Pharmacokinetic-pharmacodynamic integration of marbofloxacin against Mannheimia haemolytica in Japanese Saanen goats following intravenous and subcutaneous administration

Introduction

Mannheimia haemolytica pneumonia costs the Japanese dairy goat industry an estimated 340 million yen annually in treatment expenses, production losses, and mortality, yet the antimicrobial dosing regimens used to combat this pathogen in goats are largely extrapolated from cattle data. This extrapolation is pharmacologically hazardous because goats differ from cattle in drug disposition: they have higher metabolic rates per unit body weight, faster hepatic drug clearance, different plasma protein binding profiles, and a more alkaline rumen environment that can alter the absorption of orally administered agents. Marbofloxacin is a third-generation fluoroquinolone developed specifically for veterinary use, with broad-spectrum activity against Gram-negative bacteria including the Pasteurellaceae family (Mannheimia, Pasteurella, Histophilus). Its concentration-dependent bactericidal activity makes the AUC/MIC ratio the most relevant PK-PD index for predicting clinical outcome: an AUC/MIC of 125 or greater is generally associated with bactericidal efficacy and minimal resistance selection for fluoroquinolones against Gram-negative pathogens. While the PK of marbofloxacin has been studied in cattle, sheep, horses, dogs, and cats, caprine-specific PK data are limited to a single report in Beetal goats under tropical Indian conditions, which may not be representative of the temperate climate, breed genetics, and management practices of Saanen goat production in Hokkaido. Furthermore, no published research has integrated caprine PK data with MIC distributions derived from goatorigin M. haemolytica isolates in Japan, leaving clinicians unable to assess whether standard dosing achieves therapeutic targets. This research addressed these deficiencies by conducting a controlled PK trial in Japanese Saanen goats, establishing the MIC distribution of M. haemolytica isolates from the target patient population, and performing PK-PD integration to determine whether the 2 mg/kg dose recommended for cattle is also appropriate for goats.

Materials and Methods

Materials

Eight clinically healthy adult Saanen goats (4 males, 4 females; body weight 42-56 kg; age 2-4 years) were housed at the Hokkaido Institute of Veterinary Pharmacology research farm. Goats were fed grass hay and a commercial concentrate ration at 2% body weight (dry matter basis) with ad libitum water access. Health status was confirmed by physical examination, complete blood count, and serum biochemistry within seven days of study commencement. None of the goats had received antimicrobial treatment within the preceding 60 days. Marbofloxacin (Marbocyl 10%, Vetoquinol, Lure, France) was administered at 2 mg/kg body weight by IV injection into the jugular vein or SC injection in the lateral neck. Sixty M. haemolytica isolates were obtained from nasal swabs and transtracheal washes of goats presenting with clinical pneumonia at veterinary clinics across Hokkaido prefecture between January 2021 and December 2023. Isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) and stored at-80 degrees C in brain-heart infusion broth with 20% glycerol. The research protocol was approved by the Hokkaido Institute of Veterinary Pharmacology Animal Experiment Committee (Protocol HIVP-AEC-2023-034).

Methods

The PK trial followed a two-period, two-treatment crossover design with a 14-day washout between periods. Blood samples (3 mL) were collected from the contralateral jugular vein into lithium heparin tubes at 0 (pre-dose), 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hours postadministration. Plasma was separated by centrifugation at 3,000 g for 10 minutes and stored at-80 degrees C. Marbofloxacin concentrations were determined by highperformance liquid chromatography with ultraviolet detection (HPLC-UV; Shimadzu LC-2050C, Kyoto, Japan) using a C18 reversed-phase column (Inertsil ODS-3, 250 mm x 4.6 mm, 5 micrometres; GL Sciences, Tokyo) with a mobile phase of acetonitrile-0.01M phosphoric acid (18:82, v/v) at 1.0 mL/min, detection at 295 nm. The limit of quantification was 0.01 micrograms/mL, and intra-day and inter-day coefficients of variation were below 8%. PK parameters were calculated by non-compartmental analysis using Phoenix WinNonlin v8.3 (Certara, Princeton, NJ, USA). Parameters included AUC from zero to infinity (AUC), terminal elimination halflife (t1/2), volume of distribution at steady state (Vdss), total body clearance (Cl), peak concentration (Cmax), time to peak concentration (Tmax), and mean residence time (MRT). Absolute SC bioavailability was calculated as (AUC_SC/AUC_IV) x 100. MICs were determined by the broth microdilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines (VET01-A5) using Mueller-Hinton broth with serial two-fold dilutions of marbofloxacin from 0.008 to 64 micrograms/mL. MIC50 and MIC90 were defined as the concentrations inhibiting 50% and 90% of isolates, respectively. PK-PD integration was performed by calculating AUC/MIC, Cmax/MIC, and T>MIC for the MIC50 and MIC90 values. Statistical comparisons between IV and SC PK parameters were made by paired t-test or Wilcoxon signed-rank test as appropriate, with significance at p<0.05.

Results

All eight goats completed both treatment periods without adverse effects. Marbofloxacin plasma concentration-time profiles showed the expected biphasic decline following IV administration and a rapid absorption phase following SC injection, with peak concentrations reached within 2 hours. Following IV administration, the AUC was 35.5 + /-3.8 micrograms-h/mL, terminal half-life was 8.7 + /-1.2 hours, Vdss was 1.42 + /-0.18 L/kg (indicating extensive tissue distribution), and Cl was 0.057 + /-0.007 L/h/kg. Following SC administration, Cmax was 6.2 + /-0.7 micrograms/mL at a Tmax of 1.8 + /-0.4 hours, AUC was 30.9 + /-3.2 micrograms-h/mL, and absolute bioavailability was 87.1%. The elimination half-life after SC dosing (9.1 + /-1.3 hours) did not differ significantly from the IV value (P = 0.412), indicating that absorption was not rate-limiting. Bactericidal targets: AUC/MIC>125; Cmax/MIC>10.

Discussion

The central conclusion from this PK-PD integration is that the standard 2 mg/kg dose of marbofloxacin, administered either IV or SC, achieves bactericidal AUC/MIC ratios against the MIC90 of goat-origin M. haemolytica isolates in Hokkaido. This is a practically important result because it validates the use of a dosing regimen that clinicians in Japan are already employing based on cattle label recommendations, albeit without prior species-specific evidence.

The PK profile of marbofloxacin in Saanen goats showed several differences from published cattle data that merit discussion. The elimination half-life (8.7 hours IV) was shorter than the 10-14 hours typically reported in cattle, consistent with the faster metabolic clearance expected in a smaller-bodied ruminant species. The Vdss of 1.42 L/kg exceeded body water volume, indicating substantial tissue penetration-a property that is particularly relevant for treating pneumonia, where drug concentrations in pulmonary epithelial lining fluid are more clinically important than plasma concentrations. The SC bioavailability of 87.1% was within the 80-100% range reported across species and supports SC administration as a practical alternative to IV dosing in field conditions. A concern raised by the MIC distribution is the presence of isolates with elevated MICs (2 isolates at 1.0 micrograms/mL and 2 at 2.0 micrograms/mL or higher). For these resistant isolates, the AUC/MIC ratio at the 2 mg/kg dose would fall to 35.5 (IV) or 30.9 (SC)-well below the bactericidal target of 125 and even below the bacteriostatic threshold of 30-40. This underscores two points: first, susceptibility testing should be performed whenever possible before fluoroquinolone therapy; and second, if empirical therapy is initiated at 2 mg/kg and clinical improvement is not observed within 48 hours, the dose or the drug should be reconsidered.

The finding that Cmax/MIC ratios were high (99.2 for IV, 49.6 for SC against MIC90) is encouraging from an antimicrobial resistance perspective. High Cmax/MIC ratios are associated with reduced emergence of resistant mutant subpopulations during fluoroquinolone therapy, because the drug concentration exceeds the mutant prevention concentration for a greater proportion of the dosing interval. Maintaining these high peak concentrations through appropriate dosing is therefore important not only for clinical cure but also for stewardship of fluoroquinolone efficacy in caprine medicine.

Conclusion

The key finding of this research is that marbofloxacin at 2 mg/kg body weight, administered either intravenously or subcutaneously, achieves PK-PD indices predictive of bactericidal efficacy against the MIC90 of Mannheimia haemolytica isolates from goat pneumonia cases in Hokkaido, Japan. The AUC/MIC ratios of 284 (IV) and 247 (SC) both exceed the fluoroquinolone bactericidal target of 125 by comfortable margins, and the Cmax/MIC ratios are sufficiently high to minimise resistance selection. These findings provide the first species-specific, PK-PDbased dosing validation for marbofloxacin in Japanese Saanen goats, replacing the previous reliance on extrapolated cattle data. For clinical practice, the SC route is recommended for field use given its high bioavailability (87.1%), practical convenience, and equivalent therapeutic adequacy to IV dosing against the target pathogen. However, clinicians should remain alert to the small proportion of M. haemolytica isolates with elevated MICs, for which the standard dose may be insufficient, and should pursue susceptibility testing whenever practicable.

Acknowledgement

AcknowledgementsThe authors thank Mr. Hiroshi Watanabe and Ms. Emi Suzuki for their technical assistance with HPLC analysis and animal handling throughout the pharmacokinetic trial.

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