T.J. Bagust

Laryngotracheitis (gallid‐1) herpesvirus infection in the chicken 4. latency establishment by wild and vaccine strains of ILT virus

Tracheal organ culture (TOC) techniques utilising multiple-well plastic trays were used to detect and assay latent infection established by infectious laryngotracheitis (ILT) herpesvirus in clinically normal chickens. Between 3 and 16 months after tracheal exposure to wild strain (CSW-1, haemorrhagic tracheitis) ILT virus and 2 to 10 months after exposure to vaccine strain ILT (SA-2), groups of chickens were examined for evidence of infection. Neither the examination of tracheal swabbings in monolayer cell cultures nor the inoculation of tracheal tissue suspensions detected virus, and this result was not influenced by preliminary immunosuppressive treatment of the birds with cyclophosphamide or dexamethasone. Latent infection was detected, however, by TOC in 6 (38%) of 16 chickens 100 days to 15 months after exposure to wild strain ILT virus and in 4 (44%) of 9 chickens 2 to 10 months after exposure to the vaccine strain. These data provide the first proof that both wild and vaccine strains of ILT virus regularly establish long term latent infections. Sites of establishment of latent infection in the trachea were highly focal in distribution. Virus reactivation was demonstrated in only 20 (8.3%) of the TOC preparations established from previously infected chickens and usually from only one or two sites in each trachea. Both strains of ILT virus exhibited characteristics of latency in vitro in that virus was not detectable in supernatant fluids until 5 to 6 days after establishment of TOC. Virus shedding then usually continued for 1 to 2 weeks 10(2) to 10(4) PFU/ml being produced each 2 to 3 days. In some preparations, virus production continued for up to 30 days.

Laryngotracheitis herpesvirus infection in the chicken: The role of humoral antibody in immunity to a graded challenge infection

The clinical responses of SPF White Leghorn chickens to graded levels of infection with virulent (wild-strain) infectious laryngotracheitis (ILT) herpesvirus, administered by the tracheal route, were investigated in chickens from 1 day to 8 weeks of age. In 1-day-old chickens 40 plaque forming units (PFU) of ILT virus caused 55% mortality within 8 days. At least 500 PFU was needed to achieve comparable mortality at 3 weeks of age and this increased to 4,500 PFU of ILT virus by 6 to 8 weeks of age. A combination of surgical bursectomy at 1 day old and cyclophosphamide treatment ablated the ability of chickens to generate humoral antibody to ILT virus, but did not impair the level of protection induced by commercial ILT vaccine. Further, the passive transfer to ILT antibody-positive serum to 2-day-old or 4-week-old chickens did not significantly alter their susceptibility to tracheal challenge with virulent virus. Serum antibody was therefore discounted as a major immune mechanism in resistance to ILT virus infection. An experimental inactivated vaccine to ILT virus was also investigated. One intramuscular injection induced low serological responses, but no significant protection to ILT. A second injection of inactivated vaccine only marginally increased the titre of humoral antibody, but seemed to reduce the degree of respiratory distress. However, the levels of protection afforded by the inactivated vaccine were not significant compared with a live commercial ILT vaccine.

Avian infectious laryngotracheitis: Virus‐host interactions in relation to prospects for eradication

This review examines the virology, immunology and molecular biology of infectious laryngotracheitis virus (ILTV) and its interactions with the chicken, in the context of assessing the feasibility of eradication. Establishment of the latent phase during infection of the host, its central role in biological survival of ILTV and the host-viral events that are associated with reactivation of infection, are considered. In counterpoint there are several features of the biology of ILTV in its natural mode of infection which can be exploited in eradicating this pathogen from intensive poultry production sites. These include the high degree of host-specificity of ILTV, dependence on contact for spread, the short-lived infectivity outside the chicken and the stability of the genome and lack of significant antigenic variation. Further, ILTV cannot replicate productively in its main target organ, the trachea, in the face of local specific cell-mediated immunity. Genetically-engineered vaccines that are capable of generating immunity, but without the ILTV latent infections induced by conventional modified-live ILT vaccine strains, are now well into development. This paper postulates that, used in conjunction with specific site quarantine and hygiene measures, such vaccines can provide the technological tools required to eradicate ILTV from production sites, and then regionally, in developed poultry industries from around the year 2000.

Single and concurrent avian leukosis virus infections with avian leukosis virus-J and avian leukosis virus-A in Australian meat-type chickens

Australian broiler breeders were screened for avian leukosis viruses (ALVs) (May 2001 to December 2003) as surveillance of measures to reduce the prevalence of ALV-J. Samples of blood (4233), albumen (1122), meconium (99) and tumours (16) were obtained from 93 flocks in six Australian states. Virus isolation was performed in C/O chick embryo fibroblast cultures, which were initially screened by group-specific antigen enzyme-linked immunosorbent assay, with follow-up confirmation using polymerase chain reaction. The chronology of isolations reveals the circulation of both ALV-J and ALV-A during this period. On 16 occasions single isolations were found to contain both ALV-A and ALV-J. This is the first report of dual infections with two subgroups of ALV occurring in the same chicken. The effectiveness of ALV-J eradication measures is indicated by the absence of any ALV-J isolations in late 2003. ALV-A however, continued to be isolated from the broiler population. The detection of dual infections, as well as the ongoing occurrence of ALV-A in meat-type birds, is discussed in the context of ongoing potential for recombinations and the associated threat for the emergence of avian leukosis virus with changes in host range and pathogenicity.

Laryngotracheitis herpesvirus infection in the chicken. II. The adoptive transfer of resistance with immune spleen cells

Protection against virulent infectious laryngotracheitis (ILT) virus was successfully transferred between inbred white leghorn chickens with spleen cells or peripheral blood leukocytes from immune cock birds. Resistance to infection could be demonstrated 7 to 8 days, but not 2 days after cell-transfer. Both hyperimmune spleen cells and memory spleen cells conferred resistance to infection, while the transfer of non-immune spleen cells failed to protect the chickens. Thymocytes or bursal cells from immune donors also failed to confer protection. The majority of cyclophosphamide pretreated recipients of immune spleen cells survived the challenge with ILT virus without synthesising detectable levels of humoral antibody. These findings indicate that cell-mediated immune mechanisms are involved in vaccine-induced immunity to acute ILT infections.

Detection of avian leukosis virus with the ELISA system: evaluation of conjugation methodology and comparison of sensitivity with the phenotypic mixing test in commercial layer flocks.

Conjugates of horseradish peroxidase with rabbit IgG antibody against gs antigen (p27) of avian myeloblastosis virus were prepared using glutaraldehyde and periodate methods of coupling. Conjugates were evaluated for the detection of gs antigen of avian leukosis and sarcoma viruses in the ELISA system and the periodate conjugate was found to be superior. With an ELISA based on a periodate conjugate it was possible to detect 5 x 10(3) IU of leukosis virus propagated in cell culture. Comparisons between the PM test and ELISA on biological samples showed the PM test to be 2 to 2,000 times more sensitive than ELISA for vaginal swabs and embryo extracts. ELISA was 16 to 64 times more sensitive than the CF test when comparisons were made on albumens and embryo extracts. When the ELISA was used for testing vaginal swabs for the prevalence of LLV infection in commercial laying flocks, it appeared that the reliability of ELISA varied. In all five flocks studied, ELISA detected a higher percentage of gs+ [virus+] hens than did the PM test, the proportion of ELISA(+) PM(-) samples ranging from 3 to 53%. In some flocks ELISA failed to detect a low proportion [ 1 to 5%] of infected hens when vaginal swabs were used for screening. The suitability of ELISA for use in screening of commercial flocks for LLV infection is discussed.

Experimental infection of chickens with an australian strain of reticuloendotheliosis VIRUS. I. clinical, pathological and haematological effects

A wide range of clinical, pathological and haematological effects were found over a 40-week period in chickens inoculated at 1-day-old with a low-passage, cell-culture preparation of an Australian strain of reticuloendotheliosis virus. Feathering defects and statistically significant depression of body weights occurred in chickens up to 8 weeks of age. Other findings in birds that died or were culled during the 40-week experimental period included mild anaemia, leucopenia, heterophilia, hypoplasia of immune system organs, inflammation in visceral and nervous system organs, and bacterial or fungal infections. These results suggested that ill-thrift and death in some chickens infected with reticuloendotheliosis virus may be due to secondary infections with microorganisms subsequent to damage of immune system organs by that virus. Lymphoreticular-cell tumours of the liver, kidney or spleen were found in two birds aged 22 and 24 weeks. These results establish reticuloendotheliosis virus as a possible cause of tumours in adult fowls. Horizontal transmission of virus was demonstrated but the only abnormalities detected in the in-contact chickens were feathering defects.

Experimental infection of chickens with an Australian strain of reticuloendotheliosis virus. 2. Serological responses and pathogenesis

Fifteen SPF chickens were inoculated with an Australian strain of reticuloendotheliosis virus (REV) at 1 day of age and five uninoculated chickens were readily infected by horizontal spread from this group. Antibody detectable by the immunofluorescent antibody (IFA) test developed 3 to 6 weeks after infection, and usually persisted for 20-35 weeks, with maximum titres (40-1280) at 8 to 13 weeks. Agar gel precipitin (AGP) reactions developed more slowly and were variable in duration, the highest proportion of positive reactions being detectable 8 to 13 weeks after infection and persisting for 8 to 30 weeks. Infectious REV was readily detected in the plasma and serum of inoculated chickens 6 weeks after infection and a non-infectious REV antigenaemia usually persisted for at least a further 7 weeks, in the presence or absence of antibody. Development of a detectable REV viraemia was strongly associated with poor body development and premature mortality among the inoculated chickens. In two inoculated chickens which failed to develop detectable serological reactions, a REV viraemia occurred which persisted throughout life. At autopsy, REV was re-isolated from the kidneys of most of the inoculated chickens and from the reproductive and intestinal systems of two birds 22 and 56 weeks after infection.

Experimental infection of chickens with an Australian strain of reticuloendotheliosis virus 3. persistent infection and transmission by the adult hen.

Vertical transmission of reticuloendotheliosis virus (REV) infection was demonstrated in embryonated eggs from an adult hen with persistent REV viraemia but no serum antibody. Pooling of infected embryos from this hen with those from antibody-positive hens appeared to inhibit the infectivity of congenitally-transmitted REV. REV was detected in vaginal swabs from this hen on 11 occasions over a period of 26 weeks of adult life and infectious REV was shed from the eye, mouth and in the droppings. Direct contact between the hen and other adult hens and roosters resulted in the transmission of REV infection, with or without genital contact. These newly-established REV infections were not persistent. Transmission did not occur between the infected hen and others separated by a wire mesh barrier.

Application of a flexible-film isolator for rearing specific pathogen-free chickens and investigating poultry pathogens.

A commercially-available flexible-film isolator kit was adapted for rearing specific pathogen free poultry and for carrying out experiments with avian pathogens. By increasing the standard air hose diameter from 32 mm to 75 mm and incorporating large metal filter canisters in place of the standard plastic filter sleeves, air flow rates were increased up to 8-fold. These changes allowed a greater number of birds to be maintained for longer periods without the previous problems of condensation of water vapour on the inside surfaces of the isolator. Fibreglass mat filters were shown to be efficient in retaining Newcastle disease virus when challenged by aerosol produced experimentally. Cross-contamination by virus infections between adjacent isolators was prevented for at least 12 weeks. The use of air-tight seals between the isolator canopy and structural components, air-tight feeder and light supports, an automatic watering system and facilities to improve portability are described. The adaptations resulted in an isolator which was efficient to use and maintain.