The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nalr) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nalr and PCP separately there were no significant differences in the numbers of S1400/94 Nalr and PCP bacteria in tissues sampled on days 1, 2, and 5. By day 42 after challenge S1400/94 Nalr bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nalr. In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nalr. In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays an important role in poultry infections although this role is less important in the newly hatched chick.     

Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection

The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.     

A serological survey for pathogens in old fancy chicken breeds in central and eastern part of The Netherlands

To get an impression of the presence of pathogens in multi-aged flocks of old fancy chicken breeds in the Netherlands, plasma samples originating from 24 flocks were examined for antibodies against 17 chicken pathogens. These flocks were housed mainly in the centre and east of the Netherlands, regions with a high poultry density. The owners of the tested flocks showed their chicken at national and international poultry exhibitions. Antibodies against Avian Influenza, Egg Drop Syndrome '76 virus, Pox virus, Salmonella pullorum/gallinarum, Salmonella Enteritidis or Salmonella Typhimurium were not detected. However, antibodies against other Salmonella species, Mycoplasma gallisepticum, infectious bursal disease virus, infectious bronchitis virus, avian encephalomyelitis virus, chicken anaemia virus, infectious laryngotracheitis virus, and avian leukosis virus, subgroups A and B, and subgroup J were detected in a varying proportion of the flocks. This study shows that antibodies against many chicken pathogens are present among the flocks of old fancy chicken breeds that are exhibited at international poultry exhibitions.     

Specified pathogen-free poultry flocks: the current situation

Before the implementation of strategies to establish specified pathogen-free commercial poultry flocks, the ultimate goals need to be identified: 1) consumer protection by minimizing the risk for zoonotic diseases and food-borne pathogens, and/or 2) animal health protection against primary and secondary pathogens. The success for the establishment of specific pathogen-free poultry flocks depends on the biological characteristics, the epidemiological distribution and the transmission route of each pathogen. For zoonotic pathogens such as Salmonella Typhimurium, Salmonella Enteritidis, Campylobacter jejuni or the high pathogenic avian influenza virus, eradication has to be ultimate goal. Despite tremendous control efforts in field, only partial control of these pathogens has been achieved so far. In the future it will be necessary to continue these eradication efforts by combining optimized hygiene programs at all production levels with intensive monitoring and immunoprophylaxis. For primary pathogens affecting the health condition of poultry without known zoonotic potential, such as Salmonella Gallinarum, avian Mycoplasma or leucosis virus, specified pathogen free flocks have been established on the parent and grandparent level. In order to achieve a status free of these pathogens, rigid hygiene control, especially on the hatchery level and monitoring programs combined with elimination of pathogen- and antibody-positive birds were implemented. Nevertheless, the economically most important diseases of modern poultry production are of multifactorial origin. Ubiquitous secondary pathogens in combination with insufficient management or immunosuppressive agents induce great economic losses for the poultry producers. These secondary pathogens can not be eliminated due to their ubiquitous distribution. In the future only a reduction of these factorial diseases will be possible combining hygiene management and optimization of poultry husbandry. For the establishment of specified pathogen free poultry flocks in the field, risk analysis is necessary and the structure of poultry production has to be considered before and eradication program can be carried out successfully.     

Effect of a commercial competitive exclusion culture on reduction of colonization of an antibiotic-resistant pathogenic Escherichia coli in day-old broiler chickens

One-day-of-age broiler chickens were administered a commercial competitive exclusion (CE) product and then challenged by three different methods with an Escherichia coli O78:K80 that was pathogenic for poultry and resistant to six antibiotics. Three challenge methods were used on 2-day-old broilers: direct challenge, precolonized seeder, and instant seeder. Direct challenge was accomplished by administering the challenge E. coli per os. The precolonized seeder challenge had two chicks that had received the challenge E. coli 24 hr previously, whereas the instant seeder challenge had two chicks given the challenge E. coli per os with immediate placement with the experimental birds. One oral dose of the commercial CE product significantly reduced the colonization of the small intestine, large intestine, and ceca by the highly antimicrobial resistant poultry pathogenic E. coli O78:K80 at 7 and 14 days postchallenge by all three challenge methods. The overall mean reductions in colonization were 3.0 log10 for the large intestine, 3.0 log10 for the small intestine, and 4.0 log10 for the cecum. The most severe challenge method, on the basis of the least amount of reduction of colonization of the challenge E. coli by the CE, was by the direct oral gavage at 2 days of age.     

B型产气荚膜梭菌对补硒母羊产新生羔羊的攻击试验

采用人工攻击B型产气荚膜梭菌的方法，观察了B型产气荚膜梭菌对青海三角城羊场补硒羊只的致病性，缮果表明，B型产气荚膜梭菌不引发补硒母羊产新生羔羊的羔羊痢疾。     

华中地区鸡源沙门菌分离鉴定及耐药性分析

为了探明华中地区种鸡场沙门菌(Salmonella)的优势血清型和耐药情况,本研究从湖北、河南、湖南等省市22个规模化鸡场采集病鸡、死胚及弱雏组织样品3 724份,通过分离培养、生化试验、PCR鉴定及血清型试验确定分离菌种属及其血清型,并采用Kirby-Bauer法对分离菌株进行了耐药性分析。结果显示,本试验从3 724份病料中共分离鉴定出124株沙门菌,其中79株为D群肠炎沙门菌(63.71%,79/124),34株为D群鸡白痢沙门菌(27.42%,34/124),8株为B群鼠伤寒沙门菌(6.45%,8/124),有3株沙门菌未能确定血清型。O抗原鉴定79株肠炎沙门菌和34株鸡白痢沙门菌为O9,8株鼠伤寒沙门菌为O4。H抗原鉴定79株肠炎沙门菌为Hg,m,8株鼠伤寒沙门菌为Hi。药敏试验结果显示,124株分离菌株对萘啶酸、氨苄青霉素、四环素和多西环素耐药率分别为95.97%(119/124)、91.94%(114/124)、57.26%(71/124)和70.16%(87/124);对复方新诺明和红霉素耐药率分别为25.81%(32/124)和12.10%(15/124);对氯霉素、庆大霉素、头孢噻肟和卡那霉素耐药率分别为6.45%(8/124)、1.61%(2/124)、1.61%(2/124)和0.81%(1/124);对左氧氟沙星、阿米卡星和多黏菌素B完全敏感。99.19%(123/124)的分离株至少对一种药物耐药,87.10%(108/124)的分离株表现多重耐药。本研究为华中地区养鸡场沙门菌的诊断及防控提供了数据支撑。     

Bacillus subtilis spores competitively exclude Escherichia coli O78:K80 in poultry

Newly hatched specific pathogen-free chicks were dosed with a suspension of Bacillus subtilis spores prior to challenge with Escherichia coli O78:K80, a known virulent strain associated with avian colibacillosis, 24h later. A single oral inoculum of 2.5x10(8) spores was sufficient to suppress all aspects of E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over 2log(10) whilst colonisation of the intestine, as measured by direct caecal count, was reduced over 3log(10). Shedding of E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the duration of the experiment, 35 days. B. subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores.     

鸡沙门氏菌脉冲场凝胶电泳分型研究

为应用脉冲场凝胶电泳(PFGE)从分子水平上对禽源沙门氏菌之间的差别进行分析和研究,本研究采用自动微生物鉴定仪对12株疑似鼠伤寒沙门氏菌和15株疑似鸡白痢沙门氏菌进行了鉴定.以限制性核酸内切酶Xba I对其全基因组DNA进行酶切、PFGE分型,Info Quest FP聚类分析软件对电泳结果进行分析.结果显示,共鉴定出11株鼠伤寒沙门氏菌、1株圣保罗沙门氏菌、6株阿姆斯特丹沙门氏菌、5株亚拉巴马沙门氏菌、1株鸡白痢沙门氏菌、2株纽波特沙门氏菌和2株肠炎沙门氏菌,一共分为12个PFGE型.实验结果表明,江苏地区存在有不常见的沙门氏菌血清型,同种血清型之间的基因型差别较小(相似值为0.85～1),不同血清型之间差别较大(相似值为0.45～0.70).PFGE能从分子水平上对禽源沙门氏菌的基因组DNA进行分型.     

Interaction between probiotic lactic acid bacteria and canine enteric pathogens: a risk factor for intestinal Enterococcus faecium colonization?

Selected probiotic lactic acid bacteria (LAB) have been shown to elicit positive health effects particularly in humans. Competitive exclusion of pathogens is one of the most important beneficial health claims of probiotic bacteria. The effect of probiotic LAB on competitive exclusion of pathogens has been demonstrated in humans, chicken and pigs. In this study we evaluated the ability of certain LAB strains (Lactobacillus rhamnosus GG, Bifidobacterium lactis Bb12, Lactobacillus pentosus UK1A, L. pentosus SK2A, Enterococcus faecium M74 and E. faecium SF273) to inhibit the adhesion of selected canine and zoonotic pathogens (Staphylococcus intermedius, Salmonella Typhimurium ATCC 14028, Clostridium perfringens and Campylobacter jejuni) to immobilised mucus isolated from canine jejunal chyme in vitro. Adhesion of C. perfringens was reduced significantly by all tested LAB strains, between 53.7 and 79.1% of the control without LAB, the LAB of canine origin yielding the best reduction. The adhesion of S. Typhimurium and S. intermedius were not significantly altered by any of the LAB included in the study. Both enterococci tested significantly enhanced the adhesion of C. jejuni, to 134.6 and 205.5% of the control without LAB. E. faecium may thus favor the adhesion and colonization of C. jejuni in the dog's intestine, making it a potential carrier and possibly a source for human infection. Enhanced C. jejuni adhesion is a new potential risk factor of enterococci. Our results further emphasize the importance of safety guidelines to be established for the probiotics intended for animal use.     

枯草芽孢杆菌芽孢作为黏膜疫苗载体研究进展

枯草芽孢杆菌属于食品安全级别的微生物,因其培养简单、非致病性和遗传操作简便等特点,被广泛应用于多个领域。近年来,随着DNA重组技术快速发展,枯草芽孢杆菌被应用于黏膜疫苗载体开发,以应对细菌、病毒、寄生虫等病原体的侵入。黏膜免疫操作简便,通过口服、滴鼻、舌下等黏膜途径免疫,枯草芽孢杆菌可以有效诱导宿主机体产生体液和细胞免疫反应,因此枯草芽孢杆菌作为黏膜疫苗载体拥有巨大的潜力。本文在查阅国内外相关文献报道的基础上,介绍了枯草芽孢杆菌孢子作为黏膜疫苗载体在预防病毒、细菌和寄生虫病相关方面的应用,旨在为开发新型高效黏膜疫苗提供参考。     

Administration of autochthonous intestinal microflora--a method to prevent Salmonella infections in poultry

Administration of autochthonous intestinal microflora to chicks during the early period after hatching (Competitive Exclusion) is a widely accepted prophylactic method to control Salmonella infections in poultry. The method of competitive exclusion consists in an administration of intestinal flora from healthy adult birds to chicks during the first hours or days of their life. Use of competitive exclusion cultures will considerably enhance resistance to all Salmonella serovars colonising the chicken intestine and reduce shedding of salmonellas by infected animals. However, sole use of this method does not completely prevent Salmonella colonisation of the animals nor elimination of the agents from poultry flocks. At present, only complex competitive exclusion cultures whose composition has not been defined are capable of inducing an adequately high and reproducible efficacy. Effective preparations with a defined composition have not yet been developed because knowledge of the mechanisms of action of the competitive exclusion cultures as well as the effective species of the various bacterial genera is still inadequate. Since in approval procedures, the competitive exclusion cultures with a non-defined composition can neither be classified as medicines nor feed additives nor vaccines, WHO has proposed to establish the product category "Normal Gut Flora" (WHO, 1994). Basic prerequisites for an effective reduction of non-host-adapted Salmonella serovars in, or their elimination from poultry flocks are the performance and assurance of effective hygienic measures. Like the methods of immunisation using live or inactivated Salmonella vaccines in poultry, the method of competitive exclusion constitutes an additional prophylactic method that may be applied directly in the animal to enhance its resistance to Salmonella infection.     

Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

枯草芽孢杆菌Pab02喷雾剂对肉鸡呼吸道菌群的影响

选取48羽7日龄艾维茵肉公鸡随机分为对照组和试验组,试验组动物房内每日8时喷雾1mL/m3的枯草芽孢杆菌Pab02制剂（108 CFU/mL）,对照组同法喷雾100mL蒸馏水。28日龄时对肉鸡测体质量,采用活菌计数法和PCR-DGGE法对28,49日龄肉鸡呼吸道菌群进行检测,石蜡组织切片法对肺部结构进行观察。结果显示,喷雾枯草芽孢杆菌Pab02对28日龄肉鸡的采食量、日增重、料肉比等生长性能有所提高,但不显著（P〉0.05）;喷雾Pab02能增加49日龄呼吸道细菌数量（P〈0.01）,提高同一生态位菌群的稳定性和呼吸道菌群的多样性;Pab02喷雾剂不引起肺组织损伤,能使淋巴小结增多。结果表明,芽孢杆菌Pab02喷雾剂不能显著改善肉鸡的生长性能,但是能调节呼吸道菌群的结构,增强肺脏的免疫功能。     

Serologic detection of adenovirus infections in specific-pathogen-free chickens

A specific-pathogen-free flock of White Leghorns, which were housed conventionally and were previously serologically negative for all common poultry pathogens including avian adenoviruses, incurred an outbreak of adenovirus that was detected at about 39 weeks of age. The infection was detected serologically through the agar-gel precipitin test (AGPT) and also by a microneutralization test (MNT) adapted for 11 serotypes of avian adenovirus. The MNT detected specific antibodies to serotype-3 avian adenovirus (IBHV-Tipton) but no other serotype. While AGPT-positive sera drawn from the flock gradually declined from 54% to 17%, neutralizing-antibody levels rose sharply at 46 weeks of age to a peak that was maintained over the remaining 18 weeks of the flock's production.     

Entry and survival of Salmonella enterica serotype Enteritidis PT4 in chicken macrophage and lymphocyte cell lines

Various leukocytes are involved in the reaction to counter Salmonella infection in chicken. The various leukocyte types react differently after an infection, since some clear the infection while others may cause dissemination of Salmonella throughout the chicken. Therefore, we investigated in vitro the entry and survival of Salmonella enterica serotype Enteritidis in chicken cell lines of various cell types, including two macrophage cell lines, HD11 and MQ-NCSU (NCSU), two B-cell lines LSCC-1104-X5 (1104) and LSCC-RP9 (RP9), and a T-cell line MDCC-MSB-1 (MSB-1). The macrophages were able to internalize high numbers of S. Enteritidis. In contrast and as expected, cells of the T-cell line MSB-1 and the B-cell line RP9 internalized bacteria at a much lower level. After S. Enteritidis entered the macrophages, the number of intracellular S. Enteritidis decreased over time, so that after 48h no more than 20% of the bacteria, which had entered, survived intracellularly. In contrast to macrophages, the number of S. Enteritidis in cells of the T-cell line MSB-1 and the B-cell line RP9 increased rapidly within 12h post-inoculation. Thereafter the number of intracellular S. Enteritidis decreased only slowly. In conclusion, all three different cell types were able to control and to start clearing S. Enteritidis, although macrophages were far more effective compared to T- and B-cells. However, none of the cell lines were able to clear S. Enteritidis fully within 48h. These results suggest that the three cell types play an important but different role in the dissemination and elimination of S. Enteritidis throughout the animal.     

Prevalence of Clostridium perfringens in commercial broiler hatcheries.

Clostridium perfringens, a cause of human foodborne and poultry disease, has been isolated from the intestinal tract of poultry and from the processed carcass. Little is known about the incidence and sources of this pathogen in the poultry production environment. To determine if the broiler hatchery is a possible source of C. perfringens, we collected samples from three hatcheries, each operated by a different poultry integrator, and the presence of C. perfringens in these samples was determined. For each sampling period, eggshell fragments, chick fluff from the hatcher, and paper pads stored in the hatchery before use with chicks and after placement beneath chicks for 1 hr were evaluated. Clostridium perfringens was found in eggshell fragments, fluff, and paper pads in each of the three hatcheries. The percentages of C. perfringens-positive samples from the three hatcheries ranged from 13% to 23%, with an overall incidence of 20%. Positive samples were consistently found, i.e., detected on each of the nine sampling days (three sampling days for each of three hatcheries). These results suggest that the hatchery is a potential source/reservoir for C. perfringens in the integrated poultry operation.     

Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh

We investigated Salmonella enterica isolates from human clinical cases of gastroenteritis to determine the distribution of non-typhoidal Salmonella serovars in the human population, and compared them to isolates originating from poultry by serotyping, phage typing, plasmid profiling, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to evaluate the potential role of poultry in human non-typhoidal salmonellosis in Bangladesh. Nine different serovars were identified among the human isolates of which Salmonella Paratyphi B var Java (S. Java), S. Kentucky, S. Enteritidis, S. Virchow and S. Weltevreden also were commonly isolated from poultry. The poultry isolates belonging to S. Java, S. Kentucky and S. Enteritidis were indistinguishable from human isolates or genetically closely related, based on PFGE profiles and MLST. S. Kentucky clone ST198 and S. Java clone ST43 both well-known cause of human infections were also isolated from poultry.     

产气荚膜梭菌肠毒素的分子生物学研究进展

产气英膜梭菌(Clostridium perfringens)是一种重要的人兽共患病的病原体,它是一类革兰氏阳性产芽孢的厌氧梭菌,可引起人的食物中毒和肠毒素相关腹泻.近年来,研究人员发现由产气英膜梭菌导致腹泻的主要原因与该菌在芽孢形成过程中产生的一种肠毒素有关.文章重点针对产气英膜梭菌肠毒素的生物学特性和致病机制等方面的研究做一综述.