DIAGNOSIS AND CONTROL OF MAREK’S DISEASE (MD)

Dr. R.N. Srinivas Gowda*

Marek’s Disease is popularly called as MD by farmers, which is common in almost all the poultry flocks either in laying or broiler birds world over. It can occur in different forms in different age groups. Never seen as an outbreak like RD/ND viral disease, but there will be lingering mortality and continuous depletion of the flock. It can occur even in vaccinated flocks or sometimes cause vaccine failure. MD is going to be continuous problem in the  poultry farms  because of evolution of new pathogenic strains and these strains are difficult to control by vaccines and they also  cause immunosuppression. Veterinarians often find it difficult to  diagnosis and control ofmarek’s disease in the field, hence this article to highlight the diagnosis and control of MD.

About the disease

Marek’s Disease(MD) is one of the most common lymphoproliferative and a highly contagious oncogenic and neuropathic disease caused by a herpes virus called -Marek’s Disease Virus (MDV) of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. It is one of the examples of virus causing tumors.

Today there are evolving highly pathogenic isolates of MDV around the world capable of overwhelming the protection from currently employed vaccines. Thus MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity leading to huge economic losses. It is also an immunosuppressive disease and causes increased susceptibility to other infections.

History

It is one of the oldest disease of chicken. In 1907, the Hungarian veterinary pathologist “József Marek” described paralysis associated with a polyneuritis affecting some domestic fowls kept in his backyard. It is named after him in 1926, Pappenheimer and colleagues in USA described many features of the disease and recognized visceral lymphomas as part of the pathology. Both the neural form (formerly termed ‘neurolymphomatosis’ or ‘fowl paralysis’) and the visceral lymphomatosis form (termed ‘acute leukosis’ or ‘acute MD’. For about 50 years Marek’s disease was considered to be part of a large group of diseases referred to as the avian leukosis complex.

The Cause

Marek’s disease is caused by an alphaherpesvirus known as “Marek’s disease virus” (MDV) or Gallid alphaherpesvirus 2 (GaHV-2).

The virus is slow in cytopathic activity and remains highly cell associated, so cell-free infectious virus is virtually impossible to obtain. The oncogenic herpesvirus is immunologically unique and is closely related to the herpesvirus of turkeys (HVT).                                                                                                                                    

Presently members of the genus Mardivirus can be divided immunologically into three serotypes(Table.1.)

Table1. Serotypes, Pathotype and vaccine strains of MD (Markey et al., 2013).

Serotype	Pathotype	Abbreviation	Vaccine strain	Remarks
S.type-1 (GaHV-2)	Virulent	V	No	Includes all pathogenic and nonpathogenic strains. It varies markedly in pathogenicity
	Very virulent	VV	No
	Very virulent plus	VV+/vvv	No
	Mild	m	No
	Attenuated		Yes
S .type-2 (GaHV-3)	Non oncogenic		Yes	Contains avirulent and nononcogenic strains
S.type-3 Meleagrid herpesvirus	Non oncogenic	HVT	Yes	Includes avirulent HVT

HVT :Herpes virus of Turkeys

Table. 2, Evolution of MD strains and their pathogenicity

Sl.No	Year of occurrence	strains	Pathogenicity
1	1960	v MDV	Moderate virulence unable to induce tumors.                                                                Only induce moderate infection
2	1980	vv MD	High ability to induce tumors
3	1990	vv MD + (vvv MD)	Ability to cause Immunosuppression

Susceptibility

• Chickens are the most important natural hosts for Marek’s disease virus (MDV), Quail and Turkeys can also be infected, especially when they are raised in close proximity to chicken farms.
• Females are more susceptible than males
• Unlike leucosis, MD is more common in the early age in broilers and later at the time of onset of production (called as Late MD).

Transmission and Spread

Understanding the mode of transmission is helpful in breaking the transmission chain and control of infection.

• MD is a highly cell-associated but readily transmitted alpha herpesvirus with lymphotropic properties of gamma herpesviruses. Virus replicates in the feather follicles, where it is released into the environment through dead cells of infected feather dander and persists for long periods of time
• Infected feather follicles and dandruff act as a reservoir even after they have been shed. MDV is airborne pathogen which is transmitted via inhalation.
• Thus the virus even travel through feather follicles, dander and dead skin cells for more than 40 KM distance through wind currents.
• Horizontal transmission is the transmission of organisms between biotic and/or abiotic members of an ecosystem.
• The disease is spreading by direct and indirect contact with infected chickens and once contacted remain for life.
• MDV is not vertically transmitted from parent to offspring.

Pathogenesis

• Marek’s disease virus (MDV) infection starts with the inhalation of infectious dust.
• When chicks inhale infected dander or contaminated dust and the virus reaches the lungs where respiratory tissue and blood cells (B and T type lymphocytes) become infected.
• Mononuclear phagocytes transfer the virus to lymphoid organs, such as the spleen, thymus, and bursa, where the virus lytically replicates in lymphocytes.
• B lymphocytes are responsible for humoral (antibody mediated) immunity, whereas T lymphocytes are involved in cellular immune responses (direct defence against invading organisms).
• MDV is able to establish latency in infected T cell
• Eventually the virus reaches the feather follicles completing its replication cycle (Figure: 1.)
•  Effective vaccination prevents the development of tumours from latently (inactive or dormant state) infected T lymphocytes, and although infection and shedding of Marek’s Disease Virus can be reduced, it cannot be fully prevented by vaccination. 
• Infected chickens become healthy carriers for life and a source of infection to younger birds.

Figure 1. Schematic diagram showing the different stages of Marek’s Disease Virus pathogenesis including the virus shedding from the feather follicle epithelium and the transformation of T lymphocytes in susceptible birds. (Source: A. Gregorio Rosale Aviagen, The poultry site 2018)

• MD affects both commercial layer birdsand brroilers and may result in death or severe production loss.
• It can be seen as early as two weeks as well as in seventy weeks old chicken
• Marek’s disease is a progressive disease with variable signs and several overlapping pathological syndromes. In its clinical presentation, Marek’s disease can resemble avian leukosis, although there are key differences between the two diseases.

Clinical signs

Acute Marek’s disease or fowl paralysis occurs in explosive outbreaks in young chickens, in which a large proportion of birds in a flock show depression followed, after a few days, by ataxia and paralysis of some birds. The affected birds show one or more of the following clinical signs:

• Pale scaly and shriveled combs 
• Lethargy and labored breathing
• Lameness –one leg forward and other backward
• Frequent wing and leg paralysis; Paralysis of one or both legs
• Incoordination
• Blindness
• Greenish diarrhea
• Loss of production

Nervous disorders were observed with gasping, diarrhea and emaciation are common with  up to 15% mortalities. Marek’s disease is a progressive disease with variable signs. Six overlapping syndromes are described (Fenner et al., 1987):

• Immunosuppression(IS)  is impairment of the T-lymphocytes resulting from Marek’s disease, preventing a competent immunological response against pathogenic challenge, with the affected birds become more susceptible to disease conditions such as coccidiosis and Escherichia coli infection. Without causing gross tumors IS can occur in flocks and it is difficult to asses. Although the vaccines help on control tumor generation but do not always prevent IS.

6. Atherosclerosis only been shown to appear in chickens that were purposely infected with the virus in labs.

Gross lesions

Marek’s Disease (MD), cause tumors in heart, lungs  lymphoid organs, iris, muscle, visceral organs  -Liver, spleen, kidneys, gonads, and skin. Development of tumours in visceral organs, and peripheral nerves-leading to  paralysis of legs, wings and neck, grey eye (iris) or irregular pupil, vision impairment, blindness, skin lesions and immunosuppression, all of which can be accompanied by non-specific signs such as anorexia, weight loss and poor performance.

My first encounter of MD was from imported white Plymouth Rock chicken from USA in 1968 at AICRP Poultry for meat at  Poultry unit,  UAS Bangalore.  Out of 4,000 chicks received only 100 survived. All the dead birds showed cutaneous form of lesions, then called “Skin leucosis”. During that time specific diagnosis was based on postmortem lesions and histopathology, the only lab diagnosis was agar –gel-precipitation test(AGPT) from known feather follicle antigen.

Fig 2, Enlarered sciatic nerves, nodular tumors in liver and lung.

Diagnosis

Diagnosis mainly based on epidemiological information, age of occurrence, clinical signs, gross lesions and laboratory confirmation –histopathology, Real time PCR (RT-PCR) and immunohistochemistry.

MD induced Immunosuppression is a very complex and difficult to diagnose in the field conditions. It can occur without development of tumors or atrophy of organs.Always associated with dysregulation of immune organs.

Diagnosis is based on:

1. Postmortem examination:    

1. Typical gross lesions such as tumours in various internal organs and viscera, appearance of nodules in the feathered skin and grey, irregular eyes, Swollen enlarged brachial plexus and sciatic nerves. (fig.2)
2. The absence of bursal tumors helps distinguish this disease from lymphoid leukosis, although the presence of bursal tumors does not exclude Marek’s disease.
3. Tumour like lesions can be seen in birds as young as three weeks of age and typically are most common before flocks reach sexual maturity. tumour lesions can also be found during around peak egg production (what is called late Marek’s Disease).
4. Enlargement of sciatic nerves, irregular pupil, grayish-white nodules in the visceral organs and focal discolored areas on the skin and ovarian congestion and misshaped ova.
5. Since essentially all healthy birds are carriers of MDV without showing symptoms, virus isolation from blood samples does not have any diagnostic value.

• Histopathology:
• Histological examination of a complete set of tissues preserved  in 10% buffered formalin by a trained poultry pathologist is helpful in diagnosis of MD.  
• Histopathology  of Liver, kidney, spleen, proventriculus, Peripheral nerves (sciatic nerve), brain, Bursa, skin, eyes  show heavy pleomorphic cellular infiltrations of small lymphocytes, 4and heterophil infiltration were seen in all examined organs.
• The main picture is infiltration of Mononuclear cells in to one or more of the peripheral nerves, gonads, iris, muscle, viscera, and skin (Fig.3) .Proliferative lymphocytes (small to medium and mature cells in sciatic nerves.

Fig 3. Chicken showing accumulation of inflammatory lymphocytes in nerve sheath (white arrow) H&E, X400.(D) Peripheral nerve showing mild lymphocytic cellular infiltration in-between nerve axons. Proliferation and infiltration of neoplastic small to medium size lymphocytes that replaced liver parenchyma. pleomorphic lymphoid cell proliferation cells in liver,

1.  A conclusive diagnosis can be achieved by histochemistry of frogmen tissues. MD tumors are positive for T-cell markersCD3, CD4_, CD8 and meq+

• Molecular diagnosis of MD
• Real time PCR (polymerase chain reaction) (RT-PCR) amplification of Pathotype of Marek’s Disease Virus in suspected birds.
• Detection of large quantities of viral DNA by real-time PCR in blood, tumour cells, and feather pulp provides a specific diagnosis of Marek’s Disease Virus even in mixed infections with other viral tumour induced diseases.

Table:2 . Differential diagnosis between Marek’s disease, Lymphoid leucosis and Reticuloendotheliosis

Parameters	MAREK’S	LEUKOSIS	RETICULOENDOTHELIOSIS
Cause	Cell associated RNA Herpes Virus	DNA Retrovirus	Avian reticuloendotheliosis virus (REV)  DNA virus
Mode of infection	Horizontal -inhalation, ingestion of infected dandr, dead skin cells	Vertical, trans ovarian -transmission	Vertical, vector, transmission & contaminated vaccine
Age group	More in less than 16  weeks, Late MD occur up to 70 weeks.	Above 16 wees	First 2 week chicks clinical  signs and 14 weeks to develop tumors
Gross  lesions	-Tumors on visceral organs, –enlarged sciatic and brachial plexus, gray eyes,  Proventricular  &       skinlesions                                  -Bursa usually atrophic	Nodular Tumors on all visceral organs and gonads. No involvement of Nerves and eyes , BF-enlarged	Runting syndrome,  weight loss, paleness, occasional paralysis, and abnormal feathering, Emaciated muscles                                                 Acute neoplasia, liver, spleen, intestine, and heart. The cloacal bursa is involved in the chronic B-cell lymphomas.
Microscopic  Lesions	–Heterogeneous population of lymphoblast cells        ( T-Cells), small, Medium and large lymphocytes, Type B lesion-infiltrations of plasma cells and reticulum cells in nerves. BF: Intrafollicular tumor present	—Homogenous population of cells Uniform lymphoblast cell(B-Cells) -No nerve and skin involvement. -BF: Intrafollicular tumors absent	The nerve lesions are usually less extensive and may contain more plasma cells than in Marek’s disease  abnormal proventriculus, enteritis, or spleen and liver necrosis
Diagnosis	RT-PCR Assay, Histopathology Histochemistry , Positive  for T -cell markers	PCR –assay,	PCR assay, virus isolation, Positive for B-cell markers

Control of MD

Control of MD is based on three components

1. Biosecurity   2. Genetics and 3. Vaccination

1.Biosecurity:

1. It is critical for its control  to reduce MDV Load in the farm by proper cleaning and disinfection.
2. Mixed age group and multi age group rearing farms are more prone to infection.
3. Prevention of early environmental exposure of infection with field virus.
4. Rear the chicks in isolation away from existing old stock.
5. The main farm Workers should not enter the chick rearing (brooding) facility
6. Do not allow same vehicles supply of feed to other farms
7. Follow strictly all the biosecurity practices in the farm till they reach pullet age

2.Genetics: 

1. Selection of resistant poultry lines for certain MHC-haplocytes results in more resistant strains by parent flock suppliers.
2. the mechanisms of genetic resistance for MD are complex and not fully understood. MD-resistant line and MD-susceptible line are two highly inbred progenitor lines of White Leghorn.

3.Vaccination

The Marek’s disease vaccine is a “leaky vaccine”, which means that only the symptoms of the disease are prevented.  Vaccination protect against production of tumors, but not against infection or transmission.. This contrasts with most other vaccines, where infection of the host is prevented.

1. Vaccination has been the foundation in the protection against Marek’s Disease Virus. The majority of Marek’s Disease Virus vaccines are cell-associated, meaning the virus is present in the cells of cell cultures, grown under laboratory conditions, and used to manufacture the vaccine.
2. Vaccines are administered in-ovo  at egg transfer (18 days of incubation) and/or at hatch by subcutaneous or intra-muscular injection.
3. Commercially available vaccines include the following:
4. Traditional (live virus)
5. Serotype 1 – [CVI – 988 (Rispens)]
6. Serotype 2 – (SB-1)
7. Serotype 3 – [Herpesvirus of Turkeys (HVT)]
8. Current vaccines
9. Virulent MD (v MD) protected by HVT
10. Very virulent (vv MD) Protected byHVT+SB₁
11. Very very   very virulent MD (vv+MD) Protected by Respins CV1988)
12. Viral-vectored recombinant HVT with gene inserts for other viruses
13. rHVT – IBD
14. rHVT – ND
15. rHVT – ILT
16. rHVT – AI

Herpesvirus of Turkeys vaccines are used in combination with serotypes 1 (Rispens) and/or 2 (SB-1) to maximise preotection in breeder flocks. Today, Herpesvirus of Turkeys combined with Rispens is the most common combination used to protect breeders in very high-risk areas.

MD Vaccine consists of double Vaccination

1. First Vaccine administration of HVT In-ovo-vaccine at 18^th day embryo
2. Second Vaccine on day one at hatchery
3. After the outbreak of Md use of vaccine is no use.

Causes of Marek’s Disease Outbreaks

After vaccination in the hatchery, chicks are not protected until the Marek’s Disease Virus vaccine strain multiplies in the individual chick and starts circulating in the blood (known as viremia), which can take between 4-5 days. Therefore, it is essential to reduce the risk of early environmental exposure to Marek’s Disease Virus and/or delay the time of infection for as long as possible so the birds are fully protected.

Built-up litter and multiple age rearing farms pose a very high risk of early exposure to the field Marek’s Disease Virus and other immunosuppressive viruses. Proper selection and administration of Marek’s Disease Virus vaccines will result in adequate control; however, Marek’s Disease Virus outbreaks could occur as a result of the following factors (alone or in combination):

1. Vaccine factors:

1. Inadequate vaccine storage, handling, preparation or administration procedures
2. Suboptimal doses or dilution of vaccines
3. Additives (antibiotics) that change the pH and/or other properties of the diluent
4. Interference to Marek’s Disease Virus vaccination response caused by other vaccines
5. Early exposure to very virulent (vv) or very Virulent + (vv+) Marek’s Disease Virus strains
6. Immunosuppression caused by:
7. Infectious factors: Immunosuppressive diseases
8. Infectious Bursal Disease Virus(IBDV)
9. Infections Chicken Anemia Virus(CAV)
10. Flock management factors:
11. Overheating during hatch, processing and transport
12. Poor brooding conditions (incorrect house environmental conditions, inadequate provision of feed and water)
13. Extreme heat and cold (environmental temperatures), inadequate ventilation
14. High stocking densities, poor feed distribution, inadequate feeder space, poor body-weight development
15. Nutritional factors:
16. Poor ingredient quality and mycotoxins
17. Suboptimal levels of essential nutrients.

Take home message

MD is a complex disease because always  evolution of new pathogenic strains.

Very important aspect to consider is that without exhibiting tumors it can cause immunosuppression even in vaccinated flocks.

Since MDV is cell associated, it is difficult to control its spread through dander and feathers entering in to farm premises unless adoption of strict biosecurity measures.

Vaccine failures often occur because of not following the manufacturers recommendations.

Diagnosis is based on epidemiological data such  as , Hisotory,age and history, postmortems and by laboratory diagnosis by RT-PCR.

(Fig, credit GoogleImages)

Reference

Buscaglia C.(2013). Mixed infections of Marek’s disease and reticuloendotheliosis viruses in layer flocks in Argentina. Avian Dis. 57:569. –. [PubMed] [Google Scholar]

Bagust, T.J., Grimes, T.M. & Dennett, D.P. (1979). Infection studies on a reticuloendotheliosis virus contaminant of a commercial Marek’s disease vaccine. Australian Veterinary Journal, 55, 153157

Fadly AM , Ewer DL (2008). Enhancement of Avian Retrovirus· induced B~cell Lymphoma by Marek’s Disease, In: Saif YM; Fadly AM; Glisson IR; McDougald LR; Nolan LK ; Swayane DE; Diseases of poultry , II ed., edited by. Black wall publication, Iowa State University Press, Iowa, USA, p:558-559.

Witter RL, Schat KA(2003). “Marek’s disease.” In: Saif, Y.M., Fadly, A.M.; Glisson, I.R.; McDougald, L.R.; Nolan, L.K. ; Swayane, D.E. Diseases of Poultry, II ed., edited by Black wall publication, Iowa State University Press. Iowa, USA, p. 407-464.

(*Dr R.N.S,Gowda was former and founder VC,KVAFSU, Bidar, Former Director, IAH&VB,Bangalore and  Former Prof and University Head, Veterinary College, UAS,Banglore)