Establishment of a Bordetella avium challenge model in turkeys.

Despite the importance of Bordetella avium (BA) as a respiratory pathogen of young turkeys, no infection model for the evaluation of BA-vaccine efficacy is available. The objective of this study was to evaluate the influence of route and dose of infection on the establishment of a BA-challenge model. In our first experiment, 28-day-old turkeys were either inoculated oculonasally with 105, 107 or 109 colony forming units (CFU) of BA per bird or exposed to BA by aerosol with 105-108 CFU/m3. The respiratory tract of all inoculated birds was BA-colonized, which was confirmed by choanal swabs and samples of trachea and lung, showing the highest prevalence in the aerosol-inoculated group. BA-specific humoral immune response was detected in the form of IgG in serum from five days post infection (dpi) and IgA in lacrimal fluid from seven dpi. In the second experiment, the model was tested in a vaccination trial. Twenty-one-day-old turkeys were vaccinated with a formalin-inactivated BA vaccine intramuscularly and challenged 21 days post vaccination with 107 CFU per bird oculonasally. BA-specific IgG antibodies were detected in serum and in lacrimal fluid 14 days post vaccination. As in the first experiment, secretory BA-specific antibodies of the IgA isotype were only detected in the inoculated groups from seven dpi. Despite the lack of clinical signs or pathological alterations in both experiments, vaccine efficacy was demonstrated by significant reduction in BA colonization of the trachea (P ≤ 0.05). In our study, a reliable model for BA infection has been established and has been demonstrated to be suitable for evaluation of vaccine efficacy.

Immuno-enhancement of Taishan Pinus massoniana pollen polysaccharides on recombinant Bordetella avium ompA expressed in Pichia pastoris.

Bordetellosis, caused by Bordetella avium, continues to be an economic problem in the poultry industry of China. Vaccines with good protective ability are lacking. Thus, developing a novel vaccine against the B. avium infection is crucial. Here, we constructed a recombinant Pichia pastoris transformant capable of expressing the outer membrane protein A (ompA) of B. avium to prepare the recombinant ompA subunit vaccine and then evaluated its immune effects. To further investigate the immunomodulation effects of Taishan Pinus massoniana pollen polysaccharides (TPPPS) on this subunit vaccine, three concentrations (20, 40, and 60 mg/mL) of TPPPS were used as the adjuvants of the ompA subunit vaccine respectively. The conventional Freund's incomplete adjuvant served as the control of TPPPS. Chickens in different groups were separately vaccinated with these vaccines thrice. During the monitoring period, serum antibody titers, concentrations of serum IL-4, percentages of CD4(+) and CD8(+) T-lymphocytes in the peripheral blood, lymphocyte transformation rate, and protection rate were detected. Results showed that the pure ompA vaccine induced the production of anti-ompA antibody, the secretion of IL-4, the increase of CD4(+) T-lymphocytes counts and lymphocyte transformation rate in the peripheral blood. Moreover, the pure ompA vaccine provided a protection rate of 71.67% after the B. avium challenge. Notably, TPPPS adjuvant vaccines induced higher levels of immune responses than the pure ompA vaccine, and 60 mg/mL TPPPS adjuvant vaccine showed optimal immune effects and had a 91.67% protection rate. Our findings indicated that this recombinant B. avium ompA subunit vaccine combined with TPPPS had high immunostimulatory potential. Results provided a new perspective for B. avium subunit vaccine research.

Co-adjuvant effects of plant polysaccharide and propolis on chickens inoculated with Bordetella avium inactivated vaccine.

Taishan Pinus massoniana pollen polysaccharide (TPPPS), propolis (PP) and aloe polysaccharide (AP), used as adjuvants, have been proven to possess immunity-enhancing functions. However, their collaborative immunomodulatory effects are largely unknown. To determine which combination can induce the best effects, the three adjuvants were separately or conjointly added into Bordetella avium inactivated vaccines to investigate their co-adjuvant effects on vaccinated chickens. We found that, among all six adjuvant-treated vaccine inoculated groups (TPPPS, PP, AP, TPPPS-PP, PP-AP and TPPPS-AP), the chickens inoculated with TPPPS, PP or TPPPS-PP adjuvant vaccines showed significantly higher levels of antibody titre, cytokine, lymphocyte transformation and peripheral blood T-lymphocyte count than those of non-adjuvant vaccine inoculated groups (P < 0.05), indicating the good immune-enhancing effects of TPPPS and PP. The TPPPS-PP group showed the highest levels of antibody titres and interleukin-2 (IL-2) at 14-28 days post the first inoculation (dpi), lymphocyte transformation rates (LTRs) at 14-35 dpi, CD4(+) T-lymphocyte counts at 14-42 dpi, and CD8(+) T-lymphocyte counts at 28 dpi. The results revealed that B. avium inactivated vaccine used conjointly with TPPPS and PP induced the strongest humoral and cellular immune responses. Thus, there was a synergistic effect between TPPPS and PP on enhancing immunity, which suggests that they can be used as a novel adjuvant formulation for the development of poultry vaccines.

Effects of polysaccharide on chicks co-infected with Bordetella avium and Avian leukosis virus.

Chicks' co-infection with immunosuppressive virus and bacteria seriously threaten the development of the poultry industry. In this study, a model was established in which chicks were injected with either subgroup B ALV (ALV-B)+Bordetella avium (B. avium), or ALV-B+B. avium+Taishan Pinus massoniana pollen polysaccharide (TPPPS), or B. avium only, or B. avium+TPPPS. The data showed that the group injected with ALV-B and B. avium exhibited significant inhibition of the immune function and therefore increased pathogenicity compared with the group injected with B. avium-only. Application of TPPPS effectively alleviated immunosuppression, and body weights increased sharply in the TPPPS groups compared with non-TPPPS groups. To some extent, TPPPS may reduce the proliferation of ALV-B. These results suggest that Pinus pollen polysaccharides are beneficial treating co-infections with immunosuppressive virus and bacteria and therefore have potential for development into safe and effective immunoregulator.

Immunoregulatory effects of Taishan Pinus massoniana pollen polysaccharide on chicks co-infected with avian leukosis virus and Bordetella avium early in ovo.

In recent years, co-infection of chicken embryos with immunosuppressive viruses and bacteria occurs with an annually increasing frequency. Consequently, studies on new and safe immunoregulators, especially plant polysaccharides, have become a popular topic in the poultry industry. In the present study, we selected 300 specific pathogen free embryonated eggs, which were injected with subgroup B avian leukosis virus (ALV-B) and Bordetella avium (B. avium) to establish an artificial co-infection model. The chicks that hatched from these co-infected embryonated eggs were treated with Taishan Pinus massoniana pollen polysaccharide (TPPPS). Results indicated that relevant indices in the co-infection group were significantly lower than that in B. avium-only group. Furthermore, pathogenicity of B. avium was exacerbated, with the chicks exhibiting decreased body weights. The TPPPS groups exhibited gradual improvements in immune function and developmental status. Therefore, in terms of improving immunologic function and production performance, TPPPS could be used as immunoregulator for immune responses.

Taishan Pinus massoniana pollen polysaccharides promote immune responses of recombinant Bordetella avium ompA in BALB/c mice.

To study the effects of Taishan Pinus massoniana pollen polysaccharides (TPPPS) on Bordetella avium outer membrane protein A (ompA) recombinant protein vaccine, ompA was expressed, confirmed by Western blotting and mixed with TPPPS. Female BALB/c mice were randomly divided into six groups (I-VI). Groups I, II, and III were treated with TPPPS-ompA at doses of 200, 400, and 800 mg/ml, respectively. Groups IV, V, and VI were treated with Freund's adjuvant-ompA, pure ompA, and physiological saline, respectively. On days 3, 7, 14, 21, 28, 35, 42, and 49 after the first vaccination, antibody titers, interleukin-2 (IL-2) levels, peripheral blood CD4+ and CD8+ levels, and T lymphocyte proliferation rates in peripheral blood, as well as secreting-type immunoglobulin A (SIgA) levels in the duodenum, were measured. The antibody titers against ompA, IL-2, T lymphocyte proliferation rate, CD4+, and CD8+ in Group II were significantly (P<0.05) higher than those in other groups. However, little difference in SIgA content was observed among Groups I, II, and IV. These results indicated that TPPPS strengthened humoral and cellular immune response against recombinant ompA vaccine and 400 mg/ml TPPPS showed significance (P<0.05) compared with Freund's adjuvant. Therefore, TPPPS can be developed into an adjuvant for recombinant protein vaccines or plant-derived medicine for animal husbandry.

Monoclonal antibodies directed against the outer membrane protein of Bordetella avium.

Bordetella avium is the etiologic agent of coryza and rhinotracheitis in poultry. This respiratory disease is responsible for substantial economic losses in the poultry industry. Monoclonal antibodies (MAbs) were produced against the outer membrane proteins (OMPs) of B. avium isolated from diseased chickens. BALB/c mice were immunized with the extracted B. avium OMPs. Then the splenocytes from immunized mice and SP2/0 myeloma cells were fused using PEG 4000. Three stable hybridoma clones (designated as 3G10, 4A3, and 4E8) were produced via indirect ELISA and three rounds of subcloning. The MAbs were classified as IgG1, and can recognize the 58 kDa OMP band by Western blot assays. No MAb cross-reactivity with chicken Proteus mirabilis, Escherichia coli, and Salmonella was observed. A double antibody sandwich ELISA (DAS-ELISA) was developed using the rabbit polyclonal antibodies as the capture antibody and MAb 4A3 as the detection antibody. Under the DAS-ELISA, the minimum detectable concentration of B. avium was 1 × 10(4) CFU/mL, and no cross-reactivity occurred with chicken Proteus mirabilis, Escherichia coli, and Salmonella. Results showed that the DAS-ELISA has good sensitivity and specificity. Clinical application showed the DAS-ELISA was more sensitive than the plate agglutination test. This study may be used to develop a quick and specific diagnostic kit, analyze epitopes, and establish systems for typing B. avium.

Bordetella avium antibiotic resistance, novel enrichment culture, and antigenic characterization.

Bordetella avium continues to be an economic issue in the turkey industry as the causative agent of bordetellosis, which often leads to serious secondary infections. This study presents a broad characterization of the antibiotic resistance patterns in this diverse collection of B. avium strains collected over the past thirty years. In addition, the plasmid basis for the antibiotic resistance was characterized. The antibiotic resistance pattern allowed the development of a novel enrichment culture method that was subsequently employed to gather new isolates from diseased turkeys and a healthy sawhet owl. While a healthy turkey flock was shown to seroconvert by four weeks-of-age, attempts to culture B. avium from healthy turkey poults were unsuccessful. Western blot of B. avium strains using pooled serum from diseased and healthy commercial turkey flocks revealed both antigenic similarities and differences between strains. In sum, the work documents the continued exposure of commercial turkey flocks to B. avium and the need for development of an effective, inexpensive vaccine to control spread of the disease.

[Study on extraction and antigenicity of the outer-membrane-protein of Bordetella avium].

To study the immunogenicity of Bordetella avium OMP, ultrasonic dispersion, TritonX-100 technique were used to extract Bordetella avium OMP. Its content was determined by Bradford method and it was detected by SDS-PAGE. Then OMP immunizing antigen was prepared and 1-day chicken was vaccinated by hypodermic inoculation, with the immunizing does of 0.3mL (OMP90microg), 0.5mL (OMP150microg), 0.8mL (OMP240microg), respectively. Results showed that content of Bordetella avium OMP is 300 g/mL, the best immunizing does is 0.5mL each one and Chickens can be protected against Bordetella avium at fetal dose if antibody titer is over 1:2(8). We can see from the antibody level detected by indirect ELISA that antibody can last long enough to help chickens pass susceptible period, so OMP has good immunogenicity. Results of this study lay good foundation for the development of monoclonal antibody to OMP, rapid diagnosis kit and subunit vaccine.

Studies on the efficacy of different adjuvants in live stock specific bacterial vaccines for turkeys against Bordetella infection and onset of antibody titers in respect to the age of the turkey poults.

For evaluating the influence of the age of the vaccinated birds on the development of antibodies, five groups of turkey poults were inoculated subcutaneously at day 1, 7, 10, 14 and 21 of life with vaccine containing inactivated Bordetella avium and Freund's incomplete adjuvant. No matter which vaccine schedule was used, serum antibodies with the ELISA were first detected at the 28th day of life and increased continuously until the 49th day, when it exhibited either a peak or a plateau. Aluminium hydroxide, Freund's complete and incomplete adjuvant and a mineral oil-arlacel-tween-mixture being permitted adjuvants (appendix II EWG 2377/90) and the adjuvant Gerbu 100 were evaluated for their suitability. Turkeys were vaccinated at the age of three weeks and examined clinically as well as serologically up to the 11th week. Humoral antibodies were detected quantitatively using an ELISA for IgG and a microagglutination test for IgM and qualitatively using immunodiffusion. The damage at the application site was rated by measurement of the swelling of the tissue. In the 10th week, the animals were infected with the agent for challenge. The serological examination for IgG antibodies in the ELISA both treatments with Freund's adjuvants resulted in high titers, which differed significantly from the unvaccinated control after 21 days. IgM could be detected from day 7 onwards in all vaccinated groups and showed the highest titers when aluminium hydroxide was used as adjuvant. In the immunodiffusion assay, precipitating antibodies could be found from the first week after vaccination onwards. There was no correlation between the occurrence of precipitating antibodies and ELISA titers. The measurements of the swelling of the tissue in the beginning showed the largest swellings in the animals injected with Freund's incomplete adjuvant and differed significantly from the unvaccinated control. In the 10th week, the animals were infected with Bordetella avium for challenge. In comparison to the unvaccinated animals, all vaccinated turkeys, no matter which adjuvant was used, showed a distinct and significant reduction of the reisolation rate.

Survey of peafowl (Pavo cristatus) for potential pathogens at three Michigan zoos.

Blood samples collected from 31 free-roaming peafowl from three zoos in Michigan were tested serologically. Antibody titers were present against avian adenovirus and Bordetella avium in 19.3% and 61.3% of the samples, respectively. Serum plate agglutination tests were positive for Mycoplasma meleagridis and Mycoplasma synoviae in 3.2% and 38.7% of the samples, respectively. All birds were seronegative for avian influenza, Newcastle disease virus, West Nile virus, Mycoplasma gallisepticum, Salmonella pullorum, Salmonella typhimurium, and Giardia sp. No parasites were seen in blood smears. Cloacal swabs were cultured for anaerobic, aerobic, and microaerophilic bacteria. Clostridium perfringens type A and Escherichia coli were cultured most frequently from 64.5% and 29% of the samples, respectively, whereas Salmonella sp. and Campylobacter sp. were not isolated. Fecal samples contained moderate numbers of ascarid and Capillaria sp. ova and coccidian oocysts. Female biting lice (Goniodes gigas) were identified on three birds.