Ultrastructural pathology of Bordetella avium infection in turkeys

One-day-old turkeys were infected intranasally with Bordetella avium, and tracheas were examined by scanning and transmission electron microscopy at 1 to 5 weeks post-inoculation (PI). The predominant ultrastructural lesions were progressive loss of ciliated epithelium with replacement by nonciliated cells, bacterial colonization of ciliated cells, membrane-bound crystalline inclusions in cytoplasma of epithelial cells, depletion of mucous granules, and distortion of tracheal rings and the mucosal surface. Tracheal surface exudates consisted of mucus, necrotic cells, heterophils, and fibrin. Ciliated cells were replaced by immature cuboidal cells characterized by abundant rough endoplasmic reticulum with small numbers of electron-dense mucous granules in the apical cytoplasm. Bacterial surfaces were rough and contained numerous pleomorphic, knob-like structures, 20-50 nm in diameter. Other changes included enlarged mucosal gland openings, cell extrusion marks, pleomorphic microvilli, and cells with small numbers of short cilia.     

Tracheal lesions in young turkeys infected with Bordetella avium

Forty-six turkeys were inoculated intranasally with Bordetella avium at 3 days of age. Inoculated and non-inoculated turkeys (n = 72) were necropsied sequentially from post-exposure days (PED) 3 to 53. Tracheal lesions were studied histologically. Mild fibrinopurulent tracheitis, associated with bacterial colonization of ciliated epithelium, was seen at PED 7 to 10. At PED 14 and 21, there were numerous bacterial colonies, loss of ciliated cells, epithelial hyperplasia, depletion of mucus, and diffuse infiltration of lymphocytes and macrophages. At PED 28, the mucosa and tracheal rings were severely distorted. The mucosal epithelium, composed of immature epithelial cells and nononuclear leukocytes, had formed prominent longitudinal folds. Mucous glands were cystic and lined by low cuboidal epithelium. At PED 42 and 53, tracheas appeared normal, except for residual distortion of tracheal rings and shortened luminal epithelium.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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...     

Adherence of Bordetella avium to tracheal mucosa of turkeys: correlation with hemagglutination

Adherence to turkey tracheal mucosa and agglutination of guinea pig erythrocytes were determined for 20 strains of Bordetella avium. Ten type-I strains, 7 type-II strains, 2 transposon-induced mutants, and 1 revertant were evaluated. All type-I strains adhered readily to tracheal mucosa and agglutinated erythrocytes, whereas no type-II strains adhered to trachea or caused hemagglutination (HA). Two mutants selected for loss of HA activity were less adherent to tracheal mucosa, compared with the parent strain. Reversion of one mutant to HA-positive status was accompanied by reconstitution of much of its adherence capacity. Results of this study provide preliminary evidence that tracheal adherence and HA of B avium are closely related.     

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.     

Efficacy in turkeys of spray vaccination with a temperature-sensitive mutant of Bordetella avium (Art Vax)

Broad-breasted white turkeys were vaccinated with a temperature-sensitive mutant of Bordetella avium (Art Vax) at 2 and 15 days of age and challenged at 22 days of age by contact with infected birds. Necropsy was performed at 35 days of age. Two vaccination protocols (eyedrop/oral and spray cabinet/spray bottle) and two challenge isolates (Arkansas 105 and North Carolina [NC] isolates) were used. Neither the spray nor the eyedrop/oral methods of vaccination prevented infection of the anterior trachea with either of the virulent challenge strains. The spray and eyedrop/oral methods of vaccination were equally effective in reducing the severity of gross lesions in the trachea. The vaccine reduced the severity of gross lesions in the tracheas of turkeys challenged with the NC isolate to a level approximately equal to that observed in unchallenged vaccinated controls, but the vaccine only moderately reduced the severity of lesions in birds challenged with the 105 isolate.     

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.     

Influence of Bordetella avium infection on association of Escherichia coli with turkey trachea

Four-week-old Bordetella avium-infected and B avium-free turkeys were inoculated intratracheally with a suspension of fimbriated or nonfimbriated Escherichia coli. Numbers of E coli associated with tracheal sections were determined at postinoculation hour (PIH) 1 or 6. Significantly (P less than 0.05) greater numbers of E coli were isolated from the tracheas of B avium-infected turkeys compared with numbers in B avium-free turkeys. In B avium-free turkeys, tracheal associated E coli were 90% less at PIH 6 compared with that at PIH 1. However, in B avium-infected turkeys, numbers of E coli were not affected by postinoculation time. Seemingly, B avium-infected turkeys had reduced capacity to clear E coli from the trachea.     

Observations on colonial phenotypic variation in Bordetella avium

Two forms of Bordetella avium colonial morphology on artificial media were observed. The smooth colonial morphology is convex, with an entire edge, and has a glistening mucoid appearance when cultured on 5% bovine blood agar or tryptic soy agar. On the same media, the rough morphology has a crenated edge, a flat surface, and sometimes a wrinkled or ground-glass appearance to the surface. Colonial morphology remained stable when cultures were passaged at 37 C every 48 hours. When different salts or crystal violet were added to the media, the colonial morphology of seven different isolates did not change. Following storage at 4 C on agar, B. avium isolates switched between the smooth and rough colonial morphology, indicating that this is a reversible process. Generally, bacteria with the smooth colonial morphology were motile on sulfide indole motility (SIM) media, whereas bacteria with the rough colonial morphology were non-motile. Only the isolates with the smooth colony type colonized the upper respiratory tract of turkeys, caused clinical signs of the disease, and elicited an agglutinating antibody titer by 21 days postexposure.     

Pili of Bordetella avium: expression, characterization, and role in in vitro adherence

Electron microscopy revealed pili on all isolates of Bordetella avium and B. avium-like bacteria examined. Trypticase soy broth (TSB) and 2% peptone agar were the best media for promoting pilus expression. Cultures grown at 37 or 42 C had similar pilus production, whereas cultures grown at 18 C produced few or no pili. Pilus expression of the Art Vax strain was best when that strain was grown in TSB, but the strain yielded fewer pili than B. avium and B. avium-like isolates grown under the same cultural conditions. B. avium pili had a diameter of 2.0 nm, ranged in length from 370 nm to 1500 nm, and had a protein subunit molecular mass of about 13,100 daltons. Purified pili from B. avium did not hemagglutinate guinea pig erythrocytes, and a 1:20 dilution of hyperimmune antisera against B. avium pili did not block the hemagglutinating activity of whole-cell preparations of B. avium. In the indirect immunofluorescence test, B. avium isolates and the Art Vax strain adhered to the tracheal explants of turkeys, but B. avium-like isolates did not. Purified pili from B. avium adhered to the surface of the mucosal lining of the tracheal explants, and hyperimmune antisera against B. avium pili blocked the in vitro adherence of whole-cell preparations of B. avium. It was concluded that pili of B. avium are involved in the in vitro attachment of that bacterium to the mucosal surface of turkey tracheal explants.     

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.     

Antimicrobial sensitivity testing of Australian isolates of Bordetella avium and the Bordetella avium-like organism

SUMMARY: The in-vitro sensitivity of 16 Australian isolates of Bordetella avium and 15 isolates of B avium -like organism to 11 antimicrobial agents or combinations of agents was determined using a microtitre plate system to establish minimal inhibitory concentrations. All the B avium isolates were sensitive to ampicillin but resistant to erythromycin, lincomycin, spectinomycin, sulphamethoxazole, trimethoprim, and lincomycin + spectinomycin. Most of the B avium isolates were sensitive to tetracycline and resistant to streptomycin and sulphadiazine. All the B avium -like isolates were resistant to ampicillin, erythromycin, lincomycin, spectinomycin, streptomycin, tetracycline, trimethoprim, and lincomycin + spectinomycin. Most B avium -like isolates were sensitive to sulphadiazine, sulphamethoxazole and trimethoprim-sulphamethoxazole.