Thioglycollate broth

To induce subclinical NE infection in birds, the University of New England NE challenge procedure as described by Rodgers et al. [30 (link)] was followed. In brief, on day 9 all challenged birds were orally inoculated with 1 mL/bird field Eimeria strains (E. acervulina at 5000 oocytes/mL, E. maxima at 5000 oocytes/mL, E. brunetti at 2500 oocytes/mL); chickens in the non-challenged groups were inoculated with 1 mL of sterile PBS. Primary poultry isolates of C. perfringens (EHE-NE18) were obtained from CSIRO Livestock Industries, Geelong, Australia and was incubated overnight at 39 °C in 100 mL of sterile thioglycollate broth (USP alternative; Oxoid) followed by subsequent overnight incubation of 1 mL of the previous culture in 100 mL of cooked meat medium (Oxoid), and then in 700 mL of thioglycollate broth (USP alternative; Oxoid) containing starch (10 g/L) and pancreatic digest of casein (5 g/L) to obtain the challenge inoculum. After preparation of the inoculums, 1 mL of fresh inoculums containing approximately 10⁸ CFU/mL C. perfringens was inoculated to the chickens on day 14 and 15. Chickens in the unchallenged groups were gavaged with sterile thioglycolate medium as a sham treatment.

Native lung tissues were transferred into mixing vessels (ProbeAX; AxonLab) containing 5 mL of physiological saline and were homogenized using a dispersion instrument (ULTRA-TURRAX® Tube Drive; AxonLab). The homogenates were inoculated (0.1 mL aliquots) onto aerobic blood agar, MacConkey agar, chocolate agar, and anaerobic blood agar plates (BD Diagnostics) and into thioglycollate broth (Oxoid). Plates were incubated at 35°C and 37 °C aerobically, in an atmosphere containing 5% carbon dioxide and anaerobically (Genbox anaer, bioMérieux) for up to 14 days, respectively. Cloudy thioglycollate broths were sub-cultured onto plates. Colonies were identified using matrix-assisted-laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF MS) using the Vitek® MS (bioMérieux) or MALDI Biotyper^TM (Bruker) instruments or by 16S rRNA gene sequencing (Gorkiewicz et al., 2003 (link)).

Clostridium perfringens isolate JBCNJ055 was sub-cultured in 10 mL of thioglycollate broth (Oxoid, United Kingdom) and incubated for 6 h, aerobically at 37°C. Broth dilution MIC assays were produced in microtiter plates and prepared with ranges from 128 to 0.25 μg/mL of penicillin G, bacitracin, tetracycline and neomycin. MICs were recorded as the lowest concentration of antimicrobial agent that completely inhibits growth turbidity compared with positive controls where antibiotics were omitted. MIC testing was repeated in triplicate on three occasions.