Identification of Bordetella avium antigens recognized after experimental inoculation in turkeys

Sera and tracheal washings (TW) were used to identify antigens of Bordetella avium recognized during experimentally induced bordetellosis in young turkeys. Pooled sera and TW were examined for antibody by a microtitration agglutination test and by western immunoblotting. In addition, comparable samples collected from 1-day-old turkeys and uninoculated control turkeys also were examined. At least 8 outer membrane proteins of B avium were recognized in immunoblots of sera and TW from infected turkeys. Reactivity of TW in immunoblots was qualitatively similar but less intense, compared with reactivity of corresponding sera collected on postinoculation (PI) weeks 2, 3, and 4. Molecular weights of the major outer membrane proteins of B avium recognized by sera and TW at PI week 4 were 100,000, 97,000, 36,000, 31,000, 21,000, 18,000, 14,000, and less than 14,000. A protein with a molecular weight of 55,000 reacted nonspecifically in all samples tested. Antibody, detectable by microtitration agglutination, was in sera of 1-day-old turkeys and in sera and TW of B avium-infected turkeys during PI weeks 2 to 4.     

Development of an enzyme-linked immunosorbent assay for Bordetella avium

A Bordetella avium enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies in 1-day-old poults, experimentally infected turkeys, and naturally infected turkeys. The optimized procedure included use of a suspension of whole bacteria coated onto plastic microtiter plates, a 1:200 serum dilution, a 1:3200 dilution of commercially available goat anti-turkey IgG (heavy and light chain) conjugated with horseradish peroxidase, and 0.04% orthophenylenediamine as substrate. A sample/negative (S/N) ratio method of analysis was used to estimate antibody titer from absorbance values. The regression equation used to estimate antibody titers was derived from the testing of naturally infected turkey sera. The equation was derived by plotting the log10 titer of the sera against the S/N ratio at a 1:200 serum dilution. The ELISA was an effective method for detecting antibody to B. avium, and the procedure should prove useful for laboratories equipped for high-volume ELISA testing.     

Histologic evaluation of lung and bronchus-associated lymphoid tissue in young turkeys infected with Bordetella avium

One-day-old turkeys were inoculated intranasally with Bordetella avium. Noninoculated hatchmates were housed separately. At postinoculation weeks 1, 2, 3, 4, and 5, B avium-infected (BA+) and B avium-free (BA-) turkeys were necropsied; specimens of tracheas, intrapulmonary primary bronchi, and lung adjacent to primary bronchi were bacteriologically cultured. Lung tissue was collected for histologic examination. Lungs perfused with acetic acid were collected for evaluation to determine the size, number, and distribution of lymphoid nodules associated with primary bronchi. Bordetella avium was isolated from trachea and primary bronchi of all BA+ turkeys, but was never isolated from lung parenchyma. Acute purulent bronchitis was associated with colonization of the primary bronchi by B avium from postinoculation weeks 1 to 3. Macrophages and lymphocytes persisted in the peribronchial connective tissue for 5 weeks after inoculation. Bronchus-associated lymphoid tissue consisted of discrete lymphoid nodules protruding into the lumens of primary bronchi. Lymphoid nodules, morphologically similar in BA+ and BA- turkeys, were composed of nonciliated, cuboidal epithelium covering a zone of loosely arranged lymphocytes and macrophages and a deeper, sharply demarcated lymphoid follicle. Compared with bronchus-associated lymphoid tissue of BA- turkeys, lymphoid nodules of bronchus-associated lymphoid tissue in BA+ turkeys were more numerous and widely distributed along primary bronchi. In both BA- and BA+ turkeys, the mean diameter of lymphoid nodules doubled between 1 and 5 weeks of age.     

Passive immunization versus adhesion of Bordetella avium to the tracheal mucosa of turkeys

Three-week-old turkeys were passively immunized with convalescent serum or treated with tracheal washings from turkeys infected with Bordetella avium. Western blot analysis of the convalescent serum and tracheal washings revealed at least two bands of interaction with outer membrane protein preparations of B. avium. Adherence of B. avium in vivo to tracheal mucosa was determined and compared in treated and untreated turkeys. Passive immunization with convalescent serum reduced adherence of B. avium to the tracheal mucosa in a dose- and time-dependent manner. Adherence was significantly inhibited (P less than 0.01) when turkeys were treated intravenously with 1 ml of undiluted serum either 1 or 6 hours previously. Incubation of the bacterial inoculum with convalescent tracheal washings or application of the washings to tracheal segments before adherence determination in vivo resulted in a significant (P less than 0.01) decrease in adherence. These results indicate that adherence of B. avium to tracheal mucosa is inhibited by substances (antibody) present in both serum and tracheal secretions of convalescent turkeys.     

Isolation and characterisation of Bordetella avium and related species and an evaluation of their role in respiratory disease in poultry

A total of 24 Gram negative non fermentative bacteria obtained from poultry were compared with reference strains of Bordetella avium, Alcaligenes faecalis, Bordetella bronchiseptica and a Bordetella avium-like organism. Thirteen isolates were identified as B. avium and 11 were identified as B. avium-like. A commercial microidentification kit (the API2ONE) did not identify the field isolates but did separate them correctly into the 2 groups. A practical identification scheme, suitable for diagnostic laboratories, is proposed for these organisms. The available clinical histories suggest that B. avium is associated with upper respiratory tract disease in turkeys.     

Clinical outbreak of Bordetella avium infection in two turkey breeder flocks

An acute upper respiratory disease was observed in two broad-breasted white (BBW) turkey primary breeder flocks. Associated clinical signs included sneezing, depression, and a deep dry cough originating from large conducting airways. Morbidity reached approximately 15-20% of the hens in an affected house. None of the turkeys died, and total feed consumption was not affected. A minimal effect upon egg production was noticed. Sera from an acutely affected flock exhibited a marked rise in titer to Bordetella avium compared with preinfection sera samples. In Case 1, B. avium was isolated in pure culture from affected birds. In Case 2, B. avium was diagnosed by serological results and clinical signs; bacteriological examination was not attempted. The findings presented here are consistent with an acute clinical outbreak of B. avium-induced turkey rhinotracheitis (turkey coryza) in BBW turkey breeder hens.     

Clearance of bacteria in turkeys with Bordetella avium-induced tracheitis

Quantitative clearance of aerosolized Escherichia coli from the trachea, lung, and air sacs was measured in turkeys infected with Bordetella avium. Clearance of E. coli in turkeys with B. avium-induced tracheitis was minimally affected early in infection. Sixteen to 23 days after infection with B. avium, sporadic, mild depressions in clearance of E. coli were observed in the tracheas, which had large areas of deciliated tracheal epithelium or replacement of normal epithelium by immature hyperplastic epithelium or metaplastic squamous epithelium. Clearance of E. coli from the lung and air sacs was minimally affected in turkeys infected with B. avium.     

Effects of Bordetella avium toxin on turkey tracheal organ cultures as measured with a tetrazolium-reduction assay

Turkey tracheal organ cultures (TOCs) were exposed to one of the following Bordetella avium fractions or controls: live B. avium, formalin-killed B. avium, B. avium sonicate, heat-inactivated sonicate, culture supernatant, heat-inactivated culture supernatant, phosphate-buffered saline, or brain-heart infusion broth. After the TOCs were incubated for 2 hours with the bacterial fractions, the cellular metabolism of each TOC was evaluated using a tetrazolium chloride reduction assay, and cellular morphology was determined by light microscopy. Additionally, bacterial fractions and controls were injected into turkeys to test lethality. Although the bacterial sonicate was lethal for turkeys, neither the sonicate nor any other B. avium fraction significantly affected the metabolism or morphology of turkey TOCs.     

Genetic variation in response of turkeys to experimental infection with Bordetella avium

One hundred ninety-six male and female turkeys representing two genetic lines were experimentally infected with Bordetella avium. The lines of turkeys included a randombred control line (RBC2) and a subline (F) of RBC2 selected for increased 16-wk body weight. No difference was found between lines RBC2 and F in the number of days to onset of clinical signs, and no mortality due to B. avium infection was observed in either line. Interestingly, however, a significant depression (12%) occurred in body weight of F line poults infected with B. avium, but no significant depression occurred in body weight of RBC2 poults.     

Influence of temperature on the growth of Bordetella avium in turkeys and in vitro

Effects of temperature on growth of three strains of Bordetella avium were determined in young turkeys and in vitro. Colonization of the tracheal mucosa by two virulent strains of B. avium was significantly greater in cold-stressed turkeys than in heat-stressed turkeys. The avirulent vaccine strain, ART-VAX, colonized tracheas of cold-stressed turkeys to a limited extent but failed to colonize heat-stressed turkeys. Growth rates of the three B. avium strains were determined in brain-heart infusion broth at 30, 35, 40, and 45 C. All three strains grew best at 35 C but were killed by 45 C. Compared with virulent strains, ART-VAX grew markedly less at all temperatures, and most cultures of ART-VAX grew at 40 C only after a variable period of declining numbers of viable bacteria. This study indicates that temperature affects growth of B. avium in vivo and in vitro and that growth of the ART-VAX strain is fundamentally different from growth of virulent strains.     

Fowl cholera: influence of Bordetella avium on vaccinal immunity of turkeys to Pasteurella multocida

Turkeys exposed to Bordetella avium were vaccinated against fowl cholera with live Pasteurella multocida vaccine. Previous exposure to B. avium resulted in impairment of systemic immunity conferred by the vaccine: 86% of the vaccinated turkeys exposed to B. avium at 1 day old developed lesions or died of fowl cholera after challenge at 15 weeks old with virulent P. multocida. Of vaccinated turkeys not previously exposed to B. avium, only 26% had lesions or died of fowl cholera.     

An in vivo model for the study of Bordetella avium adherence to tracheal mucosa in turkeys

An in vivo model was developed for studies characterizing the adherence of Bordetella avium to the tracheal mucosa of turkeys. Three-week-old turkeys were anesthetized, and the cervical part of the trachea was isolated after tracheostomy was done. A hemostat was applied craniad to the tracheostomy site to occlude the tracheal lumen. Isolated tracheal segments were filled with an aqueous bacterial inoculum for 1 minute, and then excess inoculum and the hemostat were removed. After 1 hour, a 1-cm section was excised from each tracheal segment, and adherent viable bacteria were quantified. Modifications of the procedure were evaluated to produce a model that was technically simple to do, economical, and reproducible. To examine the validity of the model, adherence of B avium was compared with that of Escherichia coli and Staphylococcus aureus. Adherence of B avium to tracheal mucosa was 17 times greater than that with E coli and 1,550 times greater than that with S aureus. Colonization of the tracheal mucosa by B avium was demonstrated in tracheal sections obtained 6 hours after filling with bacterial inoculum. Because the ciliary clearance mechanism of the tracheal segments remained functional, washing of the tracheal lumen had no effect on numbers of associated bacteria. An important advantage of this model over in vitro models is the excellent preservation of the tracheal mucosal surface.     

Tracheal mucus transport rate in normal turkeys and in turkeys infected with Bordetella avium (Alcaligenes faecalis)

Using the radiopharmaceutical 99mtechnetium-sulfur colloid, the tracheal mucus transport rate (TMTR) was measured in healthy unanesthetized turkeys and in turkeys infected with Bordetella avium. The TMTR of uninfected turkeys was 35.6 +/- 14.4 cm/min. The TMTR of B. avium-infected turkeys was normal on days 0 through 14 postexposure (PE), despite heavy bacterial colonization of the tracheal epithelium. On day 21 PE, the TMTR of B. avium-infected turkeys was significantly depressed (P less than or equal to 0.01) compared with that of control turkeys. Depressed transport was associated with extensive loss of ciliated epithelium from the tracheal mucosa and replacement of the normal mucosa by immature nonciliated epithelium or metaplastic squamous epithelium.     

Isolation and characterization of Bordetella avium plasmids

Experiments were conducted to study the plasmids of Bordetella avium, B. avium-like, and B. bronchiseptica isolates from turkeys and the plasmids of the Art-Vax commercial vaccine strain. Plasmids were observed in 6 of 20 B. avium isolates, in 6 of 20 B. avium-like isolates, in all 5 B. bronchiseptica isolates, and in the Art-Vax strain. Plasmids of B. avium correlated with resistance to antibiotics but not with pathogenicity, hemagglutination of guinea pig erythrocytes, or expression of pili.     

Contribution to the taxonomy of the turkey coryza agent: cellular fatty acid analysis of the bacterium

Gas-liquid chromatography was used to analyze bacterial cellular fatty acids to elucidate the relatedness of the turkey coryza (TC) bacterium to Alcaligenes spp., Bordetella spp., and other gram-negative bacteria. The results indicated that the TC bacterium is not closely related to Alcaligenes faecalis or any of the reference strains of Alcaligenes and Bordetella studied. Most urease-positive bacterial isolates obtained from the upper respiratory tract of turkeys were identified as Bordetella bronchiseptica. It is suggested that Bordetella avium is a suitable designation for the TC bacterium formally called Bordetella-"like" and A. faecalis type I. It is also suggested that the nonpathogenic bacterium previously identified as type II A. faecalis be designated B. avium-like until further taxonomic studies are available. Furthermore, it is proposed that the term turkey coryza be used to refer to the disease induced by this bacterium.     

Turkey coryza: selected tests for detection and neutralization of Bordetella avium heat-labile toxin

Bordetella avium heat-labile toxin (HLT) was lethal for poults, mice, and embryonating chicken eggs. It produced hemorrhagic lesions in turkey and guinea pig skin. Antiserum made in turkeys neutralized the lethality of the toxin and its ability to produce hemorrhagic skin lesions. Further, antiserum against HLT of an Ohio strain neutralized lethality of HLT of strains from Iowa, North Carolina, and West Germany. The antiserum did not neutralize lethality of HLT from B. bronchiseptica. Bordetella avium HLT was not ciliostatic for turkey tracheal-ring cultures and did not stimulate adenyl cyclase activity using mouse adrenal cell cultures.     

Use of an enzyme-linked immunosorbent assay to measure antibody levels in turkey breeder hens, eggs, and progeny following natural infection or immunization with a commercial Bordetella avium bacterin

Twelve large white turkey hens were immunized with a commercially available Bordetella avium bacterin. Hens and eggs were tested using an enzyme-linked immunosorbent assay (ELISA) to determine the response to the bacterin. Three hundred poults were then obtained from two commercial flocks, the hens of one flock having been immunized with the same bacterin used on the group of 12 turkeys. Titers of the poults were monitored for 7 weeks, and poults were challenged by exposure to infected poults at 1, 7, 14, and 21 days post-hatch. Hens produced an antibody response following immunization, with a parallel antibody response being detected in eggs. Maternal antibodies were present in poults from immunized hens. Poult titers declined to near the level of poults from unimmunized hens by 14 days of age. Poults from immunized hens challenged at 1 and 7 days were resistant to development of clinical disease and gross lesions, whereas all poults from unimmunized hens exhibited clinical signs and gross lesions. After 14 days, the resistance of both groups to development of clinical disease, became near equal, neither group being affected as severely as the unimmunized hens challenged at days 1 and 7. Six commercial turkey breeding flocks and their progeny that had not been vaccinated for B. avium and had no history of B. avium infection were evaluated with the B. avium ELISA. There were variations between the flocks, with poult titers reflecting those found in the hens.     

Prevalence,virulence attributes,and antibiogram of Bordetella avium isolated from turkeys in Egypt

Tropical Animal Health and Production - Turkey coryza is a major respiratory disease caused by Bordetella avium (B. avium). It occurs in all ages of turkeys and is characterized by high morbidity...     

Detection of antibodies against Bordetella avium in turkeys by avidin-biotin enhancement of the enzyme-linked immunosorbent assay and the dot-immunobinding assay.

An avidin-biotin-enhanced enzyme-linked immunosorbent assay (AB-ELISA) and an avidin-biotin-enhanced dot-immunobinding (AB-DIB) assay for detecting antibody to Bordetella avium in turkey sera were developed and compared with the microagglutination (MA) test. Whole-cell antigen, biotin-labeled goat anti-turkey IgG conjugate, and horseradish-peroxidase-labeled streptavidin were used in the AB-ELISA and AB-DIB assay. The AB-ELISA and AB-DIB assay were sensitive, specific, and reproducible. These assays were superior to the MA test for measuring acquired and maternal antibodies against B. avium. All MA-positive sera were positive by two assays, but some sera negative by MA test had titers in the AB-ELISA and AB-DIB assay. AB-ELISA and AB-DIB titers showed a positive correlation (r = 0.866), and AB-ELISA was more sensitive than the AB-DIB assay.     

Effects of Bordetella avium infection on the pulmonary clearance of Escherichia coli in turkeys

Thirty-six 1-day-old turkeys were inoculated intranasally with Bordetella avium (BA) strain 838. Noninoculated hatchmates (n = 36) were housed separately. At 2 and 4 weeks of age, 15 inoculated (BA+) and 15 noninoculated (BA-) turkeys were exposed to an aerosol of virulent Escherichia coli. The remaining six BA+ turkeys and six BA- turkeys were used as controls (ie, not exposed to E coli). Turkeys were necropsied on postaerosolization days 0 (immediately after aerosolization), 1, 3, 5, and 7. Lung and tracheal specimens were collected from each turkey for bacterial quantitation and histologic examination. A 1-ml blood sample was collected for detection of bacteremia. Numbers of E coli in lung specimens from 2- and 4-week-old turkeys were not significantly different between BA+ and BA- groups (pooled data over time); however, numbers of E coli isolated from tracheal specimens were significantly greater in BA+ turkeys than those in BA- turkeys. Although the incidence of pulmonary abcesses and E coli bacteremia was greater in 2-week-old turkeys than in 4-week-old turkeys, the incidence was not different between BA+ and BA- turkeys. At both ages, air sacculitis developed more often and was more severe in BA+ turkeys than in BA- turkeys. Hyperplastic bronchus-associated lymphoid tissue was found more often in BA+ turkeys than in BA- turkeys and appeared to be the first site of heterophil infiltration after E coli aerosolization.