Protection provided by Rispens CVI988 vaccine against Marek's disease virus isolates of different pathotypes and early prediction of vaccine take and MD outcome.

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.

Vaccination-challenge interval markedly influences protection provided by Rispens CVI988 vaccine against very virulent Marek's disease virus challenge.

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.

Viral kinetics, shedding profile, and transmission of serotype 1 Marek's disease vaccine Rispens/CVI988 in maternal antibody-free chickens.

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.

Prevalence of Marek's disease virus in different chicken populations in Iraq and indicative virulence based on sequence variation in the ecoRI-q (meq) gene.

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.

Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism.

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.

Modelling Marek's disease virus (MDV) infection: parameter estimates for mortality rate and infectiousness.

BACKGROUND: Marek'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. RESULTS: Our 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. CONCLUSIONS: Our 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.

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.

Effects of Rispens CVI988 vaccination followed by challenge with Marek's disease viruses of differing virulence on the replication kinetics and shedding of the vaccine and challenge viruses.

Vaccination with "imperfect" vaccines that prevent disease but not infection is strongly implicated in the observed increased virulence of Marek's disease virus (MDV) over the past six decades. The current "gold standard" vaccine, Rispens CVI988 (Rispens), has maintained efficacy despite use for five decades, raising the question of whether it too favours higher virulence MDVs. To investigate this, we studied the kinetics of Rispens CVI988 (Rispens) and two MDV strains of different virulence in 236 commercial ISA Brown chickens vaccinated with Rispens at hatch and challenged with vMDV isolate MPF57 or vvMDV isolate FT158 on day five. Each treatment was replicated in two isolators and from 7 to 56 days post infection (dpi) peripheral blood leucocytes (PBL), feather and dust samples were collected and subjected to differential quantitative PCR (qPCR). Rispens vaccination significantly reduced challenge MDV viral load in a sample-dependant manner with evidence of a differentially greater inhibitory effect on the less virulent MDV. Similarly, challenge with the more virulent MDV reduced the Rispens viral load in PBL. Rispens virus load displayed a distinctive pattern of viral load that was similar in PBL and feathers, but different in dust. The clear effects of vaccination and challenge evident in PBL and feather samples were less clearly reflected in dust samples. The data are consistent with the Rispens vaccine reducing replication of lesser virulent MDVs to a greater extent like the HVT vaccine. Likely reasons for the persistent efficacy of Rispens vaccine are discussed.

Replication kinetics and shedding of very virulent Marek's disease virus and vaccinal Rispens/CVI988 virus during single and mixed infections varying in order and interval between infections.

Vaccination is thought to contribute to an evolution in virulence of the Marek's disease virus (MDV) as vaccines prevent disease but not infection. We investigated the effects of co-infections at various intervals between Rispens/CVI988 vaccine virus (Rispens) and very virulent MDV (vvMDV) on the replication and shedding of each virus. The experiment used 600 ISA Brown layer chickens in 24 isolators with all treatments replicated in two isolators. Chickens were vaccinated with Rispens and/or challenged with the vvMDV isolate 02LAR on days 0, 5, or 10 post hatching providing vaccination to challenge intervals (VCI) of -10, -5, 0, 5 or 10 days with the negative values indicating challenge prior to vaccination. Peripheral blood lymphocytes (PBL), feathers and isolator exhaust dust were sampled between 7 and 56 days post infection (dpi) and subjected to quantitative real-time polymerase chain reaction (qPCR) to differentiate the two viruses. Overall Rispens significantly reduced the viral load of vvMDV in PBL and feather cells and shedding in dust. Similarly vvMDV significantly reduced the viral load of Rispens in PBL and feather cells but not in dust. VCI significantly influenced these relationships having strong positive and negative associations with load of vvMDV and Rispens respectively. Differences between the two viruses and their effects on each other were greatest in PBL and feathers, and least in dust. This study expands our understanding of the interaction between pathogenic and vaccinal viruses following vaccination with imperfect vaccines and has implications for selection of appropriate samples to test for vaccination success.

Vaccination and reduced cohort duration can drive virulence evolution: Marek's disease virus and industrialized agriculture.

Marek's disease virus (MDV), a commercially important disease of poultry, has become substantially more virulent over the last 60 years. This evolution was presumably a consequence of changes in virus ecology associated with the intensification of the poultry industry. Here, we assess whether vaccination or reduced host life span could have generated natural selection, which favored more virulent strains. Using previously published experimental data, we estimated viral fitness under a range of cohort durations and vaccine treatments on broiler farms. We found that viral fitness maximized at intermediate virulence, as a result of a trade-off between virulence and transmission previously reported. Our results suggest that vaccination, acting on this trade-off, could have led to the evolution of increased virulence. By keeping the host alive, vaccination prolongs infectious periods of virulent strains. Improvements in host genetics and nutrition, which reduced broiler life spans below 50 days, could have also increased the virulence of the circulating MDV strains because shortened cohort duration reduces the impact of host death on viral fitness. These results illustrate the dramatic impact anthropogenic change can potentially have on pathogen virulence.