PhD Lisa Reidy

PharmD Guillaume Desoubeaux

PhD Julia Cardenas

BA Joshua Seither

PhD Kristin Kahl

MS David Chauvin

PhD Michael Adkesson

DVM, Dipl ACZM, Dipl ECZM (ZHM Pamela Govett

DVM Cynthia Stadler

DVM Dipl Aczm

DVM Maryanne Tocidlowski

BS Gayle Sirpenski

med vet, Dipl ACZM Ellen Bronson

PhD Carolyn Cray
email: carolyncrayccray@miami.edu

Desoubeaux, Cardenas, Seither Reidy

Kahl

DETECTION OF GLIOTOXIN IN PLASMA FROM BIRDS WITH CONFIRMED AND PROBABLE ASPERGILLOSIS

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Invasive infection by Aspergillus species, primarily Aspergillus fumigatus, remains a major cause of morbidity and mortality in immunocompromised humans, especially after bone marrow transplantation or cancer chemotherapy. [START_REF] Bassetti | Performance of existing definitions and tests for the diagnosis of invasive aspergillosis in critically ill, adult patients: A systematic review with qualitative evidence synthesis[END_REF] In veterinary medicine, aspergillosis is responsible for high mortality across all avian species. [START_REF] Converse | Infectious diseases of wild birds[END_REF] Similar to human medicine, antemortem diagnostic options in avian species are limited and include routine hematology, assessment of inflammation, serological testing, culture, and imaging. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF][START_REF] Savelieff | The current status of avian aspergillosis diagnoses: Veterinary practice to novel research avenues[END_REF] As many avian species show antifungal antibody reactivity in the absence of infection, the appearance of an acute phase response using plasma protein electrophoresis in tandem with clinical signalment, and imaging, is often used to aid in diagnosis. [START_REF] Cray | Galactomannan assay and protein electrophoresis findings in psittacine birds with aspergillosis[END_REF][START_REF] Cray | Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species[END_REF][START_REF] Cray | Application of galactomannan analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species[END_REF][START_REF] Desoubeaux | Application of 3hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of aspergillosis in African penguins (Spheniscus demersus)[END_REF] Over the last several years, many investigators have sought to study and implement novel test options, which have led to the development of antigen-based testing for galactomannan and beta-D-glucan for application in both human and veterinary medicine. [START_REF] Bassetti | Performance of existing definitions and tests for the diagnosis of invasive aspergillosis in critically ill, adult patients: A systematic review with qualitative evidence synthesis[END_REF][START_REF] Burco | Evaluation of plasma (1-->3) beta-D-glucan concentrations in birds naturally and experimentally infected with aspergillus fumigatus[END_REF][START_REF] Cray | Galactomannan assay and protein electrophoresis findings in psittacine birds with aspergillosis[END_REF] So far, these biomarkers were not shown to be reliable for the diagnosis of aspergillosis in birds.

Alternatively, other researchers have proposed that metabolite-based diagnostics, including gliotoxin (GT) detection, may be viable markers of aspergillosis. [START_REF] Cerqueira | Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis[END_REF][START_REF] Lewis | Detection of gliotoxin in experimental and human aspergillosis[END_REF][START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] GT is the most abundant mycotoxin produced by clinical isolates of A. fumigatus, although it can also be found in other Aspergillus species as well as other fungal genera considered non-pathogens such as Penicillium, Trichoderma, and Leptosphaera. [START_REF] Kupfahl | Gliotoxin production by clinical and environmental Aspergillus fumigatus strains[END_REF][START_REF] Lewis | Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center[END_REF][START_REF] Patron | Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes[END_REF] It has been identified as a key virulence factor that results in far-reaching immune suppression. [START_REF] Arias | Preparations for Invasion: Modulation of Host Lung Immunity During Pulmonary Aspergillosis by Gliotoxin and Other Fungal Secondary Metabolites[END_REF][START_REF] Kwon-Chung | What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus?[END_REF] Studies have demonstrated that GT inhibits phagocytosis, blocks inflammatory processes and cytokine production, and induces apoptosis in macrophages and monocytes. [START_REF] Kwon-Chung | What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus?[END_REF] Using in vivo models, survival time of immune suppressed mice was extended when A. fumigatus was used where gliotoxin was genetically deleted. [START_REF] Kwon-Chung | What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus?[END_REF] Several studies utilizing samples obtained from mammal models and banked human sera have indicated that GT detection has a strong potential as a diagnostic test for aspergillosis. [START_REF] Cerqueira | Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis[END_REF][START_REF] Lewis | Detection of gliotoxin in experimental and human aspergillosis[END_REF][START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] In addition to GT, a secondary metabolite bis(methyl)gliotoxin (bmGT) has also been detected and was reported to have better sensitivity than GT. [START_REF] Domingo | Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis[END_REF][START_REF] Vidal-Garcia | Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis[END_REF][START_REF] Vidal-Garcia | Production of the Invasive Aspergillosis Biomarker Bis(methylthio)gliotoxin Within the Genus Aspergillus: In Vitro and in Vivo Metabolite Quantification and Genomic Analysis[END_REF] In the present study, the technical parameters for detecting GT and bmGT in avian plasma through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methodology have been presented. Although GT has already been measured in tissues from turkeys and chickens naturally infected with A. fumigatus, [START_REF] De Oca | Aspergillosis, a Natural Infection in Poultry: Mycological and Molecular Characterization and Determination of Gliotoxin in Aspergillus fumigatus Isolates[END_REF][START_REF] Richard | Natural occurrence of gliotoxin in turkeys infected with Aspergillus fumigatus, Fresenius[END_REF] this is the first report of GT detection in the plasma of various avian species with confirmed and probable aspergillosis.

MATERIALS AND METHODS

Ethics

The rat study was reviewed and approved by the Institutional Animal Care and Use

Rat Model

Male Sprague-Dawley rats (n=20), Janvier Labs, Le Genest-Saint-Isle, France), 6-8 weeks old, 200-225 g, were acclimated in animal facilities eight days before the beginning of the protocol. On day 0,all animals were immunocompromised intraperitoneally with 75 mg/kg cyclophosphamide (Baxter, Guyancourt, France), and their food was changed to a low-protein diet (Safe Diets, Augy, France). To avoid opportunistic infection and limit pain, 500mg/L tetracycline (Sigma-Aldrich, Saint Quentin Fallavier, France) and 300 mg/L paracetamol (Sanofi-Aventis, Montrouge, France) were added to their drinking water. A second cyclophosphamide administration of 60 mg/kg was given on days 4, 8 and 12 to maintain immunosuppression. According to the previously published protocol, [START_REF] Chandenier | The utility of a nebulised intra-tracheal rat model of invasive pulmonary aspergillosis[END_REF][START_REF] Desoubeaux | A nebulized intra-tracheal rat model of invasive pulmonary aspergillosis[END_REF] animals were challenged intratracheally at day 5 by aerosolization of 100 µL of a PBS suspension containing 1.0x10 [START_REF] Chandenier | The utility of a nebulised intra-tracheal rat model of invasive pulmonary aspergillosis[END_REF] A. fumigatus conidia with Microsprayer IA-1B® aerosolizer (n=11, PennCentury, Philadelphia, PA, USA) or with PBS alone as a mock-infected control group (n=9). Rats were then monitored daily and euthanasia was performed before the end of the protocol if the following criteria were met: loss of weight ≥20%, discomfort score 3 (scored from 1 to 6 based on appearance changes, e.g., dirty nose, red-rimmed eyes, ruffled fur, extreme pallor; score 1, no discomfort; score 2, minor discomfort; score 3, poor discomfort; score 4, serious discomfort; score 5, severe discomfort; score 6, death), behavior changes (e.g., gasping, wheezing, prostration, instability) and decreased reaction to stimuli. These criteria were met between day 8 and 15. At necropsy, blood and lungs were collected for subsequent analysis. To verify experimental aspergillosis development, qPCR, targeting the A. fumigatus 28S ribosomal DNA 28S, was carried out in lung tissues as previously described. [START_REF] Chauvin | Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model[END_REF] Serum was analyzed for galactomannan presence using the Platelia Ag kit (Bio-Rad, Marnes-la-Coquette, France) and also banked at -80 o C for analysis of GT and bmGT.

Avian Samples

Heparinized plasma samples were obtained from routine health surveillance programs, and diagnostic investigations from birds with suspected aspergillosis were analyzed in addition to samples obtained as part of the AZA SSP program (see Acknowledgements). In some cases, repeated measures were available. Many of the samples had been stored at -80 o C for extended periods. The samples (n=119) from clinically normal penguins (n=51 individual birds) were obtained from a single facility: they were in normal condition based on physical examination and bloodwork and did not show any clinical evidence of aspergillosis. Twenty-five of these samples were obtained from penguins that did not show any clinical evidence of aspergillosis at least 6 months after sampling. Ninety-four of the samples were taken over a recent 3-month period and the penguins also remained clinically normal during this period. Two clinically abnormal groups were defined and included confirmedthose with histopathological confirmation after necropsy or consistent radiography and culture resultsand probablethose with consistent clinical signs, supporting testing including radiography, and a positive response to anti-fungal treatment.

Species represented in the confirmed sample group (n=35) ranged from 2 months to 13 years of age and included the African penguin (n=8, Spheniscus demersus), Humboldt penguin (n=6, Spheniscus humboldti), gentoo penguin (n=4, Pygoscelis papua), California condor (n=4, Gymnogyps californianus), whooping crane (n = 1, Grus americana), little blue penguin (n=2, Eudyptula minor), grey-winged trumpeter (n=2, Psophia crepitans), Magellanic penguin (n=3, Spheniscus magellanicus), helmeted curassow (n=4, Pauxi pauxi), and wreathed hornbill (n=1, Rhyticeros undulatus). Note that 17/35 samples represented repeated measures over several months in 5 cases. Those in the probable sample group (n=28) ranged from 8 months to 13 years of age and included the African penguin (n=14), Humboldt penguin (n=9), little blue penguin (n=1), Magellanic penguin (n=1), gyrfalcon (n=1, Falco rusticolus), pigeon (n=1, Columba livia domestica), and African grey parrot (n = 1, Psittacus erithacus). Note that, overall, 7/28 samples represented repeated measures over several weeks to months in 3 cases.

Chemicals and Materials

All solvents and reagents were purchased at HPLC grade unless noted. Ammonium acetate, ammonium formate, ammonium hydroxide, American Chemical Society (ACS) grade formic acid, glacial acetic acid, ACS grade diethyl ether, ACS grade ethyl acetate, LC-MS grade water, LC-MS grade acetonitrile, and methanol were purchased from VWR (Randor, PA, United States). Certified human negative serum was purchased from Bio-Rad (Hercules, CA, United States). Drug standards, which included gliotoxin and bis(methylation) gliotoxin, were purchased from Millipore Sigma (Burlington, MA, United States). Internal standards (IS), including mycophenolic acid-D4 and TFMPP-D3, were purchased from Cerilliant Corporation (Round Rock, TX, United States).

Sample Preparation

All stock solutions, negative controls, and specimens were allowed to equilibrate to room temperature for at least 30 minutes. After equilibration, a 100 µL aliquot of the specimen was fortified with 10 µL of the working internal standard solution for a final internal standard concentration of 100 ng/mL. Next, 1.2 mL of diethyl ether: ethyl acetate (50:50 v/v) was added to each tube and vortexed for approximately 30 seconds. Specimens were centrifuged at 2851 g (RCF max) for 10 min, and once complete, the supernatant was transferred to a clean tube. The eluent was then evaporated at 25°C with N 2 and reconstituted with 50 µL of the 50:50 A: B (v/v) mobile phase composition. Mycophenolic acid-D3 and TFMPP-D4 were used as internal standards.

Instrumental Analysis

An Agilent Technologies 1260 liquid chromatogram coupled to a 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, United States) equipped with a Waters XBridge BEH Shield RP18 analytical column (130Å, 5 µm, 2.1x150mm) and Waters XBridge BEH C18 Sentry guard cartridge (130Å, 5 µm, 2.1 x 10mm) (Waters Corporation, Milford, MA, United States) was utilized for chromatographic separation and identification of the gliotoxin and its metabolite. The analytical column was maintained at 40°C in the temperature-controlled column compartment. The mobile phase consisted of 5 mM ammonium formate with 0.1 % formic acid in LC-MS grade water (A) and LC-MS grade acetonitrile with 0.1% formic acid (B). The mobile phase gradient was programmed as follows: 95% A was held for 1 minute, 10 min, then decreased to 10% A over 9 minutes, with a return to 95% A at t=11 min. A 2-min post-run time was used to re-equilibrate the column before the subsequent injection. The injection volume was set at 5 L.

Mass spectral data were acquired using an Agilent Jet Stream electrospray ionization (ESI) source operated in positive ion mode. The ESI source and MS parameters are as follows: gas temp of 320°C, flow 8 L/min, nebulizer 27 psi, sheath gas heater 380°C and flow 12 L/min, the capillary voltage at 3,750 V, and the nozzle voltage 500. The instrument was operated in dynamic multiple reaction monitoring (dMRM) acquisition mode with the unit resolution was utilized for all analytes and transitions. Initially, the Agilent Optimizer software was used to identify product ions and their optimal fragmentor voltage and collision energy to determine which transitions should be used for each target analyte and internal standard. Two transitions were monitored for each analyte and internal standard. MRM transitions are shown for each analyte and internal standard in Table 1.

Each analyte minimum identification criteria included: retention time within 3% of the positive controls, a Gaussian chromatographic, peak shape, signal-to-noise greater than 3:1, and qualifier transition ratios within acceptable ranges. Figure 1 depicts a chromatogram with gliotoxin, retention time 6.7, and the IS, Retention Time 5.6 mins with the specified transitions.

Method validation parameters

Limit of Detection

The approximate LOD of the analytes was determined using a 0.5 ng/µL stock solution to fortify negative penguin sera at the following concentrations: 1, 3, 5, 10, and 15 ng/mL. Fortified specimens were prepared using six different negative penguin sera specimens over three runs (n=12). The analytes had to meet the previously stated retention time, peak shape, and qualifier ion ratio criteria in all 12 replicates to be considered detected. Additionally, the signalto-noise for both transitions had to be greater than 3:1 as calculated by the Agilent MassHunter® software using the General integrator setting.

Linearity

Calibrators were prepared in certified negative human serum to span the calibration range, analyzed over five separate batches, and statistically analyzed to establish the calibration range. In addition, multi-level positive controls prepared in species-matched specimens were analyzed alongside the calibration curve to verify accuracy in a specified matrix and the bias and precision of the analytical method.

Interferences Studies

To ensure that the target analyte itself only produces a positive identification for the target analyte, interference studies were performed. In each experiment, interference was determined to be present if a signal met the identification criteria, which included: retention time, signal-to-noise greater than 3:1, peak shape, and qualifier ion transition ratios.

To investigate if any potential interference from endogenous compounds in the matrix, six (6) sources of rat negative matrices and fifteen (15) sources of penguins were analyzed without the addition of the IS. All negative samples were pre-screened and had no history of illness or infection with Aspergillus fumigatus.

The stability and purity of the deuterated internal standards were also assessed. To verify the deuterated internal standards' purity, three different negative sera specimens were fortified with the internal standard solution at the same concentration as sample analysis to determine if any interference was observed.

Ionization Suppression/Enhancement

Suppression or enhancement of the analyte signal can occur when using LC-electrospray mass spectrometry (LC-ESI-MS). This can be caused by co-eluting compounds that can originate from the matrix itself or the extraction. Ion suppression or enhancement typically affects method parameters such as the limit of detection in qualitative assays.

To assess ionization suppression and enhancement, the average peak areas from 6 replicates of neat standards (set A) were compared with the average peak areas from ten fortified specimens (set B). The following formula was used to assess ionization suppression and enhancement:

Ionization suppression or enhancement (%) = ea rea of et ea rea of et x 100% Eq 1.

Recovery was also investigated using pre-and post-fortified samples from different penguin sera.

RESULTS

Method Validation

Limit of Detection (LOD) and linearity

Analyzing the results of the LOD study, GT and bmGT were established to have a LOD at 3 ng/mL. The appropriate calibration model was determined through statistical analysis using standardized residual plots and ANOVA to be a linear model with a weighting factor of 1/x for concentrations spanning 10-1000 ng/mL for GT and bmGT. The limit of detection (LOD) was determined as 3 ng/mL by evaluating peak shape and using the standard signal to noise (S/N) ratio of 10 and acceptable mass spectrometric criteria. This was established by utilizing six sources of rat negative matrices and fifteen sources of penguin negative matrices that were fortified with GT and bmGT. The analytical method was able to identify both compounds in all matrix specimens. The lower limit of quantitation (LLOQ) was determined through repeated measurements at 10 ng/ml for both compounds in three different matrices, duplicate, and three different batches (n=9). Chromatographic and mass spectrometric interference was not observed in the endogenous, exogenous, and internal standard studies, and no carryover was detected with a serum fortified at 500 ng/ml into the subsequent sample. There was, however, significant variation in the signal of the internal standard observed in the different penguin samples indicative of possible matrix effects and/or differences in extraction efficiencies. The observed variation appeared to affect the accuracy and precision of the calculated concentrations; therefore, it was determined that only qualitative results would be reported using this method with this internal standard.

Ionization Suppression/Enhancement (ISE) and recovery

In qualitative assays, ionization suppression or enhancement can significantly affect the LOD for an analyte. Suppression and enhancement studies using fortified penguin matrices showed an acceptable % ISE of less than 20, and recovery of GT and bmGT were established as 90%.

Rat Samples

Nine samples from the mock-infected control rats were analyzed. All had a serum galactomannan index of <0.14 (where 0.5 or greater is considered positive). Ribosomal Aspergillus DNA levels were considered insignificant. All serum samples were found to contain no interference for GT or bmGT.

Eleven infected rat samples were analyzed. In the serum, the galactomannan index ranged from 3.2 to 8.0. Infection was further confirmed by quantitation of ribosomal Aspergillus DNA levels obtained from the lung; these ranged from 4092 to 176,583 fg/µL. Serum samples from all 11 infected rats had detectable levels of GT but not bmGT.

Avian Samples

One hundred and nineteen samples were obtained from clinically normal penguins were analyzed. bmGT was not detected in any samples. GT was detected in one sample. While this penguin showed no clinical signs, during this assessment period, the electrophoresis showed abnormalities including increased globulins and there was also evidence of seroconversion by ELISA. In the absence of any treatment, the GT was not detected in any follow up samples, and the electrophoretogram returned to normal.

In birds with probable infection, 17 of 28 or 60.7% of samples had detectable GT and, in birds with confirmed infection, 30 of 35 or 85.7% of samples had detected GT (Table 2). bmGT was not detected in any of the samples.

Repeated measures were available for some of cases examined during the study (Table 3). In cases of confirmed infection, GT was consistently detected in the samples available for analysis. In the single confirmed case that was successfully treated (case 5), GT was detected on all repeated measures obtained when clinical signs were present and in one sample when the bird appeared clinically normal. In a probable case of infection (case 2), GT was not detected after 3 weeks of treatment; systemic inflammation was supported by the presence of an abnormal electrophoretogram, but clinical signs were no longer observed.

DISCUSSION

Serodiagnostic tests for avian aspergillosis are broadly limited to the same options found in human medicine. [START_REF] Savelieff | The current status of avian aspergillosis diagnoses: Veterinary practice to novel research avenues[END_REF] Antibody testing is problematic as many species of birds have been shown to have high reactivity on an ELISA based on a crude antigen preparation even when clinically normal. [START_REF] Cray | Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species[END_REF] Detection methods of circulating antigen, including galactomannan [START_REF] Cray | Galactomannan assay and protein electrophoresis findings in psittacine birds with aspergillosis[END_REF] and beta-Dglucan, [START_REF] Burco | Evaluation of plasma (1-->3) beta-D-glucan concentrations in birds naturally and experimentally infected with aspergillus fumigatus[END_REF] have been validated but have limited sensitivity for diagnosis. Currently used methods include detecting an ongoing acute-phase response via plasma protein electrophoresis. [START_REF] Cray | Galactomannan assay and protein electrophoresis findings in psittacine birds with aspergillosis[END_REF][START_REF] Desoubeaux | Application of 3hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of aspergillosis in African penguins (Spheniscus demersus)[END_REF] In a search for a more definitive marker of infection in humans, many investigators have focused on the detection of metabolites of fungal infections, [START_REF] Savelieff | Novel cutting-edge metabolite-based diagnostic tools for aspergillosis[END_REF] including GT, which has been identified as a major virulence factor of A. fumigatus, and its metabolite, bmGT. [START_REF] Arias | Preparations for Invasion: Modulation of Host Lung Immunity During Pulmonary Aspergillosis by Gliotoxin and Other Fungal Secondary Metabolites[END_REF][START_REF] Cerqueira | Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis[END_REF][START_REF] Domingo | Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis[END_REF][START_REF] Lewis | Detection of gliotoxin in experimental and human aspergillosis[END_REF][START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF][START_REF] Vidal-Garcia | Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis[END_REF][START_REF] Vidal-Garcia | Production of the Invasive Aspergillosis Biomarker Bis(methylthio)gliotoxin Within the Genus Aspergillus: In Vitro and in Vivo Metabolite Quantification and Genomic Analysis[END_REF] In the current investigation, preliminary studies were undertaken to validate an HPLC-MS/MS method using serum samples from an acute rat model of invasive aspergillosis. This model was selected as it has been well characterized and reproducible. [START_REF] Chandenier | The utility of a nebulised intra-tracheal rat model of invasive pulmonary aspergillosis[END_REF][START_REF] Desoubeaux | Proteomic demonstration of the recurrent presence of inter-alphainhibitor H4 heavy-chain during aspergillosis induced in an animal model[END_REF] This data corroborated previous reports using mouse models where GT was readily detected, which validated the implementation methodology. [START_REF] Lewis | Detection of gliotoxin in experimental and human aspergillosis[END_REF][START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] Notably, in the present study, GT was not detected in samples from mock-infected control rats thus supportive of excellent assay specificity.

Importantly, GT was not readily detected in a large cohort of clinically normal penguins, an avian species which has been shown to have a high prevalence of aspergillosis when in captive care. [START_REF] Beernaert | Aspergillus infections in birds: a review[END_REF][START_REF] Converse | Infectious diseases of wild birds[END_REF][START_REF] German | Development of an indirect ELISA for the detection of serum antibodies to Aspergillus fumigatus in captive penguins[END_REF] Although limited in sample size, the current preliminary study has detected GT in cases with confirmed or probable infection. These results are consistent with those previously reported in studies utilizing human and rat samples. [START_REF] Cerqueira | Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis[END_REF][START_REF] Lewis | Detection of gliotoxin in experimental and human aspergillosis[END_REF][START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] While the present study did not rule out infections with other fungal species, it should be noted that GT can also be produced by genera that are generally regarded as non-pathogens. [START_REF] Patron | Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes[END_REF] The sensitivity for detecting avian aspergillosis by GT has been difficult to assess, although the detection rate was higher in confirmed rather than probable cases. It has been proposed that GT would be a sensitive marker of early infection as it is produced during hyphal replication. [START_REF] Waring | The chemistry and biology of the immunomodulating 485 agent gliotoxin and related epipolythiodioxopiperazines[END_REF] Analysis of archival plasma samples pre-, during, and post-diagnosis presents challenges due to variability in the timing of collected samples that are available. Additionally, definitive diagnosis via culture or molecular diagnostics is difficult antemortem, particularly in chronic cases, and may only be confirmed by necropsy where often only histopathological analysis is completed. Cross-sectional imaging (computed tomography) and positive response to treatment are often used clinically to guide treatment and monitor advanced disease cases without a definitive diagnosis. Classification of advanced or chronic cases can be problematic as these birds may have a less severe disease (thus a positive outcome) or are either not suffering from aspergillosis or are infected with a non-or low gliotoxin-producing isolate of Aspergillus.

Thus, the scope of the present study should be considered preliminary. First, although the samples considered to be control samples (n=119) were obtained from penguins that remained in good health post sampling, there were no detailed investigations to rule out the presence of colonization with Aspergillus or aspergillosis. Second, confirmed cases were limited by histopathology reports only that were obtained from various laboratories, often in the absence of culture, special staining, and molecular confirmation. Lastly, as in many retrospective studies, the use of archived samples with variable storage methods is often suspect although, notably, stability studies of GT conducted in human sera indicated that refrigeration, freeze-thaw, and month-long freezing did not affect results. [START_REF] Cerqueira | Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis[END_REF] Ongoing collaborations with zoological institutions and aquaria are focused on advantageous screening of apparently clinically normal penguins to gauge whether this assay may aid in routine screening protocols. It is important to examine necropsy results in future studies where aspergillosis has been confirmed in the absence of gliotoxin detection. Also, while A. fumigatus is frequently observed to be the pathogen in avian aspergillosis cases; current study collaborators are urged to perform cultures and/or molecular diagnostics at necropsy with the goal of identifying the species of fungus. Previously, others have reported GT was detected in most A. fumigatus isolates but to a moderate level in A. niger and a lower number of isolates of A. terreus and A. flavus. [START_REF] Kupfahl | Gliotoxin production by clinical and environmental Aspergillus fumigatus strains[END_REF][START_REF] Lewis | Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center[END_REF] While possible false-negative results may be related to the timing of sampling vs. the course of the disease, it is recognized that many isolates of non-A. fumigatus species produced lower GT levels, and some clinical isolates of A. fumigatus in probable cases did not produce GT. [START_REF] Kupfahl | Gliotoxin production by clinical and environmental Aspergillus fumigatus strains[END_REF] In addition, it has been proposed that penguins are colonized by Aspergillus sp. So, it may be argued that the presence of GT may not necessarily reflect clinical infection. [START_REF] Reidardson | Diagnosis and treatment of aspergillosis in temperate penguins[END_REF] It could also be proposed that GT would only be detected in cases where clinical signs are evident and fungal replication is active. GT levels may also decrease with the presence of advanced disease stage where hyphal expansion is slowed. [START_REF] Vidal-Garcia | Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis[END_REF] In the present study of clinically normal African penguins, only 1 of 119 samples had detectable GT. While this penguin showed no clinical signs, other tests supported the presence of inflammation and activation of humoral immunity which appeared to resolve in follow up samples. Additional studies with focus on health assessments of clinically normal birds should be conducted to best assess the clinical applications of GT testing.

GT was detected in non-penguin species also. This supports the hypothesis that GT may be used in all avian species, but additional studies should be undertaken to validate these methods in a wider range of species. Repeated measures were available for some of the probable and confirmed cases. In 3 confirmed cases that resulted in death, GT was consistently detected.

In one confirmed case with apparent successful treatment, GT was detected at a time with improved clinical signs, so treatment was continued. Although more such cases need to be examined, this suggests that reassessment by GT testing may provide additional prognostic information to the clinician.

Other investigators demonstrated a GT metabolite, bis(methylthio)gliotoxin (bmGT), in human samples. It has been proposed that GT may become incorporated in tissues given its biological activities in apoptosis and cellular damage and that bmGT, which is non-toxic, would be a preferred marker. [START_REF] Domingo | Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis[END_REF] However, it should be noted that bmGT was not commonly detected in non-A. fumigatus isolates. [START_REF] Vidal-Garcia | Production of the Invasive Aspergillosis Biomarker Bis(methylthio)gliotoxin Within the Genus Aspergillus: In Vitro and in Vivo Metabolite Quantification and Genomic Analysis[END_REF] In human patients, the combination of GT and bmGT resulted in excellent predictive value, [START_REF] Vidal-Garcia | Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis[END_REF] although others reported bmGT and GT to be unreliable markers. [START_REF] Mercier | Gliotoxin and bis(methylthio)gliotoxin are not reliable as biomarkers of invasive aspergillosis[END_REF] While these differences may be related to the implementation of the LC-MS methods, it may also be a function of the variation in the stages of infection within the cohort of human samples examined in the studies. In the present study, bmGT was not detected in rat or avian blood samples. The lack of detection in rats is consistent with that observed in a mouse model where bmGT was detected in the lungs but not the sera from samples obtained from mouse models of aspergillosis. [START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] It was proposed that this may reflect a difference related to using an acute rodent model rather than a more prolonged natural infection often observed in humans. [START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] It could be argued that this same proposal should support the detection of bmGT in naturally infected birds where chronic infection is often observed, but it was not the case herein. The lack of detection in rats and avian species may reflect differences in biological processing of metabolites versus humans. [START_REF] Sugui | Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice[END_REF] Additional studies should be undertaken with repeated measures from infected birds or using a model of aspergillosis in a bird species to best assess the application of bmGT detection.

In the present study, penguin plasma matrices were variable which did not allow for accurate GT quantitation and may have also affected bmGT detection. This was also observed in samples from other avian species. Alternative internal standards were investigated but all showed similar variability with these matrices (data not shown). Penguins and other birds with aspergillosis have been shown to have a significant inflammatory response in conjunction with a decrease in albumin [START_REF] Desoubeaux | Application of 3hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of aspergillosis in African penguins (Spheniscus demersus)[END_REF] ; perhaps this altered plasma composition contributes to the issue of inaccurate quantitation and lack of bmGT detection. Although all criteria were acceptable in order to provide qualitative GT test results with the established 3 ng/mL LOD across multiple sources of penguin and rat matrix, there is a need for the production of a deuterated GT standard to be able to compensate for the differences observed in the penguin matrix. As decreasing GT levels during treatment may prove to be a useful prognostic marker, this is a focus of continuing studies. Extended clinical use of GT testing in birds may be limited by the cost of analysis, sample volume requirements, and turnaround time. A recent publication reported developing a fluorescent-based assay with a superior minimum detection level, which may help make GT detection more readily available for clinical applications in human and veterinary medicine. [START_REF] Gao | Development of a Fluorescently Labeled Aptamer Structure-Switching Assay for Sensitive and Rapid Detection of Gliotoxin[END_REF] The data from this preliminary investigation support the further study of the application of Aspergillus metabolite detection in avian species. While this may lead to the development of a more definitive antemortem diagnostic test for birds inclusive of many endangered species maintained under managed care, it may also promote the bird as a novel model of aspergillosis, [START_REF] Tell | Aspergillosis in mammals and birds: impact on veterinary medicine[END_REF] which could provide a further understanding of the pathogenesis of this disease.

ACKNOWLEDGMENTS:

The authors thank the African penguin Species Survival Plan (SSP) as part of the Association of Zoos & Aquariums Saving, Animals from Extinction (SAFE) conservation initiative. We also thank all the institutions that shared samples for this study including the Fort Worth Zoo, the Dallas Zoo, and the Freeport-McMoRan Audubon Species Survival Center.

  Committee at Tours University (France) as No. C37-261-3 and by the General Direction for Research and Innovation, French Ministry of Higher Education and Research through the accreditation number 01901.01. For the avian study, samples were submitted either as part of a routine or planned diagnostic analyses to the Avian & Wildlife Laboratory (University of Miami, Miami, FL, United States) or as part of SSP study protocol requesting banked samples from different AZA participating institutions. As part of the latter process, consent was received from participating institutions. No institutional approval (at the University of Miami) was required.

Figure 1 .

 1 Figure 1. Representative chromatogram of gliotoxin and internal standards (IS). The chromatogram shows the elution order and

Table 1 .

 1 MRM transitions for analytes and internal standards

	Compound Name	RT (min)	Precursor Ion (m/z)	Fragmentor (V)	Product Ion 1 (m/z)	CE 1 (V)	Product Ion 2 (m/z)	CE 2 (V)
	Gliotoxin		327.1	76	263.1	6	56.1	50
	bis(methylthio)gliotoxin		357.0	74	309.1	2	243.0	6
	TFMPP D4		235.1	125	190	21	46.1	21
	Mycophenolic acid D3		341.1	80	210	16
	490

Table 2 .

 2 Summary of Samples Analyzed for the presence of GT.

	Case Type	Detected	Not Detected	Percentage Positive
	Clinically Normal	1	119	0.8%
	Probable Infection	17	28	60.7%
	Confirmed Infection	30	35	85.7%

Table 3 .

 3 Detection of gliotoxin in probable and confirmed cases of aspergillosis with repeated assessments. change in voice noted, leukocytosis, CT scan confirmed diffuse air sacculitis with inspissated contents and pulmonary nodules, placed on itraconazole as anti-fungal treatment Not detected Clinically normal although inflammatory pattern of protein electrophoresis continued to be present, continued treatment and follow up imaging normal by

	Case Species	Day of	Gliotoxin Detection	Comments
			Sampling
	1	Humboldt		Detected	1 mo. weight loss, anorexia, leukocytosis, supportive imaging results, placed on
		penguin			itraconazole as anti-fungal treatment
		(confirmed)
				Detected	No change
				Detected	Improved appetite, decreased leukocytosis
				Not Done	Minor respiratory difficulty but appetite, attitude, and respirations were normal
				Not Done	Animal died, necropsy confirmed chronic granulomatous air sacculitis and
					multiple pulmonary nodules confirmed with intralesional fungal hyphae
	2	Humboldt penguin (probable)		Detected	Few-day day 50
	3	California		Detected	Initial respiratory signs noted (cough), placed on antibiotics
		condor
		(confirmed)
				Detected	Weight loss, increase coughing
				Not Done	CT scan showed partial collapse of right abdominal air sac, FNA nondiagnostic
				Detected	Distended air sacs, increased coughing, death day 45, confirmed infection at
					necropsy
	4	African		Detected	Leukocytosis, weight loss, lethargy, weight loss, suggestive imaging, placed on
		penguin			anti-fungal medication
		(confirmed)
				Not detected	Continued clinical signs
				Detected	Continued clinical signs, changed anti-fungal meds
				Detected	Found dead, confirmed infection at necropsy
	5	Helmeted		Detected	Respiratory signs, radiographs abnormalities present including likely granuloma,


Guillaume Desoubeaux
email: guillaume.desoubeaux@univ-tours.fr

Anne Debourgogne

Nathan P Wiederhold

Marie Zaffino

Deanna Sutton

Rachel E Burns

Salvatore Frasca

Michael W Hyatt

Carolyn Cray

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

Keywords: MLST, genotyping, Sphyrnidae sharks, outbreak, Metarhizium robertsii

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Fusariosis refers to opportunistic infections due to molds belonging to the genus Fusarium, that is often considered saprobic due to its common occurrence in soil, but also contains waterassociated lineages. [START_REF] Hoog Gs De | Atlas of Clinical Fungi[END_REF] The taxonomy of the Fusarium genera is very complex, and recently new species names have been proposed. [START_REF] Lombard | Generic concepts in Nectriaceae[END_REF] The Fusarium solani species complex (FSSC) is a phylogenetic and biological group that is currently estimated to contain at least 60 distinct -but very closely related -Fusarium species, including some cryptic and unnamed species. [START_REF] Schroers | Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex[END_REF] The members of this species complex are unified by phenotypic characters such as asexual morphs with long monophialides associated with uniloculate perithecial ascomata that are usually yellow, orange-red to brown. On the basis of molecular phylogenetic relationships, the FSSC is comprised of three major clades. FSSC clade 3 includes isolates that are pathogens of plants and animals, [START_REF] Nelson | Taxonomy, biology, and clinical aspects of Fusarium species[END_REF][START_REF] Van Diepeningen | Spectrum of Fusarium infections in tropical dermatology evidenced by multilocus sequencing typing diagnostics[END_REF][START_REF] Zhang | Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment[END_REF] and all were formerly characterized as Nectria haematococca because of a common unique sexual teleomorphic stage and their ability to reproduce with other members of the same species. [START_REF] Coleman | The Fusarium solani species complex: ubiquitous pathogens of agricultural importance[END_REF] Members of FSSC clade 3 are responsible in humans for either localized infection, especially after trauma, [START_REF] Chang | Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution[END_REF] or systemic disease with multiple internal organ involvement, as seen in immunocompromised patients. [START_REF] Nucci | Fusarium infections in immunocompromised patients[END_REF][START_REF] Lortholary | International retrospective analysis of 73 cases of invasive fusariosis treated with voriconazole[END_REF] Molecular analysis has demonstrated that the two most clinically-common species of clade 3 are F. falciforme (FSSC 3 and 4) and F. keratoplasticum (FSSC 2). [START_REF] Al-Hatmi | Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging group of human opportunists from 1958 to 2015[END_REF] In vertebrate animals, fusariosis has been described in several marine species, including sea turtles, whales, dolphins and seals [Violetta GC. A case history of Fusarium sp. in a captive population of bonnethead sharks (Syhyrna tiburo). Int. Assoc. Aquatic Anim. Med., Gulfport, 1984:64; Davis MR. Successful resolution of "bonnethead shark disease", presumptive Fusarium infection, with antifungal therapy and environmental manipulation. Int. Assoc. Aquatic Anim.

Med., Mystic, 2007: 128]. [START_REF] Abdo | Disseminated mycosis in a killer whale (Orcinus orca)[END_REF][START_REF] Crow | Fusarium solani fungal infection of the lateral line canal system in captive scalloped hammerhead sharks (Sphyrna lewini) in Hawaii[END_REF][START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Frasca | Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals[END_REF][START_REF] Garner | A retrospective study of disease in elasmobranchs[END_REF][START_REF] Naples | Successful treatment of a severe case of fusariomycosis in a beluga whale (Delphinapterus leucas leucas)[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF][START_REF] Sarmiento-Ramírez | Global distribution of two fungal pathogens threatening endangered sea turtles[END_REF][START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF][START_REF] Tuxbury | Fusarium solani species complex associated with carapace lesions and branchitis in captive American horseshoe crabs Limulus polyphemus[END_REF][START_REF] Yanong | Fungal diseases of fish[END_REF] In elasmobranchs e.g. sharks, pathologic findings during fusariosis can include dermatitis, with ulcers and hemorrhages with lesions containing white-tohemorrhagic, purulent exudates. They are primarily located within the lateral line canals, along the body, and within the cephalic canals of the cephalofoil, and can easily disseminate to other anatomical sites. [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF][START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF] Perichondritis and chondritis of the cartilaginous vertebrae, as well as chronic myositis, have been occasionally described. [START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF] Overall mortality rates are greater than 90% [Davis MR. Successful resolution of "bonnethead shark disease", presumptive Fusarium infection, with antifungal therapy and environmental manipulation. Int. Assoc. Aquatic Anim.

Med., Mystic, 2007: 128]. [START_REF] Crow | Fusarium solani fungal infection of the lateral line canal system in captive scalloped hammerhead sharks (Sphyrna lewini) in Hawaii[END_REF][START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF][START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF] To date, cases of Fusarium infections in elasmobranchs are rarely reported, as the prevalence was estimated below 0.6% in a cohort of 1,546 individuals versus 15.0% and 8.9% for bacterial and parasitic infections, respectively. [START_REF] Garner | A retrospective study of disease in elasmobranchs[END_REF] However, conventional phenotypic methods may lead to possible fungal misidentifications, and although DNA sequencing is now recognized as the most reliable method for accurate strain identification, it was not routinely practiced before the last decade, and currently it is still commonplace that veterinary samples are submitted to human laboratories for that purpose. [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF] Little is known about the transmission of Fusarium, particularly in aquatic environments, which emphasises the need for better understanding of the epidemiology of Fusarium infection.

In human medicine, several molecular tools have been developed to study the epidemiology of transmissible agents, [START_REF] Choukri | Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia[END_REF][START_REF] Gal | A cluster of Pneumocystis infections among renal transplant recipients: molecular evidence of colonized patients as potential infectious sources of Pneumocystis jirovecii[END_REF] such as multi-locus sequence typing (MLST) analysis, microsatellite genotyping, and polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP analysis. These techniques are useful in tracing the epidemiology of microbiological strains in the context of outbreaks. [START_REF] Maitte | Multilocus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used?[END_REF][START_REF] Gal | AIDS-related Pneumocystis jirovecii genotypes in French Guiana[END_REF][START_REF] Monroy-Vaca | Prevalence and genotype distribution of Pneumocystis jirovecii in Cuban infants and toddlers with whooping cough[END_REF][START_REF] Desoubeaux | Epidemiological outbreaks of Pneumocystis jirovecii pneumonia are not limited to kidney transplant recipients: genotyping confirms common source of transmission in a liver transplantation unit[END_REF] Multi-locus sequence typing enables the detection of nucleotide mutations (mostly single nucleotide polymorphisms, so called SNPs) within several specifically-chosen loci that are usually located within conserved housekeeping genes. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF] It increases the discriminatory power compared to single gene sequencing. [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF] Unlike microsatellite genotyping and PCR-RFLP, MLST is highly reproducible and facilitates the comparison of data from different laboratories. [START_REF] Maitte | Multilocus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used?[END_REF] In this study, we used MLST genotyping to evaluate isolates from eight cases of FSSC infection for possible epidemiological links. They occurred over a four-year period in Sphyrnidae sharks, i.e. five bonnethead (Sphyrna tiburo) and three scalloped hammerhead sharks (Sphyrna lewini), within a public aquarium..

MATERIALS AND METHODS

Study population

Context of the study

In addition to over 8,500 aquatic species within different habitats comprising 7,600,000 million litres (L) of water, Adventure Aquarium (Camden, NJ, U.S.A.) is home to the largest collection of sharks on the East Coast of the United States of America (U.S.A.), including rare scalloped hammerhead sharks which are listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). [START_REF]Convention on International Trade in Endangered Species of Wild Fauna and Flora. Appendice II[END_REF] In the present study, five bonnethead and three scalloped hammerhead sharks were diagnosed with Fusarium, or Fusarium-like, infections during a four-year period (04/22/2012 -11/03/2016) within a 93,860 L quarantine system (Q1) and the 2,900,000 L "Ocean Realm" (OR) exhibit. Both systems were maintained at 23 °C. The life support systems (LSS) of both Q1 and OR consisted of sand filtration, protein fractionation and ozonation.

Clustering of cases was suggested as four bonnethead shark infections occurred between February 2012 to March 2013, all within Q1, and two scalloped hammerhead shark cases were diagnosed concomitantly in February 2016, but separated in Q1 and OR. Two other cases were separately observed six and eight months after the two aforementioned onsets, respectively.

Cases definition

The diagnosis of fungal infection was confirmed by either cytological or histopathological examination of dermal lesions and internal tissues. [START_REF] Guarner | Histopathologic diagnosis of fungal infections in the 21st century[END_REF][START_REF] Anthony | A colour atlas and textbook of the histopathology of mycotic diseases[END_REF] Definitive fungal identification of Fusarium species was achieved by in vitro mycological cultures obtained from lesions and molecular sequencing as described below. [START_REF] Nelson | Taxonomy, biology, and clinical aspects of Fusarium species[END_REF][START_REF] O'donnell | Internet-accessible DNA sequence database for identifying Fusaria from human and animal infections[END_REF]

Microbiological samples

Each fungal strain, isolated from the eight diseased sharks and referred to as A -H, were subcultured onto BBL ® Sabouraud-dextrose agar supplemented with gentamicin and chloramphenicol (Becton-Dickinson, Le Pont-de-Claix, France). DNA was extracted from each by using the QIAmp ® DNA Mini kit (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions. Isolates were confirmed as belonging to the FSSC by DNA sequencing of the translation elongation factor 1-alpha (TEF1) region, as described previously. [START_REF] O'donnell | Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex[END_REF] For some isolates, additional targets were used for this purpose, including the internaltranscribed spacer region (ITS) and D1/D2 domains of the large subunit of the nuclear ribosomal RNA gene, i.e. portions of 5.8S and 28S rRNA, (ITS-nuLSU) and the RNA polymerase II second largest subunit B150 gene (RPB2). DNA extracts were stored at -20°C until subsequent analysis.

Genotyping of Fusarium solani species complex

In order to detect a clonal outbreak, molecular genotyping was performed according to the previously-reported MLST technique for the FSSC. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF] PCR mixes were prepared in a 50 μL reaction volume containing 25 μL of TaqPurple ® (Ozyme, Montigny-le-Bretonneux, France), 5 μL of each aforementioned fungal DNA extracts, and 0.5 μL of each forward and reverse oligonucleotide primer at 20 µM. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF][START_REF] Debourgogne | Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method[END_REF] Fungal DNA was amplified at five loci corresponding to the following housekeeping genes: acetylcoenzyme A carboxylase (ACC), isocitrate lyase (ICL), glyceraldehyde-3P dehydrogenase (GPD), mannitol-1P dehydrogenase (MPD), and manganese superoxide dismutase (SOD). The PCR assays were performed in a iCycler IQ Thermal ® cycler apparatus (Bio-Rad, Hercules, CA, U.S.A.), under the following conditions: an initial 2 min denaturation step at 95°C, followed by 35 cycles of hybridization/elongation, including 30 sec at 95°C, 30 sec at 55 °C, and 60 sec at 72°C, with a final extension step of 5 min at 72°C, before the unlimited cooling step at 4°C. PCR products were sequenced on both strands using the aforementioned amplification primers by the Genoscreen Company (Lille, France).

The one-letter code for nucleotides from the International Union of Pure and Applied Chemistry (IUPAC) nomenclature was used for data analyses. 36 Forward and reverse DNA sequence chromatograms were analyzed with the BioEdit ® software (Ibis Biosciences, Carlsbard, CA, U.S.A.). Consensus DNA sequences at the five aforementioned loci were registered in GenBank ® (NCBI, Bethesda, MA, U.S.A.) and compared to each other for every strain in order to determine polymorphisms and sequence type (ST). Unique numbers were assigned to allelic variants for each housekeeping gene, according to the reported method. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF][START_REF] Debourgogne | Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method[END_REF] These numbers were then combined to yield a five-digit ST genotype. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF]

Phylogenetic relationships

Phylogenetic relationships between the eight fungal strains were established by MEGA ® v6.06 software (The Biodesign Institute, Tempe, AZ, U.S.A.) according to the maximum parsimony method, using 1,000 bootstrap resampling and 50% as the cutoff value for generation of the consensus tree. The reference sequence of Fusarium staphyleae NRRL 22316 was used as the outgroup (GenBank ® accession number: JX171496.1), [START_REF] O'donnell | Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex[END_REF] and sequences of 50 epidemiologicallyunrelated FSSC isolates deposited at the Westerdijk Fungal Biodiversity Institute (formerly known as Centraalbureau voor Schimmelcultures-Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands) were downloaded from GenBank ® database to serve as controls (Suppl. Material 1). [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF][START_REF] Debourgogne | Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method[END_REF]

Statistical analysis

XLStat ® v.2016.6.04 software (Addinsoft, Paris, France) for Windows ® Microsoft, Issy-les-Moulineaux, France was used for statistical analysis. The -risk was adjusted at 0.05.

Ethics

No animal was anesthetized, euthanized, or sacrificed for the specific purposes of this study. The biological samples used herein were the result of the routine veterinary care of these sharks.

Adventure Aquarium is accredited by the Association of Zoos and Aquariums (AZA) and is held to the absolute highest standards in animal care and exhibition.

RESULTS

Study population and case confirmation

Clinical, pathological and mycological findings of the eight diseased Sphyrnidae sharks are summarized in Table 1. Gross findings at post-mortem examination included epidermal erosions, ulcers, haemorrhages, and vesicles containing white-to-hemorrhagic exudate, located on the dorsal and ventral aspects of the cephalofoil, progressing from the cutaneous, lateral line and cephalic canals, and ampullae of Lorenzini, to disseminated involvement (Figure 1A-C).

Cytological findings from skin exudate obtained from one female bonnethead shark provided evidence of fungal infection. Histopathological findings from tissue samples of the cephalofoil from the other seven sharks were very similar to one another, and consisted of severe, multifocal and focally extensive-to-regional, chronic, necrotizing dermatitis and cellulitis with ulceration of the skin, haemorrhage, and intra-lesional filamentous fungal hyphae. The inflammatory infiltrate in areas of dermatitis and cellulitis was dominated by macrophages and, in some cases, was noted to include degenerate and non-degenerate granulocytes, as well as a few multinucleate giant cells. Fungal hyphae extended into ampullae of Lorenzini, invaded blood vessels with and without vasculitis, and extended into nerves in some instances. Hyphae were 2-4 µm wide in histologic section, and appeared hyaline with parallel walls, septa, and right angle-branching (Figure 2). All eight cases were confirmed by positive mycological cultures of the lesions showing Fusarium-like features (Figure 3). Molecular identification confirmed that seven of the eight fungal isolates belonged to FSSC clade 3, further classified with three to FSSC 2 species (Fusarium keratoplasticum) and four to FSSC 9 (Fusarium haplotype 9x). Interestingly, strain C was identified as Metarhizium robertsii, although the clinical and pathologic features in the infected shark were similar to those of the other bonnetheads (Figure 1D-E). All the sharks died between one and 37 days after the onset of clinical signs, except for one scalloped hammerhead shark that is alive to date while still undergoing treatment.

Fusarium solani species complex genotyping and phylogenetic relationship

Results of MLST genotyping for each strain are reported in Table 2, except for strain C, which was excluded as it did not belong to FSSC. All isolates were successfully sequenced in the regions of the five housekeeping genes (accession numbers: KY780124 -58; Suppl. Material 1-2). No locus haplotypes were identical in all the strains, indicating that each of the five genes was useful in allowing identification of distinct genotypes. Few polymorphisms were detected with only five distinct FSSC ST identified. Unique ST1, ST4 and ST5 genotypes were found for FSSC strains D, E and H, respectively, while the same MLST sequence type ST2 was observed for strains B and F which displayed 99.8% sequence similarity; A and G strains both exhibited ST3 genotype, with 99.9% sequence similarity. Phylogenetic analysis indicated that the sequences of the 50 control FSSC clade 3 strains downloaded from the Westerdijk Institute were heterogeneously dispersed, confirming that these isolates were epidemiologically-unrelated, whereas Fusarium strains in sharks referred to as A and G were tightly clustered, supported by 95% bootstrap-value, confirming that they were not independent (Figure 4). In the same way, B and F isolates were also tightly clustered.

Transmission map

The first five cases of fusariosis or Metarhizium infection due to [A -E] strains were diagnosed in bonnethead sharks that all arrived at the same time to the aquarium (Table 1). Except for the shark infected with FSSC strain E, which was diagnosed in OR exhibit and was previously housed in Q1, all other FSSC strains were diagnosed while sharks were managed in Q1.

There was no contact between the three scalloped hammerhead sharks infected with FSSC strains [F -H] and the five bonnethead sharks, as they arrived at the aquarium three years later after the bonnethead infections and subsequent mortalities. Except for the shark with FSSC isolate F, which was diagnosed while it was still managed in Q1, the scalloped hammerhead sharks were diagnosed while in OR, after moving from Q1. The OR exhibit had also been occupied six months before by the bonnethead shark infected with the strain E, but this strain was genetically distinct from all the others.

DISCUSSION

Despite a lack of published literature, there is an increasing incidence of fusariosis in animals, especially in sharks of the genus Sphyrna. [START_REF] Crow | Fusarium solani fungal infection of the lateral line canal system in captive scalloped hammerhead sharks (Sphyrna lewini) in Hawaii[END_REF][START_REF] Garner | A retrospective study of disease in elasmobranchs[END_REF][START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF] [START_REF] Garner | A retrospective study of disease in elasmobranchs[END_REF][START_REF] Smith | Fatal Fusarium solani infections in baby sharks[END_REF][START_REF] O'donnell | Veterinary fusarioses within the United States[END_REF] and 15 in scalloped hammerhead sharks have been reported whether isolated or grouped within six clinical clusters, [START_REF] Crow | Fusarium solani fungal infection of the lateral line canal system in captive scalloped hammerhead sharks (Sphyrna lewini) in Hawaii[END_REF][START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF] including the five bonnethead and three hammerhead cases from the present study. Herein, F. solani haplotype 9x (FSSC 9) and F. keratoplasticum (FSSC 2) were most commonly isolated. In light of the previously published works, [START_REF] Al-Hatmi | Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging group of human opportunists from 1958 to 2015[END_REF] the frequent occurrence of F. keratoplasticum was expected, but that of F. solani haplotype 9x was not. Our findings in sharks may simply reflect the specific presence of these two species in this environment. Risk factors for developing fusariosis in aquarium settings are not well known, but appear to be multifactorial, including water temperature, exhibit size and design, substrate and décor, and co-exhibiting animal interactions leading to increased susceptibility to opportunistic infections. [START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF] It is noteworthy that our work is the first report of Metarhizium robertsii inciting infection in sharks. M. robertsii belongs to M. anisopliae complex which has been described as pathogen causing sclerokeratitis, [START_REF] Amiel | Atypical fungal sclerokeratitis[END_REF][START_REF] Eguchi | Severe fungal sclerokeratitis caused by Metarhizium anisopliae: a case report and literature review[END_REF][START_REF] Dorin | First unusual case of keratitis in Europe due to the rare fungus Metarhizium anisopliae[END_REF] and cutaneous infection in humans. [START_REF] Marsh | Cutaneous infection with Metarhizium anisopliae in a patient with hypohidrotic ectodermal dysplasia and immune deficiency[END_REF] A recent study evidenced that among the 11 human cases caused by M. anisopliae complex species, six were actually due to M. robertsii and none to M. anisopliae stricto sensu. [START_REF] Nourrisson | Species of Metarhizium anisopliae complex implicated in human infections: retrospective sequencing study[END_REF] Previously, molecular analysis of Fusarium strains isolated from elasmobranchs has been performed in two recent studies and consisted of ITS and D1/D2 (ITS-nuLSU gene) sequence analyses. [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF][START_REF] Pirarat | Molecular and pathological characterization of Fusarium solani species complex infection in the head and lateral line system of Sphyrna lewini[END_REF] The current study describes a different MLST analysis for the genotyping of the fungal strains isolated over a four-year period from the eight Sphyrna sharks studied herein. As shown in other infectious diseases, [START_REF] Gal | A cluster of Pneumocystis infections among renal transplant recipients: molecular evidence of colonized patients as potential infectious sources of Pneumocystis jirovecii[END_REF][START_REF] Maitte | Multilocus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used?[END_REF][START_REF] Desoubeaux | Epidemiological outbreaks of Pneumocystis jirovecii pneumonia are not limited to kidney transplant recipients: genotyping confirms common source of transmission in a liver transplantation unit[END_REF] MLST is considered to be the standard by which to investigate outbreaks. [START_REF] Van Diepeningen | Spectrum of Fusarium infections in tropical dermatology evidenced by multilocus sequencing typing diagnostics[END_REF] Recently, Debourgogne et al. demonstrated that a simple cost-efficient MLST scheme relying on sequencing fungal DNA at five loci (ACC, ICL, GDP, MPD and SOD) provided sufficient discriminatory power to reliably differentiate FSSC isolates and, thereafter, for epidemiological investigations of clustered cases. [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF] This method is not intended to support current taxonomic classification, but in comparison to sequencing of the ITS-nuLSU alone, the five-locus MLST approach allowed complete analysis of 1,616 nucleotides instead of 430; [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF] hence, its typing efficiency, i.e. ability to discriminate the highest number of isolates, was estimated at 0.48 versus 0.30. [START_REF] Debourgogne | Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method[END_REF] While ITS-nuLSU sequencing is probably sufficient for phylogenetic species recognition in other genera,, [START_REF] O'donnell | Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex[END_REF] it is too conserved to obtain a reliable species-level identification within Fusarium genus, and may not be suitable to distinguish similar strains, and to accurately address epidemiological links of clustered cases caused by a unique phylogenetic species. For example, sequencing the ITS-nuLSU region was not able to distinguish between strains A, G and B (100% similar sequences; data not shown). Likewise, it was previously shown that the MLST method is characterized by a higher discriminatory power than the reference three-locus typing scheme (0.991 versus 0.980, measured with Simpson's index of diversity, calculated according to Hunter and Gaston's modification), [START_REF] Hunter | Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity[END_REF] which means that several FSSC isolates which shared the same sequence through the three-locus scheme were correctly differentiated by the five-locus scheme. [START_REF] Debourgogne | Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method[END_REF] Using this method, the current study reliably demonstrated that two of the FSSC strains, isolates B and F, were genetically different from the other strains but were similar to each other (whereas at TEF1 region, sequences of isolates B and F were similar to strain G; data not shown), although they were isolated from different diseased sharks 1,269 days apart. On the contrary, such sharp distinction was not possible with the threelocus scheme which erroneously encompassed the strain D within the same genotype (data not shown). Because the DNA ST data for the five loci combined were sufficient to discriminate between 50 unrelated FSSC control isolates, this method could reliably characterize a single genotype as responsible for the infections in these two individuals. The presence of a common FSSC sequence type, which was found to be ST2 according to the previously reported five-digit MLST genotyping classification, [START_REF] Debourgogne | Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates[END_REF] in two sharks at different times suggested the persistence of this strain within the aquarium environment. As with other fungal genera, [START_REF] Warris | Recovery of filamentous fungi from water in a paediatric bone marrow transplantation unit[END_REF] Fusarium spp., including members of the FSSC, are well adapted to aquatic environments. For instance, 45.5% of the samples collected from coastal waters of the Mediterranean Sea yielded at least one Fusarium sp. [START_REF] Palmero | Species of Fusarium isolated from river and sea water of Southeastern Spain and pathogenicity on four plant species[END_REF] Similarly, water maintained at a constant mild-to-moderate 20-35C temperature, [START_REF] Kauffmann-Lacroix | Prevention of fungal infections related to the water supply in French hospitals: proposal for standardization of methods[END_REF][START_REF] Sautour | Fusarium species recovered from the water distribution system of a French university hospital[END_REF] commonly found in aquaria, was shown to support fungal growth. Fusarium spp. were also reported to grow from 4 to 40C, [START_REF] Marín | Water activity, temperature, and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolates from maize[END_REF] and to persist nearly six years in water distribution systems. [START_REF] Anaissie | Fusariosis associated with pathogenic Fusarium species colonization of a hospital water system: a new paradigm for the epidemiology of opportunistic mold infections[END_REF] The bonnethead and the scalloped hammerhead sharks that were infected with the strains A et G, displaying a single FSSC ST3 genotype (i.e. a distinct sequence type than ST2 that was found for strains B and F), raised an additional question, since diagnosis of fungal infections in these two sharks was separated by 1,424 days and the sharks were never maintained together in the same pool. This finding may underline a possible persistent circulation of this FSSC strain within the environment or LSS of the aquarium, although the probability of a new introduction with the same strain, but from an external source, could not be ruled out. Fusarium spp. are able to produce conidia that can remain in suspension in water, whereas the mycelium, which is generally associated with organic particles, is usually eliminated by filtration. [START_REF] Sautour | Fusarium species recovered from the water distribution system of a French university hospital[END_REF] Thus, through suspended conidia, one might hypothesize that the FSSC ST3 strain could possibly enter the plumbing and LSS of the aquarium, form biofilms on pipe surfaces, and could secondarily colonize downstream, while subsequently exposing sharks at separate times. [START_REF] Sautour | Fusarium species recovered from the water distribution system of a French university hospital[END_REF] Unfortunately due to the retrospective nature of this study, water samples were not cultured within the aquarium, and therefore the accurate determination of an environmental source of infection by various FSSC strains was not possible.

Our results suggest that reducing the risk of fungal infections caused by environmental molds should be an emphasis in the care of aquatic animals. Unlike the clinical scenarios in human hospitals, [START_REF] Groll | Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation[END_REF][START_REF] Maertens | European guidelines for antifungal management in leukemia and hematopoietic stem cell transplant recipients: summary of the ECIL 3--2009 update[END_REF][START_REF] Cornely | Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia[END_REF][START_REF] Ullmann | Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease[END_REF] systematic prophylaxis with antifungal drugs is impractical for sharks because of logistics, cost, and the risks of promoting drug resistance. Rather, prevention should be based on managing water quality and temperature, and by regularly monitoring the biofilms within the plumbing, especially those with stagnant or unused sections which can be colonized by molds. [START_REF] Kauffmann-Lacroix | Prevention of fungal infections related to the water supply in French hospitals: proposal for standardization of methods[END_REF] Some authors anecdotally attempted to decrease the water temperature of the enclosure by 5-8°C. [START_REF] Naples | Successful treatment of a severe case of fusariomycosis in a beluga whale (Delphinapterus leucas leucas)[END_REF] Installation of small-mesh filters, i.e. ≤0.2 m diameter, at different points of the water pipes provides good protection against filamentous fungi, 54 but this would not be functional with water turnover rates within LSS in the aquarium. Further information regarding the threshold of contamination at which there is an actual risk of fungal infection is needed to better define a routine monitoring plan.

Fusarium spp. are associated with pathologic lesions, morbidity and mortality in some elasmobranch species. Through this study, the clinical application of MLST genotyping at five loci has been shown to be useful in tracing FSSC strains to better understand the epidemiological aspects of fungal disease in a clustering of cases in bonnethead and scalloped hammerhead sharks, more accurately than with molecular identification to phylogenetic species level only. Specialized veterinary laboratories may wish to implement MLST for other genera of fungi as a tool for epidemiologic studies. or lateral line suggests that these could have been portals of entry for the fungi. Skin lesions have also been postulated to spread infection from one individual to another, since sensory organs along the head are used to probe the environment and to contact other individuals. [START_REF] Fernando | Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review[END_REF] Similarly, involvement of the skin has been observed in marine mammals infected with Fusarium spp. [START_REF] Naples | Successful treatment of a severe case of fusariomycosis in a beluga whale (Delphinapterus leucas leucas)[END_REF][START_REF] O'donnell | Veterinary fusarioses within the United States[END_REF][START_REF] Montali | Cyclic dermatitis associated with Fusarium sp infection in pinnipeds[END_REF][START_REF] Frasca | Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals[END_REF] ; D -Dorsal lateral aspect of the body of the bonnethead shark infected with fungal strain C identified as Metarhizium robertsii; ventral view of the head of the bonnethead shark infected with fungal strain C identified as Metarhizium robertsii; E -Multiple, severe and extensive hemorrhagic and ulcerative lesions were present on the cephalofoil involving ampullae of Lorenzini and cephalic canals. These lesions were very similar to those incited by species of the Fusarium solani species complex in the other cases.

Figure 1 :

 1 Figure 1: Gross lesions in diseased Sphyrnidae sharks with confirmed fusariosis due to

Figure 2 :

 2 Figure 2: Histopathological photomicrographs of fungal hyphae within lesions of the

Figure 3 :

 3 Figure 3: Morphologic observations of the Fusarium solani species complex (FSSC) species

Figure 4 :

 4 Figure 4: Phylogenetic tree showing the relationships of the seven Fusarium solani species

  Since the late 1980's, 19 cases in bonnethead sharks [Violetta GC. A case history of Fusarium sp. in a captive population of bonnethead sharks

	(Syhyrna tiburo). Int. Assoc. Aquatic Anim. Med., Gulfport, 1984:64; Davis MR. Successful
	resolution of "bonnethead shark disease", presumptive Fusarium infection, with antifungal
	therapy and environmental manipulation. Int. Assoc. Aquatic Anim. Med., Mystic, 2007:
	128],

ACKNOWLEDGMENTS

The authors are grateful to Martha E. O'Dowd for her technical help in handling the mycological strains, and to Manon Dominique for her protocol describing the phylogenetic study.

FUNDING

This work was supported by internal laboratory funding.

DISCLOSURE OF CONFLICT OF INTEREST

None.