Iss from a virulent avian Escherichia coli

No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols. Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability. The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E. coli Iss protein. In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E. coli, and expression was induced. The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography. The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing. Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E. coli.     

A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli

Ultrasonic inactivation of Escherichia coli followed by irradiation was found to be the most efficient method for preparation of an effective vaccine against colibacillosis. Challenge experiments revealed that this vaccine provided the best protection compared with other methods of inactivation: heat, formaldehyde, and irradiation. Preparing the ultrasonicated vaccine from O2:K1 strain increased its range and also supported adequate protection against homologous strain O78:K80. The degree of protection conferred by the vaccine was positively correlated with the antibody titer against E. coli as measured on day of challenge. Low antibody titers detected 5 days post-vaccination resulted in only 20% protection. High antibody titers detected at 8 and 15 days post-vaccination correlated with a low number of chicks with lesions. In each challenged group, the live chicks that did not develop lesions had higher antibody titers than chicks with lesions, revealing a correlation between numbers of chicks with lesions and antibody titers as measured by enzyme-linked immunosorbent assay.     

Monoclonal antibodies to avian Escherichia coli Iss

Escherichia coli infections are a major problem for the poultry industry in the United States. Yet, the virulence mechanisms operative in avian E. coli are poorly understood. In the present studies, monoclonal antibodies (MAbs) have been generated that may facilitate study of the pathogenesis of avian colibacillosis. These MAbs are directed against the Iss protein because results from our laboratory have shown that the possession of iss DNA sequences is strongly correlated with the E. coli implicated in avian colibacillosis. As part of an overall effort to explore the role of iss/Iss in colibacillosis pathogenesis, Iss protein has been purified, MAbs to Iss have been generated, and the MAbs are being evaluated. B cells from mice immunized with an Iss fusion to glutathione-S-transferase produced antibodies specifically against Iss, and these cells were used to generate the MAbs. These anti-Iss MAbs, when used in western blotting assays, can be used to distinguish iss-positive and -negative E. coli isolates, suggesting that they may be useful as reagents in the detection and study of virulent avian E. coli.     

Comparison of several challenge models for studies in avian colibacillosis

In previous studies, the embryo lethality assay (ELA) discriminated between virulent and avirulent avian Escherichia coli isolates, and also proved to be highly correlated with mortality and morbidity results of the intravenous (IV) challenge model. In the current study, the same 20 avian E. coli isolates were used in subcutaneous (subQ) and intratracheal (IT) chicken challenge models in order to determine whether the results from the prior ELA challenges and/or the IV challenge model correlate with these models. The correlation observed between the two previous ELA trials and the combined mortality/morbidity percentages of the subQ challenge model were r = 0.792, P > 0.0001 for the first ELA trial and r = 0.738, P = 0.0002 for the second ELA trial. The IV challenge results were more highly correlated with the subQ challenge results (mortality/morbidity comparison, r = 0.894, P < 0.0001). The IV challenge mortality results were slightly correlated (r = 0.4810, P=0.0319) with the IT challenge results. Several of the isolates differed in their ability to produce mortality and/or morbidity with the different challenge models. The mortality/morbidity results of the IV and subQ challenges and the mortality results of the ELA were all positively correlated with the ability of an E. coli isolate to produce Colicin V (ColV) (r = 0.7131, P = 0.0004). The IT mortality results were slightly correlated with the production of ColV (r = 0.455, P = 0.049). The IT challenge results were only slightly correlated with resulting IV mortality and ColV production. Previous results indicate that the ELA correlates extremely well with the IV challenge model. The current study demonstrates that ELA also correlates well with the subQ challenge model. Overall, the conclusion of this study is that the ELA, IV, and subQ challenge models similarly demonstrate the ability to discriminate between virulent and avirulent avian E. coli isolates.     

禽大肠杆菌病防制研究进展

禽大肠杆菌病是由致病性大肠杆菌 ( E.coli )引起的禽类不同类型疾病的总称。包括急性败血症、肉芽肿、气囊炎、肝周炎、心包炎、卵黄性腹膜炎、输卵管炎、滑膜炎、眼炎、关节炎、脐炎以及肺炎等 ,最常见的是急性败血症和卵黄性腹膜炎。引起不同禽类发病的大肠杆菌主要的血清群     

Characterization of monoclonal antibodies to avian Escherichia coli Iss

Colibacillosis accounts for annual multimillion dollar losses in the poultry industry, and control of this disease is hampered by limited understanding of the virulence mechanisms used by avian pathogenic Escherichia coli (APEC). Previous work in our laboratory has found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not commensal E. coli, making iss and the protein it encodes (Iss) candidate targets of colibacillosis-control procedures. Previously, we produced monoclonal antibodies (MAbs) against Iss to be used as a reagent in studies of APEC virulence and colibacillosis pathogenesis. Unfortunately, the utility of these MAbs was limited because these MAbs exhibited nonspecific binding. It was thought that the lack of specificity might be related to the fact that these MAbs were of the immunoglobulin M (IgM) isotype. In the present study, new MAbs were produced using a different immunization strategy in an effort to generate MAbs of a different isotype. Also, because Iss bears strong similarity to Bor, a lambda-derived protein that occurs commonly among E. coli, MAbs were assessed for their ability to distinguish Iss and Bor. For these studies, the bor gene from an APEC isolate was cloned into an expression vector. The fusion protein expressed from this construct was used to assess the potential of the anti-Iss MAbs produced in the past and present studies to distinguish Bor and Iss. The MAbs produced in this study were of the IgG1 isotype, which appeared to bind more specifically to Iss than previously generated antibodies in certain immunologic procedures. These results suggested that the MAbs generated in this study might prove superior to the previous MAbs as a reagent for study of APEC. However, both MAbs recognized recombinant Iss and Bor, suggesting that any results obtained using anti-Iss MAbs would need to be interpreted with this cross-reactivity in mind.     

Relative efficiency of techniques for detecting avian reticuloendotheliosis virus as a vaccine contaminant

Three techniques were compared for sensitivity in detecting reticuloendotheliosis virus (REV) in a deliberately contaminated Marek's disease vaccine. The most sensitive and rapid test was the indirect fluorescent antibody test (FAT). The indirect immunoperoxidase test, although simple to perform and only marginally less sensitive than the FAT, was difficult to interpret at low levels of REV. Using immunoelectron microscopy, virions were seen only after three subcultures and then not to the same level as that detected by the FAT or immunoperoxidase test. Serum raised against the HPRS-1 strain of REV detected other strains of this virus in the FAT.     

禽霍乱、大肠杆菌病蜂胶二联灭活疫苗中大肠杆菌凝集抗体消长规律的研究

应用改良大肠杆菌凝集试验对禽霍乱、大肠杆菌病蜂胶二联灭活疫苗中大肠杆菌凝集抗体消长规律的监测表明：3 d的抗体效价为2-3 Log2,5 d达到6 Log2以上,30 d达到高峰8.5-9.5 Log2,6个月仍可达到6 Log2。3批成品监测结果基本一致,表明禽霍乱、大肠杆菌病蜂胶二联灭活疫苗生产工艺已相当成熟,完全适合工厂化大规模生产。     

Use of lipopolysaccharide (LPS) as a positive control for the evaluation of immunopotentiating drug candidates in experimental avian colibacillosis models

The aetiologic agent of avian colibacillosis is Escherichia coli. Colibacillosis is a disease that causes mortality and production performance problems in chickens which results in economic losses for the poultry industry. It will be increasingly important for scientists to identify novel solutions that can be implemented which will provide poultry producers with a tool to manage this economically important disease. The purpose of this investigation was to determine whether lipopolysaccharide (LPS) could be used as a positive control to evaluate novel chemistries for immunopotentiator activity in battery or floor-pen avian colibacillosis models in chickens. In the battery study, subcutaneous administration of LPS to one-day-old broiler cockerels caused a significant reduction (P < 0.003) in all parameters of colibacillosis evaluated, i.e. mean air sac lesion scores, per cent air sac lesions, E. coli re-isolation and per cent mortality. However, in the floor-pen study, subcutaneous administration to one-day-old broiler chicks resulted in a numerical, but not statistically significant reduction (P < 0.1) in mortality associated with colibacillosis. These data indicate that LPS can be used as a positive control to evaluate the efficacy of immunopotentiator drug candidates in avian colibacillosis models.     

浅谈禽大肠杆菌病常见混合感染的类型及诊疗

近年来,家禽大肠杆菌病在大多数养殖场中均广泛流行,发病率和死亡率居高不下,很多地方已成暴发流行的趋势,严重威胁着养禽业的发展。在临床上,许多疾病可导致大肠杆菌病的继发或混合感染,如鸡沙门氏菌病、霉形体病、传染性鼻炎、传染性支气管、喉气管炎、     

禽霍乱与大肠杆菌病多价蜂胶二联灭活疫苗的研究

为研制绿色环保型的禽霍乱与大肠杆菌病多价二联灭活疫苗,本研究以禽多杀性巴氏杆菌强毒株G48-1、G48-2和禽致病性大肠杆菌常见血清型01/0154、02/086、078、018、08/093等为制苗菌株,分别以改良马丁琼脂和马丁肉汤进行固体表面培养和高密度发酵培养制备抗原,将灭活的抗原铺以具有广谱生物学活性的天然物质-蜂胶为免疫增强剂,已拥有完全自主知识产权的纳米蜂胶疫苗制造工艺技术平台制备禽霍乱与大肠杆菌病多价蜂胶二联灭活疫苗.试验结果表明,疫苗安全可靠,无明显的干扰现象,免疫后5～7 d产生坚强保护力,对禽霍乱和鸡大肠杆菌病的近期保护率均为100%.以实验动物小鼠代替本动物进行二联疫苗效检取得成功,本效检方法可缩短检验期限7 d,降低检验成本,具有更敏感、更特异、更客观、更简便等特点.禽霍乱采用攻毒法,大肠杆菌采用改良微量凝集试验测定抗体效价法测定禽-大二联疫苗的免疫期与保存期.结果表明,该疫苗的免疫期为6个月以上,-10℃保存期为18个月;4℃～8℃为12个月;15℃～30℃为6个月.田间免疫试验表明,疫苗安全可靠,现场应用已逾2亿羽份,效果良好.     

鸡大肠杆菌病的病原分离鉴定与药敏试验

由于鸡大肠杆菌血清型众多．各地流行菌株往往不同,给本病的免疫预防带来许多困难。此外．由于抗菌药物的不规范使用,造成耐药菌株不断产生,治疗效果越来越不明显：为了更好地控制鸡大肠杆菌病．2年来对送检的疑似鸡大肠杆菌病的病料进行了病原鉴定和药敏试验研究,现将情况报告如下。     

Characterization of Escherichia coli isolated from cases of avian colibacillosis.

Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin. The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable. A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed. Most isolates produced aerobactin. Ten E. coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence. All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions. The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent. Only two of the five isolates of serogroup O78 were serum resistant. No correlation between serum resistance and virulence was observed in serogroup O78.     

Clinical and microbial efficacy of antimicrobial treatments of experimental avian colibacillosis

The clinical and microbial efficacy of antimicrobial treatments of avian colibacillosis was studied, using an experimental model on chickens previously inoculated with multiresistant commensal Escherichia coli strains. One E. coli with pMG252 plasmid containing bla(FOX5) and qnrA1 genes and another E. coli with pMG298 plasmid containing bla(CTX-M15) and qnrB1 genes were first orally inoculated to chickens Both isolates were also resistant to chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin, gentamicin, kanamycin, and tetracycline. The birds were then experimentally infected with an avian pathogenic E. coli (APEC), via the air sac. Treatments (oxytetracycline (OTC), trimethoprim-sulfadimethoxin (SXT), amoxicillin (AMX) or enrofloxacin (ENR) were then offered at the therapeutic doses. Symptoms, lesions in dead or sacrificed birds, and isolation and characterization of APEC from internal organs were studied. Results showed that OTC, SXT or ENR treatments could control the pathology. AMX worsened the disease, possibly due to endotoxin shock. All APEC re-isolated from internal organs showed the same antimicrobial susceptibility as the APEC inoculated strain, except for one APEC isolate from an infected OTC-treated bird, which acquired tetracycline resistance only, and one APEC isolate recovered from the air sacs of a chicken in the infected SXT-treated group, which acquired the pMG252 plasmid and became multi-resistant. Thus three antimicrobials could control the disease but the experimental model enabled, to our knowledge, the first observation of plasmid transfer from a bacterium of the intestinal tract to a pathogenic isolate from the respiratory tract.     

吉林城区鸡大肠杆菌多价油佐剂灭活疫苗的研制

鸡大肠杆菌病发病率和死亡率均较高,对养鸡业的危害非常大.由于大肠杆菌病耐药性日趋严重,目前很多抗菌药物都不能有效控制鸡群的大肠杆菌病,研制安全、有效的大肠杆菌疫苗成为防控大肠杆菌病的有效途径.     

复方乌梅汤对禽大肠杆菌病的防治试验

孙荣华等以复方白头翁散（含有白头翁、秦皮、诃子、乌梅、白芍、黄连、大黄等）,潘树桥等以黄芩散,杨富业等以黄连、黄柏、秦皮、白头翁、白芍、茯苓制成汤剂,胡功政等以黄连、黄芩、黄柏、金银花、板蓝根、蒲公英制成丸剂,用来防治禽大肠杆菌病均获得显著疗效.     

The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants

The use of antibiotic feed additives and chemical antimicrobials in food production animals is a double-edged sword. On one hand, it helps to prevent the outbreak of disease and promotes the growth of animals, but on the other hand, concerns are mounting over the emergence of antibiotic-resistant bacteria. As a consequence, some countries have already banned the use of in-feed antibiotics which has resulted in meat producers urgently seeking environmentally friendly alternative methods to control disease. Cytokines are proteins that control the type and extent of an immune response following infection or vaccination. They therefore represent excellent naturally occurring therapeutics. The use of cytokines in poultry has become more feasible with the discovery of a number of avian cytokine genes. Since the immune system of chickens is similar to that of mammals, they offer an attractive model system to study the effectiveness of cytokine therapy in the control of disease in livestock. This review will focus on the recent advances made in avian cytokines, with a particular focus on their assessment as therapeutic agents and vaccine adjuvants.     

Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A_93–143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD₅₀ (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.