RESUMEN
Ornithobacterium rhinotracheale is a pathogen involved in respiratory infection and systemic disease in poultry. Previously, eight potential vaccine candidates were identified that induced cross-protective immunity when administered to chickens as a multi-component vaccine. In this study, we analyzed the immunogenicity of these eight recombinant proteins by subunit vaccination, and characterized the different proteins and corresponding genes more thoroughly by sequencing, in vitro expression analysis, and cellular localization experiments. We found, that all genes encoding the eight antigens were highly conserved among different O. rhinotracheale serotypes, but the different antigens were not expressed by all serotypes. Cellular fractionation experiments indicated that the majority of the antigens are predominantly located in the outer membrane fraction. Vaccination of chickens with single-antigen vaccines demonstrated that the Or77 antigen was protective against serotypes that expressed Or77 in vitro, suggesting that the protein has strong potential as a vaccine antigen. Furthermore, immunization with four-component subunit vaccines indicated the existence of immunogenic synergism between the candidate vaccine antigens.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Flavobacteriaceae/veterinaria , Ornithobacterium/inmunología , Enfermedades de las Aves de Corral/prevención & control , Sacos Aéreos/patología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/análisis , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Membrana Celular/química , Pollos , Secuencia Conservada , Reacciones Cruzadas , ADN Bacteriano/química , ADN Bacteriano/genética , Infecciones por Flavobacteriaceae/patología , Infecciones por Flavobacteriaceae/prevención & control , Expresión Génica , Variación Genética , Datos de Secuencia Molecular , Ornithobacterium/química , Ornithobacterium/genética , Enfermedades de las Aves de Corral/patología , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas , Infecciones por Flavobacteriaceae/veterinaria , Ornithobacterium/inmunología , Enfermedades de las Aves de Corral/prevención & control , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Vacunas Bacterianas/inmunología , Secuencia de Bases , Clonación Molecular , Reacciones Cruzadas , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/prevención & control , Biblioteca Genómica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Enfermedades de las Aves de Corral/inmunología , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , VacunaciónRESUMEN
Unravelling of the protective immunity acquired during a natural infection may contribute to vaccine development. To assess the role of antibody-mediated immunity in protection against Ornithobacterium rhinotracheale infection in chickens, a novel experimental method was applied that combined immune depletion and passive transfer of immunity within the same host. Administration of cyclophosphamide (CY) to broiler chickens successfully suppressed B lymphocyte development, and therefore humoral immunity, as confirmed by histological and serological analysis. Challenge of CY-treated birds with O. rhinotracheale revealed a significantly higher pathology score in comparison to immune-competent birds that received the same bacterial challenge. Measurement of serum immunoglobulin levels of immune-competent birds revealed a positive correlation between IgA and/or IgG production and protection against infection. Passive transfer of O. rhinotracheale-specific antiserum to the immune-suppressed birds prior to pathogen challenge significantly decreased morbidity. This protective effect was not observed after administration of control sera containing similar concentrations of immunoglobulins. Together, these results provide firm evidence that chicken humoral immunity to O. rhinotracheale is a key component in protection against infection. Our data confirm that the applied immune depletion and reconstitution approach is an attractive tool to analyse the nature of the protective immune response.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Infecciones por Flavobacteriaceae/prevención & control , Inmunización Pasiva , Ornithobacterium , Animales , Anticuerpos Antibacterianos/inmunología , Pollos , Infecciones por Flavobacteriaceae/veterinaria , Inmunidad Celular , InmunizaciónAsunto(s)
Pollos , Infecciones por Flavobacteriaceae/veterinaria , Ornithobacterium/aislamiento & purificación , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/epidemiología , Infecciones del Sistema Respiratorio/veterinaria , Animales , Diagnóstico Diferencial , Infecciones por Flavobacteriaceae/diagnóstico , Infecciones por Flavobacteriaceae/epidemiología , Países Bajos/epidemiología , Ornithobacterium/crecimiento & desarrollo , Enfermedades de las Aves de Corral/microbiología , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiología , Sensibilidad y EspecificidadRESUMEN
Two hundred turkey sera from eight Belgian and two French farms were tested for the presence of antibodies against avian pneumovirus (APV), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum, Mycoplasma meleagridis and Chlamydophila psittaci. At slaughter, C. psittaci, APV and ORT antibodies were detected in 94, 34 and 6.5% of the turkeys, respectively. No antibodies against M. gallisepticum or M. meleagridis were present. Additionally, turkeys on three Belgian farms were examined from production onset until slaughter using both serology and antigen or gene detection. All farms experienced two C. psittaci infection waves, at 3-6 and 8-12 weeks of age. Each first infection wave was closely followed by an ORT infection starting at the age of 6-8 weeks, which was still detectable when the second C. psittaci infection waves started. Animals on farm A were not vaccinated against APV leading to an APV subtype B outbreak accompanying the first C. psittaci infection wave. Despite subtype A APV vaccination on farms B and C, the second C. psittaci infection waves were accompanied (farm B) or followed (farm C) by a subtype B APV infection. On all farms respiratory signs always appeared together with a proven C. psittaci, APV and/or ORT infection. This study suggests an association between C. psittaci, APV and ORT, and indicates the multi-factorial aetiology of respiratory infections in commercial turkeys. All three pathogens should be considered when developing prevention strategies for respiratory disease.
Asunto(s)
Infecciones por Flavobacteriaceae/veterinaria , Metapneumovirus/inmunología , Ornithobacterium/inmunología , Infecciones por Paramyxoviridae/veterinaria , Enfermedades de las Aves de Corral/epidemiología , Psitacosis/veterinaria , Pavos , Factores de Edad , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Bélgica/epidemiología , Chlamydophila psittaci/inmunología , Chlamydophila psittaci/patogenicidad , Brotes de Enfermedades/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Infecciones por Flavobacteriaceae/complicaciones , Infecciones por Flavobacteriaceae/epidemiología , Francia/epidemiología , Mycoplasma/inmunología , Infecciones por Mycoplasma/complicaciones , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/veterinaria , Infecciones por Paramyxoviridae/complicaciones , Infecciones por Paramyxoviridae/epidemiología , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/virología , Psitacosis/complicaciones , Psitacosis/epidemiología , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/veterinaria , Infecciones del Sistema Respiratorio/virología , Estudios SeroepidemiológicosRESUMEN
The importance and prevention of the horizontal as well as the vertical transmission of Ornithobacterium rhinotracheale were investigated. In our first experiment we observed that specific-pathogen-free broiler chickens that were placed in hatching incubators at a commercial turkey hatchery during hatch showed respiratory tract lesions at postmortem examination that were positive for O. rhinotracheale by bacteriology and immunohistology. It appeared that vertical transmission occurred and that horizontal transmission of O. rhinotracheale is possible. In a second experiment, the turkeys derived from vaccinated parents showed significantly fewer respiratory tract lesions at postmortem examination at 16 days of age than the birds derived from nonvaccinated parents. In a third experiment, all vaccinated young birds, regardless of the vaccination state of their parents, showed significantly fewer respiratory tract lesions at 6 wk of age. We concluded that vaccination of the breeders reduces vertical transmission and that vaccination of the progeny is needed to resist challenge at 6 wk of age.
Asunto(s)
Pollos/microbiología , Transmisión de Enfermedad Infecciosa/veterinaria , Infecciones por Flavobacteriaceae/veterinaria , Ornithobacterium/aislamiento & purificación , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/transmisión , Infecciones del Sistema Respiratorio/veterinaria , Pavos/microbiología , Animales , Vacunas Bacterianas/uso terapéutico , Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por Flavobacteriaceae/prevención & control , Infecciones por Flavobacteriaceae/transmisión , Técnicas para Inmunoenzimas/veterinaria , Inmunohistoquímica/veterinaria , Ornithobacterium/inmunología , Enfermedades de las Aves de Corral/microbiología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/transmisión , Organismos Libres de Patógenos Específicos , Vacunación/veterinariaRESUMEN
The efficacy of a new vaccine against atrophic rhinitis in pigs was tested in the Netherlands and Denmark. The vaccine contained protein dO (a truncated Pasteurella multocida toxin which is immunogenic and non-toxic), inactivated Bordetella bronchiseptica whole cells, and an adjuvant. The sows were either vaccinated intramuscularly with 2 ml of the vaccine at six to eight and two to four weeks before expected farrowing or left unvaccinated as controls. All the piglets were challenged intranasally with B bronchiseptica when three to seven days old and with P multocida three to four days later. Pigs born to the vaccinated sows performed significantly better than pigs born to the control sows when judged on growth, average daily weight gain and snout scores. The challenge organisms were reisolated more frequently from the control pigs than from the pigs in the vaccinated group. The vaccinated sows and their progeny developed high titres of antibodies against B bronchiseptica and P multocida toxin.
Asunto(s)
Infecciones por Bordetella/prevención & control , Bordetella bronchiseptica/patogenicidad , Infecciones por Pasteurella/prevención & control , Pasteurella multocida/patogenicidad , Rinitis/prevención & control , Rinitis/veterinaria , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/prevención & control , Vacunación/veterinaria , Animales , Animales Recién Nacidos , Formación de Anticuerpos , Infecciones por Bordetella/inmunología , Bordetella bronchiseptica/inmunología , Femenino , Infecciones por Pasteurella/inmunología , Pasteurella multocida/inmunología , Porcinos , Aumento de PesoRESUMEN
Immunohistochemical techniques were used to prove that Ornithobacterium rhinotracheale was the causative agent of lesions in the air sacs and lungs in chickens, but only after infection with Newcastle Disease virus (NDV). At first, the bacteria attached to the epithelium of the air sacs. Subsequently, they infiltrated the air sacs, and caused thickening of the air sacs, the formation of oedematous and granulomatous tissue, and accumulation of macrophages. The infection peaked at 5 to 9 days, after which recovery was seen. In the lungs, some areas with bronchially-associated lymphoid tissue were affected. The other organs investigated were shown not to be affected. In the absence of NDV infection, aerosol exposure of chickens to O. rhinotracheale only resulted in minimal and temporary microscopic air sac lesions. No O. rhinotracheale cells or fragments could be detected at any time point later than 2 days post-exposure. In spite of the absence of visible lesions, chickens exposed to O. rhinotracheale without prior NDV infection reacted serologically. The duration and the titre of this immune response was indistinguishable from that obtained in chickens exposed after NDV infection. Thus, infection with O. rhinotracheale appears to be restricted to the respiratory tract, with lesions only evident in birds previously infected with NDV, even though a strong serological response can be established in the absence of prior viral infection.
RESUMEN
Ornithobacterium rhinotracheale is a relatively recently discovered bacterium of the rRNA superfamily V. It is of worldwide distribution in commercial poultry, in which it is associated with respiratory diseases, and it is also found in wild birds. Airsacculitis and pneumonia are the most common features of infection with O. rhinotracheale. These signs can be induced by aerosol in intra-tracheal or intra-thoracic administration of the organism, and can be aggravated by other factors, such as respiratory viruses, bacteria or climatic conditions. Osteitis, meningitis and joint-infections, which can be induced by intravenous application, have been associated with O. rhinotracheale, but it remains uncertain whether the organism should be regarded as a primary pathogen. The infection can be transmitted horizontally by aerosol, as well as vertically through eggs, which probably accounts for its rapid and worldwide spread. Although O. rhinotracheale is difficult to identify, some commercial identification systems have been found to be suitable, although the media used in such systems will not always support its growth. A PCR assay was also found to be suitable for identification purposes. Twelve serotypes can be distinguished within the species O. rhinotracheale, of which serotype A is the most prevalent. Genetic investigation has revealed that more species or subspecies probably exist within the genus Ornithobacterium. Therapeutic treatment of the disease can be difficult because acquired resistance against the regular antibiotics is very common within the genus. Vaccination with autogenous inactivated vaccines has been successful in reducing clinical signs, but success depends on the adjuvant used. Only potent oil adjuvants are effective in young birds with maternal antibodies, but the use of these adjuvants is known to induce some local reactions. Live vaccination is feasible, but up to now, no avirulent strains of O. rhinotracheale have been found. Vaccination of broiler breeders induced protection against experimental infection of the progeny to at least 3 weeks of age.
RESUMEN
Vaccination of young broilers with inactivated vaccines against experimental Ornithobacterium rhinotracheale challenge was found to be effective, but the results of vaccination were influenced, in a negative way, by the presence of maternal antibodies. The use of a strong adjuvant, such as mineral oil, in a bacterin was necessary to obtain good protection when maternal antibodies were present. Vaccination of broiler breeders resulted in high serologic responses and protection of their progeny against experimental O. rhinotracheale challenge up to an age of 4 wk. Vaccination of broilers with a live vaccine was found to be effective when the maternal antibody levels were low. A combination of vaccinating the breeders with a bacterin and their progeny with a live vaccine at approximately 3 wk of age seems to be the best way to protect broilers against O. rhinotracheale infection.
Asunto(s)
Pollos/inmunología , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Infecciones del Sistema Respiratorio/veterinaria , Vacunación/veterinaria , Animales , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/uso terapéutico , Pollos/microbiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/prevención & control , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/microbiología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/prevención & controlRESUMEN
In the present study 443 strains of Ornithobacterium rhinotracheale, a causative agent of respiratory disease in fowl, were investigated biochemically and serologically. In both ways O. rhinotracheale could be differentiated from other gram-negative rods and, more particularly, from the Pasteurella-like bacteria potentially pathogenic for fowl. For the biochemical characterization of O. rhinotracheale the API 2ONE identification strip proved to be useful, although O. rhinotracheale is not included in the API system. Serologically, by using monovalent antisera in agar gel precipitation (AGP) tests and enzyme-linked immunosorbent assays (ELISAs), seven serotypes (serotypes A to G) of O. rhinotracheale could be discriminated. The AGP test was chosen as the preferred method to be used for serotyping. Isolates of serotype A were found to be the most prevalent, especially in chickens. Isolates from turkeys were more heterogeneously divided over the serotypes. Some strains showed cross-reactivity between serotypes A, B, and E. Five O. rhinotracheale strains could not be serotyped with the available antisera. Relationships between the geographic origin and the serotypes were found. By the ELISA the presence of antibodies against O. rhinotracheale could be detected in 1-day-old birds as well as in birds with clinical signs, and therefore, it might be useful for diagnostic purposes.
Asunto(s)
Bacterias Gramnegativas/clasificación , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de las Aves de Corral/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Pollos/inmunología , Pollos/microbiología , Ensayo de Inmunoadsorción Enzimática , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Gramnegativas/fisiología , Infecciones por Bacterias Gramnegativas/microbiología , Pruebas de Precipitina , Serotipificación , Pavos/inmunología , Pavos/microbiologíaRESUMEN
Ornithobacterium rhinotracheale was found to cause growth retardation in both turkeys and chickens after experimental intra-air sac administration and to cause growth retardation together with airsacculitis and pneumonia after aerosol administration. Both turkey and chicken isolates of O. rhinotracheale were able to induce the same kind of respiratory inflammations and weight-gain losses in chickens as well as turkeys. Turkey rhinotracheitis virus was found to have a triggering effect on the O. rhinotracheale infection in turkeys, and Newcastle disease virus and to a lesser extent infectious bronchitis virus showed triggering effects on the O. rhinotracheale infection in chickens. Ornithobacterium rhinotracheale could be reisolated from affected organs of experimentally infected birds.
Asunto(s)
Pollos/microbiología , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de las Aves de Corral/patología , Pavos/microbiología , Aerosoles , Animales , Pollos/virología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/veterinaria , Bacterias Gramnegativas/fisiología , Infecciones por Bacterias Gramnegativas/patología , Infecciones por Bacterias Gramnegativas/fisiopatología , Trastornos del Crecimiento/etiología , Trastornos del Crecimiento/patología , Trastornos del Crecimiento/veterinaria , Virus de la Bronquitis Infecciosa/aislamiento & purificación , Virus de la Bronquitis Infecciosa/fisiología , Pulmón/microbiología , Pulmón/patología , Pulmón/virología , Enfermedad de Newcastle/patología , Enfermedad de Newcastle/fisiopatología , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Virus de la Enfermedad de Newcastle/fisiología , Pneumovirus/aislamiento & purificación , Pneumovirus/fisiología , Infecciones por Pneumovirus/patología , Infecciones por Pneumovirus/fisiopatología , Infecciones por Pneumovirus/veterinaria , Enfermedades de las Aves de Corral/etiología , Enfermedades de las Aves de Corral/fisiopatología , Tráquea/microbiología , Tráquea/patología , Tráquea/virología , Pavos/virología , Aumento de Peso/fisiologíaRESUMEN
Since August 1993 moderate to serious respiratory problems with necrotic pneumonia, growth depression and fast increasing mortality are seen in commercial turkeys (2-8 weeks of age) and broilers (4-6 weeks of age). An unidentified pleiomorphic Gram-negative rod was isolated from affected tissues. This Pasteurella-like organism, with yet unknown taxonomy, is recently named Ornithobacterium rhinotracheale gen. nov. sp. nov. or 'Taxon 28'. Experimentally severe growth depression and arthritis could be evoked in commercial turkeys and chickens. Respiratory signs caused by O. rhinotracheale could not (yet) be reproduced experimentally. This is the first report of the isolation of this organism in poultry in the Netherlands findings.