Pharmacokinetics of tildipirosin in bovine plasma,lung tissue,and bronchial fluid (from live,nonanesthetized cattle)

Menge, M., Rose, M., Bohland, C., Zschiesche, E., Kilp, S., Metz, W., Allan, M., Röpke, R., Nürnberger, M. Pharmacokinetics of tildipirosin in bovine plasma, lung tissue, and bronchial fluid (from live, nonanesthetized cattle). J. vet. Pharmacol. Therap.  35 , 550–559. The pharmacokinetics of tildipirosin (Zuprevo^® 180 mg/mL solution for injection for cattle), a novel 16‐membered macrolide for treatment, control, and prevention of bovine respiratory disease, were investigated in studies collecting blood plasma, lung tissue, and in vivo samples of bronchial fluid (BF) from cattle. After single subcutaneous (s.c.) injection at 4 mg/kg body weight, maximum plasma concentration (C_max) was 0.7 μg/mL. T_max was 23 min. Mean residence time from the time of dosing to the time of last measurable concentration (MRT_last) and terminal half‐life (T_1/2) was 6 and 9 days, respectively. A strong dose–response relationship with no significant sex effect was shown for both C_max and area under the plasma concentration–time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUC_last) over the range of doses up to 6 mg/kg. Absolute bioavailability was 78.9%. The volume of distribution based on the terminal phase (V_z) was 49.4 L/kg, and the plasma clearance was 144 mL/h/kg. The time–concentration profile of tildipirosin in BF and lung far exceeded those in blood plasma. In lung, tildipirosin concentrations reached 9.2 μg/g at 4 h, peaked at 14.8 μg/g at day 1, and slowly declined to 2.0 μg/g at day 28. In BF, the concentration of tildipirosin reached 1.5 and 3.0 μg/g at 4 and 10 h, maintained a plateau of about 3.5 μg/g between day 1 and 3, and slowly declined to 1.0 at day 21. T_1/2 in lung and BF was approximately 10 and 11 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination.     

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in 'Veterinary Fastidious Medium' (VFM) (supplemented Mueller-Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3-72 μg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.     

Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea.

A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests. Polymerase chain reactions for apxICA, apxIICA, apxIIICA, apxIBD and apxIIIBD genes were used to determine their genotype prevalence. Serotypes 2 (56 isolates), 5 (28 isolates) and 6 (11 isolates) were the most common; only two isolates belonged to serotype 7, and three were untyped. Among the 97 isolates identified by serotype, 70 had the same apx genes as their respective serotype reference strains, but 27 did not have any of the apx genes present in the corresponding serotype reference strain. Among these 27 isolates, 10 were serotype 2, 12 were serotype 5, three were serotype 6 and two were serotype 7.     

In vitro effects of Actinobacillus pleuropneumoniae on inducible nitric oxide synthase and cyclooxygenase-2 in porcine alveolar macrophages

OBJECTIVE: To determine the amount of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activity in alveolar macrophages in response to Actinobacillus pleuropneumoniae (APP) by determining nitric oxide (NO) and prostaglandin E2 (PGE2) concentrations. SAMPLE POPULATION: Freshly isolated porcine alveolar macrophages. PROCEDURE: Alveolar macrophages were incubated for 48 hours with APP (1 X 10(4) colony-forming units/mL), interleukin-1beta, (IL-1beta; 5 U/mL), tumor necrosis factor-alpha (TNFalpha; 500 U/mL), interferon-gamma (IFN-gamma, 100 U/mL), or lipopolysaccharide (LPS; 10 microg/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1beta, TNF-alpha, or IFN-gamma. In a third experiment, alveolar macrophages were incubated with fresh medium (negative control), LPS (positive control), APP alone, or APP with 1 of the following: an iNOS inhibitor (3.3 microM), a COX-2 inhibitor (10 microM); or both the iNOS and COX-2 inhibitors. Supernatant was obtained at 0, 3, 6, 9, 12, 24, and 48 hours after treatment for determination of NO and PGE2 production. RESULTS: The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE2 production. The addition of APP and IFN-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE2. Nitric oxide and PGE2 production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of iNOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.     

The influence of Actinobacillus pleuropneumoniae infection on tulathromycin pharmacokinetics and lung tissue disposition in pigs

A tulathromycin concentration and pharmacokinetic parameters in plasma and lung tissue from healthy pigs and Actinobacillus pleuropneumoniae (App)‐infected pigs were compared. Tulathromycin was administered intramuscularly (i.m.) to all pigs at a single dose of 2.5 mg/kg. Blood and lung tissue samples were collected during 33 days postdrug application. Tulathromycin concentration in plasma and lung was determined by high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method. The mean maximum plasma concentration (C_max) in healthy pigs was 586 ± 71 ng/mL, reached by 0.5 h, while the mean value for C_max of tulathromycin in infected pigs was 386 ± 97 ng/mL after 0.5 h. The mean maximum tulathromycin concentration in lung of healthy group was calculated as 3412 ± 748 ng/g, detected at 12 h, while in pigs with App, the highest concentration in lung was 3337 ± 937 ng/g, determined at 48 h postdosing. The higher plasma and lung concentrations in pigs with no pulmonary inflammation were observed at the first time points sampling after tulathromycin administration, but slower elimination with elimination half‐life t_1/2el = 126 h in plasma and t_1/2el = 165 h in lung, as well as longer drug persistent in infected pigs, was found.     

大蒜挥发油及其复方对猪胸膜肺炎放线杆菌体外抑菌试验

猪传染性胸膜肺炎(PCP)是由猪胸膜肺炎放线杆菌(APP)引起的猪的一种高度传染性疾病.以急性出血和慢性纤维素性胸膜肺炎为特征.各种年龄的猪对该病均易感,是危害养猪业的呼吸道疾病之一.20世纪90年代,用恩诺沙星、环丙沙星防治猪传染性胸膜肺炎,现今这些药物的防治效果均不理想,与猪体内的病菌产生了耐药性有关.蒜(Allium sativum L.)为百合科葱属植物蒜的鳞茎,被誉为"天然广谱抗生素".蒜中二烯丙基三硫化物(DATS)、二烯丙基二硫化物(DADS)是抑菌的主要生理活性物质[1].     

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.     

Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity

Huang, R. A., Letendre, L. T., Banav, N., Fischer, J., Somerville, B. Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity. J. vet. Pharmacol. Therap. 33 , 227–237. The pharmacokinetics (PK) and dose proportionality of gamithromycin (ZACTRAN^®), a novel azalide, after a single intravenous (i.v.) dose of 3 mg/kg or subcutaneous (s.c.) injection at 3, 6 and 9 mg/kg body weight were studied in 13 male castrate and 13 female Angus cattle. Following i.v. administration, the mean area under the curve extrapolated to infinity (AUC_inf) was 4.28 ± 0.536 μg·h/mL, and mean elimination half‐life (t_1/2) was 44.9 ± 4.67 h, with a large volume of distribution (V_ss) of 24.9 ± 2.99 L/kg and a high clearance rate (Cl_obs) of 712 ± 95.7 mL/h/kg. For cattle treated with s.c. injection of 3, 6 or 9 mg/kg, mean AUC_inf values were 4.55 ± 0.690, 9.42 ± 1.11 and 12.2 ± 1.13 μg·h/mL, respectively, and the mean elimination half‐lives (t_1/2) were 51.2 ± 6.10, 50.8 ± 3.80 and 58.5 ± 5.50 h. Gamithromycin was well absorbed and fully bioavailable (97.6–112%) after s.c. administration. No statistically significant (α = 0.05) gender differences in the AUC_Inf or elimination half‐life values were observed. Dose proportionality was established based on AUC_Inf over the range of 0.5 to 1.5 times of the recommended dosage of 6 mg/kg of body weight. Further investigations were conducted to assess plasma PK, lung/plasma concentration ratios and plasma antibacterial activity using 36 cattle. The average maximum gamithromycin concentration measured in whole lung homogenate was 18 500 ng/g at first sampling time of 1 day (～24 h) after treatment. The ratios of lung to plasma concentration were 265, 410, 329 and 247 at 1, 5, 10 and 15 days postdose. The lung AUC_inf was 194 times higher than the corresponding plasma AUC_inf. The apparent elimination half‐life for gamithromycin in lung was 90.4 h (～4 days). Antibacterial activity was observed with plasma collected at 6 h postdose with a corresponding average gamithromycin plasma concentration of 261 ng/mL. In vitro plasma protein binding in bovine plasma was determined to be 26.0 ± 0.60% bound over a range of 0.1–3.0 μg/mL of gamithromycin. The dose proportionality of AUC, high bioavailability, rapid and extensive distribution to lung tissue and low level of plasma protein binding are beneficial PK parameters for an antimicrobial drug used for the treatment and prevention of bovine respiratory disease.     

Detection of respiratory pathogens in porcine lung tissue and lavage fluid

The objective of this study was to compare the detection rate of bacterial agents in bronchoalveolar lavage fluid (BALF), taken without visual control, to that in affected lung tissue obtained from the same pig at necropsy. BALF and affected lung tissue were examined for Mycoplasma hyopneumoniae using PCR, and standard cultural methods were used for Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida and Streptococcus suis. All pigs with a history of respiratory symptoms were submitted as live animals for routine diagnostic examination. In each animal the site of lavage, marked by injecting methylene blue, differed from the site of pneumonic lesions. M. hyopneumoniae was detected more frequently in lung tissue than in BALF in cases with moderate or severe lung lesions. The detection rates of M. hyopneumoniae were higher in the BALF of pigs with mild lesions. Cultural examination of BALF was at least as satisfactory as affected lung tissue for detecting B. bronchiseptica, H. parasuis and P. multocida.     

Exposure time-dependent bactericidal activities of amoxicillin against Actinobacillus pleuropneumoniae; an in vitro and In vivo pharmacodynamic model

The pharmacodynamic effects of amoxicillin against Actinobacillus pleuropneumoniae at exposure concentration above and below minimum inhibitory concentration (MIC) were evaluated in both in vitro and in vivo. In vitro, the growth and morphological change of A. pleuropneumoniae in culture medium was observed. In vivo, the efficacy of amoxicillin on experimentally induced A. pleuropneumoniae infection in disease-free pigs was evaluated. Fifteen pigs were divided into three groups (n = 5 per group). After the onset of clinical respiratory disease symptoms, 6 h post-infection, amoxicillin sustained-release injectable formulation was injected intramuscularly at 7.5 mg/kg/day (group I) and 15 mg/kg/day (group II). Then the serum concentration of amoxicillin was measured. An untreated infected group served as controls. In each amoxicillin administration group, if symptoms were not absent after 48 h, the pig was injected with the amoxicillin sustained-release injectable formulation again using the same dosage. In vitro, the growth of A. pleuropneumoniae inhibited by amoxicillin exposure at the concentration above the MIC (1.28 x MIC), and the inhibition time was in directly proportion to the time of amoxicillin exposure. Moreover, all the cells were lysed. Whereas the bacterial growth inhibition at the amoxicillin exposure concentration below the MIC (0.25 x MIC) was not done, and the shape of cells were normal or long filamentous. In vivo, the group I clinical and pathological score was higher than the group II, and the group I weight gain was significantly less than the group II. Performance with respect to weight gain corresponded with clinical signs. The infected control group was severely affected with an 80% (4/5) mortality rate 24-96 h post-challenge. The duration of time above MIC (T > MIC) of serum amoxicillin concentration in the group I was less than group II. The present studies suggest that amoxicillin has exposure time-dependent bactericidal activity against A. pleuropneumoniae.     

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against recent field isolates of Mycoplasma bovis

The minimum inhibitory concentrations (MICS) and minimum mycoplasmacidal concentrations (MMCs) of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against 62 recent British field isolates of Mycoplasma bovis were determined in vitro by a broth microdilution method. The isolates were most susceptible todanofloxacin with MIC90 and MMC90 values of 0.5 microg/ml and 1.0 microg/ml, respectively. They were less susceptible to florfenicol with a MIC90 of 16 microg/ml and MMC90 of 32 microg/ml. Oxytetracycline and spectinomycin had only a limited effect against the majority of isolates tested with MIC50s of 32 microg/ml and 4 microg/ml, respectively and MIC90s of 64 microg/ml and more than 128 microg/ml, respectively. Nearly 20 per cent of the isolates were highly resistant to spectinomycin, and tilmicosin was ineffective, with 92 per cent of the isolates having MIC values of 128 microg/ml or greater. There was no evidence of resistance by M bovis to danofloxacin.     

Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.     

Pharmacokinetics of streptomycin with particular reference to its distribution in plasma,milk and uterine fluid of shebuffaloes

The study elucidated the pharmacokinetics of streptomycin in healthy lactating she-buffaloes after a single intramuscular (IM) injection (10 mg/kg). The drug attained its peak concentrations of 24.39±2.67, 0.45±0.05 and 5.06±0.18 g/ml at 1, 4 and 1 hour in plasma, milk and uterine fluid respectively. Calculations based on the assumption of a 2-compartment model gave a plasma t1/2 () of 4.01±0.44 h and an apparent volume of distribution [Vd(area)] of 0.47±0.06 l/kg. The drug was detectable in the plasma, milk and uterine fluid for 30, 8 and 12 hours, respectively. A therapeutic concentration of the drug was maintained for 6 to 7 hours in the plasma and for around 1 hour only in the uterine fluid. However, a therapeutic level could not be achieved in milk at any time. The results suggest that the drug can be used clinically by the IM route against streptomycin susceptible systemic infections but not those in the uterus and mammary gland.     

Estimation of sensitivity, specificity and predictive values of two serologic tests for the detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in the absence of a reference test (gold standard)

Latent-class models were used to determine the sensitivity, specificity and predictive values of a polyclonal blocking enzyme-linked immunosorbent assay (ELISA) and a modified complement-fixation test (CFT) when there was no reference test. The tests were used for detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in a survey of respiratory diseases in Danish finishing pigs. The estimates were obtained by maximum-likelihood and also by a Bayesian method (implemented with Gibbs sampling). Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence, given the true disease status.

No strong evidence of conditional dependence in either test sensitivity or specificity was found. Assuming independence, maximum-likelihood estimates and 95% confidence intervals of the sensitivity and specificity of the ELISA were 100% and 92.8% (90.1–95.5%) and the corresponding values of the CFT were 90.6% (85.8–95.4%) and 98.6% (98.0–99.3%), respectively. Bayesian estimates and posterior 95% credible intervals of the sensitivity and specificity of the ELISA were 99.7% (98.7–100%) and 92.7% (89.9–95.3%) and of the CFT were 90.6% (86.0–95.3%) and 98.7% (98.0–99.3%). The sensitivity and specificity of a combined test, where the CFT is subsequently applied to the pig sera that test positive in the ELISA, were estimated at 90.2% (85.6–95.0%) and 99.9% (99.8–100%), respectively. The cost of the combined test was less than the cost of the use of the CFT alone, at prevalences <54%. Prevalences and predictive values and their 95% limits were estimated in six sub-samples of data. The estimates of sensitivity and specificity obtained in the present investigation generally validate those reported from other sources.     

In vitro comparison of the activity of various antibiotics and drugs against new Taiwan isolates and standard strains of avian mycoplasma

Twenty-nine antibiotics or drugs were incorporated individually into mycoplasma agar to evaluate their inhibitory activity against avian mycoplasmas: 100 recent Taiwan isolates of 7 serotypes and 10 standard strains of 7 serotypes were tested. All of the standard strains were very sensitive to erythromycin, chlorotetracycline, doxycycline, minocycline, and tetracycline, but the local isolates were highly resistant to these antibiotics. The drugs or antibiotics that possessed an MIC90 of 50 micrograms/ml or less against the local isolates were tiamulin (less than 0.4 micrograms/ml), lincospectin (2.7), josamycin (2.7), lincomycin (3.0), spectinomycin (4.8), tylosin (6.0), kanamycin (6.0), chloramphenicol (6.0), gentamicin (7.5), apramycin (24.5), doxycycline (27.4), minocycline (29.0), spiramycin (30.0), colistin (44.3), leucomycin (45.0), and streptomycin (50.0). The MIC90 of the other antibiotics or drugs was greater than 50 micrograms/ml. None of the isolates or strains were sensitive to nalidixic acid, ronidazole, penicillin, ampicillin, cephalexin, carbadox, or four sulfa drugs at a concentration about 5 times the therapeutic level.     

Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.     

Comparison of plasma free-fatty-acid and blood-glycerol concentrations with measurement of lipolysis in porcine adipose tissue in vitro

Rates of adipose tissue lipid metabolism in vitro are often measured to evaluate the function in vivo of metabolic pathways and thus appraise the accretion or loss of depot fat. This study directly addressed the comparison of degradative metabolism in vitro and in vivo. The concentrations of plasma free-fatty-acids and blood-glycerol as putative representatives of lipolysis in vivo and the lipolytic rate in adipose tissue in vitro obtained at the time of blood sampling were both measured in the same pig. Concentrations of plasma free-fatty-acids and blood-glycerol were increased or decreased by infusion of the norepinephrine analog, isoproterenol or by infusion of the adrenergic antagonist, propranolol, respectively. Although lipolytic rates and sensitivity to isoproterenol in vitro changed during some acute hormonal manipulations of the pig, the modulation in vitro was usually small relative to the large changes observed in plasma free-fatty-acid and blood-glycerol concentrations. Some of the subtle changes in vitro may reflect biological responses to hormone infusion, e.g., desensitization of the response to adrenergic agonists, but the magnitude of rate changes in vitro negates prediction of the rates in vivo from rates in vitro. Extrapolation of lipolytic rates in vitro and several adipose tissue anabolic rates obtained from the literature indicate the improbability for prediction of rates and degree of fat accretion in pigs from metabolic rates in vitro.