Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells.

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

Pharmacokinetic-pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida.

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration-time curve (AUC0-∞ )/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC0-∞ /MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK-PD modelling of in vitro bacterial time-kill curves for oxytetracycline in serum established mean AUC0-24 hr /MIC ratios for 3log10 decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48-hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25- and 27-fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively.

Bactericidal effects of various concentrations of enrofloxacin, florfenicol, tilmicosin phosphate, and tulathromycin on clinical isolates of Mannheimia haemolytica.

OBJECTIVE: To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE: 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES: Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS: Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida.

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time-kill data, based on the sigmoidal Emax equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log10 reduction) and 4log10 reduction in bacterial counts. For these levels, mean AUC(0-24 h) /MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (Cmax )/MIC ratios of 13.9:1 and 25.2:1, area under concentration-time curve (AUC0-∞ )/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).

Pharmacodynamics of florfenicol for calf pneumonia pathogens.

The antimicrobial properties of florfenicol were investigated for the bovine respiratory tract pathogens, Mannheimia haemolytica and Pasteurella multocida. Three in vitro indices of efficacy and potency were determined; minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and in vitro time-kill curves for six pathogenic strains of each organism. Each was monitored in two matrices, Mueller Hinton broth (MHB) and calf serum. MBC:MIC ratios were low, 1.8 : 1 for M haemolytica in both MHB and serum and 2.4 : 1 and 2.1 : 1 for P multocida in MHB and serum, respectively. The killing action of florfenicol had the characteristics of concentration dependency against M haemolytica and codependency (on time and concentration) against P multocida. Modelling of the time-kill data after 24 hours exposure was undertaken to quantify three levels of activity for the ratio, area under concentration-time curve over 24 hours (AUC24h)/MIC; bacteriostatic action (no change in bacterial count), 3log10 reduction and 4log10 reduction in bacterial count. Mean AUC24h/MIC values for P multocida in MHB (and serum) were 22.0 (23.3) hour, 34.5 (39.9) hour and 45.8 (50.4) hour, respectively. Similar numerical values were obtained for M haemolytica. For both bacterial species, interstrain variability was low; coefficients of variation ( per cent) in serum for 3log10 and 4log10 reductions in count were, respectively, 14.3 and 24.1 for P multocida and 7.8 and 11.4 for M haemolytica. These data form a rational basis for dosage selection for treatment of calf pneumonia caused by M haemolytica or P multocida.

Pharmacodynamics of marbofloxacin for calf pneumonia pathogens.

The pharmacodynamic (PD) properties of the fluoroquinolone, marbofloxacin, were determined for the bovine respiratory tract pathogens Mannheima haemolytica and Pasteurella multocida. For six pathogenic isolates of each organism, three in vitro indices of efficacy and potency were determined, namely, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves. Each parameter was determined in two matrices, Mueller Hinton Broth (MHB) and calf serum. For serum, MBC:MIC ratios were 2.7:1 (M. haemolytica) and 2.4:1 (P. multocida). The killing action of marbofloxacin had the characteristics of concentration dependency against M. haemolytica and co-dependency (on time and concentration) against P. multocida. To confirm the characteristics of the time-kill profiles, growth inhibition produced by marbofloxacin was also established ex vivo in three biological fluids, calf serum, exudate and transudate, harvested from a tissue cage model. The in vitro time-kill data were modelled with pharmacokinetic properties of marbofloxacin, established by intramuscular administration in calves at a dose of 2 mg/kg; three levels of activity, namely bacteriostatic, 3 log10 reduction and 4 log10 reduction in bacterial counts were determined. Mean AUC(24h)/MIC values (with percentage coefficients of variation indicating inter-isolate variability) for M. haemolytica, based on serum MICs, were 31.3 (41.6), 57.7 (42.4) and 79.2 (44.6) h, respectively. Corresponding values for MHB were 20.5 (58.0), 40.5 (51.8) and 51.2 (24.30) h, respectively. When allowance was made for binding of marbofloxacin to serum protein, the AUC(24h)/MIC values for serum were similar to those for MHB. Numerical AUC(24h)/MIC values for P. multocida were slightly lower than those obtained for M. haemolytica. These data establish for the first time inter-isolate variability in AUC(24h)/MIC values required for three levels of bacterial kill for two pathogenic species and thereby provide an indication of variability in serum concentration that might be required to achieve efficacy in clinical subjects.

Pharmacokinetic-pharmacodynamic integration and modelling of oxytetracycline administered alone and in combination with carprofen in calves.

The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of oxytetracycline were investigated, when administered both alone and in the presence of carprofen, in healthy calves. The study comprised a four treatment, four sequences, and four period cross-over design and used a tissue cage model, which permitted the collection of serum, inflamed tissue cage fluid (exudate) and non-inflamed tissue cage fluid (transudate). There were no clinically relevant differences in the PK profile of oxytetracycline when administered alone and when administered with carprofen. PK-PD integration was undertaken for a pathogenic strain of Mannheimia haemolytic (A1 76/1), by correlating in vitro minimum inhibitory concentration (MIC) and time-kill data with in vivo PK data obtained in the cross-over study. Based on in vitro susceptibility in cation adjusted Mueller Hinton Broth (CAMHB) and in vivo determined PK variables, ratios of maximum concentration (Cmax) and area under curve (AUC) to MIC and time for which concentration exceeded MIC (T>MIC) were determined. The CAMHB MIC data satisfied integrated PK/PD relationships predicted to achieve efficacy for approximately 48 h after dosing; mean values for serum were 5.13 (Cmax/MIC), 49.3 h (T>MIC) and 126.6 h (AUC(96h)/MIC). Similar findings were obtained when oxytetracycline was administered in the presence of carprofen, with PK-PD indices based on MIC determined in CAMHB. However, PK-PD integration of data, based on oxytetracycline MICs determined in the biological fluids, serum, exudate and transudate, suggest that it possesses, at most, limited direct killing activity against the M. haemolytica strain A1 76/1; mean values for serum were 0.277 (Cmax/MIC), 0 h (T>MIC) and 6.84 h (AUC(96h)/MIC). The data suggest that the beneficial therapeutic effects of oxytetracycline may depend, at least in part, on actions other than direct inhibition of bacterial growth.

Direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages: inhibition of CXCL-8 secretion, induction of apoptosis, and promotion of efferocytosis.

Recent evidence indicates that immunomodulation by antibiotics may enhance their clinical efficacy. Specifically, drug-induced leukocyte apoptosis and macrophage efferocytosis have been shown to promote the resolution of inflammation in a variety of disease settings. Tulathromycin is a new macrolide antibiotic for the treatment of bovine respiratory disease. The direct antimicrobial effects of the drug alone do not fully justify its superior clinical efficacy, and we hypothesize that tulathromycin may have immunomodulating properties. We recently reported that tulathromycin promotes apoptosis and inhibits proinflammatory NF-κB signaling in bovine neutrophils. In this study, we investigated the direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages. The findings indicate that bovine monocyte-derived macrophages and alveolar macrophages readily phagocytose tulathromycin-induced apoptotic neutrophils both in vitro and in the airways of Mannheimia haemolytica-infected calves. Moreover, tulathromycin promotes delayed, concentration-dependent apoptosis, but not necrosis, in bovine macrophages in vitro. Activation of caspase-3 and detection of mono- and oligonucleosomes in bovine monocyte-derived macrophages treated with tulathromycin was observed 12 h posttreatment; pretreatment with a pan-caspase inhibitor (ZVAD) blocked the proapoptotic effects of the drug. Lastly, tulathromycin inhibited the secretion of proinflammatory CXCL-8 in lipopolysaccharide (LPS)-stimulated bovine macrophages; this effect was independent of caspase activation or programmed cell death. Taken together, these immunomodulating effects observed in bovine macrophages help further elucidate the mechanisms through which tulathromycin confers anti-inflammatory and proresolution benefits. Furthermore, these findings offer novel insights on how antibiotics may offer anti-inflammatory benefits by modulating macrophage-mediated events that play a key role in inflammation.

Pharmacodynamics of oxytetracycline administered alone and in combination with carprofen in calves.

The pharmacodynamics (PD) of oxytetracycline was investigated against a strain of Mannheimia haemolytica. In vitro measurements, comprising minimum inhibitory concentration (MIC), minimum bactericidal concentration and time-kill curves, were conducted in five matrices; Mueller Hinton Broth (MHB), cation-adjusted MHB (CAMHB) and calf serum, exudate and transudate. MICs were much higher in the biological fluids than in MHB and CAMHB. Ratios of MIC were, serum: CAMHB 19 : 1; exudate:CAMHB 16.1; transudate:CAMHB 14 : 1. Ex vivo data, generated in the tissue cage model of inflammation, demonstrated that oxytetracycline, administered to calves intramuscularly at a dose rate of 20 mg/kg, did not inhibit the growth of M haemolytica in serum, exudate and transudate, even at peak concentration. However, using in vitro susceptibility in CAMHB and in vivo-determined pharmacokinetic (PK) variables, average and minimum oxytetracycline concentrations relative to MIC (C(av)/MIC and C(min)/MIC) predicted achievement of efficacy for approximately 48 hours after dosing. Similar C(av)/MIC and C(min)/MIC data were obtained when oxytetracycline was administered in the presence of carprofen. PK-PD integration of data for oxytetracycline, based on MICs determined in the three biological fluids, suggests that it possesses, at most, limited direct killing activity against M haemolytica. These data raise questions concerning the mechanism(s) of action of oxytetracycline, when administered at clinically recommended dose rates.

Relationship of in vitro minimum inhibitory concentrations of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with signs of bovine respiratory disease.

OBJECTIVE: To determine associations between in vitro minimum inhibitory concentrations (MICs) of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with clinical signs of bovine respiratory disease (BRD). DESIGN: Observational, retrospective, cohort study. ANIMALS: 976 feeder calves with clinical signs of BRD enrolled in 16 randomized clinical trials. PROCEDURES: Records of clinical trials from October 26, 1996, to November 15, 2004, were searched to identify calves with BRD from which a single isolate of M haemolytica or P multocida was identified via culture of deep nasal swab samples prior to treatment with tilmicosin (10 mg/kg [4.5 mg/lb], SC) and for which MICs of tilmicosin against the isolate were determined. The MICs of tilmicosin against recovered isolates and response to tilmicosin treatment were evaluated. RESULTS: Tilmicosin resistance among M haemolytica and P multocida isolates was uncommon (6/745 [0.8%] and 16/231 [6.9%], respectively). Treatment outcome, defined as success or failure after tilmicosin treatment, did not vary with the MIC of tilmicosin against recovered isolates. The proportion of treatment failures attributed to M haemolytica isolates categorized as resistant (MIC of tilmicosin, ≥ 32 µg/mL) or not susceptible (MIC of tilmicosin, ≥ 16 µg/mL), was 0.2% and 0.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Recovery of tilmicosin-resistant M haemolytica or P multocida isolates was rare, and no association was detected between MIC of tilmicosin and treatment response.

Demonstration of the metaphylactic use of gamithromycin against bacterial pathogens associated with bovine respiratory disease in a multicentre farm trial.

On five commercial cattle rearing sites across Europe, a total of 802 young cattle at high risk of developing bovine respiratory disease (BRD) associated with the bacterial pathogens Mannheimia haemolytica or Pasteurella multocida and/or Mycoplasma bovis were enrolled into a multicentre, controlled field trial. Half were treated with a single dose of gamithromycin at 6 mg/kg bodyweight by subcutaneous injection and half received an injection of a saline placebo as the control. All animals were observed daily for 14 days for signs of BRD as defined by set criteria. The proportion of metaphylactic preventive treatment successes, defined as animals surviving to day 14 without signs of BRD, in the gamithromycin-treated group (86 per cent) was significantly (P=0.0012) higher than in the saline-treated controls (61 per cent). Morbidity among the treated animals was reduced by 64 per cent compared with the controls.

Determination of the duration of antibacterial efficacy following administration of gamithromycin using a bovine Mannheimia haemolytica challenge model.

The antibacterial efficacy of gamithromycin administered once 1, 5, or 10 days prior to a challenge infection with Mannheimia haemolytica serotype A1 was evaluated. Forty calves were randomly allocated on day -11, restricted by body weight, to one of three treatment groups given gamithromycin at 6 mg/kg of body weight 10, 5, or 1 days before challenge or to an untreated control group. M. haemolytica A1 challenge infections were induced on day 0 by depositing 7.4 × 10(7) CFU at the bifurcation of the main bronchus using a bronchoscope. Clinical observations were made daily from the day of allocation to day 10, when necropsy was scheduled; three calves died or were euthanized in extremis on welfare grounds prior to scheduled necropsy. At necropsy the lungs were removed, pneumonic lesions were scored, and samples of lung tissue were cultured for M. haemolytica. The three groups of animals treated with gamithromycin before challenge had significantly lower lung M. haemolytica counts and fewer clinical signs of respiratory disease than did the saline-treated group. For most of the clinical parameters, the pattern of responses differed significantly (P < 0.05) between the gamithromycin-treated groups and the control group. There were no statistically significant differences between groups in the mean lung lesion scores, partly as a result of high individual variability, particularly within the control group. The administration of gamithromycin 1, 5, and 10 days prior to M. haemolytica A1 challenge resulted in a reduction in bacterial isolation from the lungs and a reduction in the severity of clinical disease.

Field efficacy of combination vaccines against bovine respiratory pathogens in calves.

The efficacy of an inactivated bovine respiratory syncytial virus (BRSV)--bovine parainfluenza type 3 (PI3)--Mannheimia haemolytica (Mh) combination vaccine was examined in two field studies. Calves were vaccinated (i) with the inactivated vaccine, (ii) a modified live/killed viral combination vaccine, or (iii) left unvaccinated. The efficacy of the vaccines was judged by the (i) number of treated animals, (ii) number of individual antibiotic treatments per calf and (iii) mortality rates. After vaccination with the inactivated vaccine, the number of calves requiring antibiotic treatment was significantly lower than in the unvaccinated group (odds ratios: 0.26 first study and 0.53 second study), but differences between vaccination with live/killed combination vaccines and controls were not significant (odds ratios: 0.56 and 0.90, respectively). In both studies, a number of unvaccinated controls died due to respiratory disease (4.6% first and 6.7% second study). By contrast, none of the animals vaccinated with the inactivated vaccine died in the first study and only 3.3% in the second study. The mortality rates for the groups vaccinated with the live vaccine (1.3% and 7.8%) were similar to the unvaccinated controls. In summary, these data demonstrate the efficacy of the inactivated vaccine under field conditions.

Clopidogrel induced suppression of bovine platelet activation in vitro and a preliminary study of its effect on the development of Mannheimia haemolytica induced pneumonia.

We report here on the influence of the platelet antagonist clopidogrel (Plavix) on bovine platelet function. We first evaluated the capacity of clopidogrel to inhibit adenosine diphosphate (ADP)-stimulated platelet function in the bovine species, using an ex vivo approach with blood from treated animals. Platelets isolated from treated calves displayed rapid and consistent reduction in function (aggregation, thromboxane production) upon ADP, but not platelet activating factor (PAF), stimulation. We then examined the possibility that clopidogrel could influence Mannheimia haemolytica pneumonia pathobiology using an experimental challenge model. We were unable to detect significant differences between clopidogrel treated and untreated animals when challenged with intra-tracheal inoculation of M. haemolytica. There was a trend towards inhibition of platelet degranulation in the affected regions of lungs from clopidogrel treated calves, and pre-treated challenged animals had similar amounts of fibrin deposition and enhanced fibrous tissue formation in their lungs when compared with control counterparts.