Qualitative risk assessment of homogeneity, stability, and residual concentrations of antimicrobials in medicated feed and drinking water in pig rearing.

BACKGROUND: Despite the common use of oral group treatment in pig rearing, the magnitude of the factors influencing the homogeneity and stability of antimicrobial drugs in medicated feed and medicated drinking water are largely unknown, as well as the residual concentrations of the drugs after the end of the treatment. RESULTS: This study presents a qualitative risk assessment to estimate the magnitude of the risks for reduced homogeneity and stability, and increased residual concentrations of antimicrobial drugs in medicated feed and drinking water on the farm. Risk assessment was done using a questionnaire and farm visits (n = 52), combined with a second questionnaire, and concentrations of amoxicillin and doxycycline measured in medicated feed and water samples, each collected on 10 farms. For medicated feed, the duration of storage in the silo did not show to influence the concentration levels in a consistent trend, while the treatment duration had a low to negligible effect. A moderate to high risk was found caused by human error when preparing the medicated feed on the farm. Purchased medicated feed greatly reduces the risk of human error and drugs remain stable during the duration of treatment, while the risk of residual concentrations after the end of the treatment was estimated to be low to moderate. The feed intake variability was identified as a moderate to high risk factor. For medicated drinking water, the type of dosing pump, age of pre-solution, and human errors during the preparation of the pre-solution present a moderate to high risk on homogeneity and stability. Precipitation of the active substance in the absence of a stirrer in a drinking water tank was shown to be a low to moderate risk factor for residues after treatment. Waterline length had a weak correlation with the concentrations of the antimicrobials, while a moderate to high influence was detected for the water intake by the pigs. CONCLUSIONS: A considerable variation in drug concentration in both medicated feed and medicated drinking water was detected depending on their preparation. Therefore, it is important to know which factors influence the homogeneity and stability, and the residual concentrations after treatment.

The impact of therapeutic-dose induced intestinal enrofloxacin concentrations in healthy pigs on fecal Escherichia coli populations.

BACKGROUND: Knowledge of therapy-induced intestinal tract concentrations of antimicrobials allows for interpretation and prediction of antimicrobial resistance selection within the intestinal microbiota. This study describes the impact of three different doses of enrofloxacin (ENR) and two different administration routes on the intestinal concentration of ENR and on the fecal Escherichia coli populations in pigs. Enrofloxacin was administered on three consecutive days to four different treatment groups. The groups either received an oral bolus administration of ENR (conventional or half dose) or an intramuscular administration (conventional or double dose). RESULTS: Quantitative analysis of fecal samples showed high ENR concentrations in all groups, ranging from 5.114 ± 1.272 µg/g up to 39.54 ± 10.43 µg/g at the end of the treatment period. In addition, analysis of the luminal intestinal content revealed an increase of ENR concentration from the proximal to the distal intestinal tract segments, with no significant effect of administration route. Fecal samples were also screened for resistance in E. coli isolates against ENR. Wild-type (MIC≤0.125 µg/mL) and non-wild-type (0.125 < MIC≤2 µg/mL) E. coli isolates were found at time 0 h. At the end of treatment (3 days) only non-wild-type isolates (MIC≥32 µg/mL) were found. CONCLUSIONS: In conclusion, the observed intestinal ENR concentrations in all groups showed to be both theoretically (based on pharmacokinetic and pharmacodynamic principles) and effectively (in vivo measurement) capable of significantly reducing the intestinal E. coli wild-type population.

Efficacy of gamithromycin against Ornithobacterium rhinotracheale in turkey poults pre-infected with avian metapneumovirus.

Ornithobacterium rhinotracheale is an avian respiratory pathogen that affects turkeys. The objective of this study was to evaluate the clinical efficacy of gamithromycin (GAM) against O. rhinotracheale in turkeys. The birds were inoculated oculonasally with 10(8) colony-forming units (cfu) of O. rhinotracheale, preceded by infection with avian metapneumovirus. In addition to a negative (CONTR-) and a positive control group (CONTR+) there were two treated groups administered GAM (6 mg/kg) either subcutaneously (GAM SC) or orally (GAM PO) by administration as a single bolus at one-day post-bacterial infection (p.b.i.). From the start of the avian metapneumovirus infection until the end of the experiment, the turkeys were examined clinically and scored daily. In addition, tracheal swabs were collected at several days p.b.i. Necropsy was performed at 4, 8 and 12 days p.b.i. to evaluate the presence of gross lesions, and to collect trachea and lung tissue samples and air sac swabs for O. rhinotracheale quantification. The clinical score of the GAM SC group showed slightly lower values and birds recovered earlier than those in the GAM PO and CONTR+ groups. O. rhinotracheale cfus were significantly reduced in tracheal swabs of the SC group between 2 and 4 days p.b.i. At necropsy, CONTR+ showed higher O. rhinotracheale cfu in lung tissues compared to the treated groups. Moreover, at 8 days p.b.i. only the lung samples of CONTR+ were positive. In conclusion, the efficacy of GAM against O. rhinotracheale was demonstrated, especially in the lung tissue. However, the PO bolus administration of the commercially available product was not as efficacious as the SC bolus.

Quantification of 8-α-hydroxy-mutilin as marker residue for tiamulin in rabbit tissues by high-performance liquid chromatography-mass spectrometry.

For the first time, a sensitive and specific method was developed and fully validated for the quantification of the EU marker residue of tiamulin, 8-α-hydroxy-mutilin, in rabbit muscle and liver tissues using liquid chromatography combined with positive heated electrospray ionization triple quadrupole mass spectrometry. The mass spectrometer was operated in the selected reaction monitoring (SRM) mode with selection of the [M + H](+) ion in both quadrupoles 1 and 3, resulting in the SRM transition m/z 337.25 > 337.25 for quantification. Chromatography was performed using a Hypersil Gold C18 column using a gradient elution program with water and methanol as mobile phases. The sample preparation procedure for the analysis of 8-α-hydroxy-mutilin in liver and muscle samples consisted of three main steps: (1) extraction of the tissue matrix using 0.1 N hydrochloric acid/acetone (50/50, v/v), (2) hydrolysis of tiamulin and metabolites to 8-α-hydroxy-mutilin in alkaline medium at 45 °C, and (3) liquid-liquid extraction in acidic medium using ethyl acetate. This is the first method presenting fully validated results, encompassing a linearity of 50 to 2,000 µg/kg, within-run and between-run accuracy and precision, limit of quantification (50 µg/kg for both muscle and liver tissues), limit of detection (muscle, 11.9 µg/kg; liver, 20.6 µg/kg), extraction recovery (muscle, 66.2%; liver, 75.5%), signal suppression and enhancement (muscle, 51.7%; liver, 43.3%), carryover, applicability and practicability, and stability during storage and analysis. This novel method is therefore sensitive enough to be used for residue depletion studies of tiamulin in rabbits and for food safety monitoring with respect to MRL compliance of residues.

Determination of selected veterinary antimicrobials in poultry excreta by UHPLC-MS/MS, for application in Salmonella control programs.

The most important source of Salmonella spp. infection in humans is by the consumption of contaminated poultry products. Due to the risk of resistance development and its transfer from animals to humans, the Belgian Royal Decree concerning the eradication of Salmonella (C-2007/22784) prohibits treatment of poultry with antimicrobials against zoonotic Salmonella spp. To uncover illicit use, an analytical method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the determination of antimicrobial residues in poultry excreta was developed and validated for classes having an active spectrum against Salmonella spp. in poultry: ß-lactams (amoxicillin and penicillin V), fluoroquinolones (enrofloxacin, difloxacin, and flumequine), polymyxins (colistin), sulfonamides in combination with trimethoprim (sulfachloropyridazine, sulfadiazine, and sulfaclozine), and tetracyclines (chlortetracycline and doxycycline). A generic and high-throughput sample preparation was developed. Extraction of samples was performed by ultrasonication using a combination of acetonitrile and McIlvaine buffer, followed by centrifugation and filtration prior to analysis. The method was validated according to Commission Decision 2002/657/EC for linearity, apparent recovery/trueness, repeatability, reproducibility, limit of quantification, limit of detection, specificity, matrix effect, and storage stability in matrix. To demonstrate the applicability of the method, an in vivo experiment was conducted. For each antimicrobial class, one registered drug was selected and administered in the drinking water to two laying hens. Excreta samples were collected every 12 h during and until 2 days after treatment and analyzed using the developed method.

Use of LC-MS-MS as an alternative to currently available immunoassay methods to quantitate corticosterone in egg yolk and albumen.

Corticosterone (CORT) is the dominant plasma glucocorticoid in birds. There has been increasing interest in the function of CORT in avian egg yolk and in the potential to use CORT concentrations in eggs to quantify stress and to assess the effect of maternal stress on offspring. The concentration of CORT in egg yolk is most frequently assessed using enzyme or radioimmunoassays, alone or in combination with high-performance liquid chromatography. However, the quantification of CORT is frequently hampered by the presence of high concentrations of other steroid hormones which cross-react with the CORT antibody. As an alternative, we developed a sensitive and specific LC-MS-MS method. The sample-preparation procedure consisted of a protein-lipid precipitation step, followed by defatting and clean-up using a C18 SPE column. Chromatography was performed on an Acquity C18 BEH column (50 mm × 2.1 mm i.d., dp: 1.7 µm, run-time: 6 min), using 0.1% formic acid in both water (A) and acetonitrile (B) as mobile phases. The MS-MS instrument was operated in the positive-electrospray-ionization mode. The method was validated in-house according to European Guidelines (linearity, accuracy and precision, limits of quantification and detection, specificity, stability) and the results fell within the accepted ranges. The method was successfully used for the analysis of CORT in yolk and albumen of eggs collected from eight breeding lesser black-backed gulls at a Flemish coastal colony. CORT concentrations were in the range 42.4-166.3 pg g(-1) in albumen and < LOQ (75 pg g(-1))-762.5 pg g(-1) in yolk.

Pharmacokinetic and pharmacodynamic properties of gamithromycin in turkey poults with respect to Ornithobacterium rhinotracheale.

The macrolide gamithromycin (GAM) has the ability to accumulate in tissues of the respiratory tract. Consequently, GAM might be a suitable antibiotic to treat bacterial respiratory infections in poultry, such as Ornithobacterium rhinotracheale. As O. rhinotracheale infections are common in turkey flocks, the aim of this study was to determine the pharmacokinetic (PK) parameters of GAM in plasma, lung tissue, and pulmonary epithelial lining fluid (PELF) of turkeys and to correlate them with pharmacodynamic (PD) characteristics (PK/PD). The animal experiment was performed with 64 turkeys, which received either a subcutaneous (SC, n=32) or an oral (PO, n=32) bolus of 6 mg GAM/kg body weight (BW). GAM concentrations in plasma, lung tissue, and PELF were measured at different time points post administration (p.a.), and PK characteristics were determined using non-compartmental modeling. The maximum plasma concentration after PO administration was ten-fold lower than after SC injection (0.087 and 0.89 µg/mL, respectively), whereas there was no difference in lung concentrations between both routes of administration. However, lung concentrations at day 1 p.a. were significantly higher than plasma levels for both routes of administration (2.22 and 3.66 µg/g for PO and SC, respectively). Consequently, lung/plasma ratios were high, up to 50 and 80 after PO and SC administration, respectively. GAM could not be detected in PELF, although this might be attributed to the collection method of PELF in birds. The GAM minimum inhibitory concentration (MIC) was determined for 38 O. rhinotracheale strains; MIC50 and MIC90 were 2 and >32 µg/mL, respectively. PK/PD correlation for lung tissue demonstrated that the time above the MIC90 of the susceptible population (2 µg/mL) was 1 day after PO bolus and 3.5 days after SC administration. The area under the curve (AUClast)/MIC ratios for lung tissue after SC and PO administration were 233 and 90, respectively. To conclude, GAM is highly distributed to lung tissue in turkey poults, suggesting that it has the potential to be used to treat respiratory infections such as O. rhinotracheale.

Effect of administration route and dose escalation on plasma and intestinal concentrations of enrofloxacin and ciprofloxacin in broiler chickens.

BACKGROUND: The (mis)use of fluoroquinolones in the fowl industry has led to an alarming incidence of fluoroquinolone resistance in pathogenic as well as commensal bacteria. Next to simply reducing antimicrobial consumption, optimizing dosage regimens can be regarded as a suitable strategy to reduce antimicrobial resistance development without jeopardizing therapy efficacy and outcome. A first step in order to limit antimicrobial resistance is to assess the exposure of the intestinal microbiota to enrofloxacin after different treatment strategies. Therefore, a study was conducted in broiler chickens to assess the effect of route of administration (oral versus intramuscular) and dose escalation (10 and 50 mg/kg body weight) on plasma and intestinal concentrations of enrofloxacin and its main metabolite ciprofloxacin after treatment with enrofloxacin once daily for five consecutive days. Four different parts of the intestinal tract were sampled: ileum, cecum, colon and cloaca. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify both analytes in plasma and intestinal content. Sample preparation prior to LC-MS/MS analysis consisted of extraction with ethyl acetate. For intestinal content samples PBS buffer was added before extraction. The supernatant was evaporated to dryness and resuspended in water prior to analysis. RESULTS: The results in plasma and intestinal content demonstrated that biotransformation of enro- to ciprofloxacin in broiler chickens is limited. In general, the intestinal microbiota in cecum and colon is exposed to significant levels of enrofloxacin after conventional treatment (21-130 µg/g). A clear increase of intestinal concentrations was demonstrated after administration of a five-fold higher dose (31-454 µg/g). After intramuscular administration, intestinal concentrations were comparable, except for the higher levels in cloaca due to the complete bioavailability and urinary excretion. CONCLUSIONS: The intestinal microbiota is exposed to high levels of the antimicrobial, after oral as well as parenteral therapy. Furthermore, a dose and time dependent correlation was observed. The impact of the detected intestinal levels on resistance selection in the intestinal microbiota has to be further investigated.

Clinical efficacy of florfenicol administered in the drinking water against Ornithobacterium rhinotracheale in turkeys housed in different environmental conditions: a pharmacokinetic/pharmacodynamic approach.

In poultry rearing, medicated drinking water is a commonly used administration route, but drug uptake can be affected by many factors. In this study, the influence of two important parameters, the photoperiod and feeding schemes, on florfenicol uptake in turkeys was tested. First, the uptake was determined as the pharmacokinetic/pharmacodynamic profile of florfenicol; and second, we evaluated the clinical efficacy of florfenicol against Ornithobacterium rhinotracheale. Both experiments were conducted during a 5-day treatment of 30 mg/kg body weight florfenicol administered via drinking water and considering different photoperiods and feeding schemes (group 20/4L: photoperiod of 20 h, fed ad libitum; group 16/8L: photoperiod of 16 h, fed ad libitum; group 16/8R: photoperiod of 16 h, fed ad libitum but feed was withdrawn during the dark period and replaced 1 h after lighting). On day 1 of treatment, all groups showed plasma concentrations above the minimum inhibitory concentration (both MIC50 and MIC90, 1 mg/l) of 37.7%, 63.5% and 53.1% of a 24-h interval for 20/4L, 16/8L and 16/8R, respectively. Only in the 16/8L and 16/8R groups was the MIC also exceeded on day 5 (47.9% and 21.5% of a 24-h interval, respectively). In all groups, a clinical improvement could be noticed, resulting in reduction of the clinical score. However, only the 16/8L and 16/8R groups showed significant differences from the control group. The results demonstrated an important influence of the photoperiod on the pharmacokinetics of florfenicol as well as the clinical outcome in an infection model. It can be advised that the photoperiod should be <20 h to have sufficient drug intake. Nevertheless, there was no effect between fed and fasted turkeys for both the pharmacokinetics and the clinical outcome.

T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions.

The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 µg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.

Porcine intestinal epithelial barrier disruption by the Fusarium mycotoxins deoxynivalenol and T-2 toxin promotes transepithelial passage of doxycycline and paromomycin.

BACKGROUND: The gastrointestinal tract is the first target for the potentially harmful effects of mycotoxins after intake of mycotoxin contaminated food or feed. With deoxynivalenol (DON), T-2 toxin (T-2), fumonisin B1 (FB1) and zearalenone (ZEA) being important Fusarium toxins in the northern hemisphere, this study aimed to investigate in vitro the toxic effect of these mycotoxins on intestinal porcine epithelial cells derived from the jejunum (IPEC-J2 cells). Viability of IPEC-J2 cells as well as the proportion of apoptotic and necrotic IPEC-J2 cells was determined by flow cytometry after 72 h of exposure to the toxins. Correlatively, the integrity of the intestinal epithelial cell monolayer was studied using Transwell(®) inserts, in which the trans-epithelial electrical resistance (TEER) and passage of the antibiotics doxycycline and paromomycin were used as endpoints. RESULTS: We demonstrated that the percentage of Annexin-V-FITC and PI negative (viable) cells, Annexin-V-FITC positive and PI negative (apoptotic) cells and Annexin-V-FITC and PI positive (necrotic) IPEC-J2 cells showed a mycotoxin concentration-dependent relationship with T-2 toxin being the most toxic. Moreover, the ratio between Annexin-V-FITC positive and PI negative cells and Annexin-V-FITC and PI positive cells varied depending on the type of toxin. More Annexin-V-FITC and PI positive cells could be found after treatment with T-2 toxin, while more Annexin-V-FITC positive and PI negative cells were found after exposure to DON. Consistent with the cytotoxicity results, both DON and T-2 decreased TEER and increased cellular permeability to doxycycline and paromomycin in a time- and concentration-dependent manner. CONCLUSIONS: It was concluded that Fusarium mycotoxins may severely disturb the intestinal epithelial barrier and promote passage of antibiotics.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?

BACKGROUND: The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated. RESULTS: Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 µg/ml for florfenicol and 8.0 µg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 µg/ml florfenicol or 16 µg/ml trimethoprim combined with 80 µg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 µg/ml but not to 10 µg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 µg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection. CONCLUSIONS: We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Cutaneous hyalohyphomycosis in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of Nannizziopsis vriesii and successful treatment with voriconazole.

The Chrysosporium anamorph of Nannizziopsis vriesii was associated with dermatomycosis and high mortality in a group of captive giant girdled lizards (Cordylus giganteus). Treatment of one of the infected girdled lizards with voriconazole, which was selected on the basis of in vitro sensitivity testing of the isolate, resulted in resolution of lesions and negative fungal cultures from the skin. Three hours after oral administration of 10 mg/kg, the plasma level of voriconazole exceeded the 0.25-µg/mL minimal inhibitory concentration tenfold. In conclusion, administration of voriconazole at 10 mg/kg of body weight once daily for 10 weeks resulted in clinical cure and was well tolerated. A longer follow-up time and larger studies will be necessary to determine the long-term efficacy and safety of this treatment in giant girdled lizards.

Multi-residue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry.

A sensitive method for the simultaneous quantification of eight anticoagulant rodenticides (brodifacoum, bromadiolone, chlorophacinone, coumatetralyl, difenacoum, difethialone, flocoumafen and warfarin) in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) is described. The sample preparation includes a liquid-liquid extraction with acetone. The compound 7-acetoxy-6-(2,3-dibromopropyl)-4,8-dimethylcoumarin is used as an internal standard. Chromatographic separation was achieved using a Nucleodur C18 gravity column. Good linearity was observed up to 750 ng mL(-1) for chlorophacinone and up to 500 ng mL(-1) for the other compounds in plasma. In liver, good linearity was seen up to 500 ng g(-1) for brodifacoum, chlorophacinone, difenacoum and difethialone and up to 750 ng g(-1) for the other compounds. Depending on the compound, a level of 1 or 5 ng mL(-1) could be quantified fulfilling the criteria for accuracy and precision and was therefore set as limit of quantification of the method in plasma. In liver, the limit of quantification was set at 250 ng g(-1) for coumatetralyl and warfarin and at 100 ng g(-1) for the other compounds. In plasma, the limit of detection varied from 0.07 ng mL(-1) for flocoumafen to 3.21 ng mL(-1) for brodifacoum. In liver, the limit of detection varied from 0.37 ng g(-1) for warfarin to 4.64 ng g(-1) for chlorophacinone. The method was shown to be of use in a pharmacokinetic study after single oral administration to mice and in the confirmation of suspected poisoning cases in domestic animals.

Rapid method for the quantification of amoxicillin and its major metabolites in pig tissues by liquid chromatography-tandem mass spectrometry with emphasis on stability issues.

A fast method for the quantitative determination of amoxicillin (AMO), amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione (DIKETO) in pig edible tissues (kidney, liver, fat and muscle) with liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method uses a simple liquid-liquid extraction of the tissue matrix with a 10 mM potassium dihydrogen phosphate buffer (pH 4.5) as extraction solvent. After deproteinisation by ultrafiltration, the tissue extract was directly injected onto the LC column. Chromatographic separation of the components was performed on a PLRP-S polymeric column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray MS/MS mode. The method was fully validated according to EU requirements (linearity, precision, trueness, quantification limit, detection limit and specificity). The stability of the components was evaluated over the pH range from 1.2 to 8.0. Biological samples of pigs medicated with AMO and AMO/clavulanic acid were analyzed using the developed method.

Tissue depletion of amoxicillin and its major metabolites in pigs: influence of the administration route and the simultaneous dosage of clavulanic acid.

A residue depletion study of amoxicillin (AMO) and its major metabolites, amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione, was performed after a single oral (p.o.) and intravenous (i.v.) administration of amoxicillin (20 mg kg (-1)) and amoxicillin/clavulanic acid (20 and 5 mg kg (-1)) to pigs. Animals were slaughtered 12, 36, 48, 60, 72, and 84 h after dosing. Tissue samples were analyzed using liquid chromatography-tandem mass spectrometry. Kidney samples contained high concentrations of amoxicilloic acid metabolite, which depleted much slower from tissues than amoxicillin, both after p.o. (t1/2AMO = 4.5 h vs t1/2AMA = 8 h) and i.v. (t1/2AMO = 4 h vs t1/2AMA = 8 h) administration. Moreover, after oral administration, significantly higher amoxicilloic acid concentrations were measured in liver and kidney than after i.v. administration. The coadministration of amoxicillin with clavulanic acid provoked no significant differences in amoxicilloic acid tissue concentrations as compared to an amoxicillin dosing. The prolonged presence of residues of amoxicilloic acid in edible tissues can play an important role in food safety, because the compound could give rise to a possible health risk, although it is not included in the maximum residue limit legislation.

Transvenous electrical cardioversion of atrial fibrillation in six horses using custom made cardioversion catheters.

Pharmacological conversion of atrial fibrillation (AF) to sinus rhythm in horses can be difficult. The objective of this study was to investigate the feasibility of transvenous electrical cardioversion with custom made catheters in eight horses, of which three had failed cardioversion using quinidine sulfate. Two cardioversion catheters and one pacing/sensing electrode were inserted via the right jugular vein and placed using ultrasound guidance into the left pulmonary artery, the right atrium and the right ventricle, respectively. Because immediate recurrence of AF was encountered in the second horse treated, pre-treatment with amiodarone was given to each of the remaining six horses. Induction of general anaesthesia was associated with dislocation of the cardioversion catheter in three horses, requiring a second catheterisation procedure. During general anaesthesia, biphasic R wave synchronised shocks of up to 360 J were delivered between both cardioversion electrodes. In six horses (75%), including two which had failed quinidine sulfate treatment, sinus rhythm was restored with a mean energy level of 295+/-62 J. No side effects were observed. Blood analysis 3 h after cardioversion revealed normal parameters, including cardiac troponin I values. Transvenous electrical cardioversion of atrial fibrillation with custom made cardioversion catheters can be considered as a treatment option for atrial fibrillation in horses, especially when conventional drugs fail.

Similar Gastro-Intestinal Exposure to Florfenicol After Oral or Intramuscular Administration in Pigs, Leading to Resistance Selection in Commensal Escherichia coli.

Florfenicol, which is licensed for veterinary use only, proves to be a potent antimicrobial for treatment of respiratory disease. However, the subsequent exposure of the gut microbiota to florfenicol is not well described. Hence, the effect of various administration protocols on both plasma and gastro-intestinal florfenicol concentrations in pigs was evaluated. In field situations were simulated by application of different administration routes and dosages [single oral bolus at 10 or 5 mg/kg body weight (BW), medicated feed at 10 or 5 mg/kg BW and intramuscular injections at 15 or 30 mg/kg BW]. After intramuscular administration of 30 mg florfenicol/kg BW, gastro-intestinal concentrations of florfenicol, quantified 10 h after the last administration, were significantly elevated in comparison with the other treatment groups and ranging between 31.5 and 285.8 µg/g over the different gut segments. For the other treatment groups, the influence of dose and administration route was not significantly different. Bacteriological analysis of the fecal samples from the animals at the start of the experiment, demonstrated the presence of both florfenicol susceptible (with minimal inhibitory concentration (MIC) values of 2-16 µg/mL) and florfenicol resistant (MIC ≥ 256 µg/mL) Escherichia coli isolates in all treatment groups. Following, at 10 h after the last administration the susceptible E. coli population was eradicated in all treatment groups due to the high intestinal florfenicol concentrations measured. Moreover, selection of the resistant E. coli strains during treatment occurred in all groups. This is likely related to the fact that the different treatment strategies led to high gastro-intestinal concentrations albeit not reaching the high magnitude of MIC values associated with florfenicol resistance (≥256 µg/mL). Conclusively, in our experimental setup the administration route and dose alterations studied, had no influence on monitored florfenicol resistance selection in E. coli from the microbiota.

Quantitative determination of dihydrostreptomycin in bovine tissues and milk by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Dihydrostreptomycin (DHS) is an aminoglycoside antibiotic used in veterinary medicine in combination with benzylpenicillin for the treatment of bacterial infections in cattle, pigs and sheep. A method to determine its residues in edible tissues of cattle, as well as in milk, was developed and validated. Extraction of DHS from the tissues was performed using a liquid extraction with a 10 mM phosphate buffer containing 2% (w/v) trichloroacetic acid, while milk samples were treated with a 50% (w/v) trichloroacetic acid solution, followed by a solid-phase clean-up procedure on a carboxypropyl (CBA) weak cation exchange column. Ion-pair chromatography, using a mixture of 20 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase, was used to retain DHS and the internal standard streptomycin (STR) on a Nucleosil (5 microm) reversed-phase C18 column. The components were detected and quantified by electrospray ionization (ESI) tandem mass spectrometry. The method could be validated according to EC (European Community) requirements with respect to linearity, trueness and precision, the latter evaluated at the maximum residue limit (MRL) - 1000 ng g(-1) for kidney, 500 ng g(-1) for muscle, liver and fat, and 200 ng g(-1) for milk -, at one-half of the MRL and at one and a half times the MRL. A limit of quantification of 10 ng g(-1) and 1 ng ml(-1) was obtained for all tissues and for milk, respectively, which is far below one-half of the MRL as requested, while the limit of detection was in the low ppb range, varying between 1.9 and 4.2 ng g(-1) for the different tissues tested, and being 0.6 ng ml(-1) for milk. The method was used for the monitoring of DHS residues in incurred tissue and milk samples coming from cattle medicated with DHS in combination with benzylpenicillin by intramuscular injection, in order to evaluate withdrawal times.

Effect of administration route and dose alteration on sulfadiazine-trimethoprim plasma and intestinal concentrations in pigs.

Potentiated sulfonamides, such as sulfadiazine-trimethoprim (SDZ-TRIM), are frequently used antimicrobials in both human and veterinary medicine. To optimise their use in relation to the emerging problem of resistance selection, this paper studied the impact of dose and administration route of SDZ-TRIM on the exposure of the gut microbiota to these antimicrobials. An animal experiment was conducted with 36 pigs, divided into six different treatment groups (n = 6). Three different administration routes were outlined: oral (PO) gavage, intramuscular (IM) injection and medicated feed, with 5-day therapy duration. Conventional dosing (30 mg SDZ-TRIM/kg bodyweight [BW]) and half dosing (15 mg SDZ-TRIM/kg BW) was performed for the oral routes in two applications per day. For the IM route, a conventional dose of 15 mg SDZ-TRIM/kg BW or a double dose of 30 mg SDZ-TRIM/kg BW was administered once daily. After daily collection of blood and faeces, the intestinal content of all animals was sampled in different gastrointestinal tract (GIT) segments, and SDZ and TRIM were quantified. Remarkably, SDZ accumulated in distal GIT segments, independently of the administration route. High concentrations (mean ± standard deviation) up to 26.93 ± 8.36 µg/g, 11.15 ± 3.78 µg/g and 19.36 ± 1.86 µg/g after PO gavage, IM administration and medicated feed, respectively, were measured for SDZ. In contrast, TRIM concentrations decreased from proximal to distal segments and were mostly below the limit of quantification (0.025 µg/g). The high oral bioavailability of SDZ indicates gastrointestinal secretion is a substantial elimination route for SDZ.