Kudoa hypoepicardialis and associated cardiac lesions in invasive red lionfish Pterois volitans in Grenada, West Indies.

Invasive red lionfish Pterois volitans (Linnaeus, 1758) represent an ongoing ecological threat within temperate and tropical waters. Relatively little is known regarding the overall health of P. volitans and their potential for spreading pathogens in non-native regions. Lionfish collected from inshore reefs of Grenada, West Indies, in 2019 and 2021 were identified as P. volitans based on cytochrome c oxidase subunit 1 barcoding. Gross and microscopic examination of tissues revealed myxozoan plasmodia in the hearts of 24/76 (31.6%) lionfish by histopathology or wet mount cytology. Further histopathologic examination revealed severe granulomatous inflammation and myofiber necrosis associated with developing plasmodia and presporogonic life stages. Fresh myxospores were morphologically and molecularly consistent with Kudoa hypoepicardialis, being quadrate in apical view with 4 valves and 4 equal polar capsules. The spore body was 5.1-7.9 (mean: 6.0) µm long, 8.1-9.8 (8.7) µm wide, and 6.9-8.5 (7.7) µm thick. Polar capsules were 2.3-2.7 (2.5) µm long and 0.9-1.6 (1.3) µm wide. 18S small subunit rDNA sequences were 99.81-99.87% similar to sequence data from the original description of the species. Novel 28S large subunit rDNA and elongation factor 2 data, which did not match any previously reported species, were provided. This is the first account of a myxozoan parasite of P. volitans, a new host record and locality for K. hypoepicardialis, and one of few reports describing pathogen-associated lesions in invasive lionfish.

Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model.

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~106  CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted.

Mycobacterium salmoniphilum and M. chelonae in Captive Populations of Chinook Salmon.

Chinook Salmon Oncorhynchus tshawytscha is a keystone fish species in the Pacific Northwest. In 2019, unusual mortalities occurred in two different populations of cultured fingerlings from the same facility in California, USA. The systems consist of outdoor, enclosed, flow-through freshwater tanks that are maintained at 18 ± 1°C. Clinical signs and gross findings were only observed in one population and included abnormal swimming, inappetence, lethargy, skin discoloration, and the presence of multifocal nodular and ulcerative skin lesions. Microscopic lesions were infrequent and consisted of severe, locally extensive granulomatous dermatitis and myositis and mild, multifocal, granulomatous branchitis, myocarditis, and hepatitis. Intracellular acid-fast organisms were observed within areas of granulomatous myositis. Posterior kidney swabs were collected and inoculated in nutrient-rich and selective agar media and incubated at 25°C for 2 weeks. Visibly pure bacterial colonies were observed 7-10 d postinoculation. Partial sequences of 16S rRNA initially identified the recovered bacteria as members of the genus Mycobacterium. However, marked variability was observed among Mycobacterium spp. isolates by using repetitive extragenic palindromic polymerase chain reaction fingerprinting. Amplification and sequencing of the ribosomal RNA internal transcribed spacer, 65-kDa heat shock protein, and RNA polymerase ß-subunit gene of the cultured isolates identified M. salmoniphilum and M. chelonae, discrete members of the M. chelonae-abscessus complex, isolated from diseased Chinook Salmon fingerlings.

Intracoelomic- and Intramuscular-Injection Challenge Model of Piscine Streptococcosis in White Sturgeon Fingerlings.

Streptococcus iniae is a zoonotic pathogen and one of the major aetiologic agents of streptococcosis. In White Sturgeon Acipenser transmontanus, S. iniae infection typically presents as a necrotizing and heterophilic myositis, causing 30-50% mortality in infected fish. To gain a better understanding of the pathogenesis of streptococcosis in White Sturgeon, and to identify the experimental route of infection that most closely mimics the natural disease, fingerlings were challenged with a single dose of 1.3 × 108  cells/fish of S. iniae that was administered via intracoelomic/intraperitoneal (IC) or intramuscular (IM) routes. Acute mortalities were present only in the IM-challenged fish, with first mortality occurring 4 d postchallenge and the mortality rate reaching 18.3% after 9 d. The challenged fish presented erratic swimming, ulcerative skin lesions, and hemorrhages in the liver and swim bladder. Streptococcus iniae was recovered from the kidney and brain tissues of moribund and dead fish. Histopathologic analysis of fish that died acutely revealed massive proliferation of bacteria in the muscle at the injection site and within vascular organs such as the heart and spleen, with variable amounts of tissue necrosis including a necrotizing myositis. Fish that died closer to 9 d postchallenge demonstrated more pronounced multifocal to locally extensive granulomatous inflammation of skeletal muscle at the injection site, liver, kidney, and spleen. No mortality, clinical signs, or gross changes were observed in the control or IC-challenged fish. Postmortem evaluation of 10 survivors in each treatment was performed to determine carrier status in the brain and posterior kidney tissues. The prevalence of S. iniae in survivors was 10% and 0% in the IM- and IC-challenged groups, respectively. The results from this study suggest that IM-injection challenge methods are suitable for inducing streptococcosis in White Sturgeon, and they may be the preferred method for studying the pathogenesis of the naturally occurring disease in this species.

Identification of Chryseobacterium spp. isolated from clinically affected fish in California, USA.

Chryseobacterium spp. (Family Flavobacteriaceae) are emergent fish pathogens in Europe, Asia and North America. In 2016-2017, 7 bacterial isolates were recovered from posterior kidney or spleen of cultured diseased rainbow trout Oncorhynchus mykiss (n = 1), green sturgeon Acipenser medirostris (n = 1), white sturgeon A. transmontanus (n = 2), blue ram cichlid Mikrogeophagus ramirezi (n = 1), and returning fall Chinook salmon O. tshawytscha (n = 2) from different freshwater systems. Bacterial colonies were visible after 24-48 h incubation at 20°C on agar media. Isolates were Gram-negative, rod-shaped, catalase and oxidase positive. Amplification and partial sequence analysis of the 16S rRNA and gyrB genes allocated the microorganisms to the genus Chryseobacterium sharing 97.2-99.6% similarity to 6 described Chryseobacterium spp. at the 16S rRNA locus, and 87.8-99.1% similarity at gyrB. Phylogenetic analyses in conjunction with percent sequence identity suggest some of the recovered isolates may represent novel Chryseobacterium subspecies or species. The pathogenicity of 5 isolates was evaluated experimentally in rainbow trout (n = 60), brown trout Salmo trutta (n = 60) and white sturgeon (n = 36) in flow-through freshwater at 18°C. Approximately 107 CFU fish-1 was injected in the epaxial musculature of anesthetized animals. Limited mortality was observed and no bacteria were recovered from dead or moribund fish post-challenge. Thirty days post-challenge, survivors were euthanized and multiple tissues were collected and fixed for histological analysis. No consistent histopathological changes were observed in challenged or control fish. While results suggest the recovered Chryseobacterium spp. may be opportunistic pathogens, further research is warranted to better understand the role of these bacteria in fish disease.

Biochemical reference intervals and pathophysiological changes in Flavobacterium psychrophilum-resistant and -susceptible rainbow trout lines.

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.

Comparative plasma biochemistry analyte data in nesting leatherback (Dermochelys coriacea), foraging green (Chelonia mydas) and foraging and nesting hawksbill (Eretmochelys imbricata) sea turtles in Grenada, West Indies.

Blood biochemistry represents a minimally invasive tool for monitoring sea turtle health, assessing injured sea turtles and supporting conservation strategies. In Grenada, West Indies, plasma biochemical variables were examined in 33 nesting leatherback (Dermochelys coriacea), 49 foraging green (Chelonia mydas), 49 foraging hawksbill (Eretmochelys imbricata) and 12 nesting hawksbill sea turtles sampled between 2017 and 2022. Plasma biochemistry reference intervals are described herein except for nesting hawksbills, which are represented by descriptive statistics due to the low sample size. Select analyte concentrations were positively correlated with curved carapace length in leatherbacks (chloride), green turtles (total protein, albumin and globulin) and foraging hawksbills (total protein, albumin and phosphorus). Cholesterol (7.8 mmol/l ± 1.6 SD) and triglyceride (6.9 mmol/l ± 1.9 SD) concentrations were significantly higher in leatherbacks compared to foraging green turtles, foraging hawksbills and nesting hawksbills (P < 0.001 for all). Cholesterol was significantly higher in nesting hawksbills compared to foraging green turtles (P = 0.050) and foraging hawksbills (P = 0.050). Foraging hawksbills demonstrated significantly higher aspartate transaminase activities than leatherbacks (P = 0.002), green turtles (P = 0.009) and nesting hawksbills (P < 0.001). Biochemical results provide baseline population health data and support guidance for treatments during clinical sea turtle rehabilitation efforts. They also provide insight into species-specific physiologic differences and preludes further studies to better characterize the impacts of life-stage class on biochemistry reference intervals to better support wild sea turtle populations in Grenada.

Proteomic characterization of the acute-phase response of yellow stingrays Urobatis jamaicensis after injection with a Vibrio anguillarum-ordalii bacterin.

Systemic inflammatory responses of mammals and bony fish are primarily driven by coordinated up-regulation and down-regulation of plasma acute-phase proteins. Although this general principle is believed to be universal among vertebrates, it remains relatively unexplored in elasmobranchs. The objective of this study was to characterize acute changes in the plasma proteome of three yellow stingrays Urobatis jamaicensis following intraperitoneal injection with a commercial Vibrio bacterin. Changes in plasma protein levels were analyzed immediately prior to vaccination (time 0) and at 24 and 72 h post-injection by isobaric tags for relative and absolute quantitation (iTRAQ 4-plex) using shotgun-based nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and de novo peptide sequencing. Pooled 2D-LC-MS/MS and de novo sequencing data revealed differential expression of 156 distinct plasma proteins between time 0 and at least one post-vaccination time point. Using 1.5-fold change in expression as physiologically significant, 14/156 (9.0%) proteins were upregulated in at least one stingray through at least one experimental timepoint. Upregulated proteins included complement factors, Mx-protein, hemopexin, factor X and prothrombin. Seventy-six of 156 (48.7%) proteins were downregulated in the acute-phase response, including transferrin, apolipoprotein B, heparin cofactor 2, alpha2-macroglobulin, and various growth factors. Other differentially upregulated or downregulated proteins included intracellular, cell binding and structural proteins, proteins involved in physiologic processes, and unknown/hypothetical proteins. Selected bioactive factors are discussed for their putative roles in the elasmobranchs acute-phase response. These findings contribute to our understanding of disease processes in elasmobranchs, immunologic phylogeny in vertebrates, and begin the search for potential biomarkers of disease in these ecologically important fish.

Environmental and Nesting Variables Associated with Atlantic Leatherback Sea Turtle (Dermochelys coriacea) Embryonic and Hatching Success Rates in Grenada, West Indies.

Annual monitoring of leatherback sea turtle (Dermochelys coriacea) nesting grounds in Grenada, West Indies has identified relatively low hatch rates compared to worldwide trends. This study investigated the impact of selected environmental variables on leatherback sea turtle embryonic development and hatching success rates on Levera Beach in Grenada between 2015-2019. The mean number of nests per year and eggs per nest were 667.6 ± 361.6 and 80.7 ± 23.0 sd, respectively. Within excavated nests, 35.6% ± 22.0 sd of eggs successfully developed embryos and 30.6% ± 22.6 sd of eggs successfully hatched. The number of eggs per nest, along with embryo and hatching success rates, differed by nesting year. Embryo development success rate was associated with nest location, and both embryo development and hatching success rates were positively associated with nest depth and negatively associated with the percentage of eggs exhibiting microbial growth and with the presence of inspissated yolk. There was no embryo development or hatchling success association with month of the nesting season, distance from the high-water mark, distance from vegetation, nor maternal carapace length. The mean nest temperature was 31.7 °C ± 1.64 sd and mean temperatures during the middle third of egg incubation suggest clutches are highly skewed towards a preponderance of female hatchlings. Histopathologic findings in hatchling mortalities included severe, acute, multifocal, heterophilic bronchopneumonia with intralesional bacteria in 4/50 (8%) hatchlings. Data from this study guide conservation strategies by identifying risk factors and further avenues of research needed to support reproductive success of leatherback sea turtles in Grenada and the greater Caribbean region.

Plasma proteomic profiling of bacterial cold water disease-resistant and -susceptible rainbow trout lines and biomarker discovery.

Genetic variation for disease resistance is present in salmonid fish; however, the molecular basis is poorly understood, and biomarkers of disease susceptibility/resistance are unavailable. Previously, we selected a line of rainbow trout for high survival following standardized challenge with Flavobacterium psychrophilum (Fp), the causative agent of bacterial cold water disease. The resistant line (ARS-Fp-R) exhibits over 60 percentage points higher survival compared to a reference susceptible line (ARS-Fp-S). To gain insight into the differential host response between genetic lines, we compared the plasma proteomes from day 6 after intramuscular challenge. Pooled plasma from unhandled, PBS-injected, and Fp-injected groups were simultaneously analyzed using a TMT 6-plex label, and the relative abundance of 513 proteins was determined. Data are available via ProteomeXchange, with identifier PXD041308, and the relative protein abundance values were compared to mRNA measured from a prior, whole-body RNA-seq dataset. Our results identified a subset of differentially abundant intracellular proteins was identified, including troponin and myosin, which were not transcriptionally regulated, suggesting that these proteins were released into plasma following pathogen-induced tissue damage. A separate subset of high-abundance, secreted proteins were transcriptionally regulated in infected fish. The highest differentially expressed protein was a C1q family member (designated complement C1q-like protein 3; C1q-LP3) that was upregulated over 20-fold in the infected susceptible line while only modestly upregulated, 1.8-fold, in the infected resistant line. Validation of biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor H-like 1 exhibited higher abundance in the resistant line compared to the susceptible line in both control and challenged fish and thus was a baseline differentiator between lines. C1q-LP3 and STNC were elevated in Atlantic salmon plasma following experimental challenge with Fp. In summary, these findings further the understanding of the differential host response to Fp and identifies salmonid biomarkers that may have use for genetic line evaluation and on-farm health monitoring.

A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea.

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

Humoral response of captive zebra sharks Stegostoma fasciatum to salivary gland proteins of the leech Branchellion torpedinis.

Parasitism by the marine leech Branchellion torpedinis is known to cause disease and mortality in captive elasmobranchs and is difficult to control when inadvertently introduced into public aquaria. Preliminary characterization of the salivary gland transcriptome of B. torpedinis has identified anticoagulants, proteases, and immunomodulators that may be secreted into host tissues to aid leech feeding. This retrospective study examined antigen-specific serum IgM responses in captive zebra sharks Stegostoma fasciatum to leech salivary gland extract. Antibody response was examined by ELISA and Western blot assays in 20 serum samples from six zebra sharks, with a 5 year history of leech infection, and 18 serum samples from 8 captive bred zebra sharks, with no history of leech exposure. ELISA demonstrated significantly higher serum IgM titers to salivary gland extract in exposed zebra sharks compared to the non-exposed population. No obvious trends in antibody titers were appreciated in exposed zebra sharks over a four-year period. One-dimensional and two-dimensional Western blot assays revealed IgM targeted specific salivary gland proteins within the 40, 55, 70 and 90 kD range. Antigenic proteins identified by liquid chromatography-tandem mass spectrometry and de novo peptide sequencing include a secreted disintegrin, metalloproteinase and thrombospondin motif containing protein (ADAMTS), tubulin, aldehyde dehydrogenase and two unknown proteins. Humoral immune responses to leech salivary gland proteins warrants further investigation as there may be options to exploit immune mechanisms to reduce parasite burdens in aquaria.

Experimental infection of yellow stingrays Urobatis jamaicensis with the marine leech Branchellion torpedinis.

Infestations of elasmobranchs by the marine leech Branchellion torpedinis can be problematic in aquaria and negatively affect host health. To better characterize the extent and pathogenesis of disease, 12 yellow stingrays Urobatis jamaicensis were infected with 1 or 3 leeches for 14 d. Leeches were associated with anorexia, extensive cutaneous ulceration, decreased host packed cell volume (PCV) and serum total solids (TS), and mortality in 3 rays. Average decrease in host PCV positively correlated with ulcer size and parasite:host ratio. Average decrease in host serum TS positively correlated with parasite:host ratio. Blood chemistry and total white blood cell counts revealed no significant trends. Additional necropsy findings included gill and splenic pallor, pericardial edema, perirenal edema, and decreased hepatocellular lipid deposits. Microscopic evaluation of leeches demonstrated host erythrocytes and proteinaceous fluid within parasite intestines, confirming active blood feeding. Results indicate B. torpedinis has the potential to cause significant disease in elasmobranchs, including death in as few as 5 d, and identifies ulcer size and parasite:host ratio as risk factors for disease. Elucidation of this host-parasite interaction helps characterize host response to parasites and facilitate care of parasitized elasmobranchs in aquarium and wild settings.

Dermatitis and systemic mycosis in lined seahorses Hippocampus erectus associated with a marine-adapted Fusarium solani species complex pathogen.

During a 4 mo epizootic, 100% of 152 lined seahorses Hippocampus erectus in 3 separate groups died while in quarantine following shipment to a public aquarium. Twelve animals with skin depigmentation and ulceration were received by the Aquatic Pathology Service, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA, for diagnostic evaluation. Microscopically, lesions in 11 seahorses included multifocal epithelial necrosis and ulceration associated with 2 to 7 µm diameter, branching, septate fungal hyphae, typically accompanied by deeper infiltration into underlying skeletal muscle. Angioinvasion, with vascular thrombosis and tissue infarction, was a prominent feature in multiple animals. Fungal invasion of one or more internal organs was observed in 4 animals. Hyphae appeared to course freely through tissues and elicited little or no inflammatory response. Fusariosis has been reported sporadically in fish and other aquatic organisms, but identification has often been limited to the genus level based solely on morphologic features. Morphologic characteristics of the fungus isolated from this case were consistent with the Fusarium solani species complex (FSSC), which includes over 50 members that can only be identified definitively using DNA sequence data. A 3-locus typing scheme identified the isolate as a distinct species/haplotype, designated FSSC 12-a, belonging to a specific lineage that appears adapted to aquatic environments and disease in marine animals. Empirical treatment with itraconazole failed to stop mortalities, and subsequent in vitro antifungal susceptibility data explained a lack of clinical efficacy for this agent. Effective treatment in human medicine has similarly been limited by poor susceptibility to several classes of antifungal compounds.

B-glucan immunostilulation against columnaris in a white sturgeon (Acipenser transmontanus) model.

Flavobacterium columnare represent one of the most important bacterial pathogens of cultured sturgeon. However, at present there are no commercially available vaccines to prevent infection and treatment options are limited. ß-glucans have been shown to be potent immunostimulants that can provide fish protection against infectious disease. In this study, the effects of dietary ß-glucan supplementation on disease susceptibility were examined by exposing 0.3% ß-glucan-fed white sturgeon (Acipenser transmontanus) to Flavobacterium columnare in laboratory-controlled challenges. Morbidity and mortality were monitored for 15 days post-challenge (dpc). Additionally, transcript levels for pro-inflammatory cytokines, regulatory cytokines and acute phase proteins (APP) were investigated in the spleen and gills at different time points post-challenge. No evidence of protection was observed in ß-glucan-fed fish challenged with the bacteria. Moreover, significantly greater mortalities were observed in ß-glucan-fed fish challenged with F. columnare (p<0.05), likely associated with acute inflammatory response as haptoglobin and serotransferrin transcripts in the gills were significantly higher in fish within this group at 1 dpc. Transcript levels for all tested cytokines and APP in the spleen were similar amongst treatment groups. The results from this study suggest that ß-glucan supplementation at the concentration and rate investigated provides no-benefit to white sturgeon against F. columnare.

Disseminated fungal infection in two species of captive sharks.

In this report, two cases of systemic mycosis in captive sharks are characterized. These cases were progressive and ultimately culminated in terminal disease. Paecilomyces lilacinus, an uncommon pathogen in human and veterinary medicine, was associated with areas of necrosis in the liver, heart, and gill in a great hammerhead shark (Sphyrna mokarran). Fungal growth was observed from samples of kidney, spleen, spinal fluid, and coelomic cavity swabs. Dual fungal infection by Exophiala pisciphila and Mucor circinelloides was diagnosed in a juvenile zebra shark (Stegostoma fasciatum). Both fungi were present in the liver, with more severe tissue destruction associated with E. pisciphila. E. pisciphila also produced significant necrosis in the spleen and gill, while M. circinelloides was associated with only minimal tissue changes in the heart. Fungal cultures from liver, kidney, and spleen were positive for both E. pisciphila and M. circinelloides. Identification of P. lilacinus and M. circinelloides was based on colonial and hyphal morphology. E. pisciphila was identified by sequence analysis of the 28S rRNA D1/D2 region and the internal transcribed spacer (ITS) region between the 18S and 28S rRNA subunit. These cases, and a lack of information in the literature, highlight the need for further research and diagnostic sampling to further characterize the host-pathogen interaction between elasmobranchs and fungi.

Chelonid Alphaherpesvirus 5 Prevalence and First Confirmed Case of Sea Turtle Fibropapillomatosis in Grenada, West Indies.

Chelonid alphaherpesvirus 5 (ChHV5) is strongly associated with fibropapillomatosis, a neoplastic disease of sea turtles that can result in debilitation and mortality. The objectives of this study were to examine green (Chelonia mydas), hawksbill (Eretmochelys imbricata), and leatherback (Dermochelys coriacea) sea turtles in Grenada, West Indies, for fibropapillomatosis and to utilize ChHV5-specific PCR, degenerate herpesvirus PCR, and serology to non-invasively evaluate the prevalence of ChHV5 infection and exposure. One-hundred and sixty-seven turtles examined from 2017 to 2019 demonstrated no external fibropapilloma-like lesions and no amplification of ChHV5 DNA from whole blood or skin biopsies. An ELISA performed on serum detected ChHV5-specific IgY in 18/52 (34.6%) of green turtles tested. In 2020, an adult, female green turtle presented for necropsy from the inshore waters of Grenada with severe emaciation and cutaneous fibropapillomas. Multiple tumors tested positive for ChHV5 by qPCR, providing the first confirmed case of ChHV5-associated fibropapillomatosis in Grenada. These results indicate that active ChHV5 infection is rare, although viral exposure in green sea turtles is relatively high. The impact of fibropapillomatosis in Grenada is suggested to be low at the present time and further studies comparing host genetics and immunologic factors, as well as examination into extrinsic factors that may influence disease, are warranted.

Plasma proteomics of green turtles (Chelonia mydas) reveals pathway shifts and potential biomarker candidates associated with health and disease.

Evaluating sea turtle health can be challenging due to an incomplete understanding of pathophysiologic responses in these species. Proteome characterization of clinical plasma samples can provide insights into disease progression and prospective biomarker targets. A TMT-10-plex-LC-MS/MS platform was used to characterize the plasma proteome of five, juvenile, green turtles (Chelonia mydas) and compare qualitative and quantitative protein changes during moribund and recovered states. The 10 plasma samples yielded a total of 670 unique proteins. Using ≥1.2-fold change in protein abundance as a benchmark for physiologic upregulation or downregulation, 233 (34.8%) were differentially regulated in at least one turtle between moribund and recovered states. Forty-six proteins (6.9%) were differentially regulated in all five turtles with two proteins (0.3%) demonstrating a statistically significant change. A principle component analysis showed protein abundance loosely clustered between moribund samples or recovered samples and for turtles that presented with trauma (n = 3) or as intestinal floaters (n = 2). Gene Ontology terms demonstrated that moribund samples were represented by a higher number of proteins associated with blood coagulation, adaptive immune responses and acute phase response, while recovered turtle samples included a relatively higher number of proteins associated with metabolic processes and response to nutrients. Abundance levels of 48 proteins (7.2%) in moribund samples significantly correlated with total protein, albumin and/or globulin levels quantified by biochemical analysis. Differentially regulated proteins identified with immunologic and physiologic functions are discussed for their possible role in the green turtle pathophysiologic response and for their potential use as diagnostic biomarkers. These findings enhance our ability to interpret sea turtle health and further progress conservation, research and rehabilitation programs for these ecologically important species.

GENETIC AND MORPHOLOGIC CHARACTERIZATION OF DIASCHISTORCHIS PANDUS (DIGENEA: PRONOCEPHALIDAE) TREMATODES EXTRACTED FROM HAWKSBILL TURTLES, ERETMOCHELYS IMBRICATA (TESTUDINES: CHELONIIDAE), IN GRENADA, WEST INDIES.

The hawksbill turtle Eretmochelys imbricata is a critically endangered species with a worldwide distribution. Limited information is available about the naturally occurring intestinal parasites of this species and what impact these parasites may have on the health of the hawksbill turtle. Diaschistorchis pandus was identified postmortem in 5 hawksbill turtles from Grenada, West Indies, using morphologic characterization. Sanger sequencing was performed for conserved ribosomal regions (5.8S, ITS2, 28S) and the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Phylogenetic analysis of the 28S rRNA gene sequence data shows D. pandus clustering with other trematodes in the family Pronocephalidae, corroborating morphological classification. No genetic sequences have been previously reported for this trematode species, which has limited the collection of objective epidemiological data about this parasite of marine turtles.