Detection of subgroup J avian leukosis virus infection in Australian meat-type chickens.

OBJECTIVE: To determine the extent of avian leukosis virus subgroup J (ALV-J) infection in Australian broiler breeder flocks, using virus isolation and molecular biological detection. Any resultant ALV-J viral isolates to be characterised by neutralisation cross testing in order to determine antigenic relationships to overseas isolates of ALV-J. STUDY DESIGN: Samples of blood, feather pulp, albumen and tumours were obtained from broiler breeder flocks which represented four genetic strains of meat chickens being grown in Victoria, South Australia, NSW and Queensland. Dead and ailing birds were necropsied on farm and samples were collected for microscopic and virological examinations. Virus isolation was carried out in C/O and DF-1 CEF cultures and ALV group specific antigen was detected in culture lysates using AC-ELISA. Micro-neutralisation assay was used for antigenic characterisation of selected isolates. Genomic DNA was isolated from cultured cells, tumours and feather pulp. ALV-J envelope sequences were amplified by PCR using specific ALV-J primers while antibodies against ALV-J were detected by ELISA. RESULTS: A total of 62 ALV-J isolates were recovered and confirmed by PCR from 15 (31.3%) of 48 breeder flocks tested. Antibody to ALV-J was detected in 20 (47.6%) of the 42 flocks tested. Characteristic lesions of myeloid leukosis caused by ALV-J were found in affected flocks. The gross pathological lesions were characterised by skeletal myelocytomas located on the inner sternum and ribs, neoplastic enlargement of the liver, and in some cases gross tumour involvement of the spleen, kidney, trachea, skeletal muscles, bone marrow, skin and gonads. Microscopically, the tumours consisted of immature granulated myelocytes, and were present as focal or diffuse infiltrations in the affected organs. Virus micro-neutralisation assays demonstrated antigenic variation among Australian isolates and to overseas strains of ALV-J. CONCLUSION: ALV-J infection was prevalent in Australian broiler breeder flocks during 2001 to 2003. Australian isolates of ALV-J show a degree of antigenic variation when compared to overseas isolates.

Avian infectious laryngotracheitis.

Avian infectious laryngotracheitis (ILT) herpesvirus continues to cause sporadic cases of respiratory disease in chickens world-wide. Sources of transmission of ILT infection are three-fold, namely: chickens with acute upper respiratory tract disease, latently infected 'carrier' fowls which excrete infectious laryngotracheitis virus (ILTV) when stressed, and all fomites (inanimate articles as well as the personnel in contact with infected chickens). Infectious laryngotracheitis virus infectivity can persist for weeks to months in tracheal mucus or carcasses. Rigorous site biosecurity is therefore critical in ILT disease control. Furthermore, while current (modified live) ILT vaccines can offer good protection, the strains of ILTV used in vaccines can also produce latent infections, as well as ILT disease following bird-to-bird spread. The regional nature of reservoirs of ILTV-infected flocks will tend to interact unfavourably with widely varying ILT control practices in the poultry industry, so as to periodically result in sporadic and unexpected outbreaks of ILT in intensive poultry industry populations. Precautions for trade-related movements of chickens of all ages must therefore include an accurate knowledge of the ILT infection status, both of the donor and recipient flocks.

Chicken anaemia virus antibody ELISA: problems with non-specific reactions.

For accreditation of CSIRO specific pathogen-free flocks, chicken sera are routinely tested for antibody to chicken anaemia virus (CAV) using a commercially available ELISA kit. On some occasions recently, up to 18.2% positive reactions have been found in individual isolators, with an overall reaction rate of 2.7%. On subsequent bleeding the majority of the suspected birds have given negative reactions, indicating that the earlier reactions were false-positives. The protein composition of the sera that appeared CAV-negative or -positive in the ELISA were compared using various Chromatographic techniques. Separation of serum proteins by high resolution size exclusion chromatography revealed that in the false-positive sera the antigen-binding activity is associated with a protein fraction which was different from that found in the true-positive sera.

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.

An enzyme-linked immunosorbent assay for detection of reticuloendotheliosis virus infection in chickens.

An enzyme-linked immunosorbent assay (ELISA) for the detection of reticuloendotheliosis virus (REV) is described. The assay is based on antiserum produced in rabbits against the group-specific (gs) antigen of REV which has a molecular weight of 30 kDa (p30). The p30 for immunisation was obtained by electro-elution from polyacrylamide gels after separation of purified REV by electrophoresis. Western blotting indicated that the antiserum produced was specific for p30 of REV. The double antibody sandwich ELISA developed using the rabbit anti-p30 was found to be specific for REV and highly efficient for the detection of REV in a variety of samples when compared with the indirect immunofluorescence assay. The smallest amount of REV detectable was 2,000 fluorescence forming units. Serum, vaginal/ cloacal swab samples and egg albumens obtained from experimentally infected REV-viraemic chickens were all consistently positive for the ¿?i-antigen of REV. Some serum samples from non-viraemic chickens, however, gave 'false positive' reactions in the ELISA. Albumens gave higher readings in ELISA than vaginal/cloacal swab samples and should therefore be the sample of choice for screening flocks for REV.

Responses of cattle, sheep and poultry to a recombinant vaccinia virus expressing a swine influenza haemagglutinin.

Groups of cattle, sheep and poultry were inoculated with a recombinant vaccinia virus expressing the haemagglutinin of the swine influenza virus A/NJ/11/76. No adverse clinical responses were recorded and none of the animals developed a viraemia when inoculated with the recombinant or wild-type vaccinia virus. Recombinant virus reisolated from lesions in cattle was stable, maintaining its thymidine kinase negative phenotype and ability to express the swine influenza haemagglutinin. Antibodies to the influenza haemagglutinin were detected in cattle, sheep and poultry inoculated with the recombinant virus. While no animals inoculated with wild-type virus developed these antibodies, there was no detectable spread of either recombinant or wild-type virus from the inoculation sites or to in-contact uninoculated animals. The results indicate that recombinant vaccinia viruses can induce immune responses in cattle, sheep and poultry demonstrating their potential as vaccine vectors in a variety of important veterinary species.

In vitro infection studies with infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus.

Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease.

Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI.

Effect of lymphoid leukosis virus on performance of layer hens and the identification of infected chickens by tests on meconia.

The effect of subclinical infection with lymphoid leukosis virus (LLV) on the productivity traits of layer hens was investigated. In hens that shed gs-antigen of LLV to albumen, onset of sexual maturity was delayed by a mean of 11 days and the number of eggs laid was reduced -by 68 per hen up to 75 weeks of age. Shedding hens laid on average 2 g lighter eggs and of lesser specific gravity. Thirty-four % less chickens were obtained in the reproduction programmes from LLV-shedders in comparison with non-shedders. LLV had no significant effect on fertility and hatchability. Reduced egg-related performance was only directly related to LLV-shedding and dams' shedding status had no effect on the egg-related performance of their LLV-free progeny. Dams' shedding status, however, correlated with higher mortality (10% higher) among their progeny. The percentage of non-layers was also higher in progeny of LLV-shedders. Meconia were highly suitable samples for identifying both transmitting dams and infected chickens but only if the test for infectious virus was performed. ELISA on meconia was less reliable than the test for virus and therefore is not recommended for the detection of residual of infected chickens in the flocks selected for reduced gs-antigen shedding.

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.

Antibody to the 32K structural protein of infectious bursal disease virus neutralizes viral infectivity in vitro and confers protection on young chickens.

Chicken sera containing IgG antibodies specific for the 32 000 (32K) mol. wt. structural polypeptide of infectious bursal disease (IBD) virus, as assessed by Western blotting, neutralized the in vitro infectivity of tissue culture-adapted IBD virus. When injected into young chickens, the serum passively protected them from challenge with pathogenic IBD virus. Chickens immunized with the 32K structural polypeptide of IBD virus, prepared by electroelution from SDS-PAGE gels, produced antibody detectable by ELISA and the virus neutralization assay, while chickens immunized with the 37K or 41.5K viral polypeptides synthesized antibody detectable by ELISA, but only very low levels of virus-neutralizing antibody. The immunoglobulin fraction of sera obtained from chickens immunized with the 32K polypeptide, but not the 41.5K polypeptide, passively protected chickens from infection with IBD virus. It is concluded that the 32K polypeptide is a major protective immunogen of IBD virus.

Variation in susceptibility to avian sarcoma viruses and expression of endogenous avian leukosis virus antigens in specific pathogen-free chicken lines.

Five lines of chickens maintained as specific pathogen-free flocks in Australia were characterized in relation to endogenous antigens and endogenous avian leukosis virus expression. Embryos of line N were predominantly of C/E phenotype, uniformly positive for group-specific antigen and chick helper factor (gs+chf+) and 38% expressed endogenous virus at a very low titre. Embryos of line M4 were uniformly of C/ABE phenotype and were either gs+chf+ or gs-chf+. Line W19 embryos segregated for susceptibility to viruses of subgroup A, B and D and were either of C/E or C/ABE phenotype. The majority of W19 embryos were gs+chf+ with a small proportion being gs+chf-. Line I13 embryos were either of C/0 or C/ABE phenotype, uniformly gs-chf- and 44% of embryos expressed endogenous virus at a low titre. Line S segregated for susceptibility to subgroup E virus and embryos were either of C/E or C/0 phenotype, while the majority of embryos from line S were gs-chf- with some embryos being gs+chf+ or gs-chf+. The degree of interference of gs+chf+ and gs-chf+ phenotypes with subgroup E virus infection was identical with the interference patterns of classical gs+chf+ and gs-chf+ phenotypes. The resistance to infection with avian sarcoma viruses of subgroups E in lines N and M4, and to a degree in line W19, was highly associated with the presence of chf. Resistance to subgroup E virus was independent of chf in lines S and I13, probably being under the control of an independent locus. Cellular restriction of endogenous virus replication existed in all subgroup E virus-susceptible cells of line I13 in contrast to cells of line S which supported replication of endogenous virus. The phenotype of chicken cells for the expression of endogenous gs antigen and chf could accurately be predicted from the test performed on whole blood cells.

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.

Development of the normal gastrointestinal microflora of specific pathogen-free chickens.

The development of the normal intestinal microflora of the small intestine, caecum and large intestine of specific pathogen-free (SPF) chickens, was studied in the period from hatching to 84 days of age. No bacteria were detected in any of the sites at hatchery (day 1), but by day 3 significant levels of faecal streptococci and coliforms were isolated from all sites. The flora of the small intestine was limited to faecal streptococci and coliforms for the first 40 days and then lactobacilli became established and dominated the flora. A large variety of facultative and strictly anaerobic organisms colonized the caecum. Many of these species were transient and were only present for a limited period; after 40 days the flora stabilized to consist predominantly of faecal streptococci, Escherichia coli, Bacteroides spp. and Lactobacillus sp. The flora of the large intestine was composed of organisms also present in the small intestine or the caecum. These findings differ from previously published studies on conventionally reared chickens in that the number of species isolated and the population levels of organisms are much lower. This probably reflects the absence of continuous environmental challenge to the chickens because of the housing and feeding facilities in which the chickens were maintained.

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.

Practical application of ELISA for detection of vertical transmission of leukosis virus in commercial layer hens.

The efficiency of enzyme-linked immunosorbent assay (ELISA) for detection of congenital transmission of exogenous lymphoid leukosis virus (LLV) was examined using both vaginal swabs and albumen samples obtained from two layer flocks. Approximately 100 hens were studied in each flock by the ELISA and phenotypic mixing tests. ELISA testing on vaginal swabs identified 25/27 (92.5%) hens excreting LLV in their vaginal swabs and 16/17 (94.1%) of these hens that transmitted LLV to their embryos. Application of the ELISA to albumen samples was found to be as sensitive as testing vaginal swabs for detection of infected hens but also avoided 67 "false positive" reactors detected by the ELISA on vaginal swabs. Testing of albumens by the ELISA is thus recommended for use within commercial leukosis control programmes.

Inhibition of glycosylation by corynetoxin, the causative agent of annual ryegrass toxicity: a comparison with tunicamycin.

The biological activities of corynetoxins, the causative agents of annual ryegrass toxicity, were compared with those of the closely related tunicamycins and found to be essentially identical. Both showed similar antibiotic activity against Newcastle disease virus and a range of gram-positive bacteria. In preparations of rat liver rough microsomes they also strongly inhibited the uridine diphospho-N-acetylglucosamine (UDP-GlcNAc):dolichol-P N-acetylglucosamine-1-phosphate (GlcNAc-1-P) transferase, an enzyme essential for N-glycosylation of glycoproteins. Pretreatment of rats with corynetoxins resulted in dose- and time-related reduction in the level of activity of this transferase in liver microsomal preparations. The implications of this reduction are discussed with reference to annual ryegrass toxicity, the only field disease known to be caused by tunicamycin-related compounds. Both corynetoxin and tunicamycin produced similar neurological effects and increased vascular permeability in nursling rats and they showed similar LD50-values of 137 and 132 micrograms/kg, respectively, in the nursling rats.