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.

Air Sacs↗

PubMedDate not supplied

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.

Adhesins, Escherichia coli↗

PubMedDate not supplied

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli.

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Vacuolating cytotoxin produced by avian pathogenic Escherichia coli.

The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.

Animals↗

PubMedDate not supplied

Relation of plasmids to virulence and other properties of salmonellae from avian sources.

A collection of 185 isolates of 34 serovars of Salmonella from avian sources was examined for plasmids, drug resistance, biochemical properties, serum resistance, and virulence. No serovars other than S. enteritidis, S. typhimurium, and S. heidelberg showed evidence of serovar-associated plasmids. All S. enteritidis isolates carried a single plasmid of 36 Mdal and were resistant to guinea pig serum; one strain that was tested was virulent. Of 27 isolates of S. typhimurium, 11 possessed a 60-Mdal plasmid and 17 harbored a 2.3-Mdal plasmid. Among isolates of S. heidelberg, 21 of 24 carried a 2.2-Mdal plasmid. The only biochemical property that varied was fermentation of inositol, which tended to be related to serovar. Of 172 isolates, 54 were resistant to at least one drug. Multiple drug resistance was usually associated with R plasmids, and transmissible plasmids that encoded resistance to chloramphenicol and gentamicin were demonstrated. Of 117 isolates tested, 43 were resistant to guinea pig serum. Resistance appeared to be a characteristic of isolates rather than serovar and could not be related to plasmids. Twenty-five isolates highly resistant to guinea pig serum were all susceptible to the bactericidal action of chicken serum. In tests for virulence using intraperitoneally (i.p.) and orally inoculated Balb/c mice and day-old chicks, only i.p.-inoculated chicks proved useful in demonstrating large differences among isolates: LD50's ranged from 10(0) to 10(8).

Animals↗

PubMedDate not supplied

Characteristics of Escherichia coli isolates from avian cellulitis.

The purpose of this study was to characterize Escherichia coli isolates from avian cellulitis, particularly with respect to the occurrence of potential virulence factors. At slaughter, five broilers with lesions of cellulitis were selected from each of 20 farms from among the broilers processed. One hundred E. coli isolates from the lesions were characterized with respect to serotype; biotype; drug susceptibility; plasmid profile; ability to produce aerobactin, colicin, colicin V, and hemolysin; and cytotoxicity for Vero cells and chicken fibroblasts. The same properties were determined from a collection of 25 E. coli from the feces of chickens. Serotyping showed that, among the cellulitis isolates, 23 belonged to O group 78, 14 belonged to O2, eight belonged to O115, and seven belonged to O(21.83); 25 were untypable. Isolates from a single farm typically belonged to three to five O groups. More than half of the fecal isolates were untypable, and the rest were distributed among seven O groups. Biotype, drug-resistance pattern, and plasmid profile could not be used as markers of avian cellulitis E. coli. No plasmid was detected in 12% of cellulitis isolates and 48% of fecal isolates. No isolate was hemolytic or showed cytotoxic effects. Aerobactin was produced by 90% of cellulitis isolates, and colicin was produced by 85% of these isolates; the corresponding percentages for the fecal isolates were 16% and 40%. Production of colicin V was detected in 21% of cellulitis isolates and 24% of fecal isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Escherichia coli cellulitis: experimental infections in broiler chickens.

The objectives of this study were to evaluate the role of trauma to the skin in development of Escherichia coli cellulitis and to compare the abilities of three cellulitis isolates (O78, O115, O21,83), one airsacculitis isolate (untypable) and one fecal isolate (O86) of E. coli to induce cellulitis in broiler chickens. Forty-eight 4-week-old commercial broiler chickens were housed in groups of six in eight battery cages. For five groups, the skin on the left side of the abdominal region of chickens was traumatized by scratching with a 22-gauge needle, then contaminated with a swab dipped in a broth culture of one of the five E. coli isolates. For chickens in the remaining three groups, an avian cellulitis culture (O115, O21,83) or sterile broth was applied to intact skin. The experiment was duplicated. All birds were euthanatized 10-13 days postinoculation. No lesion developed in chickens in which the skin had not been traumatized. Among the traumatized birds, cellulitis isolates induced characteristic lesions of cellulitis in 86% of the birds, whereas airsacculitis and fecal isolates induced lesions in 42% and 8% of birds, respectively. Severe or moderate gross pathologic changes were found in 86% and microscopic pathologic changes were found in 88% of birds inoculated with cellulitis isolates; the corresponding percentages for the airsacculitis isolate were 25% and 17%. This study demonstrated that trauma to the skin is necessary for initiating disease and that strains of E. coli of serotypes epidemiologically associated with cellulitis are highly virulent in experimental infection.

Animals↗

PubMedDate not supplied

A prospective study of cellulitis in broiler chickens in southern Ontario.

This study investigated the associations among cellulitis and hatchery, farm, and abattoir factors. Forty-four broiler flocks from 24 farms located in southern Ontario were followed from hatching to processing. Poisson regression was used to analyze the data. Cellulitis as a count outcome (CELLCOUNT) was significantly associated (P < or = 0.05) with the hatchery of origin, strain of birds, farm size, type of litter, lighting system, total down time, prevalence of abdominal scratches, Escherichia coli-related conditions (SEROSITIS), ascites, and valgus varus deformity. However, only farm size, abdominal scratches, SEROSITIS, ascites, and valgus varus deformity were significant (P < or = 0.05) after adjusting for clustering. No significant associations were found between cellulitis and source of eggs, sex, average bird weight, feed company, growth promoter, or stocking density. Factors significantly associated with cellulitis in this study could be considered as potential risk factors for cellulitis in broiler chickens in southern Ontario.

Animal Husbandry↗

PubMedDate not supplied

Description of cellulitis lesions and associations between cellulitis and other categories of condemnation.

A total of 110 broiler flocks processed in a single processing plant in southern Ontario were studied for purposes of describing the cellulitis lesions and investigating possible associations between cellulitis and other categories of condemnation at the processing plant. Two hundred and ninety-five carcasses condemned for cellulitis were examined. They came from 65 of the 110 flocks. The lesions tended to be unilateral with most carcasses (87%) having one lesion. The majority of the lesions (92%) were located on the abdomen. Almost 65% of the lesions were large (> or = 8.1 cm2), and 27% were medium (2.1-8.0 cm2). On the basis of gross appearance, 69% of the lesions were classified as severe, 26% moderate, and 5% mild. Of 149 lesions examined histologically, 74% were classified as chronic, 21% ongoing, and 5% mild-acute. Condemnation data from the 110 broiler flocks were analyzed using Poisson regression. Simple relationships were examined between a count outcome (number of cellulitis-condemned carcasses per flock) and other categories of condemnation and average bird weight. Cellulitis was significantly associated with average bird weight (P = 0.0018), Escherichia coli-related conditions (SEROSITIS; P < or = 0.0001), ascites (P = 0.0004), cyanosis (P < or = 0.0001), valgus varus deformity (P < or = 0.0001), REJECT (combined carcass condemnations for bruising, mutilation, and contamination; P = 0.0003), and the interaction terms "average bird weight and ascites" (AVWT*ASCIT; P < or = 0.0001) and "average bird weight and cyanosis" (AVWT*CYAN; P < or = 0.0001). Average bird weight, SEROSITIS, ascites, cyanosis, valgus varus deformity, and AVWT*ASCIT were the only significant factors after adjusting for clustering. No association was observed between cellulitis and emaciation and dead on arrival. Variables significantly associated with cellulitis in the multivariate analysis could be considered as potential predictors. These predictors may share common risk factors predisposing broiler chickens to cellulitis.

Abattoirs↗

PubMedDate not supplied

A descriptive study of verocytotoxigenic Escherichia coli (VTEC) cases reported in Ontario, 1990-1994.

Characteristics of VTEC cases identified through routine surveillance in Ontario between 1990 and 1994 are described. Information was extracted from the Reportable Disease Information System (RDIS) of Ontario and was evaluated for its completeness and internal validity. A total of 2,441 VTEC cases were identified for the five-year study period corresponding to an average annual rate of 4.8 cases per 100,000. Sixteen deaths were recorded. Bloody diarrhea was reported for 546 patients (40%) and was the most frequently reported symptom. For most cases, the home was recorded as the likely risk setting (36%). Food was incriminated as the source of infection for more than 36% of cases. Nine (69%) of the thirteen data fields compulsory for transmission to the Ontario Ministry of Health had less than 10% of combined missing and unspecified values. Fields describing risk factors had greater than 56% of entries missing or unspecified.

Adolescent↗

PubMedDate not supplied

Construction and characterization of avian Escherichia coli cya crp mutants.

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.

Adenylyl Cyclases↗