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Infectious laryngotracheitis virus (ILTV) DNA has been detected in blood fractions, but the cell phenotype with which the virus is associated is unknown. This study investigated the presence of ILTV antigen in peripheral blood cells of six acutely ILTV-infected chickens (5 or 9 days post ocular inoculation with a virulent isolate) and three sham-inoculated chickens using immunofluorescent staining. Blood fractions were separated by Ficoll-Paque density gradient centrifugation, and smears were prepared from erythrocyte and leukocyte fractions. The smears were stained for ILTV glycoprotein E and the leukocyte markers CD4, CD8, Bu-1 (B cell), KUL01 (monocyte/macrophage), TCRγδ, and TCRαβ/Vβ 2 and examined under a confocal microscope. In samples from infected birds, ILTV gE-specific fluorescence was localized in B cells and all evaluated T cell types, but not in monocytes and erythrocytes. The percentage of CD4, CD8, TCRγδ, TCRαβ/Vβ 1 , TCRαβ/Vβ 2 and B cells positive for ILTV antigen ranged from 13.3% to 22.3%. None of the samples from the sham-inoculated chickens exhibited fluorescence for ILTV gE. The results of this pilot study suggest that ILTV has a tropism for peripheral blood T and B cells. Further research is required to investigate whether these cells support ILTV productive replication. RESEARCH HIGHLIGHTSSelective tropism of ILTV for peripheral blood cells was demonstrated in acutely infected birds.The ILTV antigen gE was detected in blood CD4, CD8, TCRγδ, TCRαβ and B cells but not in monocytes and erythrocytes.The highest percentage of ILTV antigen was observed in CD4 cells (22.3%) followed by TCRαβ/Vβ 1 (20.6%) , CD8 (15.4%), TCRαβ/Vβ 2 or B cells (14.4%) and TCRγδ cells (13.3%).

The objectives of this study were to compare the virulence of contemporary infectious laryngotracheitis virus (ILTV) field isolates of classes 9, 10, and 14 in meat and layer chickens, and to evaluate cloacal and oropharyngeal swabs and dust as sample types for ILTV detection. A total of 211 chickens were divided into groups and inoculated with ILTV class 9, 10, or 14, or sham-inoculated via eye drop at 15 or 22 days of age. Chickens were euthanized at 5 and 9 days post-infection. Virulence was assessed by scoring of clinical signs (conjunctivitis, dyspnoea, and demeanour), ILTV genomic copies (GC) in oropharyngeal and cloacal swabs, mortality and microscopic lesions in conjunctiva and trachea. Class 14 caused subclinical infection, while inoculation with class 9 or class 10 resulted in severe clinical signs and microscopic lesions. Compared to class 14 (2.25 ± 0.36 log 10 GC), higher viral load was observed in oropharyngeal swabs of classes 9 (7.86 ± 0.48) and 10 (7.53 ± 0.36), with a higher proportion of positive oropharyngeal and cloacal swabs in the latter groups ( P  < 0.0001). Viral detection in cloacal swabs was delayed at early stages of infection compared to oropharyngeal swabs. Dust samples from class 9- and class 10-inoculated groups showed a trend towards higher GC than that of class 14. Overall, clinical scores, mortality, viral load, and microscopic lesions were similar for classes 9 and 10, but class 9 caused more severe disease in layer chickens than meat chickens. In summary, ILTV classes 9 and 10 exhibited severe virulence, while class 14 exhibited very mild virulence. RESEARCH HIGHLIGHTS Wide variation in the virulence of three field Australian field ILTV strains. Class 9 and class 10 strains were highly virulent, while class 14 was mildly virulent. The highly virulent strains were associated with significantly higher viral genome copies in various sample types than the mildly virulent strain.

Infectious bursal disease virus (IBDV) is endemic to most poultry-producing countries worldwide. Immunosuppressive classical and variant IBDV strains endemic to Australia are genetically distinct from other international strains. We report the results of infection experiments with Australian classical strain 06/95 and variant strain 02/95 in SPF chickens. We tested the effects of strain and age of infection on bursal atrophy, viral RNA (vRNA) load in bursa of Fabricius (bursa), spleen, thymus, caecal tonsils, faeces, litter and exhaust dust as determined by real-time reverse transcriptase polymerase chain reaction. The two IBDV strains did not differ in the degree of bursal atrophy induced, lymphoid organ distribution and faecal shedding but variant strain 02/95 induced a greater antibody response to the infection than classical strain 06/95 which was associated with a more rapid decline in IBDV vRNA genome copy number (VCN) in lymphoid organs and faeces. Infection at 14 days of age induced greater bursal atrophy and higher vRNA copy number in lymphoid tissues than infection on the day of hatching, indicating true age susceptibility independent of maternal antibody (Mab) status. The direction of the association between rankings for IBDV vRNA load in bursa and relative bursal weight changed from positive at 3 and 6 days post-infection to negative at 28 days post-infection. Intra-tracheal administration of dust collected from chickens infected with IBDV resulted in successful transmission of IBDV. IBDV vRNA was detected successfully at high levels in the environmental litter and dust samples.

We tested the level of protection provided by the Rispens CVI988 (Rispens) vaccine against challenge with a virulent Marek's disease virus (MDV) pathotype (vMDV) and a very virulent pathotype (vvMDV) and the accuracy of a range of predictive measures of Marek's disease (MD) incidence and vaccine take. Commercial layer chicks (n = 236) were vaccinated (or not) with 4000 plaque-forming units (pfu) of Rispens vaccine at hatch and challenged (or not) with 500 pfu of each challenge virus five days post vaccination. The vvMDV pathotype FT158 induced higher MD incidence (65%) and mortality (33%) when compared with the vMDV pathotype MPF57 (39% and 8%, respectively). The protective index provided by the Rispens vaccine against FT158 (61%) did not differ significantly from that against MPF57 (66%). This provides additional evidence that protection provided by the Rispens vaccine is not influenced by pathotype determined in studies using vaccines of other Mardivirus species. The challenge viruses did not differ in MDV or Rispens viral load in spleen at 14 dpc (days post challenge) determined by specific quantitative polymerase chain reaction test. MDV load in peripheral blood leucocytes at 7 and 14 dpc, splenocytes at 14 dpc, feather cells at 14 and 21 dpc and isolator dust at 21 dpc were significant early indicators of subsequent MD incidence to 56 dpc. These are potentially useful as the sampling can be carried out well before the onset of MD and some measures are non-invasive. The Rispens viral load in both invasive and non-invasive samples was more useful as a measure of vaccine take.

The Rispens (CVI988) vaccine is widely used to vaccinate chickens worldwide. We tested the protective effects of the Rispens vaccine against challenge with very virulent Marek's disease virus (vvMDV) at various intervals at, before or after vaccination. The experiment used commercial ISA Brown layers and vvMDV isolate 02LAR. The protective index (PI) was measured for vaccination challenge intervals (VCI) of -10, -5, 0, 5 and 10 days, with the negative values indicating challenge prior to vaccination. Chickens were challenged by injection with 400 plaque-forming units (PFU) of 02LAR and/or vaccinated with 3200 PFU of the Rispens vaccine virus at days 0, 5 and 10 of age, with appropriate negative controls injected with diluent only. The presence of visible Marek's disease tumours was assessed up to 56 days post challenge. MDV challenge in unvaccinated chickens resulted in tumours in 52% of chickens. The Rispens vaccine provided no significant protection when challenge preceded vaccination, with PIs of -4 and 21% for VCI of -5 and -10 days respectively. On the other hand, it provided PIs of 60, 85 and 100% at VCI of 0, 5 and 10 days respectively. The study also revealed that the vvMDV load in peripheral blood lymphocytes or feather tips at 14 and 21 days post infection as determined by quantitative real-time polymerase chain reaction, which can distinguish pathogenic MDV from the Rispens vaccine strain, was an accurate early predictor of Marek's disease incidence at 56 days post challenge. The load of Rispens virus in peripheral blood lymphocytes or feathers at the same times post vaccination did not offer similar predictive power.

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Marek's disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Marek's disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Marek's disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from approximately 40% to approximately 80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log(10)MDV load in the spleen (vaccinated, 2.99+/-0.20/10(6) spleen cells; unvaccinated, 4.60+/-0.23/10(6) spleen cells) and dander (vaccinated, 5.28+/-0.13/mg; unvaccinated, 6.00+/-0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.

Much of the impact of Marek's disease in broiler chickens is considered to be due to immunosuppression induced by Marek's disease virus (MDV). The present study evaluates the effects of an Australian isolate of pathogenic MDV (strain MPF 57) and a non-pathogenic vaccinal strain of herpesvirus of turkeys (HVT) (strain FC 126) on the immune system of commercial broiler chickens for 35 days following challenge at days 0 or 3 of age. It also investigates the extent of protection provided by HVT vaccine against MDV-induced immunosuppression. Immune system variables, including relative lymphoid organ weight, blood lymphocyte phenotype (CD45+/CD3+, putatively T, and CD45+/LC+, putatively B) and antibody production following vaccination against infectious bronchitis (IB) at hatch, were used to assess the immune status of chickens. Immunosuppression was also assessed by susceptibility to secondary challenge with pathogenic Escherichia coli on day 29 post-MDV challenge. MDV infection reduced the weight of the thymus and bursa of Fabricius, the numbers of circulating T lymphocytes and B lymphocytes, and IB antibody titre. The timing of these effects varied. MDV infection greatly increased susceptibility to E. coli infection. HVT alone caused mild depletion of T and B lymphocytes but no effect on immune organ weight or IB titre. Vaccination with HVT provided good protection against most of the immunosuppressive effects of MDV but not against MDV-induced growth impairment and reduced responsiveness to IB vaccination, suggesting that recent Australian strains of MDV may be evolving in virulence to overcome the protective effects of HVT.

The polymerase chain reaction (PCR) has recently emerged as an additional tool for the monitoring and diagnosis of Marek's disease. We investigated a number of factors that may influence the interpretation of PCR results in commercial broiler chickens including the effects of route of infection and herpesvirus of turkeys (HVT)-vaccination status. We also investigated the suitability of peripheral blood lymphocytes (PBL) and spleen as tissues for Marek's disease virus (MDV) detection. HVT-vaccinated and unvaccinated commercial broiler chickens were challenged or not challenged with virulent MDV either by intraperitoneal injection or inhalation of feather dust containing the virus. Blood and spleen samples were collected at weekly intervals to day 35 post-infection for PCR of spleen or PBL. Live weight and lymphoid organ weights were also measured. Spleen and PBL were found to provide similar sensitivity of detection of MDV with a small advantage in favour of spleen. In terms of the timing of detection of MDV, intraperitoneal challenge broadly mimicked natural challenge via inhalation, although infection of birds by inhalation of infective feather dust resulted in slightly later but more complete detection of MDV in challenged birds. Vaccination with HVT delayed the detection of MDV by approximately 10 to 14 days and did not protect against the reduced growth observed in challenged chickens at day 35 post-challenge.

In ovo vaccination against Marek's disease is a widely used technology in the broiler industry.A series of experiments was carried out to determine the site of vaccine deposition in the egg during automated in ovo vaccination, and the effect of vaccine deposition site and dose on vaccine responses following vaccination with cell-associated herpesvirus of turkeys in commercial broiler chickens. Vaccine deposition site following automated in ovo vaccination was principally influenced by the age of embryo, with egg size having a smaller effect. The frequency of vaccine deposition inside the embryo body increased as incubation progressed from day 17.5 to 19.5. In experiments using manual vaccine deposition intra-embryonically (IE) or extra-embryonically (EE) at day 18.5, EE vaccine deposition resulted in a significantly delayed development of post-vaccinal viraemia relative to both IE vaccination and subcutaneous vaccination at hatch. There were no effects of vaccine dose (2000, 4000 or 8000 plaque forming units) on the timing of post-vaccinal viraemia. The timing of post-vaccinal viraemia was found to be a good indicator of the level of protection provided by the vaccine against challenge with earlier viraemia associated with better protection. IE vaccine deposition induced significantly greater protection than EE deposition against challenge with a virulent strain of Marek's disease virus. IE deposition consistently produced a high level of protection (68 to 84%) irrespective of vaccine dose or challenge day, while EE vaccine deposition produced no or low levels of protection (0 to 27%) depending on the vaccine dose and day of challenge. The growth of challenged chickens was also affected by site of vaccine deposition, with significantly higher live weights at day 56 of age in IE compared with EE vaccinated groups. These data suggest that the site of vaccine deposition within the embryo is an important determinant of the success of in ovo vaccination.