New insights into swine dysentery: faecal shedding, macro and microscopic lesions and biomarkers in early and acute stages of Brachyspira hyodysenteriae infection

Abstract

Background: Swine dysentery (SD) is a severe mucohaemorrhagic colitis in pigs caused classically by Brachyspira hyodysenteriae. Although several aspects of B. hyodysenteriae infection dynamic are already described, further research in the early stage of this infection is required. In this study, 7-week-old pigs were orally challenged with B. hyodysenteriae to obtain information about faecal shedding, macro and microscopic intestinal lesions and serum acute phase proteins in pigs at the onset of B. hyodysenteriae shedding (early infection group, n = 8), in pigs with mucohaemorrhagic diarrhoea (acute infection group, n = 8) and in non-infected controls (n = 16).

Results: First B. hyodysenteriae detection by q-PCR and first loose stools with blood and mucus occurred both at 8 days post-inoculation. The lapse between a positive q-PCR and observation of mucohaemorrhagic diarrhoea ranged from 0 to 3 days, except in a single pig in which this period lasted 5 days. Macroscopic lesions were observed in the large intestine from both infected groups although more frequent and severe in acute infection group. Microscopic observation of the apex mucosa revealed that in early infection only higher ulceration values were observed compared to healthy controls. In contrast, the acute infection group exhibited higher ulceration, neutrophils infiltration and increased mucosal thickness compared to the other two groups. Among the serum biomarkers tested, only haptoglobin, C-reactive protein, and creatine kinase showed a significant increase in pigs in the acute infection period compared to controls, whereas haptoglobin was the only factor with a significant increase at the early infection compared to non-infected animals.

Conclusions: This study provides new insights about SD and remarks the complex and limited options to perform an early detection of infected animals beyond PCR diagnosis.

Keywords: Acute phase proteins; Mucohaemorrhagic diarrhoea; Pig; Spirochaetes; Swine dysentery.

Fig. 1

Summary of faecal monitoring of Brachyspira hyodysenteriae in challenged pigs. Dotted line shows the concentration of B. hyodysenteriae in faeces estimated by qPCR (left axes); grey graph bar shows the faecal score (right axes) according to Wilberts et al. [8] in each monitored day. Positive (+) and negative (-) results in the green bar over each graph summarise the results of microbiological culture of B. hyodysenteriae. Pig IDs are indicated at the top of each panel (early infection in yellow and acute infection in dark red)

Fig. 2

Colon and apex tissue sections from control and B. hyodysenteriae infected pigs. Macroscopic pathological findings: (A) Colon without macroscopic lesion (control); (B) Colon with mild hyperaemia (early infection); (C) Colon with severe hyperaemia and/or congestion, severe meso-colonic oedema and moderate hyperplasia of the mesenteric lymph nodes (acute infection). Photomicrographs at high and low magnifications of the apex stained with haematoxylin and eosin; (D) Normal mucosal apex (control); (E) Mild necrosis of the epithelium (early infection); (F) Severe necrosis of the epithelium (acute infection); (G) No lesion, only mild haemorrhage (control); (H) Moderate number of lymphocytes in the epithelium (early infection); (I) High number of neutrophils in the lamina propria (acute infection). Arrows point areas of ulceration and necrosis (2E and 2 F) and the inflamatory infiltrate (2 H and 2I)

Fig. 3

Boxplots of apex microscopic lesions in controls and B. hyodysenteriae infected pigs. (A) Ulceration scored as 0 = no ulceration; 1 = focal ulceration 1 to 3 crypts; 2 = focal ulceration 3 to 5 crypts; 3 = focal ulceration more than 5 crypts; + 0.5 multifocal ulceration; + 1 multifocal ulceration more than 5 crypts. (B) Lamina propria haemorrhage and (C) lumen haemorrhage, scored as 0 ≤ 5 red blood cells (RBC); 1 = 6 to 10 RBC; 2 = 11 to 20 RBC; 4 = 21 to 50 RBC; 5 ≥ 51 RBC; +1 more than 3 foci of haemorrhage. (D) Neutrophilic infiltration of the lamina propria, determined as mean of ten 40x fields. (E) Mucosal thickness determined as mean of three measurements the crypts perpendicular to the mucosal surface. Each dot represents one pig. The lower, middle, and upper horizontal lines in the boxes correspond to the first quartile, median, and third quartile values. The lines extending above and below the boxes indicate the range of the upper and lower points within the 1.5 interquartile range. Results of Kruskal-Wallis test are indicated in the upper left corner of each panel. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (Wilcoxon test with Holm adjustment)

Fig. 4

Correlation between concentration of B. hyodysenteriae in faeces and faecal score or microscopic lesions in infected pigs. Concentration of B. hyodysenteriae is estimated using q-PCR in last faeces before euthanasia. (A) Faecal score, last recorded before euthanasia (0 = normal, 1 = soft but formed, 2 = semisolid, 3 = liquid to watery, + 0.5 presence of mucus and / or blood) (Spearman’s rank order correlation). (B) Ulceration (0 = no ulceration; 1 = focal ulceration 1 to 3 crypts; 2 = focal ulceration 3 to 5 crypts; 3 = focal ulceration more than 5 crypts; + 0.5 multifocal ulceration; + 1 multifocal ulceration more than 5 crypts) (Pearson correlation coefficient). (C) Lamina propria haemorrhage and (D) lumen haemorrhage (0 ≤ 5 red blood cells (RBC); 1 = 6 to 10 RBC; 2 = 11 to 20 RBC; 4 = 21 to 50 RBC; 5 ≥ 51 RBC; +1 more than 3 foci of haemorrhage) (Spearman’s rank order correlation). (E) Neutrophilic infiltration of the lamina propria, determined as mean of ten 40x fields (Spearman’s rank order correlation). (F) Mucosal thickness determined as mean of three measurements the crypts perpendicular to the mucosal surface (Pearson correlation coefficient). Correlation coefficient with the p-value is indicated in the upper left corner and the line represent linear regression line of each panel

Fig. 5

Boxplots of the measured serum biomarkers in controls and B. hyodysenteriae infected pigs. (A) Haptoglobin. (B) C- reactive protein. (C) Creatine kinase. (D) Total proteins. Each dot represents one pig. The lower, middle, and upper horizontal lines in the boxes correspond to the first quartile, median, and third quartile values. The lines extending above and below the boxes indicate the range of the upper and lower points within the 1.5 interquartile range. Results of Kruskal-Wallis (A, B, C) and ANOVA (D) test are indicated in the upper left corner of each panel. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (Wilcoxon (A, B, C) and Tukey (D) test with Holm adjustment)