Provisional epidemiological cutoff values for standard broth microdilution susceptibility testing of Flavobacterium columnare.

The gliding aquatic bacterium Flavobacterium columnare causes columnaris disease, a common problem for wild and farmed freshwater fish worldwide. Recently, a broth microdilution method was standardized to test the susceptibility of F. columnare against antimicrobials commonly used in aquaculture. We used this new method to measure the minimal inhibitory concentrations (MICs) of ten antimicrobials against 120 F. columnare isolates. The resulting MIC frequency distributions for each antimicrobial (1 MIC/isolate) were used to estimate epidemiological cut-off values (ECVs) which separate isolates with typical wild-type (WT) susceptibility from isolates with decreased non-wild-type (NWT) susceptibility. We identified 22 NWT isolates with elevated MICs relative to the ECV that covered 99.9% of the MIC distribution against one or more of the antimicrobials: ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid or oxytetracycline. Ten of the NWT isolates had decreased susceptibility to a single antimicrobial class, six isolates to two antimicrobial classes and six isolates to three or more antimicrobial classes. The MIC frequency distributions and provisional cut-off values provide data needed to set epidemiological cut-off values to monitor for the development of antimicrobial resistance among F. columnare.

Depletion of melamine and cyanuric acid in kidney of catfish Ictalurus punctatus and trout Oncorhynchus mykiss.

A risk assessment conducted in 2007 identified significant knowledge gaps about tissue residues of melamine and related triazine analogs such as cyanuric acid in animals that had eaten contaminated food. The USFDA subsequently designed studies to determine residue levels in muscle, serum, and kidneys of catfish and trout given a single gavage dose of 20 mg/kg body weight (BW) of melamine, cyanuric acid, or 20 mg/kg BW of both compounds simultaneously. Renal triazines were determined by LC-MS/MS at postdose days 1, 3, 7, 14, 28 (and day 42 for trout). When dosed individually, melamine and cyanuric acid kidney residues depleted much faster than those in fish given both compounds together. Combined dose residue depletion was punctuated by extreme outliers due to the formation of persistent renal melamine cyanurate crystals.

Renal crystal formation after combined or sequential oral administration of melamine and cyanuric acid.

We evaluated renal melamine-cyanurate crystal spherulite formation after single and repeated ingestion of both melamine (MEL) and cyanuric acid (CYA) in catfish and trout. MEL and CYA were co-administered orally over a range of doses, 0.1-20mg/kg body weight (bw) of each compound, either once or repeatedly for 4, 14 or 28 days (d). In catfish, the No Observable Adverse Effects Levels (NOAELs) for crystal formation for single, 4d or 14 d dosing were 10, 2.5 and 0.5mg/kg bw, respectively. In trout, the respective NOAELs were 2.5, 2.5 and 0.5mg/kg bw. No renal crystals formed in catfish fed 0.1mg/kg bw of each compound for 28 d. Sequential administration of 20mg/kg bw of MEL followed by 20mg/kg bw of CYA or vise-versa, with waiting periods of 1, 3, 7, 14 or 21 d between compound dosing also induced renal crystal formation in fish. These studies show that both catfish and trout are sensitive, non-mammalian models, for renal crystal formation following MEL and CYA ingestion. Since fish generally excrete chemicals more slowly than mammals, they may provide a "worst case scenario" model for higher risk populations, such as infants or persons with compromised renal function.

Residue depletion of melamine and cyanuric acid in catfish and rainbow trout following oral administration.

The intentional addition of triazines such as melamine to animal feeds and the lack of information about residue accumulation in food animals caused global concerns for food safety during 2007 and 2008. We report the results of a good laboratory practices (GLP) study to determine melamine and cyanuric acid residues in catfish and trout filets harvested at 1, 3, 7, 14, 28, and 42 days after a single oral dose of 20 mg/kg body weight of melamine, cyanuric acid, or melamine and cyanuric acid together. Peak melamine concentrations were 12.73 mg/kg (ppm) in catfish (mean = 9.98), 12.26 mg/kg in trout (mean = 7.89) on day 1. Within 7 days (catfish) or 14 days (trout) residues were <2.5 mg/kg, a level in foods accepted by many risk assessors worldwide to be unlikely to pose health risks to consumers. Peak cyanuric acid residues also occurred on day 1, 0.68 mg/kg in catfish (mean = 0.46), 2.59 mg/kg in trout (mean = 0.86). Cyanuric acid muscle residues were <2.5 mg/kg by day 3. The half-lives for melamine and cyanuric acid ranged between 1 and 4 days. Renal crystals formed in fish given both melamine and cyanuric acid, persisting for weeks after the single dose.

Metabolism and depletion of albendazole in the muscle tissue of channel catfish following oral treatment.

The residue depletion of albendazole (ABZ) and its metabolites was studied in channel catfish muscle tissue. Channel catfish were dosed once with 10 mg/kg ABZ via stomach tube with manual restraint. Muscle tissue samples were collected at 8, 16, 24, 48, 72, 96 and 120 h postdose. A high-performance liquid chromatographic method was used to assay ABZ and its major metabolites: ABZ sulfoxide (ABZ-SO), ABZ sulfone (ABZ-SO2) and ABZ aminosulfone (ABZ-2-NH2SO2) in the muscle tissue. The results indicate that ABZ and ABZ-SO were present in low concentrations, i.e. <15 and <10 microg/kg, respectively, at 8 h postdose in catfish muscle with and without skin. ABZ-SO2 was present at 1 microg/kg concentration levels until 48 h in muscle alone and 72 h in muscle with skin. ABZ-2-NH2SO2 was not detected at any withdrawal periods.

Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria.

A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.

Myxosporean plasmodial infection associated with ulcerative lesions in young-of-the-year Atlantic menhaden in a tributary of the Chesapeake Bay, and possible links to Kudoa clupeidae.

Ulcers in Atlantic menhaden Brevoortia tyrannus (Latrobe) (Clupeidae), observed along the USA east coast, have been attributed to diverse etiologies including bacterial, fungal and, recently, harmful algal blooms. To understand the early pathogenesis of these lesions, we examined juvenile Atlantic menhaden collected during their seasonal presence in Chesapeake Bay tributaries from April to October 1999 and from March to August 2000. We conducted histopathological examinations of young-of-the-year fish from the Pocomoke River tributary, which has a history of fish mortalities and high lesion prevalence. Kudoa clupeidae (Myxozoa: Myxosporea) spores were present in the muscles of fish collected in both years. Of the fish assessed by histology in April, 5 to 14% were infected, while in May 90 to 96% were infected. Infection rates remained high during the summer. Mature spores were primarily located within myomeres and caused little or no observable pathological changes. Ultrastructure showed spores with capsulogenic cells bearing filamentous projections, and a basal crescentic nucleus with mottled nucleoplasm containing cleaved, condensed chromatin. Also, a highly invasive plasmodial stage of a myxozoan was found in the lesions of juvenile Atlantic menhaden. The plasmodia were observed in fish collected between May and July, with the maximum occurrence in late June 1999 and late May 2000. Plasmodia penetrated and surrounded muscle bundles, causing grossly observable raised lesions in 73% of all fish infected with this invasive stage. Plasmodia were also detected in the visceral organs, branchial arches, and interocular muscles of some fish. Some of the invasive extrasporogonic plasmodial lesions were associated with ulcers and chronic inflammatory infiltrates. The plasmodial stage appeared to slough out of the tissue with subsequent evidence of wound healing. Ultrastructure showed plasmodia with an elaborate irregular surface, divided into distinct ectoplasm and endoplasm; the latter contained numerous spherical vegetative nuclei, secondary generative cells, and occasional cell doublets. Our ultrastructural studies indicate that the plasmodial organisms, which are important in the etiology of the skin lesions, are myxozoans, and they may represent early stages of K. clupeidae.

Metabolism and residue depletion of albendazole and its metabolites in rainbow trout, tilapia and Atlantic salmon after oral administration.

Metabolic and residue depletion profiles of albendazole (ABZ) and its major metabolites in three fish species, rainbow trout, tilapia and Atlantic salmon are reported. Based on these profiles, similarities (or dissimilarities) between species will determine the potential to group fish species. ABZ at 10 mg/kg body weight was incorporated into fish food formulated in a gelatin base or in gel capsule and fed as a single dose to six fish from each species. Rainbow trout were held three each in a partitioned 600-L tank. Tilapia and Atlantic salmon were housed in separate 20-L tanks. Samples of muscle with adhering skin were collected at 8, 12, 18, 24, 48, 72, and 96 h postdose from trout kept at 12 degrees C, at 4, 8, 12, 24, 48, 72, 96, 120, and 144 h postdose from tilapia kept at 25 degrees C and at 8, 14, 24, 48, 72, and 96 h postdose from Atlantic salmon kept at 15 degrees C. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final extracts were analyzed for parent drug ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO2) and albendazole aminosulfone using high-performance liquid chromatography with fluorescence detection. ABZ was depleted by 24 h in trout and tilapia and by 48 h in salmon; ABZ-SO, a pharmacologically active metabolite, was depleted by 48 h in tilapia, by 72 h in rainbow trout and was present until 96 h in salmon; and low levels of ABZ-SO2 and albendazole aminosulfone, both inactive metabolites, were detectable at least till 96 h in all three fish species.