RESUMO
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 (6â 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.
Assuntos
Antibacterianos/farmacologia , Infecções por Flavobacteriaceae/veterinária , Metapneumovirus/fisiologia , Ornithobacterium/efeitos dos fármacos , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/tratamento farmacológico , Perus/microbiologia , Sacos Aéreos/microbiologia , Animais , Feminino , Infecções por Flavobacteriaceae/tratamento farmacológico , Infecções por Flavobacteriaceae/microbiologia , Pulmão/microbiologia , Macrolídeos/farmacologia , Ornithobacterium/crescimento & desenvolvimento , Infecções por Paramyxoviridae/tratamento farmacológico , Infecções por Paramyxoviridae/virologia , Doenças das Aves Domésticas/microbiologia , Traqueia/microbiologiaRESUMO
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.
Assuntos
Reação de Fase Aguda/veterinária , Antibacterianos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Fatores Imunológicos/uso terapêutico , Cetoprofeno/uso terapêutico , Macrolídeos/uso terapêutico , Reação de Fase Aguda/dietoterapia , Reação de Fase Aguda/imunologia , Animais , Bovinos , Dinoprostona/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Lipopolissacarídeos , Masculino , Proteína Amiloide A Sérica/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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-1ß 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-1ß and IL-6 (p<0.05) measured in the PBMC supernatants were significantly increased. Incubation with a high concentration of both GAM and KETO significantly reduced the in vitro levels of all three cytokines. Maximal plasma concentrations of TNF-α and IL-6 were observed at 1h and 2.5h following LPS challenge in pigs, respectively. Neither GAM, nor KETO nor the combination GAM-KETO was able to inhibit the in vivo LPS-induced cytokine production. Furthermore, none of the drugs influenced the subsequent APPs production. In contrast, administration of KETO significantly reduced PGE2 production both in vitro and in vivo (p<0.05 and p<0.001, respectively) and prevented the development of fever and severe symptoms, including dyspnoea, anorexia, vomiting and lateral decubitus.
Assuntos
Inflamação/induzido quimicamente , Cetoprofeno/uso terapêutico , Lipopolissacarídeos/toxicidade , Macrolídeos/uso terapêutico , Doenças dos Suínos/induzido quimicamente , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animais , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Quimioterapia Combinada , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Cetoprofeno/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Macrolídeos/administração & dosagem , Masculino , Suínos , Doenças dos Suínos/tratamento farmacológicoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Infecções por Flavobacteriaceae/veterinária , Macrolídeos/farmacologia , Ornithobacterium/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Perus , Animais , Antibacterianos/farmacocinética , Líquido da Lavagem Broncoalveolar/microbiologia , Feminino , Infecções por Flavobacteriaceae/tratamento farmacológico , Infecções por Flavobacteriaceae/microbiologia , Pulmão/microbiologia , Macrolídeos/farmacocinética , Testes de Sensibilidade Microbiana/veterinária , Doenças das Aves Domésticas/microbiologia , Distribuição AleatóriaRESUMO
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.
Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida , Macrolídeos/análise , Macrolídeos/sangue , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Análise Química do Sangue , Galinhas , Limite de Detecção , Extração Líquido-Líquido , SuínosRESUMO
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.
Assuntos
Reação de Fase Aguda/veterinária , Doenças dos Bovinos/imunologia , Inflamação/veterinária , Lipopolissacarídeos/farmacologia , Reação de Fase Aguda/induzido quimicamente , Reação de Fase Aguda/imunologia , Animais , Bovinos , Doenças dos Bovinos/induzido quimicamente , Modelos Animais de Doenças , Febre/induzido quimicamente , Febre/imunologia , Febre/veterinária , Haptoglobinas/análise , Inflamação/induzido quimicamente , Inflamação/imunologia , Injeções Intravenosas/veterinária , Interleucina-6/sangue , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Masculino , Proteína Amiloide A Sérica/análise , Fator de Necrose Tumoral alfa/sangueRESUMO
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%.
Assuntos
Dexametasona/análogos & derivados , Glucocorticoides/farmacocinética , Sus scrofa/metabolismo , Animais , Área Sob a Curva , Disponibilidade Biológica , Cromatografia Líquida , Dexametasona/sangue , Dexametasona/farmacocinética , Feminino , Glucocorticoides/sangue , Injeções Intramusculares/veterinária , Injeções Intravenosas/veterinária , Espectrometria de Massas em TandemRESUMO
The aim of this study was to determine the pharmacokinetics of dexamethasone in broiler chickens. Dexamethasone sodium phosphate (0.3mg/kg bodyweight) was injected IV or IM and blood samples were collected at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12 and 24h after administration. Dexamethasone in the plasma samples was measured using a liquid chromatography-tandem mass spectrometry method and the pharmacokinetics analysed according to a one-compartmental model. The maximum plasma concentration after IM administration occurred at 0.37h. The elimination half-life for dexamethasone was 0.46h and 0.70h following IV and IM administration, respectively, which was shorter than other species, while the clearance (1.26L/hkg) was higher than has been reported for other species (<0.5L/hkg). The volume of distribution (â¼1L/kg) was similar to values reported for other species and the bioavailability of dexamethasone after IM administration was 100%. The results from this study will be useful in investigating whether inflammatory disease may affect the pharmacokinetic parameters of dexamethasone in chickens.
Assuntos
Galinhas/sangue , Dexametasona/farmacocinética , Animais , Área Sob a Curva , Dexametasona/administração & dosagem , Feminino , Meia-Vida , Injeções Intramusculares , Injeções Intravenosas , Masculino , Estrutura MolecularRESUMO
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-1ß (IL-1ß) 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-1ß 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 <10% and <16%, respectively. For TNF-α, IL-1ß, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.