Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

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.

EVALUATION OF A VISUAL ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR PREGNANCY DETECTION IN FOUR UNGULATE SPECIES.

This study evaluated the use of a commercially available, visual enzyme-linked immunosorbent assay (ELISA) for diagnosis of pregnancy in okapi (Okapia johnstoni), gerenuk (Litocranius walleri), eastern giant eland (Tragelaphus derbianus spp. gigas), and dama gazelle (Nanger dama). This assay has been validated for use in domestic cattle, sheep, goats, and water buffalo. Unlike other blood-based pregnancy associated glycoprotein (PAG) detection methods, this assay does not require sophisticated laboratory equipment for detection or interpretation and can therefore be utilized in many settings. Banked serum samples from gerenuk (n = 11), giant eland (n = 4), dama gazelle (n = 33) and okapi (n = 3) were tested, and a pregnant and nonpregnant sample from each individual were included. The ELISA showed 100% sensitivity and specificity in gerenuk and giant eland samples, and 0% sensitivity in dama gazelle and okapi samples. Using this assay, pregnancy was detected by 7-8 wk gestation in gerenuk and 6 wk in giant eland. These results are consistent with previous studies that were able to accurately detect pregnancy in other members of the family Bovidae, but it is possible that PAGs present in okapi and dama gazelle are structurally dissimilar relative to the intended test target, and are therefore unrecognizable using this test. The faint positivity in the dama gazelle assays may be due to cross-reactivity with other proteins in the sample, or due to inconsistent binding with the dama gazelle PAG. This ELISA appears to be an accurate, rapid, and inexpensive method of point-of-care pregnancy diagnosis in gerenuk and giant eland, but not okapi and dama gazelle. Additional studies should be pursued to further characterize the limits of pregnancy detection using this assay in gerenuk and giant eland, and to investigate the validity of this test in other nondomestic ruminant species.

SEROLOGICAL EVIDENCE OF COXIELLA BURNETII INFECTION IN THE WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN SOUTH AFRICA.

Coxiellosis, or Query (Q) fever, a disease caused by the intracellular bacteria Coxiella burnetii, was recently described in a managed breeding herd of white rhinoceros (Ceratotherium simum) in the southeastern United States. Clinical disease often results in abortion and could represent a conservation challenge for this species. In addition to the reproductive and herd management consequences, coxiellosis is also a zoonotic disease. Infection or clinical disease in any free-ranging rhinoceros species in a national park setting has not been previously described. In this study, evidence of prior infection was measured by immunofluorescent antibody titers in 89 serum samples collected from white rhinoceros within private reserves and a national park in South Africa. Total seropositivity was 48/89 (53.9% [95% CI, 43.6-63.9%]). Animals on private reserves had a seropositivity of 21/51 (41.1% [95% CI, 27.1-55.2%]), and national park rhinoceros had a higher rate of seropositivity at 71.0% [95% CI, 55.9-86.2%] (27/38; P= 0.004). Adults had a higher seropositivity compared with subadults (P= 0.03). There was no difference in seropositivity between sexes (P > 0.05). Results demonstrate that South African white rhinoceros populations are exposed to Coxiella, which could result in underrecognized reproductive consequences. Further studies should investigate potential implications for public health and conservation management of this species.

RENAL NEOPLASIA FROM A SINGLE POPULATION OF PTEROPODID BATS.

Neoplastic diseases have rarely been reported in the family Pteropodidae, and primary malignant renal neoplasms are generally uncommon across animal species. This case series describes four cases of primary renal neoplasia: three renal cell carcinomas and one nephroblastoma in three species of pteropodid bats, specifically large flying foxes (Pteropus vampyrus, n = 2), straw-colored fruit bat (Eidolon helvum, n = 1), and a little golden-mantled flying fox (Pteropus pumilus, n = 1). Two of the cases were diagnosed antemortem using ultrasonography, computed tomography, and cytology; and one of these bats with a renal cell carcinoma was treated successfully with a unilateral nephrectomy. The remaining two cases were diagnosed at necropsy.

Evaluation of Comprehensive Coelomic Fluid Analysis through Coelomic Pore Sampling as a Novel Diagnostic Tool in Elasmobranchs.

The objectives of this study were to describe a minimally invasive coelomic fluid sampling technique in elasmobranchs, to characterize the coelomic fluid composition in clinically normal and abnormal animals, and to compare findings from wild and managed populations. Fluid was collected via the coelomic pore in 89 individuals from 16 species spanning clinically normal and abnormal patients within a managed population (n = 54), a semi-managed open-lagoon population (n = 18), and a wild population (n = 17). Biochemical and cytological fluid analyses were performed on all samples, and bacterial and fungal culture, protein electrophoresis, and cholesterol electrophoresis were performed on a subset of samples. The presence of a variable volume of colorless to white and clear to slightly turbid coelomic fluid was consistent with a normal finding; however, the cytological and chemical makeup of coelomic fluid was found to provide additional clinically relevant information. The coelomic fluid from some of the abnormal samples (n = 37) contained white blood cells (n = 15) and concurrent bacteria (n = 7), the latter suggestive of bacterial coelomitis. Yolk was identified in both clinically normal and abnormal females. Of the biochemical parameters tested, calcium, chloride, cholesterol, osmolality, phosphorus, salinity, sodium, specific gravity, total protein, and urea nitrogen have clinical utility. Abnormal samples were mostly associated with reproductive disease, but to a lesser extent with coelomitis and hemocoelom. The wild and semi-managed groups had biochemical differences presumably reflective of the higher salinity of ocean water compared with that in the managed habitat. Aerobic bacteria were identified in normal (n = 7) and abnormal (n = 11) animals. Positive bacterial culture without inflammation may be normal. This study contributes to a further understanding of elasmobranch coelomic fluid analysis and its use as a diagnostic modality for the evaluation of elasmobranch health.

Plasma Bleomycin Concentrations during Electrochemotherapeutic Treatment of Fibropapillomas in Green Turtles Chelonia mydas.

Fibropapillomatosis of sea turtles is traditionally treated with surgical debulking techniques that are often associated with prolonged healing and tumor recurrence. Electrochemotherapy was recently described for green turtles Chelonia mydas and can be an alternative to surgery and even general anesthesia. The objectives of this study were to replicate an electrochemotherapy protocol from a previous report and add plasma bleomycin analysis to the treatment. After bleomycin injection into similarly sized tumors of two green turtles and immediate electroporation at two time points, plasma bleomycin reached detectable concentrations that were considerably lower than those found in human studies. At 3 months posttherapy, no healing complications or recurrences were encountered and only scar tissue remained. This study adds further support that electrochemotherapy with bleomycin has the potential to be used as an effective alternative treatment for this complex disease.

ADVANCING TRANSFUSION MEDICINE IN SEA TURTLES: OPTIMIZATION OF A CROSS-MATCHING PROTOCOL.

Whole blood transfusions are an essential treatment modality during rehabilitation of stranded sea turtles, however, standardized protocols for transfusions are not available in reptile medicine. The objective of this study was to optimize a cross-matching protocol for sea turtle blood transfusions. Fresh venous blood samples from 15 turtles (n = 14 green turtles, Chelonia mydas [Cm]; n = 1 loggerhead sea turtle, Caretta caretta [Cc]) were tested using a temperature-appropriate (i.e., reflecting body temperature), time-sensitive protocol in 26 reactions using two procedures for cross-match evaluation at 30 and 60 min at ambient air and water bath temperature. There were no significant differences between both protocols at 30- and 60-min incubation times or between microscopic evaluations at 2 or 5 min. The major cross-match identified 7/22 incompatible Cm-Cm reactions as observed by microscopic erythrocyte agglutination. Minor cross-matches resulted in 6/22 incompatible Cm-Cm reactions. About half of all Cm-Cm reactions (12/22) were compatible by major and minor cross-match. All Cc-Cm reactions (4/4) were incompatible. A higher than expected proportion of incompatible Cm cross-matching reactions suggests preexisting antibodies to nonself erythrocyte antigens in this species, or other factors promoting erythrocyte aggregation or agglutination. Preliminary data across Cm and Cc suggests cross-species incompatibility. These results indicate that sea turtles may react to donor erythrocytes even at the first transfusion. Concurrent major and minor cross-matching using the proposed protocol at 30-min incubation at room temperature should be considered a necessary and effective way to test for patient and donor incompatibilities in sea turtles.

Gastrointestinal Disease Associated with Non-albicans Candida Species in Six Birds.

A non-albicans Candida species was identified in 6 birds exhibiting clinical signs associated with gastrointestinal disease. The clinical disease signs noted in these 6 birds included diarrhea, regurgitation, and melena, and were considered concurrent or identified secondary to other comorbidities. Candida glabrata was identified in a yellow-naped Amazon parrot (Amazona auropalliata), a ring-necked dove (Streptopelia capicola), a blue-and-gold macaw (Ara ararauna), and 2 cockatiels (Nymphicus hollandicus). Candida krusei was identified in a white-crowned parrot (Pionus senilis). Fungal culture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification was correlated with results of fecal and/or crop Gram's stains, and DNA sequencing was used in one case. Three cases resolved after treatment, 2 birds died, and 1 was lost to follow-up. Non-albicans Candida infections are an emerging issue in human health care and are known to have an increased resistance to antifungal drugs. Similar to Candida albicans, these non-albicans Candida species are often identified in patients that have a history of prior antibiotic exposure. Recent data in human medicine describe a shift in species distribution away from C albicans dominance and toward other species, including C glabrata and C krusei. Both species are considered normal flora within the human and bird mycobiota and may emerge to cause disease, especially when the normal gastrointestinal environment has been disrupted.

Acute Necrotizing Pancreatitis in a Yellow-naped Amazon Parrot ( Amazona auropalliata).

A 25-year-old female yellow-naped Amazon parrot ( Amazona auropalliata) was presented for an acute onset of lethargy, ptyalism, and regurgitation. Results of hematologic testing and plasma biochemical analysis revealed severe leukopenia and heteropenia with toxic heterophils and marked hyperamylasemia and hypoproteinemia, consistent with gastrointestinal dysfunction. Survey radiograph findings were suggestive of coelomic effusion. Results of a contrast-enhanced computed tomography (CT) scan revealed a tubular, irregularly marginated structure along the medial aspect of the duodenum, consistent with an inflamed pancreas. The bird died soon after CT imaging, and acute necrotizing pancreatitis and pancreatic coagulative necrosis were observed at necropsy. This report describes the use of a less-invasive modality such as CT to aid in the antemortem diagnosis of acute pancreatitis in a psittacine bird. Pancreatitis should be considered a differential diagnosis in birds with gastrointestinal signs and coelomic effusion visualized on radiography, and the observation of an enlarged inflamed pancreas in the absence of a defined pancreatic mass on CT.

Phaeohyphomycosis resulting in obstructive tracheitis in three green sea turtles Chelonia mydas stranded along the Florida coast.

Three wild immature green sea turtles Chelonia mydas were found alive but lethargic on the shores of the Indian River Lagoon and Gulf of Mexico in Florida, USA, and subsequently died. Necropsy findings in all 3 turtles included partial occlusion of the trachea by a mass comprised of granulomatous inflammation. Pigmented fungal hyphae were observed within the lesion by histology and were characterized by culture and sequencing of the internal transcribed spacer 2 domain of the rRNA gene and D1/D2 region of the fungal 28s gene. The dematiaceous fungus species Veronaea botryosa was isolated from the tracheal mass in 2 cases, and genetic sequence of V. botryosa was detected by polymerase chain reaction in all 3 cases. Genetic sequencing and fungal cultures also detected other dematiaceous fungi, including a Cladosporium sp., an Ochroconis sp., and a Cochliobolus sp. These cases are the first report of phaeohyphomycosis caused by V. botryosa in wild marine animals.

Transition of an estuarine benthic meiofauna assemblage 1.7 and 2.8 years after a mining disaster.

Background: Estuaries are transitional coastal ecosystems that are threatened by multiple sources of human pollution. In 2015, mining tailings from an upstream dam failure caused massive metal contamination that impacted benthic assemblages on the Brazilian Rio Doce estuary. Methods: In this study, we investigate and compare meiofaunal assemblages with eDNA metabarcoding 1.7 years (2017) and 2.8 years (2018) after the initial contamination by mine tailings in order to evaluate the continued impact of sediment mine tailing contaminants on the structure of benthic assemblages after the disaster. Results: The community was dominated by Arthropoda and Nematoda 1.7 yr after the impacts (42 and 29% of meiofaunal sequence reads, respectively) but after 2.8 years Arthropoda (64.8% of meiofaunal sequence reads) and Rotifera (11.8%) were the most common taxa. This continued impact on meiofaunal assemblage revealed a lower phylogenetic diversity (7.8-fold) in 2018, despite overall decrease in metal concentration (Al, Ba, Cr, As, Fe, Zn, Mn, Pb, Cd, Co) in sediments. Our data suggests that differences in benthic assemblages and loss of diversity may be influenced by contaminants in sediments of this estuary, and indicate that broad eDNA assessments are greatly useful to understand the full range of biodiversity changes in dynamic estuarine ecosystems.

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.

Disseminated pancreatic adenocarcinoma in an eclectus parrot (Eclectus roratus).

CASE DESCRIPTION: A 15-year-old 0.412-kg (0.906-lb) sexually intact male eclectus parrot (Eclectus roratus) was evaluated because its owners found it lethargic and dyspneic at the bottom of its cage. CLINICAL FINDINGS: The parrot was thin and had generalized muscle wasting, diffuse feather loss, pale mucous membranes, and melena. The coelomic cavity was distended and soft on palpation, with coelomic effusion suspected. Results of a CBC indicated leukocytosis with left shift heterophilia, including toxic heterophils, lymphopenia, and anemia. Plasma biochemical analyses revealed severe hyperamylasemia. TREATMENT AND OUTCOME: Radiography revealed no evidence of a metallic foreign body but severe loss of coelomic detail, suggestive of a coelomic mass, coelomic effusion with coelomitis, or both. Ultrasonography and CT revealed severe accumulation of coelomic fluid; a large, heterogeneous, irregularly marginated, and moderately vascularized mass in the caudal aspect of the coelomic cavity; and multiple hepatic, coelomic, and pulmonary nodules. On the basis of a poor prognosis, the parrot was euthanized. Necropsy results confirmed exocrine pancreatic adenocarcinoma, with disseminated metastases in the liver, gastrointestinal tract, coelomic cavity, and lungs. CLINICAL RELEVANCE: In birds, pancreatic adenocarcinoma is rarely reported but should be considered a differential diagnosis for hyperamylasemia, coelomic mass, coelomic effusion, or abnormal gastrointestinal signs, alone or in combination. Ultrasonography and CT can be useful in further evaluating such patients and should be considered in the diagnostic plan.

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.

Mycobacterium haemophilum infection in a juvenile leatherback sea turtle (Dermochelys coriacea).

Mycobacteriosis is infrequently reported in free-ranging sea turtles. Nontuberculous Mycobacterium haemophilum was identified as the causative agent of disseminated mycobacteriosis in a juvenile leatherback turtle (Dermochelys coriacea) that was found stranded on the Atlantic coast of Florida. Disseminated granulomatous inflammation was identified histologically, most notably affecting the nervous system. Identification of mycobacterial infection was based on cytologic, molecular, histologic, and microbiologic methods. Among stranded sea turtles received for diagnostic evaluation from the Atlantic and Gulf of Mexico coasts of the United States between 2004 and 2015, the diagnosis of mycobacteriosis was overrepresented in stranded oceanic-phase juveniles compared with larger size classes, which suggests potential differences in susceptibility or exposure among different life phases in this region. We describe M. haemophilum in a sea turtle, which contributes to the knowledge of diseases of small juvenile sea turtles, an especially cryptic life phase of the leatherback turtle.