Development of reliable techniques for the differential diagnosis of avian tumor viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections

Authors: AHMED, HUSNAIN - Michigan State University; MAYS, JODY - US Department Of Agriculture (USDA); KIUPEL, MATTI - Michigan State University; DUNN, JOHN - US Department Of Agriculture (USDA)

Submitted to: Avian Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2018
Publication Date: 4/11/2018
Citation: Ahmed, H., Mays, J., Kiupel, M., Dunn, J.R. 2018. Development of reliable techniques for the differential diagnosis of avian tumor viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections. Avian Pathology. 47(4):364-374. https://doi.org/10.1080/03079457.2018.1451620.
DOI: https://doi.org/10.1080/03079457.2018.1451620

Interpretive Summary: There are three avian tumor viruses that can cause development of tumors in poultry. A variety of methods have been developed to diagnose which virus is causing disease, but the ideal samples for diagnosis are not always available. In many cases, the only available sample is a paraffin block, which contains formalin-fixed paraffin-embedded tissues (FFPE). We developed new reagents optimized for this type of tissue and generated a collection of tumors induced by the three avian tumor viruses. The results using these optimized techniques and newly designed reagents were highly specific and will be valuable for future research as well as in diagnostic facilities for differential diagnosis of avian tumors from FFPE tissue sections.

Technical Abstract: A variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek’s disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek’s disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analyzed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek’s disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.