SARS-CoV-2 Infection Dynamics in the Pittsburgh Zoo Wild Felids with Two Viral Variants (Delta and Alpha) during the 2021-2022 Pandemic in the United States.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in multiple animal species besides humans. The goal of this study was to report clinical signs, infection progression, virus detection and antibody response in a group of wild felids housed in adjacent but neighboring areas at the Pittsburgh Zoo. Initially, five African lions (Panthera leo krugeri) housed together exhibited respiratory clinical signs with viral shedding in their feces in March of 2021 coinciding with infection of an animal keeper. During the second infection wave in December 2021, four Amur tigers (Panthera tigris altaica) and a Canadian lynx (Lynx canadensis) showed clinical signs and tested positive for viral RNA in feces. In infected animals, viral shedding in feces was variable lasting up to 5 weeks and clinical signs were observed for up to 4 weeks. Despite mounting an antibody response to initial exposure, lions exhibited respiratory clinical signs during the second infection wave, but none shed the virus in their feces. The lions were positive for alpha variant (B.1.1.7 lineage) during the first wave and the tiger and lynx were positive for delta variant (AY.25.1. lineage) during the second wave. The viruses recovered from felids were closely related to variants circulating in human populations at the time of the infection. Cheetahs (Acinonyx jubatus) in the park did not show either the clinical signs or the antibody response.

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.

Angiostrongylus cantonensis (rat lungworm) in Florida, USA: current status.

Angiostrongylus cantonensis has been found in Florida, USA, from the panhandle in the north to Miami and surrounding areas in the southern parts of the state, in both definitive and intermediate hosts in a limited studies completed in 2015. Additional studies have identified this parasite in a variety of intermediate hosts, both native and non-native gastropod species, with new host species recorded. Many areas in Florida with higher A. cantonensis prevalence were those with a high human population density, which suggests it is a matter of time before human infections occur in Florida. Case reports in the state currently involve non-human primates and include a gibbon and orangutan in Miami. Here, we report the current status of A. cantonensis in the state, as well as the infection in a capuchin monkey and presumptive infection in a red ruffed lemur in Gainesville, Florida.

REFERENCE INTERVALS FOR ERYTHROCYTE SEDIMENTATION RATE, LACTATE, FIBRINOGEN, HEMATOLOGY, AND PLASMA PROTEIN ELECTROPHORESIS IN CLINICALLY HEALTHY CAPTIVE GOPHER TORTOISES ( GOPHERUS POLYPHEMUS).

Currently available tests for the diagnosis of inflammatory disease in reptiles are limited and poorly sensitive. However, a number of hematological and plasma biochemical analytes are validated in the diagnosis of inflammation in mammals. The objective of this study was to establish reference intervals for erythrocyte sedimentation rate, lactate, heat-precipitated fibrinogen, hematology, and plasma protein electrophoresis based on total protein by biuret method in 23 clinically healthy, captive gopher tortoises ( Gopherus polyphemus) after successful rehabilitation and to determine differences by age, sex, and season. In order to investigate biological differences, samples were collected in April, July, and November. There were no sex differences in any measured analyte; however, there were significant differences by age and season. Immature animals (<2 kg) had significantly higher total protein, albumin : globulin ratio, pre-albumin, albumin, and α-1 globulin than adults. Tortoises sampled in the spring season had significantly higher total solids (refractometer) and lower eosinophils compared with animals sampled in the summer. Further investigation is required to determine the clinical utility of these analytes in the diagnosis of inflammation in this species.

DIAGNOSTIC PERFORMANCE OF INFLAMMATORY MARKERS IN GOPHER TORTOISES ( GOPHERUS POLYPHEMUS).

The lack of sensitive and specific markers of inflammation poses a diagnostic challenge in sick or injured reptile patients. The objective of this study was to investigate the diagnostic performance of blood analytes associated with inflammation in healthy ( n = 24) and sick ( n = 38) gopher tortoises ( Gopherus polyphemus). Receiver operating characteristic analysis identified the following as the best-performing diagnostic tests: erythrocyte sedimentation rate (area under the curve [AUC] = 0.812; 95% confidence interval [CI] = 0.693, 0.900), absolute mature heterophils (AUC = 0.771; 95% CI = 0.646, 0.869), total leukocytes (AUC = 0.767; 95% CI = 0.642, 0.866), lactate (AUC = 0.766; 95% CI = 0.641, 0.864), and absolute immature heterophils (AUC = 0.755; 95% CI = 0.628, 0.856). These results support the clinical application of additional tools for the diagnosis and monitoring of inflammatory disease in gopher tortoises. Clinicians may consider adding erythrocyte sedimentation rate and lactate to the minimum database for this species.

ACUTE NONCOMPRESSIVE NUCLEUS PULPOSUS EXTRUSION CAUSING PARAPLEGIA IN A SIBERIAN TIGER ( PANTHERA TIGRIS ALTAICA).

A neutered male Siberian tiger ( Panthera tigris altaica) presented with paraplegia of 5 days' duration. Thoracolumbar magnetic resonance imaging (MRI) revealed a focal, linear T2-W hyperintense intramedullary lesion at the level of T10-11 as well as reduced size of the nucleus pulposus of the T10-11 intervertebral disc. The animal was humanely euthanatized as a result of the severity of clinical signs and the poor prognosis. Gross necropsy revealed a dark red, firm focus on the spinal cord at the level of the T11 nerve roots. Histopathology revealed liquefactive necrosis of the ventral spinal cord at the level of T9-11, with hemorrhage and vascular changes within the cord and associated meninges. Fibrovascular tissue with hemorrhage, fibrin, and fragments of cartilage consistent with nucleus pulposus material was identified extradurally at this level. The findings were consistent with acute noncompressive nucleus pulposus extrusion.

EMERGENCY ANESTHESIA AND EXPLORATORY LAPAROTOMY IN A COMPROMISED PACIFIC WHITE-SIDED DOLPHIN (LAGENORHYNCHUS OBLIQUIDENS).

Anesthesia and surgery in cetaceans have inherent risks and have rarely been utilized as viable treatment options. This report represents the first known multidisciplinary team approach to emergency laparotomy in a compromised, 22-yr-old, female Pacific white-sided dolphin ( Lagenorhynchus obliquidens ). The inciting clinical signs and ancillary diagnostics were consistent with a mechanical ileus. Although no torsion or obstruction was apparent during surgery, severe enteritis and peritonitis were noted. Postoperatively, the animal was maintained on aggressive medical management with continuous supportive care until succumbing 3 days later with clinical pathology indicative of terminal sepsis and profound inflammation. Postmortem findings included generalized vascular stasis and segmental intestinal volvulus with infarction. To the authors' knowledge, this is one of the most-complex surgical and anesthetic procedures performed in a cetacean. Though the outcome was unsuccessful, this case represents the aquatic veterinary community's collective advances in the ability to treat cetaceans under human care.

Intraperitoneal implantation of life-long telemetry transmitters in three rehabilitated harbor seal pups.

BACKGROUND: Pinnipeds, including many phocid species of concern, are inaccessible and difficult to monitor for extended periods using conventional, externally attached telemetry devices that are shed during the annual molt. Archival satellite transmitters were implanted intraperitoneally into three stranded Pacific harbor seal pups (Phoca vitulina richardii) that completed rehabilitation, to evaluate the viability of this surgical technique for the deployment of life long telemetry devices in phocids. The life history transmitters record information throughout the life of the host and transmit data to orbiting satellites after extrusion following death. RESULTS: Surgeries were performed under general anesthesia and a single transmitter was inserted into the ventrocaudal abdominal cavity via a 7-8 cm incision along the ventral midline between the umbilicus and pubic symphysis or preputial opening in each animal. Surgeries lasted from 45 to 51 min, and anesthesic times ranged from 55 to 79 min. All animals recovered well, were released into dry holding pens overnight, and were given access to water the following day. All three animals exhibited an expected inflammatory response, with acute phase responses lasting approximately three to four weeks. All three animals were tracked via externally attached satellite transmitters after release at 58 to 78 days following surgery, and minimum post-release survival was confirmed through continued movement data received over 278 to 289 days. CONCLUSION: The initial findings of low morbidity and zero mortality encountered during captive observation and post-release tracking periods support the viability of this surgical technique for the implantation of long-term telemetry devices in phocids.

Gastric Intussusceptions in a Red Corn Snake (Pantherophis guttatus) Associated with Cryptosporidiosis.

A 3-year-old female red corn snake (Pantherophis guttatus) was presented for a three-week history of anorexia and decreased defecations. On physical examination, a soft midbody intracoelomic swelling was palpated. Transcutaneous coelomic ultrasound revealed a target-like mass on a transverse section of the stomach, suggesting the presence of a gastrointestinal intussusception. On exploratory coeliotomy, a double compounded esophagogastric and gastroduodenal intussusception was diagnosed and reduced surgically. A gastropexy was also performed to prevent recurrence. On histopathology, the gastric glandular mucosa showed moderate to marked proliferation. Diffusely lining the luminal surface of glandular epithelium and free within the lumen were a myriad of protozoa consistent with Cryptosporidium sp. A diagnosis of chronic proliferative gastritis due to Cryptosporidium sp. was made based on these findings. Intussusceptions are rare in reptiles and are infrequently reported in snakes. This is the first report of a double compounded intussusception in a nonmammalian species and the first report of an intussusception involving the stomach in a snake with gastritis due to Cryptosporidium sp.

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.

Population pharmacokinetics of enrofloxacin in purple sea stars (Pisaster ochraceus) following an intracoelomic injection or extended immersion.

OBJECTIVE To determine population pharmacokinetics of enrofloxacin in purple sea stars (Pisaster ochraceus) administered an intracoelomic injection of enrofloxacin (5 mg/kg) or immersed in an enrofloxacin solution (5 mg/L) for 6 hours. ANIMALS 28 sea stars of undetermined age and sex. PROCEDURES The study had 2 phases. Twelve sea stars received an intracoelomic injection of enrofloxacin (5 mg/kg) or were immersed in an enrofloxacin solution (5 mg/L) for 6 hours during the injection and immersion phases, respectively. Two untreated sea stars were housed with the treated animals following enrofloxacin administration during both phases. Water vascular system fluid samples were collected from 4 sea stars and all controls at predetermined times during and after enrofloxacin administration. The enrofloxacin concentration in those samples was determined by high-performance liquid chromatography. For each phase, noncompartmental analysis of naïve averaged pooled samples was used to obtain initial parameter estimates; then, population pharmacokinetic analysis was performed that accounted for the sparse sampling technique used. RESULTS Injection phase data were best fit with a 2-compartment model; elimination half-life, peak concentration, area under the curve, and volume of distribution were 42.8 hours, 18.9 µg/mL, 353.8 µg•h/mL, and 0.25 L/kg, respectively. Immersion phase data were best fit with a 1-compartment model; elimination half-life, peak concentration, and area under the curve were 56 hours, 36.3 µg•h/mL, and 0.39 µg/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the described enrofloxacin administration resulted in water vascular system fluid drug concentrations expected to exceed the minimum inhibitory concentration for many bacterial pathogens.

Cryptococcus gattii Type VGIIa Infection in Harbor Seals ( Phoca vitulina ) in British Columbia, Canada.

Cryptococcosis has been reported in marine mammals in the northeastern Pacific with increasing frequency in the last 15 yr. Although a variety of cetaceans have been diagnosed with cryptococcosis, Cryptococcus gattii has not been reported in pinnipeds. We document C. gattii VGIIa in a harbor seal ( Phoca vitulina ) pup and in an unrelated adult. Both animals were presented to Vancouver Aquarium's Marine Mammal Rescue Centre (VAMMRC) with generalized weakness, dehydration, respiratory compromise, minimally responsive mentation, and suboptimal body condition. Necropsy and histopathology findings were consistent in both animals and featured generalized lymphadenopathy, bronchopneumonia, and meningoencephalitis with intralesional yeast and fungemia. Cryptococcal serum antigen titers were ≥1,024 in both animals. Fungal culture of lung and lymph nodes confirmed C. gattii . Exposure was likely via inhalation prior to presentation to VAMMRC, and C. gattii infection was the proximate cause of death. This report expands the range of susceptible host species as C. gattii continues to emerge as a pathogen of concern in marine mammals in the northeastern Pacific.

POPULATION PHARMACOKINETICS OF ENROFLOXACIN AND ITS METABOLITE CIPROFLOXACIN IN THE GREEN SEA URCHIN (STRONGYLOCENTROTUS DROEBACHIENSIS) FOLLOWING INTRACOELOMIC AND IMMERSION ADMINISTRATION.

Sea urchin mass mortality events have been attributed to both infectious and noninfectious etiologies. Bacteria, including Vibrio spp. and Pseudoalteromonas spp., have been isolated during specific mortality events. Aquarium collection sea urchins are also subject to bacterial infections and could benefit from antimicrobial treatment, but pharmacokinetic studies have been lacking for this invertebrate group until recently. This study evaluated the pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin in the green sea urchin (Strongylocentrotus droebachiensis) after intracoelomic injection and medicated bath immersion administration. The utility of a population pharmacokinetic method using nonlinear mixed effects modeling (NLME) was also evaluated. Thirty sea urchins were assigned to either the injection or immersion group. Twelve study animals and three untreated controls were utilized for each administration method: enrofloxacin 10 mg/kg intracoelomic injection or a 6-hr enrofloxacin 10 mg/L immersion. Each animal was sampled four times from 0 to 120 hr. Water samples were collected during immersion treatment and posttreatment time points in both groups. Hemolymph and water sample drug concentrations were analyzed using high-performance liquid chromatography, and pharmacokinetic parameters were determined using an NLME population pharmacokinetic method. Enrofloxacin concentrations were fit to a two-compartment model with first-order input for the intracoelomic injection group. The enrofloxacin elimination half-life (t½), peak hemolymph concentration (CMAX), and area under the curve (AUC) were 38.82 hr, 90.92 µg/ml, and 1,199 hr·µg/ml, respectively. Enrofloxacin was modeled to a one-compartment model with first-order input for the immersion treatment. The enrofloxacin t½, CMAX, and AUC were 33.46 hr, 0.48 µg/ml, and 32.88 hr·µg/ml, respectively. Ciprofloxacin was detected in trace concentrations in all hemolymph samples, indicating minimal production of this metabolite. The concentrations of enrofloxacin achieved far exceeded minimum inhibitory concentrations reported for teleost pathogens. No adverse effects were associated with enrofloxacin administration by either treatment method or from hemolymph sampling.

Novel heterocyclic analogues of firefly luciferin.

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.

Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism.

According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.

Chemically modified firefly luciferase is an efficient source of near-infrared light.

Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of near-infrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.

Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain.

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.