Population structure, intergroup interaction, and human contact govern infectious disease impacts in mountain gorilla populations.

Infectious zoonotic diseases are a threat to wildlife conservation and global health. They are especially a concern for wild apes, which are vulnerable to many human infectious diseases. As ecotourism, deforestation, and great ape field research increase, the threat of human-sourced infections to wild populations becomes more substantial and could result in devastating population declines. The endangered mountain gorillas (Gorilla beringei beringei) of the Virunga Massif in east-central Africa suffer periodic disease outbreaks and are exposed to infections from human-sourced pathogens. It is important to understand the possible risks of disease introduction and spread in this population and how human contact may facilitate disease transmission. Here we present and evaluate an individual-based, stochastic, discrete-time disease transmission model to predict epidemic outcomes and better understand health risks to the Virunga mountain gorilla population. To model disease transmission we have derived estimates for gorilla contact, interaction, and migration rates. The model shows that the social structure of gorilla populations plays a profound role in governing disease impacts with subdivided populations experiencing less than 25% of the outbreak levels of a single homogeneous population. It predicts that gorilla group dispersal and limited group interactions are strong factors in preventing widespread population-level outbreaks of infectious disease after such diseases have been introduced into the population. However, even a moderate amount of human contact increases disease spread and can lead to population-level outbreaks.

Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri.

Briefly considered extinct in the wild, the future of the Wyoming toad (Anaxyrus baxteri) continues to rely on captive breeding to supplement the wild population. Given its small natural geographic range and history of rapid population decline at least partly due to fungal disease, investigation of the diversity of key receptor families involved in the host immune response represents an important conservation need. Population decline may have reduced immunogenetic diversity sufficiently to increase the vulnerability of the species to infectious diseases. Here we use comparative transcriptomics to examine the diversity of toll-like receptors and major histocompatibility complex (MHC) sequences across three individual Wyoming toads. We find reduced diversity at MHC genes compared to bufonid species with a similar history of bottleneck events. Our data provide a foundation for future studies that seek to evaluate the genetic diversity of Wyoming toads, identify biomarkers for infectious disease outcomes, and guide breeding strategies to increase genomic variability and wild release successes.

Implications of comparative ventral body wall histology on selection of abdominal surgical approach and closure in 12 species of fish.

A ventral midline surgical approach for fish celiotomy is commonly performed in veterinary clinical medicine and research, although the relevant ventral body wall anatomy of many fish species is not well documented. Histological evaluation of tissue samples from the ventral body wall of 12 fish species was performed to provide a reference for surgical approach and closure decisions. The width between muscle bundles running parallel to the long axis and total thickness of tissue layers varied among species. An appreciable space between longitudinal muscles of the ventral body wall and a lack of muscle, vessels and nerves on midline in all species examined supports recommendations of ventral midline incisions to spare important structures. Dense connective tissue consistent with an aponeurosis between musculature along the ventral body wall was not observed in any species evaluated. Connective tissue was concentrated within the dermis of all species evaluated, with an additional layer of collagen along the coelomic membrane in Russian sturgeon Acipenser gueldenstaedtii, koi Cyprinus carpio, goldfish Carassius auratus, black drum Pogonias cromis, black seabass Centropristis striata, tomtate Haemulon aurolineatum and scup Stenotomus caprinus. A sufficiently wide space on ventral midline for practical targeting during the surgical approach is present in A. gueldenstaedtii, C. carpio, striped bass Morone saxatilis, H. aurolineatum, P. cromis, rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta. Sand perch Diplectrum formosum, C. auratus, S. caprinus, grey triggerfish Balistes capriscus and black sea bass Centropristis striata have a negligible space between longitudinal muscles on midline. The variation in ventral body wall structure observed in this study helps inform surgical decision making for celiotomy incision and closure in these species.

EVALUATION OF EUTHANASIA OF MOON JELLYFISH (AURELIA AURITA) USING SIMPLE SALT SOLUTIONS.

Immersion euthanasia methods reported over the most recent decades for aquatic invertebrates use organic alcohols or halogenated hydrocarbons that can interfere with nuclear magnetic resonance (NMR) analysis. A rolling study design evaluated potassium chloride (KCl), magnesium chloride (MgCl2), and magnesium sulfate (MgSO4) as potential ion-based euthanasia methods for moon jellyfish (Aurelia aurita) destined for metabolomic analysis by NMR spectroscopy. Death was defined as the cessation of autonomous bell pulsing and response to external stimulus. MgCl2 applied at a dose of 142 g/L provided euthanasia within 32 sec of applications without the untoward effects observed with the other two salts. Euthanasia with KCl at the doses tested was associated with abnormal behavior and tissue degradation during dissection. MgSO4 at the doses tested resulted in abnormal behavior and failed to provide rapid euthanasia.

THE MESOPTERYGIAL VEIN: A RELIABLE VENIPUNCTURE SITE FOR INTRAVASCULAR ACCESS IN BATOIDS.

Intravascular access in batoid species is commonly achieved using the ventral coccygeal or radial wing vessels. However, these approaches can be difficult because of the presence of cartilage, lack of specific landmarks, species variation, and small vessel size in many species. This study used postmortem contrast radiography and gross dissection to develop landmarks for a new, dependable vascular access in three Myliobatiform species commonly maintained in captivity: Atlantic stingray (Hypanus sabinus), cownose ray (Rhinoptera bonasus), and smooth butterfly ray (Gymnura micrura). The mesopterygial vein provides quick vascular access and is suitable for administration of large fluid volumes and intravascular drugs. It is located immediately ventrolateral to the metapterygium cartilage, which sits adjacent to the coelomic cavity and supports the caudal half of the pectoral fin. Using the pectoral girdle and cranial third of the metapterygium cartilage as landmarks, vascular access can be achieved by directing a needle medially at approximately a 30° (adult cownose rays) or 45° angle (Atlantic stingrays, juvenile cownose rays, smooth butterfly rays) toward the metapterygium cartilage. Differences in the degree of needle direction are due to species and age-specific shapes of the metapterygium cartilage. The mesopterygial vein is an alternate site of quick and reliable venous access in batoid species.

ASSESSMENT OF A VISUAL SCORING SYSTEM FOR IDENTIFYING AND QUANTIFYING ANEMIA IN MALE EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA).

The North Carolina State University College of Veterinary Medicine Turtle Rescue Team (TRT) regularly admits wild eastern box turtles (EBT) ( Terrapene carolina carolina) for treatment and rehabilitation. Adult males of this species generally have an impressive red coloration of the iris, a feature commonly used to differentiate them from their brown-eyed female conspecifics. Male EBTs that present often display marked pallor of the eyes, which has accompanied clinical anemia. The aim of this study was to examine the relationship between eye color and packed cell volume (PCV) in adult male EBTs, and to assess the accuracy of using eye color to predict PCV. Eye-color scoring cards were created using Adobe Creative Cloud Photoshop®. Each card was composed of blocks displaying progressively lighter tints of a red hue, with a number (score) assigned to each block. Five-block, six-block, and nine-block scoring cards were prepared, with lower numbers (scores) corresponding to lighter hues. To determine the variation in subjective color scores assigned by different human observers, 85 individuals were asked to score EBT eye color in three animals using each of the three scoring cards. Eye color of adult male EBTs that presented to the TRT ( n = 18) and those housed at the North Carolina Museum of Natural Sciences ( n = 6) were scored using each of the three cards, immediately preceding venipuncture of the occipital sinus for determination of PCV. All animals were scored by at least two individuals. Turtles with low scores (pale eyes) using all three scoring cards consistently had lower PCVs. The five- and six-block scorecards had a positive predictive value (PPV) of 100% while the nine-block score card had a PPV of 71.4%. This specific and noninvasive method for estimating PCV of male EBTs is clinically desirable in both rehabilitation and hospital settings.

Nephrolithiasis in free-ranging North American river otter (Lontra canadensis) in North Carolina, USA.

The North American river otter (Lontra canadensis) serves as an indicator species for environmental monitoring, is prized as a valuable furbearer, and is a popular display animal in zoologic collections. Nephrolithiasis has been reported as a frequent problem in other free-ranging and captive otter species but is rarely reported in North American river otters. In this study, we compared the prevalence of nephrolithiasis diagnosed using routine gross pathologic examination techniques with the use of computed tomography (CT) of excised kidneys. We also evaluated whether otter nephroliths could be accurately classified by their CT densities, and we examined the renal tissue uric acid concentrations in free-ranging otters in North Carolina, USA. Kidneys were collected from carcasses of legally trapped, free-ranging animals. Nephroliths were observed in 16.2% of the individuals (n = 229). Associations were found between age and nephrolith status and between capture location and nephrolith status (P = 0.026 and < 0.001, respectively). Computed tomography Hounsfield unit density measurements were not useful in determining nephrolith chemical composition in this study. Renal tissue uric acid concentrations were similar across genders, age groups, and stone status. The chemical composition of the nephroliths was determined by scanning electron microscopy-energy dispersive X-ray spectroscopy to be calcium phosphate in the carbonate form.

Anaerobic oral flora in the North American black bear (Ursus americanus) in eastern North Carolina.

Microbial flora can provide insight into the ecology and natural history of wildlife in addition to improving understanding of health risks. This study examines the anaerobic oral flora of hunter killed black bears (Ursus americanus) in eastern North Carolina. Oral swabs from the buccal and lingual supragingival tooth surfaces of the first and second mandibular and maxillary molars of 22 black bears were inoculated onto Brucella Blood Agar plates supplemented with hemin and vitamin K after transport from the field using reduced oxoid nutrient broth. Sixteen anaerobic bacterial species, representing nine genera were identified using the RapID ANA II Micromethod Kit system and a number of organisms grown that could not be identified with the system. The most frequently identified anaerobes were Peptostreptococcus prevotii, Streptococcus constellatus, and Porphyromonas gingivalis. The diversity in the anaerobic oral flora of black bear in eastern North Carolina suggests the importance of including these organisms in basic health risk assessment protocols and suggests a potential tool for assessment of bear/habitat interactions.

Use of thermography and fluorescein angiography in the management of a Chilean flamingo with avascular necrosis of the wing.

A Chilean flamingo (Phoenicopterus chilensis) was presented to the veterinary clinic at the North Carolina Zoological Park for evaluation of acute weakness of the right wing. Results of a physical examination revealed a lack of a palpable pulse in the radial artery, which suggested occlusion or obstruction of the vessel. Radiography, thermography, and fluorescein angiography confirmed right wing injury and vascular compromise. Based on the poor prognosis for return to function associated with irreversible vascular damage, the wing was amputated. After a period of observation and treatment, the bird was returned to public exhibit.

Observable Metabolites and Metabolomic Sampling Protocols for Managed African Savanna Elephant (Loxodonta africana) Whole Blood Using H-NMR Spectroscopy.

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolomics of heparinized whole blood drawn from six African savanna elephants (Loxodonta africana) maintained on a well characterized diet. Whole blood samples obtained under behavioral restraint, then quickly frozen in liquid nitrogen, were stored at -80 °C until analysis. Frozen samples were thawed under controlled conditions and extracted with methanol and chloroform to separate the polar and non-polar metabolites. We identified 18 polar metabolites and 14 non-polar lipids using one-dimensional (1D) and two-dimensional (2D) NMR spectra. Despite unexpected rouleaux formation in the thawed frozen samples, spectra were consistent among animals and did not vary dramatically with age or the sex of the animal.

Histological, chemical, and morphological reexamination of the "heart" of a small Late Cretaceous Thescelosaurus.

A three-dimensional, iron-cemented structure found in the anterior thoracic cavity of articulated Thescelosaurus skeletal remains was hypothesized to be the fossilized remains of the animal's four-chambered heart. This was important because the finding could be interpreted to support a hypothesis that non-avian dinosaurs were endothermic. Mammals and birds, the only extant organisms with four-chambered hearts and single aortae, are endotherms. The hypothesis that this Thescelosaurus has a preserved heart was controversial, and therefore, we reexamined it using higher-resolution computed tomography, paleohistological examination, X-ray diffraction analysis, X-ray photoelectron spectroscopy, and scanning electron microscopy. This suite of analyses allows for detailed morphological and chemical examination beyond what was provided in the original work. Neither the more detailed examination of the gross morphology and orientation of the thoracic "heart" nor the microstructural studies supported the hypothesis that the structure was a heart. The more advanced computed tomography showed the same three areas of low density as the earlier studies with no evidence of additional low-density areas as might be expected from examinations of an ex situ ostrich heart. Microstructural examination of a fragment taken from the "heart" was consistent with cemented sand grains, and no chemical signal consistent with a biological origin was detected. However, small patches of cell-like microstructures were preserved in the sandstone matrix of the thoracic structure. A possible biological origin for these microstructures is the focus of ongoing investigation.

A comparison of multiple esterases as biomarkers of organophosphate exposure and effect in two earthworm species.

Two different earthworm species, Eisenia fetida and Lumbricus terrestris, were exposed to 5 µg/cm(2) of malathion to evaluate their usefulness as sentinels of organophosphate exposure and to assess three different esterases, as biomarkers of malathion exposure and effect. Tissue xenobiotic burdens and esterase activity were determined for each species and each esterase in order to assess variability. E. fetida exhibited 4-fold less variability in tissue burdens than did L. terrestris and had less variable basal esterase activities. An attempt was made to correlate malathion and malaoxon tissue burdens with esterase activity post-exposure. There was no malaoxon present in the earthworm tissues. No significant correlations were determined by comparing acetylcholinesterase, butyrylcholinesterase, nor carboxylesterase activities with malathion burdens.

Retinal cone photoreceptor distribution in the American black bear (Ursus americanus).

The distribution of cone photoreceptor subtypes (important for color vision and vision quality) varies widely in different carnivore species, but there have been limited studies on bear (ursid) cone distribution. A previous behavioral study suggests that American black bears (Ursus americanus) are dichromatic, indicating that they possess two cone subtypes, although the retinal distribution of cones is unknown. The purpose of this study was to examine the subtype and topography of cones in American black bear retinas to further predict the nature of their color vision and image resolution. We studied 10 eyes from seven individual legally hunted black bears in northeastern North Carolina. Cryosections and retinal wholemounts were labeled using antibodies targeting two cone opsin subtypes: long/medium (L/M) wavelength sensitive and short (S) wavelength sensitive. Cones in fluorescent microscopy images were counted and density maps were created for retinal wholemounts. The black bear retina contains both cone subtypes and L/M cones outnumber S cones by at least 3:1, a finding confirmed in retinal frozen sections. There are higher concentrations of S cones present than typically seen in other carnivores with some evidence for co-expression of L/M and S cones. A cone-dense area centralis is present dorsotemporal to the optic nerve, similar to other carnivores. These results confirm that American black bears are predicted to have a dichromatic vision with high acuity indicated by the presence of a dorsotemporally located area centralis.

Comparison of African savanna elephant (Loxodonta africana) fatty acid profiles in whole blood, whole blood dried on blood spot cards, serum, and plasma.

BACKGROUND: African elephants in managed care have presented differences in the balance between omega-3 and omega-6 fatty acids, a situation primarily thought to be due to dietary differences between the managed animals and their free-ranging counterparts. Because of this, circulating fatty acid status is included in routine monitoring of elephant health. A method of blood collection that requires only a few drops of whole blood, dried on filter paper (DBS) and can be used for analyzing full fatty acid profiles offers advantages in clinical application. METHODS: This study compared the use of whole blood, and whole blood DBS, serum or plasma for use in evaluating circulating fatty acid composition in African savannah elephants. Samples from six African elephants (two males and four females) were collected during the same week at the NC Zoo, Asheboro, NC. RESULTS: Results found only 2 of 36 individual fatty acids and none of the 10 fatty acid groupings were different when comparing the four blood fraction sample types to each other with Mann-Whitney U-Test pairwise comparisons. Myristic acid (14:0) was lower in the DBS samples than in whole blood, serum, and plasma and pentadecaenoic acid (15:1) was slightly more concentrated in DBS and whole blood. DISCUSSION: Results indicate that fatty acid profile of serum, plasma, whole blood, and DBS are comparable in African elephants. The DBS method offers advantages in acquisition and handling and may be preferable to other methods in both routine health assessment of captive animals and field research on free ranging animals.

Investigation of Dried Blood Spot Cards for Fatty Acid Analysis Using Porcine Blood.

Fatty acids, especially omega-3 and omega-6 fatty acids, are important for reproductive and cardiovascular health in animals. While monitoring fatty acids is traditionally conducted using frozen blood fractions such as serum and plasma, advancements in analytical technology have developed a method of collecting microsamples of dried whole blood on Ahlstrom 226 grade filter paper that can provide information on long-term fatty acid status of animals. Blood samples were collected from five male pigs in both the traditional frozen method and on dried blood spot cards (DBS). The DBS samples were collected with untreated syringes and tubes, and approximately 320 µL of blood was placed on each card with approximately 80 µL per spot (4 spots). Statistical analysis was performed to compare the two sample groups to each other using the Mann-Whitney U-test and determine if DBS samples were similar to traditional whole blood samples. Of the 30 fatty acids and fatty acid groups with measurable concentrations, only four individual fatty acids, behenic acid, omega-3 docosapentaenoic acid, nervonic acid, and adrenic acid, had statistical differences. Most of these differences were minor and could be due to analytical errors or contamination. Comparisons between sample types found similar concentrations of key omega fatty acids and PUFAs and support the use of DBS collection as a less invasive method of blood collection and fatty acid analysis.

Applications of chemical shift imaging to marine sciences.

The successful applications of magnetic resonance imaging (MRI) in medicine are mostly due to the non-invasive and non-destructive nature of MRI techniques. Longitudinal studies of humans and animals are easily accomplished, taking advantage of the fact that MRI does not use harmful radiation that would be needed for plain film radiographic, computerized tomography (CT) or positron emission (PET) scans. Routine anatomic and functional studies using the strong signal from the most abundant magnetic nucleus, the proton, can also provide metabolic information when combined with in vivo magnetic resonance spectroscopy (MRS). MRS can be performed using either protons or hetero-nuclei (meaning any magnetic nuclei other than protons or ¹H) including carbon (¹³C) or phosphorus (³¹P). In vivo MR spectra can be obtained from single region of interest (ROI or voxel) or multiple ROIs simultaneously using the technique typically called chemical shift imaging (CSI). Here we report applications of CSI to marine samples and describe a technique to study in vivo glycine metabolism in oysters using ¹³C MRS 12 h after immersion in a sea water chamber dosed with [2-¹³C]-glycine. This is the first report of ¹³C CSI in a marine organism.

Metabolomic investigations of American oysters using H-NMR spectroscopy.

The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance ((1)H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding (1)H NMR peak assignments for future NMR-based metabolomic studies in oysters.

Digestible Energy Intake and Digestive Efficiency of Human-Managed North American River Otters (Lontra canadensis).

Diets currently provided to captive North American river otters (Lontra canadensis) are highly variable with different institutions providing various whole foods, commercial complete prepared diets, or combinations of both. This study investigated the digestible energy intake, gastrointestinal transit time, and digestive efficiency of three different diets being fed at three North Carolina institutions. Otters housed at Institution A (n = 3) were fed strictly fish. Otters housed at Institutions B (n = 3) and C (n = 2) were fed a majority fish based diet (58.5 and 74.1%, respectively), supplemented with fruits, vegetables, and supplemental protein sources as enrichment. There was an apparent trend between increased percentage of fish in the diet and faster transit time and higher digestive efficiency. As less fish was included in the diets, the GI transit time was longer (Institution A, 106 minutes; Institution B, 145 minutes; Institution C, 208 minutes). Median digestive efficiency was high for all three groups (A, 91.4%; B, 87.8%; C, 89.8%) but was higher for the institutions feeding fish. Additionally, the overall median gross energy intake for the eight animals in this study was 163.1 kcal/kgBM0.75/day (range: 92.2 to 260.7 kcal/kgBM0.75/day). While all three institutions had healthy otter populations, it appears that a higher fish diet should be further studied as the model North American river otter diet.

Biochemical Effects of Routine Gonadectomy on Blood of Domestic Ferrets (Mustela putorius furo).

We studied domestic ferrets (Mustela putorius furo) to evaluate the physiologic effects of routine surgery. Standard plasma biochemistry panels and 1H-NMR spectroscopy of heparinized whole blood were performed on samples taken 24 h prior to and immediately after surgery from female and male ferrets undergoing routine gonadectomy. Increases in plasma glucose, phosphorus, potassium, and creatine kinase concentrations associated with the duration of surgery were identified on plasma biochemistry panels. Whole-blood NMR spectra allowed us to identify 42 metabolites and one drug residue. Variations between pre- and postoperative metabolite concentrations were most pronounced for female ferrets, which underwent more prolonged surgery than males. Affected metabolites included organic acids and osmolytes (betaine, methylmalonate, D-lactate), fatty acids and lipids (2-hydroxy-3-methylbutyric acid), and amino acid groups (acetylglycine, alloisoleucine, leucine, and isoleucine). These findings indicate that 1H-NMR spectroscopy of whole blood provides insight into metabolic perturbations in domestic ferrets undergoing surgery that are not detected in routine clinical chemistry panels.

Raccoon roundworm prevalence (Baylisascaris procyonis) at the North Carolina Zoo, USA.

Baylisascaris procyonis is an important zoonotic nematode of raccoons (Procyon lotor). Infection with this parasite has important health implications for humans, zoo animals, and free-ranging wildlife. As a large, natural habitat zoo, the North Carolina Zoo (NC Zoo) coexists with native wildlife. Raccoons are abundant at the NC Zoo and the prevalence of B. procyonis is unknown. Raccoon latrines were located through employee reporting and systematic searching throughout the zoo and sampled for B. procyonis in October and November of 2018 and 2019. Parasite prevalence, latrine location, substrate category and latrine persistence were recorded. Thirty-three latrines were located in 2018 and eight new latrines in 2019 while four latrines from the prior year were no longer available to be sampled. Of the 29 latrines sampled over the two years, 16 (55%) persisted for at least one year. The majority of the latrines were found on natural substrate with rock showing the highest preference. Just over half (n = 21 of 41 total) of the active latrines in the study were in or immediately adjacent to animal enclosures. Two latrines were found in public areas including one contaminating children's play equipment. Additionally, fresh fecal samples were collected from five adult raccoons presented to the zoo's veterinary clinic in 2018 and 2019. All fecal samples tested by centrifugal flotation for both years were negative for B. procyonis. The results of this study show the value of field sampling to properly assess risk and enable informed decision-making regarding public health and wildlife management.