Rustrela Virus in Wild Mountain Lion (Puma concolor) with Staggering Disease, Colorado, USA.

We identified a rustrela virus variant in a wild mountain lion (Puma concolor) in Colorado, USA. The animal had clinical signs and histologic lesions compatible with staggering disease. Considering its wide host range in Europe, rustrela virus should be considered as a cause for neurologic diseases among mammal species in North America.

A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Evaluation of repRNA vaccine for induction and in utero transfer of maternal antibodies in a pregnant rabbit model.

Mother-to-child transmission is a major route for infections in newborns. Vaccination in mothers to leverage the maternal immune system is a promising approach to vertically transfer protective immunity. During infectious disease outbreaks, such as the 2016 Zika virus (ZIKV) outbreak, rapid availability of vaccines can prove critical in reducing widespread disease burden. The recent successes of mRNA vaccines support their evaluation in pregnant animal models to justify their use in neonatal settings. Here we evaluated immunogenicity of self-amplifying replicon (repRNA) vaccines, delivered with our clinical-stage LION nanoparticle formulation, in pregnant rabbits using ZIKV and HIV-1 as model disease targets. We showed that LION/repRNA vaccines induced robust antigen-specific antibody responses in adult pregnant rabbits that passively transferred to newborn kits in utero. Using a matrixed study design, we further elucidate the effect of vaccination in kits on the presence of pre-existing maternal antibodies. Our findings showed that timing of maternal vaccination is critical in maximizing in utero antibody transfer, and subsequent vaccination in newborns maintained elevated antibody levels compared with no vaccination. Overall, our results support further development of the LION/repRNA vaccine platform for maternal and neonatal settings.

Intramural coronary artery and myocardial pathology in captive tigers (Panthera tigris) and African lions (Panthera leo).

There is limited published data regarding cardiovascular disease in nondomestic felid populations. To address this knowledge gap, necropsy cases of tigers and lions with representative myocardial samples submitted to a diagnostic laboratory were histologically assessed with hematoxylin and eosin and Sirius red stains. A total of 32 submissions (15 tigers, 17 lions) were identified in a 4-year period. All tigers and lions had some degree of coronary artery lesions in the left ventricle and/or interventricular septum. Major findings included moderate to marked arteriosclerosis in 8 tigers (53%) and 4 lions (24%) and moderate to marked perivascular fibrosis in 10 tigers (67%) and 9 lions (53%). Moreover, 10 tigers (67%) and 8 lions (47%) had coronary artery lesions with variable degrees of perivascular cardiomyocyte degeneration and/or loss. To our knowledge, this is the first report describing coronary artery pathology in captive tigers and lions.

California sea lion (Zalophus californianus) lymph-node explant reveals involvement and possible transcriptional regulation of SLAM and nectin-4 during phocine distemper virus infection.

Phocine distemper virus (PDV) is a significant cause of mortality for phocid seals; however, the susceptibility of otariids to this virus is poorly understood. The authors used a lymph-node explant culture system from California sea lions (Zalophus californianus, CSL) to investigate: (1) the role of signaling lymphocyte activation molecule (SLAM) and nectin-4 in PDV infection and their cellular expression patterns, (2) if PDV induces transcriptional regulation of cell-entry receptors, and (3) the involvement of apoptosis in PDV infection. PDV replicated in the lymph-node explants with peak replication 3 days post-infection (dpi), but the replication was not sustained 4 to 5 dpi. The PDV+ cells co-localized SLAM and nectin-4. These cells expressed IBA1, indicating a histiocytic lineage. Comparison of receptor expression between infected and mock-infected lymph nodes suggested transcriptional downregulation of both receptors during the initial stage of infection and upregulation during the late stage of infection, but the values lack of statistical significance. Cleaved caspase-3+ cells were slightly increased in the infected lymph nodes compared with the mock-infected lymph node from 1 to 4 dpi, but without statistical significance, and a few apoptotic cells co-expressed PDV. The results suggest that lymph-node explants might be an important model to study PDV pathogenesis. CSLs have the potential to be infected with PDV, as they express both cell-entry receptors in histiocytes. The lack of statistical significance in the PDV replication, transcriptional regulation of viral receptors, and changes in apoptosis suggest that although CSL might be infected by PDV, they might be less susceptible than phocid species.

Mortality associated with SARS-CoV-2 in nondomestic felids.

Between September and November 2021, 5 snow leopards (Panthera uncia) and 1 lion (Panthera leo) were naturally infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) and developed progressive respiratory disease that resulted in death. Severe acute respiratory syndrome coronavirus 2 sequencing identified the delta variant in all cases sequenced, which was the predominant human variant at that time. The time between initial clinical signs and death ranged from 3 to 45 days. Gross lesions in all 6 cats included nasal turbinate hyperemia with purulent discharge and marked pulmonary edema. Ulcerative tracheitis and bronchitis were noted in 4 cases. Histologically, there was necrotizing and ulcerative rhinotracheitis and bronchitis with fibrinocellular exudates and fibrinosuppurative to pyogranulomatous bronchopneumonia. The 4 cats that survived longer than 8 days had fungal abscesses. Concurrent bacteria were noted in 4 cases, including those with more acute disease courses. Severe acute respiratory syndrome coronavirus 2 was detected by in situ hybridization using probes against SARS-CoV-2 spike and nucleocapsid genes and by immunohistochemistry. Viral nucleic acid and protein were variably localized to mucosal and glandular epithelial cells, pneumocytes, macrophages, and fibrinocellular debris. Based on established criteria, SARS-CoV-2 was considered a contributing cause of death in all 6 cats. While mild clinical infections are more common, these findings suggest that some SARS-CoV-2 variants may cause more severe disease and that snow leopards may be more severely affected than other felids.

Golden lion tamarin metapopulation dynamics five years after heavy losses to yellow fever.

The golden lion tamarin (GLT) is an Endangered primate endemic to Brazil's lowland Atlantic Forest. After centuries of deforestation and capture for the pet trade, only a few hundred individuals survived, all in isolated forest fragments 85 km from Rio de Janeiro city. Intensive conservation actions, including reintroduction of zoo-born tamarins, increased numbers to about 3700 in 2014. The most severe yellow fever epidemic/epizootic in Brazil in 80 years reduced two of the largest GLT populations by over 90%. Herein we report the results of a 2023 survey of GLTs designed to examine the dynamics of population recovery following yellow fever. Results indicate that populations hard hit by yellow fever are recovering due in part to immigration from adjacent forest fragments. No local extirpations were observed. About 4800 GLTs live in the survey area. This represents a 31% increase since the baseline survey completed in 2014. Two factors explain most of the increase: four large areas that had no GLTs or very low-density populations in 2014 are now at moderate density (three areas) or low density (one area), explaining 71% of overall increase since 2014. Increase in forest area within our survey area may explain up to 16% of the increase in GLT numbers since 2014. Results of computer simulations suggest that strengthening forest connectivity will facilitate metapopulation resilience in the face of mortality factors such as yellow fever.

Retrospective comparison of thyroid hormones in Steller sea lions Eumetopias jubatus under professional care with and without thyroid disease.

An evaluation of morbidity and mortality in Steller sea lions (SSLs) Eumetopias jubatus under professional care identified a high prevalence of thyroid disease in aged animals. While studies have investigated the effects of age, season, nutrition, stressors, and pollutants on thyroid hormones in pinnipeds, data from individuals with known thyroid disease are lacking. Histopathological reports of adult SSLs in North American aquariums from 1979 to 2022 with banked serum (n = 14) were reviewed. Radioimmunoassays, which have been validated in this species, were used to determine triiodothyronine (TT3), total thyroxine (TT4), and free thyroxine (fT4). A solid-phase chemiluminescent immunometric assay for thyroid-stimulating hormone (TSH) quantification was validated by testing inter- and intra-assay repeatability, recovery, and parallelism. Lesions included follicular cysts (n = 5), adenoma with thyroglossal duct or follicular cysts (n = 2), medullary or follicular C-cell carcinoma with follicular cysts and nodular hyperplasia (n = 1), neuroendocrine carcinoma with follicular cysts (n = 1), and sarcomatoid carcinoma metastasis to the thyroid (n = 1). Significantly lower concentrations of TT3, TT4, and fT4 in SSLs with thyroid disease (n = 10) versus those without (n = 4) were attributed to space-occupying lesions, advanced age, and/or season. TSH was not significantly different between groups. Fifty percent of diseased SSL profiles were within reference ranges, and all individuals with lesions were clinically euthyroid. These data expand the current understanding of thyroid disease in SSLs and underscore the need for additional diagnostics (e.g. cervical ultrasound) concurrently with hormone assays to effectively screen for underlying abnormalities.

Prevalence and pattern of congenital clubfoot among less than 5-year-old children in Ethiopia; cross-sectional based study.

BACKGROUND: Clubfoot is one of the most common congenital malformations, but it is also one of the most neglected public health problems among less than five-year-old children, mainly in middle- and low-income countries. Approximately 80% of clubfoot cases are found in low- and middle-income countries. In this study setting, no epidemiological studies have been conducted to assess clubfoot deformity. Due to this gap, the study aimed to assess prevalence, and pattern of congenital club foot among less than 5-year-old children. MATERIALS AND METHODS: An institutional-based cross-sectional study was carried out at Black Lion Specialized Hospital at the pediatric orthopedic clinic. The sample size was 261 to determine the prevalence and pattern of congenital clubfoot. Terms like frequency, percentage, and mean were used for data presentation. RESULT: A total of 36,303 pediatric patients visited Black Lion Specialized Hospital during the study period, and clubfoot prevalence was 7.2 per 1000. The largest subclassification of congenital clubfoot was idiopathic clubfoot, which accounted for 6.2 per 1000, whereas syndromic clubfoot was 0.3 per 1000, and neuropathic clubfoot was shared at 0.36 per 1000. Most of the cases in this study were bilateral clubfoot, with males having more dominance. CONCLUSION: In the area under investigation, a significant prevalence of congenital clubfoot was observed, especially among male children. The majority of cases were bilateral, with idiopathic clubfoot being the dominant form.

Comparative pharmacokinetics of a single oral dose of meloxicam in the California sea lion (Zalophus californianus) and Pacific harbor seal (Phoca vitulina richardii).

Pharmacokinetics studies have investigated meloxicam, a non-steroidal anti-inflammatory drug, dosing strategies in a wide variety of non-domestic animals; however, there is no prior study examining well-founded dosing for pinnipeds. To develop dosing protocols, pharmacokinetic information is needed, with an examination of differences between pinniped species. Apparently, healthy California sea lions (Zalophus californianus: CSL; n = 13) and Pacific harbor seals (Phoca vitulina richardii: PHS; n = 17) that had completed rehabilitation were enrolled into a population-based pharmacokinetic study. Each animal was administered a single oral dose of meloxicam at 0.1 mg/kg, and two blood samples were collected from each animal at varying intervals during a 96-h study period. Plasma concentrations of meloxicam were determined by high-pressure liquid chromatography. Data were analyzed with nonlinear mixed effects modeling (Phoenix® NLME™, Certara, St. Louis, MO 63105, USA). The results indicated that in PHS, peak plasma concentration (Cmax) was 0.33 µg/mL with an elimination half-life (Ke t½) of 31.53 h. In CSL, Cmax was 0.17 µg/mL with Ke t½ of 32.71 h. All animals enrolled completed the study without outward adverse clinical signs. The elimination half-life was longer than previously recommended dosing intervals for pinnipeds; however, we cannot speculate in the optimum clinical dose from these results.

From Caves to the Savannah, the Mitogenome History of Modern Lions (Panthera leo) and Their Ancestors.

Lions (Panthera leo) play a crucial ecological role in shaping and maintaining fragile ecosystems within Africa. Conservation efforts should focus on genetic variability within wild populations when considering reintroduction attempts. We studied two groups of lions from two conservation sites located in Zambia and Zimbabwe to determine their genetic make-up, information that is usually unknown to the sites. In this study, we analysed 17 specimens for cytb and seven microsatellite markers to ascertain family relationships and genetic diversity previously obtained by observational studies. We then produced a standardised haplogroup phylogeny using all available entire mitogenomes, as well as calculating a revised molecular clock. The modern lion lineage diverged ~151 kya and was divided into two subspecies, both containing three distinct haplogroups. We confirm that Panthera leo persica is not a subspecies, but rather a haplogroup of the northern P.l. leo that exited Africa at least ~31 kya. The progenitor to all lions existed ~1.2 Mya, possibly in SE Africa, and later exited Africa and split into the two cave lion lineages ~175 kya. Species demography is correlated to major climactic events. We now have a detailed phylogeny of lion evolution and an idea of their conservation status given the threat of climate change.

Vaccine Platform Comparison: Protective Efficacy against Lethal Marburg Virus Challenge in the Hamster Model.

Marburg virus (MARV), a filovirus, was first identified in 1967 in Marburg, Germany, and Belgrade, former Yugoslavia. Since then, MARV has caused sporadic outbreaks of human disease with high case fatality rates in parts of Africa, with the largest outbreak occurring in 2004/05 in Angola. From 2021 to 2023, MARV outbreaks occurred in Guinea, Ghana, New Guinea, and Tanzania, emphasizing the expansion of its endemic area into new geographical regions. There are currently no approved vaccines or therapeutics targeting MARV, but several vaccine candidates have shown promise in preclinical studies. We compared three vaccine platforms simultaneously by vaccinating hamsters with either a single dose of an adenovirus-based (ChAdOx-1 MARV) vaccine, an alphavirus replicon-based RNA (LION-MARV) vaccine, or a recombinant vesicular stomatitis virus-based (VSV-MARV) vaccine, all expressing the MARV glycoprotein as the antigen. Lethal challenge with hamster-adapted MARV 4 weeks after vaccination resulted in uniform protection of the VSV-MARV and LION-MARV groups and 83% of the ChAdOx-1 MARV group. Assessment of the antigen-specific humoral response and its functionality revealed vaccine-platform-dependent differences, particularly in the Fc effector functions.

Unlocking synergies of drawdown operation: Multi-objective optimization of reservoir emergency storage capacity.

Optimizing reservoir drawdown operations holds significant implications for hydropower generation, water supply, and drought mitigation strategies. However, achieving multi-objective optimization in reservoir drawdown operations poses fundamental challenges, particularly considering emergency storage capacity and seasonal drought patterns. This study introduces a novel multi-objective optimization framework tailored for a mega reservoir, focusing on drawdown operations to enhance hydropower generation and water supply reliability. A drawdown operation model leveraging a multi-objective ant lion optimizer is developed to simultaneously maximize reservoir hydropower output and minimize water shortage rates. China's Three Gorges Reservoir (TGR), situated over the upper reaches of the Yangtze River, constitutes the case study, with the standard operation policy (SOP) serving as a benchmark. Results showcase the efficacy of the proposed method, with substantial improvements observed: a 10.6% increase in hydropower output, a 6.0% reduction in water shortage days, and a 9.5% decrease in minimal reservoir water release compared to SOP. This study provides robust technical and scientific bolster to optimize reservoir ESC and enhance the synergy between hydropower generation, water supply, and drought resilience. Additionally, it offers decision-makers actionable strategies that account for emergency water supply capacities. These strategies aim to support mega reservoir's resilience against extreme drought events facilitating the collaboration between modelers and policy-makers, by means of intelligent optimization and decision-making technologies.

Quantifying animals' perception of environmental predictability using anticipatory behavior.

Animals under human care often experience predictable daily husbandry events, which can promote the development of anticipatory behavior. Previous research suggests even short delays in the arrival of a predictable, desired outcome can lead to negative welfare outcomes for animals. As such, providing reliable information to animals regarding the onset of important events may be a simple but useful method to support positive welfare. Here we evaluated the quantitative characteristics of anticipatory behavior of a California sea lion at the San Francisco Zoo in three situations (1) when the animal had accurate information about the occurrence of the event (temporally predictable training sessions), (2) when the information was semi-reliable (unscheduled training session), and (3) when a reliable signal was offered before unscheduled sessions. Results showed that providing a reliable cue resulted in a stronger temporal correlation of anticipation before the beginning of the unscheduled session, similar to the temporally predictable training session. However, providing a reliable cue did not reduce the intensity of the anticipatory behavior. We propose to take into account two aspects of the quantitative characteristics of anticipatory behavior: correlation with time until a desired event (correlation) and intensity, where the correlation indicates the ability of an individual animal to predict the occurrence of an event, and the intensity indicates the degree of sensitivity of the animal to reward. We discuss the implications for animal welfare and husbandry.

Anthropogenic Transmission of SARS-CoV-2 from Humans to Lions, Singapore, 2021.

In Singapore, 10 captive lions tested positive for SARS-CoV-2 by real-time PCR. Genomic analyses of nanopore sequencing confirmed human-to-animal transmission of the SARS-CoV-2 Delta variant. Viral genomes from the lions and zookeeper shared a unique spike protein substitution, S:A1016V. Widespread SARS-CoV-2 transmission among humans can increase the likelihood of anthroponosis.

Probable Transmission of SARS-CoV-2 from African Lion to Zoo Employees, Indiana, USA, 2021.

We describe animal-to-human transmission of SARS-CoV-2 in a zoo setting in Indiana, USA. A vaccinated African lion with physical limitations requiring hand feeding tested positive for SARS-CoV-2 after onset of respiratory signs. Zoo employees were screened, monitored prospectively for onset of symptoms, then rescreened as indicated; results were confirmed by using reverse transcription PCR and whole-genome virus sequencing when possible. Traceback investigation narrowed the source of infection to 1 of 6 persons. Three exposed employees subsequently had onset of symptoms, 2 with viral genomes identical to the lion's. Forward contact tracing investigation confirmed probable lion-to-human transmission. Close contact with large cats is a risk factor for bidirectional zoonotic SARS-CoV-2 transmission that should be considered when occupational health and biosecurity practices at zoos are designed and implemented. SARS-CoV-2 rapid testing and detection methods for big cats and other susceptible animals should be developed and validated to enable timely implementation of One Health investigations.

Can smaller predators expand their prey base through killing juveniles? The influence of prey demography and season on prey selection for cheetahs and lions.

Smaller predators may overcome body size restrictions on their prey base by selecting for juveniles of larger prey species. However, traditional prey selection models ignore demographic classes within prey species. We refined these models for two predators with contrasting body sizes and hunting strategies, by including seasonal consumption and availability of prey demographic classes. We predicted that cheetahs would select for smaller neonate and juvenile prey especially of larger species, while lions would select for larger, adult prey. We further predicted seasonal diet shifts in cheetah, but not lion. We recorded species-specific demographic class prey use (kills) via direct observation and GPS cluster of cheetahs and lions fitted with GPS collars. Species-specific demographic class prey availability was estimated from monthly driven transects, and species-specific demographic class prey preferences were estimated. The availability of prey demographic classes varied seasonally. Cheetahs preferred neonates, juveniles, and sub-adults during the wet season, but adults and juveniles during the dry season. Lions preferred adult prey irrespective of season, with sub-adults, juveniles, and neonates killed relative to their abundance. This confirms that traditional prey preference models do not adequately account for demographic-specific prey preference. This is particularly important for smaller predators, like cheetahs, that focus on smaller prey but can expand their prey base by killing juveniles of larger species. For these smaller predators, prey availability will vary strongly seasonally, making them more vulnerable to processes that influence prey reproduction, like global change.

SCSLnO-SqueezeNet: Sine Cosine-Sea Lion Optimization enabled SqueezeNet for intrusion detection in IoT.

Security and privacy are regarded as the greatest priority in any real-world smart ecosystem built on the Internet of Things (IoT) paradigm. In this study, a SqueezeNet model for IoT threat detection is built using Sine Cosine Sea Lion Optimization (SCSLnO). The Base Station (BS) carries out intrusion detection. The Hausdorff distance is used to determine which features are important. Using the SqueezeNet model, attack detection is carried out, and the network classifier is trained using SCSLnO, which is developed by combining the Sine Cosine Algorithm (SCA) with Sea Lion Optimization (SLnO). BoT-IoT and NSL-KDD datasets are used for the analysis. In comparison to existing approaches, PSO-KNN/SVM, Voting Ensemble Classifier, Deep NN, and Deep learning, the accuracy value produced by devised method for the BoT-IoT dataset is 10.75%, 8.45%, 6.36%, and 3.51% higher when the training percentage is 90.

Numt Parser: Automated identification and removal of nuclear mitochondrial pseudogenes (numts) for accurate mitochondrial genome reconstruction in Panthera.

Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program Numt Parser, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from 2 ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. Numt Parser decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of Numt Parser to 2 other bioinformatic approaches that can be used to account for numt contamination. We found that Numt Parser outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. Numt Parser therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.

Linking glucocorticoid variations to monthly and daily behavior in a wild endangered neotropical primate.

Identifying the factors swaying physiological stress levels in wild animals can help depict how they cope with environmental and social stressors, shedding light on their feeding ecology, behavioral plasticity, and adaptability. Here, we used noninvasive methods to explore the link between glucocorticoid levels and behavior in an endangered neotropical primate facing habitat fragmentation pressure, the black lion tamarin (Leontopithecus chrysopygus). We investigated monthly and day-to-day glucocorticoid variations independently to attempt to disentangle the complex nature of the adrenocortical activity. Between May 2019 to March 2020, we followed two groups of black lion tamarins in two different areas, a continuous forest and a small fragment, and gathered behavioral data (over 95 days in total; 8.6 ± 3.9 days/month) and fecal samples (Nsamples = 468; 4.93 ± 3.5 samples/day) simultaneously. Preliminary analyses enabled us to identify circadian variations linked to the biological rhythm, which were taken into account in subsequent models. Monthly analyses revealed that black lion tamarin fecal glucocorticoid metabolite levels vary according to changes in activity budget associated with the fruit consumption, movement, and resting time of the groups. At a day-to-day level, while intergroup encounters led to increases in fecal glucocorticoid metabolite concentrations, we found that changes in food intake or activity level did not trigger physiological stress responses. These findings suggest that diet and ranging patterns, driven by food availability and distribution, influence physiological stress at a seasonal scale, while acute stressors such as interspecific competition trigger short-term stress responses. Exploring fecal glucocorticoid metabolite variations over different timescales can help uncover the predictive and reactive facets of physiological stress in wild species. Moreover, having a comprehensive understanding of the physiological state of species is a valuable conservation tool for evaluating how they cope in changing environments.