Twenty-four, 4-week old, specific pathogen free, female CF-1 mice (Charles River Laboratories, Kingston, NY were obtained from a commercial supplier and randomly assigned to one of three inoculation groups: sham (Ctrl; n = 8), “Brachyspira hampsonii” clade II strain 30,446 (Bhamp; n = 8), or positive control B. hyodysenteriae (Bhyo; n = 8). Each inoculation group was divided into two plastic cages (6 cages total, n = 4 per cage). One cage per group was fed normal rodent chow, RMH 3000 (RMH; Federated Co-Operatives Limited, Saskatoon, SK, Canada), and the other was fed a defined low zinc, Teklad diet TD85420 (TD) for the duration of the experiment. Each mouse was assigned a unique identity by tail-mark and was allowed to acclimate for 7 days prior to inoculation. Mice were housed in ventilated, biocontainment level 2 cage racks with aspen shavings, which were changed once per week. Mouse houses and crinkle bedding were provided in each cage for enrichment.
Bacterial strains and culture
The “B. hampsonii” strain 30,446 and B. hyodysenteriae strain G44 (generously provided by Boehringer Ingelheim Vet Medica, St Joseph, MO) used for inoculation were cultured in JBS broth (BHI plus 0.5% (v/v) sheep blood and 0.5% (v/v) glucose) . To detect Brachyspira in samples from mice, faecal samples and terminal caecal content swabs were cultured on BJ agar  at 42 °C using an anaerobic gas pack (Oxoid Anaerogen™, Thermo Fisher Scientific, Waltham, MA) for a total of 4 days, with readings taken at 48 and 96 h post-plating. Appearance of zones of strong β-haemolysis on selective BJ agar was considered a presumptive positive culture result.
Molecular identification and quantification of Brachyspira
Total DNA was purified from 1 mL samples of broth cultures using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Mississauga, ON) and the concentration of “B. hampsonii” 30,446 or B. hyodysenteriae in inoculum preparations was determined using clade- or species-specific SYBR green quantitative real-time PCR with primers as previously described . Results were expressed as logarithm base 10 genome equivalents (GE) per mL.
For confirmation of species identity from culture-positive faecal or terminal caecal samples, zones of strong β-haemolysis were sampled with a sterile toothpick and subjected to genus-specific Brachyspira PCR using previously published primers targeting the NADH oxidase (nox) gene . PCR products were purified and sequenced using the amplification primers. Raw sequence data was assembled and edited using Pregap4 and Gap4  and the resulting sequences compared to published nox sequences for identification.
The mice underwent a 7-day acclimatization period, during which they were fed exclusively the appropriate diet for their experimental group. Water and food were provided ad libitum throughout the experiment. For each of two daily inoculations (2:00 p.m.), mice were restrained using the double handed manual technique, then held vertically by the scruff and inoculated intra-gastrically using a straight, 20 gauge blunt-ended feeding needle. The inocula was 0.2 mL of a 24 h old broth culture of the relevant Brachyspira species on day 0 (Bhyo = 1.6 × 108, Bhamp = 1.2 × 109 GE per 200 μL dose) and one (Bhyo = 1.0 × 108, Bhamp = 1.56 × 109 GE per 200 μL dose) day post-inoculation (dpi). The negative control (Ctrl) mice were sham inoculated with sterile broth in the same manner. The mice were then allowed to recover in their original cages. No sedation was used.
Mice were monitored by research personnel twice daily (8:30 a.m. and 3:00 p.m.) to assess signs of illness based on physical appearance and faecal consistency. At each observation, mice were held by the tail and placed on the examiner’s palm to facilitate simultaneous defecation. Faeces were observed for the presence of mucus and/or blood, and graded using a scoring system modified from previous pig challenge experiments conducted in our laboratory [2, 23](Additional file 1): Score 0 = formed faecal pellet, 1 = formed pellet with a trace mucous tail, 2 = soft, mucoid faeces, 3 = faeces with blood (+/− mucus). From each mouse, faecal pellets were collected at −2 and 0 dpi for culture and nox PCR to confirm that mice were Brachyspira-free prior to inoculation. Faecal samples were also collected from individual mice after inoculation at 5, 7, 9, and 12 dpi to monitor Brachyspira shedding.
The experiment was terminated at 15 dpi. At this time, mice were euthanized by way of anaesthetic overdose (isoflurane gas inhalation; AErrane™ Baxter Corporation, Mississauga, ON, Canada) followed by exsanguination. A necropsy was performed immediately thereafter with focus on the gastrointestinal tract. Fresh samples of caecum and colon were collected for culture and nox PCR. The carcass was placed in 10% neutral buffered formalin for 24 h. and samples of ileum, colon, caecum, heart, lung, salivary gland, rectum, liver, spleen, kidney and stomach were processed routinely for histological analysis of 5 μm haematoxylin and eosin stained (H and E) sections by a board certified veterinary pathologist blinded to treatment group. For Experiment 1, the presence or absence of catarrhal inflammation and epithelial regeneration in the colon and caecum were recorded.
Fluorescent in situ hybridization (FISH)
To better visualize the interaction of Brachyspira organisms on the colonic mucosae and crypts, 2 cases from each Brachyspira group, selected on the basis of high fecal consistency score, lesion severity and Brachyspira DNA concentration in colon, along with 1 control case were selected for FISH targeting a Brachyspira generic probe. The staining protocol was adapted from previous work [24, 25]. Paraffin embedded colonic tissue sections were cut at 4 μm and mounted on histology slide (FisherBrand Superfrost plus, Thermo Fisher Scientific Inc., MA., USA). Tissue sections were deparaffinized by baking in a rotary oven (BK200 Roto-Dry Slide Dryer, Mopec, MI, USA) for one hour at 65 °C, followed by three 10 min. Xylene serial passages. Tissues were dehydrated by passage through 100% ethanol (2 passages, 10 min. Each), 95% ethanol (5 min.), and 70% ethanol (5 min.), and allowed to air dry at room temperature. A previously described , a custom fluorophore-labelled DNA probe, SER1410 (Alexa 546–5′-GTCATTCCATCGAAACATA- 3′) designed for Brachyspira spp. was obtained from a commercial source (Invitrogen Corporation, CA, USA) and reconstituted using nuclease free-water to a working concentration of 5 ng/μl in hybridization buffer (20 mM Tris [pH 7.2], 0.9 M NaCl, 0.1% sodium dodecyl sulfate [w/v],40% formamide [w/v], 10% dextran sulfate). Tissue sections were covered with 200 μl of hybridization solution, placed in a hybridization oven (Boekel Scientific, Feaesterville, PA, USA), and hybridized overnight at 45 °C. Following hybridization, tissue sections were washed in wash buffer (20 mM Tris [pH 7.2], 0.9 M NaCl, 10% dextran sulfate [w/v]) pre-warmed to 45 °C for 20 min., rinsed with sterile water, and air dried. Slides were mounted on antifading agent (ProLong® Gold Antifade Reagent, Life Technologies, Carsbad, CA, USA) and covered with 0.13–0.17 mm thickness coverglass (Fisherbrand #1.5 cover glass, Thermo Scientific, MI, USA). Mounted slides were kept at 4 °C until analyzed.
The slides were visualized using a wide-band epifluorescence microscope (Olympus IX83, Olympus Corporation, Tokyo, Japan) under 100× objective lens (Olympus UPlanSApo 100×, 1.40, oil, Olympus Corporation, Tokyo, Japan). Filters corresponding to GFP/Green (LED excitation - 460 nm, emission - ET525/50) and RFP/Red (LED excitation - 565 nm; emission – ET630/75) were used for multicolour imaging. Images were captured using Andor Zyla sCOMS camera (Andor Technologies, Belfast, UK) and an imaging software (Olympus cellSens imaging software, Olympus Corporation, Richmond Hill, ON, Canada).
Procedures for Experiment 2 were similar to above with a few exceptions described below.
Four-week old, specific pathogen free, female CF-1 (n = 8) and C3H/HeN (“C3H”, n = 9) mice were randomly assigned to one of two inoculation groups as follows: CF-1-Ctrl (n = 4), C3H-Ctrl (n = 4), CF-1-Bhamp (n = 4), and C3H-Bhamp (n = 5). All mice were fed TD85420 diet and allowed to acclimate for 14 days prior to inoculation.
Because the mice were 1 week older at inoculation than those used in Experiment 1, the inoculum dose was increased to 0.3 mL per mouse of “B. hampsonii” broth culture containing 2.31 × 108, 2.22 × 108, 1.33 × 108 GE per 300 μL dose, on D0, D1, D2, respectively. The mice also underwent a 6-h fasting period prior to each of three intra-gastric inoculations to decrease gastric transit time. Inoculation procedures were identical to Experiment 1.
Molecular identification and quantification of Brachyspira
To confirm the detection of “B. hampsonii” 30,446 in pre-mortem faecal samples and terminal caecal samples, zones of strong β-haemolysis were sampled with sterile toothpicks and subjected to clade-specific conventional PCR using the same primers employed in the quantitative PCR assay used in Experiment 1 . PCR reactions contained 2.5 U Taq DNA polymerase (Quanta Bio Sciences, MD, USA), 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl pH 8.3, 250 μM of each dNTP and 20 pmol each of primers JH0224 and JH0225. PCR reactions were incubated at 94 °C for 3 min. Followed by 40 cycles of (15 s. at 94 °C, 15 s. at 60 °C and 30 s. at 72 °C) and a final extension of 5 min at 72 °C. No template controls were run with each set of PCR reactions. A visible band of 215 bp on a 1% agarose gel confirmed detection of “Brachyspira hampsonii” stain 30,446.
Clinical assessments were performed twice daily as previously described for Experiment 1. The faecal scoring system was refined to distinguish “mild” from “severe” soft, mucoid faeces. Slightly soft and mucoid faeces were scored as 2.0, whereas very soft and mucoid faeces were scored 2.5 (Additional file 1). Faecal samples were collected from each mouse at −2 and 0 dpi for culture and nox PCR to confirm that the mice were Brachyspira-free prior to inoculation.
The experiment was terminated at 14 dpi. The entire colon was linearized and five sections throughout its length were preserved in Carnoy’s solution, instead of 10% neutral buffered formalin, to allow for better preservation of the mucous layer and structural integrity of intestinal tissue . Alternating sections were collected for histopathology, Brachyspira culture and PCR. The depth of crypts was assessed in H and E stained sections of colon by measuring the distance between the bottom and top of intact crypts using an eyepiece reticle. Measurements were performed by an observer blinded to treatment group on the available colonic sections (range 9–16) for each mouse, with 5 measurements per section. The following histological changes in the colon and caecum were semi-quantitatively graded from 0 to 3 (Additional files 2, 3, 4 and 5): catarrhal inflammation, epithelial regeneration, degree of mucus accumulation and neutrophilic infiltration.
All statistical analyses were performed using Stata v14 (StataCorp, College Station, TX). Group differences were deemed statistically different if P < 0.05, and a trend towards significance if 0.05 ≤ P < 0.1 a priori.
A mean faecal score per mouse was calculated by averaging the twice daily scores from 3 dpi to termination. Fecal consistency scores from 0 to 2 dpi were not considered in this calculation because the intra-gastric infusion of liquid media (200–300 μl) periodically results in a loose pellets. Mean faecal scores and the number of days that the inoculated Brachyspira (Bhyo or Bhamp) was cultured from faeces (total shedding days) were compared between diet and Brachyspira spp. using a Kruskal Wallis test and post-hoc Wilcoxon rank-sum test where needed. The incubation period was defined as the number of days required for a mouse to develop consistent soft mucoid faeces (score 2) during the post-inoculation period. Mice that did not develop soft mucoid faeces were assigned a value of 15 d (i.e. censored at termination). The incubation period was compared between Brachyspira spp. and diet using a Kaplan Meier survival curve followed by Wilcoxon and log-rank tests for equality of survivor functions. Finally, the relationship of histopathologic lesions (catarrhal inflammation, epithelial regeneration) in caecum and colon to Brachyspira group, diet and clinical outcome was assessed using cluster analysis (Ward’s linkage; Matched Similarity Measure). The appropriate number of clusters were determined using the post-hoc Calinski-Harabasz pseudo-F index and Duda-Hart Je (2)/Je (1) index stopping rules.
Similar analyses was undertaken to determine potential differences between mouse strains following “B. hampsonii” challenge. In addition, separate multilevel, linear regression models (XTMIXED) were used to investigate differences in crypt depth between mouse strains and inoculation status. For these, mouse identification was included as a random intercept. The relationship of histopathologic lesions (catarrhal inflammation, neutrophil infiltration, mucus accumulation, mucus epithelial regeneration) in caecum and colon to Brachyspira spp., mouse strain and clinical outcome was assessed using cluster analysis (Ward’s linkage; Euclidean Dissimilarity Measure). Because the histopathology data was ordinal, cluster analysis was performed on scaled-ranks as described by Kaufman and Rousseeuw .