T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8 h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

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.

Experimental infectious challenge in pigs leads to elevated fecal calprotectin levels following colitis, but not enteritis.

BACKGROUND: Fecal calprotectin is largely applied as a non-invasive intestinal inflammation biomarker in human medicine. Previous studies in pigs investigated the levels of fecal calprotectin in healthy animals only. Thus, there is a knowledge gap regarding its application during infectious diarrhea. This study investigated the usefulness of fecal calprotectin as a biomarker of intestinal inflammation in Brachyspira hyodysenteriae and Salmonella Typhimurium infected pigs. RESULTS: Fecal samples from pigs with colitis (n = 18) were collected from animals experimentally inoculated with B. hyodysenteriae (n = 8) or from sham-inoculated controls (n = 3). Fecal samples from pigs with enteritis (n = 14) were collected from animals inoculated with Salmonella enterica serovar Typhimurium (n = 8) or from sham-inoculated controls (n = 4). For both groups, fecal samples were scored as: 0 = normal; 1 = soft, wet cement; 2 = watery feces; 3 = mucoid diarrhea; and 4 = bloody diarrhea. Fecal calprotectin levels were assayed using a sandwich ELISA, a turbidimetric immunoassay and a point-of-care dipstick test. Fecal calprotectin levels were greater in colitis samples scoring 4 versus ≤ 4 using ELISA, and in feces scoring 3 and 4 versus ≤ 1 using immunoturbidimetry (P < 0.05). No differences were found in calprotectin concentration among fecal scores for enteritis samples, regardless of the assay used. All samples were found below detection limits using the dipstick method. CONCLUSIONS: Fecal calprotectin levels are increased following the development of colitis, but do not significantly change due to enteritis. While practical, the use of commercially available human kits present sensitivity limitations. Further studies are needed to validate the field application of calprotectin as a marker of intestinal inflammation.

Poly-ε-Caprolactone Microsphere Polymers Containing Usnic Acid: Acute Toxicity and Anti-Inflammatory Activity.

Usnic acid (UA) has been studied by its pharmacological properties; however, it presents moderate toxicity, low solubility, and absorption by biological membranes. The aim of this study was to develop poly-ε-caprolactone microsphere polymers containing UA (UA-micro) and evaluate their acute toxicity and anti-inflammatory activity. The microspheres were prepared by multiple emulsion technique (water/oil/water) and characterized by the encapsulation efficiency, particle size, polydispersity index, and zeta potential. The acute toxicity of UA and UA-micro (25-50 mg/kg; p.o.) was evaluated in mice. The anti-inflammatory activity of UA and UA-micro was evaluated by subcutaneous air pouch and carrageenan-induced paw edema in rat, with measurement of inflammatory cytokines and MPO levels. The UA presented encapsulation efficiency of 97.72%, particle size of 13.54 micrometers, polydispersity index of 2.36, and zeta potential of 44.5 ± 2.95 mV. The UA-micro presented lower acute toxicity (LD50 value up to 2000 mg/kg; p.o.) when compared to UA. UA-micro and UA (25 mg/kg) significantly reduced paw volume and decreased MPO levels, whereas only UA-micro (50 mg/kg) reduced significantly IL-1ß, TNF-α, and NO levels in inflammatory exudate. These results suggest that controlled release systems, as microspheres, can be a promising alternative to reduce the toxicity of UA, making it a viable compound for inflammation therapy.

Molecular modeling and cytotoxicity of diffractaic acid: HP-ß-CD inclusion complex encapsulated in microspheres.

In this pioneer study, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was used to improve the solubility of the diffractaic acid (DA) via inclusion complex (DA:HP-ß-CD). Subsequently, DA:HP-ß-CD was incorporated into poly-ε-caprolactone (PCL) microspheres (DA:HP-ß-CD-MS). Microspheres containing DA (DA-MS) or DA:HP-ß-CD (DA:HP-ß-CD-MS) were prepared using the multiple W/O/W emulsion-solvent evaporation technique. The phase-solubility diagram of DA in HP-ß-CD (10-50mM) showed an AL type curve with a stability constant K1:1=821M-1. 1H NMR, FTIR, X-ray diffraction and thermal analysis showed changes in the molecular environment of DA in DA:HP-ß-CD. The molecular modeling approach suggests a guest-host complex formation between the carboxylic moiety of both DA and the host (HP-ß-CD). The mean particle size of the microspheres were ∅DA-MS=5.23±1.65µm and ∅DA:HP-ß-CD-MS=4.11±1.39µm, respectively. The zeta potential values of the microspheres were ζDA-MS=-7.85±0.32mV and ζDA:HP-ß-CD-MS=-6.93±0.46mV. Moreover, the encapsulation of DA:HP-ß-CD into microspheres resulted in a more slower release (k2=0.042±0.001; r2=0.996) when compared with DA-MS (k2=0.183±0.005; r2=0.996). The encapsulation of DA or DA:HP-ß-CD into microspheres reduced the cytotoxicity of DA (IC50=43.29µM) against Vero cells (IC50 of DA-MS=108.48µM and IC50 of DA:HP-ß-CD-MS=142.63µM).