Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection

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Abstract

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5 mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24 h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and α-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and α-tocopherol reverted significantly towards normalisation within 24 h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and α-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and α-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.

Introduction

The outcome of antibiotic treatment of bacterial infections is traditionally evaluated by subjective clinical observations, e.g. combined in clinical score systems (Wallgren et al., 1999; Fodor et al., 2000). The clinical evaluation is often supported by more objective microbiological and pathological observations, especially in experimental studies. During the last decades, objective biochemical infection markers, e.g. the acute-phase protein C-reactive protein, have been extensively used as evaluators of the condition of human patients with acute infections (van Leeuwen and van Rijswijk, 1994). In contrast, objective biochemical markers of infection are not used routinely in veterinary medicine, although they might be a valuable supplement to the classically used methods of evaluation. To be usable for evaluation of antibacterial treatment of acute bacterial infection, an infection marker must respond rapidly and changes in the plasma concentration of the marker must parallel the development of the disease and reflect changes in the underlying pathology.

The objective of the present study was to evaluate objective biomarkers of infection for monitoring antibiotic treatment of bacterial infections, using an experimental model of acute Actinobacillus pleuropneumoniae (Ap)-infection in pigs. Thus, the changes in plasma concentration of biochemical infection markers, C-reactive protein (CRP), haptoglobin, interleukin-6 and zinc, were monitored during the course of the infection. Furthermore, plasma ascorbic acid and α-tocopherol were quantified as a measurement of the oxidative stress in the infected animals. To investigate the discriminative properties of the markers, the effect of the macrolide-like antibiotic tiamulin and the fluoroquinolone danofloxacin was compared, using dosage regimens expected to result in different therapeutic efficacy in favour of danofloxacin. The changes in the biochemical variables were subsequently compared with classic clinical, haematological and pathological observations.

Section snippets

Animals

Sixteen male, castrated pigs (Danish landrace × Yorkshire × Duroc) weighing from 28 to 38 kg were obtained from a herd, shown to be free of Ap and Mycoplasma hyopneumoniae by clinical and serological examination in accordance with the Danish SPF (specific pathogen free) surveillance system. The pigs were housed in individual pens in an isolated unit with a natural light schedule. The animals were fed twice daily with antibiotic-free commercial feed (Organica Promax, KFK, Præstø, Denmark, 1.12 Feed

Clinical and haematological observations

Within 20 h after challenge, the infected animals developed clinical and haematological signs of pneumonia with increased rectal temperature, respiratory rate and leukocyte number (WBC) (Figs. 1A–C). At this time-point, the infected animals received a single dose of either danofloxacin or tiamulin. Body temperature remained elevated in the tiamulin group (TG) throughout the experiment, while returning to the initial level in the danofloxacin group (DG) 24 h after treatment (Fig. 1A). Likewise,

Discussion

In recent years, the use of antibiotics has been the subject of much debate. In striving for a more rational use of antibiotics, well-validated and clinically relevant infection models provide a valuable tool for testing various antibiotics and dosage regimens. In the aerosol model used, the pigs were infected by voluntary inhalation of a relative low dose of bacteria, mimicking the natural infection with Ap. Ap is a common and severe cause of pleuropneumonia in pigs and comprises an important

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