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.

INTRAVENOUS IOPAMIDOL PHARMACOKINETICS IN COMMON CARP (CYPRINUS CARPIO).

Koi carp (Cyprinus carpio), a variety of common carp, has gained popularity as an ornamental fish worldwide. Their high monetary and sentimental value has necessitated the development of antemortem diagnostic options. Contrast-enhanced computed tomography (CT) scanning with intravenous iopamidol has been shown to be safe and diagnostically effective at a minimum dose of 480 mg iodine (I)/kg in koi. The purpose of this study was to evaluate the pharmacokinetic parameters of this dose of iopamidol, as well as excretory mechanisms specific to fish, using common carp as a model. Blood, posterior kidney, gill, and bile were collected, necessitating sacrificial sampling. Thirty-five adult fish were randomly divided into six sampling groups. Five sampling groups (n = 6/group) received 480 mg I/kg; the control group (n = 5) received an equivalent volume of saline. The iopamidol groups were sampled at the following time points postinjection: 5 min, 1 hr, 6 hr, 24 hr, and 48 hr. The control group was sampled at 48 hr. Concentrations of iopamidol were determined using liquid chromatography tandem mass spectrometry; noncompartmental analysis was used to calculate pharmacokinetic parameters. Total clearance (3.04 ml/hr per kilogram) was slower, the volume of distribution smaller (79.92 ml/ kg), and the elimination half-life (20.39 hr) prolonged compared to similar studies in mammals. The time-concentration profiles of kidney and gill were similar; these organs appear to be responsible for the majority of iopamidol excretion. However, that of bile was much different, showing slower, low-level accumulation with time, suggesting that in fish, multiple organ systems play a role in elimination beyond just the kidney. In particular, they may rely more heavily upon biliary excretion, which thus far has been noted only in mammals with renal impairment. Further research is warranted to investigate if the slower elimination allows diagnostic CT images to be acquired at different time points postinjection.

Validation of environmental DNA sampling for determination of Ceratonova shasta (Cnidaria: Myxozoa) distribution in Plumas National Forest, CA.

Ceratonova shasta is the etiological agent of myxozoan-associated enteronecrosis in North American salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and a freshwater fabriciid annelid. The success and survival of annelids can be enhanced by flow moderation by dams, and through the erosion of fine sediments into stream channels following wildfires. In this study, the presence of C. shasta environmental/ex-host DNA (eDNA) in river water and substrate samples collected from areas affected by recent fire activity in California, USA, was investigated. Additionally, DNA loads in the environment were compared to C. shasta infection in sentinel-exposed rainbow trout (Oncorhynchus mykiss). Significant associations between C. shasta detection in environmental samples and location within a wildfire perimeter (p = 0.002), between C. shasta detection in sentinel fish and exposure location within a wildfire perimeter (p = 0.015), and between C. shasta detection in fish and locations where water temperature was above the median (p < 0.001) were observed. Additionally, a higher prevalence of C. shasta infection in fish was detected where C. shasta was also detected in environmental samples (p < 0.001). Results suggest that pathogen eDNA sampling can be used as a non-invasive, rapid, specific, and sensitive method for establishing risk of C. shasta infection in wild populations. Knowledge of the complete life cycle of the target parasite, including ecology of each host, can inform the choice of eDNA sampling strategy. Environmental DNA sampling also revealed a novel species of Ceratonova, not yet observed in a host.

Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites.

OUTBREAK AND TREATMENT OF CARP EDEMA VIRUS IN KOI ( CYPRINUS CARPIO) FROM NORTHERN CALIFORNIA.

Carp edema virus (CEV) is the causative agent of carp edema virus disease (CEVD), also referred to as koi sleepy disease, which is an emerging disease of global concern that may cause high rates of morbidity and mortality in common carp and ornamental koi ( Cyprinus carpio). This article reports the third confirmed outbreak of CEVD in California. In June 2015, three koi presented with clinical signs of cutaneous lesions, severe lethargy, and signs of hypoxia. All fish tested positive for CEV by polymerase chain reaction (PCR). Euthanasia and complete necropsy were performed on two fish. The most significant necropsy findings included necrotizing branchitis with marked interstitial edema, multifocal cutaneous ulcerations, and severe cutaneous edema. Treatment of the pond with 0.3-0.5% salt was recommended to the owner. Approximately 7 wk later, a recheck visit was made to the pond. No mortalities had been noted since the initiation of the salt treatment. Physical examination revealed a vast improvement but not complete elimination of the clinical signs of hypoxia and intermittent lethargy in the affected fish. Gill biopsy samples from the two most affected fish were tested and remained PCR positive for CEV. Subsequent recheck visits over 11 mo postdiagnosis and initiation of treatment showed continued improvement in most fish. Gill samples from all fish in the pond ( n = 9) were repeatedly tested by quantitative PCR for CEV, and all samples were negative. This case series further confirms the global spread of CEV and the need for practitioners to be vigilant for outbreaks of this disease. If CEVD is suspected, treatment with 0.3-0.5% salt can be recommended to potentially mitigate the effects of this disease. However, fish may remain potential carriers of this pathogen, and strict biosecurity measures should continue to be enforced for any pond that has had a confirmed CEV outbreak.

Co-infection of Acipenserid herpesvirus 2 (AciHV-2) and Streptococcus iniae in cultured white sturgeon Acipenser transmontanus.

A mortality event in cultured white sturgeon Acipenser transmontanus (Richardson, 1836) sub-adults was investigated. After transfer between farms, high mortality was observed in fish, associated with back arching, abnormal swimming, and ulcerative skin lesions. Necropsy of moribund individuals revealed hemorrhagic ascites and petechial hemorrhages in the coelomic peritoneum and serosa of internal organs. Acipenserid herpesvirus 2 (AciHV-2) was isolated from external tissue samples, then identified and genotyped by sequencing of the terminase and polymerase genes. In addition, Streptococcus iniae was recovered from internal organs of affected fish. Histologic changes were limited to interstitial hematopoietic areas of the kidney and consisted of small foci of necrosis accompanied by fibrin deposition, minimal inflammatory response, and small numbers of bacterial cocci compatible with streptococci. Identity was confirmed by partial sequencing of the 16S rRNA, rpoB, and gyrB genes. Genetic fingerprinting demonstrated a genetic profile distinct from S. iniae isolates recovered from previous outbreaks in wild and cultured fish in North America, South America, and the Caribbean. Although the isolates were resistant to white sturgeon complement in serum killing assays, in vivo challenges failed to fulfill Koch's postulates. However, the clinical presentation, coupled with consistent recovery of S. iniae and AciHV-2 from moribund fish, suggests viral and bacterial co-infection were the proximate cause of death. To our knowledge, this represents the first report of AciHV-2 and S. iniae co-infection in cultured white sturgeon.

Diversity of Veronaea botryosa from different hosts and evaluation of laboratory challenge models for phaeohyphomycosis in Acipenser transmontanus.

Veronaea botryosa has been identified as a pathogen of cultured white sturgeon Acipenser transmontanus. In 2015, samples from 19 white sturgeon were received for diagnosis, of which 14 cultured positive for V. botryosa. Intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions was investigated using repetitive extragenic palindromic PCR fingerprinting (rep-PCR). The rep-PCR profiles of 16 V. botryosa isolates from a human, sea turtles, and cultured fish were distinct from those of other phaeoid fungi belonging to the genera Cladophialophora and Exophiala. To gain a better understanding of the pathogenesis of V. botryosa mycosis, 5 laboratory challenge methods were evaluated in white sturgeon fingerlings. Intramuscular (IM) and intracoelomic (IC) injection challenges produced cumulative mortalities of 13.3% (8/60) and 3.3% (2/60), respectively, and V. botryosa was recovered from 100% (10/10) of dead fingerlings. Affected fish exhibited abnormal orientation and/or failure to maintain neutral buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerated skin. After 6 wk, surviving fish were euthanized, and samples of liver were taken for mycological evaluation. Viable fungus was detected in 90% and 100% of fish surviving IM and IC challenge, respectively. No V. botryosa-associated mortality was detected in other groups challenged by immersion, immersion with abrasion, or orally. Both IM and IC challenge routes appear suitable for the induction of V. botryosa infection in white sturgeon and can serve as models for the study of disease pathogenesis associated with this emergent pathogen.

Expression of immune markers of white sturgeon (Acipenser transmontanus) during Veronaea botryosa infection at different temperatures.

Systemic phaeohyphomycosis caused by Veronaea botryosa is one of the most important emergent diseases to affect sturgeon aquaculture in North America. White sturgeon (Acipenser transmontanus) cultured at temperatures above 15 °C are at higher risk of severe disseminated disease and higher mortalities. Despite this, little is known regarding disease pathogenesis and the immune response to infection. The objective of this study was to investigate the acute (2 days post-challenge [dpc]) and chronic (32 dpc) response of white sturgeon at 13 °C and 18 °C challenged with V. botryosa via intramuscular injection, using gene expression analysis of a diverse array of soluble immune and inflammatory mediators. Significantly greater amounts of irf8 (p < 0.05) and tfg-ß (p < 0.05) genes were detected in gills of exposed fish at 18 °C when compared to those at 13 °C 32 dpc. Transcript levels of haptoglobin, serotransferrin, serum amyloid, cathelicidin, tnf-α, and il-17 were significantly increased in splenic tissues of challenged fish maintained at 18 °C late in infection (p < 0.05). However, only haptoglobin and serotransferrin transcript abundance were significantly greater in exposed fish when compared to controls 32dpc. Moreover, haptoglobin transcripts at this time point were significantly greater in exposed fish at 18 °C when compared to those challenged at 13 °C. Fewer differences were detected in fish kept at 13 °C. In agreement with transcript quantification, western blot assessment of haptoglobin showed increased levels in the challenged fish maintained at 18 °C.

Prevalence and phenotypic characterization of Salmonella enterica isolates from three species of wild marine turtles in Grenada, West Indies.

BACKGROUND AND AIM: Salmonella enterica causes enteric disease in mammals and may potentially be transmitted from marine turtles that shed the pathogen in the environment. Marine turtle-associated human salmonellosis is a potential public health concern in Grenada, as the island supports populations of leatherback turtles (Dermochelys coriacea), hawksbill turtles (Eretmochelys imbricata), and green turtles (Chelonia mydas) that interface with veterinarians and conservation workers, the local population, and the thousands of visitors that frequent the island yearly. To date, the prevalence of S. enterica has only been examined in a small subset of marine turtles in the Caribbean and no studies have been conducted in Grenada. The aim of this study was to quantify the prevalence of S. enterica in leatherback, hawksbill and green turtles in Grenada, characterize phenotypes and DNA profiles, and explore the potential risk to human health in the region. MATERIALS AND METHODS: A total of 102 cloacal swabs were obtained from nesting leatherback turtles and foraging hawksbill and green turtles. Samples were cultured on enrichment and selective media and isolates were phenotypically characterized using serotyping, pulsed-phase gel electrophoresis, and antibiotic susceptibility. Enrichment broths were additionally screened by polymerase chain reaction (PCR) using S. enterica-specific primers. RESULTS: S. enterica was cultured from 15/57 (26.3%) leatherback turtles, 0/28 hawksbill, and 0/17 green turtles. This included S. enterica serovars Montevideo, S. I:4,5,12:i:-, Salmonella Typhimurium, Salmonella Newport, S. I:6,7:-:-, and S. I:4,5,12:-:-. Five/15 leatherback turtles carried multiple serovars. Eight pulsotype groups were identified with multiple clustering; however, there was no clear association between pulsotype group and serotype profile. Five/71 isolates showed resistance to streptomycin or ampicillin. Twenty-one/57 leatherback turtles, 14/28 hawksbill turtles, and 8/17 green turtles tested positive for S. enterica by quantitative PCR. CONCLUSION: Nesting leatherback turtles actively shed S. enterica and poses a risk for zoonosis; however, the presence of viable pathogen in green and hawksbill species is unclear. These findings help elucidate the role of marine turtles as potential sources of zoonotic S. enterica and provide baseline data for one health research in Grenada and the wider Caribbean region.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.