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To date, information about reasons to select and kill poultry on-farm and which method veterinarians and poultry producers preferably use is scarce. Little is also known about their knowledge of the legislation regarding on-farm killing methods and of methods alternative to the one(s) they use, as well as their perception of those alternatives. In this study, Flemish poultry veterinarians (n = 13), broiler chicken producers (n = 27), and turkey producers (n = 4) were surveyed on killing methods they currently use in practice and alternative methods, on their opinion about what constitutes an appropriate method for on-farm killing of poultry, and on their reasons for killing. All poultry veterinarians and chicken producers who filled out the survey kill poultry by manual cervical dislocation (CD), whereas some turkey farmers also indicated killing by percussive blow to the head (n = 1) or exsanguination (n = 1). Turkey producers seem to be more inclined not to kill animals with injuries or symptoms of disease as compared to veterinarians or chicken producers, such that moribund turkeys are more likely to remain in the flock. Veterinarians considered the following properties of a killing method important: animal friendliness, applicability inside the stable, a high success rate, and time efficiency. Producers ranked the properties similarly, but for them, ease of performance and cost efficiency were more important than applicability inside the stable. Producers scored those killing properties rather positively for manual as well as mechanical CD. Veterinarians and chicken producers considered the captive bolt method to be easy to perform, to have a high success rate, to be feasible to perform in the stable, and to be animal friendly. Turkey producers, however, had doubts about the latter 2 properties. Gas, injection, and electrocution were inferior methods to kill poultry according to producers. In conclusion, manual CD is the most common method for killing broiler chickens and turkeys, and knowledge of, and experience with, alternative methods is very limited, both among veterinarians and producers. Informing them about legislation and training for the use of alternative killing techniques are recommended. Flemish Government [LNE/STG/DWZ/16/07] The authors would like to thank the Flemish Government for financing this project (LNE/STG/DWZ/16/07) and Michael Plante-Ajah (Animal Sciences Unit, ILVO, Melle, Belgium) for proof-reading the manuscript as a native English speaker.

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.

The immunomodulatory properties of gamithromycin (GAM), ketoprofen (KETO) and their combination (GAM-KETO) were investigated after both in vitro and in vivo lipopolysaccharide (LPS)-induced inflammation. The influence of these drugs was measured on the production of prostaglandin E2 (PGE2) and the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in both LPS-stimulated porcine peripheral blood mononuclear cells (PBMCs) and LPS-challenged pigs. Additionally, effects on the production of acute phase proteins (APPs), including pig major acute phase protein (pig-MAP) and C-reactive protein (CRP), as well as on the development of fever, pulmonary symptoms and sickness behaviour were investigated. Dexamethasone was included as a positive control in the in vitro research. Following an 18h-incubation period with 1.25μg/mL LPS, the levels of TNF-α, IL-1β and IL-6 (p

A sensitive and specific method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid (PELF) using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis(®) Ostro™ 96-well plate (chicken, turkey and calf plasma) or HybridSPE(®)-Phospholipid SPE cartridges (pig plasma and turkey lung tissue), while a liquid-liquid extraction with diethyl ether in alkaline medium was used for PELF of turkey poults. Chromatography was performed on a C18 Hypersil GOLD column using 0.01M ammonium acetate in water with a pH of 9, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the following selected reaction monitoring transitions were monitored for gamithromycin (protonated molecule>product ion): m/z 777.45>619.35 and m/z 777.45>157.80 for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration ranges tested (2.5-10,000ngmL(-1) for chicken, pig and calf plasma; 5.0-2500ngmL(-1) for turkey plasma; 50-10,000ngg(-1) for turkey lung tissue and 20-1000ngmL(-1) for turkey PELF). Limits of quantification (LOQ) were 2.5ngmL(-1) for chicken, pig and calf plasma and 5.0ngmL(-1) for turkey plasma, while the limits of detection (LOD) ranged between 0.007 and 0.07ngmL(-1). For lung tissue and PELF, respective LOQ and LOD values of 50ngg(-1) and 0.76ngg(-1) (lung tissue) and 20ngmL(-1) and 0.1ngmL(-1) (PELF) were obtained. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values. The accuracy fell within -30% to +10% (concentrations 1-10ngmL(-1)) or -20% to +10% (concentrations>10ngmL(-1) or ngg(-1)) of the theoretical concentration. The method was successfully applied for the quantitative determination of gamithromycin in plasma samples of chickens, turkeys, pigs and calves; and in lung tissues and PELF of turkeys, all derived from pharmacokinetic studies in these animal species.

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n=3) and an LPS-challenged group (n=11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5h p.a.) and tachycardia (5h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.

The aim of this study was to investigate the pharmacokinetic properties of gamithromycin in pigs after an intravenous (i.v.) or subcutaneous (s.c.) bolus injection of 6 mg/kg body weight. The plasma concentrations of gamithromycin were determined using a validated high-performance liquid chromatography-tandem mass spectrometry method, and the pharmacokinetics were noncompartmentally analysed. Following i.v. administration, the mean area under the plasma concentration-time curve extrapolated to infinity (AUCinf) and the mean elimination half-life (t1/2λz) were 3.67 ± 0.75 μg.h/mL and 16.03 h, respectively. The volume of distribution at steady state (Vss) and the plasma clearance were 31.03 ± 6.68 L/kg and 1.69 ± 0.33 L/h.kg, respectively. The mean residence time (MRTinf) was 18.84 ± 4.94 h. Gamithromycin administered subcutaneously to pigs demonstrated a rapid and complete absorption, with a mean maximal plasma concentration (Cmax) of 0.41 ± 0.090 μg/ml at 0.63 ± 0.21 h and a high absolute bioavailability of 118%. None of the reported pharmacokinetic variables significantly differed between both administration routes.

Contamination of feeds with mycotoxins is a worldwide problem and mycotoxin-detoxifying agents are used to decrease their negative effect. The European Food Safety Authority recently stated guidelines and end-points for the efficacy testing of detoxifiers. Our study revealed that plasma concentrations of deoxynivalenol and deepoxy-deoxynivalenol were too low to assess efficacy of 2 commercially available mycotoxin-detoxifying agents against deoxynivalenol after 3 wk of continuous feeding of this mycotoxin at concentrations of 2.44±0.70 mg/kg of feed and 7.54±2.20 mg/kg of feed in broilers. This correlates with the poor absorption of deoxynivalenol in poultry. A safety study with 2 commercially available detoxifying agents and veterinary drugs showed innovative results with regard to the pharmacokinetics of 2 antibiotics after oral dosing in the drinking water. The plasma and kidney tissue concentrations of oxytetracycline were significantly higher in broilers receiving a biotransforming agent in the feed compared with control birds. For amoxicillin, the plasma concentrations were significantly higher for broilers receiving an adsorbing agent in comparison to birds receiving the biotransforming agent, but not to the control group. Mycotoxin-detoxifying agents can thus interact with the oral bioavailability of antibiotics depending on the antibiotic and detoxifying agent, with possible adverse effects on the health of animals and humans.

Macrolide antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to be modulators of the innate immune response, irrespectively of their antimicrobial and anti-inflammatory actions. Therefore, it was our objective to evaluate whether the macrolide gamithromycin (GAM) and the NSAID ketoprofen (KETO) attenuate the acute-phase response in calves, and whether their combined administration is beneficial due to synergistic and/or additive effects. To this end, both drugs, as well as their combination, were studied in a previously developed inflammation model, i.e., the induction of an acute-phase response by an intravenous lipopolysaccharide (LPS) challenge (0.5 μg/kg body weight). Sixteen 4-week-old Holstein-Friesian calves were randomized into 4 groups: a positive control (+CONTR) group, receiving LPS but no pharmacological treatment (n=4) and a GAM (n=4), a KETO (n=4) and a GAM-KETO (n=4) group, receiving the respective drugs 1h prior to LPS administration. Clinical scoring and blood collection were performed at regular time points until 72 h post LPS challenge. Plasma concentrations of the selected cytokines (tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6)), acute-phase protein (serum amyloid A (SAA)) and prostaglandin E2 (PGE2) were subsequently quantified. Pre-treatment with GAM had no effect in the inflammation model compared to the +CONTR group. KETO, on the other hand, completely inhibited depression, anorexia and fever. This remarkable influence was associated with a significant reduction of PGE2 synthesis by KETO, while the effect on TNF-α, IL-6 and SAA was not straightforward. The combined administration of GAM and KETO provided no synergistic or additive effects in this model, neither clinically nor regarding the studied inflammatory mediators. In conclusion, KETO entirely inhibited PGE2 synthesis, fever development and depression, while GAM did not exert any effect in this model. These results promote the concomitant use of an antimicrobial drug and a NSAID in the treatment of calf diseases associated with LPS, both to enhance clinical recovery and to improve animal welfare.

The aim of this study was to define the in vivo immunomodulatory properties of the macrolide antibiotic gamithromycin in calves, with respect to the acute phase response. Additionally, the corticosteroid dexamethasone was included as a positive control immunomodulatory drug. Both drugs, as well as their combination,were studied in a previously developed inflammation model,which was initiated by an intravenous lipopolysaccharide (LPS) challenge (0.5 μg/kg body weight). Twenty-four 4-week-old male Holstein Friesian calves were randomized into four groups: no pharmacological treatment (n = 6) or a pharmacological treatment with gamithromycin (n= 6), dexamethasone (n= 6) or their combination (n= 6) 1 h prior to LPS administration. Blood collection and clinical scoring were performed at regular time points until 72 h post LPS challenge. Plasma concentrations of selected cytokines (tumour necrosis factor-α (TNF-α) and interleukin 6 (IL-6)) and acute phase proteins (serum amyloid A and haptoglobin) were subsequently determined. Gamithromycin did not have any beneficial effect on the LPS-induced clinical signs (dyspnea, fever, anorexia and depression), nor on the studied inflammatory mediators. In the dexamethasone and combination groups, the occurrence of dyspnea and fever was not prominently influenced, although the calves recovered significantly faster from the challenge. Moreover, dexamethasone significantly inhibited the levels of TNF-α and IL-6, suggesting a key role for these cytokines in sickness behaviour. In conclusion, unlike dexamethasone, gamithromycin did not directly reduce cytokine release in an LPS inflammation model in calves.

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 mu g/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 mu g/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 mu g/mL, respectively. PK/PD correlation for lung tissue demonstrated that the time above the MIC90 of the susceptible population (2 mu g/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.

The pharmacokinetic properties of ketoprofen were determined in 4-week-old calves after intramuscular (i.m.) injection of a racemic mixture at a dose of 3 mg/kg body weight. Due to possible enantioselective disposition kinetics and chiral inversion, the plasma concentrations of the R(-) and S(+) enantiomer were quantified separately, using a stereospecific HPLC-UV assay. A distinct predominance of the S(+) enantiomer was observed, as well as significantly different pharmacokinetic parameters between R(-) and S(+) ketoprofen. More in specific, a greater value for the mean area under the plasma concentration-time curve (AUC(0→∞)) (46.92 ± 7.75 and 11.13 ± 2.18 μg·h/mL for the S(+) and R(-) enantiomer, respectively), a lower apparent clearance (Cl/F) (32.8 ± 5.7 and 139.0 ± 25.1 mL/h·kg for the S(+) and R(-) enantiomer, respectively) and a lower apparent volume of distribution (V(d)/F) (139 ± 14.7 and 496 ± 139.4 mL/kg for the S(+) and R(-) enantiomer, respectively) were calculated for the S(+) enantiomer, indicating enantioselective pharmacokinetics for ketoprofen in calves following i.m. administration.

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

Gamithromycin is a new macrolide antibiotic that is only registered for use in cattle to treat respiratory disorders such as bovine respiratory disease. The aim of this study was to determine the pharmacokinetics of gamithromycin in broiler chickens. Gamithromycin (6 mg/kg of BW) was injected intravenously (IV) or subcutaneously (SC) to six 4-wk-old chickens in a parallel study design, and blood was collected at different time points postadministration. Quantification of gamithromycin in plasma was performed using an in-house validated liquid chromatography-tandem mass spectrometry method and the pharmacokinetics analyzed according to a 2-compartmental model. Following IV administration, the mean area under the plasma concentration-time curve (AUC0→∞), and α and β half-life of elimination (t1/2el α and t1/2el β) were 3,998 h•ng/mL, 0.90 h, and 14.12 h, respectively. Similar values were obtained after a SC bolus injection, i.e., 4,095 h•ng/mL, 0.34 h, and 11.63 h, for AUC0→∞, t1/2el α, and t1/2el β, respectively. The mean maximum plasma concentration (889.46 ng/mL) appeared at 0.13 h. Gamithromycin showed a large volume of distribution after IV as well as SC administration, 27.08 and 20.89 L/kg, respectively, and a total body clearance of 1.61 and 1.77 L/h•kg, respectively. The absolute bioavailability was 102.4%, showing that there is a complete absorption of gamithromycin after a SC bolus injection of 6 mg/kg of BW.

The pharmacokinetics of dexamethasone (DEX) were investigated after an intravenous (IV) or intramuscular (IM) bolus injection of 0.3mg/kg bodyweight DEX sodium phosphate in pigs. The plasma concentrations of DEX were determined using a validated high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and the pharmacokinetics were determined by one-compartmental analysis. The mean area under the plasma concentration-time curve and the mean elimination half-life were 133.07 ± 39.59 ng.h/mL and 0.77 h, and 173.24 ± 53.59 ngh/mL and 1.06 h following IV and IM administration, respectively. The volume of distribution and clearance recorded after IV administration were 2.78 ± 0.88 L/kg and 2.39 ± 0.57 L/hkg, respectively. An IM bolus injection of DEX sodium phosphate in pigs resulted in a fast and complete absorption, with a mean maximal plasma concentration of 80.94 ± 21.29 ng/mL at 0.35 ± 0.21 h and a high absolute bioavailability of 131.06 ± 26.05%.

Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were