Brian A. Stacy

Genomic characterization of two novel reptilian papillomaviruses, Chelonia mydas papillomavirus 1 and Caretta caretta papillomavirus 1

In this paper we describe the characterization of the genomes of two sea turtle papillomaviruses, Chelonia mydas PV (CmPV-1) and Caretta caretta PV (CcPV-1). The isolation and sequencing of the first non-avian reptilian PVs extend the evolutionary history of PVs to include all amniotes. PVs have now been described in mammals, birds and non-avian reptiles. The chelonian PVs form a distinct clade most closely related to the avian PVs. Unlike the avian PVs, both chelonian PVs have canonical E6 and E7 ORFs, indicating that these genes were present in the common ancestor to mammalian and non-mammalian amniote PVs. Rates of evolution among the non-mammalian PVs were generally slower than those estimated for mammalian PVs, perhaps due to lower metabolic rates among the ectothermic reptiles.

HEMATOLOGY AND BLOOD BIOCHEMISTRY OF CAPTIVE MUGGER CROCODILES (CROCODYLUS PALUSTRIS)

Abstract Comprehensive hematologic and biochemical analyses were conducted on blood from 23 male and 31 female clinically stable captive mugger crocodiles (Crocodylus palustris). Erythrocyte mean corpuscular volume (MCV), potassium, cholesterol, and calcium concentrations were significantly greater in juvenile males than in juvenile females, but no significant differences were determined between parameters of subadult males and subadult females. The mean WBC count and mean heterophil count were significantly higher in adult males than in adult females. Mean uric acid concentration was significantly greater in adult females than in males. Mean erythrocyte count was significantly higher in adults than in juveniles. Adult mean WBC and lymphocyte counts were significantly lower than those of both juveniles and subadults. Subadults had significantly lower mean eosinophil counts than both adults and juveniles. Subadults had significantly lower mean alkaline phosphatase activities than juveniles, whereas the adults had significantly lower aspartate aminotransferase and alanine aminotransferase activities than other groups. Lactate dehydrogenase activities were significantly lower for subadults than for juveniles and adults. Cholesterol concentrations were significantly higher for subadults and juveniles compared with adults. Triglyceride concentration was significantly lower for subadults and highest for juveniles. Glucose concentrations were significantly higher for adults. Blood urea nitrogen was significantly lower for subadults than for both adults and juveniles. Uric acid concentrations were significantly higher for juveniles than for the subadults and adults. The subadult animals also had a significantly lower potassium concentration. The results obtained were then compared with known values for other crocodilian species.

Spirorchiidiasis in stranded loggerhead Caretta caretta and green turtles Chelonia mydas in Florida (USA): host pathology and significance

Spirorchiid trematodes are implicated as an important cause of stranding and mortality in sea turtles worldwide. However, the impact of these parasites on sea turtle health is poorly understood due to biases in study populations and limited or missing data for some host species and regions, including the southeastern United States. We examined necropsy findings and parasitological data from 89 loggerhead Caretta caretta and 59 green turtles Chelonia mydas that were found dead or moribund (i.e. stranded) in Florida (USA) and evaluated the role of spirorchiidiasis in the cause of death. High prevalence of infection in the stranding population was observed, and most infections were regarded as incidental to the cause of death. Spirorchiidiasis was causal or contributory to death in some cases; however, notable host injury and/or large numbers of parasites were observed in some animals, including nutritionally robust turtles, with no apparent relationship to cause of death. New spirorchiid species records for the region were documented and identified genera included Neospirorchis, Hapalotrema, Carettacola, and Learedius. Parasites inhabited and were associated with injury and inflammation in a variety of anatomic locations, including large arteries, the central nervous system, endocrine organs, and the gastrointestinal tract. These findings provide essential information on the diversity of spirorchiids found in Florida sea turtles, as well as prevalence of infection and the spectrum of associated pathological lesions. Several areas of needed study are identified with regard to potential health implications in the turtle host, and findings caution against over-interpretation in individual cases.

Cryptosporidiosis caused by two distinct species in Russian tortoises and a pancake tortoise.

Cryptosporidiosis in squamates is well documented, but there is very limited information available on cryptosporidiosis in testudines. We describe three cases of cryptosporidiosis in tortoises with associated pathology. Two Russian tortoises (Agrionemys [Testudo] horsfieldii) and a pancake tortoise (Malacochersus tornieri), all from separate collections, were found dead. At necropsy, two had histological evidence of intestinal cryptosporidiosis and one had gastric cryptosporidiosis. Consensus Cryptosporidium sp. PCR and sequencing was used to identify the Cryptosporidium sp. present in these three tortoises. In the juvenile Russian tortoise with gastric cryptosporidiosis, the organism had 98% homology with a previously reported sequence from an Indian star tortoise isolate. A second chelonian Cryptosporidium sp. was identified in the pancake tortoise and the second Russian tortoise. This sequence was 100% identical to a shorter gene sequence previously reported in a marginated tortoise. This is the first report coordinating pathology with Cryptosporidium characterization in chelonians. The two Cryptosporidium sp. found in tortoises segregate according to site of infection, and there may be further differences in pathology, host range, and transmission. These Cryptosporidium sp. appear to be able to infect diverse tortoise host species. This may be an under-recognized problem in tortoises.

BREVETOXIN IN BLOOD, BIOLOGICAL FLUIDS, AND TISSUES OF SEA TURTLES NATURALLY EXPOSED TO KARENIA BREVIS BLOOMS IN CENTRAL WEST FLORIDA

Abstract:u2003 In 2005 and 2006, the central west Florida coast experienced two intense Karenia brevis red tide events lasting from February 2005 through December 2005 and August 2006 through December 2006. Strandings of sea turtles were increased in the study area with 318 turtles (n = 174, 2005; n = 144, 2006) stranding between 1 January 2005 and 31 December 2006 compared to the 12-yr average of 43 ± 23 turtles. Live turtles (n = 61) admitted for rehabilitation showed clinical signs including unresponsiveness, paresis, and circling. Testing of biological fluids and tissues for the presence of brevetoxin activity by enzyme-linked immunosorbent assay found toxin present in 93% (52 of 56) of live stranded sea turtles, and 98% (42 of 43) of dead stranded sea turtles tested. Serial plasma samples were taken from several live sea turtles during rehabilitation and toxin was cleared from the blood within 5–80 days postadmit depending upon the species tested. Among dead animals the highest brevetoxin levels were found in feces, stomach contents, and liver. The lack of significant pathological findings in the majority of animals necropsied supports toxin-related mortality.

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.

Yolk Coelomitis in Fiji Island Banded Iguanas (Brachylophus fasciatus)

Abstract Yolk coelomitis is a major cause of death in captive sexually mature female Fiji Island banded iguanas (Brachylophus fasciatus) maintained by the Zoological Society of San Diego. The medical records, breeding histories, and pathology archival materials from this group were reviewed to study this health problem. From 1987 through 2004, deaths of nine of 21 adult females were due to yolk coelomitis. Most iguanas had a history of reproduction-related problems, which included reproductive failure, episodes of lethargy associated with ovarian activity, folliculostasis, ovostasis, and behavioral abnormalities. Most affected iguanas either were found dead or presented moribund and subsequently died or were euthanized. Clinical signs were nonspecific and included lethargy, cutaneous discoloration, and coelomic effusion. Yolk leakage in most cases was associated with the presence of large vitellogenic follicles undergoing atresia and resulted in coelomitis characterized by florid mesothelial proliferation.

Characterization of an erythrocytic virus in the family Iridoviridae from a peninsula ribbon snake (Thamnophis sauritus sackenii)

A wild peninsula ribbon snake (Thamnophis sauritus sackenii) in Florida was found to have hypochromic erythrocytes containing two different types of inclusions: purple granular inclusions, and pale orange or pink crystalloid inclusions that were round, oval, rectangular, or hexagonal in shape. Transmission electron microscopy revealed hexagonal or pleomorphic, homogenous inclusions and enveloped particles morphologically consistent with a member of the Iridoviridae. Histopathology of the animal revealed necrotizing hepatitis consistent with sepsis. Consensus PCR was used to amplify a 628-bp region of iridoviral DNA-dependent DNA polymerase. Bayesian and maximum likelihood phylogenetic analysis found that this virus was distinct from other known iridoviral genera and species, and may represent a novel genus and species.

Detection of Spirorchiid Trematodes in Gastropod Tissues by Polymerase Chain Reaction: Preliminary Identification of an Intermediate Host of Learedius learedi

Abstract Marine spirorchiid trematodes are associated with morbidity and mortality in sea turtles worldwide. The intermediate hosts remain unknown, and discovery efforts are hindered by the large number and great diversity of potential hosts within sea turtle habitats, as well the potential for low prevalence and overdispersion. A high-throughput DNA extraction and polymerase chain reaction–based method was developed to detect the internal transcribed spacer 2 (ITS2) region of the ribosomal gene of 2 spirorchiid genera, Learedius and Hapalotrema, within pooled samples of gastropod tissues. A model system consisting of freshwater snail (Pomacea bridgesii) tissues and DNA extracts spiked with adult Learedius learedi and known quantities of spirorchiid DNA was used to develop and test the technique. Threshold of detection was found to be equivalent to an early prepatent infection within 1.5 g of gastropod tissue. This technique was used to screen approximately 25 species of marine gastropods at a captive facility where green turtles (Chelonia mydas) become infected by L. learedi. The parasite was detected in a sample of knobby keyhole limpet (Fissurella nodosa), thus providing the first evidence of an intermediate host for a marine spirorchiid trematode. This technique has many potential applications in trematode life cycle discovery studies.

Mast cell tumour in a giant Galapagos tortoise (Geochelone nigra vicina).

A well-differentiated cutaneous mast cell tumour was diagnosed in a subadult female giant Galapagos tortoise. The tumour was a pedunculated, verrucose mass located near the base of the neck. The histological features, which were diagnostic for a mast cell tumour, included abundant intracytoplasmic granules that were stained metachromatically with Giemsa and toluidine blue stains. Mast cell tumours are rare in reptiles, and this is the first description of a mast cell tumour in a chelonian.