Cutaneous ulcerative lesions of unknown etiology affecting lionfish Pterois spp. in the Gulf of Mexico.

OBJECTIVE: Cutaneous ulcerative skin lesions in a complex of invasive Gulf of Mexico lionfish (Red Lionfish Pterois volitans, Devil Firefish P. miles, and the hybrid Red Lionfish × Devil Firefish) became epizootic beginning in mid-August 2017. Herein, we provide the first pathological descriptions of these lesions and summarize our analyses to elucidate the etiology of the disease. METHODS: We examined ulcerated and normal fish through gross pathology and histopathology, bacterial sampling, and unbiased metagenomic next-generation sequencing. We tracked prevalence of the disease, and we used biological health indicators (condition factor, splenosomatic and hepatosomatic index) to evaluate impacts to health, while considering sex and age as potential risk factors. RESULT: Typical ulcerative lesions were deep, exposing skeletal muscle, and were bordered by pale or reddened areas often with some degree of scale loss. Only incidental parasites were found in our examinations. Most fish (86%; n = 50) exhibited wound healing grossly and histologically, confirmed by the presence of granulation tissues. A primary bacterial pathogen was not evident through bacterial culture or histopathology. Metagenomic next-generation sequencing did not reveal a viral pathogen (DNA or RNA) but did provide information about the microbiome of some ulcerated specimens. Compared with clinically healthy fish, ulcerated fish had a significantly lower condition factor and a higher splenosomatic index. Disease prevalence at monitored sites through July 2021 indicated that ulcerated fish were still present but at substantially lower prevalence than observed in 2017. CONCLUSION: Although some common findings in a number of specimens suggest a potential role for opportunistic bacteria, collectively our suite of diagnostics and analyses did not reveal an intralesional infectious agent, and we must consider the possibility that there was no communicable pathogen.

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.

Gastrointestinal entamoebiasis in captive anurans in North America.

Infections with Entamoeba spp. are recognized as a cause of clinical disease in many species including humans and reptiles; however, cases in amphibians are under-reported. Investigation of a mortality event among a captive population of Cranwell's horned frogs Ceratophrys cranwelli at a production facility in Florida, USA, revealed that deaths were due to the newly described Entamoeba species CT1. Infection caused severe necroulcerative gastroenterocolitis with a predilection for the colon. To date, this Entamoeba species has only been described in invasive cane toads Rhinella marina in Australia. Retrospective screening of archived anuran cases from a zoological pathology service identified 8 cases from captive populations that had histological evidence of gastrointestinal entamoebiasis. Molecular characterization was positive in 3 cases. Two cases, 1 in a Puerto Rican crested toad Peltophryne lemur and 1 in an Amazon milk frog Trachycephalus resinifictrix, showed 100% homology to E. ranarum and 1 case in a White's tree frog Litoria caerulea showed 100% homology to Entamoeba sp. CT1. This is the first report of novel Entamoeba sp. CT1 being associated with clinical disease in anurans within North America and also the first report of this Entamoeba species causing disease within managed collections as far back as 2003.

Reference Intervals for Blood Analytes of Adult Aquarium-Housed Russian Sturgeon Acipenser gueldenstaedtii.

Russian Sturgeon Acipenser gueldenstaedtii are an important, critically endangered, roe-producing species. Despite a wealth of knowledge pertaining to other members of family Acipenseridae, there is very limited published information regarding baseline blood analytes in Russian Sturgeon. The objectives of this study were (1) to establish reference intervals for a suite of hematological and biochemical data and (2) to compare plasma chemistry data to two point-of-care (POC) cartridges, tested on the VetScan iSTAT 1 analyzer, that use heparinized whole blood for the assessment of clinically normal, aquacultured adult Russian Sturgeon sedated with eugenol (AQUI-S 20E) at a single institution. Reference intervals are reported. The calculated hematocrit measured by the POC analyzer tended 4-5% lower than the spun packed cell volume, confirming the importance of spun packed cell volume as a reliable measurement of red blood cell mass. Various analytes, notably whole-blood urea nitrogen, glucose, sodium, total carbon dioxide, chloride, ionized calcium, and anion gap, were significantly different by both POC cartridges. This study successfully produced reference intervals for blood analytes in adult Russian Sturgeon under managed care and creates a foundation for future studies into the effects of extrinsic and intrinsic factors and variations of analytical methodologies on blood analytes in this species.

Characterization of a novel picornavirus isolated from moribund aquacultured clownfish.

Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolayer lysis in the initial inoculation and upon repassage. Transmission electron microscopy of infected SSN-1 cells revealed small round particles (mean diameter=20.0-21.7 nm) within the cytoplasm, consistent with the ultrastructure of a picornavirus. Full-genome sequencing of the purified virus revealed a novel picornavirus most closely related to the bluegill picornavirus and other members of the genus Limnipivirus. Additionally, pairwise protein alignments between the clownfish picornavirus (CFPV) and other known members of the genus Limnipivirus yielded results in accordance with the current International Committee on Taxonomy of Viruses criteria for members of the same genus. Thus, CFPV represents a proposed new limnipivirus species. Future experimental challenge studies are needed to determine the role of CFPV in disease.

Phylogenomic characterization of red seabream iridovirus from Florida pompano Trachinotus carolinus maricultured in the Caribbean Sea.

Between 2010 and 2016, six mortality events were observed in Florida pompano (Trachinotus carolinus) maricultured in the Dominican Republic. Histopathological examination and conventional PCR confirmed a megalocytivirus (MCV) infection in each case. Subsequently, next-generation sequencing and phylogenomic analyses confirmed that MCV DNA was present in the infected pompano tissue samples from 2010, 2014, and 2016, and each was determined to be red seabream iridovirus (RSIV). Annotation of the RSIV genome sequences identified 121 open reading frames, and BLASTN analysis revealed the highest nucleotide sequence identity (> 99%) to a RSIV clade 1 MCV isolated from a moribund red seabream (Pagrus major) maricultured in Japan. These cases represent the first fully sequenced RSIV genomes detected outside of Asia and are the earliest reports of MCV infections in Florida pompano. This recent geographical expansion of RSIV warrants further attention to determine its potential economic and ecological impact.

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.

Identification of Francisella noatunensis in novel host species French grunt (Haemulon flavolineatum) and Caesar grunt (Haemulon carbonarium).

Francisella noatunensis is an emerging pathogen of fish that has been isolated from several cultured species worldwide. Here presented is a case involving several hundred marine grunts that were caught near the Florida Keys for display in public aquaria. These fish were maintained in a recirculating system where they began to experience mortalities approximately two weeks post-stocking. Postmortem examination revealed disseminated systemic granulomatous disease most severely affecting spleen and kidney. Splenic and renal tissue homogenates inoculated in modified Thayer Martin agar media yielded colonies consistent with F. noatunensis 4 days post inoculation. Bacterial colonies and tissues were confirmed positive after real-time PCR amplification of the intracellular growth loci gene (iglC) specific for F. noatunensis subspecies orientalis. Consequently, multiple novel host species for this pathogen were identified, including the French grunt (Haemulon flavolineatum) and the Caesar grunt (Haemulon carbonarium).

Occurrence of Piscirickettsiosis-like syndrome in tilapia in the continental United States.

From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 +/- 0.1 x 0.8 +/- 0.2 microm, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.

Molecular and immunohistochemical diagnosis of Francisella noatunensis subsp. orientalis from formalin-fixed, paraffin-embedded tissues.

Members of the genus Francisella (viz., F. noatunensis subsp. orientalis [Fno] and F. noatunensis subsp. noatunensis) have been described as causative agents of chronic granulomatous and pyogranulomatous lesions in wild and cultured fish species. In the present study, 68 archived formalin-fixed, paraffin-embedded (FFPE) tissues from several fish species, collected at different geographical locations from 2000 to 2011, were analyzed using a real-time polymerase chain reaction assay for the detection of the Fno intracellular growth loci C (iglC) gene and by immunohistochemistry for the demonstration of Fno antigens. The results revealed a high correlation between these 2 diagnostic techniques validating their use for the diagnosis of Fno infection in archived FFPE tissues and confirming the presence of Fno in fish species from the Cari y years of the present century.

Association of mycobacteria in recirculating aquaculture systems and mycobacterial disease in fish.

Mycobacterium marinum isolates cultivated from tissue containing granulomatous lesions in Florida pompano Trachinotus carolinus and from biofilm samples collected from their tank and water recirculating system had identical (L1 of 11 bands) repetitive-sequence-based polymerase chain reaction (rep-PCR) DNA fingerprints. A second M. marinum clone sharing 4 of 11 rep-PCR bands with the first clone was isolated from some fish tissues but not from system samples. Water samples yielded low numbers of colonies of mycobacteria (0.08-1.3/mL), but high numbers were recovered from biofilms (260-12,000/swab) and filters (63-21,000/ filter). Mycobacterium hemophilum, M. chelonae, M. trivale, M. gastri, and M. gordonae were isolated from system samples alone.