Synergy between avian pneumovirus and Ornithobacterium rhinotracheale in turkeys

The purpose of this study was to assess the possible synergism between Ornithobacterium rhinotracheale (ORT) and avian pneumovirus (APV), inoculated into turkeys via the natural route, for the reproduction of respiratory disease. Three-week-old specific pathogen free turkeys were inoculated oculonasally with either APV subtype A, ORT or both agents using two different time intervals (3 and 5 days) between APV and ORT. The birds were observed clinically on a daily basis and swabbed intratracheally at short, regular intervals. They were killed at 1, 3, 5, 8 and 15 days post single or dual inoculation and examined for gross lesions at necropsy. Samples of the turbinates, trachea, lungs, air sacs, heart, pericardium and liver were taken for bacteriological and/or histological examination. Combined APV/ORT infections resulted in overt clinical signs and a longer persistence of ORT in the respiratory tract and aggravated the macroscopic and histological lesions in comparison with the groups given single infections. In all ORT-challenged turkeys, ORT was isolated from the turbinates, trachea and lungs, but in turkeys infected with both agents ORT was frequently found in the air sacs and on a single occasion in the heart and pericardium. The time interval between APV and ORT inoculation did not have a significant effect on the outcome of the dual infection. A conspicuous important feature was the attachment of ORT to the cilia of the epithelium of the turbinates and trachea of both ORT-infected and APV/ORT-infected birds. In conclusion, the results show that ORT is able to adhere to and colonize the respiratory tract but, under the circumstances used in this study, is not capable of inducing respiratory disease without viral priming.     

Preliminary antigenic characterization of an avian pneumovirus isolated from commercial turkeys in Colorado,USA

An avian pneumovirus (APV) isolated from turkeys showing respiratory disease in Colorado, USA, shared some characteristics with earlier subgroup A and B APV strains. This virus, designated the Colorado isolate (Colorado), when used after either seven passages in chick embryo fibroblasts (CEF), or seven passages in CEF followed by six turkey passages, induced clinical signs in turkeys that were similar to those caused by earlier APV strains. Although it induced an antibody response in specific pathogen free chickens, clinical signs were not seen. Unlike subgroups A or B, Colorado did not cause ciliostasis in tracheal organ cultures, but produced a cytopathic effect in chick embryo fibroblasts typical of that seen with other APV strains. Monospecific antisera to A or B strains did not neutralize Colorado and vice versa; nor did monoclonal antibodies, which neutralize subgroup A or B strains, neutralize Colorado. However, it was partially neutralized by a subgroup A hyperimmune serum. A homologous enzyme-linked immunosorbent assay (ELISA) antigen was essential for the detection of Colorado antibodies, since ELISAs in which subgroup A or B strains were used detected antibody to Colorado very poorly. Subgroup A and B vaccines protected turkeys against challenge with Colorado. However, while Colorado protected turkeys, and to some extent chickens, against subgroup A strains, protection against a subgroup B challenge was less good in both species. These results indicate that Colorado should be classified as an APV, but the antigenic differences suggest that it does not belong to subgroups A or B, and represents a separate subgroup (subgroup C) or possibly a separate serotype.     

Dual infection of turkey poults with avian pneumovirus and Mycoplasma synoviae

Poults were infected with avian pneumovirus (APV) at 1-day-old, followed by Mycoplasma synoviae (Ms) 3 days later. Dual infection did not cause an increase in severity of clinical disease, or gross and microscopic lesions due to APV. The patterns of virus and Ms isolations from tracheal swabs or tissues from single and dual infected groups were similar. Ms infection did not induce Ms antibodies, nor did it affect seroconversion to APV in the dual infection.     

Swollen head syndrome associated with E. coli and infectious bronchitis virus in the Central Valley of California

A commercial broiler flock in the Central Valley of California experienced a sudden increase in mortality due to heavy culling. Clinical signs included a snick, swollen heads and severe depression. Necropsy and histology revealed tracheitis, rhinitis, facial cellulitis, blepharitis, episcleritis, otitis media and caseous exudate within the air spaces of cranial bones. Escherichia coli serotype O78 was isolated from all lesions. Infectious bronchitis virus (Massachusetts serotype) was isolated from trachea/nasal cavity tissue pool. Adenovirus group 1 was isolated from trachea/nasal cavity and caecal tonsil tissue pools. Serum samples were positive for infectious bronchitis using ELISA and haemagglutination inhibition tests, but negative for turkey rhinotracheitis by an ELISA using the British antigen. This case suggests that swollen head syndrome can be associated with viruses other than pneumovirus.     

Growth of vaccine strains of avian pneumovirus in different cell lines.

The isolation of avian pneumovirus (APV) (avian metapneumovirus) is usually performed in embryonated chicken eggs or chicken embryo fibroblast cell cultures followed by adaptation in continuous cell lines such as Vero cells. This study was conducted to find a suitable cell line that could be used to propagate vaccine strains of APV to high titre. For this purpose, we compared the growth of two Vero cell-adapted vaccine strains of APV (P63 and ca-APV) in seven different cell types with their growth in Vero cells. The cell types used were BGM-70, MA-104, QT-35, BHK-21, McCoy and DF-1 cells and primary turkey embryo fibroblast cells. When compared with growth in Vero cells, both viruses yielded higher titres in BGM-70 cells, while P63 also produced higher titres in MA-104 cells. In another experiment, the two viruses were grown and titrated in Vero cells under various cell culture conditions, such as age of cells, seeding concentration, and time of harvest. None of these cell culture variables were found to affect virus titres.     

Antigenic differentiation of avian pneumovirus isolates using polyclonal antisera and mouse monoclonal antibodies

Avian pneumovirus (AVP) isolates F83, CC220 and 1260 from Great Britain and 1556, 657/4, 2119 and 872/S from France, Hungary, Italy and Spain, respectively, were compared in ELISA and virus neutralization (VN) tests for reactions with chicken polyclonal sera against each of the viruses and monoclonal antibodies (MAbs) against two British isolates. ELISA test results using the polyclonal antisera indicated that all seven viruses were antigenically related, but some variation between strains could be detected, especially when antigens were prepared from infected cells using Nonidet P40 (NP40) rather than freezing and thawing. In VN tests results also showed that all viruses tested were related but there was evidence that the three British isolates showed closer relationships with each other than with the viruses from Italy, Hungary and Spain. In ELJSA tests, isolates F83 and 1556 bound all 11 MAbs and 1260 reacted with 10/11 MAbs. Isolate CC220 showed reaction with all the MAbs but for 8/11 MAbs the optical density differences were low. Isolates 2119 and 872/S both reacted only with MAb 4 and none of the MAbs reacted with 657/4.     

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR

Summary.  This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 × 10⁻⁵ TCID⁵⁰ (0.0323 μg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota. Received June 15, 1999 Accepted January 3, 2000     

Comparative pathogenesis of a subtype A with a subtype B avian pneumovirus in turkeys

This paper describes a study in which the pathogenesis of avian pneumovirus strains, isolated in Belgium, and belonging to the two subtypes A and B, were compared in 2-week-old turkeys. After oculonasal inoculation, animals were either observed for clinical signs or killed for pathological and virological examination. Virus titration and immunofluorescence were performed on the conjunctivae, turbinates, sinuses, upper and lower part of the trachea, lungs and air sacs. No differences were seen between the two subtypes concerning respiratory signs, or macroscopic and microscopic lesions in the respiratory tract. Slight variations were found in site and extent of virus replication. First, only subtype A was able to invade the lower parts of the respiratory tract (bronchi), whereas viral antigens were not detected in the lungs with subtype B. Secondly, the subtype A strain infected two times more epithelial cells at all levels of the upper respiratory tract compared to subtype B. Thirdly, the amount of virus produced at different sites along the respiratory tract was lower in subtype B-inoculated turkeys than in subtype A-inoculated ones.     

In vitro and in vivo studies on the pathogenicity of avian pneumovirus for the chicken oviduct

The pathogenicity of avian pneumovirus (APV) for the chicken oviduct was studied in vitro using oviduct organ cultures (OOC), in vivo in young chicks with artificially enlarged oviducts and in adult female chickens. The OOCs were prepared from artificially enlarged oviducts of young chicks induced by oestrogen treatment and from normal oviducts of adult hens. The pathogenicity of APV for OOCs in vitro and oviducts in vivo were assessed by virus titrations and immunofluorescence. While in vitro experiments revealed an intrinsic susceptibility of oviduct epithelium to APV infection, in vivo studies failed to show APV replication in the hen oviduct, even after intravenous inoculation.     

Nucleotide sequence of the matrix protein gene of a subgroup B avian pneumovirus

The nucleotide sequence of the gene encoding the matrix protein of a subgroup B avian pneumovirus has been determined. The gene shows 73.5% homology with that of a subgroup A virus, with most differences occurring in the third codon position. Comparison with pneumovirus matrix proteins shows that the APV matrix protein retains the hydrophobic domain common to the others. The analysis indicates that the matrix protein gene can be used to differentiate the two APV subgroups.The sequence reported here has been submitted to the EMBL databank under accession number U37586.     

Virulence factors of avian Escherichia coli associated with swollen head syndrome.

Virulence characteristics of 50 strains of Escherichia coli isolated from chickens with swollen head syndrome were examined. The results were the following: in the absence of D-mannose, 74% of strains agglutinated guinea pig erythrocytes, but in the presence of D-mannose 32% agglutinated guinea-pig erythrocytes and 62% agglutinated human erythrocytes. When slide agglutination assays were carried out with antisera to adhesin of bovine and swine origin (K88, K99, F41, F42 987P and 2134P), only 14% of strains agglutinated with antiserum to F41. Colicin V was produced by 78% of the E. coli strains and 80% produced aerobactin. In the serum resistance test, 36 (72%) of strains showed resistance to normal chicken serum. Only seven (14%) strains expressed K1 capsular antigen, while motility was found in 62% of the strains.     

Interaction between live avian pneumovirus and Newcastle disease virus vaccines in specific pathogen free chickens.

One-day-old specific pathogen free White Leghorn chicks were vaccinated with live avian pneumovirus (APV) vaccine, live Newcastle disease virus (NDV) vaccine or both. At intervals up to 28 days after vaccination, distribution of the virus in the tissues was studied, together with humoral and mucosal antibody responses in lachrymal fluid and tracheal washes. APV vaccine was detected for almost twice as long in the dual vaccinates as in the single vaccinates. Higher numbers of isolations of ND virus vaccine were obtained from the dual rather than the single vaccinates at 7 days post-vaccination but the reverse occurred at 14 days. APV serum antibodies were significantly lower in the dual rather than the single vaccinates. However, there were similar levels of local APV-specific IgA in the lachrymal fluids of both single and dual APV vaccinates. NDV serum antibody titres in the dual vaccinates were significantly higher than in the singly NDV-vaccinated chickens. It appears that simultaneous vaccination of chicks with live APV and NDV vaccines causes temporary suppression of APV vaccine proliferation and reduces humoral antibody responses to it, although the antibody response to NDV is enhanced.     

Cloning,expression and immunogenicity of the avian pneumovirus (Colorado isolate) F protein

The F protein of the Colorado isolate of avian pneumovirus (APV), expressed from a DNA plasmid, was recognized by antiserum to both A and B subgroup APVs. After two intramuscular injections of turkeys with this plasmid, a homologous antibody response was detected by enzyme-linked immunosorbent assay. This antibody also recognized subgroup A APV. However, there was no neutralization of the Colorado isolate or of subgroup A or B viruses. Although no significant clinical protection was detected following homologous challenge of poults, an anamnestic serological response was seen, suggesting that a systemic antibody response but no local mucosal immunity was induced.     

Expression of recombinant small hydrophobic protein for serospecific detection of avian pneumovirus subgroup C

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.     

Detection and serological identification of adeno-associated virus in avian adenovirus stocks.

Eleven avian adenovirus strains were tested for the presence of avian adeno-associated viruses (AAAV). Six strains contained AAAV. Electron microscopy using rabbit anti-AAAV serum was useful in detecting the satellite virus. The AAAV previously isolated from guail bronchitis virus was related to each of the six new isolates by immunoagglutination, complement fixation, immunodiffusion, and neutralization tests.     

Microimmunofluorescence for the serological diagnosis of avian mycoplasma infections

A microimmunofluorescence test was used to detect antibodies to mycoplasmas in avian sera. When the specificity of the system was established, the test was applied to the detection of M. synoviae antibodies in experimentally and naturally infected chickens and turkeys. The test is considerably more sensitive than the rapid serum agglutination test, detects reactors earlier in the experimentally infected birds and produces titres that are markedly greater. In naturally infected birds the titres were also very much higher than rapid serum plate test titres.