Adenovirus infection associated with respiratory disease in commercial chickens

The lesions and etiologic agents associated with 13 outbreaks of respiratory disease in commercial chickens were investigated. Adenoviruses were isolated from tracheal and lung tissues of affected chickens in all 13 outbreaks. Escherichia coli was isolated from the lung of an occasional bird. The tracheal specimens were consistently negative for Bordetella avium, but E. coli and occasionally Staphylococcus aureus were isolated. There was also serological evidence in one outbreak, and pathological evidence in another, of a concurrent infectious bursal disease virus (IBDV) infection of chickens affected with the disease. Gross and microscopic alterations in the tracheas and lungs of affected chickens were similar in all outbreaks and consisted of catarrhal tracheitis and occasionally multifocal pneumonia with mononuclear cell infiltrates. Hepatitis and splenitis with heterophil infiltrates occasionally were seen in birds with coliform septicemia. The tracheal and lung lesions in the present investigation were considered primarily of adenovirus etiology, complicated by secondary bacterial infection.     

Dual infections of Eimeria tenella and Escherichia coli in chickens

The interactive effects of Eimeria tenella and Escherichia coli infection in chickens were investigated. Specific pathogen free chickens inoculated orally with E tenella and challenged four days later with E coli via the air sac showed more severe acute septicaemic lesions and subacute serositis than chickens given E coli alone. Moreover, caecal lesions induced by E tenella were more severe in chickens given both E tenella and E coli than in those given E tenella alone. In contrast, oral inoculation of E coli did not result in acute septicaemic lesions or subacute serositis and had no effect on the severity of the caecal lesions caused by E tenella.     

Factors affecting the development of respiratory disease complex in chickens

Factors playing a part in the development of respiratory disease complex in chickens were investigated in a series of experiments. The experimental infection was produced by exposing chickens to Mycoplasma gallisepticum and the B1 vaccine strain of Newcastle disease virus and later exposing them to aerosols containing the O1:K1 serotype of Escherichia coli. Chickens became susceptible (pericarditis or death) to E. coli 8 days after mixed respiratory disease challenge. One day after respiratory disease challenge, lesions consisted of edema and infiltration with lymphoid cells and heterophils. At the time of susceptibility to E. coli, the lesions were strongly lymphoid with many dense follicular areas and very few heterophils. The incidence of pericarditis and death was similar when the concentration of bacteria in the aerosol inoculum ranged between 10(9)/ml and 10(5)/ml. At the time of maximum susceptibility to aerosol challenge, chickens were less susceptible to intravenously administered E. coli than were the uninfected controls. Resistance of chickens that had been selectively bred for a high (HA) or low (LA) antibody response to sheep erythrocytes was compared. HA chickens were more resistant to respiratory agents and less resistant to E. coli than LA line chickens. When the lines were exposed to respiratory disease followed by exposure to aerosols containing E. coli, the HA line had the lowest incidence of pericarditis and death.     

Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli, and treated homogenates obtained from broilers

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkuhn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.     

Influence of Clostridium perfringens and its toxin in germ-free chickens

Twenty-one of 56 germ-free chickens died after receiving an oral inoculation of a broth culture of Clostridium perfringens. At necropsy there was detachment and disruption of the epithelial layer at the tips of villi and sloughed epithelial cells in the duodenum. These are characteristic lesions of necrotic enteritis. Germ-free chickens receiving either purified alpha-toxin or the supernatant of broth cultures of C perfringens died, but no bird died after receiving supernatant of broth cultures neutralised by anti-alpha-toxin serum. It was concluded that alpha-toxin of C perfringens was the cause of death in young germ-free chickens.     

Kidney lesions associated with mortality in chickens inoculated with waterfowl influenza viruses

Seventy-six type A influenza viruses recovered from waterfowl in Wisconsin, California, South Dakota, Florida, Texas, Alabama, and Nebraska were tested for virulence in chickens. The challenge to chickens was intravenous inoculation of first-, second-, or third-egg-passage virus. Each of the virus strains was tested separately in three or four chickens. Eighteen of the 76 viruses caused the death of one or more chickens following inoculation. Postmortem lesions were similar in all dead birds. In decreasing order of frequency, gross lesions included: swollen kidneys evident as accentuated lobular patterns, urates in the pericardial sac, and urates on the surface of the liver. Microscopic lesions present in kidneys were consistent with visceral gout. Mortality was associated with inoculations having higher concentrations of infectious virus. These results indicate that the influenza A viruses circulating in duck populations may include strains potentially pathogenic for chickens.     

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.     

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.     

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。     

Characterization of Escherichia coli isolates from peritonitis lesions in commercial laying hens

Five clinically normal chickens from three farms (farm A, farm B, and farm C), for a total of 15 clinically normal chickens, were examined bacteriologically. In a similar manner, five dead chickens with lesions of peritonitis from each of the same three commercial egg-laying operations were selected for bacterial culturing. Escherichia coli were isolated from the cloaca in 14 of 15 healthy chickens and from all 15 chickens with peritonitis. Oviducts of normal chickens did not contain E. coli (0/15) whereas oviducts from 13 of 15 hens with peritonitis were positive for this pathogen. No lesions and no E. coli (0/15) were found in the peritoneal cavity of healthy hens, but peritonitis lesions from 13 of 15 dead chickens yielded E. coli. On farm A and farm B, a flock consisted of all chickens within a single house and all chickens in each flock were of the same age and same genetic strain. In flock 1 from farm A, all five E. coli isolates from the oviduct and all five isolates from the peritoneal cavity were serogrouped as O78; contained the virulence genes iroN, sitA, iutA, tsh, and iss; and belonged to phylogenetic group A. In flock 2 from farm B, all four E. coli isolates from the oviduct and all four isolates from the peritoneal cavity were serogrouped as O111; contained virulence genes iroN, sitA, iutA, traT, iss, and ompT; and belonged to phylogenetic group D. These data suggest that all chickens with peritonitis in a single flock on farms A and B were likely infected by the same E. coli strain. Escherichia coli isolates from the magnum and peritoneum had the same serogroup, virulence genotype, and phylogenetic group, which is consistent with an ascending infection from the oviduct to the peritoneal cavity.     

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.     

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。     

Comparative infectivity for axenic and specific-pathogen-free chickens of O2 Escherichia coli strains with or without virulence factors

Adhesion to epithelial respiratory cells, iron acquisition, and production of K1 polysaccharide capsules have been proposed as potential virulence factors of avian Escherichia coli. These factors were studied by inoculating groups of axenic or specific-pathogen-free (SPF) chickens intratracheally with O2 E. coli strains after previous challenge with a wild strain of infectious bronchitis virus (IBV). In all experiments, the association between IBV and an E. coli strain endowed with the three virulence factors previously mentioned resulted in the most severe pathological effects, as measured by mortality, weight gains, lesions, and reisolation of E. coli from internal organs. An E. coli strain devoid of virulence factors was able only to induce mild pathological effects restricted to the respiratory tract when combined with IBV. Both E. coli strains were more invasive in axenic chickens than in SPF chickens. These results confirm the probable involvement of the three factors studied in the pathogenic properties of avian E. coli. This model can be used to assess the role of virulence factors, by comparing pairs of positive and negative isogenic strains.     

大肠杆菌病患鸡肝、脾结构的病理学研究

为了更好地了解大肠杆菌病患鸡肝、脾结构的病理变化,从云南省昆明地区养鸡场采集发病鸡,通过对其进行细菌分离鉴定,确诊为感染鸡大肠杆菌病后,采集发病鸡的肝、脾等制作组织切片,显微观察其病理变化。结果发现,肝脏组织的病理学变化主要为肝组织发生水肿、凝固样坏死,病变严重区域发生玻璃样变;脾组织的病理学变化主要为脾脏组织间隙增大,脾脏淋巴细胞稀疏,脾组织中间质细胞发生增生,出现大量长杆状细胞,脾组织中淋巴细胞出现皱缩,吞噬细胞数量增多。     

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.     

Escherichia coli multiplication and lesions in the respiratory tract of chickens inoculated with infectious bronchitis virus and/or E. coli.

Escherichia coli numbers and histopathological changes were studied in the respiratory tract of line 151 chickens intranasally inoculated with infectious bronchitis virus (IBV) and/or virulent E. coli; this line is highly susceptible to IBV. Chickens inoculated with IBV alone showed increased numbers of E. coli in the trachea and had tracheitis, airsacculitis, and bronchiolitis. One of 17 chickens inoculated with IBV alone died with fibrinopurulent serositis. Chickens inoculated with IBV and E. coli had more severe and persistent respiratory lesions than those inoculated with IBV alone. E. coli was isolated from tracheas of chickens inoculated with IBV and E. coli more frequently than from chickens inoculated with IBV alone. In this group, 14 of 27 chickens died with tracheal plugs or with fibrinopurulent serositis. There was neither increased numbers of E. coli nor significant lesions in the respiratory tract of the group inoculated with E. coli alone. These results suggest that IBV may facilitate E. coli invasion into the lower respiratory tract of the chicken.     

The effect of sodium chloride extract and commercial lipopolysaccharides of Escherichia coli and Salmonella typhimurium on chickens.

In chickens inoculated into the heart with a sodium chloride extract of Escherichia coli strain (serotype O2) isolated from a chicken with colibacillosis, characteristic hemorrhages into the anterior chamber of the eyes (hyphema) were found. Significant lesions were limited to the eyes. Cyclophosphamide-treated chickens were more sensitive to the extract than untreated chickens and hyphema was usually seen in association with hemorrhages of the iris. These activities were not reduced by heating the extract at 60 degrees C for one hour or by trypsin digestion. Chickens inoculated into the heart with commercial lipopolysaccharides of E. coli (serotypes O111:B4 and O55:B5) and Salmonella typhimurium showed similar lesions in the eyes as the chickens inoculated with the sodium chloride extract. These findings suggest that the endotoxin may induce hyphema in chickens.     

A comparison of the efficacy against Marek's disease of cell-free and cell-associated turkey herpesvirus vaccine.

One-day-old White Leghorn and broiler chicks with maternal antibody to turkey herpesvirus (HVT) were vaccinated with 300 or 1,000 plaque-forming units (PFU) of cell-free or cell-associated HVT vaccine and challenged with virulent Marek's disease virus (MDV) by contact exposure. Broiler chicks receiving 300 PFU of cell-associated HVT had a 3.3% incidence of MD lesions, whereas only 2.0% of those receiving 1,000 PFU had macroscopic lesions. Broiler chicks vaccinated with 300 PFU of cell-free vaccine had 6.8% gross lesions, and 0.67% of the birds receiving 1,000 PFU had MD lesions. Unvaccinated broiler chickens had a 28.3% incidence of MD lesions. Unvaccinated White Leghorn chickens had a 48.9% incidence of macroscopic lesions, whereas 5.4% of the birds receiving 300 PFU of cell-associated HVT had gross lesions, and 8.3% of the birds vaccinated with 1,000 PFU had lesions. In contrast, 6.7% of the chicks vaccinated with 300 PFU of cell-free HVT had MD lesions, and only 4.0% of those receiving 1,000 PFU of cell-free HVT had macroscopic lesions.     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

Immunogenicity of an oil-emulsified Escherichia coli bacterin against heterologous challenge

Immunogenicity of an oil-emulsified Escherichia coli bacterin against heterologous challenge was investigated. In Expts. 1 and 2, chickens were vaccinated with E. coli serotype O1 bacterin and challenged with E. coli serotype O2 (Expt. 1) and O78 (Expt. 2). Positive control chickens were not vaccinated but challenged with E. coli serotype O2 or O78; unvaccinated unchallenged chickens served as negative controls. When challenged with E. coli serotype O2, unvaccinated chickens showed a higher morbidity than vaccinated chickens. There was no mortality in either group. Although average gross lesion scores were generally higher in the unvaccinated chickens, they were not significantly different (P greater than or equal to 0.05) from those in the vaccinated chickens. In Expt. 2, morbidity was slightly higher in the unvaccinated challenged chickens. No mortality occurred in either group. There was no significant difference (P greater than or equal to 0.05) between vaccinated and unvaccinated chickens in average gross lesion scores. In general, E. coli recovery was higher in the unvaccinated challenged chickens, being highest in the air sacs followed by the liver, heart blood, and pericardial sacs. There was no morbidity, mortality, or gross lesions in the unvaccinated unchallenged chickens. No E. coli was recovered from the tissues cultured. The results of these laboratory trials revealed that an oilemulsified monovalent E. coli bacterin did not protect chickens against other E. coli serotypes associated with colibacillosis.