Chlortetracycline

Antimicrobial susceptibility testing was performed by using a customized 96-well Sensititre plate (Trek Diagnostics, Oakwood, GA, USA) with the following antimicrobials: enrofloxacin (ENRO), gamithromycin (GAM), tulathromycin (TUL), tildipirosin (TIP), tilmicosin (TIL), tylosin tartrate (TYL), florfenicol (FFN), oxytetracycline (OXY), chlortetracycline (CTET), and penicillin (PEN). Serial two-fold dilutions were prepared as follows: ENRO, 0.12–128 µg/mL; TIP, 0.12–128 µg/mL; GAM, 0.25–256 µg/mL; TUL, 0.25–256 µg/mL; TIL, 1–256 µg/mL; TYL, 1–128 µg/mL; FFN, 0.25–256 µg/mL; OXY, 0.5–256 µg/mL; and CTET, 1–256 µg/mL. PEN (2–8 µg/mL) served as a control. Growth was assessed by using a color redox indicator, alamarBlue (Invitrogen, Fisher Scientific), based on a blue-to-pink color change.
For AST, the reserve culture was inoculated into the PPLO broth with 0.5% sodium pyruvate and incubated for 72 h. Following incubation, the actively growing broth cultures of isolates were subcultured into a neat PPLO broth and incubated for 24 h. Next, the optical density (OD) at 450 nm was determined by using a NanoDrop One Spectrophotometer (Fisher Scientific) and cultures were normalized to an OD₄₅₀ = 0.1. Cultures were further diluted up to 10× in neat PPLO media prior to preparation of the inoculum. This inoculum (culture: media, 1:50) was prepared in a 20% alamarBlue solution in neat PPLO media and added to each well according to the manufacturer’s procedure (Trek Diagnostics, Thermo Fischer Scientific, Oakwood, OH, USA). This provided a final concentration of 10³ to 5 × 10⁵ CFU/mL in a 10% alamarBlue solution (as per manufacturer’s instructions). The plates were sealed with a CO₂ permeable film and incubated for 48–72 h. Minimum inhibitory concentrations (MIC) were visually determined at 48 and 72 h and reported as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [24 ].
If growth was observed in the positive control wells (no antibiotics), then the MIC values for that isolate were accepted. The MIC was defined as the lowest concentration of antimicrobial that prevented visible growth of the inoculated M. bovis culture. A M. bovis reference strain (Mycoplasma bovis ATCC^® 25523™) was tested five times for quality control.

For assessment of Fusarium spp. on residues, straw of oats was collected each year at all field locations within a week of sowing. In 2010, the 1st year of the experiment, residues were collected across the whole field area, in order to calculate the background level of Fusarium spp.. In 2011 and 2012, oat straw residues were collected from each treatment. Within each experimental plot, residues were collected from four 1 m × 1 m quadrats outside the area designated to be harvested. The residues were dried at 25°C for 24 h and stored at room temperature until used for the recovery of Fusarium spp.
For the recovery of Fusarium species, 50 pieces of straw from each plot were analyzed, except from Solør in 2011 where 100 pieces were used. The straw pieces, 1.5–2 cm long and mostly including a node, were surface disinfected in 0.5% NaOCl for 30 s, transferred to 70% alcohol for 15 s, then rinsed three times with sterile distilled H₂O and then finally transferred to sterile filter paper to remove surface water. The straw pieces were then plated onto Petri dishes containing a modified CZID (Abildgren et al., 1987 (link)) in which iprodione was replaced with propiconazole (0.75 mg/l). The plated residues were then incubated for 7–10 days, under alternating 12 h darkness and 12 h near ultra violet light (‘black light’) and white light at 20°C. Fusarium mycelium, observed following the incubation period, was transferred to SNA (Nirenberg, 1976 ) containing chlortetracycline to reduce bacterial growth. A small piece of sterile filter paper was placed on the agar surface to promote sporulation. The SNA cultures were incubated for 10–14 days (incubation conditions as above) and used for the morphological identification of Fusarium species (Leslie and Summerell, 2006 ). The percentage of Fusarium-infested straw residues was calculated as the number of residue pieces infested with Fusarium as a proportion of the total number of residue pieces analyzed.
Inoculum potential (IP) was calculated for each plot as the percentage of the residues infested with Fusarium spp. multiplied with the proportion (0–1) of the plot surface covered by residues after sowing. The percentage of the plot surface for the four treatments covered with residue after sowing are presented in Table 1, and also in Seehusen (2014) .

This procedure was previously described [31 (link)] and modified for guinea pig spermatozoa [39 (link),41 (link)] as follows: The stain solution was prepared by dissolving 250-μM chlortetracycline (CTC)-HCl in TN buffer (20 mM Tris, 130 mM NaCl, and 5 mM cysteine at pH 7.8); fresh CTC stock was prepared daily. At the time of the assay, 20 μL of non-capacitated or capacitated spermatozoa, treated or not with VAS2879, were mixed with 20 μL of pre-warmed CTC stock solution and incubated for 20 s in a water bath at 37 °C. Immediately after incubation, the CTC-sperm suspension was fixed adding 3.5 µL of 12.5% glutaraldehyde in 1.25 M Tris (pH 7.5), immediately followed by gentle mixing. Fixed samples were kept in a dark box. After 1–4 h of fixation, slides were prepared and examined under fluorescence microscope (Ex330-380/Em420 nm). All fluorescence images were obtained using an Olympus BX500 fluorescence microscope. In each sample, 100 spermatozoa were classified as expressing one of three CTC staining patterns: F pattern, a faint fluorescence in the acrosome region, which is characteristic of non-capacitated acrosome-intact cells; B pattern, a bright fluorescence in the acrosomal region with a band along the equatorial segment, which is typical of capacitated, acrosome-intact cells; AR pattern, a fluorescence in the equatorial segment and/or post-acrosomal region, which is characteristic of physiologically capacitated acrosome-reacted cells. The presence or absence of the acrosomal cap on each cell was verified using phase-contrast illumination.

CZID media [6 (link)] was modified to Czapek Dox propiconazole dichloran agar (CZPD) as described by Hofgaard et al. [20 (link)]. The CZPD media contained (per L distilled water): 48 g of Czapek Dox agar (Sigma-Aldrich, Buchs, Switzerland), 1 mL of 0.2% dichloran (Aldrich, Steinheim, Germany) solution in ethanol, 1 mL of 5% chloramphenicol (Sigma, Shanghai, China) solution in ethanol, 1 mL of trace metal solution (1 g ZnSO₄·7H₂O [Fisher Scientific, Loughborough, UK] + 0.5 g CuSO₄·5H₂O [Fisher Scientific] per 100 mL distilled water), 10 mL of filter-sterilised 0.5% chlortetracycline hydrochloride (Sigma, Rimini, Italy) solution and 1 mL of 0.3% Bumper^® suspension (containing 750 µg propiconazole). Chlortetracycline and Bumper^® solution were added after autoclaving and cooling the media to 55 °C.

A total of 108 Duroc × Landrace × Yorkshire crossbred piglets (54 barrows and 54 gilts) were weaned at 21 ± 1 d of age (average initial BW 6.50 ± 0.21 kg), divided into 3 treatments (6 replicate pens/treatment, 6 piglets/pen according to BW and sex), and provided ad libitum access to feed and water. The 3 treatments included: a control group, an antibiotic group (a basal diet containing 40 g/t of virginiamycin and 500 g/t of chlortetracycline), and a probiotic group (basal diet + 1 × 10¹¹ CFU/kg BL−S6). Feed was given for 14 days. The test strain BL−S6 used in the present study was screened and preserved in the laboratory. The dry powder product of BL−S6 contained live bacteria at a concentration of 1 × 10¹¹ CFU/g. Diets for piglets were formulated based on the National Research Council (2012) and detailed in Supplementary Table S1.