Characterization of Alabama Red Leg Lesions Induced by Marek’s Disease Virus

Brandon T. Armwood, Aneg L. Cortes, Arun K. R. Pandiri, Isabel M. GimenoCollege of Veterinary Medicine, North Carolina State University

ObjectiveThe objectives of this study were to evaluate the incidence of ARL in infected meat type chickens and to characterize the histopathological features, immunophenotypes of the infiltrative cells, and MDV transcripts in ARL lesions.

Materials and methods

Discussion

References1.Cho, K.O. et al. Avian Pathol. 25:325-343. 1996.2.Cho, K.O. et al. J.Vet.Med.Sci. 60:843-847. 1998.3.Cui, Z. et al. Acta Virol. 43:169-173. 1998.4.Gimeno, I.M. et al. Avian Pathology 34:332-340. 2005.5.Gimeno, I.M. et al. Avian Pathology 39:71-79. 2010.6.Gimeno, I.M. et al. Marek’s disease. In: AAAP slides study sets. AAAP. 2003.7.Lee, L.F. et al. J.Immunol. 130:1003-1006. 1983.8.Pandiri, A.K. et al. Avian Diseases 52:572-580. 2008.9.Payne, L.N., and P.M. Biggs. J.Natl.Cancer Inst. 39:281-302. 1967.10.Witter, R.L. Avian Dis. 41:149-163. 1997.

Incidence of skin lesions in shanks. 14% of the MDV challenged chickens developed gross lesions in the shanks; 45% of them had bright red skin in the legs (Figure B) and 55% skin tumors (Figure C). Histopathology and cell phenotype: Histopathological evaluation of the ALR demonstrated edema and severe infiltration of mononuclear cells with plasma cell-like morphology and marked cellular fragmentation (Figures B1-B4). Lesions observed in ARL differ greatly from those detected in the shanks with skin tumors (Figure C1-C2).

Results

A B C

A1

A2

B1

B2

B3

B4

C1

C2

Cell infiltrates, viral antigen expression and viral transcripts (Table 1). MDV-induced tumors consisted on CD4+ cells, had low expression of MDV pp38 antigens, and high levels of meq transcripts. By contrast, ARL lesions had fewer CD4+ infiltrates cells, higher expression of pp38 antigen and lower level of meq transcripts than tumors. MDV DNA load present in the lesions was very similar for both ARL and tumor lesions

Figure A: Uninfected control chicken. Skin on the shank is yellow and glistening; A1 (20X) and A2 (40X): H&E stained section of the shank skin from image A demonstrating a thick cornified uniform epidermis and a dermis consisting of loose fibrous connective tissue stroma with scant cellularity. Figure B: ARL in a MDV-infected chicken. The shanks from the same live chicken had varying degrees of cutaneous erythema and edema. B1 (20X) and B2 (40X) H&E stained section of the less severely affected shank skin demonstrating a raised papule due to an expanded edematous dermis with numerous capillaries distended with blood and minimal dermal inflammatory infiltrate. B3 (20X) and B4 (40X): H&E stained section of the more severely affected shank skin demonstrating a markedly expanded dermis with abundant lymphoplasmacytic infiltrate. Figure C: MDV-induced skin tumors in the shank. C1 (20X) and C2 (40X): H&E stained section shank in image C demonstrating marked expansion of the dermis with a very heterogeneous, pleomorphic, mononuclear cell population that is characteristic of an MDV lymphoma. This neoplastic cell population is distinct from the predominantly inflammatory cell infiltrate see in figure B1-4.

Feature

ARL Tumors

Cell infiltrates CD4+ 1+ 3+Viral antigen expression

pp38 2+ 1+

gB - -

Viral transcripts

ICP4 6.70E+03 5.90E+04pp38 7.80E+03 1.80E+03meq 9.60E+06 1.00E+08gB 3.90E+01 1.70E+01

MDV DNA load pp38-onc 2.12E+02 3.73E+02

Table 1

This study demonstrated that ARL is a relatively common lesion in meat type chickens when inoculated with vv+MDV strain 645. Furthermore, ARL has unique features that differ from MDV-induced tumors. Unlike MDV-induced tumors, only few cells infiltrating ARL lesions were CD4+ cells. Furthermore, in ARL expression of pp38 antigen was higher and transcription of meq was lower than in MDV-induced tumors. ARL was characterized by hypervascularization, edema, and infiltration of plasma cells. Similar lesions have been reported in MDV-induced B type lesions in the nerves (9) , in the eye (8), and in the feather pulp of white leghorn chickens (1). Similar to those lesions, it is likely that ARL is also of inflammatory nature. However, the high level of transcription of oncogene meq, albeit lower than in MDV-induced tumors, warrant further studies. ARL lesions have been only described in meat type chickens raised in the floor. At this point, it is unknown if this is due to genetic differences between meat type and egg type chickens. However the fact that meat type chickens are raised in the floor, and a mild level of dermatitis is common, might contribute to the development of ARL. Further studies to better understand the pathogenesis of ARL are needed.

Introduction

Chickens. Female commercial broilers were used in this studyVirus. Very virulent plus (vv+) MDV strain 645 (10) and a commercial vaccine strain of serotype 3 HVT.Experimental design. 400 chickens were used in this experiment; 300 of them were vaccinated at day of age via subcutaneous with HVT and challenged by contact with very virulent plus strain 645 few hours later. The remaining 100 chickens were vaccinated but not challenged with 645 as negative control. Chickens were evaluated for clinical signs and development of skin lesions daily. Necropsy was conducted in chickens that died before the experiment and at termination (8 weeks). Skin of the shanks were collected at termination (5 ALR and 5 tumors)Histopathology. Skin of the shanks was collected and fixed in buffered fixed formalin. Routine hematoxilin and eosin stain was conducted.Immunohistochemistry. Skin of the shanks were collected and snap freeze in liquid nitrogen as described (4). Monoclonal antibodies H19 (3), IAN 86.17 (7) were used to evaluate expression of viral antigens pp38 and gB, respectively, as reported (4). Monoclonal antibody CT4 (Southern Biotech, Birmingham, AL) was used to evaluate the immunophenotype of the infiltrates. Real time PCR and RT-PCR. Load of viral DNA in skin of the shanks as well as viral transcripts (ICP4, pp38, gB, and meq) were evaluated as reported (5).

Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by an alpha-herpesvirus, Marek’s disease virus (MDV). MD is characterized by the development of lymphoma in nerves, skin, and various viscera. MDV is able to induce a variety of lesions in the skin besides of tumors (1, 2). Pathogenesis of skin lesions have been studied mainly in SPF white leghorn chickens in feathered skin. In meat type chickens, MDV can induce a unique skin lesion in the shanks named Alabama Red Leg (6). ARL has been reported in old broilers and roasters and it does not seem to appear in white leghorn chickens. The lesions are poorly characterized. Hyperemia of the shanks that can disappeared after the chicken died has been described (6). In some more severe cases hyperemic lesions can appear combined with skin tumors in the shank (6). It is unclear if the hyperemic lesions are related or not to the development of tumors.

Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by an alpha-herpesvirus, Marek’s disease virus (MDV). MD is characterized by the development of lymphoma in nerves, skin, and various viscera. MDV is able to induce a variety of lesions in the skin besides of tumors (1, 2). Pathogenesis of skin lesions have been studied mainly in SPF white leghorn chickens in feathered skin. In meat type chickens, MDV can induce a unique skin lesion in the shanks named Alabama Red Leg (6). ARL has been reported in old broilers and roasters and it does not seem to appear in white leghorn chickens. The lesions are poorly characterized. Hyperemia of the shanks that can disappeared after the chicken died has been described (6). In some more severe cases hyperemic lesions can appear combined with skin tumors in the shank (6). It is unclear if the hyperemic lesions are related or not to the development of tumors