Virulence and lienotoxicity of Bordetella bronchiseptica in mice

Whole-cell suspensions (WCSs) and cell-free sonicated extracts (SEs) of seven Bordetella bronchiseptica strains were studied for lethality and lienotoxicity in mice. Lethality was assessed after intravenous and intracerebral inoculation, and lienotoxicity by splenic atrophy after intravenous inoculation. The strains represented phase I isolates with or without cytotoxin production, their phase III subcultures and a phase IV variant. The lethality and lienotoxicity of the SEs were in close positive correlation with cytotoxin production. The WCSs of all phase I strains were lethal, irrespective of their cytotoxin- and lienotoxin-producing ability. The only difference was that cytotoxic phase I strains caused splenic atrophy while the noncytotoxic phase I strain induced splenic hypertrophy in the surviving mice. The WCSs of phase III and IV variants were non-lethal and caused splenic hypertrophy even though all but one of them showed some cyto- and lienotoxic activity when their SEs were tested. The results indicate that B. bronchiseptica possesses two different mouse lethal factors: one seems to be identical with the cytotoxin, the other is associated with cell integrity and viability and, presumably, propagation in vivo. It also follows from the results that only the SEs are suitable for accurate determination of the lienotoxin-producing ability of B. bronchiseptica.     

Effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice

OBJECTIVE: To evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus. ANIMALS: 2,213 mice. PROCEDURE: Inactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus. RESULTS: Vaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.     

Immunogenicity and safety of an attenuated Bordetella bronchiseptica vaccine in pigs

The immunogenicity and safety of an attenuated Bordetella bronchiseptica vaccine for swine atrophic rhinitis (AR) was evaluated in 22 hysterectomy-produced, colostrum-deprived pigs and 18 conventional pigs. None of 8 pigs inoculated at 7 days of age intranasally with greater than or equal to 3 X 10(5) colony-forming units (CFU) of vaccinal strain/pig and 2 of 5 pigs inoculated at 7 days of age intranasally with 3 X 10(4) CFU of the vaccinal strain/pig developed AR after intranasal challenge exposure with a virulent strain at postinoculation week (PIW) 3. The remaining 3 vaccinated pigs and 4 nonvaccinated pigs developed AR. Thirteen pigs were inoculated intranasally with 3 X 10(6) to 3 X 10(9) CFU of the vaccinal strain at 7 days of age. At PIW 12, the pigs were killed and necropsied. None of the pigs had clinical signs of AR and/or pneumonia. Virulence was studied by transmission of vaccinal strain through 3 serial growing passages on the nasal mucosa of a litter of hysterectomy-produced colostrum-deprived pigs. Inoculum (nasal swab samples from 2 pigs 4 days after inoculation with 10(8) CFU of vaccinal strain at 5 days of age) was inoculated into the nasal cavity of 2 nonvaccinated pigs. This procedure was repeated 3 times. After the 1st passage, the vaccinal strain was recovered on postinoculation day 4, but after postinoculation day 4, the vaccinal strain was not recovered until the end of the 3rd passage. Turbinate atrophy or pneumonia was not recognized in these inoculated pigs. The vaccinal strain provided immunogenicity without ill effects.     

Inhibitory effect of sulfamonomethoxine on capsule formation of Bordetella bronchiseptica.

Bordetella bronchiseptica phase I organism possesses a capsule and has the ability to agglutinate with K antiserum, although phase III organism lacks both. The present study examines the effect of sulfamonomethoxine (SMMX) on capsule formation of B. bronchiseptica. I also investigated whether or not the organisms possessed a capsule by bacterial agglutination with K antiserum. Three SMMX-resistant strains of B. bronchiseptica phase I organisms showed loss of agglutinability with K antiserum by culturing them at a higher concentration of 1.56 micrograms/ml of SMMX. These results indicated that capsule formation of SMMX-resistant B. bronchiseptica is inhibited by SMMX.     

Devitalization of Bordetella bronchiseptica in vitro using vapors of Persterile and triethyl glycol]

Using the method of bacterial carriers, the devitalizing effect was studied of Persteril and triethylene glycol vapours, acting in vitro, on a 24-hour culture of a mixture of 39 strains of Bordetella bronchiseptica, isolated from the nasal cavity of pigs which came from five stocks suffering from the clinical form atrophic rhinitis. After exposure of the carriers of Bordetella bronchiseptica to the action of spontaneously developing vapours o the mentioned chemicals, the devitalizing effect was determined by bacteriological examination. The vapours of the water solution of Persteril, containing 0.1% of peracetic acid, showed a 90.91 to 93.83% devitalizing effect after 48-hour exposure at room temperature. When the vapours of warmed concentrated triethylene glycol were used, a 98.57 to 100% devitalizingeff ect was obtained after a 72-hour exposure under the same temperature conditions. In experiements performed at a temperature of 37 degrees C, a 24-hour exposure sufficed for obtaining 91.67 to 100% devitalizing effectiveness. There is a discussion concerning the importance of the obtained results and the need for testing them experimentally by a methodically simple and cheap continual disinfection of the air and stable surfaces in premises for pig stocks affected by atrophic rhinitis.     

Effect of vaccination on experimental infection with Bordetella bronchiseptica in dogs

OBJECTIVE: To determine comparative efficacy of vaccines administered IM and intranasally, used alone or sequentially, to protect puppies from infection with Bordetella bronchiseptica and determine whether systemic or mucosal antibody response correlated with protection. DESIGN: Randomized controlled trial. ANIMALS: 50 specific-pathogen-free Beagle puppies. PROCEDURE: In 2 replicates of 25 dogs each, 14-week-old puppies that were vaccinated against canine distemper virus and parvovirus were vaccinated against B bronchiseptica via intranasal, IM, intranasal-IM, or IM-intranasal administration or were unvaccinated controls. Puppies were challenge exposed via aerosol administration of B bronchiseptica 2 weeks after final vaccination. Clinical variables and systemic and mucosal antibody responses were monitored for 10 days after challenge exposure. Puppies in replicate 1 were necropsied for histologic and immunohistochemical studies. RESULTS: Control puppies that were seronegative before challenge exposure developed paroxysmal coughing, signs of depression, anorexia, and fever. Vaccinated puppies (either vaccine) that were seronegative before challenge exposure had fewer clinical signs. Puppies that received both vaccines had the least severe clinical signs and fewest lesions in the respiratory tract. Vaccinated dogs had significantly higher concentrations of B bronchiseptica-reactive antibodies in serum saliva before and after challenge. Antibody concentrations were negatively correlated with bacterial growth in nasal cavity and pharyngeal samples after challenge exposure. CONCLUSIONS AND CLINICAL RELEVANCE: Parenterally and intranasally administered vaccines containing B bronchiseptica may provide substantial protection from clinical signs of respiratory tract disease associated with infection by this bacterium. Administration of both types of vaccines in sequence afforded the greatest degree of protection against disease.     

An agglutination test for the detection of Bordetella bronchiseptica infection in swine.

An agglutination test with the use of formalin-killed antigen of the cell carrying the capsule was developed and used for the detection of antibody in swine naturally infected with Bordetella bronchiseptica. Under optimum antigen concentration and reaction temperature 210 or 60% of 342 serum samples tested from 42 conventional swine herds were positive for Bordetella infection. In contrast, only 34 or 10% of 342 nasal swabs from the same animals were positive for Bordetella by culture technique. The test was relatively free of cross-reactivity to related organism. However, 2.7 and 13.0% of sera from growing pigs and mature hogs, respectively, reacted with antigen of Pasteurella multocida. Because of this, only agglutinin reactions in 1:20 dilutions or higher to Bordetella were considered positive. The bulk of the antibody activity of selected sera tested from various age ranges of swine was mercaptoethanol sensitive, suggesting that serum antibody in Bordetella infection may be associated with immunoglobulin IgM. Because of the high agglutinability and stability of formalin-killed antigen the test may be useful as an auxiliary aid for the diagnosis of Bordetella infection where the organism cannot be identified by culture means.     

Histological changes in the nasal ventral conchae of piglets infected with Bordetella bronchiseptica.

Piglets infected intranasally with Bordetella bronchiseptica were injected with two fluorochrome markers. Transverse sections of undecalcified nasal conchae (cut between the third incisor and the third premolar teeth) were examined by microradiography and fluorescence microscopy; surface-stained sections were evaluated by light microscopy. The fluorescent surface of the nasal ventral conchae from the infected piglets was increased as compared with the controls. This was due to an increased amount of fluorescent mineralization fronts as well as to the presence of abnormal fluorescent areas within trabeculae. Trabecular mineral content of the microradiographs was irregular and varied from hypo- to hypermineralized. When compared with the corresponding surface-stained sections, no correlation could be made between the mineral content and the type of tissue. These findings suggest that an increased number of osteoblasts which secrete prebone matrix but are modified so that mineralization does not occur normally.     

Interaction between Bordetella bronchiseptica and toxigenic Pasteurella multocida on the nasal mucosa of SPF piglets.

The interaction between Bordetella bronchiseptica and type D toxigenic Pasteurella multocida was studied in five groups of 4 specific-pathogen-free (SPF) piglets each. At 28 days of age, piglets of groups 3 and 4 were inoculated into both nostrils with 10(8) colony-forming-units (CFU) of a non-dermonecrotic toxin (DNT)-producing, phase I strain of B. bronchiseptica. Piglets of groups 1 and 3 were treated intranasally with a sonic extract of the non-toxic strain of B. bronchiseptica and those of groups 2 and 4 with B. bronchiseptica DNT into the left nostril. Sonic extract and DNT treatment was started at 33 days of age and lasted for 5 days. Piglets of group 5 served as controls. At the age of 37 days, piglets of all groups except group 5 were inoculated into both nostrils with 5 x 10(7) CFU of toxigenic P. multocida. At slaughter at 50 days of age, P. multocida was recovered from the left nasal cavity of 3 piglets of group 2 and all piglets of group 4. In piglets inoculated with B. bronchiseptica DNT the mucosal epithelial cells of the left nasal cavity showed loss of cilia, regressive lesions such as vacuolation, karyopycnosis and necrosis, hypertrophy of the epithelium, infiltration of the epithelium and submucosa by inflammatory cells, could also be seen. The results suggest that action of the B. bronchiseptica DNT on the nasal mucosa is a precondition of the growth of P. multocida in the nasal cavity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ribotyping and restriction endonuclease analysis reveal a novel clone of Bordetella bronchiseptica in seals.

The goal of the present study was to characterize, by ribotyping and restriction endonuclease analysis (REA), 35 phocine Bordetella bronchiseptica isolates and to ascertain their relationship to one another and to isolates acquired from other host species. Thirty-four isolates were obtained in Scotland during a 10-year period encompassing the 1988 epizootic; the remaining isolate was obtained independently in Denmark. All phocine isolates had an identical Pvu II ribotype unique from the 18 ribotypes previously detected in strains from heterologous hosts. Alternative restriction enzymes, useful for subgrouping strains within Pvu II ribotypes, also failed to discriminate among isolates from seals. The exclusive occurrence of a single ribotype of B. bronchiseptica in a particular host species has not been previously observed. Similarly, REA based on either HinfI or Dde I profiles did not reveal detectable polymorphisms, although unique patterns were readily distinguished among a limited number of isolates from other host species. This is the first report demonstrating the utility of REA using frequently cutting enzymes for discrimination of B. bronchiseptica strains. These data suggest that B. bronchiseptica-induced respiratory disease in seals along the Scottish shore may be due to the circulation of a single, unique clone.     

Distribution of atrophy in the nasal ventral conchae of piglets infected experimentally with Bordetella bronchiseptica.

The distribution and extent of atrophy caused by Bordetella bronchiseptica in the nasal ventral conchae were investigated by intranasally infecting piglets at day 4 of life. Experimental and control piglets were killed at one, two, three, four, five and six weeks of age. Serial transverse 80 microns thick nondecalcified sections were cut throughout the entire length of the nasal ventral conchae, and microradiographed. It was observed that the distribution and extent of atrophy was not uniform along the ventral conchae. The articular and transverse laminae as well as the connecting zone were not atrophied, except at their rostral extremity. The entire dorsal scroll was atrophied. This atrophy was moderate, except at the rostral and caudal extremities of the dorsal scroll, where its size was less than half that of the controls. The ventral scroll was less atrophied than the dorsal scroll, although a marked atrophy was present at its rostral extremity. From these observations, it is suggested that two transverse sections should be made through the nose of pigs in order to establish a diagnosis of atrophic rhinitis at slaughter: one section at the level of the first-second premolar tooth and one more rostral section, at the level of the canine tooth.     

Comparison of the inhibitory effect of sulfamonomethoxine and other sulfonamides on capsule formation of Bordetella bronchiseptica.

The inhibitory effect of sulfamonomethoxine and other sulfonamides on the capsule formation of sulfonamide-resistant Bordetella bronchiseptica was investigated. All the sulfonamides having MeO(-OCH3) groups inhibited the capsule formation of B. bronchiseptica. Strong inhibition was obtained with sulfamonomethoxine. Inhibition was not seen with sulfonamides having no MeO groups.     

Studies on linear epitopes of Semliki Forest virus in protection studies against lethal challenge virus infection.

Purified reduced and non-reduced glycoproteins E1 and E2 of Semliki Forest Virus (SFV) were used to investigate the protection potency to prevent clinical disease after lethal virus challenge. In parallel synthetic oligopeptides deduced from conserved regions of the nucleotide sequences coding for the glycoproteins E1 and E2 were included. It could be demonstrated that both reduced and non-reduced glycoprotein preparations induced protection against lethal virus challenge, whereas the oligopeptides did not. The role of linear epitopes in immunity and their potential use as synthetic vaccines against Alphaviruses are critically discussed.     

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.     

猪支气管败血波氏杆菌菌毛fimD基因的克隆表达及间接ELISA检测方法的建立

参照GenBank上支气管败血波氏杆菌的菌毛fimD基因序列（X75811）,设计1对引物,从本实验室分离鉴定的猪源支气管败血波氏杆菌中扩增出fimD编码区1098bp的片段,将其克隆至原核表达载体pET-28a,转化到大肠杆菌BL21（DE3）中进行融合表达。SDS-PAGE和Western blot检测证实该表达产物以包涵体形式存在,大小约42ku,与用支气管败血波氏杆菌菌体制备的阳性血清能发生特异性反应。将包涵体变性和复性后包被酶标板建立间接ELISA（fimD-ELISA）,特异性良好。用fimD-ELISA检测临床送检的668份血清,阳性率为30．7％。用该法与微量凝集试验平行检测102份血清,fimD-ELISA的敏感性高于微量凝集试验。     

Crystal violet staining of Bordetella bronchiseptica colonies for differentiation of phase-I strains from variant strains in degraded phases.

After 2 days of growth on Brain heart infusion agar (BHIA) at 38 degrees C, phase-I colonies and degraded-phase colonies of Bordetella bronchiseptica could be differentiated by their ability to take up crystal violet (CV). Phase-I colonies in X mode, but not colonies in degraded phases (phases II, III, and rough) bound CV. Phenotypically-altered C-mode colonies (grown at 32 degrees C or lower temperatures) also lacked this ability. CV staining offers an easy method for the recognition of different colony types that appear identical when observed on BHIA.