The efficacy of an avian metapneumovirus vaccine applied simultaneously with infectious bronchitis and Newcastle disease virus vaccines to specific-pathogen-free chickens.

Avian metapneumovirus (aMPV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) are important respiratory pathogens of chickens. To achieve early posthatch protection against all three diseases it would be helpful to deliver live aMPV, IBV, and NDV vaccines simultaneously at 1 day of age. However, previous work has indicated that the efficacy of aMPV vaccines may be affected when codelivered with IBV or NDV vaccines. The efficacy of an aMPV vaccine when codelivered to chickens in a trivalent combination with an NDV and an IBV vaccine was examined. The serological antibody response to the aMPV vaccine given with the IBV and NDV vaccine was significantly lower than when the aMPV vaccine was given alone. However, the aMPV vaccine did not affect the serological response to the IBV and NDV vaccines. Irrespective, the efficacy of the aMPV vaccine was not affected based on clinical signs postchallenge. This is the first report showing aMPV, IBV, and NDV vaccines can be codelivered without affecting the efficacy of the aMPV vaccine.

Onset of immunity following in ovo delivery of avian metapneumovirus vaccines.

Avian metapneumovirus (aMPV) is an important cause of disease in chickens and turkeys. As infection can occur early in life and spread of the virus throughout a flock is rapid, an early onset of immunity post-vaccination would be advantageous. We have studied the serological immune response and the onset of protective immunity of an aMPV vaccine delivered to chickens via the in ovo route compared to oculonasal delivery at day old. A 1000-fold lower dose delivered in ovo to chicken specific pathogen free (SPF) embryos, than vaccination at day old, provided a significantly higher antibody response. In the presence of maternally derived antibody (MDA), there was no significant difference in antibody response between the vaccination routes. However, the onset of immunity (OOI) for the vaccine delivered to MDA positive chicken embryos was 5 days post-hatch in comparison to 8 days post-hatch for the same dose of vaccine given at day old indicating that chicks would be protected against disease earlier in the field if vaccinated by the in ovo route. In further experiments the OOI for a turkey vaccine delivered to MDA positive turkey embryos was shown to be 8 days post-hatch.

Hatchability, serology and virus excretion following in ovo vaccination of chickens with an avian metapneumovirus vaccine.

The present investigation describes for the first time the effect of an avian metapneumovirus vaccine administered in ovo to 18-day-old chicken embryos. The application of the vaccine had no adverse effect on the hatchability or the health of the chicks post hatch. The antibody titres achieved were higher than those determined for birds vaccinated at 1 day old. Not only were the mean titres in the in ovo vaccinated groups higher, but many more birds developed a measurable antibody response than birds vaccinated at 1 day old. Variation of the vaccine dose used in ovo had little effect on the serological responses that peaked 21 to 28 days post hatch. Re-isolation of the vaccine virus was much more successful from birds vaccinated in ovo than from birds vaccinated at 1 day old, and detection of the nucleic acid by polymerase chain reaction correlated with the results of live virus isolation.

Avian metapneumovirus excretion in vaccinated and non-vaccinated specified pathogen free laying chickens.

Vaccinated and non-vaccinated specified pathogen-free White Leghorn laying chickens were challenged at peak of lay by the intravenous or oculonasal route with a virulent avian metapneumovirus (aMPV) subtype B chicken strain. Severe clinical signs and a drop in egg production were induced in the non-vaccinated intravenously challenged birds whereas the vaccinates were not affected. Live virus excretion was demonstrated in the faeces and respiratory tract of non-vaccinated hens for up to 7 days post intravenous challenge. After oculonasal challenge, virus excretion could only be demonstrated in the respiratory tract for up to 5 days. No live virus excretion was found in either the faeces or the respiratory tract of vaccinated birds. Concurrent with live virus isolation, the presence of viral RNA was demonstrated by single reverse transcription-polymerase chain reaction (RT-PCR). Nested RT-PCR was more sensitive and viral RNA could be detected in non-vaccinated birds up to 28 days post either intravenous or oculonasal challenge, at which time the experiment was terminated. Viral RNA was detected for up to 12 days in vaccinated birds. This is the first study investigating excretion of aMPV and viral RNA in vaccinated and non-vaccinated laying hens challenged under experimental conditions. The results are of importance with regard to the persistence of aMPV and the appropriate diagnostic detection method in laying birds.

Infectious bronchitis virus vaccine interferes with the replication of avian pneumovirus vaccine in domestic fowl.

Experiments were performed in chickens to ascertain whether application of infectious bronchitis (IB) H120 vaccine had an effect on the replication of an attenuated avian pneumovirus (APV) strain, using as indicators virus detection, humoral antibody responses and clinical protection against in vivo APV challenge. A preliminary experiment demonstrated that pharyngeal swabs were as efficient for recovery of APV as were buccal cavity swabs, and that either site was superior to swabbing the nasal cavity. APV was detected to a similar extent by both a reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation; therefore, RT-PCR was used in subsequent experiments. In chickens vaccinated with APV alone, APV was detected by RT-PCR in most birds for 1 week after vaccination. When IB vaccine had been applied 1 week earlier, APV detection was delayed and much reduced. This interference by IBV resulted in a lower APV antibody response to vaccination. Following challenge with virulent APV, birds that had been vaccinated with APV alone were fully protected both clinically and virologically. Chickens that had received both vaccines were still protected clinically, but challenge virus could be detected in some pharyngeal swabs 4 days after challenge. In contrast, the APV vaccine had no effect on either the antibody response to the IB vaccine or the level of protection against IB challenge. It is concluded that IB vaccination interferes with the replication of APV, resulting in a reduction in the antibody response but with no adverse effect on the induction of protective immunity.

Protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus.

The ability of the infectious bronchitis (IB) Ma5 and 4/91 live-attenuated vaccines to protect against kidney damage caused by a nephropathogenic strain of IB virus (B1648) was investigated. Protection parameters considered were gross and microscopic renal pathology, and the use of a polymerase chain reaction to detect IB RNA in kidney tissue. By each parameter, Ma5 vaccine alone provided poor protection, but 4/91 alone or the combined program both protected well.

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.

Characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in Brazil.

Fifteen isolations of infectious bronchitis (IB) virus were made from a total of 126 Brazilian poultry flocks of all ages that were examined. These flocks (14 chicken and 1 quail) were experiencing a variety of IB-like conditions including respiratory disease, digestive and kidney problems, and drops in egg production. One of the isolates was of the Massachusetts serotype. The remainder were examined by means of cross-neutralization tests in tracheal organ cultures and were shown to belong to at least four antigenic groups, all different from ones described previously in other countries. Some, but not all, of the flocks from which they were isolated had been vaccinated against IB with vaccines of the Massachusetts serotype. In vivo protection studies showed that the MA5 vaccine (of the Massachusetts serotype) protected well against challenge with four of these isolates, representing the different serotypes reported in this study.

Avian pneumovirus infection of laying hens: experimental studies.

Administration of a virulent strain of avian pneumovirus (APV) to specific pathogen free laying hens by the oculonasal route failed to induce a drop in egg production or any adverse effects on eggshell quality. However, intravenous (i.v.) inoculation of the same strain caused a substantial drop in egg production and a high incidence of soft and thin-shelled eggs. Some respiratory signs were also observed and the hens appeared sick, with diarrhoea being observed in approximately one-half of the hens between 4 and 11 days post-inoculation (p.i.). APV antigen was detected in the oviduct epithelium up to 9 days p.i. This challenge model was then used to investigate the efficacy of live attenuated turkey rhinotracheitis (TRT) vaccine administered alone at 1 day old, or an inactivated TRT vaccine (at 16 weeks), or a combined programme using both vaccines, in protecting against this challenge. Neither the live nor the inactivated vaccine alone protected against clinical signs (respiratory infection or diarrhoea). However, the inactivated, but not the live, vaccine did protect against the effect of the i.v. challenge on laying performance. In contrast, the combined vaccination programme protected completely against both clinical signs and poor egg-laying performance. This protection lasted until at least 60 weeks of age. On the basis of the results with this experimental model, it is concluded that the use of live priming followed by administration of inactivated TRT vaccine is necessary to provide complete protection of laying chickens against APV challenge.

Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes.

The regime of administering the infectious bronchitis (IB) Ma5 vaccine (Massachusetts serotype) at 1 day old and the heterologous 4/91 vaccine at 2 weeks of age, was shown to be highly effective in protecting the respiratory tract of specified pathogen free chickens. Protection, as measured by assessing ciliary activity of the tracheal epithelium following challenge, was excellent against challenge at 5 weeks of age with IB strains of many serotypes, isolated from disease outbreaks in different parts of the world. This vaccination protocol was more effective than revaccination with a vaccine of the same serotype as the first vaccine. Furthermore, significantly better protection was seen when the Ma5 vaccine was given before, rather than at the same time as or following, the 4/91 vaccine. It is suggested that the use of these two IB vaccines will frequently broaden the protection possible against challenge with IB isolates of many different serotypes, without the need to develop a new IB vaccine to combat each new IB serotype that emerges.

The use of virus isolation, histopathology and immunoperoxidase techniques to study the dissemination of a chicken isolate of avian pneumovirus in chickens.

A subgroup B isolate of turkey rhinotracheitis virus (TRTV) or avian pneumovirus (APV), obtained from a flock of commercial breeding chickens experiencing poor egg production, mortality and swollen head syndrome, was shown to cause substantial respiratory signs in both young SPF chickens and chicks with high levels of maternally derived TRT antibodies. This isolate replicated to high titre in the respiratory tract of experimentally inoculated SPF chickens for approximately 5 days after inoculation, but was recovered only occasionally after that time. It was never recovered from non-respiratory tract tissues. A detailed, sequential histological and immunoperoxidase study was performed. This revealed that, whilst TRT virus could be demonstrated consistently in the epithelium of upper respiratory tract tissue, although only for a short time after inoculation, the damage which it caused was minimal and recovery was rapid. This study, using a pathogenic TRT isolate obtained from diseased chickens, provides clear evidence that TRT virus can cause damage to the respiratory tract of chickens and that this damage is both localized and short lived.

An experimental turkey rhinotracheitis (TRT) infection in breeding turkeys and the prevention of its clinical effects using live-attenuated and inactivated TRT vaccines.

The experimental inoculation of 38-week-old turkey hens with a pool of field isolates of turkey rhinotracheitis virus (TRTV) induced a marked respiratory infection and a substantial drop in egg production. Administration of a live-attenuated TRT vaccine at 1 week of age did not protect the layers against respiratory infection, but provided good protection against the effects of challenge on laying performance. However, a combination of live priming followed by injection of inactivated vaccine provided excellent protection against both respiratory infection and drops in egg production.

A survey of the presence of a new infectious bronchitis virus designated 4/91 (793B).

On the basis of virus isolation and the demonstration of specific neutralising antibody in sera, infectious bronchitis virus (IBV) 4/91 (commonly called 793B) has been shown to be present in broiler, breeder and layer flocks of chickens in many parts of western Europe and also in Thailand and Mexico. These flocks had all been vaccinated against infectious bronchitis and the need for improved methods to control this new virus, still prevalent at least four years after it was first isolated, is discussed.

Host specificity of Salmonella infection in chickens and mice is expressed in vivo primarily at the level of the reticuloendothelial system.

By experimental infection, host-specific Salmonella serotypes were shown to demonstrate specificities for chickens, mice, and other laboratory animals. Following oral inoculation, four strains of Salmonella gallinarum and two S. pullorum strains, isolated from diseased poultry, were more virulent for chickens than for mice. By contrast, four strains each of S. choleraesuis and S. dublin, isolated from diseased pigs and cattle, respectively, were more virulent for mice than for chickens. These results were also reflected in the degree of virulence expressed after parenteral inoculation. In addition, S. choleraesuis, but not other serotypes, killed rats, guinea pigs, and rabbits. S. typhimurium strains varied widely in their virulence, and some strains were virulent for both mice and chickens. Four other serotypes isolated from poultry or human food poisoning cases and a nonpathogenic Escherichia coli strain were much less virulent for both experimental host species. Most of the host-specific Salmonella serotypes studied were able to colonize the distal alimentary tract and invade the tissues in both mice and chickens to various degrees. There was, however, a greater difference in the ability to survive and multiply in the visceral organs, particularly the spleen and the liver, once invasion had occurred which correlated with the virulence for the host species involved.

A comparison of three serological methods to detect chicken and turkey antibodies to avian nephritis virus and the use of virus-specific monoclonal antibodies.

Avian nephritis virus (ANV) strain G-4260 was inoculated orally in to 1-day-old and 3-week-old chickens and the sequential antibody response to the virus was monitored by serum neutralization, ELISA and immunofluorescence tests. The order of sensitivity of the serological tests was in the sequence given above, with the neutralization test being by far the most sensitive. There was no obvious difference in the antibody titres produced by either age group. Virus was recovered from kidney tissue, the highest titres being obtained 3 to 5 days post-inoculation. Histological examination revealed mainly lymphocytic infiltration of the interstitium and degenerative changes in the tubular epithelium of the kidney. The G-4260 strain of ANV was given orally to 1-day-old turkey poults, but no serological response was induced. Virus was not recovered from the kidneys and no histological lesions were produced in this organ. Monoclonal antibodies were produced which neutralized the infectivity of ANV. An antigen trap ELISA was developed using a monoclonal antibody and infected chicken kidney tissue cultures. However, this assay did not detect ANV in kidney samples taken directly from infected chickens, with the exception of one sample which was shown to contain the highest concentration of infectious virus.

The secretory antibody response of inbred lines of chicken to avian infectious bronchitis virus infection.

Two-week-old chicks of a line highly resistant (line C) or highly susceptible (line 151) to infectious bronchitis virus (IBV) were inoculated with the Massachusetts-41 strain of IBV. Tracheal washings, saliva, lachrymal fluid and serum were collected at intervals after inoculation and titrated for their antibody content using neutralization tests and ELISAs. There was no marked difference in antibody concentrations in tracheal washings of the two lines, nor in neutralizing antibody or IBV-specific IgG titres in serum or respiratory tract secretions. However, more IBV-specific IgA was detected in both saliva and lachrymal fluid of the line C chicks following either intranasal or eyedrop inoculation. The different IgA response in lachrymal fluid, but not saliva, was also observed in another pair of inbred chicken lines of different susceptibility to IBV infection, namely lines 6, and 7(2).

Use of an infectious bronchitis virus and Escherichia coli model infection to assess the ability to vaccinate successfully against infectious bronchitis in the presence of maternally-derived immunity.

Using as challenge, a mixed infectious bronchitis virus (IBV) and Escherichia coli infection, specified pathogen-free (SPF) chicks and chicks with maternally-derived immunity (MDI) could be protected by a single intra-nasal vaccination with a live-attenuated IB vaccine given at 1 day of age against intranasal challenge with either a homologous or a heterologous IBV strain for at least 7 weeks. However, protection was incomplete if challenge occurred within approximately 14 days of vaccination. Vaccination at 7 or 14 days of age was also equally effective in protecting both SPF and MDI chicks for at least 4 weeks. The value of using in challenge studies a mixed IBV and E. coli infection, which takes account of the response of the intact chicken to challenge, is discussed.

Comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry.

Infectious bronchitis virus (IBV) was inoculated intranasally into line C and line 151 chickens, which were resistant and highly susceptible, respectively, to IBV infection, and the resulting respiratory lesions compared. Histologically, damage to the mucociliary system of the respiratory tract in line 151 persisted longer than in line C. Using the avidin-biotin-peroxidase complex method, IBV antigens were detected in histological sections of the tracheas of line 151 for a longer time than in line C. Ultrastructural studies confirmed the histological findings. Immuno-globulin G-, IgM- and IgA-containing cells were seen in the lamina propria and between epithelial cells in the trachea of both lines from at least 7 to 12 days after IBV inoculation but in greater numbers in line 151.

The pathogenesis of turkey rhinotracheitis virus in turkey poults inoculated with the virus alone or together with two strains of bacteria.

Turkey rhinotracheitis virus (TRTV) was recovered in large amounts from the upper respiratory tract of young turkey poults for about 5 days after experimental eyedrop inoculation with either a virulent or an attenuated strain. Thereafter, small amounts of virus were isolated, but only for up to 14 days after inoculation. Virulent TRTV could be recovered infrequently from the internal organs or the intestinal tract but only if bacteria (Bordetella avium and a Pasteurella-like organism) were administered contemporaneously. The results of transmission studies were in agreement with the virus isolation results since they showed that virus could be transmitted from inoculated poults to TRTV-free poults placed in direct contact with them only during the first 9 days after inoculation. Whether inoculated alone or together with TRTV, B. avium colonised the upper respiratory tract to the same degree but apparently did not invade. In contrast, the Pasteurella-like organism did not colonise when inoculated alone but could be recovered from the respiratory tract when administered simultaneously with either a virulent or an attenuated strain of TRTV.