Sample collection and isolation of C. novyi
Diagnostic samples, primarily livers, collected from sows with sudden death within 12 h after death were submitted to the Jeonbuk National University Veterinary Diagnostic Center in 2015 (case no. 150557) . Swab samples taken from affected sites on the livers were inoculated in Reinforced clostridial medium (RCM) (BD Biosciences, New Jersey, USA) and incubated anaerobically at 37 °C for 72 h in a gas jar. Then, the incubated broth medium was heated at 100 °C for 10 min, reinoculated in 5% sheep blood agar, and again incubated under the same conditions. Colonies suspected of being C. novyi that exhibited beta hemolysis on sheep blood agar plates were subsequently identified as C. novyi type B based on differential PCR, Gram staining and 16S rRNA gene sequencing .
The morphology of the bacteria in the identified colonies was further confirmed using endospore staining. Spores and vegetative cells were identified using a 5% malachite green staining solution (Thermo Fisher Scientific Inc., MA, USA) and a safranin staining solution (Gram stain kit solution), respectively, as described previously .
DNA extraction from isolates
DNA was extracted from colonies grown on 5% sheep blood agar for differential PCR and 16S rRNA gene sequencing by Patho Gene-spin™ (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer’s instructions.
Differential PCR for detection of the C. novyi isolate
The suspected colonies were confirmed using C. novyi specific flagellin gene (filC) primers, where fliC was amplified using differential primers for C. novyi types A and B, as well as C. haemolyticum, which is genetically close to C. noyvi [14, 19].
The PCR assays in this study were performed in a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in a 20 μl reaction containing: 1× PCR buffer; 2 mM MgCl2; 250 μM each dNTP; 1 unit of DNA polymerase (AccuPower Multiplex PCR kit; Bioneer Inc., Alameda, CA, USA); and 10 pmol of each primer. The PCR conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56.5 °C for 1 min and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. The amplified PCR products were separated by electrophoresis in 2% (w/v) agarose gels and stained with Red Safe™ (iNtRON Biotechnology, Seongnam, Korea). The strains ATCC 17861 (C. novyi type A; 472 bp), ATCC 25758 (C. novyi type B; 551 bp), and KCTC 5570 (C. haemolyticum; 819 bp) were used as positive controls.
Histopathological examination of the liver
Approximately 2-cm3 liver tissue samples were fixed in 10% phosphate-buffered formalin, routinely processed, and embedded in paraffin. Tissue sections (4 μm) were prepared using a microtome (HM-340E, Thermo Fisher Scientific Inc., MA, USA), and the sections were placed onto glass slides. Hematoxylin and eosin (H&E) staining was performed according to standard techniques, and Gram staining was performed on liver tissue collected from the dead sow using a Gram stain kit according to the manufacturer’s instructions.
Identification and characterization of the C. novyi isolate
The biochemical characteristics of the C. novyi isolate were determined using an API 20A kit (BioMerieux, USA) according to the manufacturer’s instructions. The lecithinase activity of the C. novyi isolate was confirmed on an egg yolk agar plate (Kisan Biotech. Co., Seoul, Korea), where the isolate was inoculated onto the plate using a sterilized loop and incubated anaerobically for 72 h at 37 °C. The presence of opalescence around a colony was taken to indicate lecithinase activity. Additionally, a catalase test was performed using pure C. novyi isolates smeared on a microscope slide. A drop of 3% hydrogen peroxide (Wako, Osaka, Japan) was added, and the production of copious bubbles was taken to indicate that the bacteria were positive for catalase.
16S rRNA gene sequencing analysis
16S rRNA gene sequencing was performed by a sequencing facility (Biofact Co., Daejun, Korea) using a pure culture of the C. novyi isolate. The 16S rRNA gene sequence of the C. novyi isolate was identified by a BLAST search and compared with other Clostridium species sequences downloaded from the NCBI database. Multiple alignment of the 16S rRNA gene sequences was performed using MegAlign (DNASTAR, Madison, Wisconsin, USA), and a phylogenetic tree was constructed with the neighbor joining method using MEGA 6 .
Cell culture assay of the C. novyi isolate on Vero cells
Vero 76 cells (ATCC CRL-1587) were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Korea) supplemented with heat-inactivated 5% fetal bovine serum (FBS; Invitrogen, USA), 2 mM L-glutamine, and a 100× antibiotic-antimycotic solution [Anti-Anti, Invitrogen; 1× solution contains 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml Fungizone® (amphotericin B)] at 37 °C under a humidified 5% CO2 atmosphere.
The cell culture assay was performed in Vero 76 cells as described previously [26, 31]. Briefly, C. novyi cell-free supernatant obtained after centrifugation was prepared by filtrating the broth through a 0.2-μm cellulose acetate syringe filter (Corning, Germany). Two-fold serial dilutions of the filtered supernatant were prepared with cell medium for up to 8 dilutions. Cell culture assays were performed in 96-well tissue culture plates (Falcon, NY, USA). Subsequently, the supernatant of cultured Vero 76 cells was discarded, and the cells were washed twice with 1× PBS. Then, the diluted cell-free supernatant of the C. novyi isolate was added to the Vero 76 cells, which were then incubated at 37 °C under a humidified atmosphere with 5% CO2 for 5 days. The cell-free culture supernatants of reference strains of C. novyi type A (ATCC 17861) and C. novyi type B (ATCC25758) were used as positive controls.
DNA was extracted from cell pellets of C. novyi isolate from 200-ml cultures using Patho Gene-spin™, according to the manufacturer’s instructions. A sample of high-quality, high-molecular-weight DNA was used to prepare size-selected SMRTbell templates of approximately 20 kb. A NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., MA, USA) and a Qubit fluorometer (Thermo Fisher Scientific Inc., MA, USA) were used to measure the concentration of gDNA, and the sample passed quality control (QC) screening criteria (≥200 ng). For PacBio RS sequencing, 8 g of input gDNA was used for the 20-kb library preparation. For gDNA where the size range was less than 17 kb, we used a Bioanalyzer 2100 (Agilent Technologies, CA, USA) to determine the actual size distribution. If the apparent size of the gDNA was greater than 40 kb, the gDNA was sheared with a g-TUBE (Covaris Inc., Woburn, MA, USA) to produce library fragments in the optimal size range and purified using AMPure PB magnetic beads (Beckman Coulter Inc., CA, USA). Then, the concentration of the gDNA was measured using both a NanoDrop spectrophotometer and a Qubit fluorometer, and approximately 200 ng/μl gDNA was run on a field-inversion gel. A library was prepared in a total volume of 10 μl using a PacBio DNA Template Prep Kit 1.0 (for 3–10 kb), and SMRTbell templates were annealed using a PacBio DNA/Polymerase Binding Kit P6. A PacBio DNA Sequencing Kit 4.0 and 1 SMRT Cell was used for sequencing. SMRT Cells (Pacific Biosciences, CA, USA) with C4 chemistry were used, and 240-min movies were captured for each SMRT cell using the PacBio RS II (Pacific Biosciences, CA, USA) sequencing platform. The subsequent steps were based on the PacBio Sample Net-Shared Protocol, which is available at http://pacificbiosciences.com/.
All runs were performed with diffusion-based loading and analyzed using standard primary data analysis methods by implementing HGAP and Quiver . The coding DNA sequences were predicted with Prokaryotic Genome Annotation Pipeline version 4.5 on the NCBI website (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Additional functional annotation was performed with the Rapid Annotation using Subsystem Technology server .