Adherence of Bordetella avium to turkey tracheal mucosa: effects of culture conditions

Adherence of Bordetella avium to the tracheal mucosa of turkeys was evaluated, using bacteria grown under different culture conditions. Several solid and liquid media were used at incubation times of 12, 24, 36, or 48 hours with incubation temperatures of 18, 26.5, or 35 C. Adherence of B avium was greatest when the bacteria were grown on solid media at 35 C. Use of Bordet-Gengou or brain-heart infusion agar was associated with significantly greater (P less than 0.05) adherence compared with adherence of bacteria grown on other media. Adherence was greatest, using cultures in the stationary phase of growth; however, with some media, adherence diminished when incubation was extended beyond 36 hours. Adherence of B avium was reduced but not completely prevented when cultures were incubated at 18 C.     

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.     

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.     

Turkey coryza: toxin production by Bordetella avium

Twelve strains of Bordetella avium representing isolates from turkeys in the United States, the Federal Republic of Germany, and the Republic of South Africa were tested for toxin production. Sterile filtered sonicates from 9 of 12 strains contained a toxin that was lethal for 7-to-10-day-old poults. Mice were also susceptible to the lethal effects of the toxin. No differences in susceptibility to the toxin were found between Beltsville small white and broad-breasted white poults. The toxin was solubilized by sonication and inactivated by heating at 56 C for 30 min. Treatment with formalin or proteolytic enzymes inactivated the toxin, indicating that it is probably a protein. The evidence suggests that the toxin is involved in the pathogenesis of turkey coryza.     

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.     

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.     

Effect of Bordetella avium infection on electrocardiograms in turkey poults.

Electrocardiograms (ECGs) were recorded from turkey poults infected with the W isolate of Bordetella avium. Strip chart data (amplitudes and intervals) were measured at 7, 14, 21, and 28 days postinoculation and compared with data from uninoculated controls. B. avium infection altered P-, RS-, and T-wave amplitudes, as well as P-R and S-T intervals throughout the 4-week experimental period. Heart rates were similar over the 4-week period in control poults, while rates in the infected birds were variable. Alterations in ECGs associated with B. avium infection appeared to be the result of disturbances in the cardiovascular system and may be associated with the pathogenicity of the organism in turkey coryza.     

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.     

直接荧光抗体法检测禽波氏杆菌

制备了兔抗禽波氏杆菌特异性荧光抗体并建立了直接荧光抗体检测方法 ,对禽波氏杆菌在人工感染雏鸡内的致病机理作了初步研究。结果 ,该荧光抗体只与其相应菌株发生特异性荧光反应 ,而不与其他病原菌株发生反应。对人工感染发病雏鸡的检测结果表明 ,禽波氏杆菌主要定植于上呼吸道黏膜 ,并造成损害。该技术具有简便、快速、敏感和特异性强等优点     

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.     

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.     

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。     

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.     

Absence of mouse-lethal toxins in Australian isolates of Bordetella avium

A total of 18 Australian isolates of Bordetella avium and seven reference B. avium strains from Europe and the USA were tested for the presence of a mouse-lethal toxin. Five of the seven reference strains of B. avium, but none of the 18 Australian isolates, produced the toxin.     

禽波氏杆菌分离株的16S rRNA基因序列分析

采用PCR方法对本实验室分离保存的10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,扩增其16SrRNA基因的5′端片段（783bp）,并测定所得片段的DNA序列。用DNAStar分析软件将所获得的序列与GenBank中的禽波氏杆菌序列进行比较,由此构建禽波氏杆菌菌株的系统发育树。结果显示,10株禽波氏杆菌核苷酸序列与GenBank中收录的AF177666株禽波氏杆菌核苷酸序列的同源性为82.0%～82.5%,与兔支气管败血波氏杆菌和猪支气管败血波氏杆菌核苷酸序列的同源性均为99.2%～99.9%,其中P9（山鸡波氏杆菌）、P11（兔支气管败血波氏杆菌）与P14（猪支气管败血波氏杆菌）分别发生1个核苷酸的缺失。本试验结果表明,16SrRNA序列分析是鉴定禽波氏杆菌的一种快速而准确的方法。     

The effects of heat-labile Bordetella avium toxin on turkey poults

Intraperitoneal exposure of turkey poults to various concentrations of Bordetella avium sonicate containing heat-labile toxin indicated a high degree of toxicity: poults died after exposure to sonicate containing as little as 6.32 micrograms of protein. The toxic effects were dose-related: poults that received sonicate containing 158 micrograms of protein died in 4 to 6 1/2 hours, those that received 31.6 and 6.32 micrograms of protein died in 25 to 30 hours, and those that received 1.2 micrograms survived the 6-day course of the study and were apparently unaffected. Histologic examination of poults that received lethal doses of the toxin revealed degeneration and necrosis of parenchymal cells of the liver and pancreas as well as hyperemia, hemorrhage, and necrosis of the mucosa of the small intestine. No lesions were observed in poults that received the sublethal dose of toxin or in unexposed poults. Repeated intranasal exposure of poults to the sonicate did not produce clinical signs of disease or gross lesions.     

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.     

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.