Carnobacterium maltaromaticum associated with meningoencephalitis and otitis in stranded common thresher sharks (Alopias vulpinus).

Juvenile common thresher sharks (Alopias vulpinus) have been recently stranding along the California coastline. Using Illumina sequencing of the bacterial 16S rRNA gene along with necropsy, cytological, bacteriological, and histological techniques, we screened microbial communities and described lesions characterizing affected sharks with the purpose of identifying potential pathogen sources and pathologic processes. Histopathological assessment of moribund sharks revealed severe meningoencephalitis, as previously described in stranded salmon sharks (Lamna ditropis), along with inflammation of the inner ear and subcutaneous tissues surrounding the endolymphatic ducts. Furthermore, inflamed areas were characterized by the prevalence of Carnobacterium maltaromaticum, suggesting this bacterium as a potential pathogen that gains access to the inner ear through the endolymphatic ducts, with subsequent spread into the brain. The absence or low abundance of this bacterium in the spiral valve in both healthy and infected sharks suggests that Carnobacterium is not a commensal member of their digestive communities and the spiral valve is unlikely to be the source of the pathogen. Furthermore, phylogenetic analysis suggests that C. maltaromaticum strains isolated from diseased sharks have minimal genetic variation and differ from other strains originating from food or diseased teleosts. While a C. maltaromaticum-like organism has previously been associated with meningoencephalitis in salmon shark strandings, this is the first study to report common thresher shark strandings associated with C. maltaromaticum, involving the endolymphatic ducts as portals of entry to the brain.

A practical guide to necropsy of the elasmobranch chondrocranium and causes of mortality in wild and aquarium-housed California elasmobranchs.

Elasmobranchs are common, iconic species in public aquaria; their wild counterparts are key members of marine ecosystems. Post-mortem examination is a critical tool for disease monitoring of wild elasmobranchs and for management of those under human care. Careful necropsy of the head, with a focus on clinically relevant anatomy, can ensure that proper samples are collected, increasing the chance of presumptive diagnoses prior to slower diagnostic workup. Immediate feedback from a thorough head necropsy allows for faster management decisions, often identifying pathogens, routes of pathogen entry, and pathogenesis, which are current shortcomings in published literature. This article proposes a protocol for necropsy of the elasmobranch chondrocranium, emphasizing unique anatomy and careful dissection, evaluation, and sampling of the endolymphatic pores and ducts, inner ears, brain, and olfactory system as part of a complete, whole-body necropsy. Extensive use of cytology and microbiology, along with thorough sample collection for histology and molecular biology, has proven effective in identifying a wide range of pathogens and assisting with characterization of pathogenesis. The cause of mortality is often identified from a head necropsy alone, but does not replace a thorough whole-body dissection. This protocol for necropsy and ancillary diagnostic sample collection and evaluation was developed and implemented in the necropsy of 189 wild and aquarium-housed elasmobranchs across 18 species over 13 years (2011-2023) in California. Using this chondrocranial approach, meningoencephalitis was determined to be the primary cause of mortality in 70% (118/168) of stranded wild and aquarium-housed elasmobranchs. Etiology was largely bacterial or protozoal. Carnobacterium maltaromaticum bacterial meningoencephalitis occurred in salmon sharks (Lamna ditropis), shortfin mako sharks (Isurus oxyrinchus), common thresher sharks (Alopias vulpinus), and one Pacific electric ray (Tetronarce californica). Miamiensis avidus was the most common cause of protozoal meningoencephalitis and found almost exclusively in leopard sharks (Triakis semifasciata) and bat rays (Myliobatis californica) that stranded in San Francisco Bay. Bacterial pathogens were found to use an endolymphatic route of entry, while protozoa entered via the nares and olfactory lamellae. Trauma was the second most common cause of mortality and responsible for 14% (24/168) of wild shark strandings and deaths of aquarium-housed animals.

METAGENOMIC NEXT-GENERATION SEQUENCING REVEALS MIAMIENSIS AVIDUS (CILIOPHORA: SCUTICOCILIATIDA) IN THE 2017 EPIZOOTIC OF LEOPARD SHARKS ( TRIAKIS SEMIFASCIATA) IN SAN FRANCISCO BAY, CALIFORNIA, USA.

During March to August of 2017, hundreds of leopard sharks ( Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, US. Similar mass stranding events occurred in 1967 and 2011, but analysis of those epizootics was incomplete, and no etiology was confirmed. Our investigation of the 2017 epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. We pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening. We showed that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard ( n=12) and other shark species ( n=2) that stranded in San Francisco Bay but was absent in leopard sharks caught elsewhere. This ciliated protozoan has been implicated in devastating outbreaks in teleost marine fish but not in wild elasmobranchs. Our results highlight the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease.

Draft Genome Sequences of Nine New Carnobacterium maltaromaticum Strains Isolated from Diseased Sharks.

Here, we report the draft genome sequences of 9 strains of Carnobacterium maltaromaticum (SK_LD1 to SK_LD3 and SK_AV1 to SK_AV6), a member of the Carnobacteriaceae family (phylum Firmicutes). These strains were isolated from the brain and the inner ear of three diseased thresher sharks and two diseased salmon sharks. The genome assembly resulted in an average of 3,306,205.9 ± 29,143.9 bp and 3,085 ± 32.67 coding DNA sequences (CDS).

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis.

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.

Effects of husbandry practices, gender, and normal physiological variation on growth and reproduction of Japanese medaka, Oryzias latipes.

Japanese medaka, Oryzias latipes, are currently used in a variety of research applications for toxicological and carcinogenesis research, yet the impact of certain husbandry factors on study outcome has received limited attention. In this study, we demonstrated significant effects of stocking density (SD), dietary restriction (DR) and photoperiod on somatic growth in medaka. Higher stocking densities significantly inhibited somatic and hepatic growth in females, while having no similar effects on males. Daily egg production declined in a step-wise manner in groups of fish stocked at densities ranging from 0.38 to 2.6 fish per l. Significantly slower somatic growth was observed in fish fed on a mildly restrictive dietary protocol compared with those fish fed to excess. Further significant declines were observed in somatic growth of fish fed at a rate comparable to that used in research studies (3-4% body weight (BW) per day). Fish reared at an 8-h light:16-h dark (8L/16D) photoperiod were significantly smaller than cohorts reared at a 16-h light:8-h dark (16L/8D) photoperiod and female sexual maturity was delayed in the short photoperiod cohort. In normal female medaka, a high degree of individual variation was observed in daily egg production, as well as diurnal fluctuations in ovarian weight, gonadosomatic index (GSI) and hepatosomatic index (HSI). These results indicate some husbandry factors affect growth, sexual maturation and egg production in medaka and should be taken into consideration in studies evaluating endpoints that may be impacted by growth and reproductive performance, i.e. carcinogenesis and endocrine disruption studies. The high degree of individual variation among normal females and daily fluctuations in organ weights should also be considered in study design.

Retrospective analysis: bile hydrocarbons and histopathology of demersal rockfish in Prince William Sound, Alaska, after the Exxon Valdez oil spill.

Demersal rockfish are the only fish species that have been found dead in significant numbers after major oil spills, but the link between oil exposure and effect has not been well established. After the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska, several species of rockfish (Sebastes spp.) from oiled and reference sites were analyzed for hydrocarbon metabolites in bile (1989-1991) and for microscopic lesions (1990 and 1991). Biliary hydrocarbons consistent with exposure to Exxon Valdez oil were elevated in 1989, but not in 1990 or 1991. Significant microscopic findings included pigmented macrophage aggregates and hepatic megalocytosis, fibrosis, and lipid accumulation. Site differences in microscopic findings were significant with respect to previous oil exposure in 1991 (P=0.038), but not in 1990. However, differences in microscopic findings were highly significant with respect to age and species in both years (P<0.001). We conclude that demersal rockfish were exposed to Exxon Valdez oil in 1989, but differences in microscopic changes in 1990 and 1991 were related more to age and species differences than to previous oil exposure.

Pathology of ocular lesions associated with gas supersaturation in white seabass.

Cultured juvenile white seabass Atractoscion nobilis (WSB) can suffer from intraocular emphysemas and exophthalmia in the hatchery environment. To identify the cause, two size-groups of WSB were exposed to five gas saturation levels, ranging from 98% to 122% total gas pressure (TGP), over a 96-h exposure period in 18 degrees C and 23 degrees C seawater. Histological examination revealed that the gross and subgross lesions associated with gas supersaturation included corneal and orbital emphysema, along with subretinal, optic nerve, and iridial hemorrhage. Corneal emphysema was the most prominent gross lesion, with the severity and prevalence increasing between size-groups and water temperatures as TGP increased. Following the same pattern was orbital emphysema, which affected more than 93% of the fish examined and caused hemorrhage in the subretinal space, around the optic nerve, in the iris, or a combination thereof. Iridial hemorrhage occurred in 91% of the fish examined and decreased significantly with fish size. The prevalence and severity of hemorrhage in the subretinal space increased significantly with TGP and fish size but not with temperature. Optic nerve hemorrhage was absent in small fish exposed at 18 degrees C but increased significantly with temperature and fish size. The reverse was true for the large fish.

An artificial fertilization method with the Japanese medaka: implications in early life stage bioassays and solvent toxicity.

An in vitro fertilization method was used to study the effects on medaka (Oryzias latipes) embryos reared either from 0.5h (early blastodisc) or 6.5h (early blastula) post-fertilization for 200 h in varying concentrations of dimethyl sulfoxide (DMSO), methanol, or ethanol (0.06, 0.13, 0.25, 0.50, 1.00, 1.50, and 2.00% v/v). Physiological and anatomical parameters in embryos and larvae were examined and compared across groups. Among the three solvents, ethanol induced the most severe effects in embryos and larvae. Based on anatomical abnormalities, no differences were observed between both windows of exposure to DMSO. Similarly, no differences were observed at concentrations of methanol or ethanol 0.25% v/v. Only two endpoints, hatching success for methanol (EC50 1.84% v/v), and spinal deformities in larvae for ethanol (EC50 0.60% v/v) pointed the earlier window of exposure as significantly more sensitive. Further research is needed to investigate if using this exposure methodology for chemicals with more specific modes of action may result in increased sensitivities.