实验性鸡大肠杆菌病病理学动态变化

用致病性大肠杆菌O18分离株和／或低致病性禽流感病毒（Mildly pathogenic avian influenza virus ,MPAIV)接种10－12日龄SPF鸡。在接种后1－96h进行临床症状与大体病理变化、组织学观察发现：大肠杆菌接种组、MPAIV接种组和健康接种组除扑杀鸡外未见鸡死亡，MPAIV与大肠杆菌混合接种组除扑杀鸡外死亡率为24％。混合接种组的病变比大肠杆菌接种组出现的时间早，恢复也慢，各脏器的病理变化更严重。MPAIV主要引起各实质器官的坏死，结果表明，大肠杆菌经气管内接种后试验鸡主要表现为呼吸道的炎症反应；MPAVI可使鸡大肠杆菌病严重化。     

Experimental Escherichia coli respiratory infection in broilers

This study determined optimal conditions for experimental reproduction of colibacillosis by aerosol administration of avian pathogenic Escherichia coli to 2-to-4-wk-old broiler chickens. The basic model for reproducing disease was intranasal administration of approximately 10(4) mean embryo infectious dose of infectious bronchitis virus (IBV) followed by aerosol administration of an 02 or an 078 strain of E. coli in a Horsfall unit (100 ml of a suspension of 10(9) colony-forming units/ml over 40 min). Scores were assigned to groups of infected chickens on the basis of deaths; frequency and severity of lesions in the air sacs, liver and heart; and recovery of the challenge E. coli 6 days post-E. coli infection. An interval of 4 days between the IBV and E. coli challenges was best whether the chickens received the IBV at 8 or 20 days of age. Typically, 50%-80% of the chickens developed airsacculitis and 0 to 29% of the chickens developed pericarditis or perihepatitis, with little or no mortality. Escherichia coli alone resulted in no deaths and 0 to 20% airsacculitis, but these percentages increased to 0 to 5% and 52%-60% when the E. coli aerosol was administered through a cone-shaped chamber. Administration of IBV alone failed to induce lesions. Recovery of the challenge E. coli from chickens did not correlate well with lesions. On the basis of these data, administration of IBV to 20-day-old chickens followed 4 days later by exposure to an avian pathogenic E. coli reproduces avian colibacillosis with the low mortality, high percentage of airsacculitis, and low percentage of septicemic lesions characteristic of the conditions seen in the natural disease.     

Comparative efficacy of enrofloxacin, oxytetracycline, and sulfadimethoxine for the control of morbidity and mortality caused by Escherichia coli in broiler chickens

The purpose of the present study was to compare the ability of enrofloxacin, oxytetracycline, and sulfadimethoxine to reduce morbidity and mortality caused by Escherichia coli (colibacillosis) in broiler chickens. The chickens were raised in 80 pens (20 birds per pen) with 20 pens representing each treatment group under simulated commercial conditions that produced a colibacillosis challenge scenario. Each group of 20 randomized pens (replicates) was given one of four water treatments. Chickens that received enrofloxacin had significantly less mortality (P < 0.01), lower average gross pathology (colibacillosis) scores (P < 0.01), and better feed-conversion ratios (P < 0.05) than did chickens that received either oxytetracycline or no medication. Chickens that received enrofloxacin had significantly less mortality and lower pathology scores than those that received sulfadimethoxine and numerically lower feed conversion than the sulfadimethoxine group. Results from the present study show that enrofloxacin is superior to oxytetracycline and sulfadimethoxine for the control of morbidity and mortality caused by E. coli in broiler chickens. Our findings will help veterinarians choose and prescribe the most efficacious antimicrobial when treating colibacillosis.     

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.     

Clinical and pathological features of Aspergillus fumigatus infections in poultry in southern Nigeria

This study was undertaken to supply information on Aspergillus fumigatus infection of poultry in Nigeria. The disease in broiler chicks was characterized by gasping, droopiness, emaciation and heavy mortality while affected grower chickens showed emaciation, weakness, diarrhoea and 17 per cent mortality. The disease was sporadic in laying flocks. Granulomatous nodules were observed in birds that died in each outbreak. The nodules were numerous and affected mainly the lungs and thoracic air sacs in the broiler chicks while only few large nodules were observed mainly in the abdominal air sacs in the layers.     

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.     

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。     

实验性鸡大肠杆菌病的超微动态病理变化

10～ 12日龄 SPF鸡 180只 ,随机分为 4组 ,分别气管内注射致病性大肠杆菌 O18分离株 (大肠杆菌接种组 )、低致病性禽流感病毒 (mildly pathogenic avian influenza virus,MPAIV) H9N2株 (MPAIV接种组 )、先接种 MPAIV再接种大肠杆菌 (混合接种组 ) ,并设健康对照组 ,分别于接种后不同时间 ,取气管、肺、气囊、胸腺、法氏囊、脾、肝和肾组织 ,制作超薄切片 ,电镜观察。结果表明 ,MPAIV与大肠杆菌混合接种组比大肠杆菌接种组出现病变的时间早、恢复慢 ;在大肠杆菌接种组 ,接种后 3h气囊上皮和间质细胞中都可见典型的大肠杆菌 ;在混合接种组 ,接种后 3h,气囊间质细胞的吞噬泡中可见多个 MPAIV粒子。由此认为 ,MPAIV可使鸡大肠杆菌病严重化 ,大肠杆菌对 MPAIV的入侵和在鸡体内的复制可能有促进作用。     

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.     

Experimental factors affecting mortality following inoculation of chickens with avian nephritis virus (G-4260).

Groups of approximately 20 one-day-old chickens were inoculated with G-4260, the reference strain of avian nephritis virus (ANV), or saline. Based on mortality rates from severe nephritis in comparable experiments, light Sussex chickens generally were more susceptible than Rhode Island red (RIR) chickens. Mortality was greater in those given broiler starter than those given other feeds, and was greater when light Sussex chickens were given broiler starter feed and cold-stressed at 15 +/- 1 C for 2 hr daily during the first week rather than brooded normally. Inoculation with G-4260 either orally or by intraperitoneal injection produced similar results in RIR chickens. Thirty-three inoculated chickens died of severe nephritis between 4 and 12 days postinoculation, and 24 (73%) of them had visceral urate deposits. Inoculated inbred white leghorn Line 15 chickens with maternal antibody to ANV were brooded normally and given broiler feed: they were susceptible to infection as evidenced by subsequent histological lesions in the kidneys and serology, but mortality was not a feature. There were no deaths from nephritis in inoculated non-inbred white leghorn chickens free of maternal antibody to ANV that were given broiler feed and brooded normally. These results have implications in standardizing experimental conditions for the study of mortality induced by G-4260 and similar viruses.     

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     

Congo red medium to distinguish between invasive and non-invasive Escherichia coli pathogenic for poultry

In the course of our molecular studies of virulence factors associated with invasive avian Escherichia coli infections, it was first necessary to distinguish between common E. coli and those that cause septicemia in poultry. We found a direct correlation between the ability of clinical isolates of E. coli to bind Congo red dye (CR) and their ability to cause septicemic infection in chickens. This finding was supported by bacteriological studies of 30 broiler flocks (26 sick and 4 healthy) and by virulence studies in chickens and mice. All 144 isolates of E. coli from internal tissues of diseased birds were determined to be CR-positive (red colonies). Congo-red-positive E. coli colonies were isolated from air sacs, pericardium, liver, lung, joint fluid, and heart blood of chickens with lesions of colisepticemia. In contrast, of 170 E. coli isolates from the poultry house environment and from the trachea and cloaca of healthy birds, more than half were CR-negative (white colonies). No CR-negative (white) E. coli colonies were found in internal organs from birds with typical lesions of colisepticemia. We feel that these preliminary findings suggest that the CR dye binding could be used as a phenotypic marker to distinguish between invasive and noninvasive isolates.     

Isolation of ureaplasmas from poultry and experimental infection in chickens

Ureaplasmas were isolated from the oropharynxes of 47 of 247 (19 per cent) Leghorn chickens (Gallus gallus domesticus) and from five Japanese bantams but none was isolated from the oropharynx or cloaca of other poultry comprising 10 Japanese game, 75 common quails (Coturnix coturnix japonica), 17 turkeys (Meleagris gallopavo) and 10 guinea fowls (Numida galeata). In apparently healthy chickens, ureaplasmas were found at various sites, including the conjunctiva, nasal cavity, oropharynx, upper and lower tracheas, but not from the air sac, lungs, yolk, oviduct, urine or cloaca. All the isolates were antigenically similar but had no serological relation to those isolated from man, monkey, cattle, goat, sheep, dog and cat. In chickens experimentally infected with an avian ureaplasma, the organisms infected the oropharynx and nasal cavity but none of the birds inoculated demonstrated any clinical signs or macroscopic lesions.     

山东省禽致病性大肠杆菌菌株的分离与鉴定

从山东省临沂、烟台、滨州、济南、聊城、德州等15地、市的解剖诊断门诊部收集临诊上有典型病变鸡(鸭)病的病料230余份中分离出大肠杆菌165株,115株大肠杆菌进行了O血清型鉴,共鉴定出84个分离株的O血清型,鉴定结果表明O78、O36、O6与O107是本次调查的优势血清型.挑选59株进行致病性试验.动物试验结果表明94.9%的菌株对7日龄内雏鸡具有很强的致病作用,多数菌株可使接种雏鸡100%死亡     

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.     

Isopathic and pluralist homeopathic treatment of commercial broilers with experimentally induced colibacillosis

This study sought to determine the efficacy of isopathic and pluralist homeopathic treatment of colibacillosis in broiler chickens and thereby contribute to the evaluation of homeopathy in general. In each of two experiments three groups of broilers, infected intratracheally at 8 days of age with E. coli (O78:K80), were treated with different combinations of homeopathic remedies. Control groups and an infected, doxycyline-treated group were included. Experiments differed only in the dose of E. coli. Efficacy of treatment was evaluated based on the parameters mortality, body weight gain and colibacillosis lesions. In both experiments doxycyline prevented mortality and reduced E. coli lesions and stunting. None of the homeopathically treated groups differed significantly with respect to any of the parameters from the non-medicated, infected control group. It is concluded that the results of this study do not justify use of these homeopathic remedies for treatment of colibacillosis in broilers. Furthermore, no significant effects of this homeopathic treatment were established.     

Cloning and sequencing of the iss gene from a virulent avian Escherichia coli

Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control.     

Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion

Several studies suggest that the expression of F1 fimbriae could be involved in the virulence of Escherichia coli for chickens. F1 fimbriae display multivalent properties such as adhesion to epithelia or interaction with the immune system that imply specific interactions between the adhesin FimH and different cell receptors. We constructed a delta fimH mutant of the avian pathogenic E. coli MT78 and evaluated its in vivo colonization and pathogenicity, as compared to that of the parent strain. The generated mutant PA68 was unable to adhere in vitro to chicken epithelial pharyngeal or tracheal cells; mutant bacteria were mostly afimbriated although a minority of them displayed altered piliation phenotypes. Two inoculation routes were used to compare the ability of MT78 and PA68 to colonize the respiratory tract and to induce colibacillosis in chickens. In the first model, 2-wk-old axenic chickens were inoculated intratracheally with one or both E. coli strains, after primary infection with infectious bronchitis virus. In the second model, 3-wk-old specific-pathogen-free chickens were inoculated via the caudal thoracic air sac. After intratracheal inoculation, the delta fimH mutant was found to be a better colonizer than MT78 in the trachea of inoculated chickens. Furthermore, when both strains were inoculated simultaneously, the delta fimH mutant constituted 98% of the bacterial population in the trachea at day 7 postinoculation. Irrespective to the inoculation route, MT78 and PA68 showed similar abilities to induce macroscopic lesions in chickens, to provoke bacteremia, and to colonize the internal organs. However, 4 days after intra-air sac inoculation, bacterial counts of the mutant were lower in the spleen and liver than those of MT78. Our results show that FimH is not required for colonization of the trachea of axenic chickens by E. coli and that it is not a major determinant of bacterial pathogenicity. On the contrary, the lack of expression of FimH seems to favor the in vivo colonization of the trachea of chickens by E. coli.     

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.     

Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions.

The objective of this study was to characterize virulence factors of Escherichia coli isolates from broilers with simultaneous occurrence of cellulitis and other colibacillosis lesions. Thirty flocks were sampled and 237 birds with cellulitis were examined. Eighty-two (34.6%) of 237 birds condemned for cellulitis had gross lesions in the heart, air sacs, joints, or liver. In 58 chickens, E. coli was isolated from both the cellulitis and other lesions of colibacillosis, and 18.9% of the E. coli isolates from the 2 types of lesions belonged to the same O group. Escherichia coli of serogroups O78, O1, and O2 predominated. Isolates of the same serogroup that were derived from different lesions in the same birds had similar patterns of biotype, aerobactin production, serum sensitivity profile, antibiotic sensitivity, and K1 capsule production. Escherichia coli derived from cellulitis lesions produced virulence factors similar to those found in E. coli isolated from other colibacillosis lesions in poultry.