Ocular lesions of captive cephalopods.

Ocular lesions are uncommonly reported and described in invertebrate species. In this study, cases from 2 diagnostic laboratories, in which lesions were noted in 33 diagnostic specimens from various species of cephalopods, including octopuses, squid, nautiluses, and cuttlefish, were reviewed. Clinical information and gross lesions were described in a minority of cases. The most common lesion was inflammation of varying severity and was most commonly within the anterior uvea (iris and ciliary papilla), followed by the posterior chamber and lens. More than half of the cases with inflammation had concurrent hyperplastic lesions of the iris and ciliary papilla, including posterior iris epithelial hyperplasia, cystic adenomatous hyperplasia, and/or posterior epithelial cysts. The most common clinical observation was cloudy eyes, which correlated histologically to anterior uveitis in all cases where it was documented. Dermatitis and cutaneous ulceration were the most frequent comorbidities in cases where clinical information was available.

Myxobolus lentisuturalis infection in a farmed population of goldfish Carassius auratus from the USA.

Myxobolus lentisuturalis is a myxozoan parasite of piscine muscle that has been described in goldfish Carassius auratus and Prussian carp Carassius gibelio. This report documents a naturally occurring infection of M. lentisuturalis in a population of farmed goldfish in the USA. Postmortem examination was performed on 4 affected goldfish. Gross findings included large cystic cavities along the dorsal midline filled with caseous exudate. Histopathology revealed myxozoan plasmodia and spores in the epaxial muscles with varying degrees of granulomatous and necrotizing myositis accompanied by lymphohistiocytic meningoencephalitis. Spore morphology and dimensions were consistent with M. lentisuturalis, as observed by light microscopy. PCR and sequence analysis of the small subunit ribosomal DNA of infected muscle samples from 2 goldfish confirmed the parasite to have 99-100% nucleotide identity to M. lentisuturalis sequences recovered from similar cases of this parasite infecting goldfish in China and Italy and Prussian carp in China. This is the first reported case of M. lentisuturalis in the USA and furthers the understanding of the pathogenicity of this under-described parasite.

Characterization of an alloherpesvirus from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA).

In the spring of 2017, 2 adult lake sturgeon (LS) Acipenser fulvescens captured from the Wolf River, Wisconsin (USA), presented with multiple cutaneous plaques that, upon microscopic examination, indicated proliferative epidermitis. Ultrastructural examination of affected keratinocytes revealed particles in the nucleus having a morphology typical of herpesviruses. A degenerate PCR assay targeting the DNA polymerase catalytic subunit (pol) gene of large double-stranded DNA viruses generated amplicons of the anticipated size from skin samples, and sequences of amplicons confirmed the presence of a novel alloherpesvirus (lake sturgeon herpesvirus, LSHV) related to acipenserid herpesvirus 1 (AciHV1). The complete genome (202660 bp) of this virus was sequenced using a MiSeq System, and phylogenetic analyses substantiated the close relationship to AciHV1. A PCR assay targeting the LSHV DNA packaging terminase subunit 1 (ter1) gene demonstrated the presence of the virus in 39/42 skin lesion samples collected from wild LS captured in 2017-2019 and 2021 in 4/4 rivers in Wisconsin. Future efforts to isolate LSHV in cell culture would facilitate challenge studies to determine the disease potential of the virus.

Diffuse large B cell lymphoma and a novel gammaherpesvirus in northern elephant seals Mirounga angustirostris.

Two emaciated male northern elephant seal (NES) Mirounga angustirostris pups were admitted to The Marine Mammal Center (Sausalito, California, USA) and treated for malnutrition. Complete blood counts showed a progressive moderate to marked leukocytosis characterized by a predominance of large monomorphic mononuclear cells of probable lymphoid origin, frequently with flower-shaped nuclei. Both seals were euthanized due to suspected lymphoid neoplasia. At necropsy, most lymph nodes in both pups were markedly enlarged, some with distinct white nodules, the spleens were diffusely enlarged, and the intestinal mucosae were thickened. Histopathologic features consistent with disseminated large cell lymphoma were identified to varying degrees of severity in lymph nodes, bone marrow, liver, tonsils, spleen, liver, intestines, kidneys, lower urinary tract, and several other organs. Immunohistochemical staining of neoplastic cells was most consistent with B lymphocyte origin, with most cells staining positively for Pax 5 and CD20 with admixed small CD3-positive T lymphocytes and CD204-positive macrophages. PCR and sequencing identified a novel gammaherpesvirus, herein called miroungine gammaherpesvirus 3, from affected tissues. This virus is in a clade outside of named genera that utilize hosts in the suborder Caniformia. The present study is the first description of diffuse large B cell lymphoma with leukemic manifestation and concomitant detection of a novel gammaherpesvirus in free-living NESs. Further research regarding the prevalence of this new gammaherpesvirus and its associated pathogenesis in this species is indicated.

Effect of water temperature on frog virus 3 disease in hatchery-reared pallid sturgeon Scaphirhynchus albus.

Ranaviruses are large double-stranded DNA viruses within the genus Ranavirus (family Iridoviridae) that are being detected with increasing frequency among aquacultured and wild fishes. In the USA, multiple sturgeon hatcheries have experienced ranavirus epizootics resulting in significant morbidity and mortality in young-of-year (YOY). Significant economic losses have resulted from repeated outbreaks of frog virus 3 (FV3), the type species for the genus Ranavirus, in YOY pallid sturgeon Scaphirhynchus albus reared at a hatchery within the Missouri River Basin. Water temperature and stocking density are known to influence the severity of ranavirus disease in ectothermic vertebrates. To determine the effect of water temperature on ranavirus disease in hatchery-raised S. albus, we conducted FV3 challenges at 2 temperatures (17 and 23°C) and compared cumulative survival over a 28 d study period. A mean (±SE) survival rate of 57.5 ± 13.2% was observed in replicate tanks of sturgeon maintained at 23°C, whereas no mortality was observed among sturgeon maintained at 17°C. In a second challenge study, we compared the effect of water temperature on disease progression by regularly sampling fish over the study period and evaluating lesions by histopathology and in situ hybridization, and by assessing viral titer and load in external and internal tissues using virus isolation and qPCR, respectively. Results suggest that temperature manipulation may be an effective mitigation strategy that sturgeon hatcheries can employ to minimize ranavirus-associated disease.

Complete genome sequences of infectious spleen and kidney necrosis virus isolated from farmed albino rainbow sharks Epalzeorhynchos frenatum in the United States.

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

Diagnosis and treatment of multi-species fish mortality attributed to Enteromyxum leei while in quarantine at a US aquarium.

Enteromyxum leei is an enteric myxozoan parasite of fish. This myxozoan has low host specificity and is the causative agent of myxozoan emaciation disease, known for heavy mortalities and significant financial losses within Mediterranean, Red Sea, and Asian aquaculture industries. The disease has rarely been documented within public aquaria and, to our knowledge, has never been confirmed within the USA. This case report describes an outbreak of E. leei in a population of mixed-species east African/Indo-Pacific marine fish undergoing quarantine at a public aquarium within the USA. Four of 16 different species of fish in the population, each of a different taxonomic family, were confirmed infected by the myxozoan through cloacal flush or intestinal wet mount cytology at necropsy. Clinical and histopathological findings in this case are similar to previous findings describing myxozoan emaciation disease, e.g. severe emaciation, cachexia, enteritis, and death. Sequence analysis of the 18S rDNA of intestinal samples from a powder blue tang Acanthurus leucosternon and an emperor angelfish Pomacanthus imperator confirmed the parasite to have 99-100% identity with other E. leei sequences. Spore morphology and ultrastructure were consistent with previous reports of E. leei. Treatment of clinically affected fish by oral administration of the coccidiostats amprolium and salinomycin led to reduction of mortalities and resolution of clinical signs. This case report highlights the importance of thorough examination and surveillance of fish during quarantine, particularly with respect to enteric myxozoans.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

In Vitro Exposure of Harbor Seal Immune Cells to Aroclor 1260 Alters Phocine Distemper Virus Replication.

In the last 30 years, several large-scale marine mammal mortality events have occurred, often in close association with highly polluted regions, leading to suspicions that contaminant-induced immunosuppression contributed to these epizootics. Some of these recent events also identified morbillivirus as a cause of or contributor to death. The role of contaminant exposures regarding morbillivirus mortality is still unclear. The results of this study aimed to address the potential for a mixture of polychlorinated biphenyls (PCBs), specifically Aroclor 1260, to alter harbor seal T-lymphocyte proliferation and to assess if exposure resulted in increased likelihood of phocine distemper virus (PDV USA 2006) to infect susceptible seals in an in vitro system. Exposure of peripheral blood mononuclear cells to Aroclor 1260 did not significantly alter lymphocyte proliferation (1, 5, 10, and 20 ppm). However, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), lymphocytes exposed to 20 ppm Aroclor 1260 exhibited a significant decrease in PDV replication at day 7 and a significant increase at day 11 compared with unexposed control cells. Similar and significant differences were apparent on exposure to Aroclor 1260 in monocytes and supernatant. The results here indicate that in harbor seals, Aroclor 1260 exposure results in a decrease in virus early during infection and an increase during late infection. The consequences of this contaminant-induced infection pattern in a highly susceptible host could result in a greater potential for systemic infection with greater viral load, which could explain the correlative findings seen in wild populations exposed to a range of persistent contaminants that suffer from morbillivirus epizootics.

A RETROSPECTIVE STUDY OF THE LESIONS ASSOCIATED WITH IRON STORAGE DISEASE IN CAPTIVE EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

Egyptian fruit bats (Rousettus aegyptiacus) are one of many species within zoologic collections that frequently develop iron storage disease. The goals of this retrospective multi-institutional study were to determine the tissue distribution of iron storage in captive adult Egyptian fruit bats and the incidence of intercurrent neoplasia and infection, which may be directly or indirectly related to iron overload. Tissue sections from 83 adult Egyptian fruit bats were histologically evaluated by using tissue sections stained with hematoxylin and eosin, trichrome, and Prussian blue techniques. The liver and spleen consistently had the largest amount of iron, but significant amounts of iron were also detected in the pancreas, kidney, skeletal muscle, and lung. Hepatocellular carcinoma (HCC; 11) was the most common neoplasm, followed by cholangiocarcinoma (4). Extrahepatic neoplasms included bronchioloalveolar adenoma (3), pulmonary carcinosarcoma (1), oral sarcoma (1), renal adenocarcinoma (1), transitional cell carcinoma of the urinary bladder (1), mammary gland adenoma (1), and parathyroid adenoma (1). There were also metastatic neoplasms of undetermined primary origin that included three poorly differentiated carcinomas, a poorly differentiated sarcoma, and a neuroendocrine tumor. Bats with hemochromatosis were significantly more likely to have HCC than bats with hemosiderosis (P = 0.032). Cardiomyopathy was identified in 35/77 bats with evaluable heart tissue, but no direct association was found between cardiac damage and the amount of iron observed within the liver or heart. Hepatic abscesses occurred in multiple bats, although a significant association was not observed between hemochromatosis and bacterial infection. To the authors' knowledge, this is the first publication providing evidence of a positive correlation between hemochromatosis and HCC in any species other than humans.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

New disease records for hatchery-reared sturgeon. II. Phaeohyphomycosis due to Veronaea botryosa.

A series of fungal cases in hatchery-reared juvenile and young adult Siberian sturgeon Acipenser baerii and white sturgeon A. transmontanus occurred at production facilities in Florida and California, USA, respectively. Affected fish exhibited abnormal orientation and/or buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerative skin and eye lesions. Necropsies revealed haemorrhage throughout the coelom, serosanguinous coelomic effusion and organomegaly with nodular or cystic lesions in multiple organs. Fungal hyphae were observed in 27 fish (24 A. baerii and 3 A. transmontanus) via microscopic examination of tissue wet mounts and on slides prepared from colonies grown on culture media. Histopathological examination of these infected tissues revealed extensive infiltration by melanised fungal hyphae that were recovered in culture. Phenotypic characteristics and sequencing of the fungal isolates with the use of the internal transcribed spacer region and 28S rRNA gene confirmed the aetiological agent as Veronaea botryosa. To our knowledge, this is the first documentation of V. botryosa infection in fish, although melanised fungi of the closely related genus Exophiala are well-known pathogens of freshwater and marine fishes.

Valvular dysplasia and congestive heart failure in a juvenile African penguin (Spheniscus demersus).

Abstract: An aquarium-housed, 6-mo-old African penguin (Spheniscus demersus) presented with acute respiratory distress. Auscultation revealed a grade II-III systolic murmur in the absence of adventitial sounds, and an enlarged heart without pulmonary edema was seen radiographically. Echocardiographic evaluation revealed atrioventricular (AV) valvular dysplasia and ventricular enlargement. The penguin was treated with enalapril, furosemide, and pimobendan but died within 3 wk of detection of the murmur. Congenital dysplasia of the right AV valve with right atrial and ventricular dilation and ventricular hypertrophy were diagnosed on postmortem examination.

Conjunctivitis, rhinitis, and sinusitis in cliff swallows (Petrochelidon pyrrhonota) found in association with Mycoplasma sturni infection and cryptosporidiosis.

Fledgling cliff swallows were cared for at a rehabilitation facility when clinical signs of ocular disease, characterized by conjunctivitis, epiphora, and hyperaemia of palpebrae and nictitans, were recognized. Treatment consisted of topical and oral antibiotic therapy and one topical steroid administration. However, one cliff swallow died and three were killed due to poor therapeutic response. Conjunctival swabs were obtained ante-mortem from the three cliff swallows and were submitted for mycoplasma culture and molecular diagnostics. Heads of the three birds were fixed in 10% neutral buffered formalin and submitted for histopathologic examination of oculonasal tissues. Mycoplasma cultures and molecular evaluation of isolates identified Mycoplasma sturni, but not Mycoplasma gallisepticum, from each specimen. Histopathologic examination revealed lymphoplasmacytic conjunctivitis, rhinitis and infraorbital sinusitis with follicular lymphoid hyperplasia, epithelial hyperplasia, and protozoal stages compatible with Cryptosporidium spp. arranged in and along the apical surfaces of epithelial cells. Identification of concurrent M. sturni and Cryptosporidium spp. infections in these cliff swallows demonstrates an alternative infectious condition that can produce gross and microscopic lesions comparable with those commonly observed in M. gallisepticum infections of house finches and other passerine species. Conjunctivitis associated with M. sturni and Cryptosporidium spp. in cliff swallows may represent an emerging disease risk to a naïve, high-density and colonial species such as colony-nesting cliff swallows.

Systemic mycosis in a California sea lion (Zalophus californianus) with detection of cystofilobasidiales DNA.

A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.

Sulawesi tortoise adenovirus-1 in two impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota).

Sulawesi tortoise adenovirus-1 (STAdV-1) is a newly discovered virus infecting endangered and threatened tortoises. It was initially described from a confiscated group of 105 Sulawesi tortoises (Indotestudo forsteni) obtained by the Turtle Survival Alliance and distributed to five sites with available veterinary care across the United States. In a 3-yr period from the initial outbreak, one multi-species collection that rehabilitated and housed adenovirus-infected Sulawesi tortoises experienced deaths in impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota). Impressed tortoises that died had evidence of systemic viral infection with histopathologic features of adenovirus. Adenovirus was identified by consensus nested polymerase chain reaction (PCR) testing and subsequent sequencing of PCR products. Sequencing indicated that the adenovirus infecting these impressed tortoises and Burmese star tortoise was STAdV-1. In one impressed tortoise, viral infection was confirmed using transmission electron microscopy. In situ hybridization using a semiautomated protocol and fluorescein-labeled riboprobe identified STAdV-1 inclusions in spleen, liver, kidney, and testis of one impressed tortoise. The impact of this virus on captive and wild populations of tortoises is unknown; however, these findings indicate that STAdV-1 can be transmitted to and can infect other tortoise species, the impressed tortoise and Burmese star tortoise, when cohabitated with infected Sulawesi tortoises.

B cells oppose Mycoplasma pneumoniae vaccine enhanced disease and limit bacterial colonization of the lungs.

Development of an effective vaccine for Mycoplasma pneumoniae has been hindered by reports of Vaccine Enhanced Disease (VED) in test subjects vaccinated and challenged in studies conducted in the 1960s. The exact mechanism of disease exacerbation has yet to be fully described, but host immune responses to Lipid-Associated Membrane Proteins (LAMPs) lipoprotein lipid moieties have been implicated. LAMPs-induced exacerbation appears to involve helper T cell recall responses, due in part to their influence on neutrophil recruitment and subsequent inflammatory responses in the lung. Herein, we characterized the functions of host B cell responses to M. pneumoniae LAMPs and delipidated-LAMPs (dLAMPs) by conducting passive transfer and B cell depletion studies to assess their contribution to disease exacerbation or protection using a BALB/c mouse model. We found that antibody responses to M. pneumoniae LAMPs and dLAMPs differ in magnitude, but not in isotype or subclass. Passive transfer, dLAMP denaturation, and monoclonal antibody studies indicate that antibodies do not cause VED, but do appear to contribute to control of bacterial loads in the lungs. Depletion of B cells prior to LAMPs-vaccination results in significantly enhanced pathology in comparison to B cell competent controls, suggesting a possible regulatory role of B cells distinct from antibody secretion. Taken together, our findings suggest that B cell antibody responses to M. pneumoniae contribute to, but are insufficient for protection against challenge on their own, and that other functional properties of B cells are necessary to limit exacerbation of disease in LAMPs-vaccinated mice after infection.

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

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