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.     

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.     

Turkey coryza: lack of correlation between plasmids and pathogenicity of Bordetella avium

Plasmids were removed from pathogenic Bordetella avium using a variety of treatments. The plasmid-cure rates depended on the treatment and isolate. Pathogenicity of B. avium in turkey poults was not altered by removal of plasmids.     

Antibiotic susceptibility of canine Bordetella bronchiseptica isolates

The antimicrobial sensitivities of 78 recent (1995-1998) canine isolates of Bordetella bronchiseptica from 13 separate sources were determined. Minimum inhibitory concentrations were assessed using the E-test method or by agar dilution. All 78 isolates were sensitive to tetracycline, doxycycline, enrofloxacin, and amoxycillin/clavulanic acid; the majority were sensitive to ampicillin (63/78; 81%), trimethoprim (57/78; 73%), and sulphadiazine (63/78; 81%). Plasmids were detected in 14 out of the 24 isolates tested. There was no correlation between the presence of plasmids and antibiotic resistance, but there was some correlation between the presence of plasmids and the origin of the isolates. Three sizes of plasmid were found: 20, 14, and 5.5 kb. Eight of the isolates contained all three plasmids, the remainder one or two, Thirteen isolates demonstrated beta-haemolysis, of which six produced a soluble haemolysin. Except for one isolate, haemolysin production correlated with plasmid carriage. Pulsed-field gel electrophoresis showed that all except one isolate could be grouped in the same genotype. Within this genotype isolates could be divided into three subtypes, generally corresponding to their place of origin.     

Detection of urease-negative Bordetella bronchiseptica from the field

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs? assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.     

Isolation of Bordetella avium from poultry

Four Australian isolates of Gram-negative nonfermentative bacteria obtained from poultry were compared with reference strains of Bordetella avium, Bordetella bronchiseptica and Alcaligenes faecalis. The Australian isolates were identified as Bordetella avium. A routine procedure for the identification of this recently recognised poultry pathogen is described.     

A comparison of outer membrane proteins and surface characteristics of adhesive and non-adhesive phenotypes of Bordetella avium

The outer membrane protein profiles of four adherent and one reduced-adherence mutant phenotype of Bordetella avium were compared; a non-adherent B. avium-like organism isolated from turkeys was also examined. The organisms were grown on brain-heart infusion agar at 35 C for 36 hours. In addition, one of the adherent phenotypes was grown at 18 C and 40 C. The outer membrane proteins were isolated by sonication and detergent extraction with Triton X-100. Surface characteristics of intact bacteria were examined using negative stain and transmission electron microscopy. The adherent phenotypes had identical protein profiles by electrophoresis. The non-adherent B. avium-like organism lacked at least five of the proteins present on the adherent strains. The non-adherent mutant phenotype had a protein profile similar to that of the adherent organisms, although several proteins were present in much lower concentrations. Fimbriae were found on both adherent and non-adherent organisms. By comparing protein profiles of adherent and non-adherent B. avium we were able to make a preliminary determination of the membrane proteins that lack adhesive properties.     

应用23S rRNA对禽波氏杆菌分离株的进化分析

通过PCR扩增分离保存的8株禽波氏杆菌、3株兔支气管败血波氏杆菌及1株猪支气管败血波氏杆菌菌株的23S rRNA基因的片段(710bp),分别克隆到载体pMD18-T后测序。利用BLAST工具进行同源性搜索;用DNAStar分析软件进行同源核苷酸序列的多重比较分析并构建禽波氏杆菌菌株的系统生物进化树。结果显示,8株禽波氏杆菌核苷酸序列之间同源性为99.2%～99.7%,与GenBank中收录的NC_010645株禽波氏杆菌核苷酸序列同源性为92.4%～92.5%,与兔支气管败血波氏杆菌和猪支气管败血波氏杆菌的核苷酸序列同源性均为98.5%～99.2%。国内分离的禽波氏杆菌菌株和支气管败血波氏杆菌菌株均与国外分离菌株在遗传基因上有一定的差异。结果表明,23S rRNA序列分析可以作为鉴定禽波氏杆菌的一种快速简便的方法。     

Molecular epidemiology of feline bordetellosis in two animal shelters in California,USA

"Kennel cough" in dogs in animal shelters is readily transmissible, reduces adoption rates, and commonly leads to the euthanasia of affected dogs. In cats, tracheobronchitis, conjunctivitis, and pneumonia have been associated with Bordetella bronchiseptica infection-but most cases of upper-respiratory infection (URI) probably are caused by herpesvirus and calicivirus, and many B. bronchiseptica culture-positive cats are clinically normal. Our prospective observational study was undertaken to document the contribution of B. bronchiseptica to disease in cats and dogs from two animal shelters undergoing outbreaks of canine kennel cough, to evaluate whether cross-species transmission might have occurred, and to determine if the presence of infected cats represented a risk to dogs. Clinically defined cases of kennel cough in dogs and URI in cats were investigated in two shelters by calculating clinical-disease incidence, alveolar-lavage cytological examination, bacterial and viral cultures, antibiotic-susceptibility testing, and molecular fingerprinting by pulsed-field gel electrophoresis.In a 40-cat and 40-dog "no-kill" shelter, the prevalences of culture positivity were 47% for B. bronchiseptica and 36% for calicivirus at the same time as two resident dogs demonstrated clinical cough. When no dogs had kennel cough 3 months later, 10% of cats were B. bronchiseptica-culture-positive and 63% calicivirus positive. In a large traditional shelter, the incidence of kennel cough in dogs increased over 12 weeks to a maximum of 19 cases/week/120 dogs, during which time the culture prevalence was 23% for B. bronchiseptica in dogs and 47% in cats. Three to 6 months before the kennel-cough epidemic, no dogs or cats were B. bronchiseptica positive. Very little genetic variability was detected in isolates from these shelters; all isolates except one corresponded to a single strain type which was identical to the pattern in a vaccine used in these shelters. Isolates from other cats, a horse, a llama, and a sea otter were genetically distinct from the shelter isolates. There was widespread resistance to cephalosporins and ampicillin, but low or no resistance to amoxicillin/clavulanate, trimethoprim-sulfamethoxazole, tetracycline, and enrofloxacin. Greater percent resistance was observed in the traditional shelter than in the no-kill shelter and feline isolates were more likely to be resistant than canine isolates.     

Polymorphism of pertactin gene repeat regions in Bordetella bronchiseptica isolates from pigs

Bordetella bronchiseptica pertactin (prn) is an outer membrane protein which has been implicated as both an adhesin and a protective antigen that induces immunity against atrophic rhinitis in pigs. Previous studies demonstrated extensive heterogeneity of the prn sequence within two distinct regions of amino acid repeats for B. bronchiseptica isolated from the United States and Europe. By deducing the amino acid sequences of the repeat regions of the prn gene from recent isolates from Korea, two region 1 variants and five region 2 variants were identified. Five pertactin types were distinguished based on combinations of variants of both regions. Interestingly, none of the field isolates have the same pertactin type as the B. bronchiseptica P4 strain widely used to vaccinate pigs.     

Evaluation of canine Bordetella bronchiseptica isolates using randomly amplified polymorphic DNA fingerprinting and ribotyping

Bordetella bronchiseptica is a respiratory tract pathogen in a variety of species. Previous studies suggest little genetic variation among canine B. bronchiseptica isolates. The degree of genetic diversity in 26 canine B. bronchiseptica strains was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprinting and ribotyping. Strains evaluated include historic reference strains (N=3). vaccine strains (N=5) and clinical isolates (N=18). RAPD fingerprinting with the 10-nucleotide primer OPA-4 resulted in four distinct fingerprint patterns. RAPD fingerprinting consistently separated four previously characterized electromorphotype (EMT) 6 strains into two fingerprint types. Ribotyping, using the restriction endonuclease PvuI, resulted in six distinct ribotypes. With the exception of vaccine strains, considerable genetic diversity exists in the canine B. bronchiseptica isolates examined. These findings indicate the genetic variability within canine strains of B. bronchiseptica is greater than appreciated previously. Additionally, OPA-4 RAPD fingerprinting and PvuI ribotyping will be useful tools in epidemiologic studies of canine B. bronchiseptica isolates.     

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.     

Strain- and growth condition-dependent variability in outer membrane protein expression by Bordetella bronchiseptica isolates from dogs.

OBJECTIVE: To characterize strain-dependent and growth condition-dependent variability in outer membrane protein (OMP) expression of Bordetella bronchiseptica isolates from dogs and evaluate the systemic immune response to OMP of B bronchiseptica among infected dogs. SAMPLE POPULATION: 8 strains of B bronchiseptica isolated from dogs, including a historic reference strain, 2 commercially available vaccine strains, and 5 field strains, and serum samples collected from 3 specific-pathogen-free (SPF) dogs before and 1 month after infection with B bronchiseptica. PROCEDURE: OMP were isolated from cultures in the late exponential phase of growth and compared among strains and, within strains, among growth conditions by means of polyacrylamide gel electrophoresis and immunoblotting. Serum samples were probed with OMP from 1 of the field strains. RESULTS: Strain-dependent variability in OMP profiles and growth condition-dependent and strain-dependent variability in expression of filamentous hemagglutinin (FHA) and pertactin was found, along with heterogeneity of the pertactin proteins produced by these B bronchiseptica strains. All 3 SPF dogs seroconverted to proteins with estimated molecular masses of 200 and 66 kDa, suggesting that FHA and pertactin were involved in the immunologic response of these dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there is growth condition and strain variability in expression of OMP, FHA, and pertactin proteins produced by B bronchiseptica. This information could be useful in the improvement of vaccines for prevention of bordetellosis in dogs.     

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.     

Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs

Three strains of Bordetella bronchiseptica were compared for their ability to assist colonisation of the nasal cavity of gnotobiotic pigs by toxigenic Pasteurella multocida. Toxigenic P multocida (counted in nasal washings) colonised the cavity in large numbers in pigs previously infected with a cytotoxic phase I strain of B bronchiseptica (B58), whereas it colonised only in small numbers in those previously infected with B65, a phenotypic phase III variant of B58. Toxigenic P multocida colonised pigs infected with a non-cytotoxic phase I strain of B bronchiseptica (PV6) in fewer numbers than were seen in pigs infected with the cytotoxic phase I strain but in greater numbers than in pigs infected with the phase III strain. The turbinates of pigs infected with the cytotoxic phase I strain of B bronchiseptica and toxigenic P multocida were most severely affected and those in pigs infected with the non-cytotoxic phase I strain and toxigenic P multocida were moderately reduced in size. The turbinates of pigs infected with the phase III strain and toxigenic P multocida were slightly reduced in size except for one piglet whose turbinates were severely affected. Pigs infected with the non-cytotoxic phase I strain of B bronchiseptica alone showed no signs of atrophy and their turbinates were used to calculate reductions (per cent) in those infected with P multocida. The reduction (per cent) in size of turbinates and total numbers of P multocida isolated from the nasal washings of each pig were linearly related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of protection against Bordetella avium in turkey poults exposed to B. avium-like bacteria

Six laboratory experiments were designed to determine whether poults infected with the nonpathogenic Bordetella avium-like (BAL) bacteria would develop immunity to B. avium (BA), the causative agent of turkey coryza. The BAL bacteria were isolated from poults given that organism, but few colonies were observed by 3 weeks postexposure. No serum-agglutinating antibody to the BAL bacterium was detected in poults exposed to that organism. Poults exposed to BAL bacteria either once or twice at different ages were not protected from infection or disease following experimental challenge between 1 and 7 weeks of age with pathogenic BA.     

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.     

Cross protection studies on Bordetella bronchiseptica in mice using an intracerebral challenge model.

Protective activities of heat-inactivated (60 degrees C for 30 min) merthiolate preserved Bordetella bronchiseptica and B. pertussis bacterins were compared in intraperitoneally immunized mice challenged intracerebrally (i.p./i.c.) or intraperitoneally (i.p./i.p.). In the i.p./i.c. assay (Kendrick test), a B. pertussis bacterin protected mice against challenge with B. pertussis 18-323, as well as against phase I cytotoxic and non-cytotoxic strains of B. bronchiseptica. A B. bronchiseptica bacterin, prepared from a phase I cytotoxic strain, gave protection against two phase I B. bronchiseptica strains, irrespective of their cytotoxin-production. A non-cytotoxic phase I strain of B. bronchiseptica elicited protection against the homologous strain only. Neither cytotoxic nor non-cytotoxic B. bronchiseptica strains protected mice challenged with B. pertussis 18-323. Vaccines prepared from phase III strains of B. bronchiseptica were not protective at all against any of the challenge strains. No such differences in the protective activities of the bacterins could be detected by the i.p./i.p. method. They seem to cross-protect equally well. The results indicate that the Kendrick test may be useful in testing potency of different B. bronchiseptica bacterins.     

Serological Investigations of Mycobacterium Avium and M. Avium-Like Bacteria Isolated from Domestic and Wild Animals

One hundred and two Mycobacterium avium and M. avium-like strains isolated from domestic and wild animals were submitted to serological typing by means of the agglutination method developed by Schaefer. Of the 82 porcine strains M. intracellulare serotype 8 dominated, followed in order of frequency by serotypes 1, 4, 2, 10 and 6. Of the 20 strains originating from other animals 7 were typed as either serotype 2, 3, 8 or 10. The 3 strains isolated from wild birds were all serotype 2. As a considerable number of the wild animal strains were autoagglutinable, the suitability of the agglutination test for typing such strains is discussed.     

Adherence of Bordetella bronchiseptica and Pasteurella multocida to porcine nasal and tracheal epithelial cells.

The ability of 19 different Bordetella bronchiseptica isolates and 25 Pasteurella multocida isolates to adhere in vitro to porcine nasal and tracheal epithelial cells was examined. It was found that B. bronchiseptica adhered well to upper respiratory tract cells. In contrast the number of P. multocida organisms which adhered was four to six times less than the number of B. bronchiseptica adherent organisms. This difference was statistically significant (p less than 0.0001). Both microorganisms adhered in greater numbers to nasal cells than to tracheal cells (p less than 0.005). The data indicated that B. bronchiseptica possesses a greater ability than P. multocida to attach to porcine upper respiratory tract cells.