An optimized swine dysentery murine model to characterize shedding and clinical disease associated with "Brachyspira hampsonii" infection

Abstract

Background: The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with "Brachyspira hampsonii" clade II (Bhamp).

Methods and results: In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores.

Conclusion: In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of "Brachyspira hampsonii" clade II.

Keywords: Brachyspira hyodysenteriae; C3H; CF-1; Inoculation; Mouse; Swine dysentery; TD85420; Typhlocolitis; “Brachyspira hampsonii”.

Fig. 1

Representative histopathologic lesions in Experiment 1 and 2 mice. a catarrhal inflammation showing groups of degenerative epithelial cells (arrow) in the lumen of colon; 40×. b epithelial regeneration showing very frequent mitotic figures (arrows) are noted in the hyperplastic colonic crypts; 40×. c neutrophilic infiltration, showing neutrophils (arrow) in the lumen and in the lamina propria of the colon; 40×. d mucoid exudate showing large lakes of basophilic mucus (arrow) in the lumen of colon; 20 ×

Fig. 2

Representative fluorescence in situ hybridization images of colon from Experiment 1 mice. Brachyspira cells are observed with strong positive signal (red serpentine-shaped cells), whereas non-specific cellular fluorescence appears green/yellow. a Large numbers of spirochaetes are observed within the luminal mucus of “B. hampsonii” infected mouse (#2) with no lesions fed RMH diet; b Large numbers of spirochaetes are observed within the colonic crypts of a B. hyodysenteriae infected mouse (#19) fed RMH diet with catarrhal inflammation in colon. c & d) Spirochaetes in close proximity with colonic epithelium and goblet cells from a B. hyodysenteriae infected mouse (#16) fed TD85420 diet with catarrhal inflammation in colon

Fig. 3

Cluster dendrogram and data table for Experiment 1 showing clustering of histopathology. Three clusters (Ward’s linkage, Matched Similarity Measure) are evident; all unrelated to Brachyspira species, diet or clinical outcome. Cluster 1 (blue) distinguishes mice with epithelial regeneration in caecum. Two of three mice also have a catarrhal inflammation in colon, as do all mice in cluster 2 (red). Cluster 3 (green) includes mice with no pathology in caecum and colon. R = regeneration of epithelium; CI = presence of catarrhal inflammation; TD = low zinc TD85420 diet; RMH = RMH3000 chow diet (normal zinc); spp. = Brachyspira species used for inoculation; hamp = “B. hampsonii”; hyo = B. hyodysenteriae; ctrl = non-inoculated control; ID = mouse identification; + = lesion or soft mucoid faeces present; − = lesion or soft mucoid faeces absent

Fig. 4

Cluster dendrogram and data table for Experiment 2 showing clustering of histopathology. Five clusters (Ward’s linkage of rank-scaled scores, Euclidean dissimilarity measure) are evident; unrelated to mouse strain, but associated with clinical outcome. Values in lesion columns represent the lesion severity score of colonic and caecal tissue sections, with more intense grey scale indicating mice with more prevalent lesions. The single mice in cluster 3 (blue) and 4 (orange) had the highest mean faecal scores and most severe colonic and caecal lesions. One inoculated mouse in cluster 2 (red) also had severe intestinal pathology and high faecal score. Cluster 5 (purple) included mice with severe pathology but both had low mean faecal scores. Mice with the least severe clinical disease and pathology were grouped in cluster 1 (green). Clusters R = regeneration of epithelium; NI = neutrophilic inflammation; IM = increased mucoid exudate; CI = catarrhal inflammation; CF1 = CF-1 mouse strain; C3H = C3H mouse strain; Spp. = Brachyspira species used for inoculation; hamp = “B. hampsonii”; ctrl = non-inoculated control; ID = mouse identification; + = soft mucoid faeces present; − = soft mucoid faeces absent