Epidemiological Cutoff Values for Standard Broth Microdilution Susceptibility Testing of Flavobacterium columnare Isolated from Fishes.

Flavobacterium columnare, the causative agent of columnaris disease in a large variety of freshwater fish, is a major problem in commercial aquaculture. A limited number of antimicrobial therapies are available to control this disease; therefore, these agents must be used judiciously. To facilitate effective monitoring for changes in susceptibility, the Clinical Laboratory Standards Institute (CLSI) has a standard broth microdilution test method specific for F. columnare. However, there are no CLSI-approved criteria (termed epidemiological cutoff values [ECVs]) to interpret results. Nevertheless, researchers have developed provisional ECVs based on testing by one laboratory. To satisfy CLSI data requirements, three laboratories used the standard method to generate additional antimicrobial susceptibility data against ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfadimethoxine/ormetoprim, and sulfamethoxazole-trimethoprim using 109 F. columnare isolates. The new data combined with previously published data from 120 F. columnare isolates were analyzed and ECVs proposed to CLSI. Of the 10 antimicrobials, ECVs were approved for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline, which were published in the 2020 edition of the CLSI document VET04 performance standards. These ECVs will help microbiologists categorize decreased antimicrobial susceptibility among F. columnare and will help in surveillance efforts to ensure judicious antimicrobial use.

Epidemiological Cutoff Values for Standard Broth Microdilution and Disk Diffusion Susceptibility Testing of Aeromonas hydrophila Isolated from Fish.

Aeromonas hydrophila and other closely related Aeromonas species cause motile aeromonad septicemia, a common fish disease. The disease affects many aquaculture sectors potentially requiring antimicrobial treatments. Therefore, researchers and laboratory diagnosticians need criteria called epidemiological cutoff values (ECVs) to determine whether a bacterial isolate has developed decreased susceptibility to an antimicrobial. To generate ECVs for this bacterium, we assembled a diverse collection of 245 isolates previously identified as A. hydrophila from fish. Using rpoD sequencing, we confirmed that 97 of the 245 isolates were A. hydrophila. We allocated the isolates among three laboratories and tested their susceptibility against eight antimicrobials using standard Clinical and Laboratory Standards Institute (CLSI) disk diffusion and broth microdilution methods. The resulting frequency distributions were statistically analyzed to determine wild-type cutoff estimates, which, along with scatterplots, were used to estimate potential ECVs. In collaboration with the CLSI, aquaculture working group, we proposed ECVs for six of the eight antimicrobials tested. Subsequently, the CLSI Subcommittee on Veterinary Antimicrobial Susceptibility Testing reviewed our data and approved the ECVs to be added to the 2020 edition of the VET04 performance standards for antimicrobial susceptibility testing of aquatic bacteria.

Analysis of enterotoxigenic Bacillus cereus strains from dried foods using whole genome sequencing, multi-locus sequence analysis and toxin gene prevalence and distribution using endpoint PCR analysis.

Bacillus cereus strains were isolated from dried foods, which included international brands of spices from South East Asia, Mexico and India purchased from several retail stores, samples of powdered infant formula (PIF), medicated fish feed and dietary supplements. The genetic diversity of 64 strains from spices and PIF was determined using a multiplex endpoint PCR assay designed to identify hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Thirteen different B. cereus toxigenic gene patterns or profiles were identified among the strains. Randomly selected B. cereus strains were sequenced and compared with reference Genomic Groups from National Center Biotechnology Information using bioinformatics tools. A comprehensive multi-loci sequence analysis (MLSA) was designed using alleles from 25 known MLST genes specifically tailored for use with whole genome assemblies. A cohort of representative genomes of strains from a few FDA regulated commodities like dry foods and medicated fish feed was used to demonstrate the utility of the 25-MLSA approach for rapid clustering and identification of Genome Groups. The analysis clustered the strains from medicated fish feed, dry foods, and dietary supplements into phylogenetically-related groups. 25-MLSA also pointed to a greater diversity of B. cereus strains from foods and feed than previously recognized. Our integrated approach of toxin gene PCR, and to our knowledge, whole genome sequencing (WGS) based sequence analysis, may be the first of its kind that demonstrates enterotoxigenic potential and genomic diversity in parallel.

Draft Whole-Genome Sequences of Chryseobacterium piscicola and Chryseobacterium shigense.

We report the draft whole-genome sequences for Chryseobacterium piscicola and Chryseobacterium shigense type strains, bacteria that have been associated with fish gill disease.

Application and evaluation of a high-resolution mass spectrometry screening method for veterinary drug residues in incurred fish and imported aquaculture samples.

The ability to detect chemical contaminants, including veterinary drug residues in animal products such as fish, is an important example of food safety analysis. In this paper, a liquid chromatography high-resolution mass spectrometry (LC-HRMS) screening method using a quadrupole-Orbitrap instrument was applied to the analysis of veterinary drug residues in incurred tissues from aquacultured channel catfish, rainbow trout, and Atlantic salmon and imported aquacultured products including European eel, yellow croaker, and tilapia. Compared to traditional MS methods, the use of HRMS with nontargeted data acquisition and exact mass measurement capability greatly increased the scope of compounds that could be monitored simultaneously. The fish samples were prepared for analysis using a simple efficient procedure that consisted of an acidic acetonitrile extraction followed by solid phase extraction cleanup. Two different HRMS acquisition programs were used to analyze the fish extracts. This method detected and identified veterinary drugs including quinolones, fluoroquinolones, avermectins, dyes, and aminopenicillins at residue levels in fish that had been dosed with those compounds. A metabolite of amoxicillin, amoxicillin diketone, was also found at high levels in catfish, trout, and salmon. The method was also used to characterize drug residues in imported fish. In addition to confirming findings of fluoroquinolone and sulfonamide residues that were found by traditional targeted MS methods, several new compounds including 2-amino mebendazole in eel and ofloxacin in croaker were detected and identified. Graphical Abstract Aquacultured samples are analyzed with a high-resolution mass spectrometry screening method to detect and identify unusual veterinary drug residues including ofloxacin in an imported fish.

Draft Whole-Genome Sequences of 18 Flavobacterium spp.

We report here the draft whole-genome sequences for 18 Flavobacterium species type strains that have historically been associated with fish gill disease.

Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

Depletion of melamine and cyanuric acid in serum from catfish Ictalurus punctatus and rainbow trout Onchorhynchus mykiss.

Melamine and its triazine analogs, such as cyanuric acid, have been used to artificially inflate protein content both in animal feed ingredients, as well as in milk products produced for human consumption. We report here a LC-MS/MS method to quantify and confirm melamine and cyanuric acid in serum from channel catfish and rainbow trout with a limit of quantification of 0.8 µg/mL. The method was applied to serum samples from a residue depletion study in which fish were given a single oral dose of 20 mg/kg body weight melamine, cyanuric acid, or both compounds together. Samples were taken at 1, 3, 7, 14, and 28 days (an additional 42 day was added for trout). When given alone or in combination with cyanuric acid, melamine residues were highest on day 1 in both catfish and trout. Cyanuric acid was only quantifiable at day 1 in trout when given alone, and not at all in catfish. The serum half life of melamine in catfish was 1.50-1.62 days and 3.09-3.67 days in trout. This work highlights the differences of depletion kinetics in fish, which can be measured in days, as compared to the depletion in mammals, measured in hours.

Dose-response assessment of nephrotoxicity from a twenty-eight-day combined-exposure to melamine and cyanuric acid in F344 rats.

The adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of cats and dogs in the USA and other countries. In a previous 7-day dietary study in F344 rats, we established a no-observed-adverse-effect level (NOAEL) for a co-exposure to melamine and cyanuric acid of 8.6 mg/kg bw/day of each compound, and a benchmark dose lower confidence limit (BMDL) of 8.4-10.9 mg/kg bw/day of each compound. To ascertain the role played by the duration of exposure, we treated F344 rats for 28 days. Groups of male and female rats were fed diet containing 0 (control), 30, 60, 120, 180, 240, or 360 ppm of both melamine and cyanuric acid. The lowest dose that produced histopathological alterations in the kidney was 120 ppm, versus 229 ppm in the 7-day study. Wet-mount analysis of kidney sections demonstrated the formation of melamine cyanurate spherulites in one male and two female rats at the 60 ppm dose and in one female rat at the 30 ppm dose, establishing a NOAEL of 2.1mg/kg bw/day for males and <2.6 mg/kg bw/day for females, and BMDL values as low as 1.6 mg/kg bw/day for both sexes. These data demonstrate that the length of exposure is an important component in the threshold of toxicity from a co-exposure to these compounds and suggest that the current risk assessments based on exposures to melamine alone may not reflect sufficiently the risk of a co-exposure to melamine and cyanuric acid.

Development of a fast screening and confirmatory method by liquid chromatography-quadrupole-time-of-flight mass spectrometry for glucuronide-conjugated methyltestosterone metabolite in tilapia.

This paper describes the development of a fast method to screen and confirm methyltestosterone 17-O-glucuronide (MT-glu) in tilapia bile. The method consists of solid-phase extraction (SPE) followed by high-performance liquid chromatography-mass spectrometry. The system used was an Agilent 6530 Q-TOF with an Agilent Jet stream electrospray ionization interface. The glucuronide detected in the bile was characterized as MT-glu by comparison with a chemically synthesized standard. MT-glu was detected in bile for up to 7 days after dosing. Semiquantification was done with matrix-matched calibration curves, because MT-glu showed signal suppression due to matrix effects. This method provides a suitable tool to monitor the illegal use of methyltestosterone in tilapia culture.

Marker residue determination of tritium-labeled ivermectin in the muscle of aquacultured largemouth bass, hybrid striped bass, and yellow perch following oral treatment.

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered ³H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 µCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of ³H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.

A No Observable Adverse Effects Level (NOAEL) for pigs fed melamine and cyanuric acid.

Ingesting melamine adulterated milk products led to kidney stones in many infants in 2008. This differs from the renal failure caused by intratubular crystal formation after co-ingestion of melamine (MEL) and cyanuric acid (CYA) in adulterated pet foods in 2007. To better understand the potential risk of developing crystal nephropathy following co-ingestion of MEL and CYA, we fed 16 weanling pigs 0, 1, 3.3, 10, 33, or 100 mg/kg bw/day of each MEL and CYA, or 200 mg/kg bw/day of either compound individually for 7 days. Crystals were found in the renal medulla and cortex and urine sediments of all pigs fed both MEL and CYA each at 10 mg/kg bw/day (or greater). Crystals were also found in one of the two pigs fed 200 mg/kg bw/day MEL-only. In a 28 day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kg bw/day of MEL and CYA or 200 mg/kg bw/day MEL-only. Only one of the 3.3 mg/kg MEL and CYA pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed MEL and CYA for 28 days was concluded to be 1.0 mg/kg bw/day corresponding to 25 mg/kg (ppm) MEL and 25 mg/kg (ppm) CYA in dry feed.

Bioaccumulation of melamine in catfish muscle following continuous, low-dose, oral administration.

In this study, catfish muscle was analyzed for melamine (MEL) and cyanuric acid (CYA) residues following experimental feeding with low doses of MEL and MEL and CYA (MEL+CYA) and with the insoluble melamine-cyanurate complex (MEL=CYA). Catfish were daily fed 0.1 mg/kg BW of MEL for 15, 28, or 42 days, 0.1 mg/kg BW of MEL+CYA for 28 days, 2.5 mg/kg BW of MEL+CYA for 14 days, or 400 mg/kg BW of MEL=CYA for 3 days. Residues in the tissue were determined by LC-MS/MS. MEL was extracted with acidic acetonitrile, followed by defatting with dichloromethane, and isolated with cation exchange solid phase extraction (SPE). For CYA analysis, fish were extracted with dilute acetic acid, defatted with hexane, and cleaned up with a graphitic carbon SPE. Catfish fed 0.1 mg/kg BW of MEL reached a maximum muscle residue concentration of 0.33 ± 0.04 mg/kg (ppm) after 28 days of continuous feeding. The same concentration was found for MEL+CYA feeding at the 0.1 mg/kg BW level for 28 days. Feeding at 2.5 mg/kg BW of MEL+CYA yielded muscle concentrations above the 2.5 mg/kg level of concern for most of the study fish. Finally, catfish fed high levels of the MEL=CYA complex (400 mg/kg BW) accumulated relatively little MEL in the muscle (0.14 ± 0.07 mg/kg) and, unlike treatment with MEL+CYA, did not form renal melamine-cyanurate crystals. Appreciable concentrations of CYA were not detected in any of the muscles tested. These studies provide data to model the bioaccumulation of triazine residues into edible fish tissue as a result of the continuous consumption of adulterated feed.