Detection of lymphoproliferative disease virus in domestic and wild turkeys through RNAscope in situ hybridization and immunohistochemistry

Diagnosis of lymphoid hyperplasia and neoplasia due to lymphoproliferative disease virus (LPDV; <i>Retroviridae</i>, <i>Alpharetrovirus</i>) infection in turkeys is challenging due to histopathologic similarities with nonspecific inflammation and cellular responses to other retroviral infections. Localization of LPDV RNA or protein antigens within affected tissues, which has previously not been shown, would allow for more definitive diagnoses. We evaluated formalin-fixed paraffin-embedded tissues from 32 turkeys, including 15 naturally infected wild turkeys, 11 experimentally infected domestic turkeys, and 6 uninfected wild and domestic turkeys, using RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC). ISH probes targeted a segment of the <i>gag</i> gene, and IHC antibodies were designed to recognize the capsid protein. Tissues from 4 infected turkeys were subjected to concurrent ISH and IHC labeling. All infected turkeys had ISH and IHC cytoplasmic and/or nuclear labeling of lymphocytes in at least one tissue, including within lymphoid tumors. ISH labeling was widely scattered in lymphocytes, whereas IHC labeling distribution was more limited. Spleen consistently exhibited the strongest and most widespread ISH and IHC labeling in both wild and domestic turkeys. Labeled lymphocytes typically were localized to splenic germinal centers and around periarteriolar lymphoid sheaths in the thymic cortex. Lymphocytes occasionally had simultaneous ISH and IHC labeling in selected cases. No uninfected turkeys had ISH or IHC labeling. Our 2 methods of LPDV detection in formalin-fixed paraffin-embedded tissues can aid in distinguishing lymphoid proliferation due to LPDV from other etiologies and further characterize pathogenesis.