In vitro susceptibility testing for the emerging pathogenic mould Veronaea botryosa and pharmacokinetic parameters of natamycin in white sturgeon (Acipenser transmontanus).

Systemic phaeohyphomycosis caused by the dematiaceous mould Veronaea botryosa is an important emergent disease affecting captive sturgeons (Acipenser spp.). The disease, colloquially known as "fluid belly," causes morbidity and mortality in adult animals resulting in significant economic losses to the aquaculture industry. Advancements in therapeutic and prophylactic protocols have been partially hampered by the lack of basic protocols to grow and manipulate the fungus in the laboratory. In this study, microbroth kinetic protocols were established to analyse V. botryosa growth in seven nutrient media at different temperatures. Generated area under the curve (AUC) indicates that potato flake dextrose broth (PFD-B) and Sabouraud dextrose broth (SD-B) incubated at 25°C provided the greatest growth. The generated protocol was then used to test the susceptibility of V. botryosa isolates to natamycin, a macrolide polyene antifungal agent used as a food preservative. SD-B and RPMI with l-glutamine (+RPMI-B) containing different concentrations of natamycin were inoculated with V. botryosa conidia and the generated growth curves were compared using cubic smoothing spline model. The non-inhibitory concentration and minimal inhibitory concentration (MIC; decrease of AUC by 90% compared with control) were determined to be <1 µg/mL and 16 µg/mL of natamycin in SD-B media. To gain an understanding of the tissue distribution of natamycin in white sturgeon, pharmacokinetics was tested. Based on pharmacokinetic parameters determined in this study and targeting a blood concentration >16 µg/mL for 24 h, an intravenous dose >1 g/kg would be needed, making the use of this drug unrealistic. The information presented in this study can be used to investigate susceptibility of pathogenic fungus to antimicrobials and disinfectants as well as support future therapeutic protocols against emerging fungal diseases like fluid belly.

Analysis of the white sturgeon (Acipenser transmontanus) immune response during immunostimulation and Veronaea botryosa infection.

Systemic phaeohyphomycosis caused by Veronaea botryosa is regarded as an important emerging mycotic disease of sturgeon aquaculture. However, no vaccines or treatments are currently available. The effects of dietary ß-glucan supplementation on resistance to V. botryosa infection was examined in controlled challenges by exposing immunostimulated and control fish to ~7.25 × 105 fungal spores/fish via intra-muscular injection. Six weeks post-challenge, cumulative mortality was determined and antibodies to acute phase-proteins (APP) were used to quantify the conserved APP peptides in the serum of challenged and control fish using Western blot. Transcript levels for all tested pro-inflammatory cytokines, APP, and regulatory cytokines in the spleen were similar amongst treatments at the end of the three-week feeding period. However, significantly higher survival occurred in fingerlings fed 0.3% ß-glucans compared to non-immunostimulated fish groups (p < 0.05) six weeks post-challenge. A strong proinflammatory response was detected in exposed treatment groups, and greater survival at 6 weeks was associated with higher transcript abundance of Il-17 in fish fed ß-glucans. Findings support the important role of this cytokine in response to fungal infection.

Assessment of clinical outcomes associated with mercury concentrations in harbor seal pups (Phoca vitulina richardii) in central California.

Monomethyl mercury (MeHg+) from the diet can cause mild to severe neurotoxicosis in fish-eating mammals. Chronic and low-level in utero exposure also can be neurotoxic, as documented in laboratory animal studies and epidemiologic investigations. In free-ranging animals, it is challenging to study low-level exposure related neurotoxicosis, and few studies have investigated the relationship between mercury (Hg) and adverse outcomes in wild populations. Relative to Hg concentrations on admission we evaluated different types of behaviors for 267 Pacific harbor seal (HS; Phoca vitulina richardii) pups at The Marine Mammal Center from 2015 to 2019 during rehabilitation after stranding and maternal separation. Admitted HS pups underwent a clinical exam; including sex and weight determination, and hair (partly lanugo grown in utero) and blood samples were collected for total Hg concentration ([THg]) determination. All pups were monitored weekly (behavior assessments included response to tactile stimulation, movement, swimming, interactions with other seals, hand feeding, and feeding independently), and days in rehabilitation and survival were recorded. There was a significant negative correlation between [THg] and responses to tactile stimulation and movements, measured in both hair and whole blood (p < 0.05). This relationship was found both during the intensive care unit (ICU) stage, and during the pool stage of rehabilitation. Additionally, there was a significant association between greater [THg] and number of days spent in rehabilitation, although there was no relationship between [THg] and survival. There was a significant sex difference, with greater [THg] in female pups, which contrasts with previously published findings in juvenile and adult harbor seals. Our findings support small, but significant associations between gestational THg exposure and clinical effects for tactile sensory response and movement, and longer rehabilitation durations for HS pups, although there was considerable variability among animals.

Experimental Infection of Mice with Veronaea botryosa as a Model for Human Phaeohyphomycosis.

Veronaea botryosa is a ubiquitous, dematiaceous mold capable of causing cutaneous and subcutaneous lesions in humans. In the last decade, V. botryosa has been associated with emergent systemic fungal infections in aquatic animals, including cultured sturgeon (Acipenser spp.), captive amphibians, and wild reptiles. Recently, repetitive extragenic palindromic PCR (rep-PCR) fingerprinting has demonstrated intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions. However, little is known regarding the pathogenic potential of the different genetic clades, and no mammalian model currently exists to investigate V. botryosa phaeohyphomycosis. In this study, we inoculated immunocompetent heterozygotic (nu/+) and immunodeficient homozygotic (nu/nu) Hsd:Athymic Nude-Fox1nu mice subcutaneously or through orogastric gavage with 1 of 3 representative V. botryosa strains that had been recovered from white sturgeon (Acipenser transmontanus), green sea turtle (Chelonia mydas), and human hosts and typed by using rep-PCR analysis. Daily mortality and morbidity were recorded, and dissemination of the fungus was investigated through culture of splenic samples and histologic analysis of the injection site, regional lymph nodes, salivary gland, spleen, liver, mesenteric lymph node, and gastrointestinal tract. No differences in survival, fungal burden, or dissemination were observed between fungal strains, routes of inoculation, or host immune status. Fungal infection was observed after subcutaneous inoculation only, was localized to the inoculation site, and was identified in both nu/nu and nu/+ mice. Fungal strain variability was not associated with virulence in a murine model of infection, and this novel mouse model of V. botryosa phaeohyphomycosis recapitulates the human clinical condition.

Effects of temperature on Veronaea botryosa infections in white sturgeon Acipenser transmontanus and fungal induced cytotoxicity of fish cell lines.

Veronaea botryosa is a melanized mold and cause of systemic fungal infections in cultured sturgeon (Acipenser spp.). Mortality in adult female sturgeon caused by this emergent pathogen results in significant economic losses for the caviar industry. Little is known regarding environmental conditions conducive to V. botryosa infection. This study evaluated the effect of temperature on V. botryosa infectivity and dissemination following intramuscular injection challenge of white sturgeon maintained at 13 or 18 °C for 40 days. Daily mortality was recorded and persistence of the fungus in the livers of moribund and surviving fish was investigated using culture and histopathological analysis. Fish maintained at 18 °C developed systemic phaeohyphomycosis and had significantly greater mortality than controls and fish maintained at 13 °C (p < 0.05). Challenged fish, regardless of temperature, exhibited lesions in multiple organs. However, muscle lesions, angioinvasion, and systemic dissemination were more severe and widespread in fish challenged at the higher temperature. In vitro cytotoxicity of V. botryosa was evaluated in white sturgeon skin (WSSK-1) and epithelioma papulosum cyprini (EPC) cell lines inoculated at spore:cell ratios of 1:10, 1:1 and 10:1, then incubated 15, 20 and 25 °C. Cytotoxicity, as indicated by quantifying the release of lactate dehydrogenase into culture supernatants, increased with increasing spore dose and incubation temperature in both fish cell lines. Findings suggest that temperature significantly influences the development of systemic V. botryosa infection in white sturgeon and that WSSK-1 and EPC cells are suitable in vitro models for the study of host-pathogen interactions between V. botryosa and fish epithelial cells.