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.

Marek's disease virus (MDV),

BackgroundMarek's disease virus (MDV) is an economically important oncogenic herpesvirus of poultry. Since the 1960s, increasingly virulent strains have caused continued poultry industry production losses worldwide. To understand the mechanisms of this virulence evolution and to evaluate the epidemiological consequences of putative control strategies, it is imperative to understand how virulence is defined and how this correlates with host mortality and infectiousness during MDV infection. We present a mathematical approach to quantify key epidemiological parameters. Host lifespan, virus latent periods and host viral shedding rates were estimated for unvaccinated and vaccinated birds, infected with one of three MDV strains. The strains had previously been pathotyped to assign virulence scores according to pathogenicity of strains in hosts.ResultsOur analyses show that strains of higher virulence have a higher viral shedding rate, and more rapidly kill hosts. Vaccination enhances host life expectancy but does not significantly reduce the shedding rate of the virus. While the primary latent period of the virus does not vary with challenge strain nor vaccine treatment of host, the time until the maximum viral shedding rate is increased with vaccination.ConclusionsOur approach provides the tools necessary for a formal analysis of the evolution of virulence in MDV, and potentially simpler and cheaper approaches to comparing the virulence of MDV strains.

Probably the most effective current vaccine against Marek's disease is the live Rispens (CVI988) attenuated serotype 1 Marek's disease virus (MDV). It is unknown whether the currently available Rispens vaccines transmit effectively between chickens. To investigate the kinetics and shedding of three commercially available strains of this virus and the extent of lateral transmission, we measured the shedding rate in dander and the viral load in peripheral blood lymphocytes (PBLs) and feather tips over time. Four identical climate-controlled rooms were stocked with a total of 70 specific-pathogen-free chickens for 56 days. In each of three rooms, 10 chickens were vaccinated with one of the commercial vaccines at day old and left in contact with 10 unvaccinated chickens. The fourth room contained 10 unvaccinated control chickens. As determined by MDV-specific quantitative real-time polymerase chain reaction of weekly room dust and individual PBLs and feather tip samples, the vaccine virus was shed from the vaccinated chickens in dander from day 7 postvaccination and transmitted effectively from vaccinated to in-contact chickens with a lag period of 2-3 wk. Viral load in PBLs and feather tips peaked at days 7 and 14, respectively, and declined thereafter, whereas viral load in dust increased rapidly to day 21 and then increased gradually thereafter. Antibody titer at day 56 was correlated with earlier measures of MDV load in PBLs but not feather tips or dust. These results show that currently available Rispens CVI988 vaccine virus is shed in significant quantities from vaccinated chickens and transmits effectively between chickens.

A cross-sectional survey was conducted in six provinces in southern Iraq to determine the point prevalence of Marek's disease virus (MDV) in different chicken populations followed by sequencing the meq gene for phylogenetic analysis and virulence-associated polymorphisms. A total of 109 samples from unvaccinated flocks were analyzed comprising 52 dust and 30 spleen samples from commercial broiler farms and 27 spleens from local layer chickens purchased in the town markets. The overall prevalence of MDV was 49.5% with no significant differences between provinces (P = 0.08) or sample types (P = 0.89). Prevalence ranged from 36.8% in Karbala and Nasiriyah to 65% in Amarah. The percentages of positive samples were 59.1%, 46.7%, and 48.1% in broiler dust, broiler spleen, and layer spleen, respectively. The overall mean (+/- SEM) Log10 MDV viral copy number per milligram of dust or spleen as determined by quantitative PCR was 1.78 +/- 0.19, with no significant differences between provinces (P = 0.10) or sample types (P = 0.38). In positive samples only, the overall mean was 3.43 +/- 0.18. Sequencing of the meq gene from samples that showed high levels of MDV target in qPCR testing was attempted. Nine samples were sequenced. These sequences were compared with meq sequences of MDVs of different pathotype. All the Iraqi MDVs had a short meq gene of 897 base pairs because of the deletion of 123 bp relative to the reference strain Md5. The Iraqi meq sequences also contained single-nucleotide polymorphisms, resulting in differences in the amino acid sequence. All of the nine Iraqi meq genes encoded two repeats of four-proline sequences. The published negative association between four-proline repeat number and MDV virulence suggests that the Iraqi MDVs are likely to be highly virulent, but this needs to be confirmed by in vivo testing. Taken together, these results indicate that MDV is common in unvaccinated commercial and village chickens in southern Iraq, that there is limited meq gene sequence variation, that all sequenced samples had a short meq with two four-proline repeats, and that this is consistent with a high level of virulence.

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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.

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