Pathogenesis Of Swine Dysentery Research Articles

Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysentery.

Swine dysentery (SD) is a worldwide production-limiting disease of growing-finishing pigs in commercial farms. The importance of the large intestinal microbiota in the swine dysentery pathogenesis has been established, but not well characterized. The objective of this study was to characterize the fecal bacterial microbiota of pigs immediately prior to developing clinical signs of swine dysentery. A total of 60 fecal samples were collected from 15 pigs with SD. Sampling times included a time point prior to SD (d0, n=15), 2 days before mucohaemorrhagic diarrhea was observed (d-2SD, n=15), 1 day before mucohaemorrhagic diarrhea was observed (d-1SD, n=15), and the day when pigs developed mucohemorragic diarrhea (MHD, n=15). Sequencing of cpn60 amplicons was used to profile the microbiome, and analyses were performed on QIIME2. Increased Chao1 index in d-1SD and MHD samples when compared to the d0 was the only change observed in alpha diversity. No differences between sampling times on beta diversity (Bray-Curtis dissimilarity) were found. Although a small sample size was investigated, differential abundance analysis revealed that Alistipes dispar and Parabacteroides gordonii were increased in MHD fecal samples when compared to d-2SD and d-1SD. It is suggested that these taxa may play a role in the pathogenesis of SD, which is known to require the presence of Brachyspira spp. and an anaerobe for severe disease development.

Acute infection with Brachyspira hyodysenteriae affects mucin expression, glycosylation, and fecal MUC5AC.

Infection with strongly β-hemolytic strains of Brachyspira hyodysenteriae leads to swine dysentery (SD), a production-limiting disease that causes mucohemorrhagic diarrhea and typhlocolitis in pigs. This pathogen has strong chemotactic activity toward mucin, and infected pigs often have a disorganized mucus layer and marked de novo expression of MUC5AC, which is not constitutively expressed in the colon. It has been shown that fucose is chemoattractant for B. hyodysenteriae, and a highly fermentable fiber diet can mitigate and delay the onset of SD. We used lectins targeting sialic acids in α-2,6 or α-2,3 linkages, N-acetylglucosamine (GlcNAc), α-linked L-fucose, and an immunohistochemical stain targeting N-glycolylneuraminic acid (NeuGc) to investigate the local expression of these mucin glycans in colonic tissues of pigs with acute SD. We used a commercial enzyme-linked immunosorbent assay (ELISA) to quantify fecal MUC5AC in infected pigs and assess its potential as a diagnostic monitoring tool and RNA in situ hybridization to detect IL-17A in the colonic mucosa. Colonic mucin glycosylation during SD has an overall increase in fucose, a spatially different distribution of GlcNAc with more expression within the crypt lumens of the upper colonic mucosa, and decreased expression or a decreased trend of sialic acids in α-2,6 or α-2,3 linkages, and NeuGc compared to the controls. The degree of increased fucosylation was less in the colonic mucosa of pigs with SD and fed the highly fermentable fiber diet. There was a significant increase in MUC5AC in fecal and colonic samples of pigs with SD at the endpoint compared to the controls, but the predictive value for disease progression was limited. Fucosylation and the impact of dietary fiber may play important roles in the pathogenesis of SD. The lack of predictive value for fecal MUC5AC quantification by ELISA is possibly due to the presence of other non-colonic sources of MUC5AC in the feces. The moderate correlation between IL-17A, neutrophils and MUC5AC confirms its immunoregulatory and mucin stimulatory role. Our study characterizes local alteration of mucin glycosylation in the colonic mucosa of pigs with SD after B. hyodysenteriae infection and may provide insight into host-pathogen interaction.

Confirmation of the Role of Rapidly Fermentable Carbohydrates in the Expression of Swine Dysentery in Pigs after Experimental Infection

Two experiments were conducted to test the hypothesis that soluble non-starch polysaccharides (NSP) and resistant starch (RS) cause swine dysentery (SD) in pigs experimentally infected with the spirochete Serpulina hyodysenteriae. In Experiment 1, a source of soluble NSP (guar gum; GG), insoluble NSP (oat chaff; OC), resistant starch (retrograde cornstarch; RS) or a combination of GG and RS (GG + RS) was added to a diet containing cooked white rice (R), soybean meal (SBM) and animal protein (meat and bone meal, bloodmeal, fishmeal). A diet containing only cooked white rice, SBM and the sources of animal protein (AP) was also fed. In Experiment 2, three rice-based diets containing different levels of RS were fed to pigs. In Experiment 1, the pH of digesta in the cecum, proximal colon and distal colon of pigs fed diets R-GG, R-RS and R-GG + RS was lower (P < 0.001), and volatile fatty acid concentration higher (P < 0.001), than in pigs fed diets R-OC and R-AP. Pigs fed diets with RS and GG + RS had greater (P < 0.05) concentrations of ATP in the large intestine than pigs fed other diets. There were no significant differences in any fermentation indices measured in Experiment 2. In Experiment 1, pigs fed diets R-GG, R-RS and R-GG + RS were colonized with S. hyodysenteriae after experimental infection. However, only pigs consuming diets R-GG (4 of 5) and R-GG + RS (5 of 5) showed clinical signs of SD. Spirochetes were isolated from the feces of all pigs fed diets containing RS in Experiment 2. However, and in contrast to Experiment 1, 80–100% of pigs infected with S. hyodysenteriae displayed clinical signs of SD. These data confirm the role of fermentable carbohydrate in the pathogenesis of SD.

Cell membrane permeability and mitochondrial dysfunction-inducing activities in cell-free supernatants from Serpulina hyodysenteriae serotypes 1 and 2

Membrane permeability (MP) and mitochondrial dysfunction-inducing (MDI) activities were detected in cell-free supernatants (CFS) of Serpulina hyodysenteriae, using either hemoglobin release from porcine red blood cells (RBC) or cytoplasmic lactate dehydrogenase release from porcine peripheral blood lymphocytes (PBL), and reduction of the 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide dye by porcine PBL. The MP and MDI activities of CFS correlated with each other for serotype 1 and 2 isolates taken at different population densities; however, the kinetics of toxin production varied between each serotype. The loss of enteropathogenicity of two field isolates with nonpathogenic phenotypes and pathogenic isolates passaged up to 45 times in vitro was not attributable to a loss of either membrane permeability or mitochondrial dysfunction-inducing activity of cell-free supernatants. Results from this study suggested the potential for two separate toxins being involved in the pathogenesis of swine dysentery, with the MDI activity correlating with age susceptibility to clinical disease.

Swine dysentery: more unknown than known

Swine dysentery (SD) is an economically important disease. It is caused by the spirochete Serpulina hyodysenteriae. In order to minimize the economic damage of SD, several methods to control this disease are recommended. Whereas hygienic measures and use of antimicrobials are actually practised for prevention, detection and exclusion of carriers of S. hyodysenteriae and vaccination against the desease still needs further attention. The last two methods require reliable and sensitive diagnostic tests and understanding of the pathogenesis of and immune development against SD. At present the detection of all individual carriers of S. hyodysenteriae is not yet assured, since the tests for screening individual animals are not satisfactorily evaluated as far as sensitivity and/or specificity are concerned. Studies on the pathogenesis of SD have been performed to develop a vaccine. Besides hemolysin/cytotoxin production, no other virulence factors have been identified with certainty for S. hyodysenteriae. Recently however, further indications for a role of motility in the pathogenesis of SD have been obtained. In this manuscript we summarize the most relevant recent findings.

Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examine the effect of Ca 2+, Co 2+, Cu 2+, Fe 2+, Mg 2+, Mn 2+, Ni 2+, and Zn 2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO 4 or CuSO 4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn 2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn 2+ by EDTA indicated that Zn 2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn 2+. When the spirochetes were washed after incubation in the presence of ZnSO 4 for 2 cycles and incubated in fresh PBS-GR without Zn 2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO 4 was due to a direct action of ZnSO 4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO 4, the inhibitory effect of Zn 2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn 2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO 4 revealed clumping of ribosomes and clearing of cell cytoplasm.

Reduced virulence of Serpulina hyodysenteriae hemolysin-negative mutants in pigs and their potential to protect pigs against challenge with a virulent strain

The role of the Serpulina hyodysenteriae hemolysin encoded by the tlyA gene in the pathogenesis of swine dysentery (SD) was studied. tlyA mutants of two S. hyodysenteriae strains (B204 and C5) were tested for virulence in pigs. None of the animals developed SD. However, after infection with wild-type strain B204 or C5, the incidence of SD was 100 or 60%, respectively. Thus, the tlyA-encoded hemolysin of S. hyodysenteriae is an important virulence factor in SD. The potential of these mutants to protect pigs against challenge with a virulent S. hyodysenteriae strain was also studied. After challenge with wild-type strain B204, 50% of pigs previously inoculated with the B204 tlyA mutant were protected, whereas all control pigs contracted SD. None of the pigs previously inoculated with the C5 tlyA mutant developed SD upon challenge with wild-type strain B204, whereas 40% of the control pigs developed SD in this experiment. Thus, previous colonization with S. hyodysenteriae tlyA mutants in pigs provides partial protection against challenge with a virulent S. hyodysenteriae strain.

The Role of Hemolysin(s) in the Pathogenesis of Serpulina hyodysenteriae

Serpulina (Treponema) hyodysenteriae, an anaerobic beta hemolytic spirochaete, is the etiologic agent of swine dysentery. Not much is known at present about the virulence factors of S. hyodysenteriae. However, the hemolysin production of this bacterium is generally accepted to be a virulence factor. To study the exact role of hemolysin production in the pathogenesis of swine dysentery, the gene encoding a hemolysin, tly, was cloned and its nucleotide sequence determined. After inactivation of this gene, the virulence of a tly-minus mutant in mice was tested. The mutant had reduced hemolysis indicating that the tly-encoded hemolysin was not the only hemolysin produced by S. hyodysenteriae. Mice infected with the tly-minus mutant had fewer cecal lesions than mice infected with the wild-type S. hyodysenteriae. It was concluded that the tly-encoded hemolysin might be an important virulence factor, but not the only one. Since it was demonstrated that spirochaetes can be transformed through electroporation, this has made a genetic approach to elucidate the pathogenesis of spirochaetal infections possible.

Inactivation of a Serpula (Treponema) hyodysenteriae hemolysin gene by homologous recombination: Importance of this hemolysin in pathogenesis of S. hyodysenteriae in mice

The role of Serpula hyodysenteriae hemolysin in the pathogenesis of swine dysentery was studied. The inactivation of a gene, tly, encoding a hemolysin, and the virulence of a tly − mutant in mice is reported. We obtained chromosomal integration of the distrupted tly gene via homologous recombination in a S. hyodysenteriae wild-type strain following electroporation. A plasmid construct, in which the tly gene was disrupted by a kanamycin-resistance gene, was used for the electroporation. The mutants obtained had reduced hemolysis indicating that the tly encoded hemolysin is not the only hemolysin produced by S. hyodysenteriae. Mice infected with the tly − mutant had fewer cecal lesions than mice infected with the wild-type S. hyodysenteriae. From these results it was concluded that the tly encoded hemolysin may be an important virulence factor, but not the only one. It was demonstrated that gene transfer to spirochaetes can be achieved through electroporation.

Inactivation of aSerpula (Treponema) hyodysenteriaehemolysin gene by homologous recombination: Importance of this hemolysin in pathogenesis ofS. hyodysenteriaein mice

The role of Serpula hyodysenteriae hemolysin in the pathogenesis of swine dysentery was studied. The inactivation of a gene, tly, encoding a hemolysin, and the virulence of a tly− mutant in mice is reported. We obtained chromosomal integration of the distrupted tly gene via homologous recombination in a S. hyodysenteriae wild-type strain following electroporation. A plasmid construct, in which the tly gene was disrupted by a kanamycin-resistance gene, was used for the electroporation. The mutants obtained had reduced hemolysis indicating that the tly encoded hemolysin is not the only hemolysin produced by S. hyodysenteriae. Mice infected with the tly− mutant had fewer cecal lesions than mice infected with the wild-type S. hyodysenteriae. From these results it was concluded that the tly encoded hemolysin may be an important virulence factor, but not the only one. It was demonstrated that gene transfer to spirochaetes can be achieved through electroporation.

Biochemical and immunochemical characterisation of strains of Treponema hyodysenteriae

The protein composition of 18 clinical isolates of Treponema hyodysenteriae from pigs with swine dysebtery in Australia were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunublot analysis. Coomassie Blue stained SDS-PAGE-profiles of whole cell and outer membrane (OM) proteins demonstrated the same gel pattern among the T. hyodysenteriae isolates, particularly the OM proteins in the molecular mass (M r) range of 30 kDa to 40 kDa. The T. hyodysenteriae isolates were categorised into two distinct groups (A and B) based on the strain-variability in the 37 kDa OM protein. Immunoblotting of whole cell proteins after SDS-PAGE using serum from rabbits and pigs immunised with known T. hyodysenteriae serotypes revealed a number of common immunoreactive bands in all isolates. LPS typing of the T. hyodysenteriae isolates by immunoblotting with the rabbit antiserum revealed one additional serotype emphasising the LPS heterogeneity among the strains isolated from geographic locations in Australia, Great Britain and the U.S.A. Immunoblotting of the OM preparations revealed several common immunoreactive polypeptides corresponding to M r values of 34 kDa to 30 kDa among the T. hyodysenteriae and T. innocens isolates but a distinct 39 kDa found only in the T. hyodysenteriae isolates. Trypsin proteolysis of intact T. hyodysenteriae cells caused selective loss of these and other major abundant proteins identifying the location of the 39 kDa, 36 kDa,and 30 kDa proteins on the cell surface and suggesting a possible role of these proteins in the pathogenesis of swine dysentery.

Analysis of lipopolysaccharide antigens ofTreponema hyodysenteriae

Lipopolysaccharide (LPS) extracts obtained from Treponema hyodysenteriae of serogroups A, B, D and E, and from T. innocens were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), silver-staining, and immunoblotting with hyperimmune rabbit sera. All organisms possessed multiple LPS bands, but their position and number differed. Immunoblotting of LPS with grouping sera identified three or four major antigenic LPS components in the 10-42 kDa range in all organisms: these components were largely specific to each type-organism of a serogroup, and presumably represented group antigens. Although some minor cross-reactivity occurred between LPS from organisms in the different groups, this was insufficient to merit changes to the current LPS serogrouping system for T. hyodysenteriae. Besides this LPS 'complex', other higher-molecular-weight material which appeared to be a common component of the treponemes examined was present in low concentrations. Organisms with different serotypes within a serogroup apparently possessed common LPS bands, but also had unique LPS bands which may account for their serotype specificity. One 'untypable' organism lacked group-specific LPS and was thought to be a mutant of a group B organism. The loss of serogroup LPS by the isolate suggested that this material is an external component of the cell wall. The availability of an atypical organism lacking LPS components may facilitate further studies on the pathogenesis of swine dysentery.

Studies on the pathogenesis of swine dysentery. II. Search for a cytotoxin in spirochetal broth cultures and colon content.

Broth cultures of Treponema hyodysenteriae and colonic content from pigs with swine dysentery were tested for cytotoxicity in cell cultures, erythrocyte suspensions and in ligated segments of pig colon. Live cells of T. hyodysenteriae attached to the surface of cells in all cultures tested but did not penetrate them nor cause morphologic change detectable by light microscopy. Only live T. hyodysenteriae caused erythrolysis. Broth cultures or colonic content sterilized by filtration or by disruption with ultrasound had no visible effect on the cell cultures, erythrocyte suspensions or the mucosa of ligated colonic segments.

Studies on the Pathogenesis of Swine Dysentery

Swine dysentery was induced in pigs and in ligated colonic segments by inoculation of pure cultures of, or colonic contents containing, Treponema hyodysenteriae. The mildest changes, best seen in ligated segments 48 or 72 hours after inoculation, were congestion and leucocytic margination in mucosal capillaries and depletion of mucigen from goblet cells lining the base of the crypts of Lieberkühn. Superficial mucosal necrosis and crypt cell hyperplasia were later changes. Perfusion studies with India ink did not demonstrate occlusive mucosal ischemia in acute swine dysentery. Mucosa with lesions of swine dysentery contained at least 10(5) colony forming units of T. hyodysenteriae per gram. Mucosa without lesions had 10(5) or fewer T. hyodysenteriae per gram. Segments with acute swine dysentery were distended with clear mucoid fluid with electrolyte composition indicative of net colonic secretion. No increase in the concentration of volatile fatty acids was detected in content from intact colons or colonic segments with lesions of acute swine dysentery.

Aetiology and pathogenesis of swine dysentery -- recent advances.

Aetiology and pathogenesis of swine dysentery -- recent advances.