Brachyspira-associated colitis and salmonellosis in pigs: novel approaches and host-pathogen interactions

Abstract

This thesis aimed to verify the association of the intestinal microbiota preceding the clinical signs of swine dysentery (SD) on disease onset; to investigate the porcine T-independent B-cellresponse to Brachyspira spp. and Salmonella enterica serovar Typhimurium; and to assess the usefulness of fecal calprotectin (FC) as a biomarker of intestinal inflammation in B. hyodysenteriae and S. Typhimurium infected pigs.In the first trial, 60 fecal samples were collected from 15 contact pigs at: one day after contact with seeder pigs inoculated intra-gastrically with B. hyodysenteriae(d0); 2 days (d-2SD) and 1 day (d-1SD) before mucohaemorrhagic diarrhea was observed, and at the day when pigs developed mucohemorragic diarrhea (MHD). Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target.Only an increase in the Chao1 index in d-1SD and MHD compared to d0 samples was observed at the genus level for the alpha diversity index. Differential abundance analysis revealed that amplicon sequence variants (ASV) were modulated in the days prior to diarrhea observation. An increase in Alistipes dispar and Parabacteroides gordonii was detected in MHD. In summary, there was an alteration in the fecal microbiota in the days prior to the development of clinical SD.In the second trial, immortalized porcine B-cells were co-incubated for 8 hours with sham-inoculum (n=6), E. coli LPS (n=6), B. hyodysenteriae (n=6),B. hampsonii (virulent and avirulent strains, n = 3 for each), B. pilosicoli (n=6), B. pilosicoli dead (n=3), S. Typhimurium (n =6). B-cell mortality was evaluated using Trypan blue, and the expression levels of B-cell activation-related genes were assessed using RT-PCR.Only S. Typhimurium and LPS led to increased B-cell mortality follow the exposure period.B. pilosicoli downregulated CD19, syk, lyn, and TNF-α, when compared to the negative control group. Our findings suggest that B. pilosicoli does not elicit a B-cell response, , nor does it trigger antigen presentation mechanisms. All other bacteria could activate different triggers within the T-independent B-cell pathway. In the third trial, fecal samples from pigs with colitis were collected from animals experimentally inoculated with B. hyodysenteriae or from sham-inoculated controls (n =18). Fecal samples from pigs with enteritis werecollected from animals inoculated with S. Typhimurium or from sham-inoculatedcontrols (n = 14). For both groups, fecal samples were scored as: 0 = normal; 1 = soft, wet cement; 2 = watery feces; 3 = mucoid diarrhea; and 4 = bloody diarrhea. FC levels were analyzed using a sandwich ELISA, a turbidimetric immunoassay and a point-of-care dipstick test. FC concentration were higher in colitis samples scoring 4 compared with ≤ 4 fecal scores using ELISA, and in feces scoring 3 and 4 than ≤ 1 using immunoturbidimetry. Regardless of the assay used, no differences in FC levels were found among fecal scores for enteritis samples. This initial data suggest that FC only peaks at detectable levels following colitis but not enteritis. Hence, this indicates its potential role as a biomarker of infectious colitis in pigs and possible support in judicious therapeutic interventions.