Evaluation of magnetic resonance imaging for detection of internal tumors in green turtles with cutaneous fibropapillomatosis

OBJECTIVE: To describe the gross cross-sectional anatomy of green turtles (Chelonia mydas) and evaluate magnetic resonance imaging (MRI) for detection of internal tumors in green turtles with cutaneous fibropapillomatosis. DESIGN: Prospective study. ANIMALS: 3 dead green turtles, 1 healthy green turtle, and 8 green turtles with cutaneous fibropapillomatosis. PROCEDURES: Gross cross-sectional anatomy of a dead turtle was described. Each live turtle underwent a complete physical examination, and dorsoventral whole-body survey radiographic views were obtained. Magnetic resonance imaging was performed in dorsal and transverse planes. Radiographs and magnetic resonance images were examined for evidence of internal nodules. Results were compared with necropsy findings in 5 of 8 turtles. RESULTS: Nodules in the lungs of 2 turtles were detected via radiography, whereas pulmonary nodules were detected in 5 turtles via MRI. No other visceral nodules were detected via radiography; however, masses in the stomach and adjacent to the bladder and kidneys were detected in 1 turtle via MRI. Other extrapulmonary abnormalities observed at necropsy were not detected on MR images. CONCLUSIONS AND CLINICAL RELEVANCE: MRI may be valuable for detection of internal tumors in green turtles with cutaneous fibropapillomatosis. Nodules were more apparent in the lungs than in other organs. Results of MRI may serve as prognostic indicators for sea turtles undergoing assessment, treatment, and rehabilitation. Clinical application may be limited by cost and availability of MRI technology.     

Proliferative dermatitis in a loggerhead turtle, Caretta caretta, and a green turtle, Chelonia mydas, associated with novel papillomaviruses

A subadult loggerhead turtle, Caretta caretta, presented with generalized small, white, raised lesions over its neck, shoulders, and all four flippers. A juvenile green turtle, Chelonia mydas, recently treated for fibropapillomatosis, presented with four similar localized lesions on one flipper. To diagnose the conditions, biopsies of the lesions were taken for histopathology, electron microscopy, and molecular diagnostics. Histopathologic findings were similar in the two turtles and skin lesions were characterized by multifocal areas of epidermal hyperplasia accompanied by variation and abnormalities in the nuclear morphology of keratinocytes and a few intranuclear inclusions in some cells. Transmission electron microscopy revealed multiple epithelial cells with large intranuclear aggregates of virions consistent in morphology with papillomavirus. Papillomavirus was detected in samples from both turtles by polymerase chain reaction (PCR). Sequence analysis of the partial sequence of the papillomavirus E1 gene revealed two viruses (CcPV and CmPV) that were distinct from each other and from other species in Papillomaviridae, and likely represent two novel species and perhaps a new genus.     

Conjunctivitis, tracheitis, and pneumonia associated with herpesvirus infection in green sea turtles

Fourteen juvenile (15- to 20-month-old) green sea turtles (Chelonia mydas), representative of a group of sea turtles with clinical signs of respiratory tract disease, were euthanatized and submitted for necropsy. Macroscopically, lesions included periglottal necrosis, tracheitis with intraluminal caseous and laminated necrotic debris, and severe pneumonia. Several turtles had caseous conjunctival exudate covering the eyes. Microscopically, the turtles had fibrinonecrotic inflammation around the glottal opening, tracheitis, and severe bronchopneumonia and interstitial pneumonia. In multifocal areas, periglottal and tracheal epithelial cells adjacent to areas of necrosis had hypertrophic nuclei with amphophilic intranuclear inclusions. A mixed population of primarily gram-negative microorganisms was isolated from the tracheal and glottal lesions. Attempts at viral isolation in cultures of green sea turtle kidney cells resulted in the development of cytopathic effects characterized by giant cell formation and development of intranuclear inclusions. Using electron microscopy, intranuclear viral particles (88 to 99 nm in diameter) were seen in inclusion-containing tracheal and glottal epithelial cells and infected green sea turtle kidney cells; particles were consistently seen enveloping from nuclear membranes, and mature particles (132 to 147 nm) were found in the cytoplasm. On the basis of size, conformation, location, and presence of an envelope, the particles most closely resembled those of herpes-viruses.     

Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta)

Herpesviruses are associated with lung-eye-trachea disease and gray patch disease in maricultured green turtles (Chelonia mydas) and with fibropapillomatosis in wild sea turtles of several species. With the exception fibropapillomatosis, no other diseases of wild sea turtles of any species have been associated with herpesviral infection. In the present study, six necropsied Atlantic loggerhead sea turtles (Caretta caretta) had gross and histological evidence of viral infection, including oral, respiratory, cutaneous, and genital lesions characterized by necrosis, ulceration, syncytial cell formation, and intranuclear inclusion bodies. Nested polymerase chain reaction targeting a conserved region of the herpesvirus DNA-dependent-DNA polymerase gene yielded two unique herpesviral sequences referred to as loggerhead genital-respiratory herpesvirus and loggerhead orocutaneous herpesvirus. Phylogenetic analyses indicate that these viruses are related to and are monophyletic with other chelonian herpesviruses within the subfamily alpha-herpesvirinae. We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.     

Potential Noncutaneous Sites of Chelonid Herpesvirus 5 Persistence and Shedding in Green Sea Turtles Chelonia mydas

Abstract

Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, in which productive virus replication and virion shedding occurs in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples from green turtles Chelonia mydas. These samples were taken from the carcasses of 10 juvenile green turtles with and without clinical signs of FP that stranded in Florida during 2014. Quantitative PCR for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from five out of five FP-positive and three out of five FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle.

Received September 8, 2016; accepted April 17, 2017 Published online July 26, 2017     

Pathologic changes of experimental simian varicella (Delta herpesvirus) infection in African green monkeys (Cercopithecus aethiops)

Gross and microscopic lesions of experimental simian varicella (Delta herpesvirus) infection in African green monkeys (Cercopithecus aethiops) are described. In stratified squamous epithelium, such as the epidermis, tongue, and esophagus, focal areas of epidermal hyperplasia, ballooning degeneration, and blood-filled vesicle formation with ulceration were apparent. Most visceral organs were involved, and the changes included necrosis, hemorrhage, and characteristic intranuclear eosinophilic inclusions in a variety of cells. A generalized vascular involvement was present with intranuclear inclusions in the endothelial and smooth muscle cells in various organs. The nervous system was normal on gross and microscopic examination. Virus titer and serum transferase values were correlated with the clinical signs of infection.     

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

Abstract

Fibropapillomatosis (FP) is a neoplastic disease that primarily affects green turtles Chelonia mydas in epidemic proportions worldwide. Although several infectious agents (herpesvirus, retrovirus, and papillomavirus) have been associated with the condition, the etiologic agent has not been isolated or characterized. Recently, FP has been reported in other sea turtle species including confirmed cases in loggerhead turtles Caretta caretta in Florida and field observations in olive ridley turtles Lepidochelys olivacea in the Pacific coasts of Mexico and Costa Rica. Skin and tumor specimens were collected from 72 olive ridley turtles nesting in Ostional Wildlife Refuge, Costa Rica, between July and September 1997. In all, 50 tumor biopsies were examined from 25 of the affected turtles. In addition, six biopsies were examined from five turtles that did not have visible masses and served as controls. Grossly, masses were 25 mm or less 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 fibropapillomas and eight were classified as chronic active dermatitis and not tumors. Twenty of 42 fibropapillomas were in stages of regression and 9 of the remaining 22 tumors had histological changes that suggested early degeneration within the tumor. During field surveys based on gross lesions, prevalences of 1–10% have been reported in this nesting population. This is considered the first histopathologic confirmation of FP in olive ridley turtles.     

First Record of Fibropapillomatosis in a Green Turtle Chelonia mydas from the Baja California Peninsula

Fibropapillomatosis (FP) is characterized by multiple fibroepithelial tumors in all parts of the skin and has been reported in sea turtles worldwide. Clinically infected individuals are often emaciated and anemic. In Mexico, however, there are few records of this disease. In this study of green turtles Chelonia mydas in Laguna San Ignacio in Baja California Sur (BCS), we noted one juvenile with multifocal fibropapilloma lesions on the external upper surface of its eyes and hind flippers. Light microscopy revealed hyperkeratosis, epidermal hyperplasia, dermal papillary projections, and fibroblast proliferation. Electron microscopy revealed viral particles. Biopsies of normal skin were done to determine the origin of the turtle through genetic analysis. Its mitochondrial DNA matched that of a haplotype (CMP2) from a Hawaiian green turtle population. Finding FP in a turtle captured in BCS elucidates the need for further monitoring along the west coast of Mexico. Further investigation should include testing tumors to detect and characterize any chelonid herpesviruses and explore any association with FP and other diseases that pose a health risk to other sea turtle species.

Received March 26, 2016; accepted August 3, 2016 Published online October 27, 2016     

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.     

Phylogenetic Variation of Chelonid Alphaherpesvirus 5 (ChHV5) in Populations of Green Turtles Chelonia mydas along the Queensland Coast,Australia

Abstract

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north–south trend.

Received October 22, 2016; accepted May 7, 2017 Published online July 26, 2017     

Diseases of the respiratory tract of chelonians.

Diseases of the respiratory tract commonly occur in captive chelonians, and several diseases also have occurred in wild chelonians. Infectious causes include viruses, bacteria, fungi, and parasites. Herpesviruses have surfaced as important pathogens of the oral cavity and respiratory tract in Hermann's tortoise (Testudo hermanii), spur-thighed tortoise (Testudo graeca), and other tortoises in Europe and the United States. Herpesvirus-associated respiratory diseases also have been reported in the green turtle, Chelonia mydas, in mariculture in the Cayman Islands. Of diseases caused by bacteria, an upper respiratory tract disease caused by Mycoplasma sp has been reported in free-hanging and captive gopher tortoises in the southeastern United States and in desert tortoises in the Mojave Desert of the southwestern United States. Mycotic pulmonary disease is commonly reported in captive chelonians, especially in those maintained at suboptimal temperatures. An intranuclear coccidia has been seen in several species of captive tortoises in the United States, and, in one case, a severe proliferative pneumonia was associated with organisms in the lung. The most common noninfectious cause of respiratory disease in chelonians results from trauma to the carapace. Although pulmonary fibromas commonly occur in green turtles with fibropapillomatosis, for the most part, tumors of the respiratory tract are uncommon in chelonians.     

Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection

Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifiable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids.     

A herpesvirus-type agent associated with skin lesions of green sea turtles in aquaculture.

Nine successive groups of green sea turtles (Chelonia mydas) were observed in aquaculture during the posthatchling period. During the first 6 months of growth, each group underwent an epizootic of skin lesions, named gray-patch disease. Two types of skin lesions are associated with gray-patch disease: papules and, more characteristically, spreading gray patches which appear 7 to 8 weeks after hatching. In both types of lesions, intranuclear inclusions are found in keratinocytes in the malpighian layer of the epidermis. Electron microscopic examination of scrapings from lesions and biopsies revealed many viral particles, with features characteristic of the herpesvirus group. Transmission of gray-patch disease is possible with bacteria-free preparations of viral particles.     

Geographic variation in marine turtle fibropapillomatosis.

We document three examples of fibropapillomatosis by histology, quantitative polymerase chain reaction (qPCR), and sequence analysis from three different geographic areas. Tumors compatible in morphology with fibropapillomatosis were seen in green turtles from Puerto Rico and San Diego (California) and in a hybrid loggerhead/ hawksbill turtle from Florida Bay (Florida). Tumors were confirmed as fibropapillomas on histology, although severity of disease varied between cases. Polymerase chain reaction (PCR) analyses revealed infection with the fibropapilloma-associated turtle herpesvirus (FPTHV) in all cases, albeit at highly variable copy numbers per cell. Alignment of a portion of the polymerase gene from each fibropapilloma-associated turtle herpesvirus isolate demonstrated geographic variation in sequence. These cases illustrate geographic variation in both the pathology and the virology of fibropapillomatosis.     

Altered in vitro immune responses in green turtles (Chelonia mydas) with fibropapillomatosis.

The immune competence of green sea turtles (Chelonia mydas) with fibropapillomatosis was assessed using in vitro techniques to measure lymphocyte proliferation in response to mitogens. In comparison with captive, healthy green sea turtles, those afflicted with fibropapillomas demonstrated diminished proliferation with Concanavalin A, phytohemagglutinin (T-cell mitogens), and lipopolysaccharide (B-cell mitogen). Also, markedly decreased proliferative responses to the lymphocyte polyclonal stimulator combination of ionomycin and phorbol myristate acetate were observed. Total circulating white blood cell counts were not statistically different between the two groups, although an overall decrease in lymphocyte number was observed in the papilloma group. The albumin/globulin ratio was decreased in the papilloma group because of decreased albumin and increased gamma globulins.     

Review paper: preclinical models of psoriasis

Psoriasis is the most common autoimmune disease in man and is characterized by focal to coalescing raised cutaneous plaques with consistent scaling and variable erythema. The specific pathogenesis of psoriasis is not completely understood, but the underlying mechanisms involve a complex interplay between epidermal keratinocytes, T lymphocytes as well as other leukocytes (including dendritic cells and other antigen presenting cells [APCs]), and vascular endothelium. Mirroring the complexity of mechanisms that underlie psoriasis, there are a relatively large number of models of psoriasis. Each model is based on a slightly different pathogenic mechanism, and each has its similarities to psoriasis as well as its limitations. In general, psoriasis models can be very broadly divided on the basis of the pathogenic mechanisms that interplay to cause psoriasis, with the addition of several relatively poorly defined spontaneous murine mutant models. Other than the spontaneous mutant models, murine models of psoriasis can be divided into those that are genetically engineered (transgenic and knockout-with manipulation of either the epidermis, leukocytes, or the endothelium), and those that are induced (either by immune transfer or by xenotransplantation of skin from psoriatic patients). In addition to the murine models, in vitro human epidermal models have recently become more widely utilized. While no one single model of psoriasis is ideal, many have proven to be extremely valuable in investigating and better understanding the molecular mechanisms that underlie the complex interplay between epidermal keratinocytes, the innate and adaptive immune system, and the vascular endothelium in psoriasis.     

Experimental viral pneumonia in guinea pigs: an ultrastructural study

Homogenized lung tissue was used to experimentally reproduce lethal viral pneumonia in guinea pigs. The resultant lesions corresponded with those of the spontaneous disease. Pneumonia with necrotic bronchiolar epithelium accompanied by basophilic intranuclear inclusion bodies was the primary finding. With transmission electron microscopy, numerous viral particles were found--mainly within the nucleus of pulmonary epithelial cells but also within the cytoplasm and in extracellular space. The appearance of viral particles, in particular their paracrystalline and crystalline deposition within the nucleus, indicates that our experimentally induced pneumonia was caused by an adenovirus.     

Cerebellar degeneration in cattle grazing Solanum bonariense ("Naranjillo") in Western Uruguay.

Cattle in western Uruguay that were eating Solanum bonariense developed periodic episodes of ataxia, hypermetria, hyperesthesia, head and thoracic limb extension, opisthotonus, nystagmus, and falling to the side or backward. Similar clinical signs were experimentally reproduced in cattle by administration of S. bonariense via rumen cannula at a dose of 1,024 g/kg body mass. No significant gross lesions were observed in field cases or experimentally induced cases. Spontaneous and induced histologic lesions were similar and included vacuolation, degeneration, and loss of Purkinje cells. Axonal spheroids, microcavitations, and other changes of wallerian-type degeneration in cerebellar white matter were also observed. Ultrastructural changes included increased number of electron-dense residual storage bodies in membrane-bound vesicles in affected Purkinje cells, and similar vesicles and mitochondria in axonal spheroids. No histologic lesions were detected in the other examined tissues. The Purkinje-cell swelling and vacuolation with subsequent cerebellar degeneration are suggestive of Purkinje-cell specific toxin that produces abnormal lysosome function and cell specific axonal transport. This is the first report of S. bonariense toxicity.     

Cytology of canine cutaneous round cell tumors. Mast cell tumor, histiocytoma, lymphosarcoma and transmissible venereal tumor.

Sixty-four canine cutaneous round cell tumors were divided into 25 mast cell tumors, 15 histiocytomas, nine cutaneous lymphosarcomas and 15 transmissible venereal tumors. The final diagnosis was made from cytologic, clinical and histologic findings. Cytologic features were significantly distinctive in mast cell tumor, transmissible venereal tumor, and most cases of histiocytoma and lymphosarcoma to allow a diagnostic opinion. This opinion was supported by subsequent histologic examination. In some instances cytology was considered essential in rendering a diagnostic opinion even though histology was available.     

Evaluation of hematology and serum biochemistry of cold-stunned green sea turtles (Chelonia mydas) in North Carolina, U.S.A

Hypothermia or cold-stunning is a condition in which the body temperature of an animal decreases below normal physiologic range and which has been linked to severe morbidity in sea turtles. Reports have focused on the physiologic changes caused by cold-stunning in Kemp's Ridley sea turtles (Lepidochelys kempii) and loggerhead sea turtles (Caretta caretta), but few have evaluated the green sea turtle (Chelonia mydas). This study evaluated hematologic and serum biochemical profiles of cold-stunned green sea turtles in North Carolina, USA. When compared with healthy, free-ranging juvenile green turtles from the same region, cold-stunned turtles exhibited hypoglycemia, hypocalcemia (both total and ionized calcium), hyponatremia, hypokalemia, hypoproteinemia, hypoalbuminemia, hyperphosphatemia, and elevations in uric acid and blood urea nitrogen. These findings contrast with some previously reported changes in cold-stunned Kemp's Ridley and loggerhead sea turtles. These results emphasize the importance of basing therapeutic regimens on biochemical analyses in cold-stunned sea turtles.