Abstract
Fibropapillomatosis (FP), a transmissible neoplastic disease of marine turtles characterized by a likely herpesviral primary etiology, has emerged as an important disease in green sea turtles (Chelonia mydas) over the past three decades. The objectives of this study were to determine the suitability of three different chelonid fibropapilloma-associated herpesvirus (CFPHV) gene targets in polymerase chain reaction (PCR) assays of affected tissues; to explore the presence of CFPHV in non-affected skin from turtles with and without tumors; and to better understand tissue localization of the CFPHV genome in a tumor-free turtle by evaluating CFPHV presence in microanatomic tissue sites. Two aggregations of green sea turtles (Chelonia mydas) in Puerto Rico were evaluated, with six sampling intervals over the three-year period 2004–2007. Primary and nested PCR for three different herpesviral gene targets- DNA polymerase, capsid maturation protease, and membrane glycoprotein B- were performed on 201 skin biopsies taken from 126 turtles with and without external tumors. Laser capture microdissection and nested PCR were used to identify tissue localizations of CFPHV in skin from a normal turtle. Of the turtles sampled in Manglar Bay, 30.5% had tumors; at the relatively more pristine Culebrita, 5.3% of turtles sampled had tumors. All three PCR primer combinations successfully amplified CFPHV from tumors, and from normal skin of both tumored and tumor-free turtles. Via nested PCR, the polymerase gene target proved superior to the other two gene targets in the positive detection of CFPHV DNA. CFPHV infection may be common relative to disease incidence, supporting the idea that extrinsic and/or host factors could play a transforming role in tumor expression. Laser capture microdissection revealed CFPHV in skin from a tumor-free turtle, harbored in both epidermal and dermal tissues. Identification of CFPHV harbored in a non-epidermal site (dermis) of a tumor-free turtle indicates that virus is latent in a non-tumored host.
Highlights
Fibropapillomatosis (FP) is a transmissible neoplastic disease of marine turtles that is characterized by a variable number of cutaneous and conjunctival growths, with occasional appearance in visceral organs
Three different primer sets were designed for three chelonid fibropapilloma-associated herpesvirus (CFPHV) genes– deoxyribonucleic acid (DNA) polymerase catalytic subunit (UL30, pol), capsid maturation protease (UL26), and membrane glycoprotein B (UL27, gB), and these nested CFPHV polymerase chain reaction (PCR) targets were confirmed according to gene fragment size
The gene targets were further verified by the results of the Clustalb multiple sequence alignments, which were conducted on the three nested PCR products- one sample of each type of nested amplicon
Summary
Fibropapillomatosis (FP) is a transmissible neoplastic disease of marine turtles that is characterized by a variable number of cutaneous and conjunctival growths, with occasional appearance in visceral organs. The tumors often cause affected animals to become debilitated by hampering feeding and movement, obscuring vision, or in the case of visceral tumors, leading to organ failure (Balazs 1986; Herbst 1994; Jacobson et al 1989; Quackenbush et al 1998; Smith & Coates 1938). Visceral tumors are typically identified as fibromas, myxofibromas, or fibrosarcomas (Norton et al 1990; Work et al 2004). Visceral tumors tend to develop late in the course of disease, and are generally perceived as a more chronic lesion (Harshbarger 1991; Herbst 1994; Herbst et al 1999; Jacobson et al 1989; Lucke 1938)
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