Abstract

Orogenital papillomatosis and squamous cell carcinoma are significant health issues in Atlantic bottlenose dolphins (Tursiops truncatus). Since the first documented case in 1998, lesions have been reported in both free-ranging animals, as well as those under managed care.^1,2 Clinical disease progression ranges from early-stage lesions, often characterized as glossitis and mucosal ulcerations, to infiltrative neoplastic masses, indicating advanced disease.³ Successful treatment of squamous cell carcinoma in dolphins has proven difficult, and treatment is usually aimed at improving clinical presentation and reducing the chance of recurrence. Current therapeutic modalities include cryotherapy and surgical removal of discrete lesions, as well as laser therapy, oral and intralesional chemotherapy, and NSAIDs, such as piroxicam.^1,4-6 Previous studies have indicated a potential viral etiology based on PCR screening for papillomaviruses and herpesviruses and immunoassays; however, the scope of current viral diagnostics for dolphins remains limited.^7,8 Although both virus families can be involved in persistent infection and oncogenesis in other species, the role of these species-specific viruses in bottlenose dolphin tumor progression has not yet been elucidated. Developing a comprehensive understanding of this novel disease is critical in assessing individual and population health, as well as the potential role of environmental factors, such as anthropogenic stressors.

The objectives of this study were to 1) define the viruses that are potentially responsible for the development of papillomatosis and squamous cell carcinoma in dolphins, 2) compare the efficacy of swab samples and biopsy samples for virus detection, and 3) develop an immortalized dolphin cell line from primary cells obtained from biopsy tissue. Our study utilized next-generation sequencing (NGS), along with a custom bioinformatics pipeline, to screen tissue and swab samples from wild and captive dolphins. A critical advantage of NGS over PCR, in terms of virus identification, is the ability to detect novel viruses without prior knowledge of sequence information via de novo assembly. Our preliminary results indicate that gammaherpesviruses, rather than papillomaviruses, appear to be the driving factor in this disease process. As viral DNA reads were detected from oral and genital swabs using NGS, we demonstrated the efficacy of swabs as a potentially important non-invasive diagnostic and screening tool. Additionally, in order to attempt to isolate viruses from NGS-positive samples, we developed three immortalized dolphin cell lines from oral frenulum primary cell cultures via retroviral transduction. As bottlenose dolphin herpesviruses and papillomaviruses appear to be species-specific, access to immortalized dolphin cell lines is essential for viral isolation as a potential diagnostic modality. Future directions for this research include the creation of RNAscope assays with virus-specific probes, viral culture and subsequent viral characterization, and the development of a polyclonal antibody for diagnostic assays. Elucidating the etiologic agent and malignant transformation process of this disease may ultimately lead to the advancement of treatment protocols, including more targeted therapies and preventative recommendations.

Acknowledgments

The authors wish to thank Georgia Aquarium, The Dolphin Company, Marineland Dolphin Adventure, Gulfarium Marine Adventure Park, and the Sarasota Dolphin Research Program, as well as other participants, for their contributions to this study. Biological sampling was conducted under NMFS Permit No. 23802.

*Presenting author
+Student presenter

Literature Cited

1.  Renner MS, Ewing R, Bossart GD, Harris D. Sublingual squamous cell carcinoma in an Atlantic bottlenose dolphin (Tursiops truncatus). J Zoo Wildlife Med. 1999;30(4):573–576.

2.  Bossart GD, Ghim S, Rehtanz M, et al. Orogenital neoplasia in Atlantic bottlenose dolphins (Tursiops truncatus). Aquat Mamm. 2005;31(4):473–480.

3.  Walsh MT, Pelton CA, de M Souza CH, et al. Revisiting dolphin orogenital papillomatosis and squamous cell carcinoma: diagnosis, clarification and therapeutic considerations. IAAAM 48th Annual Conference Proceedings; 2017; Cancun, Mexico.

4.  McKinnie CJ, Dover SR. Diagnosis and treatment of lingual carcinoma in an Atlantic bottlenose dolphin (Tursiops truncatus). IAAAM 34th Annual Conference Proceedings; 2003; Kohala Coast, HI.

5.  McKinnie CJ, Dover SR, Ogilvie G, Bossart GD. Treatment of oral squamous cell carcinoma in Atlantic Bottlenose dolphin (Tursiops truncatus). IAAAM 32nd Annual Conference Proceedings; 2001; Tampa, FL.

6.  Duan T, March, DT, Bossart GD. Use of piroxicam as a treatment for an oral squamous cell carcinoma in a bottlenose dolphin (Tursiops aduncus). IAAAM 45th Annual Conference Proceedings; 2014; Gold Coast, Australia.

7.  Rehtanz M, Bossart GD, Fair PA, Reif JS, Ghim S, Jenson AB. Papillomaviruses and herpesviruses: who is who in genital tumor development of free-ranging Atlantic bottlenose dolphins (Tursiops truncatus)? Vet Microbiol. 2012;160(3):297–304.

8.  Rehtanz M, Shin-je G, McFee W, et al. Papillomavirus antibody prevalence in free-ranging and captive bottlenose dolphins (Tursiops truncatus). J Wildlife Dis. 2010;46(1):136–145.