Journal of Aquatic Animal Health 11:283-289, 1999@ Copyright by the American Fisheries Society 1999

Pathology of Fibropapillomatosis inOlive Ridley Turtles Lepidochelys olivacea Nesting in Costa Rica

A. ALONSOAGUlRRE*'Joint Institute for Marine and Atmospheric Research, University of Hawaii,

2570 Dole Street, Honolulu, Hawaii 96822-2396, USA

TERRY R. SPRAKER

State VeterinaryDiagnostic Laboratory, Colorado State University,Fort Collins, Colorado 80523, USA

ANNY CHAVES AND LESLIE DU TOIT

Douglas Robinson Marine Turtle Research Center,Ostional, Costa Rica

WHITNEY EURE

2449 Laurens Road, Greenville, South Carolina 29607, USA

GEORGE H. BALAZSNational Marine Fisheries Service, Southwest Fisheries Science Center,

Honolulu Laboratory, 2570 Dole Street, Honolulu, Hawaii 96822-2396, USA

Abstract.-Fibropapillomatosis (FP) is a neoplastic dis-ease that primarily affects green turtles Chelonia mydasin epidemic proportions worldwide. Although several in-fectious agents (herpesvirus, retrovirus, and papilloma-virus) have been associated with the condition, the etio-logic agent has not been isolated or characterized. Re-cently, FP has been reported in other sea turtle speciesincluding confirmed cases in loggerhead turtles Carettacaretta in Florida and field observations in olive ridleyturtles Lepidochelys olivacea in the Pacific coasts of Mex-ico and Costa Rica. Skin and tumor specimens were col-lected from 72 olive ridley turtles nesting in OstionalWildlife Refuge, Costa Rica, between July and September1997. In all, 50 tumor biopsies were examined from 25of the affected turtles. In addition, six biopsies were ex-amined from five turtles that did not have visible massesand served as controls. Grossly, masses were 25 mm orless in diameter, white to gray, smooth to verruciform,raised tumors of the integument of the neck and flippers.Histologically, 42 of 50 were diagnosed as fibropapillo-mas and eight were classified as chronic active dermatitisand not tumors. Twenty of 42 fibropapillomas were instages of regression and 9 of the remaining 22 tumorshad histological changes that suggested early degenera-tion within the tumor. During field surveys based on grosslesions, prevalences of 1-10% have been reported in thisnesting population. This is considered the first histopath-ologic confirmation of FP in olive ridley turtles.

* Corresponding author: aguirre@wpti.orgI Present address: Tufts University School of Veteri-

nary Medicine, Wildlife Clinic, 200 Westboro Road,North Grafton, Massachusetts 01536, USA.

Received September 16, 1998; accepted April I, 1999

Fibropapillomatosis (FP) is a disease character-ized by multiple cutaneous and internal tumors,ranging from 0.1 cm to more than 30 cm in di-ameter, that has primarily been reported in greenturtles Chelonia mydas worldwide (Jacobson et al.1989; Balazs and Pooley 1991; Aguirre et al. 1994;Herbst 1994; Williams et al. 1994; Adnyana et aI.1997). The disease has a circumtropical distribu-tion and has been observed in all major oceans;where present, prevalence varies among locations,ranging from as low as 1.4% to as high as 90%(Herbst 1994). Several infectious agents have beenidentified as associated with 'the tumors, includingretroviruses (Casey et al. 1997), herpesviruses (Ja-cobson et al. 1991; Herbst et al. 1995; Quacken-bush et al. 1998; A. A. Aguirre and T. R. Spraker,unpublished data), and papillomaviruses (Y. Lu,University of Hawaii, unpublished data), yet theprimary etiologic agent remains to be proven.

The olive ridley turtle Lepidochelys o/ivacea isthe most abundant species of marine turtle in theworld as a result of continuous massive nestings("arribadas") and their recent protection. Thelargest nesting aggregation in the world occurs inthe Orissa, India, followed by three major sites inthe eastern Pacific off the coasts of Mexico and

Costa Rica. As many as 100,000 females may nestin one arribada in Ostional, Costa Rica. The spe-cies is classified as endangered by the IUCN RedData Book (Pritchard 1997).

283

284 AGUIRRE ET AL.

Causes of disease or natural mortality are mostlyunknown for olive ridley turtles. The first reportof fleshy tumors on the head, neck, and front flip-pers of nesting females occurred in Costa Rica in1982 (Cornelius and Robinson 1983). The first sus-pected case ofFP in the Ostional olive ridley turtlepopulation was reported during the arribada ofOctober 1987. A nesting female was photographeddemonstrating grossly multiple cutaneous tumorswith the largest measuring 30 mm in diameter.Since that time, and based on field observations,both the prevalence and the size of skin tumors inindividual animals have increased. The objectiveof the present study was to describe the histopa-thology of skin masses surgically removed fromadult female olive ridley turtles nesting at OstionalWildlife Refuge and to determine whether or notthey were fibropapillomas.

Methods

An intense field survey was undertaken on Os-tional beach between July and September 1997.Turtles were sampled during the arribadas oc-curring during those months. Turtles were mea-sured and tagged following techniques previouslydescribed (Lutz and Musick 1997). All turtleswere thoroughly examined for the presence of FPand a description of mass size, number, and lo-cation was recorded. Biopsies were collectedfrom fibropapillomas of neck and flippers witheither a disposable 6-mm Baker's dermal punch(Webster, Inc., Sterling, Massachusetts) or by ex-cisional biopsy. Pigmentation, surface, attach-ment, and location of growths were Tecorded. Inaddition, normal skin biopsies were taken fromthe left brachial and neck regions of clinicallyhealthy turtles, which served as controls. Skinand tumor areas were thoroughly prepared withBetadine solution before surgical excision. A top-ical double antibiotic ointment was applied post-biopsy with wounds left open for drainage. Allmasses and skin biopsies were immersed in 10%neutral phosphate-buffered formalin. In the lab-oratory, tissues were embedded in paraffin, sec-tioned to 5-6-fJ.m thick, and stained with hema-toxylin and eosin, Masson's trichrome stain, andGomori methenamine-silver nitrate stain for the

identification of fungi.

Results

Field Observations

An average of 300,000 turtles nest at Ostionaleach month. It is calculated that approximately6-10% of these nesting females present cutane-

ous FP with 1% being severely affected. Duringthe most recent surveys performed by two of us(A.C. and L.d.T.), 30 of 300 (January 1998) and5 of 125 (February 1998) turtles were grosslyidentified with FP. Field observations providedevidence that the size and number of tumors has

increased on individual turtles among seasons.For example, between 1991 and 1993, the averagesize of tumors found was 10 mm in diameter with

an average number of three per turtle. In 1997,the average size of tumors observed was 25 mmin diameter, with the largest measuring 140 mm,and the average number of tumors per turtle wasmore than 20 (A. Chaves and L. du Toit, unpub-lished data).

Gross Pathology

Skin and tumor specimens were collected from72 nesting female olive ridley turtles for histo-pathologic and virologic studies; from these, 50tumor biopsies were examined from 25 of theaffected turtles. In addition, six biopsies were ex-amined from five turtles that did not have visiblemasses and served as controls. Mean :t SE curvedcarapace length for all 30 females with and with-out FP was 68.2 :t 0.5 cm (range, 63-73 cm) andcurved carapace width averaged 73 :t 0.5 cm(range, 67-77 cm); they appeared strong, in goodbody condition, and were actively depositingeggs. All turtles were lightly affected, demon-strating single or multiple masses measuring from1 to 34 cm, except for two turtles that were se-verely affected with FP. Tumors were observedin the front flippers (25 turtles), neck (6 turtles),carapace margins (6 turtles), eyes (3 turtles), andseams and scutes (2 turtles). Gr.ossly, tumors werefrom 5 mm to several cm in diameter, white togray (pigmented), smooth to verruciform, wellcircumscribed, and pedunculated masses on theintegument of the neck and front flippers. Bothof the heavily affected turtles presented multi-lobulated, cauliflower-like masses, some with ul-cerated or necrotic tissue. None of the turtles ex-

amined presented tumors in the rear flippers orthe tail area.

Histopathology

Epidermis of skin biopsies (5) from control tur-tles was 5-8 cells in thickness and had no epi-dermal or dermal pathology (Figure 1). In all, 50tumor biopsies 30 mm or less in diameter wereexamined histologically from 25 other turtles.Numerous histological features of the epidermis,dermis, and subcutaneous tissues were evaluated

COMMUNICA nONS 285

FIGUREI.-Light micrograph of normal skin stainedwith hematoxylin and eosin from olive ridley turtle nest-ing in Ostional-Wildlife Refuge, Costa Rica. Observethe uniformity in the thickness of the epidermis ap-proximately 6-7 cells thick. The thin layer of superficialkeratin, the interweaving of mature collagen of the der-mis, and pigmentation are normal. Bar = 333 /-Lm.

(Tables 1, 2). In all, 42 of 50 masses were di-agnosed as fibropapillomas based on criteria pre-viously published (Jacobson et al. 1989; Aguirreet al. 1994). Eight of the 42 masses that weregrossly classified as FP were histologically di-agnosed as small foci of chronic active dermatitisand not tumors. These areas of chronic active

dermatitis were characterized by mild acanthosis(hyperplasia -of the stratum spino sum) and or-throkeratotic hyperkeratosis (thickening of thestratum corneum) but not pseudoepitheliomatoushyperplasia. The epidermis covering the tumorswas characterized by acanthosis from 10 to 15cells in thickness. A mild to moderate degree oforthrokeratotic hyperkeratosis and a mild to mod-erate degree of pseudoepitheliomatous hyperpla-

FIGURE2.-Light micrograph of cutaneous fibropa-pilloma tissue section stained with hematoxylin and eo-sin from olive ridley turtle. The epidermis is character-ized by mild orthrokeratotic hyperkeratosis and mod-erate acanthosis. The dermis is composed of spindle cellsforming haphazard fascicles. Bar = 133 /-Lm.

sia characterized by elongation of rete ridges in-terdigitating with the underlying n~oplastic con-nective tissue of the dermis were observed (Fig-ure 2). Mild intercellular (spongiosis) andintracellular edema were identified within the epi-dermis. In areas of extensive spongiosis a milddegree of cytoplasmic vacuolar degeneration wasobserved. Mild individual cellular necrosis and

degeneration associated with a mild infiltrationof lymphoid cells were observed in the stratumbas ale. Mitotic figures were not observed. Smallto extensive areas of necrosis associated with un-

derlying inflammation were found within the epi-dermis. Margination of chromatin with intranu-clear inclusion bodies was not found except inone tumor that had a few squamous epithelialcells that contained intranuclear bodies of viral

286 AGUIRRE ET AL.

TABLE I.-Histopathological changes of the epidermisobserved in 42 integumental fibropapilloma biopsies from

olive ridley turtles nesting at Ostional, Costa Rica.

Histopathological change

Papillar patternLinear patternAcanthosisOrthrokeratotic hyperkeratosisPseudoepitheliomatous hyperplasiaIntercellular edemaIntracellular edema

Cytoplasmic vacuolar degenerationIndividual cell necrosisNecrosis (full depth) with underlying

inflammationLymphocytic infiltration, st. basaleBacteria on surface of epidermisFungus and algae on surface of epidermisMites or amphipods on surface of epidennis

Number (%)

19 (45)21 (50)41 (98)39 (93)39 (93)23 (38)13 (31)13 (31)2 (5)

6 (14)35 (83)33 (79)0(0)

30 (71)

FIGURE3.-Light micrograph of fibroblastic portionof a cutaneous fibropapilloma from olive ridley turtle.This portion of the tumor is composed of plump spindlecells forming a relatively haphazard arrangement of fas-cicles. The degree of cellularity is moderate. Bar = 66fLm; stained with hematoxylin and eosin.

inclusions. Infiltration of lymphoid cells wascommonly observed within the stratum basale.

The portion of the tumor within the dermis pre-dominated in most tumors and was characterized

by a proliferation of plump spindle-shaped cellsof fibroblastic or mesenchymal origin (Figure 3).Two patterns of growth were observed. Papillary,the most common pattern, was characterized bypapillary projection of epidermal-and fibroblastictissue over a fibrovascular stalk or core in largertumors. The second pattern, linear, was a moreflattened pattern of dermal proliferation that ap-peared to be just beneath the epidermis in smallertumors. This linear pattern was also present aroundthe edges of most papillary tumors.

Three patterns of cellular architecture of the fi-broblastic portion of the tumors were observed in-

FIGURE4.-Light micrograph of fibroblastic portionof a cutaneous fibropapilloma, interpreted as regressing,from olive ridley turtle. Note the extensive inflammationcharacterized by lymphoid infiltration with loss of neo-plastic fibroblastic tissue. Bar = 66 fLm; stained withhematoxylin and eosin.

COMMUNICATIONS 287

cluding a relatively haphazard arrangement ofwhorls and fascicles of proliferating cells, sheetsof cells, and interweaving bundles of spindle-shaped fibroblastic cells. In some areas the inter-weaving bundles were thin and tightly interwoven,whereas in other areas the interweaving fascicleswere in wide bands. Often all three patterns couldbe found in the same tumor, especially if it was15 mm or larger in diameter. The degree of dermalcellularity was relatively low in most tumors andmoderate in approximately one-third. Mitotic fig-ures were not observed. A mild to moderate degreeof neovasculation was found in all tumors. Thesevessels were often cuffed by a thin layer of lym-phocytes. Melanin was found to some degree inmost tumors.

Masson's trichrome stain verified the presenceof collagen in tumors. Granulomas surrounded byminimal inflammation and containing blood trem-atode eggs were occasionally observed within tu-mors. Keratine "pearls" were characterized bysmall circular foci of keratin surrounded by a thinlayer of epidermal cells. The margins of most tu-mors (83%) showed evidence of spread by expan-sion and not infiltration into surrounding tissues.Twenty of 42 tumors demonstrated an immuno-logically mediated reaction characterized bymarked neovascularization and inflammation as-sociated with extensive lymphoid infiltration andloss of fibroblastic tissue (Figure 4). Four of thesebiopsies were collected from sessile, smooth, pe-dunculated tumors. Nine of the remaining 22 tu-mors presented early changes of degeneration.Changes in the underlying dermis or subcutis werecharacterized by mild to moderate lymphoid cuff-ing of vessels. Occasionally granulomas contain-ing spirorchid trematode eggs were observed with-in the underlying subcutaneous tissues. Fibroblast-ic hyperplasia was a feature of the underlying con-nective tissue (Table 2).

Discussion

The present study reports the first histopatho-logic confirmation of FP in olive ridley turtles.Smith and Coates (1938) first reported FP on agreen turtle captured at Key West, Florida, in 1934.A detailed description was made suggesting a pos-sible viral etiology. The same year a brief reportfollowed from the Tortugas (Lucke 1938:92-94).Then, Schlumberger and 'Lucke (1948) first de-scribed internal tumors from specimens collectedat the Dry Tortugas.

Since the early 1980s, FP has reached epidemicproportions throughout the world. Detailed his-

TABLE 2.-Histopathological changes of the dermis andsubdermis-subcutis observed in 42 integumental fibropap-ilIoma biopsies collected from olive ridley turtles nestingat Ostional, Costa Rica.

Histopathologic change

Dermis

Number (%)

Fibroblastic proliferation patternsHaphazardSheets

Interweaving bundlesCellularity of tumor

LowModerate

Neovascularization in tumorLowModerate

Vessels cuffed with lymphocytesFoci lymphocytic inflammationPigment, melaninGranulomas containing parasitic ovaKeratin pearlsMargins spread by expansionRegression

19 (45)13 (31)3 (7)

28 (67)13 (31)

14 (33)27 (64)36 (86)32 (76)24(57)

7 (17)2 (5)

35 (83)20(48)

Subdermis-subcutis

Vessels cuffed with lymphocytesGranulomas containing trematode eggs

33 (79)6 (14)

topathologic description of cutaneous FP has beenmade for green turtles in the Caribbean (Williamset al. 1994), Florida (Jacobson et al. 1989), theHawaiian Islands (Aguirre et al. 1994), and In-donesia (Adnyana et al. 1997). Gross evidence ofFP has been also reported in loggerhead turtles(Caretta caretta), olive ridley turtles (Lepidochelysolivacea), Kemp's ridley turtles (L. kempii), andflatback turtles (Natator depressus) (Limpus andMiller 1994). From these, histopathologic confir-mation of FP has occurred for loggerheads and astranded Kemp's ridley turtle from Florida (J. C.Harshbarger, George Washington University Med-ical Center, unpublished data).

The basic histological features of FP in oliveridley turtles presented many similarities to theones described in Hawaiian green turtles (Aguir-re et al. 1994). One major difference, however,was that 48% of the tumors (20 of 42) from theolive ridleys had extensive areas of lymphocyticinflammation within fibroblastic tumor tissue,and nine of the remaining tumors had histologicchanges of inflammation with mild degenerationwithin the tumor. This is in comparison to 2%(1 of 52) of the tumors from juvenile green tur-tles similarly collected during field situations inthe Hawaiian Islands (Aguirre et al. 1994, 1998).These histological changes suggest regression.Similar structural changes have been described

288 AGUIRRE ET AL.

in canine cutaneous histiocytomas that includednecrosis of tumors with marked limphocytic in-filtration (Pulley and Stannard 1990). One otherfactor that may playa role in the state of re-gression is the possible viral etiology of FP. Itis common to find inflammation in the virus-

induced papillomas of young cattle, horses, anddogs. This inflammation is believed to be as-sociated with regression (Schneider 1978). Ifthese tumors in nesting olive ridley turtles werein stages of regression, this may explain why 8of the 50 surgically removed masses that grosslywere suspected to be tumors were instead areasof inflammation (chronic dermatitits). Anothermarked difference observed between turtles ofCosta Rica and the Hawaiian Islands was related

to age and sex of turtles collected. Generally,turtles captured in Hawaii were juvenile or sub-adult turtles (40-80 cm in standard carapacelength) of both sexes. During the Costa Ricanstudy, only nesting females were sampled. Adultturtles may be more resistant to FP or might havesurvived the infection with their tumors in stagesof regression. Further controlled studies will an-swer some of these questions.

The appearance of this disease in olive ridleyturtles constitutes an added threat to sea turtles of

the Pacific. The disease has been reported in otherolive ridley populations, even though encouragingsigns of recovery of the species have been ob-served worldwide. Biopsy specimens from six ol-ive ridley turtles from Ostional were subjected tomolecular study using the seminested PCR (poly-merase chain reaction) assay with degenerateprimers. A herpesvirus DNA polymerase sequencewas identified in four tumors (Quackenbush et al.1998). Further studies are underway including ef-forts to isolate and characterize the etiologic agentto provide possible insights for the epidemiology,control, treatment, and prevention of this disfig-uring and debilitating disease.

Acknowledgments

Olive ridley turtle biopsies were collected andtransported between Costa Rica and the UnitedStates under CITES permit U.S.786600 issued tothe National Marine Fisheries Service and CITESpermit CR912085 to the Douglas Robinson MarineTurtle Research Center. The support of S. Abbott-Stout, S. K. K. Murakawa, and D. M. Parker isgreatly appreciated.

References

Adnyana, W., P. W. Ladds, and D. Blair. 1997. Obser-vations of fibropapillomatosis in green turtles (Che-lonia mydas) in Indonesia. Australian VeterinaryJournal 75:737-742.

Aguirre, A. A., G. H. Balazs, B. Zimmerman, and T. R.Spraker. 1994. Evaluation of Hawaiian green turtles(Chelonia mydas) for potential pathogens associatedwith fibropapillomas. Journal of Wildlife Diseases30:8-15.

Aguirre, A. A., T. R. Spraker, G. H. Balazs, and B.Zimmerman. 1998. Spirorchidiasis and fibropapil-10matosis in green turtles from the Hawaiian Is-lands. Journal of Wildlife Diseases 34:91-98.

Balazs, G. H., and S. G. Pooley, editors. 1991. Researchplan for marine turtle fibropapilloma. NOAA Tech-nical Memorandum NMFS-SWFSC-156.

Casey, R. N., and five coauthors. 1997. Evidence ofretrovirus infections in green turtles Chelonia mydasfrom the Hawaiian Islands. Diseases of Aquatic Or-ganisms 31:1-7.

Cornelius, S. E., and D. C. Robinson. 1983. Abundance,distribution, and movements of olive ridley sea tur-tl.es in Costa Rica, III. U.S. Fish and Wildlife Ser-vice, Endangered Species Repo.rt 13, Albuquerque,New Mexico.

Herbst, L. H. 1994. Fibropapillomatosis of marine tur-tles. Annual Review of Fish Diseases 4:389-425.

Herbst, L. H., and five coauthors. 1995. Experimentaltransmission of green turtle fibropapillomatosis us-ing cell-free tumor extracts. Diseases of AquaticOrganisms 22: 1-12.

Jacobson, E. R., and seven coauthors. 1989. Cutaneousfibropapillomas of green turtles (Chelonia mydas).Journal of Comparative Pathology 101:39-52.

Limpus, C. J., and J. D. Miller. 1994. The occurrenceof cutaneous fibropapillomas in marine turtles inQueensland. Pages 186-188 in R. James, compiler.Proceedings of the Australian marine turtle conser-vation workshop. Queensland' Department of En-vironment and Heritage and The Australian NatureConservation Agency, Brisbane, Australia.

Lucke, B. 1938. Studies on tumors in cold-blooded ver-

tebrates. Carnegie Institution, Annual Report of theTortugas Laboratory, Washington, D.C.

Lutz, P. L., and J. A. Musick, editors. 1997. The biologyof sea turtles. CRC, Marine Science Series, BocaRaton, Florida.

Pritchard, P. C. H. 1997. Evolution, phylogeny, and cur-rent status. Pages 1-28 in P. L. Lutz and J. A. Mu-sick, editors. The biology of sea turtles. CRC, Ma-rine Science Series, Boca Raton, Florida.

Pulley, L. T., and A. A. Stannard. 1990. Tumors of theskin and soft tissue. Pages 23-82 in J. E. Moulton,editor. Tumors in domestic animals, 3rd edition.University of California Press, Los Angeles.

Quackenbush, S. L., and ten coauthors. 1998. Threeclosely related herpes viruses are associated withfibropapillomatosis in marine turtles. Virology 246:392-399.

COMMUNICA nONS 289

Schlumberger, H. G., and B. Lucke. 1948. Tumors offishes, amphibians, and reptiles. Cancer Research8:657-753.

Schneider, R. 1978. General considerations. Pages 1-15 in J. E. Moulton, editor. Tumors in domesticanimals, 2nd edition. University of California Press,Los Angeles.

Smith, G. M., and C. W. Coates. 1938. Fibro-epithelialgrowths of the skin in large marine turtles, Cheloniamydas (Linneaus). Zoologica (New York) 23:93-98.

Williams, E. H., Jr., and ten coauthors. 1994. An epi-zootic of cutaneous fibropapillomas in green turtlesChelonia mydas of the Caribbean: part of a pan-zootic? Journal of Aquatic Animal Health 6:70-78.