Absorption, tissue distribution and excretion of flumequine and oxolinic acid in corkwing wrasse (Symphodus melops) following a single intraperitoneal injection or bath treatment

The pharmacokinetic properties of the antibacterial agents oxolinic acid and flumequine were studied in corkwing wrasse (Symphodus melops) after either intraperitoneal injection or bath treatment. Following intraperitoneal administration the peak plasma concentrations (Cmax) and the time to peak plasma concentrations (Tmax) were estimated to be 2.0 microg/mL and 12 h, respectively, for oxolinic acid and 2.6 microg/mL and 12 h, respectively, for flumequine. In muscle, Cmax and Tmax were estimated to 6.7 microg/g and 12 h, respectively, for oxolinic acid with corresponding values of 8.5 microg/g and 13 h, respectively, for flumequine. In liver, Cmax and Tmax were calculated to 7.0 microg/g and 12 h, respectively, for oxolinic and 12.2 microg/g and 11 h, respectively, for flumequine. Elimination half-lives (t1/2 beta) of 26, 24 and 29 h, respectively, for plasma, muscle and liver were calculated for flumequine. For oxolinic acid two distinct elimination phases were found and calculated to be 16 h (t1/2 beta) and 57 h (t1/2 gamma) in plasma, 15 and 59 h, respectively, in muscle and 20 and 72 h, respectively, in liver. Bath treatment using 150 mg/L of flumequine or 200 mg/L of oxolinic acid for 72 h resulted in flumequine concentrations of 1.0 microg/mL in plasma, 5.0 microg/g in muscle and 12.4 microg/g in liver. Corresponding values for oxolinic acid were 1.0 microg/g in plasma, 2.5 microg/g in muscle and 4.9 microg/g in liver.     

Experimental Treatment of Aeromonas salmonicida Infections with Enrofloxacin and Oxolinic Acid: Field Trails

Abstract

The antimicrobials enrofloxacin and oxolinic acid were evaluated under field conditions for treatment of clinical Aeromonas salmonicida infections in salmonids by feeding medicated diets. Treatment of Atlantic salmon Salmo salar with enrofloxacin or oxolinic acid at 5 mg/ kg per day for 5 d was not successful. Monitoring of enrofloxacin-medicated fish revealed low tissue and serum antimicrobial activities. Lack of efficacy in the Atlantic salmon trial may have been due to the low dose, lack of bioavailability of the drug, or lack of acceptance of the medicated ration. Treatment of hybrid brook trout Salvelinus fontinalis × lake trout S. namaycush was successful when enrofloxacin was used at 10 mg/kg per day for 10 d, but not when oxolinic acid was used at 5mg/kg per day for 10 d. Antimicrobial activity in tissues of enrofloxacin-medicated fish was elevated during the 10-d treatment period. No antimicrobial activity could be detected in muscle, skin, or liver of the enrofloxacin-medicated fish at 20 d after the last day of medication.     

In Vitro Resistance by Fish Pathogens to Aquacultural Antibacterials,Including the Quinolones Difloxacin (A-56619) and Sarafloxacin (A-56620)

Abstract

Two new quinolone antibacterials, difloxacin (A-56619) and Sarafloxacin (A-56620), were compared with oxolinic acid, oxytetracycline, and ormetoprim-sulfadimethoxine in vitro for their activities against common bacterial pathogens of fish. The objectives were to determine (1) the frequencies of in vitro resistance to antibacterials at eight times the minimum inhibitory concentration (MIC), (2) the rates and extents of decrease in antibacterial susceptibility when organisms were serially transferred to increasing concentrations of drug, (3) the stability of the decreased susceptibility, and (4) cross-resistance to other antibacterials by organisms with developed resistance. The frequency of spontaneous resistance to all antibacterials at eight times the MIC was low, 10^-7-10^-10. Quinolone-selected mutants that showed low-level resistance would be inhibited by achievable in vivo levels of difloxacin or Sarafloxacin. Oxolinic acid would not inhibit such mutants. The rate of susceptibility decrease during serial transfer was gradual and stepwise for all organism-drug combinations. In contrast to difloxacin and Sarafloxacin, MICs of oxolinic acid, oxytetracycline, and ormetoprim-sulfadimethoxine for some final transfer cultures were above achievable serum levels. Developed resistance was stable for all antibacterials. Cross-resistance was seen among the three quinolones but was not seen with the other antibacterials, except for oxytetracycline. Based on results, resistance to difloxacin and Sarafloxacin by fish pathogens will not develop easily during proper therapeutic use.     

Evaluation of the dorsal aorta cannulation technique for pharmacokinetic studies in Atlantic salmon (Salmo salar) in sea water

The antimicrobial drug flumequine was given intravascularly and orally to cannulated and non-cannulated Atlantic salmon (Salmo salar) in sea water at 11°. The cannulated fish were divided into two groups, which were given flumequine (25 mg/kg) intravenously into the caudal vein (n = 8) and orally via a stomach tube down the oesophagus (n= 8). After a washout period of 2 days, the intravenously administered fish were given the drug orally, and the orally administered fish were given the drug intravenously. Blood samples were taken at different time points after drug administration through a cannula inserted into the dorsal aorta. The fish in the non-cannulated group were either given flumequine intravenously or orally, and blood samples were collected by killing five fish at predetermined time points after administration. The haematocrit values were measured in all the fish daily for 4 days after drug administration and thereafter, in all the collected blood samples throughout the whole experiment. The haematocrit values differed significantly between the cannulated and the non-cannulated fish. We found low haematocrit values and slow drug elimination in the cannulated groups, compared with higher haematocrit values and faster drug elimination in the non-cannulated groups, but further investigations are needed to prove any causal relations of this observation. The volume of distribution (V_d(ss)) was twice as large in the cannulated groups compared with the non-cannulated group, in the fish administered the drug intravenously. In the last part of the elimination phase, the half-lives differed considerably between the cannulated and the noncannulated groups both after oral and intravenous administration. The slower depletion of the drug concentration in the plasma of the cannulated fish is due to the large V_d(ss) as there are only small differences in clearance (Cl_T) between the groups. In this study the elimination of flumequine in cannulated Atlantic salmon differed from the elimination of flumequine in non-cannulated Atlantic salmon.     

Antibiotic Resistance of Vibrio anguillarum,in Relation to Serovar and Plasmid Contents

A total of 520 Vibrio anguillarum strains, isolated from fish and the environment, were tested for their sensitivity to 20 different antibiotics. Most isolates were of European origin. The results were compared with data on the O-serogroup and plasmid contents. All strains were sensitive to neomycin, spectinomycin, nitrofurantoin, flumequine and oxolinic acid, while most strains were sensitive to streptomycin, Oxytetracycline, chloramphenicol, sulphonamides, trimethoprim, sulphonamides with trimethoprim, nalidixan, rifampicin, novobiocin and O/129. A major part of the strains were resistant to the macrolides, spiramycin and lincomycin. For ampicillin, cephalothin, and Colistin marked differences were recorded with respect to O-serogroup. Most O1 strains were resistant to Colistin and sensitive to ampicillin and cephalothin, while most O2 strains were sensitive to Colistin but resistant to ampicillin and cephalothin. Some antibiotic resistant strains carried plasmids but no conjugation experiments were carried out to detect possible R factors.     

The efficacy of a single intraperitoneal injection of oxolinic acid in the treatment of bacterial infections in goldsinny wrasse (Ctenolabrus rupestris) and corkwing wrasse (Symphodus melops) studied under field and laboratory conditions

The efficacy of a single intraperitoneal injection of oxolinic acid to control an outbreak of atypical Aeromonas salmonicida infection in goldsinny wrasse (Ctenolabrus rupestris) and in the treatment of systemic vibriosis in corkwing wrasse (Symphodus melops) was examined. In addition a field study was performed to examine the effect of medication on the survival rate of goldsinny wrasse in Atlantic salmon cages. Four groups of wild caught goldsinny wrasse, each of 50 fish, were treated with an intraperitoneal injection of propylene glycol:saline (50:50) (control) or 50 mg/kg oxolinic acid at a concentration of 50 mg/mL. Three days after medication the fish in all groups were treated by an intraperitoneal injection of prednisolone acetate and an increase in seawater temperature from 9.0 to 11.5 degrees C. Cumulative mortalities were 18% in the two groups treated with oxolinic acid and 94 and 100% in the unmedicated control groups, giving a 'relative percentage survival' (RPS) value of 82%. A laboratory maintained population of originally wild caught corkwing wrasse experiencing high daily mortality was treated with oxolinic acid (50 mg/kg) or propylene glycol:saline (control). Cumulative mortalities were 84% (control) and 42% (oxolinic acid medicated group) giving an RPS value of 50%. In a field investigation using goldsinny wrasse approximately 30% were medicated with oxolinic acid (50 mg/kg) prior to stocking in cages with Atlantic salmon. In two of three cages the cumulative mortality was significantly lower (P = 0.025 and P < 0.001) in the medicated groups.     

Pharmacokinetics of oxolinic acid in sea-bass, Dicentrarchus labrax (L., 1758), after a single rapid intravascular injection

The pharmacokinetics of oxolinic acid was studied in sea-bass ( Dicentrarchus labrax ). The fish were kept in seawater at 15.2°C with a 12 h/12 h photoperiod. Oxolinic acid was injected in the caudal vein of anaesthetized sea-bass in a single rapid intravascular administration at a dose of 10 mg/kg of body weight. Plasma concentrations of oxolinic acid were determined using two analytical methods, a classic plate diffusion bioassay using Escherichia coli and a high performance liquid chromatography (HPLC) using solid phase extraction with an internal standard and a U.V. detection. The mean recoveries were 99.6% and 110.8% and determination limits were 0.04 μg/mL and 0.02 μg/mL, for the bioassay and the HPLC respectively. Compared to other fish species, the oxolinic acid was rapidly (absorption half life, t_a1/2= 0.69 h) distributed to body tissues outside the blood volume (volume of central compartment, V_c= 0.4 L/kg) and presented a large volume of distribution (V_dss= 2.55 L/kg). Considering its disappearance from the central compartment (rate constant: central-eliminated, k ₁₀= 0.16 h^–1) and its total body clearance ( Cl _t= 0.066 L/kg.h), the elimination phase of the oxolinic acid in sea-bass was shorter than in trout kept in freshwater, and longer than in salmon in seawater. Consequently, the area under the concentration–time curve ( AUC = 157 μg.h/mL) and the mean residence time ( MRT = 42 h) were relatively low and short, respectively.     

Pharmacokinetics of flumequine in sheep after intravenous and intramuscular administration: bioavailability and tissue residue studies

The pharmacokinetic properties of flumequine and its metabolite 7-hydroxyflumequine were determined in six healthy sheep after single intramuscular (i.m.) and intravenous (i.v) injections at a dose of 6 mg/kg body weight. The tissue residues were determined in 20 healthy sheep after repeated i.m. administration with a first dose of 12 mg/kg and nine doses of 6 mg/kg. The flumequine formulation used was Flumiquil 3% Suspension Injectable®. The mean plasma concentrations of flumequine after i.v. administration were described by a three-compartment open model with a rapid distribution and a relatively slow elimination phase. The low value of volume of distribution at steady state (V_dss) (0.52 ± 0.24 L/kg) and high value of volume of distribution (V_dλ₃) (5.05 ± 3.47 L/kg) emphasized the existence of a small compartment with a slow rate of return to the central compartment. The mean elimination half-life was 11.5 h. The 7-hydroxyflumequine plasma levels represented 2.3% of the total area under the curve. The mean plasma concentrations of flumequine after i.m. administration were characteristic of a two-compartment model with a first order absorption. The mean maximal plasma concentration (1.83 ± 1.15 μg/mL) was obtained rapidly, i.e. 1.39 ± 0.71 h after the i.m. administration. The fraction of dose absorbed from the injection site was 85.00 ± 30.13%. The minimal concentrations of flumequine during repeated treatment were significantly lower in females than in males. Eighteen hours after the last repeated i.m. admini-stration, the highest concentration of flumequine was observed at the injection sites followed by kidney, liver, muscle and fat. The highest concentration of 7-hydroxyflumequine was observed in the kidney and was ten times lower than the flumequine concentration. The longest flumequine elimination half-life was observed in the fat.     

Bacterial resistance to quinolone antibiotics in Poland

The resistance of 167 pathogenic bacteria of animal origin to quinolones was determined by the disc diffusion method, and by the minimum inhibitory concentration (MIC) test. The highest resistance of Escherichia coli was found to be against nalidixic acid (NA), 49.1% and flumequine (FLU), 38.2%. The sensitivity of the strains were: ciprofloxacin (CIP; 81.8%); enrofloxacin (ENR; 81.8%); norfloxacin (NOR; 80.0%); and pefloxacin (PE; 76.4%). Salmonella spp. showed 100% sensitivity to CIP, ENR, NOR and PE. A high resistance percentage in the cases of: FLU (86.7%); PE (50.0%); and CIP (26.65%) distinguished the Streptococcus spp. The highest percentage sensitivity of Staphylococci was found with three fluoroquinolones: CIP, ENR and NOR, 94.3% each (66 strains). The studies did not indicate that a total cross-resistance might occur between the examined quinolones.     

Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics.

Received October 27, 2015; accepted April 13, 2016 Published online August 2, 2016     

Depletion study of three formulations of flumequine in edible tissues and drug transfer into chicken feathers

Cornejo, J., Lapierre, L., Iragüen, D., Pizarro, N., Hidalgo, H., San Martín, B. Depletion study of three formulations of flumequine in edible tissues and drug transfer into chicken feathers. J. vet. Pharmacol. Therap. 34 , 168–175. To ensure the delivery of safe animal products to consumers, withdrawal times (WDT) of drugs must be respected. Drugs administered in therapies can also reach nonedible tissues (for humans) such as feathers; this transfer is of concern as feather meal is used in diets of food producing animals, being this a possible source of residue contamination of final products for human consumption. WDTs of three flumequine formulations (10%, 80% premix powder and 20% solution) as well as the transfer of this drug into feathers were determined. One hundred and twenty broiler chickens were allocated into four experimental groups (36 birds each). Three of them were treated with 24 mg/kg bw orally for five consecutive days of each flumequine formulation, whereas one group remained untreated (12 birds as control group). After the treatment ended, six chickens of each experimental group and two controls were slaughtered daily for 6 days. Samples of muscle, liver and feathers were collected and analyzed by liquid chromatography tandem mass spectrometry (LC MS/MS). The WDTs showed differences between formulations. Flumequine concentrations found in feathers remained high during WDT and after this period, thus suggesting that the WDTs estimated for the pharmaceutical formulation of flumequine do not guarantee the absence of this drug in chicken nonedible tissues such as feathers.     

Isolation and molecular characterization of quinolone resistant Salmonella spp. from poultry farms

The antimicrobial susceptibility of 94 Salmonella strains isolated from different poultry farms in Chile (broiler and laggin hens) were analyzed by the dilution plates method. Thirty-nine of them were resistant to flumequine, nalidixic acid and oxolinic acid with MIC values higher than 64 μg/ml. These quinolone resistant strains were analyzed in order to determine the presence of mutations in the QRDR region of gyrA gene by AS-PCR-RFLP analysis. 51.3% of the strains showed mutations at codon Ser 83 and 41.0% showed mutations at codon Asp 87. No mutations were observed on codon Gly 81. These mutations were confirmed by sequenciation of one representative strain from different RFLP pattern. Likewise, no double mutations were observed. Over 90% of the quinolone resistant strains presented mutations at the QRDR region of the gyrA gene. Three phenotypically resistant strains did not show any mutations on the QRDR region of gyrA gene. However, other molecular resistant mechanism could be involve. This is the first study that demonstrate the emergency of quinolone and fluoroquinolone resistance in Chilean Salmonella strains isolated from poultry thus indicating the requirement of monitoring programmes in veterinary medicine.     

Comparisons of nonspecific and specific immunomodulation by oxolinic acid, oxytetracycline and levamisole in salmonids

Oxolinic acid, a promising drug for the treatment of bacterial fish disease agents, was tested for possible immunomodulatory effects on fish. Another antibiotic oxytetracycline, known to be immunosuppressive at higher treatment doses, and levamisole, a known immunostimulator for higher vertebrates, were also compared for causing changes in the nonspecific defense compartment and the specific immune system in rainbow trout. Groups of fish were immunized with Yersinia ruckeri O-antigen bacterin in combination with selected doses of the drugs. The nonspecific defense activity was measured by demonstrating neutrophil metabolic activity by the nitroblue tetrazolium assay, by counting engulfed bacterial cells for a phagocytic index and by counting leukocytes with adherent bacterial cells for the adherence index. The specific immune response was monitored by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. The results showed that oxolinic acid, used at recommended doses for the treatment of bacterial diseases, did not cause immunosuppression in either the nonspecific defense or specific immune system compartments, whereas tetracycline at 10 mg/kg caused reduced activity in both. Fish given levamisole injections before the antigen injection showed a stimulated nonspecific defense but a much reduced specific immune response.     

Chlorinated pesticide residues and polychlorinated biphenyls in fish from various locations in Czechoslovakia

Samples of 14 fishes (175 samples on the whole) were examined in 1984 to 1986 for residues of chlorinated pesticides and polychlorinated biphenyls. Residues of the given substances were present in all the samples. The contents of chlorinated pesticide residues in fish are influenced mainly by the food spectrum; significant influence was also exerted by the contamination of the environment where the fish lived, by the age of the fish, and by the amount of their body fat in which the lipophile chlorinated carbohydrates are stored. No tolerance limits have been proposed in Czechoslovakia for the residues of chlorinated carbohydrates in fish bodies, except for the sum of DDT = 2.00 mg per kg of fat. When the data on the fish samples were compared with the highest admissible content of chlorinated pesticides per unit mass of fish muscle tissue tolerated in the FRG and the content of PCB tolerated in the USA, the fish from the Vltava river downstream of Prague fail to comply with these FRG and US regulations; all fish from all other places comply with them. Owing to large differences in the contents of fat in muscle between different fishes, it is more conclusive from the point of view of food hygiene to express the results as data per 1 kg of muscular tissue; these data represent real contents in fish as foodstuff.     

Pathogenicity and drug susceptibility of the Pasteurella anatis isolated in chickens in Taiwan

A strain of Pasteurella anatis (PA) was isolated from the sinus of an adult leghorn laying chicken with sinusitis, nasal discharge, drop in egg production, and low mortality, symptoms initially thought to indicate infectious coryza. The tiny, smooth, whitish colonies were identified as PA. To compare its pathogenicity with that of commercial broilers, nine groups, 10 birds per group, of 10-day-old broilers were individually inoculated with the strain of PA, Pasteurella multocida (PM), or Escherichia coli (EC) by intravenous, intraperitoneal, intramuscular, or subcutaneous inoculation. The PA was determined to cause the signs, lesions, and septicemic death, which are similar to the symptoms of PM or EC infection. At 1 wk postinfection (PI), the mortality rate was between that of PM and EC infection at 1 wk PI. Twenty antimicrobial-containing discs were evaluated, and the isolate was highly sensitive to cetiofer, amoxicillin, lincopectin, and furazolidone. Furthermore, it was moderately sensitive to tetracycline and enrofloxacin and only slightly sensitive to cephalothin, chloramphenicol, flumequine, nalidixic acid, neomycin, oxolinic acid, streptomycin, and trimethoprim. The PA infection was treated successfully with amoxicillin.     

Antibacterial effect of chloramphenicol, thiamphenicol and florfenicol against aquatic animal bacteria

The minimum inhibitory concentration (MIC) was measured to evaluate the antibacterial activities of chloramphenicol (CP), thiamphenicol (TP) and florfenicol (FFC) against the aquatic bacterial isolates from soft-shell turtles, fish and shellfish. Amoxicillin (AMPC), oxytetracycline (OTC) and oxolinic acid (OA) were included to compare with above protein synthesis inhibitors. The results showed that the order of MIC range of the isolates from soft-shell turtles for tested drugs was OA>FFC, CP>TP> AMPC, OTC. The percentage of the resistant strains indicated that OA was the lowest (7.14%) and OTC was the highest (85.07%). The order of antibacterial activity against the isolates from fish was OA>FFC>CP>AMPC>OTC>TP. The percentage of the resistant strains revealed that OA (13.64%) and OTC (80.91%) were the lowest and the highest, respectively. For the isolates from shellfish, the order of antimicrobial activity was OA>CP, FFC>AMPC, OTC, TP. TP showed the greatest percentage of the resistant strains (58.7%), but that of OA was the lowest (4.35%). The most common resistant patterns of the isolates from turtles, fish and shellfish were AMPC-OTC, CP-TP-AMPC-OTC, and FFC-CP-TP-AMPC-OTC, respectively. There were partially-complete resistance of the resistant isolates among CP, TP and FFC. The findings indicated that previous treatment might affect the choice of drug to use for aquatic bacterial diseases.     

Characteristics of Biceps femoris and Longissimus thoracis muscles of five cattle breeds grown in a feedlot system

Our experiment was conducted to study physical quality and chemical composition of meat of Angus (A), Hereford (H), Friesian Holstein (FH), Australian Commercial Cross (ACC) and Murray Grey (MG) cattle breeds grown in a feedlot system. Significant differences were observed among five cattle breeds for cooking loss, tenderness, fatty acid composition, collagen and cholesterol content of meat. However, pH, water‐holding capacity, water, ash, fat and crude protein contents were not different. Cholesterol content of meat of A and FH was lower (P < 0.01) than that of H, ACC and MG. Acidity (pH), cooking loss, shear force, water, ash, crude protein, collagen and cholesterol content of Biceps femoris muscle were higher than those of Longissimus thoracis muscle, but water‐holding capacity and fat content were lower. Unsaturated fatty acid content of Biceps femoris muscle was higher (P < 0.01) than that of Longissimus thoracis. In conclusion, the differences among cattle breeds had a great effect on cooking loss, shear force, collagen, fatty acid composition and cholesterol of meat, but had little effect on water‐holding capacity, pH and proximate composition of meat. The meat of Hereford cattle was the best quality, because of the lowest shear force, cooking loss and collagen. Longissimus thoracis muscle was relatively better in cooking loss, tenderness and collagen content than Biceps femoris muscle.     

食品中喹诺酮类兽药多残留检测技术的应用与发展

喹诺酮类兽药在食品中的残留问题引起各国的重视,建立高效灵敏的多残留检测方法是喹诺酮类兽药检测方法研究的热点和趋势.通过对微生物抑制法、色谱法、免疫法、蛋白芯片法在多残留检测技术的解析,概述食品中喹诺酮类药物多残留检测技术的应用现状与发展,为开展药物残留检测的研究和监测提供借鉴.     

食品中(氟)喹诺酮类兽药多残留检测技术的应用与发展

喹诺酮类兽药在食品中的残留问题引起各国的重视,建立高效灵敏的多残留检测方法是喹诺酮类兽药检测方法研究的热点和趋势。通过对微生物抑制法、色谱法、免疫法、蛋白芯片法在多残留检测技术的解析,概述食品中喹诺酮类药物多残留检测技术的应用现状与发展,为开展药物残留检测的研究和监测提供借鉴。