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Marek's disease in Australia — developments in monitoring and control

Stephen Walkden-Brown1 and Peter Groves2

1School of Environmental and Rural Science, University of New England, Armidale NSW 2351; 2Zootechny P/L, 975 The Northern Rd, Bringelly NSW, 2171

Posted Flock & Herd April 2013

Introduction

Marek's disease (MD) is a common disease of chickens worldwide, including Australia, caused by the Marek's disease virus (MDV), a cell-associated alphaherpesvirus. The virus is lymphotrophic, but also infects cells of the feather follicle epithelium in which it replicates rapidly and is shed in feather dander, being transmitted laterally via inhalation of this material. The disease as first described by Josef Marek in 1907 was characterised by paralysis and lymphocytic infiltration of peripheral nerves in older chickens1. This form of the disease, known as "classical" MD was superseded in the 1950s and 60s by "acute" MD characterised primarily by lymphomas in multiple organs in younger chickens. This development, associated with the intensification of the poultry industry, was a major threat to the industry until the concurrent discovery of the causative agent and development of live vaccines in the UK and USA in 1969. Since that time, there has been an ongoing evolution of virulence of MDV in countries such as the USA, associated with sequential vaccine failure and greater virulence in unvaccinated chickens with or without maternal antibody directed against MDV 2. Marek's disease vaccines are "imperfect" in that they protect against MD but not infection and shedding of virus, and as such are implicated in the evolution of virulence of the virus3. Recent modelling work, based on our Australian research has shown that both vaccination and the reduced host lifespan that has occurred with the intensification of the chicken meat industry are implicated as causes of the observed increase in virulence4. MDV and two other closely associated viruses belong to the alphaherpesvirus genus Mardivirus and the 3 species, also known as MDV serotypes, are classified as follows:

The Australian situation regarding MD up to 2000 has been well summarized5. The disease was under relatively good control until a major epidemic between 1992 and 1997. Prior to this, layers and breeders were vaccinated with HVT and or MDV-2 vaccines with maternal antibody providing sufficient protection for broilers. Relaxation of Australian quarantine protocols in 1990 saw the importation of new strains of both layer and broiler chickens from 1992 onwards and their complete dominance of the Australian industry due to improvements in efficiency. However, between 1992 and 1997 MD caused enormous losses in both the layer and broiler industries as conventional Australian vaccines and vaccination programs failed to control MD in the imported genotypes with mortalities in the range of 20-40% of birds being common in layer and breeder flocks. The problem in layers and broiler breeders was brought under control by the importation in 1997 of seed for the Rispens CVI988 serotype 1 vaccine and MD remains well controlled by this vaccine at present. Broilers had not traditionally been vaccinated against MD in Australia, but during 1992-97 clinical MD was appearing in birds from 35 days onwards associated with reduced flock productivity, typically around 8 points (0.08) in feed conversion ratio, and increased intercurrent disease. These problems were generally responsive to vaccination with HVT and in 1996 Embrex® machines for in-ovo vaccination of broiler eggs with high titre cell-associated HVT vaccine were introduced to Australia. This helped to bring the immediate problem in broilers under control, although at considerable cost. Currently about 50% of Australia's broilers are vaccinated with HVT in ovo or at hatch, while all long-lived commercial chickens (layers and breeders) are vaccinated at hatch with the Rispens CVI-988 vaccine.

Our group's research into Marek's disease commenced in 1998 and in a succession of projects has had the following broad aims:

Overview of Australian research and field studies

Pathotyping and evolution of virulence: Early studies into the virulence of Australian isolates of MDV using non-standardised methods, suggested that some isolates were highly virulent and able to overcome the protective effects of vaccination with HVT6-8. To assess virulence more formally we undertook isolator studies using an adaptation of a formal MDV pathotyping method based on the protective index provided by HVT and HVT/MDV2 bivalent vaccines developed at the USDA Avian Diseases and Oncology Laboratory (ADOL) (Witter 1997). The original method involves administration of a fixed challenge of MDV to a defined susceptible genotype of chicken (line 15x7 cross) having maternal antibody against MDV. MDVs are classified as mild (mMDV), virulent (vMDV), very virulent (vvMDV) or very virulent plus (vv+MDV) on the basis of MD lesions induced in unvaccinated chickens and those vaccinated with HVT and HVT/MDV2 bivalent vaccine over a 56-day post challenge period. We adapted this method to pathotype a range of Australian MDVs in maternal antibody free, specific pathogen free, white leghorn layer birds9, commercial Cobb broiler birds10, and commercial ISA Brown layer birds11 with the results of all 3 studies summarised in an early report12. The main findings of this work were as follows:

Image of chickens exhibiting paralysis
Figure 1. Photographs of chickens exhibiting early paralysis/mortality syndrome induced by MDV. The syndrome occurs at days 9-15 post challenge with very virulent MDV in chicks free of MDV-specific maternal antibody. Affected chickens exhibit depression, ataxia, altered head and wing carriage (including torticollis), progressing to marked paresis/paralysis, sternal recumbency, coma and death over 2-3 days. Diarrhoea is also a feature. At necropsy, marked thymic and bursal atrophy are the most consistent findings. In some cases no trace of the thymus can be found.

Development and application of molecular tests for MDV: Major challenges in the diagnosis of MD historically have been the difficulty in culturing the virus, and in differentiating vaccinal from wild-type strains of MDV. The latter is a particular problem when vaccination does not prevent infection and chickens may harbour all 3 serotypes of MDV concurrently. Initially we developed or adapted standard PCR tests to differentiate the different MDV serotypes, later using fully quantitative taqman real-time PCR (qPCR) tests14, 15.

Given that MDV is shed in copious amounts in feather dander, and is stable in this material at normal temperatures16 we worked on the idea of quantifying MDV in dust collected from chicken sheds as a means of monitoring and diagnosing MD. Our extensive work on developing and validating this methodology, and field application with a commercial chicken company over 7 years has recently been detailed17. Early field studies with unvaccinated broiler chickens revealed an exponential increase in MDV load in dust over time, MDV isolation from the affected farms, and a negative association between MDV load and chicken performance. In isolator studies, concurrent shedding of MDV of all 3 serotypes was demonstrated18 and clear association between MDV load in various tissues (including dust) and subsequent MDV load was demonstrated12. Since late 2004, a major chicken processing company has implemented routine quantitative testing for MDV in dusts in many sections of its broiler grow-out operations (Table 1). The testing to date has been conducted at our laboratory at the University of New England (now NATA accredited) and the results are used to develop vaccination and other MD control strategies on a regional basis. Of the 2390 samples, 26.3% were positive for MDV with significant regional and year-to-year variation. Overall regional prevalence of positives ranged from 15% in QLD, to 83% in the Riverina region of NSW, where an ongoing outbreak of delayed paralysis, and other unusual features, has been diagnosed as MD. Vaccination with HVT reduced the overall percentage of positives from 28.4% for known unvaccinated flocks (n=285) to 19.9% (n=899 flocks) with 30.6% positive for flocks of unknown vaccination status (n=1206). This clearly demonstrates that the virus is not as ubiquitous as is commonly thought.

Table of dust samples
Table 1. Number of dust samples submitted for commercial qPCR testing for MDV by region and year

Epidemiology and modeling of MD: Based on the results of early field and isolator challenge studies with MDV a deterministic model of MD transmission was developed19. A major study into the epidemiology of MDV in 72 Australian broiler flocks based on testing of dust for MDV in 288 sheds20 revealed that 50% of farms had detectable MDV. Factors which were found to have no association with the presence of MDV1 in dust included state, company, HVT vaccination status, bird strain, terminal disinfection program, turnaround time, disinfection of vehicles entering site, water source or sanitation, age of parent flocks, final age of birds and overall farm performance. Factors identified as significant risk factors for the presence of MDV1 in dust were birds hatched in summer-autumn (odds ratio 3.73), farms with more than 4 sheds (3.02) and farms with another chicken farm within 2 km (2.98). The provision of clothing for visitors on farm (odds ratio 0.48) and using wood-based litter material (compared to straw or rice hulls) (0.65), were found to be protective against the presence of MDV1 in dust. Factors which were significantly associated with higher viral load detected in dust samples were chicken strain, sexed flocks, proximity of another poultry farm and higher average live weight (>2764 gm), while in ovo HVT vaccination, wood-based litter material and processing at an older age were associated with lower viral loads. However, we have recently confirmed significant inhibition of PCR when DNA is extracted from litter based on hardwood shavings.

In MDV transmission studies we have found comparatively low rates of transmission in positive pressure isolator rooms21 presumably due to constant removal of infective dander, but in floor pens working with sentinel chickens, transmission rates of 8.4%/day and 0.8% per day were obtained to in-contact unvaccinated and HVT-vaccinated chickens respectively.

More recent modelling work with international collaborators based upon our chicken studies in Australia has shown that increased MDV virulence is associated with increased shedding from the host22, and that both vaccination and shorter host lifespan favours the selection of more virulent, rather than less virulent MDV strains4.

Differentiation of wild type MDV-1 from the Rispens CVI988 and viral kinetics: We

Have recently developed qPCR tests to differentiate between Australian strains of MDV and the serotype 1 Rispens CVI988 vaccine used routinely in Australian layers and breeder hens23. Initial studies have confirmed that the Rispens vaccine virus is shed in significant quantities from vaccinated birds24 and can be detected early in lymphocytes, feathers or dust samples. Depending on the order they are inoculated into birds, both wild-type and Rispens MDV interfere with the kinetics of each other, with the initially inoculated virus having an inhibitory effect on the later virus. Vaccinal protection is strongly influenced by the vaccination-challenge interval25, 26.

Discussion and implications

Marek's disease is an evolving disease characterised by failure of control and sustained outbreaks in different parts of the world at any given time. The last major Australian epidemic in the 1990s led to significant investment in MD research in this country. This enabled significant contributions to our understanding and control of MD at an international level. Of particular significance and novelty is the Australian approach of monitoring MDV levels in poultry dust - an easily collected, transported and stable material that integrates information from a large number of chickens into a single informative sample. Tactical vaccination based on the results of dust testing is likely to reduce the selection pressure for increased virulence of MDV induced by blanket vaccination of broilers. MD is considered to be "under good control" in Australia currently. However, an outbreak of apparently atypical MD in one region is a salutary warning of the need to maintain vigilance of, and diagnostic and research capability for, this disease.

Acknowledgements

We are grateful for funding over the years from the Australian Research Council, Baiada Select Poultry, Rural Industries Research and Development Corporation, Australian Egg Corporation Ltd and the Poultry CRC. Key collaborators in the research reported here include Peter Young, Greg Underwood, Greg Tannock, Tim Mahony, Ton Schat, Fakhrul Islam, Aminul Islam, Katrin Renz, Zahid Hussain, Tanzila Islam, Salih Wajid and Sithara Ralapanawe. We are also indebted to the technical support of Sue Burgess, Paul Reynolds, Sue Sharpe, Julie Cooke, Nadeene Clarke and Gary Taylor.

References

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  2. Witter RL. The changing landscape of Marek's disease. Avian Pathology 1998;27:S46-S53
  3. Gandon S, Mackinnon MJ, Nee S, Read AF. Imperfect vaccines and the evolution of pathogen virulence. Nature 2001;414:751-755
  4. Atkins KE, Read AF, Savill NJ, et al. Vaccination and reduced cohort duration can drive virulence evolution: Marek's Disease virus and industrialized agriculture. Evolution 2013:In Press
  5. Jackson CAW. Lessons from 30 years of Marek's disease control in Australia for 2000 and beyond. Proceedings of Australian Poultry Science Symposium 2000;12:186-189
  6. McKimm-Breschkin JL, Faragher JT, Withell J, Forsyth WM. Isolation of very virulent Marek's disease viruses from vaccinated chickens in Australia.Australian Veterinary Journal 1990;67:205-209
  7. Zerbes M, Tannock GA, Jenner RJ, Young PL. Some characteristics of a recent virulent isolate of Marek's disease virus. Australian Veterinary Journal 1994;71:21-22
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  9. Renz KG, Cooke J, Clarke N, et al. Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism. Avian Pathology 2012;41:161-176
  10. Walkden-Brown SW, Islam A, Islam AFMF, et al. Pathotyping of Australian isolates of Marek's disease virus in commercial broiler chickens vaccinated with HVT or bivalent (HVT/SB1) vaccine and association with viral load in spleen and feather dander. Australian Veterinary Journal 2013:Submitted Feb 2012. Accepted subject to minor revision July 2012. Resubmit 2014/2018/2012
  11. Renz K. In vitro and in vivo characterization of selected Australian isolates of Marek's disease virus. Animal Science. University of New England, Armidale, 2008:256
  12. Walkden-Brown SW, Cooke J, Islam A, et al. Pathotyping of Australian Isolates of Marek's disease virus. Proceedings of the Australian Veterinary Poultry Alliance 2007;2007:32-37
  13. Witter RL, Gimeno IM, Reed WM, Bacon LD. An acute form of transient paralysis induced by highly virulent strains of Marek's disease virus. Avian Diseases 1999;43:704-720
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  15. Islam A, Cheetham BF, Mahony TJ, Young PL, Walkden-Brown SW. Absolute quantification of Marek's disease virus and herpesvirus of turkeys in PBL, feather tip and dust samples using real-time PCR. Journal of Virological Methods 2006;132:127-134
  16. Carrozza JH, Fredrickson TN, Prince RP, Luginbuhl RE. Role of desquamated epithelial cells in transmission of Marek's disease. Avian Dis 1973;17:767-781
  17. Walkden-Brown SW, Islam A, Groves PJ, et al. Development, application and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR. Avian Diseases 2013:Accepted
  18. Islam A, Walkden-Brown SW. Quantitative profiling of the shedding rate of the three Marek's disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal viruses. Journal of General Virology 2007;88:2121-2128
  19. Gao Z, Walkden-Brown SW, Islam AFMF, et al. A model for Marek's Disease transmission in broiler chickens. Proceedings of Australian Poultry Science Symposium 2004;16:145-148
  20. Groves PJ, Walkden-Brown SW, Islam AFMF, et al. An epidemiological survey of MDV in Australian broiler flocks. James Cook University, 2008
  21. Crabb JR, Walkden-Brown SW, Baigent SJ, Smith LP, Nair VK. Rate of natural transmission of Marek's disease virus (MDV) to susceptible chickens in the post-challenge period: effect of vaccination with HVT and association with MDV genome count in air. Proceedings of Australian Poultry Science Symposium 2009;20:184-187
  22. Atkins KE, Read AF, Savill NJ, et al. Modelling Marek's disease virus (MDV) infection: parameter estimates for mortality rate and infectiousness. BMC Veterinary Research 2011;7:1-12
  23. Renz KG, Cheetham BF, Walkden-Brown SW. Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis. Journal of Virological Methods 2013;187:144-152
  24. Islam T, Renz KG, Walkden-Brown SW. Viral load, shedding rate and lateral transmission of Marek's disease vaccinal virus (Rispens/CVI988) in SPF chickens. In: Roberts JR, editor. The Poultry Research Foundation University of Sydney, Sydney, 2011
  25. Ralapanawe S, Walkden-Brown SW, Islam AMFM, Renz KG. Protection provided by Rispens CVI988 vaccine against very virulent Marek's disease virus challenge in IsaBrown chickens. Proceedings of Australian Poultry Science Symposium 2013;23:Submitted
  26. Islam T, Walkden-Brown SW, Renz KG, Islam AMFM. Effect of widely divergent vaccination-challenge intervals on protection provided Rispens CVI988 vaccine against very virulent Marek's disease virus challenge in IsaBrown chickens. Proceedings of Australian Poultry Science Symposium 2013;23: Submitted

 

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