Experimental transmission of enzootic nasal adenocarcinoma in sheep

Walsh et al. Veterinary Research 2013, 44:66 http://www.veterinaryresearch.org/content/44/1/66 RESEARCH VETERINARY RESEARCH Open Access Experimental transmission of enzootic nasal adenocarcinoma in sheep Scott R Walsh1, Nicolle M Linnerth-Petrik1, Darrick L Yu1, Robert A Foster1, Paula I Menzies2, Andrés Diaz-Méndez1,3, Heather J Chalmers3 and Sarah K Wootton1* Abstract Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus. Introduction Enzootic nasal adenocarcinoma (ENA) is a neoplasm of the secretory epithelial cells of the nose of sheep and goats [1]. ENA tumors can arise unilaterally or bilaterally, originating from the ethmoid turbinate and often expanding to occlude the nasal cavity. No metastasis has been reported in ENA cases, but disruption of the nasal septum structure as well as erosion of the cribiform plate has been reported [2,3]. Due to the cell type transformed and the space occupying nature of the tumor, the clinical signs of ENA include production of copious nasal exudate, open mouth breathing, dyspnea and facial asymmetry [1]. A neoplasm of the secretory epithelial cells of the distal lung of sheep, called * Correspondence: 1 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Full list of author information is available at the end of the article ovine pulmonary adenocarcinoma (OPA), is known to be caused by jaagsiekte sheep retrovirus (JSRV) [4] and a similar etiology is suspected for ENA [5,6]. ENA is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), which is genetically very similar to JSRV [7]. ENTV is divided into two distinct sub species, one infecting sheep (ENTV-1) [7] and the other infecting goats (ENTV-2) [8]. Studies involving ENTV are hindered by the fact that there is no cell culture system for propagating the virus. Although a causal relationship between ENTV-1 infection and the development of ENA in sheep has not been proven, reverse transcriptase activity, ENTV-1 specific nucleotide fragments and antigens that cross react with antibodies against JSRV proteins are consistently found in nasal exudate as well as nasal tumor tissue [3,5,9,10]. ENA has been shown to be infectious in goats through intrasinus or © 2013 Walsh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Walsh et al. Veterinary Research 2013, 44:66 http://www.veterinaryresearch.org/content/44/1/66 intranasal inoculation of newborn goat kids with clarified nasal exudates pooled from three ENA affected goats [11]. In 1953, Cohrs reported transmission of ENA in sheep using cell and bacteria-free tumor filtrate [12], but more recently, similar experiments attempting transmission of ENA in sheep were unsuccessful (Dr James DeMartini, personal communication). In this study, we tested the hypothesis that ENA can be induced in healthy l4-day-old lambs after exposure to nebulized cell-free tumor homogenate derived from sheep with ENA. We show that while the rate of tumor induction was low, clinical signs could be detected as early as 12 wpi. The clinical signs, histopathology, and tissue distribution of ENTV-1 provirus in experimentally and naturally infected animals were similar, thereby validating the experimental infection method used in this study and providing further support for the hypothesis that ENTV-1 is the causative agent of ENA in sheep. Materials and methods Page 2 of 13 Animals were euthanized after the onset of clinical signs or, in the absence of clinical signs, at 45 wpi. At necropsy, the nose was serially cut transversely at ~2 cm intervals and samples of normal conchae and lesions were collected. Tissue samples collected at necropsy included, trachea, lung, submandibular lymph node, liver, spleen, and kidney. These samples were divided in half and stored at −80°C for subsequent isolation of nucleic acids and protein or fixed in 10% neutral buffered formalin for 24 h prior to embedding in paraffin wax and sectioning. Computed tomography (CT) Computed tomography of the head was performed using a GE Bright Speed 16-slice helical CT scanner. The scan parameters were 120 kvp, 200 mA, 1.25 mm slice thickness, and 0.75 pitch. For the CT scan, lambs were anesthetized using routine veterinary methods for this species. Images were interpreted by a board certified veterinary radiologist (HC). Animals, inoculum, and sample collection The Animal Care Committee at the University of Guelph approved all animal use and related procedures. Five lambs born to dams from a research flock at the University of Guelph with no previous history of ENA were infected at 14 days of age with two mL of ENA inoculum. Two lambs were mock infected with vehicle alone and were housed with the experimen (...truncated)