six hours post-dose, total SDM concentrations ranged from 1.4–24.8 μg/mL and 0.6–12.6 μg/g, with mean total SDM concentrations of 9.1 μg/mL and 5.3 μg/g for plasma and muscle, respectively. However, a mean plasma:muscle concentration ratio of 1.8:1 ± 0.3:1 was obtained over all concentrations and sampling periods. The plasma:muscle 95% t distribution interval for individual fish was 1.2:1 to 2.4:1. A correlation coefficient of 0.967 was obtained for the relationship between plasma and muscle total SDM concentration among individual fish ( n = 25). Results of this study indicate that plasma total SDM concentration may be used to identify samples containing violative SDM muscle residue. No fish contained total SDM muscle residues greater than the FDA tolerance (0.1 μg/g) by 48 hours following the final dose.     

丙型副伤寒沙门氏菌脂多糖提取纯化及活性分析

为了提取、纯化天鹅源丙型副伤寒沙门氏菌脂多糖（LPS）并检测其活性,试验通过热酚水法提取丙型副伤寒沙门氏菌LPS,采用DNaseⅠ、RNase A和蛋白酶K及醇沉法纯化LPS,测定LPS提取物中多糖、蛋白及核酸的含量,鲎试剂检测凝集活性,显色基质法测定其活性。结果显示,纯化的丙型副伤寒沙门氏菌LPS平均产率为1.48%,多糖含量为3.84%,蛋白含量为1.49%,核酸含量为 5.45%,且核酸片段低于100 bp,SDS-PAGE电泳和银染结果显示条带主要集中在10～15 ku范围内,与2 EU/mL鲎试剂的最小凝集浓度是10.99 ng/mL,显色基质法测定其活性为9.82×10⁵ EU/mg。该试验提取、纯化的丙型副伤寒沙门氏菌LPS纯度较高,生物活性良好。     

Lincomycin-induced severe colitis in ponies: association with Clostridium cadaveris.

Four groups of two ponies, free of fecal Salmonella and Clostridium cadaveris, were treated as follows: Group A, control group; B, single nasogastrically administered dose of lincomycin (25 mg/kg) followed 48 h later by 3 L of C. cadaveris (10(9) organisms/mL); C, the same dose of lincomycin as group B; D, the same dose of C. cadaveris as group B on each of three occasions at 12 h intervals. Groups A and D remained healthy, but groups B and C developed severe colitis 48-56 h (B) or 72 h (C) after administration of lincomycin. Three ponies were euthanized and one in group B died. Clostridium cadaveris was isolated at about 10(6)/mL of colonic contents from these ponies, but one pony in group B also yielded Salmonella typhimurium from the colon. Subsequent challenge of group A ponies (3 L of C. cadaveris 10(9)/mL, three times at 12 h intervals) did not produce colitis. Nasogastric administration of lincomycin (25 mg/kg) to group A and D ponies, 20 days after administration of C. cadaveris, resulted in severe colitis in all ponies within 48-72 h. Salmonella agona was isolated from the colonic contents of one pony and C. cadaveris (10(6)/mL) from all four ponies. Clostridium cadaveris was not isolated from the colonic content of 45 healthy horses examined immediately after death. These studies confirm the potential for lincomycin to induce severe enterocolitis in ponies and implicate C. cadaveris further as a cause of "idiopathic colitis" in ponies.     

Diversity of Salmonella serovars in feedyard and nonfeedyard playas of the Southern High Plains in the summer and winter

OBJECTIVE: To compare Salmonella isolates cultured from feedyard and nonfeedyard (control) playas (ie, temporary shallow lakes) of the Southern High Plains. SAMPLE POPULATION: Water and muck (sediment) samples were obtained from 7 feedyard playas and 3 nonfeedyard playas in the winter and summer. PROCEDURE: Each water and muck sample was enriched with sulfur-brilliant-green broth and incubated in a shaker at 37 degrees C for 24 hours. A sample (100 mL) of the incubated bacterial-enriched broth was then mixed with 100 mL of fresh sulfur-brilliant-green enrichment broth and incubated in a shaker at 37 degrees C for 24 hours. After the second incubation, a swab sample was streaked on differential media. Suspect Salmonella isolates were further identified by use of biochemical tests, and Salmonella isolates were confirmed and serovar determinations made. RESULTS: Salmonella isolates were not recovered from the 3 control playas. Seven Salmonella enterica serovars were isolated from 5 of 7 feedyard playas in the summer, and 13 S. enterica serovars were isolated from 7 of 7 feedyard playas in the winter. In the summer, 296 isolates were cultured, and 47 were Salmonella organisms. In the winter, 288 isolates were cultured, and 171 were Salmonella organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that feedyard playas are frequently contaminated with many Salmonella serovars. These pathogens should be considered whenever feedyard managers contemplate the use of water from these playas. Water from feedyard playas should not be used to cool cattle in the summer or for dust abatement.     

Prevalence and antimicrobial resistance of Salmonella spp. in pet mammals, reptiles, fish aquarium water, and birds in Trinidad

The prevalence of Salmonella spp. was determined in 970 animals comprising 423 pet birds, 485 fish aquaria water and 62 other pets (40 pet mammals, 14 reptiles, eight others - crustaceans, snail, stingray) from both pet shops and households throughout Trinidad. The serotypes of Salmonella spp. isolated were identified and the resistance to various antimicrobial agents was determined. Overall nine (0.9%) of 970 pet animals were positive for Salmonella spp. Six isolates of Salmonella spp. were recovered from all pet birds with two isolates of serotype Aberdeen and one isolate each of Thompson, Rubislaw, Panama and Newport. The prevalence of Salmonella spp. in birds was 0.9%. Four isolates of Salmonella spp. were recovered from fish aquaria water, serotypes included Panama (two isolates), Newport (one isolate) and Virchow (one isolate). Prevalence of Salmonella spp. from fish aquaria was 0.4%. No isolate of Salmonella spp. was detected in pet mammals sampled while two isolates were recovered from reptiles, S. Enteritidis and S. Montevideo. One isolate of Salmonella spp. was recovered from a stingray, serotype unknown. Antimicrobial resistance was present is all animal types. The highest prevalence of resistance was to streptomycin among isolates from birds (83.3%) and other pets (100.0%) while isolates from fish aquarium water exhibited comparatively high resistance to cephalothin (50.0%). It was concluded that the isolation of Salmonella spp. from apparently healthy birds, fish aquarium water and other pet animals may pose a health risk to their owners and contacts as all serotypes are known to be potentially pathogenic depending on the oral dosage of the organism and the immune status of those in contact. The high prevalence of resistance to antimicrobial agents among Salmonella isolates across pet species may pose chemotherapeutic consequences to their owners and contacts.