Supplementary file 1
Description
Detailed overview of the antimicrobial susceptibility test results for each Pasteurellaceae isolate and WGS results from a randomized clinical trial evaluating the efficact of oxytetracycline (OTC) and florfenicol (FF) treatment for pneumonia guided by quick thoracic ultrasound. Regression of maximum consolidation depth <1cm was used as a criterion for cure and to stop antimicrobial treatment. Additionally, the study assessed the associations of consolidation depth at treatment initiation with cure and treatment duration. The trial involved 320 veal calves, randomly assigned into one of two groups: one receiving OTC (n=160) and the other FF (n=160) on day 1 (2-day metaphylaxis). Clinical scoring and qTUS were done on day 1 and every 48 hours for a 10-day period. After day 1, only calves with consolidations ≥1cm were given further treatment. On each time point, maximum consolidation depth was used to categorize calves into four qTUS categories: healthy (no consolidation), mild pneumonia (consolidation <1cm), moderate pneumonia (consolidation 1–3cm) and severe pneumonia (consolidation ≥3cm). Cure, treatment duration and the number of antimicrobial dosages (NAD) were compared between treatment groups. In addition, pathogen identification and antimicrobial susceptibility testing was performed on isolates from non-endoscopic broncho alveolar lavage fluid. On day 1, 30.0% (96/320) of the calves had consolidation ≥1cm, which increased to 50.9% (162/318) by day 9. After single metaphylactic treatment, cure was 20.9% (9/43) and 20.9% (9/43) in the OTC and FF group, respectively. Calves with severe pneumonia had lower odds to be cured after first treatment than calves with moderate pneumonia (Odds ratio (OR) = 0.17; 95% Confidence interval (CI): 0.04 - 0.63). By day 9, final cure of the initial cases was 27.9% in both the OTC- and FF-group. both groups, cure was similar at all observation points (P > 0.05). Overall, final cure of all calves with either moderate or severe pneumonia during the trial was 41.2% (52/102) and 19.0% (12/63), respectively (P = 0.004). Median treatment duration was 4 days (Interquartile range (IQR) = 2-6; Minimum (Min)=2; Maximum (Max)=8) and was similar in both treatment groups (P = 0.59). Treatment duration for calves with moderate pneumonia (Med=6; IQR= 4-6; Min=2; Max=8) was lower than the median treatment duration of calves with severe pneumonia (Med=8; IQR= 4-8; Min=2; Max=8) (P = 0.004). When compared to calves with mild pneumonia on day 1, calves with moderate (P = 0.01) and severe pneumonia (P < 0.001) had significantly longer treatment durations. In this study, cure was low and not different between both antimicrobials. Categorizing calves based on consolidation depth appears useful as both cure and treatment duration were different for the mild, moderate and severe group.
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Identification of the pathogens present at the beginning and end of the trial period was done by sampling a total of 15 calves on 2 separate time points. The first samples were taken when the outbreak of BRD was first identified, before treatment was initiated (day 7 after arrival, 5 samples). On this time point, a calf from each compartment was conveniently selected based on the presence of clearly defined lung consolidation ≥1cm. Next, 10 animals were sampled at the end of the trial period (day 9). At this point, 5 animals were randomly selected, using the Excel RAND function, from calves with consolidation ≥1cm that did not have a consolidation ≥1cm on any of the previous observation points (day 1, 3, 5, 7 and 9). At the same time, 5 other calves were randomly selected, using the same RAND function, out of the calves that had a consolidation ≥3cm at all previous observations points (day 1, 3, 5, 7 and 9). In all calves, respiratory sampling was done using nonendoscopic bronchoalveolar lavage (nBAL), performed on unsedated calves using a sterilized catheter, as previously described (Van Driessche et al., 2017; Pardon and Buczinski, 2020). At each sampling point, the nBAL samples from each group of 5 calves were pooled and sequenced using nanopore sequencing to identify all involved viral pathogens and Mycoplasma spp. (Theuns et al., 2018; Bokma et al., 2021b). To identify bacterial infections, individual nBAL samples were sent to an external accredited laboratory (DGZ Vlaanderen, Flanders, Belgium), and were directly inoculated on Colombia agar supplemented with 5% sheep blood (blood agar; Oxoïd, Hampshire, UK) and on M. bovis selective indicative agar (Bokma et al., 2020a). Bacteria detected on blood agar were identified by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Brüker Daltonik GmbH, Bremen, Germany) and M. bovis was identified based on lipase activity, as described previously (Bokma et al., 2020a). At the external laboratory, disk diffusion was used to test antimicrobial resistance in all Pasteurellaceae that were isolated. The clinical breakpoints used were based on the recommendations of references except tildipirosin and gamithromycin, which were assessed using the suppliers’ recommendations (Mast Group, Liverpool, UK; Christensen et al., 2018; Amara et al., 2024). When present, M. bovis was isolated and cultured in broth for additional strain typing. When possible, the genome of the involved strains was fully sequenced using nanopore sequencing and screened for the presence of known point mutations previously associated with antimicrobial resistance against enrofloxacin, gamithromycin, tylosin, tilmicosin, tetracycline, spectinomycin and gentamycin (Vereecke et al., 2020; Bokma et al., 2021a).