Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

HEMATOLOGY AND PLASMA CHEMISTRY COMPARISONS AMONG JUVENILE AMERICAN BLACK BEARS (URSUS AMERICANUS) UNDERGOING REHABILITATION.

The American black bear (Ursus americanus) is an opportunistic and adaptable species with high rehabilitation success rates. Injured, ill, and orphaned bears across the southeastern United States are examined and treated at the University of Tennessee College of Veterinary Medicine followed by rehabilitation at Appalachian Bear Rescue (ABR). Hematology and biochemistry reference ranges exist for healthy adult black bears; however, most bears presenting to ABR are young and of variable health status. Thus, further investigation into the difference of blood values at varying ages and presentations is warranted. ABR records from 1996 to 2022 included 106 bears with completed hematology and plasma biochemistry panels (22 paired samples at intake and release, 84 at intake only). Intake-only samples consisted of 12 neonates (<3 mon old), 64 cubs (3-12 mon), and 30 yearlings (1-2 yr). Bears presented as orphaned neonates (22%), orphaned cubs (45%), malnourished yearlings (24%), and injured/ill (9%) during fall (16%), winter (13%), spring (32%), and summer (39%). Changes in hematology and plasma biochemistry results between intake and release included an increase in hematocrit and glucose. Injured/ill bears presented with higher total leukocyte count (WBC), absolute neutrophils (ABS segs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (P < 0.05). Positive correlation between ALT, AST, proteins, and blood urea nitrogen and negative correlation between absolute lymphocytes and alkaline phosphatase were noted with age. Both WBC and ABS segs were lower during winter (P < 0.05). Understanding what factors affect juvenile black bear blood values improves clinical expectations and evaluation upon intake, clinical evaluation, and treatment.

INTRAOCULAR PRESSURES OF AQUARIUM-HOUSED COWNOSE RAYS (RHINOPTERA BONASUS) WITH NORMAL AND ABNORMAL OPHTHALMIC EXAMS.

Cownose rays (Rhinoptera bonasus) are common elasmobranchs in zoos and aquaria; however, there is a lack of published information regarding ocular findings in this species. Intraocular pressure (IOP) was measured in a total of 52 cownose rays (Rhinoptera bonasus) from two unrelated aquaria (n = 22 from A1, n = 30 from A2) using a TonoVet rebound tonometer on two settings (dog = D, and unidentified species = P) as part of a full ophthalmologic examination. Adult (n = 38) and juvenile (n = 14) rays were sampled out of water briefly in sternal recumbency. Intraocular pressure (mean ± SD [range]) in the D setting (9.10 ± 2.57 [4-18] mmHg) was higher than the P setting (5.21 ± 2.32 [0-12] mmHg) (P<0.001). Statistical analysis revealed no difference in IOP between right and left eyes, and no correlation between body weight and IOP. No differences in IOP between sex, age group, and location were identified in either setting. However, a significant difference was observed between levels of severity of corneal disease in IOP D setting (P=0.006) and P setting (P=0.024), and levels of severity of intraocular disease in IOP D setting (P=0.034) only. This study provides baseline IOP values using rebound tonometry in aquarium-housed cownose rays with apparent corneal and intraocular lesions and reveals that the D setting may be more sensitive in identifying IOP changes in eyes with intraocular disease.

CLINICAL AND HISTOPATHOLOGIC OCULAR FINDINGS IN AQUARIUM-HOUSED COWNOSE RAYS (RHINOPTERA BONASUS).

Cownose rays (Rhinoptera bonasus) are susceptible to ocular disease with their prominent globes, but despite being popular animals housed in aquaria, there is little published information about their normal ocular anatomy and common pathologic ocular findings. A total of 63 live cownose rays (CNR) from three unrelated, separately housed groups had ocular examinations, and 5 adult rays were selected for ocular ultrasound. All examinations were performed out of the water, and most without anesthesia. Clinical findings were described, categorized, and scored by severity. Sixty-two of 63 rays (123 eyes) had clinical abnormalities, including 110 eyes with corneal pathology (mild = 76, moderate/severe = 34) and 74 eyes with intraocular pathology (mild = 44, moderate/severe = 30). Grey-to-white corneal opacities were the most common pathology (n = 58 rays/100 eyes) followed by cataracts (n = 41 rays/58 eyes), then persistent (or dysplastic) pupillary membranes (n = 14 rays/15 eyes). Most pathologic findings appeared inactive, but one aquarium had several CNR with active ocular pathology. There was a significant association between the diagnosis of moderate/severe corneal and intraocular pathology with age (P = 0.008 and P = 0.014, respectively) and weight (P = 0.001 and P = 0.039, respectively), as well as moderate/severe corneal pathology and group sampled (P = 0.03). There were no other significant variables identified. Additionally, histopathology of 14 eyes (11 rays) from two different facilities were examined, with keratitis (n = 8) and uveitis (n = 2) as the most common lesions. This study shows a high prevalence of pathologic ocular findings in cownose ray eyes with heavier adults more likely to be affected than lighter juveniles. Comprehensive ocular evaluation is important in this species and serial ocular exams and future studies should be pursued to monitor ocular disease progression and better understand possible etiologies.

UTILITY OF URINE ANTIGEN TESTING FOR DIAGNOSIS OF BLASTOMYCOSIS IN NONDOMESTIC FELIDS.

Blastomycosis caused by the fungus Blastomyces dermatitidis has been reported to cause disease in numerous species of nondomestic felids. Diagnosis of blastomycosis in domestic species often relies on the combination of clinical signs, radiographic findings, and commercial urinary antigen testing. In this report, the sensitivity, specificity, and positive and negative predictive values for urine blastomyces antigen testing for use in nondomestic felids were examined and compared with findings on postmortem examination. The study showed a sensitivity of 100%, specificity of 91.86%, positive predictive value of 50%, and negative predictive value of 100% for urine antigen testing. Furthermore, radiographic and hematologic findings were compared with those of animals diagnosed with blastomycosis. Radiographic evidence consistent with blastomycosis was found in those animals diagnosed via urine antigen testing, but no significant differences in plasma biochemistry parameters between diseased and nondiseased animals were found. This study provides evidence that a positive blastomycosis antigenuria test result should be combined with other diagnostic methods to confirm the presence of infection with B. dermatitidis, whereas a negative antigenuria test result is 100% effective in predicting the absence of disease.

RABIES DIAGNOSIS AND RESPONSE TO VACCINATION IN SOUTHERN TAMANDUA (TAMANDUA TETRADACTYLA).

Rabies has rarely been described in Xenarthra, and rabies vaccine response has not been documented. A southern tamandua (Tamandua tetradactyla) presented with nonspecific clinical signs and was euthanatized. Subsequently, immunohistochemistry and RT-PCR confirmed a rabies diagnosis. Following these tests, a group of eight captive tamanduas were vaccinated with a killed rabies vaccine, and titers were measured at the time of vaccination and 23 d later. One animal had day 0 titers suggestive of previous vaccination or exposure. All animals had detectable neutralizing rabies virus antibody titers after vaccination, but one animal failed to meet the World Organization for Animal Health's definition for adequate vaccination (≥0.5 IU/ml), and two other animals had low antibody titers (0.56 and 0.6 IU/ml). Rabies should be considered as a possible cause of illness in tamanduas, and rabies vaccination may be a useful preventative measure when anthropic interaction through medical care or ambassador roles is occurring.

Disseminated Mycobacterium genavense infection in a guinea pig (Cavia porcellus): a case report.

BACKGROUND: Mycobacteria are found in many environmental conditions and infect a variety of species, including rodents and rabbits. Guinea pigs are used experimentally as a model for Mycobacterium tuberculosis, but natural mycobacteriosis in guinea pigs has not been reported. CASE PRESENTATION: A 1.5-year-old female guinea pig was found acutely deceased with no premonitory illness. On gross post-mortem examination, multifocal to coalescing, raised, firm, pale tan nodules with discrete, irregular margins were noted over the surfaces of all lung lobes. Histopathology revealed nodules composed of clustered foamy macrophages and multinucleated giant cells containing numerous bacterial rods. Similar bacteria-laden macrophages were noted within sections of the liver, heart, palpebral conjunctiva, duodenum, and cecum. Polymerase chain reaction was performed on tissues collected during post-mortem examination. The 16S rRNA gene product was sequenced and was identical to the Mycobacterium genavense type strain. CONCLUSIONS: To the best of the author's knowledge, this report details the first documented case of Mycobacterium genvaense infection in a guinea pig and a follow up investigation of close-contact animals. Given their experimental susceptibility and this clinical case report, mycobacteriosis should be considered as a differential in guinea pigs exhibiting weight loss in the absence of other clinical signs. With the potential for zoonotic transmission in immunosuppressed individuals, precautions should be taken to safeguard human health in cases of guinea pigs with suspected M. genavense infection.

MEASUREMENT OF INTRAOCULAR PRESSURE USING REBOUND TONOMETRY IN ANESTHETIZED PERUVIAN FUR SEALS (ARCTOCEPHALUS AUSTRALIS) FROM PUNTA SAN JUAN, PERU.

Ocular disease in pinnipeds under human care is well described, and intraocular pressure (IOP) can be impacted by a variety of ophthalmic conditions. Species-specific reference parameters from clinically normal animals are instrumental for understanding how ophthalmic diseases may impact ocular pressures. IOP measurements were obtained using rebound tonometry from free-ranging Peruvian fur seals (Arctocephalus australis unnamed subspecies) at Punta San Juan, Peru, over a 6-yr period (2010-2016). Retrospective data obtained from 108 (81 adults and 27 neonates comprising 69 females and 39 males) anesthetized fur seals with normal anterior segment ophthalmic examinations was included in the analysis. Differences in IOP from each eye were compared to categorical variables (age, year, sex, restraint) using an independent-samples t test. All univariate results with a significance of P < 0.05 were included in multivariate analysis. Of the 13 general linear models evaluated, the top two for both the right and the left eye included age class when all variables were evaluated simultaneously. Neonates had significantly lower IOP values than adults in both the right eye (17.5 mm Hg; 95% confidence interval [CI]: 14.0-21.1 mm Hg compared to 33.5 mm Hg; 95% CI: 31.0-36.1 mm Hg, respectively) and the left eye (18.4 mm Hg; 95% CI: 14.4-22.5 mm Hg compared to 32.3 mm Hg; 95% CI: 29.3-35.3 mm Hg, respectively). Anesthesia method was not statistically significant (P > 0.05). This is the first report of normal IOP measurements for any fur seal species. Described data can be used to improve diagnosis and management of ocular alterations in pinnipeds.

Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus.

OBJECTIVE AND DESIGN: Human stem cell-derived hepatocyte-like cells (HLCs) have shown high potential as authentic model for dissection of the HCV life cycle and virus-induced pathogenesis. However, modest HCV replication, possibly due to robust innate immune responses, limits their broader use. To overcome these limitations and to dissect the mechanisms responsible for control of HCV, we analysed expression of key components of the interferon (IFN) system in HLCs, assessed permissiveness for different HCV strains and blocked innate immune signalling by pharmacological intervention. RESULTS: Transcriptional profiling revealed that HLCs constitutively express messenger RNA of RLRs, and members of the IFN pathway. Moreover, HLCs upregulated IFNs and canonical interferon-regulated genes (IRGs) upon transfection with the double-stranded RNA mimic poly(I:C). Infection of HLCs with Jc1-HCVcc produced only limited viral progeny. In contrast, infection with p100, a Jc1-derived virus population with enhanced replication fitness and partial resistance to IFN, resulted in robust yet transient viraemia. Viral titres declined concomitant with a peak of IRG induction. Addition of ruxolitinib, a JAK/STAT inhibitor, permitted chronic infection and raised p100 infectious virus titres to 1×105 FFU/mL. IRGs expression profiling in infected HLCs revealed a landscape of HCV-dependent transcriptional changes similar to HCV-infected primary human hepatocytes, but distinct from Huh-7.5 cells. Withdrawal of ruxolitinib restored innate immune responses and resulted in HCV clearance. CONCLUSION: This authentic human cell model is well suited to examine acute and chronic host-HCV interactions, particularly IFN-triggered antiviral effector functions and mechanisms of innate immune control of HCV infection.

Natural Canine Distemper Virus Infection in Linnaeus's 2-Toed Sloths (Choloepus didactylus).

An outbreak of canine distemper virus in a private zoo in eastern Tennessee in July 2016 led to fatal clinical disease in 5 adult, wild-caught Linnaeus's 2-toed sloths (Choloepus didactylus). Clinical signs included hyporexia, lethargy, mucopurulent nasal discharge, and oral and facial ulcers. At necropsy, affected animals had crusts and ulcers on the lips, nose, tongue, and oral cavity. Microscopically, all sloths had widespread, random, hepatic necrosis; lymphoid depletion; and bronchointerstitial pneumonia. The central nervous system did not contain gross or histopathologic lesions in any of the 5 sloths, although immunoreactivity for viral antigen was present within vessel walls. Epithelial cells and histiocytes within numerous organs contained intranuclear and intracytoplasmic inclusions and occasional syncytial cells. Canine distemper virus was confirmed with immunohistochemistry and virus isolation. Viral sequencing identified the novel American-4 strain prevalent in eastern Tennessee wildlife. This is the first pathologic characterization of canine distemper virus infection in sloths (family Choloepodidae, order Pilosa) and emphasizes the significant morbidity and mortality in this species.

OBJECTIVE GAIT ANALYSIS IN HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI) USING A PRESSURE-SENSITIVE WALKWAY.

Assessment of pododermatitis, osteoarthritis, and other causes of lameness in penguins can be challenging. Subjective gait analysis using visual observation and response to analgesic therapy can be affected by observer variation and caregiver placebo bias. A pressure-sensitive walkway (PSW), however, allows for objective gait analysis and assessment of analgesic therapeutic response. In this study, a 3-m-long PSW was used to analyze gait in 21 adult Humboldt penguins (Spheniscus humboldti). Medical record reviews and comprehensive examinations were performed on all penguins; five penguins were considered abnormal, with either right-sided (n = 3) or bilateral historical lameness-causing disease (n = 2) and were analyzed separately from the normal data set. All penguins walked across the PSW four times and gait parameters (step and stride distances and velocities, maximum force, impulse, and peak pressure) were calculated for each foot in each penguin. Statistical comparisons were made between right and left feet, sexes, and normal and abnormal penguins for each gait parameter. Among normal penguins, there were no significant differences between feet or sex. Left step width was shorter in abnormal penguins than that of normal penguins. Study results established baseline values for Humboldt penguins. This will allow objective monitoring of progression and response to therapy in penguin lameness cases, both current and future. The data also provide a foundation to compare gait parameters with other penguin populations and species.

Pharmacokinetics of orally administered tramadol in Muscovy ducks (Cairina moschata domestica).

This study documents the pharmacokinetics of oral tramadol in Muscovy ducks. Six ducks received a single 30 mg/kg dose of tramadol, orally by stomach tube, with blood collection prior to and up to 24 hr after tramadol administration. Plasma tramadol, and metabolites O-desmethyltramadol (M1), and N,O-didesmethyltramadol (M5) concentrations were determined by high-pressure liquid chromatography (HPLC) with fluorescence (FL) detection. Pharmacokinetic parameters were calculated using a one-compartment model with first-order input. No adverse effects were noted after oral administration. All ducks achieved plasma concentrations of tramadol above 0.10 µg/ml and maintained those concentrations for at least 12 hr. Elimination half-life was 3.95 hr for tramadol in ducks, which is similar to other avian species. All ducks in this study produced the M1 metabolite and maintained plasma concentrations above 0.1 µg/ml for at least 24 hr.

EFFECTS OF OXYGEN AND ISOFLURANE ANESTHESIA ON HEMOLYMPH GAS ANALYSIS AND RIGHTING REFLEX OF ASIAN FOREST (HETEROMETRUS LONGIMANUS) AND DICTATOR SCORPIONS (PANDINUS DICTATOR).

Large arachnids are commonly managed under professional care, and anesthesia is occasionally required for physical examination and diagnostic and therapeutic procedures. Anesthetic responses and hemolymph gas analysis have been studied previously in spiders, but scorpions have yet to be investigated. This study measured hemolymph gas values with an i-STAT point of care blood gas analyzer in healthy adult Asian forest scorpions (Heterometrus longimanus = HL, n = 8) and dictator scorpions (Pandinus dictator = PD, n = 12) breathing: 1) room air (RA), 2) 100% oxygen for 10 min in a chamber (OX), and 3) 5% isoflurane and oxygen (ISO) in a chamber until induction or loss of righting reflex. All scorpions recovered without complications, and there were no cartridge failures. Analysis of hemolymph gas values revealed that pH was lower in OX compared with RA and ISO and was lower in PD compared with HL scorpions. The partial pressure of carbon dioxide did not differ between inhaled gases but was higher in PD compared with HL. The partial pressure of oxygen (pO2) was higher in ISO compared with OX, and both were higher than when breathing RA. Despite a lack of species difference in pO2, PD had a more dramatic increase in pO2 in ISO compared with HL (significant species and inhalant interaction). PD had a significantly shorter induction time than HL, but recovery time (return of righting reflex) did not differ between species. Subjectively, HL exhibited rough inductions compared with PD, characterized by violent whole-body and tail movements. The unexpected increase in pO2 in ISO compared with OX, along with the species-specific differences and anesthetic effects, emphasizes the unique respiratory physiology of scorpions and demonstrates that further species-specific studies of anesthetics are warranted.

Analgesic Efficacy of Tramadol Compared With Meloxicam in Ducks (Cairina moschata domestica) Evaluated by Ground-Reactive Forces.

The purpose of this study was to determine the efficacy of tramadol and meloxicam in an induced, temporary arthritis model in ducks as assessed by ground-reactive forces measured by a pressure-sensitive walkway (PSW) system. Twelve ducks (Cairina moschata domestica) were randomly separated into 3 equal groups of 4 birds each: water control, tramadol treatment, and meloxicam treatment. Baseline measurements were collected by having all ducks walk along a 3-m-long PSW in a custom-built corral before anesthesia and induction of arthritis. Arthritis was induced in all groups through injection, under anesthesia, of a 3% monosodium urate (MSU) solution into the intertarsal joint. One hour after MSU injection, birds were orally gavage fed 1 mL of tap water (control), tramadol (30 mg/kg), or meloxicam (1 mg/kg). After treatments, all ducks were reevaluated on the PSW at 1, 2, 3, 4, 8, and 24 hours post-MSU injection. The difference in maximum force was significantly greater in the control group than in both the tramadol- (P = .006) and meloxicam-treated (P = .03) individuals. Post hoc comparisons revealed differences between control and treated birds occurred only at the 3- and 4-hour time points after administration. No differences were found in the absolute difference in maximum force between tramadol- and meloxicam-treated birds at any time point (P > .05). Results of this study support the hypothesis that tramadol (30 mg/kg PO) and meloxicam (1 mg/kg PO) improve certain objective variables in an induced arthritis model in ducks. Our findings also support studies in other avian species that determined that both tramadol and meloxicam are effective analgesic drugs in some birds.

REPRODUCTIVE HORMONE PATTERNS IN MALE AND FEMALE COWNOSE RAYS ( RHINOPTERA BONASUS) IN AN AQUARIUM SETTING AND CORRELATION TO ULTRASONOGRAPHIC STAGING.

Reproductive management of cownose rays ( Rhinoptera bonasus) under professional care plays an important role in conservation of the species, but hormone and ultrasonographic analyses of their 12-mo reproductive cycle have not been documented previously. Plasma reproductive hormone concentrations (17B-estradiol, progesterone, testosterone, and androstenedione) were measured monthly via radioimmunoassay for 1 yr in an aquarium-managed population of adult females ( n = 15) and males ( n = 5). Ultrasounds of the uterus were performed each month at the time of sample collection to identify gestation stage (0-5) based on a previously developed in-house staging system. Stages were correlated to hormone concentrations to track progression through pregnancy. Thirteen females were reproductively active, and each produced one pup in March-June, similar to timing for free-ranging populations. Female estradiol increased steadily throughout gestation from stages 0 to 5, while progesterone, testosterone, and androstenedione were increased only in early gestation (stages 1 and 2). Unlike month of year, gestation stage strongly predicted hormone concentration, but specific values to predict parturition date were not identified. Male testosterone and progesterone were higher in March-June (mating season) than July-January, while estradiol and androstenedione did not exhibit a seasonal pattern. Aquarium-managed cownose rays have similar reproductive patterns to what is reported in wild populations. Ultrasonographic monitoring with serial hormone analysis and accurate mating records will provide the most useful information for managing a reproductive population of cownose rays in an aquarium setting.

ACUTE-PHASE RESPONSES IN HEALTHY, MALNOURISHED, AND OTOSTRONGYLUS-INFECTED JUVENILE NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Acute-phase proteins (APPs) are utilized to detect early inflammation in many domestic and nondomestic species, but variability exists between species and inflammatory diseases as to which APPs are most useful. Stranded juvenile northern elephant seals (NESs; Mirounga angustirostris) undergoing rehabilitation at the Marine Mammal Center experience high mortality rates due to severe arteritis caused by the lungworm, Otostrongylus circumlitis (OC), and there are currently no effective antemortem diagnostic tools for this disease. To characterize patterns of the acute-phase response in the NES, two APPs-serum amyloid A (SAA) and C-reactive protein (CRP)-were measured, and serum protein electrophoresis was performed to measure albumin and globulin fractions in 81 serum samples from 58 NESs in four different health states: healthy, malnourished, preclinical for OC infection, or clinical for OC infection. Compared to healthy NESs (median, 11.2 mg/L), SAA concentrations were significantly increased in malnourished (33.9 mg/L), preclinical (247 mg/L), and clinical OC-infected NESs (328 mg/L) (P < 0.05). CRP concentrations were increased only in clinical OC-infected NESs (median, 53.9 mg/L) and were below detectable limits in the other three groups (<0.01 mg/L). These results show that SAA and CRP are positive APPs in NESs with OC infection, and that SAA may serve as the major APP for this species. Albumin : globulin ratios were significantly increased in malnourished NESs (median, 1.26) and decreased in clinical OC-infected NESs (0.53). As a result, albumin is a negative APP in the NES, similar to other mammalian species. APP monitoring can be helpful in detecting and monitoring inflammation in rehabilitating juvenile NESs.

SEROLOGIC RESPONSE TO CANINE DISTEMPER VACCINATION IN CAPTIVE LINNAEUS'S TWO-TOED SLOTHS ( CHOLOEPUS DIDACTYLUS) AFTER A FATAL CANINE DISTEMPER VIRUS OUTBREAK.

Canine distemper virus (CDV) affects many wild and captive, nondomestic species worldwide but has not been previously reported in Xenarthra. Paucity of information on vaccination safety and efficacy presents challenges for disease prevention in captive collections. CDV infections and subsequent mortalities in five captive Linnaeus's two-toed sloths ( Choloepus didactylus) in eastern Tennessee are reported. Clinical signs included oculonasal discharge, oral ulcerations, and diarrhea, and the diagnosis was confirmed by necropsy, histopathology, immunohistochemistry, virus isolation, and polymerase chain reaction. Viral sequencing identified the strain to be consistent with a new CDV lineage currently affecting domestic dogs and wildlife in Tennessee. Seven sloths were examined and vaccinated using a recombinant CDV vaccine on days 0 and 21. Subsequent blood samples showed increased titers in 3/4 sloths. Based on the outbreak and serologic findings postvaccination without adverse effects, the authors recommend recombinant CDV vaccination in sloths exposed to known carriers of CDV.

Determination of Tear Production and Intraocular Pressure With Rebound Tonometry in Wild Humboldt Penguins ( Spheniscus humboldti ).

Tear production and intraocular pressures (IOPs) were determined in 38 and 102 wild Humboldt penguins (Spheniscus humboldti), respectively, from the Punta San Juan Marine Protected Area in Ica, Peru. Tear production was measured by Schirmer tear test, and IOP was measured with a TonoVet rebound tonometer. Adult (n = 90) and chick (n = 12) penguins were sampled from 2 different beaches (north and south facing) during 2 sampling years (2010 and 2011). Results showed a mean ± SD (range) of 9 ± 4 (2-20) mm/min for tear production and 28 ± 9 (3-49) mm Hg for IOP. Tear production in penguins differed between beach and sex, whereas IOP differed between age, year, and beach. The IOPs were negatively correlated with packed cell volume. Tear production and IOP values had greater variation in this population than it has in other avian species. Previous investigations of IOP and tear production in Spheniscus species were conducted with birds housed under professional care in artificial marine and freshwater environments. This is the first study, to our knowledge, investigating tear production and IOP in wild penguins and establishes valuable reference intervals for this species.

Barrier-Independent, Fitness-Associated Differences in Sofosbuvir Efficacy against Hepatitis C Virus.

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvir-specific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvir-specific resistance mutations, linked to in vivo fitness of pretreatment viral populations.