Pharmacokinetics of gentamicin in channel catfish (Ictalurus punctatus)

The pharmacokinetics of gentamicin were determined in male and female Ictalurus punctatus weighing between 0.3 kg and 1.7 kg. Plasma gentamicin concentrations were measured by radioimmunoassay technique. In the 1st experiment, an intracardiac bolus dosage of gentamicin (1 mg/kg) was given to 10 channel catfish. Samples of blood were obtained (by cardiac puncture) immediately before gentamicin was given and at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0003 minute-1). The maintenance therapeutic IV dosage of gentamicin was calculated to be 1.6 mg/kg given at 33.2-hour intervals. In the 2nd experiment, an IM dosage of the drug (1 mg/kg) was given to 9 channel catfish, and samples of blood were obtained at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0002 minutes-1). The maintenance therapeutic IM dosage of gentamicin was calculated to be 3.5 mg/kg given at 33.2-hour intervals. The IM bioavailability of gentamicin in channel catfish was estimated to be 60%. Other pharmacokinetic values were also determined. It was concluded that the therapeutic regimen of choice for gentamicin in channel catfish was 3.5 mg/kg given IM with 33-hour intervals between doses.     

Characterization of a mannose-binding lectin from channel catfish (Ictalurus punctatus)

Mannose-binding lectin (MBL) is an important component of innate immunity capable of activating the lectin pathway of the complement system. A MBL gene was isolated from channel catfish (Ictalurus punctatus). The deduced protein contains a canonical collagen-like domain, a carbohydrate recognition domain (CRD), and a neck region similar to mammalian mannose-binding lectin. The catfish mannose-binding lectin CRD contains the EPN motif shown previously to mediate mannose specificity. The catfish mannose-binding lectin showed 30-43% identity with MBL protein sequences of rainbow trout, zebrafish, common carp, and goldfish, and 33-35% identity with sequences of mammalian species. In this study, while liver was the predominant source of mannose-binding lectin gene expression in healthy tissues, mannose-binding lectin expression in spleen rose sharply following challenge with a Gram-negative bacterium.     

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus)

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.     

Molecular and functional characterization of channel catfish (Ictalurus punctatus) neutrophil collagenase

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.     

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 (C_ss(max)), time of C_ss(max) (T_max), minimal concentration at steady‐state (C_ss(min)), and V_c/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.     

Plasma/muscle ratios of sulfadimethoxine residues in channel catfish (Ictalurus punctatus)

Channel catfish ( n = 84) maintained at a water temperature of 27°C were used in a feeding study to determine the plasma to muscle concentration ratios of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine residues. Sulfadimethoxine medicated feed was provided free choice at 42 mg SDM/kg body weight once daily for 5 days and the plasma and muscle concentrations of SDM were determined at selected withdrawal times (6, 12, 24, 48, 72, and 96 hours) following the last dose. Considerable variation in total SDM tissue concentration among fish within a sampling period was observed. For fish ( n = 12) at six hours post-dose, total SDM concentrations ranged from 1.4–24.8 μg/mL and 0.6–12.6 μg/g, with mean total SDM concentrations of 9.1 μg/mL and 5.3 μg/g for plasma and muscle, respectively. However, a mean plasma:muscle concentration ratio of 1.8:1 ± 0.3:1 was obtained over all concentrations and sampling periods. The plasma:muscle 95% t distribution interval for individual fish was 1.2:1 to 2.4:1. A correlation coefficient of 0.967 was obtained for the relationship between plasma and muscle total SDM concentration among individual fish ( n = 25). Results of this study indicate that plasma total SDM concentration may be used to identify samples containing violative SDM muscle residue. No fish contained total SDM muscle residues greater than the FDA tolerance (0.1 μg/g) by 48 hours following the final dose.     

A light and electron microscopic study of the leukocytes of channel catfish (Ictalurus punctatus)

A light and electron microscopic study of channel catfish (Ictalurus punctatus) white blood cells was carried out. The white blood cells of channel catfish consist of lymphocytes, neutrophils, thrombocytes and monocytes. A fifth cell type is illustrated and described which could not be characterized. These leukocytes are characterized and compared with their mammalian counterparts.     

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.     

Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes

Four monoclonal antibodies (MAbs) were evaluated for specific reactions to various catfish peripheral blood leucocytes and anterior kidney cells. The MAb 9E1 served as a standard and positive control for all test reactions because of its defined reactivity with channel catfish immunoglobulin and immunoglobulin bearing cells. Of the four MAbs, two have been characterized as being specific for a non-immunoglobulin marker on lymphocytes, thus marking T lymphocytes and two were specific for catfish neutrophils. Morphological, flow cytometric and functional analysis of the reactive cells verified these findings.     

Mobilization and activation of nonspecific cytotoxic cells (NCC) in the channel catfish (Ictalurus punctatus) infected with Ichthyophthirius multifiliis

Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.     

饲粮中添加DL-肉碱复合物和复合胆汁酸对斑点叉尾鮰生长性能的影响

本试验研究了在斑点叉尾鮰饲粮中添加DL-肉碱复合物和复合胆汁酸对其生长性能的影响.采用单因子三重复试验设计,以135尾平均体重(30.6±0.2)g的斑点叉尾鮰随机分为3组.对照组饲喂基础饲粮,DL-肉碱复合物组饲喂在基础饲粮中分别添加DL-肉碱复合物200 mg/kg的饲粮,DL肉碱复合物-复合胆汁酸组饲喂在基础饲粮中添加DL-肉碱复合物200 mg/kg+复合胆汁酸37.5 mg/kg的饲粮.试验期为8周.结果表明:与对照组相比,在饲粮中添加DL-肉碱复合物能够显著提高饲料效率17.28%(P<0.05),显著降低饲料系数15.43%(P<0.05),提高特定生长率12.50%;补充添加DL-肉碱复合物+复合胆汁酸后特定生长率和饲料转化效率较DL-肉碱复合物单独添加组分别降低4.27%和5.76%,差异不显著.     

Specific serum antibody responses in channel catfish (Ictalurus punctatus) provide limited protection against Streptococcus ictaluri challenge

Passive immunization studies were conducted to determine the role of specific antibodies in immunity to Streptococcus ictaluri. Adult channel catfish (Ictalurus punctatus) were injected i.p. with tryptic soy broth as control or with 1.5 × 10(7)colony-forming units (cfu) S. ictaluri/fish at 0, 30, and 60 d, and serum was collected 90 d after the original challenge. Fish were passively immunized by i.p. injection with serum from the tryptic soy broth (TSB) control group, anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (SSI), or heat-inactivated anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (HISSI). These passively immunized fish were then challenged 72 h later with 1.5 × 10(8)cfu S. ictaluri/fish. Over 21 d, the mean cumulative percent survival was 43.3 (TSB), 63.3 (SSI), and 50.0 (HISSI). A significant difference in cumulative percent survival was noted between the TSB and the HISSI groups, and significant differences were noted between these groups and the SSI group. Serum obtained from immunized fish 72 h after passive immunization exhibited increased anti-S. ictaluri antibody levels. Twenty-one days after the challenge, the HISSI and SSI group antibody levels significantly increased above their corresponding pre-challenge levels. No significant (r(2)=0.0806; P<0.5985) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results indicate that both specific anti-S. ictaluri antibodies and non-specific immune responses are important for protection against S. ictaluri.     

Influence of route of administration on the humoral response of channel catfish (Ictalurus punctatus) to Yersinia ruckeri

Channel catfish were inoculated intraperitoneally, intramuscularly, or intraesophageally with Yersinia ruckeri. Three antigen doses were administered by each route of injection. Four fish from each treatment were sacrificed at 5-day intervals for 40 days. Serum from each individual was tested for antibody activity against Y. ruckeri by indirect enzyme-linked immunosorbent assay (ELISA). High titers of anti-Y. ruckeri antibody were elicited by all doses (10(5) to 10(9) cells/g of fish) regardless of the route of administration. Mean titers for saline injected fish ranged up to 1:64 for each route of inoculation while mean titers for bacteria injected fish ranged up to 1:4096. Mean titers of 1:128 or greater were observed by day 15 post injection; titers peaked about day 30 and diminished thereafter. Channel catfish were most responsive to the antigen (10(7) to 10(9) cells/g of fish) when administered intramuscularly although lower doses (10(5) to 10(6) cells/g of fish) administered intraperitoneally elicited a substantial response. There was little evidence of dose-dependent responses for any of the routes of immunization. The rapid onset of relatively high serum titers suggests that the fish were mounting a secondary response to Yersinia ruckeri.     

Lipopolysaccharide regulates myostatin and MyoD independently of an increase in plasma cortisol in channel catfish (Ictalurus punctatus)

The effects of lipopolysaccharide (LPS) on plasma cortisol and the expression of MyoD and myostatin (MSTN) mRNAs were evaluated in channel catfish. In addition, the effect of dexamethasone (Dex) on MyoD and MSTN mRNAs was examined. For the LPS injection experiments, juvenile channel catfish were injected intraperitoneally with 1.5 mg/kg LPS or sterile PBS. Blood was collected at 1, 3, 12, and 24 h post-injection for cortisol determination, and muscle samples were collected at 3, 12, and 24 h for mRNA analysis. For the Dex injection experiment, fish were injected with 1.0 mg/kg Dex or saline and muscle samples were collected at 12 and 24 h. There was no effect of LPS on plasma cortisol at any of the time points measured. Injection with LPS increased the abundance of MyoD mRNA at 3 and 12 h, and decreased the abundance of MSTN mRNA at 24 h. There was no effect of Dex injection on the abundance of MyoD mRNA. However, Dex injection decreased the abundance of MSTN mRNA at 12 h post-injection. These results suggest that LPS regulates the expression of MyoD and MSTN independently of an increase in plasma cortisol, and that the regulation of MyoD in the channel catfish differs from mammals in response to inflammatory stimuli. These results also confirm that exogenous glucocorticoids decrease the expression of MSTN as shown in other fish species.     

Effects of fasting on circulating IGF-binding proteins, glucose, and cortisol in channel catfish (Ictalurus punctatus)

The effects of fasting on IGF-binding proteins (IGFBPs), glucose, and cortisol in channel catfish were examined. Fed fish (controls) were compared to 14-, 30-, and 45-day fasted fish and 45-day fasted fish refed for 15 additional days. Body length and weight changes, condition factor (CF), hepatosomatic index (HSI), and plasma glucose and cortisol were assessed to determine growth and metabolic status. Body length and growth rates were inhibited (P<0.05) after 14, 30, and 45 days of fasting. The 14-, 30-, and 45-day fasted fish exhibited hypoglycemia and reduced CF and HSI. Cortisol levels were increased (22.8 +/- 15.2 ng/ml versus 4.7 +/- 3.9 ng/ml) in 30-day fasted fish compared to fed controls (P<0.05). Associated with the increase in cortisol in fasted fish was a concomitant increase in plasma levels of a 20-kDa IGFBP through day 45. A 35- and a 45-kDa IGFBP were also identified but were similar between fed and unfed fish throughout the experiment. At the end of 15 days of refeeding, 20-kDa IGFBP, glucose, and cortisol levels were similar to fed controls. Refeeding also caused an increase in growth rates. These results suggest the existence of a catfish counter part to mammalian IGFBP-1, similar to lower molecular mass IGFBPs reported in other species of fish. These results also suggest that a 20-kDa IGFBP is upregulated during fasting-induced growth inhibition of channel catfish and provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in fish.     

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 Aquaculture 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 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Effect of size and temperature on the quantity of immunoglobulin in channel catfish, Ictalurus punctatus

Concentrations of serum immunoglobulin (Ig) were determined for 30 channel catfish from pond water at 10 degrees C. These values were compared to measurements of 15 channel catfish from pond water at 30 degrees C. Channel catfish from 10 degrees C pond water had no significant (P greater than 0.05) different Ig concentrations (mean, 398 mg/dl) than catfish from 30 degrees C pond water (mean, 367 mg/dl). Serum Ig concentrations appear not to be different in cold (10 degrees C) vs warm (30 degrees C) pond water for 37.5-45 cm catfish. Channel catfish, 7.5-15 cm (n = 24) had significantly (P less than 0.05) lower Ig levels (mean, 104 mg/dl) than catfish either 7.5-25.5 cm (n = 57, mean, 232 mg/dl) or 37.5-45 cm (n = 45, mean, 388 mg/dl). Also, catfish 17.5-25.5 cm had a significantly (P less than 0.05) less Ig than catfish 37.5-45 cm. The concentrations of serum Ig increase with size (P = 0.0001) of catfish. The mean Ig concentration for 7.5-45 cm catfish (n = 126) was 263 mg/dl. The Ig concentration range was 44 to 650 mg/dl of serum.