USE OF VISCOELASTIC COAGULATION TESTING IN MEGACHIROPTERA (PTEROPUS HYPOMELANUS AND PTEROPUS VAMPYRUS) REVEALS HIGH VARIABILITY IN CLOT KINETICS.

Megachiroptera is a mammalian suborder that includes old world fruit bats. Common clinical problems among captive Megachiroptera, such as liver disease (e.g., iron storage disease), kidney disease (e.g., protein-losing nephropathy), and heart disease (e.g., dilated cardiomyopathy), carry elevated risk for hemostatic derangements. The assessment of viscoelastic coagulation assays, however, has not yet been reported in bats. The main objective of the study was to describe viscoelastography data using the Viscoelastic Coagulation Monitor (VCM) Vet in captive large flying foxes (Pteropus vampyrus) (n = 20) and variable flying foxes (Pteropus hypomelanus) (n = 10). Additional objectives were to compare viscoelastic and clotting parameters (1) between healthy P. vampyrus and P. hypomelanus bats and (2) between untreated bats and those treated with meloxicam or aspirin, and (3) to examine relationships between activated partial thromboplastin time (aPTT) and potentially homologous viscoelastic parameters clotting time (CT) and clot formation time (CFT). The results showed marked variability among clinically normal bats. The intrinsic pathway, as measured by aPTT, had prolonged times compared with most terrestrial mammals, but similar times to birds, marine mammals, and sea turtles. A search of P. vampyrus genome found stop codons present in two exons of the factor XI gene; alterations in factor XI expression would be expected to alter intrinsic coagulation. Because of the high variability, no statistically significant findings were noted in the secondary objectives. Correlation between aPTT and CT or CFT was not strong (rs = 0.406 or 0.192, respectively). The results from this study suggest that clot kinetics vary widely among Megachiroptera when using the VCM Vet with untreated blood. A prolonged intrinsic coagulation pathway, as has been found in other megachiropteran species, and activation of the extrinsic coagulation pathway during venipuncture may be responsible for the inconsistent results.

A mystery revealed: an update on eosinophil and other blood cell morphology of the Argentine black and white tegu (Salvator merianae).

Reptile white blood cell (WBC) morphological features are strikingly variable across species. In the Argentine black and white tegu (Salvator merianae), red tegu (Salvator rufescens), and Savannah monitor (Varanus exanthematicus), previous reports described a WBC type with a single distinct, clear, linear- to ovoid- to crescent-shaped inclusion of presumptive monocytic origin. The objective of this study was to further investigate the origin of this unique WBC type with crescent-shaped inclusions. Blood samples from two Argentine black and white tegus, tegu 1, a 4-year-old female, and tegu 2, a 2-year-old presumed male, were submitted for routine hematological evaluation. Additional blood films were prepared and stained with these cytochemical stains: alkaline phosphatase (ALP; naphthol AS-MX phosphate substrate), alpha-naphthyl butyrate esterase, alpha-chloroacetate esterase, myeloperoxidase, Periodic acid-Schiff, and Sudan black B. Blood films from tegu 1 were also stained with a second ALP stain (5-bromo-4-chloro-3-indoxyl-phosphate and nitroblue tetrazolium substrate), Luna, luxol fast blue, and toluidine blue. The blood from tegu 1 was cytocentrifuged to isolate and fix the buffy coat in glutaraldehyde 2.5% aqueous solution for transmission electron microscopy. Six morphologically distinct WBC types were identified from tegu 1, including heterophils, basophils, monocytes, azurophils, lymphocytes, and the unique WBC type, which were identified as eosinophils with inclusions. WBC types in tegu 2 were similar; however, eosinophils lacked a discernable inclusion. Proper WBC identification will be useful in obtaining accurate hemogram data for this species.

Considerations on the Evolutionary Biology and Functions of Eosinophils: What the 'Haeckel'?

The origins and evolution of the eosinophilic leukocyte has received only scattered attention since Paul Ehrlich first named this granulocyte. Studies suggest that myeloperoxidase, expressed by granulocytes, and eosinophil peroxidase diverged some 60-70 million years ago, but invertebrate to vertebrate evolution of the eosinophil lineage is unknown. Vertebrate eosinophils have been characterized extensively in representative species at light microscopic, ultrastructural, genetic, and biochemical levels. Understanding of eosinophil function continues to expand and includes to date regulation of "Local Immunity And/Or Remodeling/Repair" (the so-called LIAR hypothesis), modulation of innate and adaptive immune responses, maintenance of tissue and metabolic homeostasis, and under pathologic conditions, inducers of tissue damage, repair, remodeling, and fibrosis. This contrasts with their classically considered primary roles in host defense against parasites and other pathogens, and involvement in T-helper 2 inflammatory and immune responses. The eosinophils' early appearance during evolution and continued retention within the innate immune system across taxa illustrate their importance during evolutionary biology. However, successful pregnancies in eosinophil-depleted humans/primates treated with biologics, host immune responses to parasites in eosinophil-deficient mice, and the absence of significant developmental or functional abnormalities in eosinophil-deficient mouse strains under laboratory conditions, raise questions of the continuing selective advantages of the eosinophil lineage in mammals and humans. The objectives of this review are to provide an overview on evolutionary origins of eosinophils across the animal kingdom, discuss some of their main functions in the context of potential evolutionary relevance, and highlight the need for further research on eosinophil functions and functional evolution.

DIAGNOSTIC PERFORMANCE OF BLOOD ANALYTES FOR THE DIAGNOSIS OF RENAL DISEASE IN BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AT THE PHOENIX ZOO (2001-2020).

Renal disease is an important cause of morbidity and mortality in managed black-footed ferrets (BFF; Mustela nigripes).4,6,12 The objectives of this study were to establish reference intervals for blood analytes of clinically normal BFF (1-2 yr old), summarize the frequency of various renal histopathologic findings in a managed population of BFF, assess the diagnostic performance of blood analytes and urine specific gravity (USG) for the diagnosis of renal disease, and assess if comorbidities or age affects the performance of these analytes in diagnosing renal disease. Reference intervals were established using a cohort (n = 35) of clinically normal, young adult BFF. Postmortem records for all BFF at the Phoenix Zoo between 2001 and 2020 were reviewed, and those with available blood analyte data within 2 wk of death were included (n = 89). Ferrets were placed into one of three groups, based on the organ location of histopathologic abnormalities following necropsy: renal disease as the primary change; those with renal disease and at least one other affected major organ system; or absence of abnormalities in the kidneys. In ferrets with substantial renal changes, the primary diagnosis was amyloidosis (29 of 39; 74.4%). Creatinine, blood urea nitrogen, phosphorus (P), calcium (Ca), Ca:P ratio, USG, globulins, and cholesterol were the best-performing analytes for the diagnosis of renal disease, with an area under the curve of at least 0.90 (95% CI $ 0.80, 1.00). Serum renal markers were within reference intervals in BFF that died without histologic evidence of renal disease. Several blood analytes were significantly affected by age in animals that died of renal disease. This study provides reference intervals for blood analytes in young adult clinically normal BFF and illustrates the clinical utility for the diagnosis of renal disease in this species, particularly creatinine, USG, and P.

Lactococcus garvieae-associated septicemia in a central bearded dragon.

Lactococcus garvieae is the causative agent of lactococcosis in fish and an emerging zoonotic pathogen with high levels of antimicrobial resistance. We report a case of L. garvieae-associated septicemia in a central bearded dragon (Pogona vitticeps) confirmed via whole-blood PCR and direct sequencing. Following a 30-d course of ceftazidime (20 mg/kg IM q72h), the animal's clinical condition had not resolved; leukopenia persisted, with heterophil toxic change. Coelomic ultrasound findings were consistent with preovulatory follicular stasis, folliculitis, and coelomitis. Following surgical ovariectomy and an additional 30-d course of ceftazidime, the animal's behavior and appetite returned to normal, the animal tested negative via whole-blood PCR assay, and the CBC was unremarkable. To our knowledge, L. garvieae with L. garvieae-associated clinical disease has not been reported previously in a bearded dragon. We conclude that L. garvieae should be considered as a possible etiologic agent in cases of septicemia in bearded dragons, with the potential for zoonotic transmission warranting further investigation.

Blood analyte reference intervals and correlations with trace elements of immature and adult Eastern Pacific green turtles (Chelonia mydas) in coastal lagoons of Baja California Sur, México.

Sea turtles can bioaccumulate high concentrations of potentially toxic contaminants. To better understand trace element effects on sea turtles' health, we established reference intervals for hematological and plasma biochemical analytes in 40 in-water, foraging immature and adult Eastern Pacific green turtles (Chelonia mydas) from two coastal lagoons in Baja California Sur, quantified whole blood concentrations of eight trace elements, and assessed their correlations. Rank-order trace element concentrations in both immature and adult turtles was zinc > selenium > nickel > arsenic > copper > cadmium > lead > manganese. Immature turtles had significantly higher copper and lower nickel and zinc concentrations. Additionally, a number of relationships between trace elements and blood analytes were identified. These data provide baseline information useful for future investigations into this population, or in other geographic regions and various life-stage classes.

Blood analytes of clinically normal and diseased neonatal and weaned farmed white-tailed deer (Odocoileus virginianus) fawns.

Recent research focused on farmed deer has exposed many knowledge gaps regarding health assessment protocols for white-tailed deer (WTD). The objectives of this study were to establish de novo blood analyte reference intervals for farmed WTD fawns at birth (1-2 days of age; n = 84) and again at weaning (76-125 days of age; n = 28), to compare data at birth and at weaning to understand how these analytes are affected by the intrinsic factors age and sex in clinically normal WTD fawns, and to compare between clinically normal and sick WTD weanlings (respiratory disease n = 12; orbivirus-infected n = 6). Reference intervals were established for WTD fawns at birth and weaning. Female WTD neonates had significantly higher red blood cell counts, hematocrit, and hemoglobin compared to males. Most blood analytes were significantly different in clinically normal WTD neonates compared to weanlings, suggesting an effect of age. The observed sex- and age-related variations in WTD highlight the need to establish reference intervals that account for intrinsic factors. The comparison of clinically normal and sick WTD weanlings in this study identified higher MCHC and absolute monocytes in sick weanlings but these findings were presumably not biologically relevant given the small sample size for sick fawns. While the reference interval data presented herein will be useful for the veterinary care of WTD fawns at critical time periods in a high-density farm setting, this study also demonstrates the need to identify more sensitive and specific biomarkers for the assessment of health status in farmed WTD with specific underlying diseases.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

Coelomic fluid of asteroid echinoderms: Current knowledge and future perspectives on its utility for disease and mortality investigations.

Coelomic fluid surrounds the internal organs of asteroid echinoderms (asteroids, otherwise known as sea stars or starfish) and plays an essential role in the immune system, as well as in the transport of respiratory gases, nutrients, waste products, and reproductive mediators. Due to its importance in physiology and accessibility for nonlethal diagnostic sampling, coelomic fluid of asteroids provides an excellent sample matrix for health evaluations and can be particularly useful in disease and mortality investigations. This is especially important in light of recent increases in the number of affected individuals and species, larger geographic scope, and increased observed frequency of sea star wasting events compared with historic accounts of wasting. This review summarizes the current knowledge about coelomocytes, the effector cell of the asteroid immune system; coelomic fluid electrolytes, osmolality, acid-base status and respiratory gases, and microbiota; and genomic, transcriptomic, and proteomic investigations of coelomic fluid. The utility of coelomic fluid analysis for assessing stressor responses, diseases, and mortality investigations is considered with knowledge gaps and future directions identified. This complex body fluid provides an exciting opportunity to increase our understanding of this unique and ecologically important group of animals.

IDENTIFICATION AND RETROSPECTIVE EVALUATION OF A FILARIOID NEMATODE SPECIES IN MANAGED GRASSHOPPER SPARROWS (AMMODRAMUS SAVANNARUM).

The grasshopper sparrow (Ammodramus savannarum) is a species of ground-dwelling passerine bird with 12 different subspecies. The Florida subspecies (Ammodramus savannarum floridanus) is classified as federally endangered, with the most common threats including habitat loss, nest predation, and floods. A managed breeding program was established at White Oak Conservation (Yulee, FL) in 2015 with eastern grasshopper sparrows (Ammodramus savannarum pratensis) as a model for breeding Florida grasshopper sparrows as part of an assurance colony. A filarioid parasite species (Aproctella sp.) was characterized by PCR after identification by blood films and postmortem examinations of both subspecies housed at White Oak Conservation. This Aproctella species was distinct from others with available sequence. Records from 157 eastern and Florida grasshopper sparrows were reviewed, and correlations between presence of filariasis and subspecies, sex, body condition score, and presence of systemic isosporosis, squamous metaplasia, coelomitis, airsacculitis, or a combination of conditions were investigated. Twenty-nine (18.5%) birds (13 of 71 Florida grasshopper sparrows; 16 of 86 eastern grasshopper sparrows) were positive for filariasis by blood film review, grossly or by tissue imprint at postmortem examination, or histologically. Filariasis was significantly correlated with systemic isosporosis, coelomitis, and airsacculitis; was not correlated with subspecies, sex, or squamous metaplasia; and had a questionable correlation with body condition score. This report provides evidence that this Aproctella species has potential to contribute to morbidity and mortality in the grasshopper sparrow. This information will be helpful for implementing effective measures against suspected vectors and for the development of best practice strategies for the health management of the species in breeding programs.

Perspective: Opportunities for advancing aquatic invertebrate welfare.

Welfare considerations and regulations for invertebrates have lagged behind those for vertebrates, despite invertebrates comprising more than 95% of earth's species. Humans interact with and use aquatic invertebrates for exhibition in zoos and aquaria, as pets, research subjects, and important food sources. Recent research has indicated that aquatic invertebrates, in particular cephalopod mollusks and decapod crustaceans, experience stress and may be able to feel pain. With this article, we present results of a survey on attitudes of aquatic animal health professionals toward aquatic invertebrate welfare and provide practical recommendations for advancing aquatic invertebrate welfare across four areas of opportunity: use of anesthesia, analgesia, and euthanasia; development of less invasive diagnostic and research sampling methods based on 3R principles; use of humane slaughter methods for aquatic invertebrates; and reducing impacts of invasive procedures in aquaculture and fisheries. We encourage consideration of these opportunities to achieve far-reaching improvements in aquatic invertebrate welfare.

CLINICOPATHOLOGIC EVALUATION OF AN EX SITU POPULATION OF ADULT AFRICAN WHITE-BELLIED PANGOLIN (PHATAGINUS TRICUSPIS).

Death due to illegal trade and trafficking serves as the largest threat to pangolins in their geographic range countries. Appropriate veterinary care is essential to the welfare of sick or debilitated pangolins. Given the challenge of comprehensive physical examination of this species due to its unique anatomic features and poorly studied physiology, laboratory diagnostics can provide essential information to guide appropriate medical care. This retrospective study included previously analyzed blood and urine samples collected from adult (n = 17) African white-bellied pangolins (Phataginus tricuspis) under managed care that were sampled over a 4-yr period as part of routine veterinary examinations. Samples from clinically normal animals were used to summarize results for this species. Pangolins had a lymphocyte-predominant leukogram with a mean neutrophil to lymphocyte ratio of 0.83 (min-max: 0.13-4.81). Seasonal, sex, and age differences were present for several hematologic and serum biochemical analytes. Urine samples had a median urine specific gravity (USG) of 1.040 (min-max: 1.006-1.060), a neutral-to-basic urine pH (6.0-8.6), and contained mainly struvite crystals or infrequently calcium oxalate dihydrate crystals. Winter samples had higher USG than summer samples. The clinicopathologic data provided herein offers applicable information useful for the clinical management of pangolins.

Cultivating epizoic diatoms provides insights into the evolution and ecology of both epibionts and hosts.

Our understanding of the importance of microbiomes on large aquatic animals-such as whales, sea turtles and manatees-has advanced considerably in recent years. The latest observations indicate that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful approach to culture putatively obligate epizoic diatoms without their hosts. That some taxa can be cultured independently from their epizoic habitat raises several questions about the nature of the interaction between these animals and their epibionts. This insight allows us to propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultured strains, we found that several unique diatom taxa have evolved independently to occupy epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Morphologic and physiologic characteristics of green sea turtle (Chelonia mydas) hatchlings in southeastern Florida, USA.

The ability of sea turtle hatchlings to survive into adulthood is related, in part, to their individual health status. Documenting a variety of health data is essential for assessing individual and population health. In this study, we report health indices for 297 green sea turtle (Chelonia mydas) hatchlings that emerged from 32 nests deposited on Juno Beach, Florida, USA in June-July, 2017. Results of physical examination, morphometrics, and infectious disease testing (chelonid alphaherpesvirus 5, ChHV5), and blood analyte reference intervals (hematology, plasma protein, glucose) are presented. Carapacial scute abnormalities were observed in 36% (108/297) of all hatchlings, including abnormal vertebral (86/297, 29%), lateral (72/297, 24%), and both vertebral and lateral (50/297, 17%) scutes. Hatchlings from nests laid in July, which was ~ 1.6 °C warmer than June, had significantly shorter incubation periods, and higher body mass, straight carapace length, body condition index, packed cell volume, and heterophil:lymphocyte ratios compared to hatchlings from nests laid in June. These results suggest that incubation temperatures are linked to hatchling developmental factors and size, nutritional and/or hydration status, and/or blood cell dynamics. Blood samples from all 297 hatchlings tested negative for ChHV5 DNA via quantitative PCR, including 86 hatchlings from the nests of 11 adult females that tested positive for ChHV5 via qPCR or serology in a separate study, lending support to the hypothesis that ChHV5 is horizontally (rather than vertically) transmitted among green turtles. Information resulting from this study represents a useful dataset for comparison to future health assessment and population monitoring studies of green turtle hatchlings in the northwestern Atlantic Ocean.

Disseminated Histiocytic Sarcoma in Two Free-Living Florida Manatees (Trichechus manatus latirostris).

During necropsy of two manatees, masses in multiple organs including liver and spleen were identified. They were composed of neoplastic round to spindle-shaped cells, positive for vimentin, Iba-1, lysozyme, and Mac387, consistent with histiocytic sarcoma. One manatee also had an undifferentiated sarcoma that may have arisen from the histiocytic sarcoma.

Correction: Turner et al. Steatitis in Cold-Stunned Kemp's Ridley Sea Turtles (Lepidochelys kempii). Animals 2021, 11, 898.

In the original publication [...].