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.     

Safety,immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.     

表达IBDV VP2蛋白重组减毒沙门菌活载体疫苗株的构建及免疫原性分析

通过PCR克隆出IBDV VP2基因,将其插入到表达载体pYA3341中,构建重组质粒pYA3341-VP2。将重组质粒电转入鼠伤寒沙门菌疫苗株X4550（缺失Asd、Cya、Crp基因）,获得重组疫苗菌株X4550（pYA3341-VP2）。进行重组菌VP2蛋白表达的鉴定;测定重组菌的稳定性、生长曲线、安全性以及小鼠免疫试验。结果表明,酶切鉴定证实重组质粒构建成功;SDS-PAGE和Western blot证实重组菌表达的VP2蛋白能与鸡抗IBDV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用;口服重组菌免疫小鼠,ELISA检测产生了抗IBDV抗体;中和试验表明产生的抗体具有中和活性。本试验成功构建了能稳定表达IBDV VP2蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550（pYA3341-VP2）,为研究IBD口服基因工程疫苗奠定了基础。     

表达猪繁殖与呼吸综合征病毒GP5蛋白减毒鼠伤寒沙门氏菌疫苗株的构建及动物免疫试验

本试验旨在构建表达猪繁殖与呼吸综合征病毒(PRRSV) GP5蛋白的口服重组减毒鼠伤寒沙门氏菌活载体疫苗株.PCR克隆除去信号肽序列的PRRSV ORF5基因,将其插入到表达载体pYA3341中,构建重组质粒pYA3341-ORF5.将重组质粒电转入鼠伤寒沙门氏菌疫苗株X4550(缺失Asd、Cya、Crp基因),获得重组疫苗菌株X4550(pYA3341-ORF5).鉴定重组菌GP5蛋白的表达;测定重组菌定居特性及安全性;检测免疫小鼠血清抗体;流式细胞仪检测重组菌对小鼠T淋巴细胞CD4+和CD8+亚群的影响;最后进行免疫猪血清抗体检测.结果表明,酶切鉴定证实重组质粒构建成功;Western blot证实重组菌表达的GP5蛋白能与PRRSV阳性血清特异性结合;重组菌在体内可较稳定地定居于小鼠的肠系膜淋巴结和脾脏中,并在其中表达出GP5蛋白;小鼠口服试验证实重组菌无毒性作用;重组菌口服免疫小鼠可以产生抗GP5蛋白抗体且抗体具有中和活性;重组菌株能不同程度地使CD4+、CD4+/CD8+升高,而使CD8+下降,表明重组菌对细胞免疫功能具有调节作用;淋巴细胞增殖试验表明,重组菌能诱发小鼠产生较强的细胞免疫应答;重组菌口服免疫猪可以产生抗GP5蛋白抗体.本试验成功构建了能稳定表达PRRSV GP5蛋白的口服减毒鼠伤寒沙门氏菌疫苗株,为研究PRRSV口服基因工程疫苗奠定基础.     

The effect of exposure to a delta cya/delta crp mutant of Salmonella typhimurium on the subsequent colonization of swine by the wild-type parent strain.

This study characterizes the clinical response and colonization pattern of caesarean-derived, colostrum-deprived swine exposed to a delta cya/delta crp mutant (chi 4233) of S. typhimurium and challenged with the wild-type parent strain. chi 4233 was mildly virulent in swine and induced transient fever and soft stools. Chi 4233 colonized the ileum, cecum, liver, spleen, tonsils, and mandibular and ileocolic lymph nodes of swine in a manner similar to the parental wild-type, but the numbers of S. typhimurium (chi 4233) in the ileum were 100- to 1000-fold less than those of pigs exposed to the parental wild-type. Pigs exposed to chi 4233 21 days before parental wild-type challenge demonstrated a milder clinical response to challenge than did pigs that did not receive chi 4233. The wild-type populations in the ilea of chi 4233-exposed pigs after challenge were 100- to 10,000-fold less than those in pigs not receiving chi 4233. The liver, spleen, and ileocolic lymph nodes were cleared of wild-type S. typhimurium more quickly after challenge in chi 4233-exposed pigs. The populations of chi 4233 in the ilea of exposed pigs after wild-type challenge were also less than would have been expected in unchallenged pigs. Thus, exposure of swine to a delta cya/delta crp mutant of S. typhimurium modulated the subsequent response to parental wild-type challenge and reduced carrier populations of wild-type S. typhimurium in infected swine.     

Reciprocal competitive exclusion of salmonella and Escherichia coli by native intestinal microflora of the chicken and turkey

Both the native intestinal microflora of chickens that protected chicks against salmonellae and Escherichia coli and native turkey intestinal microflora were evaluated for their reciprocal protective capacity in both species against Salmonella typhimurium and a pathogenic strain of E. coli. Nalidixic-acid-resistant forms of the S. typhimurium and E. coli strains were used in seeder-bird and individual-bird challenge tests. Reciprocal protection was provided by native chicken and turkey intestinal microflora in chicks and poults against S. typhimurium and the pathogenic strain of E. coli. The chicken and turkey microflora appeared to be equally effective in protecting the two species from S. typhimurium, but protection against E. coli was somewhat greater in the chicken than in the turkey.     

禽大肠杆菌病免疫保护机理的研究

以禽病原性大肠杆菌O18、O78分离株制成超声波裂解铝佐剂灭活苗免疫14日龄鸡，以相同或不同外膜蛋白型（Outer membrane protein pattern,OMP型）的O18、O78分离株攻毒。结果表明：O78血清相同和不同OMP型分离株间能获得最大保护；O18血清型相同OMP型分离株间获得最大保护，而不同OMP型分离株间不能保护；上述两个血清型的分离株间不论OMP型是否相同，均缺乏保护。以间接ELISA试验、间接血凝试验分别测定了试验鸡临攻毒前针对大肠杆菌OMPs和脂多糖(Lipopolysaccharide,LPS）的抗体。结果表明：免疫组鸡血清上述两种抗体明显高于攻毒对照组；在免疫组，存活鸡临攻毒前血清中上述两种抗体滴度恒高于死亡鸡，但除3个组外，多数组差异不显著。攻毒对照组这一关系不稳定。结果说明：禽大肠杆菌疫苗的免疫保护，主要与O血清型有关，部分与OMP型有关，如O18分离株，免疫保护性抗原含OMPs,LPS等多个抗原表位。     

Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.     

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.     

Recombinant Iss as a potential vaccine for avian colibacillosis

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.     

乳酸杆菌对病原菌粘附肉鸡肠粘液的影响

研究嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同肠段粘液糖蛋白的粘附性能,探讨嗜酸乳杆菌对四种病原菌的粘附排斥作用.结果表明,在不同的肠道部位,嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白的粘附能力不同,而禽大肠杆菌O78、大肠杆菌ATCC 25922在各肠段粘液上的粘附性能相近;在相同的肠道部位,所试菌与肠粘液糖蛋白的粘附能力有差异,其中嗜酸乳杆菌的粘附作用最强;嗜酸乳杆菌对所试病原菌均有不同程度的粘附排斥作用,其中对鸡白痢沙门氏菌的粘附排斥较强,而对大肠杆菌ATCC25922的则较弱.     

鸡大肠杆菌Ⅰ型菌毛亚单位苗交叉保护的初步研究

含Ⅰ型菌毛的３ 个不同血清型（Ｏ１ 、Ｏ７８ 及Ｏ８８） 的菌株， 大容量培养后提取菌毛制备３ 种单价菌毛油乳苗。用１ 日龄雏鸡分别免疫３ 种单价菌毛油乳剂苗， 每雏免疫量为１２５ μｇ ， 隔离饲养至２ 周龄经气囊攻毒， 并评价疫苗的免疫原性。结果未免疫鸡出现８７－５ ％ ～１００ ％ 的死亡率， 免疫鸡用同源菌株攻毒后死亡率仅１２－５ ％ ， 用异源菌株攻毒出现３７－５％ ～６２－５ ％ 的死亡率。免疫鸡攻毒后未死亡者， 经扑杀观察， 可见在气囊、心包及肝脏的病变非常轻微， 且攻毒后比非免疫鸡能更有效地清除攻入气囊的大肠杆菌     

Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens

Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive exclusion approaches being used widely. Previous studies showed that Bacillus subtilis PY79(hr) was an effective competitive exclusion agent for use in poultry to control avian pathogenic Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of B. subtilis PY79(hr) in the control of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of B. subtilis spores prior to challenge with S. Enteritidis (S1400) and C. perfringens, respectively. For both challenge models, a single oral inoculum of 1x10(9) spores given 24h prior to challenge was sufficient to suppress colonisation and persistence of both S. Enteritidis and C. perfringens. In particular, the faecal shedding of S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment. B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.     

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.     

Cellular defense of the avian respiratory system: protection against Escherichia coli airsacculitis by Pasteurella multocida-activated respiratory phagocytes

The concept of nonspecific cellular defense of the respiratory system of poultry against respiratory pathogens by "preventive activation" of avian respiratory phagocytes (ARPs) was tested in an in vivo protection trial. Chickens were stimulated intratracheally by Pasteurella multocida Choloral vaccine strain. Seven hours later, these and mock-inoculated control chickens were challenged with pathogenic Escherichia coli via the air-sac route. Stimulated chickens had a 25-fold-elevated number of ARPs compared with mock-inoculated control chickens. The proportion of active phagocytes and the phagocytic capacity of these cells was higher in the ARP populations of stimulated chickens than in the ARP populations of control chickens. In vivo protection against E. coli air-sac infection was demonstrated by reduction of morbidity and mortality rates, diminished weight loss, and lower scores of gross and histopathological lesions of P. multocida-stimulated chickens compared with mock-inoculated controls.     

B cell and macrophage response in chicks after oral administration of Salmonella typhimurium strains

Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis. In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.     

鸡源大肠埃希氏菌部分生物学特性研究

对从天津地区分离到的71株鸡大肠杆菌的部分生物学特性包括致病性、血清型、耐药特性和免疫原性等进行了研究。结果表明其中60株为致病性菌株,占分离菌株的84.5%;60个致病性菌株共定型出45个菌株,分属O1、O2、O5、O6、O20、O45、O53、O74、O75、O78、O88、O89、O92、O107、O111、O145等16个血清型,其中O78、O88、O2、O45、O53和O145为优势血清型,占定型菌株的73.4%;试验菌株具有广泛的耐药性,60个致病性菌株均为多重耐药。免疫原性测定试验结果表明,O2、O78、O88血清型菌株均可对相同血清型菌株提供很好的保护,但3个血清型菌株之间缺乏有效的保护。     

几种病毒与禽病原性大肠杆菌的人工联合感染

以2种剂量的低致病性禽流感病毒(lowly pathogenic avian influenza virus,LPAIV)，传染性支气管炎病毒（infectious bronchitis virus,IBV)疫苗株H120和H52，新城疫病毒（Newcastle disese viurs,NDV)Lasota株分别于气管内注射10日龄易感鸡，2d后，气管注射禽病原性大肠杆菌O37株（O78），连续观察5d，结果，除LPAIV单独感染组有6．25％的死亡率外，其余各病毒单独接种组均健活；大肠杆菌O37株单独接种组的死亡率为62．50％，较高剂量的LPAIV，IBV H120和H52，NDV Lasota株与大肠杆菌O37株有效强的协同致病作用，死亡率分别达到81.25%,100.00%,93.75%和87.50%，而较低剂量的上述病毒则无明显的协同作用，IBV，NDV疫苗株与大肠杆菌联合接种组的多数死亡鸡病程推迟。     

鸡毒支原体SC克隆致弱株免疫原性测试——开放动物模型的建立与应用

对罗斯鸡和 Ｓ Ｐ Ｆ来航鸡用鸡毒支原体强毒攻击时,以鸡致病性大肠杆菌 Ｏ７ ８ 血清型菌株 １６ 小时培养物 ０２ｍ ｌ／羽皮下注射作人工诱导发病,试验鸡出现明显气囊病变,初步建立了以鸡致病性大肠杆菌为诱导因子的 Ｍ Ｇ 野外环境人工发病的动物模型。以此开放模型检测 Ｓ Ｐ Ｆ来航鸡以鸡毒支原体 Ｓ６ 克隆致弱株 Ｆ１５６ 代培养物点眼、滴鼻免疫后 ３０ 日、６０ 日龄的攻毒保护率,结果保护率分别达 ９０％ （１８／２０）、８４％ （４２／５０）,而对照组为 ３０％ （６／２０）、５０％ （１５／３０）,中期免疫效果证明,鸡毒支原体 Ｓ６ 克隆致弱株有良好的免疫保护作用。     

Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.