Diagnostic Considerations for Marek s Disease. Frederic J. Hoerr, DVM, PhD AMEVEA - Argentina Colon, Entre Rios May 15, 2013

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1 Diagnostic Considerations for Marek s Disease Frederic J. Hoerr, DVM, PhD AMEVEA - Argentina Colon, Entre Rios May 15, 2013

2 Control of Marek s Disease Accurate diagnosis Apply appropriate procedures to prevent the disease FOCUS A practical approach to Marek s disease and tumor diagnostics for the diagnostician

3 Resources for Marek s Disease and Tumor Diagnostics $40 USD Online $0 USD - PDF

4 Diagnostic Strategy for Marek s and Tumors History, epidemiology, clinical observations, necropsy Characterization of the tumor cell Characterization of the virus Presence Quantity Absence The pathogenesis and diagnosis viral tumor disease is complicated. A practical diagnostic strategy helps to make the correct diagnosis.

5 Marek s disease - Broilers Also young breeders and layers

6 Marek s Disease in Broilers Clinical presentation Immunosuppression Skin lesions at slaughter Lymphoma at slaughter Paralysis classic presentation Transient paralysis Causes Errors in vaccine administration Emergence of new challenge strain

7 Marek s Disease Immunosuppression Occurs at a young age Difficult to confirm Assume - if problems exist in Marek s protection Marek s Disease Control

8 Condemnations Skin Lymphosarcoma

9 Marek s Disease Paralysis Range Paralysis - Classic Transient Paralysis

10 Broilers or Young Breeders & Layers History and Epidemiology Age of birds affected Date first observed Breeder flock Hatchery source Farm history Necropsy Examine multiple birds Record the incidence and severity of lesions Examine skin, viscera, and nerves Photograph representative lesions cell phone cameras

11 Broilers or Young Breeders & Layers Vagus nerve Histopathology Skin lesions with 1 cm margin Nerves from each chicken, Vagus, brachial plexus, and sciatic Select enlarged nerves if possible Tumors Spleen, liver, kidney, gonad, brain, and proventriculus from each chicken One eye from each affected chicken Use neutral-buffered formalin! 10 formalin:1 tissue Pelvic plexus and sciatic

12 Broilers or Young Breeders & Layers FTA Cards If possible, collect from euthanized affected chicken Label the cards!! Enlarged feather follicle Feather pulp Tumors blot the central portion Whirl-Pak bags (optional) Feather pulp, skin lesions, and tumors in separate bags Ice for transport Freeze to hold for testing. Store in a freezer with no automatic defrost, or on dry ice (CO2), or liquid nitrogen

13 Marek s Disease in Breeders and Layers

14 Tumor diagnosis in breeders and layers Tumor or a non-neoplastic lesion? Lymphoma? Marek s disease, avian leukosis, or reticuloendotheliosis Spontaneous lymphoma with no viral association Not lymphoma but viral etiology? Myelocytomatosis, histiocytic sarcoma, hemangiosarcoma Lymphoma : Common lesion with different etiologies, each requiring different prevention and control responses Diagnosis is possible if the correct specimens are available.

15 Tumor diagnosis in breeders and layers History and Epidemiology Age of birds affected Date first observed Breeder flock Hatchery source Farm history Clinical Examination Select cull or sick chickens With gloved hand, palpate the bursa of Fabricius through the dorsal wall of the cloaca Chickens with enlarged bursa are valuable for necropsy If all bursae are normal, proceed with necropsy

16 Tumor diagnosis in breeders and layers Necropsy Examine multiple birds Record the incidence and severity of lesions Examine skin, viscera, and nerves, brain, bursa of Fabricius, thymus, and eyes Photograph representative lesions cell phone cameras

17 Tumor diagnosis in breeders and layers Histopathology Skin lesions with 1 cm margin Nerves from each chicken, Vagus, brachial plexus, and sciatic Select enlarged nerves if possible Tumors Spleen, liver, kidney, gonad, brain, and proventriculus, bursa, (thymus) from each chicken One eye from each affected chicken Use neutral-buffered formalin! 10 formalin:1 tissue Marek s - ocular lymphoma Avian leukosis bursa intrafollicular lymphoma

18 Lesion distribution Tissue Marek s Leukosis Reticuloendotheliosis Spontaneous Lymphoma Viscera Eye (-) Nerve + - (+) (-) Bursa Interfollicular (initial) Intrafollicular (initial) Intrafollicular (initial) Brain (-) (-)

19 Breeders, 38 Weeks Eye - Iris Liver Liver Eye - Iris

20 Brain Breeder 40wks Liver Ovary Eye

21 Lymphoid tumors nerve and eye not received Spleen Ovary

22 Tumor diagnostics in breeders and layers FTA Cards If possible, collect virus specimens from euthanized affected chickens Tumors blot the incised central portion Enlarged feather follicle tissue Feather pulp Label the cards!! Whirl-Pak bags (optional) Feather pulp, skin lesions, and tumors in separate Whirl-Pak bags Place on ice for transport Freeze to hold further testing. Store in a freezer without automatic defrost, or on dry ice (CO2), or liquid nitrogen.

23 Tumor diagnostics in breeders and layers FTA Cards If possible, collect from euthanized affected chicken Label the cards!! Enlarged feather follicle Feather pulp Tumors blot the central portion Whirl-Pak bags (optional) Feather pulp, skin lesions, and tumors in separate bags Ice for transport Freeze to hold for testing. Store in a freezer with no automatic defrost, or on dry ice (CO2), or liquid nitrogen

24 Tests and Procedures Standard Gross pathology Histology Impression smear cytology Autolysis decreases cytological detail; however, if sufficient tissues are available (eye, nerve, bursa), the lesion distribution can support a diagnosis Advanced Immunohistochemistry DNA Analysis PCR Virus Isolation Serology

25 Lymphosarcoma = Avian Leukosis Chicken B-cell IHC (Bu-1) Bu-1 B-Cell MAb CD4 T-Cell MAb S. B. Lockaby

26 Marek s Disease Neuritis IHC Bu-1 B-Cell MAb CD3 T-Cell MAb S. B. Lockaby

27 Tumor virus detection and diagnosis Dual or multivalent infections are common Exogenous and endogenous retroviruses Negative tests = negative Positive tests = Identification of virus by sequencing Marek s disease vaccine virus infects for life Positive tests = expected result (vaccine) Viruses differentiated by specific primers or sequencing Tumors have more virus than normal tissue Tumor viruses MUST be differentiated by exclusion. Dr. Guillermo Zavala

28 Frequently Asked Questions AAAP Tumor Virus Committee 2012

29 Vaccination Current practices for broilers HVT HVT+SB1 (very virulent MDV challenge) Current practices for large broilers with severe challenge HVT+CV1988 (Rispens), or HVT+SB1+CV1988 (Rispens) Current practices for breeders and layer HVT+CV1988 (Rispens) With vv+mdv challenge, HVT+SB1+CV1988 (Rispens) Double vaccination (variable application) Effective if second application of higher protective vaccine Recommended: (1) in ovo + (2) 1 day -- use vaccine in hatchery

30 Vaccine Failure - Audit Checklist Cold management of vaccine Vaccine titration specification and procedure Measure vaccine DNA in feather pulp of vaccinated chicks Quantitative PCR Monitor early challenge with field viruses Pathotyping of prevalent field strains Immunosuppression differential Marek s disease Chicken infectious anemia Infectious bursal disease Reovirus Mycotoxins Stress

31 Acknowledgements Dr. Gregorio Rosales Dr. Guillermo Zavala Dr. Susan Lockaby

32 Thank You