Marine turtle fibropapillomatosis is associated with chelonid fibropapilloma-associated herpesvirus (C-FP-HV) and commonly affects juvenile green turtles (Chelonia mydas) in neritic (nearshore) habitats. Green turtles have a complex life history, characterized by shifts in trophic level as well as habitat during ontogeny. Thus, several hypotheses can be proposed for when turtles become infected with C-FP-HV. They may acquire the virus at an early stage in the life cycle, including prenatal, hatchling, or the posthatchling pelagic stages. Alternatively, they may become infected later in life after they emigrate from the open ocean to neritic habitats. Each hypothesis generates predictions about the spatial distribution of genetic variants of C-FP-HV among nearshore sites within a region. Sequencing of polymerase chain reaction-amplified viral DNA from fibropapillomas of individual turtles was used to genotype the viral variants present in marine turtles from different coastal areas in Florida. We found four distinct virus variants (A, B, C, and D), two of which (A and C) were present in multiple turtle species. Green turtles in Florida were infected with variants A, B, and C. Variant A was found in green turtles from all three areas. Outside the Indian River Lagoon, variant A was most commonly detected and was found in >94% of diseased green turtles and 70% of loggerhead sea turtles (Caretta caretta) in the Florida Bay/Florida Keys. However, in the Indian River Lagoon, variant B was found in >94% of affected green turtles. Variant B was not detected outside of the Indian River system. Chi-square analysis strongly supported (P<0.001) an association between viral variant distribution in green turtles and location. On the basis of the assumption that juvenile green turtles found in Florida's west-central coast, Florida Keys, and Indian River Lagoon areas represented recruits from a mixed pelagic population, we expected that the distribution of viral variants in these turtles would be relatively homogeneous among locations; this would correspond to infection in the earlier phases of their life cycle. The heterogeneous distribution of viral variants in green turtle tumors from different Florida coastal locations strongly supports the hypothesis that, during epizootics, turtles are infected with specific C-FP-HV variants after they arrive as juveniles in neritic habitats. The conclusion that C-FP-HV is acquired after turtles recruit to nearshore habitats should help focus further research efforts on understanding the mechanisms of transmission and raises the possibility that the effect of fibropapillomatosis on turtle populations might be reduced by management strategies designed to break the cycle of transmission in these locations.

Green turtle fibropapillomatosis (GTFP), characterized by multiple benign fibroepithelial tumors on the skin and eyes, has become a growing threat to green turtle Chelonja mydas populations worldwide. The cause of GTFP 1s unknown, but a viral etiology is suspected. This study investigated whether GTFP could be experimentally transmitted to young captive-reared green turtles using cell-free fibropapilloma extracts prepared from free-ranging turtles with spontaneous disease Turtles raised from eggs collected from 4 separate clutches in the wild were assigned to 4 expenmental groups and 1 control group. For each experiment a crude homogenate (33 % w/v) was prepared from fibropapillomas removed from a free-ranging turtle with spontaneous disease. The crude tumor homogenates were freeze-thawed and centrifuged to yield cell-free extracts that were used (both filtered and unaltered) for inoculation. Recipients were inoculated by intradermal injection or by scarification; control turtles were not treated but \yere housed with treated turtles. Fibropapillomas developed in all 12 turtles receiving 3 of the 4 tumor extracts, and were first detected between 15 and 43 wk post inoculation. Both filtered and unfiltered tumor extracts successfully induced tumor development. During the 10 and 12 mo monitoring periods. fibropapillomas did not develop in control turtles or in any turtles inoculated with the fourth tumor extract. Although 2 sets of experiments were performed 8 wk apart, most of the tumors in both sets became evident simultaneously after water temperatures rose Experimental tumors were h~stologically i n d~s -tinguishable from spontaneous fibropapillomas found In free-living turtles but lacked evidence of endoparasites. Scattered foci of epidermal degeneration were found in most sections of experimentally induced fibropapillomas and within some sections taken from donor turtles. Electron rnicroscopy revealed virus-like particles conforming in size, morphology, and intranuclear location with herpesvirus. Negativestaining electron microscopy of transmission-positive tumor extracts failed to demonstrate intact virus particles. This study demonstrates that the etiology of GTFP is an infectious filterable subcellular agent. The herpesvirus identified in this study is 1 possible candidate for the etiology of GTFP.

Sea turtle fibropapillomatos~s (FP) is a disease marked by proliferat~on of b e n~g n but debilitating cutaneous fibropapillomas and occasional visceral fibromas Transmission experiments have implicated a chloroform-sensltlve transforming agent present in filtered cell-free tumor homogenates in the etiology of FP In t h~s study, consensus pnmer PCR methodology was used to test the a s s o c~a t~o n of a chelonian herpesvirus with f~bropapillomatosis Fibiopap~lloma and skin samples were obtalned f~o m 17 green and 2 loggerhead turtles affected iwth FP stranded along the Flor~da coastline Ninety-three cutaneous and vlsceral tumors fiom the 19 turtles, and 33 skln samples from 16 of the turtles, were tested All turtles affected with FP had herpesvlrus associated w t h thelr tumors as detected by PCR N~nety-six percent (89/93) of the tumors but only 9 % (3/33) of the skin samples from affected turtles contained detectable herpesvirus The skin samples that contained herpesvirus were all within 2 cm of a flbropapillo~na Also. 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contalned detectable herpesvirus None of 18 normal skln samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus The data indicated that heipesvlrus was detectable only withln or close to tumors To determine if the same vlrus infected both turtle specles, partial nucleotide sequences of the herpesvlrus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples The sequences predicted that herpesviius of loggerhead turtles dlffered from those of green turtles by only 1 of 60 a m~n o acids In the sequence examined, Indicating that a chelonlan herpesvlrus exhibltlng minor intratyplc vanation was the only helpesvlrus present in tumors of both green and loggerhead turtles The FP-assoc~ated herpesvlrus reslsted cultlvat~on on chelonian cell hnes whlch support the replicat~on of other chelonian herpesvlruses These results lead to the conclus~on that a chelonian herpesvirus IS regularly associated with f~bropap~llomatosis and 1s not merely an ~ncldental flndlng in affected turtles

Abstract. Tumor biopsy samples from 25 Floridian and 15 Hawaiian green turtles (Chelonia mydas) with spontaneous green turtle fibropapillomatosis (GTFP) and from 27 captive-reared green turtles with experimentally induced GTFP were examined microscopically to differentiate the histologic features that result from GTFP pathogenesis and those that result from incidental factors that may vary according to geographic region. Common histologic features for spontaneous and experimentally induced tumors included fibroblast proliferation in the superficial dermis, epidermal acanthosis and hyperkeratosis, epidermal basal cell degeneration with dermalepidermal cleft formation, spinous layer degeneration with intraepidermal vesicle and pustule formation, and ulceration. Visceral tumors, found in eight of 10 (80%) free-ranging turtles with cutaneous disease that were examined after death, had extensive interstitial fibrous proliferation. The presence of spirorchid trematode eggs and associated foreign body granulomas, common secondary findings within spontaneous tumors, varied by geographic location, and these findings were not observed in experimentally induced tumors. Eosinophilic intranuclear inclusions and intranuclear herpesvirus-associated antigen immunoreactivity were found in 18 of 38 (47%) experimentally induced cutaneous tumors and nine of 119 (7.5%) spontaneous tumors from Floridian but not Hawaiian turtles. The possible involvement of GTFP-associated herpesvirus in the pathogenesis of epidermal degenerative changes and GTFP pathogenesis is discussed.Key words: Chelonia mydas; dermatitis; fibropapilloma; green turtles; herpesvirus; immunohistochemistry; tumor.Green turtle fibropapillomatosis (GTFP) is a disease of green turtles (Chelonia mydas) that is characterized by multiple cutaneous papillomas, fibromas, and fibropapillomas, as well as occasional visceral fibromas. Concern over recent increases in the prevalence of GTFP around the world has focused attention on identifying the cause and understanding the pathogenesis of this disease so that management strategies can be developed to minimize its impact on populations of endangered green turtles. 18 A thorough description of the disease process should identify the relevant histologic features seen in spontaneous lesions and distinguish these from features that are incidental or result from secondary processes. In addition, any proposed etiologic hypothesis should account for the major histologic features of the disease, and the pathogenesis of these lesions should be consistent with what is known about the pathobiology of closely related pathogens. Therefore, careful systematic evaluation of lesions from different populations can sometimes yield the etiologic agent but more effectively can eliminate candidates not present in all groups.The first histologic descriptions of GTFP were published about 60 years ago. 41,58,59 Subsequently, Jacobson et al. 31 provided a more detailed description of light and electron microscopic features of GTFP. These basic histolog...

Beginning in October 2000, subadult loggerhead sea turtles Caretta caretta showing clinical signs of a neurological disorder were found in waters off south Florida, USA. Histopathology indicated generalized and neurologic spirorchiidiasis. In loggerhead sea turtles (LST) with neurospirorchiidiasis, adult trematodes were found in the meninges of the brain and spinal cord of 7 and 3 affected turtles respectively, and multiple encephalic intravascular or perivascular eggs were associated with granulomatous or mixed leukocytic inflammation, vasculitis, edema, axonal degeneration and occasional necrosis. Adult spirorchiids were dissected from meningeal vessels of 2 of 11 LST brains and 1 of 10 spinal cords and were identified as Neospirorchis sp. Affected LST were evaluated for brevetoxins, ciguatoxins, saxitoxins, domoic acid and palytoxin. While tissues from 7 of 20 LST tested positive for brevetoxins, the levels were not considered to be in a range causing acute toxicosis. No known natural (algal blooms) or anthropogenic (pollutant spills) stressors co-occurred with the turtle mortality. While heavy metal toxicosis and organophosphate toxicosis were also investigated as possible causes, there was no evidence for their involvement. We speculate that the clinical signs and pathologic changes seen in the affected LST resulted from combined heavy spirorchiid parasitism and possible chronic exposure to a novel toxin present in the diet of LST.KEY WORDS: Spirorchiidiasis · Brain · Spinal cord · Neuropathy · Loggerhead sea turtle · Caretta caretta Resale or republication not permitted without written consent of the publisherDis Aquat Org 70: [139][140][141][142][143][144][145][146][147][148][149][150][151][152][153][154] 2006 be under-diagnosed rather than being a rare occurrence.Digenetic trematodes of the family Spirorchiidae utilize freshwater turtles and sea turtles as their definitive hosts, and are known to cause neurologic complications. At least 8 genera and 20 species of spirorchiids infect the loggerhead Caretta caretta, green Chelonia mydas and hawksbill Eretmochelys imbricata sea turtles (Lauckner 1985), and are the most pathogenic of sea turtle parasites (George 1997). Spirorchiids are vascular system generalists, with a preference for the heart and arterial system of their turtle hosts (Platt & Brooks 1997). Adult parasites may cause endocarditis, arteritis and thrombosis of the blood vessels (Gordon et al. 1998). In addition to direct pathological effects induced by the adult parasite, eggs released within the vascular system may be transported to remote areas, such as the central nervous system (CNS), where they lodge in small vessels, often initiating a mild to severe granulomatous inflammatory response. The eggs can also migrate through blood vessel walls, causing tissue damage and inflammation in adjacent tissues (Gordon et al. 1998). Spirorchiid parasitism may promote secondary gram-negative bacterial infections (Raidal et al. 1998). Meningitis and encephalitis have been reported in green...