Patricia S. Gaunt

Preliminary Assessment of the Tolerance and Efficacy of Florfenicol against Edwardsiella ictaluri Administered in Feed to Channel Catfish

Abstract A tolerance study was conducted to determine the palatability of florfenicol to channel catfish Ictalurus punctatus. Four tanks of fish (20 fish/tank) were assigned to each of five treatments distinguished by the amount of florfenicol given in feed per kilogram of body weight, namely, 0, 10, 20, 40, or 100 mg. Fish were fed at a rate of 2.5% of body weight per day for 10 consecutive days. On day 11, all surviving fish were euthanatized, counted, and weighed as a group. Florfenicol- medicated feed was palatable to fish at doses of 10, 20, 40, and 100 mg for 10 consecutive days. All 400 fish were necropsied and examined by histopathology, and no treatment-related changes were observed. In a separate exploratory efficacy study, four tanks (20 fish/tank) were assigned to each of the following treatments: (1) not challenged with Edwardsiella ictaluri and fed unmedicated feed, (2) challenged with E. ictaluri and fed unmedicated feed, (3) challenged with E. ictaluri and fed florfenicol at 10 mg per kilo...

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aqua-culture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 μg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.

Efficacy of Florfenicol for Control of Mortality Caused by Flavobacterium columnare Infection in Channel Catfish

The studied in channel catfish Ictalurus punctatus fingerlings held in 80-L aquaria. Nonabraded fish were challenged by immersion on day 0. Thirty 80-L tanks were randomly assigned in equal numbers to two treatment groups, one in which fish were fed a commercial diet without florfenicol (unmedicated feed) and one in which they were fed a diet containing 10 mg of florfenicol/kg of body weight (medicated feed) for ten consecutive days. Mortality was monitored during the treatment period and a 14-d posttreatment observation period. At the end of the posttreatment period, all fish were euthanized, examined for gross lesions, and cultured for F. columnare. Significantly fewer fish fed the medicated diet died (8.0%) than fish fed the unmedicated diet (54.2%). Flavobacterium columnare was cultured from 15.0% of the medicated fish, compared with 68.9% of the unmedicated fish. The gross lesions in the fish were consistent with columnaris disease, and F. columnare was cultured from 99.5% of the dead fish. No differences were observed in weight gain and appetence between the medicated and unmedicated groups. For the F. columnare strain used in this study, the minimal inhibitory concentration of florfenicol ranged from 0.5 to 1.0 mg/mL in the 30 bacterial cultures obtained from infected fish, and the mean disk diffusion zone of inhibition was 40 mm. There were no adverse effects among the medicated fish. Administration of florfenicol at a dosage of 10 mg/kg body weight for 10 d was efficacious and safe for the control of mortality from F. columnare infection in channel catfish.

Determination of Florfenicol Dose Rate in Feed for Control of Mortality in Nile Tilapia Infected with Streptococcus iniae

A dose titration study was conducted to determine the dosage of florfenicol (FFC) in feed to control Streptococcus iniae-associated mortality in Nile tilapia Oreochromis niloticus. Six tanks were assigned to each of five treatments: (1) not challenged with S. iniae and fed unmedicated feed; (2) challenged with S. iniae by injection and fed unmedicated feed; (3) challenged with S. iniae and given FFC at 5 mg/kg of body weight (bw) in medicated feed; (4) challenged with S. iniae and given 10 mg FFC/kg bw; and (5) challenged with S. iniae and given 15 mg FFC/kg bw. Treatment was initiated the day after inoculation, and feed was administered for 10 d. Cumulative mortality was 0% in the unchallenged, untreated group; 35.8 +/- 4.4% (mean +/- SE) in the challenged, unmedicated group; 19.2 +/- 2.7% in the 5-mg/kg treated group, 12.5 +/- 3.8% in the 10-mg/kg group, and 2.5 +/- 1.1% in the 15-mg/kg group. The cumulative mortality was significantly less in each challenged, FFC-treated group than in the challenged, unmedicated controls (5 mg/ kg: P = 0.0156; 10 mg/kg: P = 0.0007; 15 mg/kg: P < 0.0001). The efficacy of the 10- and 15-mg/kg FFC dosages was studied in a separate dose confirmation study. Fish in all tanks were injected with S. iniae. At 4 h postinoculation, 10 tanks were assigned to each of three feed treatments: (1) unmedicated feed; (2) 10 mg FFC/kg bw; and (3) 15 mg FFC/kg bw. Cumulative mortality was 20.5 +/- 2.0% in the challenged, unmedicated group; 11.0 +/- 2.1% in the 10-mg/kg group; and 5.5 +/- 2.4% in the 15-mg/kg group. Mortality was significantly less in the medicated groups than in the challenged, unmedicated control group (10 mg/kg: P = 0.0270; 15 mg/kg: P = 0.0007). Fish in both studies were necropsied, cultured for bacteria, and examined for gross lesions. The minimum inhibitory concentration of FFC against S. iniae in both studies ranged from 0.5 to 1.0 microg/mL. Florfenicol was palatable, safe, and efficacious for control of Nile tilapia mortality due to S. iniae infection.

Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.

Response of Channel Catfish to Diets Containing T-2 Toxin

Abstract The T-2 toxin is a trichothecene mycotoxin produced by certain molds of the genus Fusarium that infect the grains, wheat by-products, and oilseed meals used in the production of animal feeds. An aquarium study was conducted with juvenile channel catfish Ictalurus punctatus. Experimental diets were prepared by replacing the untreated casein in a semipurified diet with casein treated with pure T-2 toxin in the amounts necessary to produce five levels of the toxin. Dietary concentrations were 0, 0.625, 1.25, 2.5, and 5.0 mg/kg of diet. A concurrent study with similarly sized channel catfish was conducted to substantiate the toxicity of the T-2 toxin. This study required feeding the control diet at the same levels of daily consumption (pair-feeding) as in the treatments with the three highest levels of T-2 toxin (1.25, 2.5, and 5.0 mg/kg) to compare differences in weight gain, feed conversion, hematocrit, and survivability. After 8 weeks, significant (P < 0.05) reductions in growth were observed for ...

New Data on Henneguya pellis (Myxozoa: Myxobolidae), a Parasite of Blue Catfish Ictalurus furcatus

Abstract The original description of Henneguya pellis, a myxozoan parasitizing blue catfish Ictalurus furcatus, is supplemented with new data on histopathology, spore morphology, and 18S small subunit (SSU) ribosomal DNA (rDNA) sequence. Plasmodia presented as both internal and external, raised, cyst-like lesions on the body wall of the peritoneal cavity and on the skin. The cysts contained numerous elongate, lanceolate myxospores, flattened parallel to the suture line. The spore body was 14.8 ± 1.1 µm (range 13.0–17.1) long and 4.8 ± 0.8 µm (range 4.0–7.4) wide in frontal view. The caudal appendages were 77.7 ± 8.8 (range 57.4–96.4) in length. There were 2 pyriform polar capsules, unequal in length, with the longer capsule measuring 7.2 ± 0.6 µm (range 6.2–8.4) in length and the shorter capsule measuring 6.5 ± 0.5 µm (range 5.5–8.0). The polar capsules were not significantly different in width, measuring 1.7 ± 0.2 µm (range 1.4–1.9). There were 8 turns in the polar filament coil. The total length of the spore was 92.5 ± 9.2 µm (range 73.3–113.5). Spore morphology and site of development are similar to that of Henneguya sutherlandi from channel catfish; however, 18S rDNA sequence data support previous findings that identify H. pellis and H. sutherlandi as 2 distinct species.

Multidose pharmacokinetics of orally administered florfenicol in the channel catfish (Ictalurus punctatus).

Plasma disposition of florfenicol in channel catfish was investigated after an oral multidose (10 mg/kg for 10 days) administration in freshwater at water temperatures ranging from 24.7 to 25.9 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After the administration of florfenicol, the mean terminal half-life (t(1/2)), maximum concentration at steady-state (Css (max)), time of Css (max) (T(max)), minimal concentration at steady-state (Css (min)), and Vc /F were 9.0 h, 9.72 μg/mL, 8 h, 2.53 μg/mL, and 0.653 L/kg, respectively. These results suggest that florfenicol administered orally at 10 mg/kg body weight for 10 days could be expected to control catfish bacterial pathogens inhibited in vitro by a minimal inhibitory concentration value of <2.5 μg/mL.

Single intravenous and oral dose pharmacokinetics of florfenicol in the channel catfish (Ictalurus punctatus)

Plasma distribution and elimination of florfenicol in channel catfish were investigated after a single dose (10 mg/kg) of intravenous (i.v.) or oral administration in freshwater at a mean water temperature of 25.4 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After i.v. florfenicol injection, the terminal half-life (t(1/2)), volume of distribution at steady state (V(ss)), and central volume of distribution (V(c)) were 8.25 h, 0.9 and 0.381 L/kg, respectively. After oral administration of florfenicol, the terminal t(1/2), C(max), T(max), and oral bioavailability (F) were 9.11 h, 7.6 μg/mL, 9.2 h, and 1.09, respectively. There was a lag absorption time of 1.67 h in oral dosing. Results from these studies support that 10 mg florfenicol/kg body weight in channel catfish is an efficacious dosage following oral administration.

Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials

A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for the fish pathogens Flavobacterium columnare and F. psychrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted (4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of the 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC ± 1 two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.