Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

双重PCR检测肠炎沙门氏菌方法的建立

沙门氏菌是引起人类食物中毒事件的主要病原之一,其中,肠炎沙门氏菌因其可感染家禽、污染禽蛋而受到广泛关注。本试验根据沙门氏菌属特异性基因片段fimY和肠炎沙门氏菌特异性基因片段sdfI分别设计一对引物,对25株沙门氏菌和大肠杆菌进行双重PCR扩增,结果显示22株沙门氏菌均出现与理论值大小497bp相符的属特异性条带,作为对照的3株大肠杆菌则未显示该条带,并且7株肠炎沙门氏菌均出现与理论值大小293bp相符的特异性条带。另外,敏感性试验结果显示肠炎沙门氏菌50336和鸡白痢沙门氏菌SP1的模板浓度分别为18ng和15ng时仍能清晰扩增出特异性条带。上述结果表明建立的PCR方法具有快速简便、灵敏性高、特异性强等特点,为公共卫生、食品安全、畜牧兽医及出入境安检等多部门检测和监测沙门氏菌提供了前提基础和技术保障。     

肠炎沙门氏菌快速检测方法的建立

利用针对肠炎沙门氏菌主要O因子抗原O9的3－47－26单克隆抗体建立了快速检测肠炎沙门氏菌的竞争ELISA方法（C－ELISA）。将被检样品与McAb作用，竞争性抑制了McAb与包被肠炎沙门氏菌的结合，同时，也减弱了酶标二抗与McAb的结合，以降低底物反应的颜色，从而达到检测样品的目的。试验结果表明，本方法只选择性地与肠炎沙门氏菌反应，而与其它沙门氏菌均不反应。通过与国标法对210份样品检测结果比较表明，竞争ELISA方法的敏感性和特异性分别为94．4％和97．7％，从而为肠炎沙门氏菌的检测提供了一种快速、敏感、特异的检测方法。     

肠炎沙门氏菌感染及其检测方法研究进展

该文主要介绍了宿主对肠炎沙门氏菌(SE)的防御能力、SE感染与宿主细胞的关系、SE在禽类中多呈隐性感染的原因分析,并对当前SE检测方法的研究进展作了一综述.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

应用改良阻断ELISA检测禽网状内皮组织增殖病血清抗体

应用禽网状内皮组织增殖病病毒纯化抗原和抗ＲＥＶ单克隆抗体建立了改良阻断ＥＬＩＳＡ用于鸡血清中ＲＥＶ抗体检测，并对北京地区鸡群中随机采样的３６份血清样本进行了检测，阳性率为５．６％。与间接ＥＬＳＩＡ的检测结果进行了统计学比较，两种方法的阳性率无显著差异。结果表明本试验所建立的改良阻断ＥＬＩＳＡ可以用于鸡群ＲＥＶ感染的血清学调查。     

Adsorption of Salmonella enteritidis by cetylpyridinium-exchanged montmorillonite clays

Recent experiments in our laboratory have suggested that certain montmorillonite clays, when exchanged with the cationic surfactant cetylpyridinium (CP), may be useful in removing bacteria from aqueous solution. During an initial study, screening various CP-exchanged products for potential antibacterial activity, three CP-exchanged clays - CP*AAM (acid-activated montmorillonite), CP*STx-1 (Ca(++)-montmorillonite), and CP*SWy-2 (Na(+)-montmorillonite), proved to be the most effective. Binding studies were performed using 1mg each of CP-exchanged AAM, STx-1, and SWy-2 with a standardized Salmonella enteritidis solution containing approximately 40,000 colony forming units (CFU)/ml. The modified clays reduced bacterial numbers 98.1, 97.6, and 95.2%, respectively. In contrast, the parent clays only produced reductions of 39.8, 16.9, and 16.6%, respectively. Attempts were made to desorb CP from the modified clays by washing in sterile physiological saline for 24h. The resulting wash solutions failed to produce any significant reduction in bacterial colony counts; while, the washed clays retained their full antimicrobial activity. These findings suggested that the antibacterial effect of the clays is localized on the clay surface and is not due to CP dissociating from the clay. Electron microscopy revealed that the bacteria adhered to the surface of the CP-exchanged clays, but not the parent clays. Results from timed binding studies showed that the antibacterial effect was stable over the period observed. Rates of binding were positively influenced by increasing temperature, not affected by changes in pH, and negatively influenced by the presence of organic contaminants. The mechanism by which bacterial counts are reduced may involve the enhanced hydrophobicity and affinity of the CP-exchanged clay for Salmonella and the antibacterial activity of CP.     

肠炎沙门菌LAMP检测方法的建立

为建立一种能快速检测肠炎沙门菌(SE)的方法,本研究根据基因库中SE种特异性基因(sdfⅠ)的保守序列,设计一套特异性环介导等温扩增(LAMP)引物,建立了SE的LAMP可视化检测方法.该方法的敏感性可达100 fg DNA,高于常规PCR方法100倍;全部反应可在1h内完成;可通过肉眼观察颜色直接判定结果;对其他常见病原体的检测结果均为阴性.结果表明建立的LAMP方法简便、快速、灵敏、特异,可用于SE感染的快速检测.     

Detection of antibodies to S. enteritidis in broilers by means of indirect ELISA and chemiluminescent immunoassay (CLIA)

This study was conducted to develop a serological detection system for the monitoring of broiler flocks for Salmonella enteritidis infections. A specific S. enteritidis antigen (FG-Antigen) was used to compare the sensitivity and the specificity of the chemiluminescent immunoassay (CLIA) with those of the indirect ELISA. This comparison was performed using a total of 578 sera, which, depending on the microbiological and vaccination history, were categorized into groups. Most of the serum samples which were classified as positive showed higher titers in CLIA than in ELISA. Using the prevalence of positive reactors, significant differences between Groups were additionally demonstrated. The absorbance values of the passively immunized group showed the highest and those of the Salmonella-negative group the lowest correlation-coefficient. Using the mean net absorbance of the prevalence group, the ELISA system exhibited a sensitivity of 100% and a specificity of 96.2%, while CLIA had a sensitivity and a specificity of 85.7% and 96.2%, respectively. ELISA and CLIA can be used in the examination of non vaccinated flocks for S. enteritidis-infections as alternative to the bacteriological culture method. CLIA is distinguished for its fast and convenient procedure as well as for its wider measurement spectrum, while the indirect ELISA is almost as efficient as CLIA and requires less investment.     

Characterization of Salmonella enteritidis strains.

A study was conducted to characterize 318 Salmonella enteritidis strains that were mainly isolated from poultry and their environment in Canada. Biotype, phagetype (PT), plasmid profile (PP), hybridization with a plasmid-derived virulence sequence probe, antibiotic resistance, outer membrane proteins (OMPs), and lipopolysaccharide (LPS) profiles were determined. Relationships of these properties to one another, and their diagnostic and pathogenic significance were assessed. Biotyping indicated that failure to ferment rhamnose was sometimes useful as a marker for epidemiologically related strains. Phagetyping was the most effective method for subdividing S. enteritidis; it distinguished 12 PTs. Phagetype 13 was occasionally associated with septicemia and mortality in chickens. The strains belonged to 15 PPs. A 36 megadalton (MDa) plasmid was found in 97% of the strains. Only the 36 MDa plasmid hybridized with the probe. Seventeen percent of the strains were drug resistant; all strains were sensitive to ciprofloxacin. Thirty-five of 36 strains possessed the same OMP profile, and 36 of 41 strains contained smooth LPS.     

Salmonellosis in young calves due to Salmonella enteritidis.

The clinical and epidemiological features of an outbreak of salmonellosis due to Salmonella enteritidis in a group of calves are described. The major clinical signs were dullness, pyrexia and diarrhoea. Five of the 15 calves died but deaths were mainly confined to the younger members of the group. The recovery of salmonella organisms from rectal swabs was maximal shortly before four of the five deaths occurred and declined rapidly thereafter. Only two of the surviving 10 calves developed significant flagellar agglutination titres.     

Establishment of an enzyme-linked immunosorbent assay with a coated deflagellated Salmonella enteritidis antigen for detection of a specific chicken antibody

We attempted to establish an enzyme-linked immunosorbent assay (ELISA) for field monitoring/profiling purposes for Salmonella enteritidis (SE) infection of poultry flocks. Serotyping rabbit sera, commercially obtained, specific for Salmonella identification sera to O2, O4, O7, O8, S. Vi, S. Hm, and O9, showed negative ELISA (E)-values (< 0.2) on ELISA, except the O9 identification serum (E-value > 0.5). Similar negative E-value results were obtained for antisera to Echerichia coli (E. O antigen). Field serum samples originating from SE-isolated flocks yielded similar positive ratios on both ELISAs including the present coated deflagellated SE antigen and a commercially obtained flagellated SE antigen and that of rapid plate aggregation with a pullorum antigen (PD-RPA). About 100 days after the first monitoring, no SE isolation in the same flock was observed resulting in a carrier state of SE infection. Although both the monitoring results with commercially obtained ELISA and PD-RPA showed lower positive or negative ratios, the present ELISA showed a higher positive ratio than that of the first monitoring. The present ELISA is suggested to be a suitable method to do accurate profiling on the carrier state of infection.     

Duration of immunity induced in chickens by an attenuated live Salmonella enteritidis vaccine and an inactivated Salmonella enteritidis/typhimurium vaccine

The aim of this study was to examine the duration of immunity of different vaccination schemes using the S. enteritidis live vaccine Gallivac Se and the S. enteritidis-S. typhimurium inactivated vaccine Gallimune Se+St. Three groups of Lohman Brown chickens were used. Group one was vaccinated three times orally with Gallivac Se at weeks one, seven and 13 of age. Group two was vaccinated twice orally with Gallivac Se in weeks one and seven and once i.m. with Gallimune Se+St in week 14 of age. A third group was not vaccinated and served as the control group. Eight randomly selected chickens from each of the three groups were challenged with a nalidixic acid resistant S. enteritidis PT4 strain in weeks 24, 51 and 71 of age and the same number of animals were challenged with a S. typhimurium DT 104 strain in weeks 26, 54 and 73 (75) of age.The chickens were euthanised seven days post challenge and the number of challenge strain organisms (log10 cfu) in the liver and on caecal mucosa was determined.The quantitative investigation of the challenge strain in the liver and caecal mucosa revealed a statistically significant (p < 0.05) lower challenge strain burden in the vaccinated groups compared with the non-vaccinated control group up to week 71 (73) of age. The protective effects were demonstrated for both challenge strains.     

Detection of airborne Salmonella enteritidis in the environment of experimentally infected laying hens by an electrostatic sampling device

Bacteriologic culturing of environmental samples taken from sources such as manure pits and egg belts has been the principal screening tool in programs for identifying commercial laying flocks that have been exposed to Salmonella enteritidis and are thus at risk to produce contaminated eggs. Because airborne dust and aerosols can carry bacteria, air sampling offers a potentially efficient and inexpensive alternative for detecting S. enteritidis in poultry house environments. In the present study, an electrostatic air sampling device was applied to detect S. enteritidis in a room containing experimentally infected, caged laying hens. After oral inoculation of hens with a phage type 13a S. enteritidis strain, air samples were collected onto agar plates with the electrostatic sampling device, an impaction air sampler, and by passive exposure to the settling of aerosols and dust. Even though the floor of the room was cleaned once per week (removing most manure, dust, and feathers), air samples were positive for S. enteritidis for up to 4 wk postinoculation. On the basis of both the number of S. enteritidis colonies observed on incubated agar plates and the frequency of positive results, the efficiency of the electrostatic device was significantly greater than that of the passive exposure plates (especially at short collection intervals) and was similar to that of the far more expensive impaction sampler. The electrostatic device, used for a 3-hr sampling interval, detected airborne S. enteritidis on 75% of agar plates over the 4 wk of the study.