Otariid gammaherpesvirus 1 in South American fur seals (Arctocephalus australis) and a novel related herpesvirus in free-ranging South American sea lions (Otaria byronia): Prevalence and effects of age, sex, and sample type.

Otariid gammaherpesvirus 1 (OtGHV1) is associated with high rates of urogenital carcinoma in free-ranging California sea lions (Zalophus californianus; CSL), and until recently was reported only in the Northern Hemisphere. The objective of this study was to survey free-ranging South American sea lions (Otaria byronia; SASL) and South American fur seals (Arctocephalus australis: SAFS) in Punta San Juan, Peru for OtGHV1 and to determine prevalence characteristics. Twenty-one percent (14/67) of urogenital swabs collected over three years (2011, 2014, 2015) from live pinnipeds of both species tested positive with a pan-herpesvirus conventional PCR. Sequencing of SAFS amplicons revealed 100% homology to OtGHV1 at the DNA polymerase, glycoprotein B, and viral bcl2-like genes. Sequencing of SASL amplicons revealed a novel related virus, herein called Otariid gammaherpesvirus 8 (OtGHV8). For comparison of sample sites, urogenital, conjunctival, and oropharyngeal swabs collected from 136 live pinnipeds of both species at Punta San Juan between 2011-2018 were then assayed using quantitative PCR for a segment of the OtGHV1/8 DNA polymerase gene using a qPCR assay now determined to cross-react between the two viruses. In total, across both species, 38.6% (51/132) of urogenital swabs, 5.6% (4/71) of conjunctival swabs, and 1.1% (1/90) of oropharyngeal swabs were positive for OtGHV1/8, with SASL only positive on urogenital swabs. Results from SASL were complicated by the finding of OtGHV8, necessitating further study to determine prevalence of OtGHV1 versus OtGHV8 using an alternate assay. Results from SAFS suggest a potential relationship between OtGHV1 in SAFS and CSL. Though necropsy surveillance in SAFS is very limited, geographic patterns of OtGHV1-associated urogenital carcinoma in CSL and the tendency of herpesviruses to cause more detrimental disease in aberrant hosts suggests that it is possible that SAFS may be the definitive host of OtGHV1, which gives further insight into the diversity and phyogeography of this clade of related gammaherpesviruses.

Health Assessment of Free-Ranging Eastern Indigo Snakes (Drymarchon couperi) from Hydrologic Restoration Construction Sites in South Florida, USA.

There is a paucity of information regarding the health status of free-ranging eastern indigo snakes (EIS; Drymarchon couperi) in heavily modified and developing landscapes. As a component of regional Florida Everglades restoration efforts, several areas occupied by EIS are being converted from agricultural lands to reservoirs. From 2020 to 2022, 28 EIS were opportunistically captured at two of these sites and brought into captivity to join a captive breeding colony; however, 11 snakes died within 5 mo of capture. Health assessments were performed on 28 individuals and included hematology and plasma biochemistry analysis, as well as screening for pesticide contaminant levels, parasites, and other pathogens. Overall, the presence of pathogens was relatively high, suggesting immunosuppression secondary to stress: 25/28 (89.4%) Kalicephalus sp.; 12/28 (42.9%) Raillietiella orientalis; 11/28 (39.2%) Ochetosoma validum; 7/28 (25.0%) Cryptosporidium serpentis; 3/28 (10.7%) snake adenovirus 1; and 1/28 (3.6%) Ferlavirus genotype C. Stress may have been caused by physical displacement, habitat modification, and noise pollution. These potential stressors (including the presence of remnant harmful chemicals from previous land use and the impacts on this federally threatened species) should be considered further when making restoration or construction decisions.

Chronic mandibular osteomyelitis associated with a novel probable Kocuria sp in a sugar glider (Petaurus breviceps).

Dental disease in sugar gliders (Petaurus breviceps) is understudied, with only limited clinical and pathological data available. An approximately 7-year-old female sugar glider presented to its primary care veterinarian for a decline in food intake, rapid weight loss and a mass involving the rostral mandible. At necropsy, the mandibular mass effaced most of the rostral mandible and adjacent musculature. Histologically, the mandible was disrupted by nodular infiltrates of variably degenerate neutrophils and macrophages encased in granulomatous inflammation and fibrous connective tissue. Within the neutrophilic cell population were segments of fragmented, necrotic bone and cloud-like colonies of gram-positive cocci. Aerobic culture yielded a heavy, pure growth of a gram-positive coccus morphologically consistent with those identified in the lesions, which was identified as a presumptively novel Kocuria sp by polymerase chain reaction and sequencing of the 16S rRNA gene. This is the first description of Kocuria infection in association with clinically significant pathology in an animal. Although isolated as a pure growth, Kocuria sp cannot be confirmed as the sole cause of lesion formation due to the case chronicity and potential for unculturable, polymicrobial infections. This report adds to our understanding of the clinical and pathological aspects of dental disease in sugar gliders.

Multisystemic Enterococcosis in Brown Anoles (Anolis sagrei) from Florida, USA.

Beginning in July 2019, numerous free-ranging brown anoles (Anolis sagrei), an invasive lizard species in Florida, USA, were reported with large, soft, subcutaneous masses and disfiguring facial swellings. Postmortem evaluations of six affected animals, including cytology, histology, and electron microscopy, identified the presence of myriad chain-forming coccoid bacteria surrounded by a prominent clear capsule and abundant lightly basophilic matrix material with minimal associated granulomatous inflammation and effacement of normal tissue. Standard PCR and sequencing of the lesions revealed 100% nucleotide identity to Enterococcus lacertideformus. This bacterium was first observed in 2014 as the cause of a severe, multisystemic infection in several species of lizards (geckos and skinks) on Christmas Island, an Australian external territory in the Indian Ocean. Previously, analysis of E. lacertideformus had been hindered by an inability to grow the bacterium in standard culture conditions. We successfully cultured the organism on primary anole kidney cells. Given the growing recognition of host species diversity and geographic distribution noted for this organism, there is potential concern for spread to native North American lizards, especially the green anole (Anolis carolinensis), whose population numbers have apparently decreased due to introduced brown anoles.

IDENTIFICATION AND RETROSPECTIVE EVALUATION OF A FILARIOID NEMATODE SPECIES IN MANAGED GRASSHOPPER SPARROWS (AMMODRAMUS SAVANNARUM).

The grasshopper sparrow (Ammodramus savannarum) is a species of ground-dwelling passerine bird with 12 different subspecies. The Florida subspecies (Ammodramus savannarum floridanus) is classified as federally endangered, with the most common threats including habitat loss, nest predation, and floods. A managed breeding program was established at White Oak Conservation (Yulee, FL) in 2015 with eastern grasshopper sparrows (Ammodramus savannarum pratensis) as a model for breeding Florida grasshopper sparrows as part of an assurance colony. A filarioid parasite species (Aproctella sp.) was characterized by PCR after identification by blood films and postmortem examinations of both subspecies housed at White Oak Conservation. This Aproctella species was distinct from others with available sequence. Records from 157 eastern and Florida grasshopper sparrows were reviewed, and correlations between presence of filariasis and subspecies, sex, body condition score, and presence of systemic isosporosis, squamous metaplasia, coelomitis, airsacculitis, or a combination of conditions were investigated. Twenty-nine (18.5%) birds (13 of 71 Florida grasshopper sparrows; 16 of 86 eastern grasshopper sparrows) were positive for filariasis by blood film review, grossly or by tissue imprint at postmortem examination, or histologically. Filariasis was significantly correlated with systemic isosporosis, coelomitis, and airsacculitis; was not correlated with subspecies, sex, or squamous metaplasia; and had a questionable correlation with body condition score. This report provides evidence that this Aproctella species has potential to contribute to morbidity and mortality in the grasshopper sparrow. This information will be helpful for implementing effective measures against suspected vectors and for the development of best practice strategies for the health management of the species in breeding programs.

Identification of a novel Neorickettsia species in a Kemp's ridley sea turtle with granulomatous nephritis and development of a quantitative PCR assay.

An adult male Kemp's ridley turtle was found dead on the coast of Kenedy County, Texas, in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene polymerase chain reaction (PCR) and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed and detected the organism within the liver and colon of the index case. A quantitative PCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp's ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

AUSTWICKIA CHELONAE IN A WILD GOPHER TORTOISE (GOPHERUS POLYPHEMUS) AND EVIDENCE OF POSITIVE SELECTION ON THE DIPHTHERIA-LIKE TOXIN GENE.

Austwickia (Dermatophilus) chelonae is a filamentous, Gram-positive Actinobacteria in the Dermatophilaceae family. It has caused fatal granulomatous disease in diverse captive reptile species on three continents, but its presence in wild or free-ranging populations was unknown. An adult female gopher tortoise (Gopherus polyphemus) was presented euhydrated, but cachectic and infested with ticks, with two firm, encapsulated masses over the cranioventral neck and right stifle. The tortoise had moderate nonregenerative anemia and evidence of inflammation; plasma biochemistry data was within normal limits. Fine needle aspirate of the neck lesion revealed abundant necrosis and aggregates of cocci. Computed tomography delineated the masses and revealed an additional mass adjacent to the left zygomatic bone. After surgical excision, histology identified chronic granulomas with intralesional filamentous bacteria. Pan-bacterial 16S rRNA PCR and sequencing of the masses identified A. chelonae. Despite treatment with oxytetracycline and ceftazidime, the tortoise deteriorated and was euthanatized. An esophageal lesion consistent with A. chelonae was seen on postmortem examination, although it was determined that the tortoise ultimately succumbed to fungal pneumonia caused by Metarhizium robertsii, an entomopathogenic biotoxin sprayed as insect control. This case reveals A. chelonae is present in free-ranging chelonians in North America. This organism produces a toxin gene similar to diphtheria toxin, one of the most potent known biotoxins, which has not been previously identified outside the genus Corynebacterium. Novel PCR primers were designed for the toxin and rpoB genes, which were amplified and sequenced from two cases and compared with two available genomes. Selection analysis revealed that the toxin gene is under positive selection, which implies it interacts significantly with the immune system, making it a good candidate for immunodiagnostic test development.

Identification of a novel mortality-associated Helicobacter species in gopher tortoises (Gopherus polyphemus), qPCR test development and validation, and correlation with mortality in a wildlife rehabilitation population.

The genus Helicobacter includes spiral-shaped bacteria in the phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacteriales, that have been associated with disease in animals, including reptiles. Three wild gopher tortoise (Gopherus polyphemus) index cases presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytological examination of nasal discharge from all 3 tortoises identified marked heterophilic and mild histiocytic rhinitis with abundant extracellular and phagocytized spiral shaped bacteria that stained positive with Warthin-Starry stain. Polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two tortoises died despite treatment attempts, and the third was moribund and was euthanized. Histological examination of the nasal mucosa (n = 3) showed granulocytic to lymphocytic rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P < 0.0001). These assays were used to survey nasal wash samples from 31 rehabilitating gopher tortoises. Mortality of tortoises significantly correlated with higher Helicobacter loads detected by qPCR (P = 0.028). Appropriate quarantine protocols for tortoises during rehabilitation should consider this organism. Upper respiratory disease in tortoises may involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account.

Clinical Manifestation and Molecular Characterization of a Novel Member of the Nannizziopsiaceae in a Pulmonary Granuloma From a Galapagos Tortoise (Chelonoidis nigra).

Nannizziopsiaceae is a family of fungal organisms within the order Onygenales containing two genera of important reptile pathogens, Nannizziopsis and Paranannizziopsis. A captive Galapagos tortoise (Chelonoidis nigra) from Boca Raton, Florida, United States, was presented for a clinical history of chronic progressive lethargy and inappetence. At initial presentation, the tortoise had a moderate non-regenerative anemia, leukocytosis, whip-like heterophil projections, erythrocyte fragmentation, and fibrin strands, with the latter two raising concern for disseminated intravascular coagulation. A single large encapsulated pulmonary granuloma was identified through imaging, including plain film radiography and bronchoscopy. Direct intralesional samples were obtained from transcarapacial celioscopy for fungal culture, cytology, histopathology, and polymerase chain reaction. Amplification and sequencing of the ITS2 region of the rRNA genes with Bayesian and maximum likelihood analyses placed the fungus in the family Nannizziopsiaceae within the order Onygenales, representing a novel fungal species.

Oral, Cloacal, and Hemipenal Actinomycosis in Captive Ball Pythons (Python regius).

Ball pythons (Python regius) are one of the most commonly kept and bred reptiles in captivity. In a large ball python breeding colony, a unique syndrome characterized by granulomatous inflammation of the cloaca and hemipenes (phalli) was observed in 140 of 481 (29.1%) breeding males, but only one of 1,446 breeding females. Lesions were absent in virgin males (n = 201) and virgin females (n = 293). On postmortem examination (n = 13, 12 males, 1 female), numerous well-defined mucosal and submucosal granulomas were present in the hemipenes (males) and cloaca (males and female). Extension into the coelomic cavity and liver was noted in a subset of these animals. An additional small subset of breeder animals (6/2027; 0.3%) presented with oral and mandibular swellings. Postmortem examination (n = 4, all female) showed oral lesions histologically indistinguishable from the cloacal/hemipenal lesions. Aerobic bacterial culture of a hepatic granuloma of one snake resulted in the isolation of filamentous, Gram-positive bacilli; amplification, and sequencing of the 16S rRNA gene and subsequent phylogenetic analysis of the isolate identified the bacterium as a novel species of Actinomyces. Screening of cloacal and oral granulomas using a specific, heminested 16S rRNA PCR assay confirmed the presence of the agent in all 17 snakes, as well as in cloacal swabs taken at the time of necropsy in 11/13 snakes. The Actinomyces sp. was also identified by PCR of cloacal swabs of unaffected snakes (n = 94) from the affected colony and two unrelated, grossly unaffected breeding colonies. In the affected colony, 65.5% of breeding animals (n = 23) but only 11.9% of virgin animals (n = 42) tested PCR positive, with breeding status being a significant predictor of bacterium presence (P < 0.00001). This study characterizes a granulomatous mucosal disease syndrome of breeding male ball pythons associated with a novel Actinomyces. In stark contrast to male snakes, the presence of the bacterium in both breeding and virgin females was very rarely associated with clinical disease. Though additional studies are necessary, these data suggest a role for the novel bacterium in the disease process, a predilection for clinical disease in male snakes, and the potential for sexual transmission of the disease.

IDENTIFICATION OF HELODERMATID ADENOVIRUS 2 IN A CAPTIVE CENTRAL BEARDED DRAGON (POGONA VITTICEPS), WILD GILA MONSTERS (HELODERMA SUSPECTUM), AND A DEATH ADDER (ACANTHOPHIS ANTARCTICUS).

Adenoviruses are medium-sized DNA viruses with very high host fidelity. The phylogenetic relationships of the adenoviruses strongly resemble that of their hosts, consistent with evolutionary codivergence. The genus Atadenovirus appears to have evolved in squamate hosts. Perhaps the best known of the squamate adenoviruses is Agamid adenovirus 1 (AgAdV1), found most commonly in central bearded dragons (Pogona vitticeps), where it is a prevalent cause of hepatitis/enteritis, especially in young animals. All previous reports of adenoviruses in bearded dragons were AgAdV1. Helodermatid adenovirus 2 (HeAdV2) was first seen in Mexican beaded lizards (Heloderma horridus). Subsequently, partial adenoviral polymerase gene sequence from a western bearded dragon (Pogona minor) in Australia was found to share 99% nucleotide homology with HeAdV2. This article reports the discovery of a virus identical to HeAdV2 in a captive central bearded dragon in Florida and wild Gila monsters (Heloderma suspectum) in Arizona. Additionally, a partial adenoviral polymerase gene sharing 98% homology with this HeAdV2 was discovered in a death adder (Acanthophis antarcticus) in Australia. These findings call into question the provenance of HeAdV2. Further studies of atadenoviral host range, diversity of adenoviruses in captive animals, and characterization of adenoviruses from wild squamates are indicated.

Intrapericardial Encephalitozoon pogonae-associated arteritis with fatal hemopericardium in two juvenile central bearded dragons.

Two male juvenile central bearded dragons ( Pogona vitticeps) were submitted for postmortem examination after dying at their respective homes. Dragon 1 had marked hemopericardium with restrictive epicarditis. The inner aspect of the distended pericardial sac was lined by a fibrinoheterophilic membrane. In addition, granulomas abutted the testes. Dragon 2 had acute hemopericardium and granulomatous arteritis of the great vessels exiting the heart. Histologically, both animals had granulomatous arteritis of the large arteries with intrahistiocytic gram-positive, slightly elongated, up to 2 µm long microorganisms that contained a vacuole. These microorganisms were also present in the paratesticular granulomas. On transmission electron microscopy, the microorganisms were identified as microsporidians given the presence of exospore, endospore, vacuole, nucleus, and a filament with 4-6 coils. The microsporidia were identified as Encephalitozoon pogonae based on sequencing of the internal transcribed spacer 1 of the ribosomal RNA genes. Microsporidia are agents of disease in bearded dragons. Intrapericardial arteritis of large arteries with hemopericardium or restrictive epicarditis is a fatal manifestation of this infection.

A novel herpesvirus associated with chronic superficial keratitis and proliferative conjunctivitis in a great horned owl (Bubo virginianus).

An adult great-horned owl (Bubo virginianus; GHOW) presented with a history of recurrent corneal ulceration of the right eye (OD). Findings included ulcerative superficial keratitis, proliferative conjunctivitis, and iris pigmentary changes. The ulcer was initially nonresponsive to medical therapy, but showed rapid and appropriate healing following diamond burr debridement. Proliferative conjunctivitis markedly improved following topical antiviral therapy with cidofovir 1%, interferon alpha 2B ophthalmic solutions, and oral l-lysine. Histopathologic evaluation of a conjunctival biopsy revealed epithelial features suspicious for viral cytopathic changes and intranuclear structures suspicious for viral inclusions, suggestive of a possible viral-induced papillomatous conjunctivitis. A novel alphaherpesvirus, referred to as Strigid Herpesvirus 1 (StrHV1), was identified using PCR and gene sequencing. This case represents a new clinical manifestation of a previously unreported herpesvirus in the GHOW. Identification of the herpes virus was critical to administration of appropriate therapy and resolution of the conjunctivitis, and corneal epithelial debridement promoted resolution of the chronic corneal epithelial defect.

Management of Ocular Human herpesvirus 1 Infection in a White-faced Saki Monkey (Pithecia pithecia).

A 20-y-old male intact white-faced saki monkey (Pithecia pithecia) presented with an acute ocular disease of the right eye. Clinical signs included periocular swelling, conjunctivitis, and anisocoria with a miotic right pupil. Conjunctival swabs were positive for Human herpesvirus 1 (HHV1) according to PCR amplification with sequencing. Initial clinical signs resolved with supportive treatment, and the animal was managed chronically by using acyclovir (5 mg/kg PO twice daily) during flare-ups. After more than 2 y, the progression of clinical disease led to enucleation of the right eye. At 2 mo after surgery, acute presentation of severe neurologic signs, including ataxia and blindness, resulted in euthanasia. Histopathology, PCR analysis, and sequencing results were consistent with viral encephalitis due to HHV1; coinfection with Pithecia pithecia lymphocryptovirus 1 was identified. This report describes the first case of managed HHV1 infection in a platyrrhine primate and the first case of HHV1 in a white-faced saki monkey that was not rapidly fatal.

Pneumonia in a Captive Central Bearded Dragon With Concurrent Detection of Helodermatid Adenovirus 2 and a Novel Mycoplasma Species.

A 4-year-old captive male central bearded dragon ( Pogona vitticeps) was presented for recurrent episodic dyspnea and anorexia with occasional expulsion of oral mucoid discharge. Despite empirical antimicrobial therapy and supportive care, the animal died and was submitted for autopsy. Defining histologic features included heterophilic and lymphocytic interstitial pneumonia, with occasional amphophilic intranuclear inclusions and prominent type II pneumocyte hyperplasia. Transmission electron microscopy revealed intranuclear 80-nm, nonenveloped, hexagonal viral particles within pneumocytes. Helodermatid adenovirus 2 (HeAdV2) was determined as the etiologic agent through pan-adenoviral consensus polymerase (PCR) chain reaction and sequencing. Nucleic acid from a novel Mycoplasma sp. (provisionally called Mycoplasma pogonae) was identified by pan-generic PCR targeting the mycoplasma 16S ribosomal RNA gene with sequencing and phylogenetic analysis. As bacteria morphologically consistent with Mycoplasma sp. were not observed by special stains and transmission electron microscopy, the detection of M. pogonae nucleic acid is of indeterminate significance; however, M. pogonae and HeAdV2 coinfection may have exacerbated disease.

GRANULOMATOUS ENCEPHALOMYELITIS IN A FALSE GHARIAL (TOMISTOMA SCHLEGELII) ASSOCIATED WITH A NOVEL CHLAMYDIA SPECIES.

A 5-yr-old, captive, hatched, female false gharial (Tomistoma schlegelii) presented with a 1-mo history of cervical spinal curvature. Antemortem diagnostics, including blood work, electromyography, muscle biopsies, and advanced imaging tests, were either within reference ranges or did not identify any specific etiology. Necropsy revealed extensive, marked, chronic granulomatous encephalomyelitis along with neuronal necrosis, rarefaction, gliosis, and astrocytosis of the white and gray matter of the cerebrum, cerebellum, brainstem, and spinal cord. Pan-chlamydiae polymerase chain reaction protocols for the 16S ribosomal RNA and ompA genes were performed on samples of spinal cord and brain, and both resulted in amplicons. Sequencing of the products revealed that they were positive for a novel Chlamydia species. Infections by members of the phylum Chlamydiae have been reported in a diverse range of vertebrate hosts, including crocodilians. Chlamydia spp. infections are likely underdiagnosed because of a paucity of diagnostic techniques specific for detection. This is the first case report of a novel Chlamydia species associated with severe granulomatous encephalomyelitis in a false gharial.

Antemortem Diagnosis of Coxiellosis in a Blue and Gold Macaw ( Ara ararauna).

A 15-year-old female blue and gold macaw ( Ara ararauna) was presented for evaluation after being found laterally recumbent, reluctant to move, and lethargic. Results of a complete blood count showed an increased number of immature heterophils with increased cytoplasmic basophilia and degranulation and the presence of a left shift. Radiographs and a computed tomography scan were performed and revealed a markedly enlarged spleen. An ultrasound-guided fine-needle aspirate of the spleen was submitted for cytologic examination and aerobic bacterial culture. While the culture revealed no growth, cytologic examination identified mononuclear phagocytes with cytoplasmic vacuoles containing structures consistent with bacteria. Pan-bacterial 16S rRNA polymerase chain reaction of the splenic sample followed by direct sequencing identified a Coxiella-like agent identical to one previously isolated in the liver of a golden-mantled rosella ( Platycercus eximius). Phylogenetic analysis shows that avian coxiellosis agents and Coxiella burnetii, the agent of Q fever, represent 2 independent events of development of vertebrate pathogenicity in this group of tick endosymbionts. This report suggests diagnostic and treatment directions for coxiellosis in avian patients and indicates where further study is needed.

Coinfection with a novel fibropapilloma-associated herpesvirus and a novel Spirorchis sp. in an eastern box turtle (Terrapene carolina) in Florida.

Herpesviruses are important pathogens of chelonians, and include Chelonid herpesvirus 5, which is associated with fibropapillomatosis in sea turtles. Spirorchid trematodes are blood flukes that reside within the cardiovascular system of marine turtles and may be associated with severe disease. An eastern box turtle (Terrapene carolina) at the South Florida Wildlife Care Center (Fort Lauderdale, Florida) was presented to the facility with papillomatous growths behind both rear legs. Surgical removal resulted in remission for 8 months; however, lesions recurred, prompting a second surgery and acyclovir therapy. Surgical biopsies revealed subacute superficial inflammation associated with the supporting stroma of the cutaneous papillomas and granulomas within the superficial dermis containing fragmented and collapsed brown trematode eggs surrounded by multinucleated giant cells and epithelioid macrophages. Pan-herpesviral and pan-trematode consensus polymerase chain reaction and sequencing were run on tissue samples. Comparative sequence analysis revealed a novel alphaherpesvirus and a novel trematode in the genus Spirorchis. The animal became anorexic and was euthanized due to poor quality of life. While we do not yet have a complete understanding of the effects of herpesvirus and trematode infections in eastern box turtles, the findings thus presented provide initial insights into the disease relationships among these chelonians.

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps).

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe-based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons.

A novel siadenovirus detected in the kidneys and liver of Gouldian finches (Erythura gouldiae).

A novel siadenovirus was found in six captive Gouldian finches (Erythrura gouldiae) in the United States and Hungary. Histopathological examination revealed inclusions in the kidney of the captive Gouldian finch in the United States, and virions morphologically consistent with adenoviruses were seen by electron microscopy. Partial sequence of the DNA-dependent DNA polymerase gene was gained by consensus PCR and sequencing in all six finches, and all proved to be identical. In one Hungarian finch, additional sequence was obtained from the DNA polymerase gene, the pre-terminal protein (pTP) gene, the 52k gene, and the hexon gene. Bayesian, maximum likelihood, and distance-based analyses showed the novel virus clusters with the siadenoviruses, and is herein referred to as Gouldian finch adenovirus 1. The genes looked at in this study had low G+C percentages, which is common in the genus Siadenovirus, and suggestive of recent host switch. The significance of this virus' presence is unknown at this time as clinical signs of positive birds varied.