Combined in-vitro and on-farm evaluation of commercial disinfectants used against Brachyspira hyodysenteriae

Background Swine dysentery (SD) is a severe infectious disease with a relevant impact on pig production usually caused by Brachyspira hyodysenteriae, although B. hampsonii causes an identical clinical picture. SD control relies on antimicrobials, good management practices and strict biosecurity with cleaning and disinfection as crucial tools to avoid the pathogen transmission. This study evaluates the in-vitro efficacy of an array of commercial disinfectants against a collection of B. hyodysenteriae isolates using broth tests. The efficacy of cleaning and disinfection protocols was also evaluated on two farms with endemic SD using surface swabs collected in emptied pens before and after cleaning and disinfection procedures, using both real-time PCR and bacterial microbiological culture. Results Most of the commercial disinfectants evaluated were effective against all B. hyodysenteriae isolates tested, with a reduction of more than 5.00 log10 CFU/mL (bactericidal efficacy of 99.999%). However, some isolates exhibited reduced susceptibility to Virkon-S and Limoseptic disinfectants. The evaluation of cleaning and disinfection protocols on farms with SD outbreaks showed that approximately half the pens tested (n = 25) were positive by real-time PCR after pigs removal (mean B. hyodysenteriae counts 5.72 ± 1.04 log10 CFU/mL) while almost 20% of the pens remained positive after cleaning (n = 7) and disinfection (n = 5) procedures although with significantly lower, mean estimates (4.31 ± 0.43 log10 CFU/mL and 4.01 ± 0.55 log10 CFU/mL, respectively). Conclusions These results show the efficacy of disinfectants against B. hyodysenteriae but also stress the need to implement adequately the cleaning and disinfection protocols on pig farms and review and revise their efficiency periodically.

Preparation and Characterization of a New Monoclonal Antibody Specific Against Lawsonia intracellularis and Its Application in Indirect Immunofluorescence and Immunocytochemistry Assay

Proliferative enteropathy (PE) is an infectious enteric disease caused by Lawsonia intracellularis (L. intracellularis) and is endemic in pig herds worldwide. However, a L. intracellularis-specific monoclonal antibody plays an important role in the evaluation of L. intracellularis infection in vitro. Therefore, the objective of this study was to produce and identify the characteristics of a new monoclonal antibody against the outer membrane protein (Omp2) of L. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC). The results indicated that three highly specific monoclonal antibodies against the Omp2 protein (4D9, 3G2, and 7G5) of L. intracellularis were obtained by using purified Omp2 as an immunogen, the titers of ascitic fluids of 4D9, 3G2, and 7G5 cells were 1:2,048,000, 1:512,000, and 1:256,000, respectively. IFA analysis showed that the 4D9, 3G2, and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs or closely related to L. intracellularis, such as Desulfovibrio, Bilophila wadsworthia (B. wadsworthia), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), and Brachyspira hyodysenteriae (B. hyodysenteriae). IFA and IHC results indicated that the monoclonal antibodies can be successfully used as primary antibodies to detect L. intracellularis in infected cells and in the crypt of the ileum from infected tissues of PE. Our findings suggested that the new monoclonal antibody specific against L. intracellularis will be useful for the evaluation of L. intracellularis infection in vivo and in vitro.

Detection of Swine Dysentery (SD) caused by Brachyspira hyodysenteriae via PCR in Northern Vietnam

This study aimed to diagnose swine dysentery (SD) caused by Brachyspira hyodysenteriae in pigs by the PCR method in Vietnam. Of the 250 samples, 29 isolates of B. hyodysenteriae (11.60%) were identified by PCR in seven provinces of Northern Vietnam, and the infection rate differed from region to region. From the positive cases of B. hyodysenteriae, we analyzed B. hyodysenteriae infected cases according to the ages of the pigs, farm sizes, and veterinary hygiene practices to get more information about the disease in Vietnam. The results showed that the positive B. hyodysenteriae samples were commonly seen in post-weaning pigs (32.14%) in households (20.73%) with poor hygiene (24.69%). Clinical signs of SD included high fever (100%); anorexia (100%); watery, bloody diarrhea, usually gray to brown in color (100%); and weight loss (86.42%). Gross lesions of SD were limited to the large intestine were described as having a fibrinous, blood-flecked membrane covering the mucosa (93.75%), swollen with hemorrhaged colon and cecum (75.00%), and mesenteric lymph nodes (81.25%).

Complete Circular Genome Sequences of Brachyspira hyodysenteriae Isolates of the Four Different Sequence Types Causing Swine Dysentery in Switzerland

The complete genomes of four Brachyspira hyodysenteriae isolates of the four different sequence types (STs) (ST6, ST66, ST196, and ST197) causing swine dysentery in Switzerland were generated by whole-genome sequencing and de novo hybrid assembly of reads obtained from second (Illumina) and third (Oxford Nanopore Technologies and Pacific Biosciences) high-throughput sequencing platforms.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection is Species-Specific and Varies between Strains

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact in the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with a massive mucus induction, which substantially increases the amount of B. hyodysenteriae binding sites in the mucus. We have previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level ( B. innocens, B. pilosicoli, B. murdochii, B. hampsonii and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae , and within this species, the binding ability to mucins varies between strains/isolates, increases to mucins from pigs with SD, and is associated to sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon including a shift in sialic acid containing structures. Thus, we demonstrate through hierarchical cluster analysis and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans, that sialic acid containing structures in the mucin O -glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Whole-Genome Sequencing of Brachyspira hyodysenteriae Isolates From England and Wales Reveals Similarities to European Isolates and Mutations Associated With Reduced Sensitivity to Antimicrobials

Brachyspira hyodysenteriae is the principal cause of swine dysentery, a disease that threatens economic productivity of pigs in many countries as it can spread readily within and between farms, and only a small number of antimicrobials are authorized for treatment of pigs. In this study, we performed whole-genome sequencing (WGS) of 81 B. hyodysenteriae archived at the Animal and Plant Health Agency (APHA) from diagnostic submissions and herd monitoring in England and Wales between 2004 and 2015. The resulting genome sequences were analyzed alongside 34 genomes we previously published. Multi-locus sequence typing (MLST) showed a diverse population with 32 sequence types (STs) among the 115 APHA isolates, 25 of them identified only in England; while also confirming that the dominant European clonal complexes, CC8 and CC52, were common in the United Kingdom. A core-genome SNP tree typically clustered the isolates by ST, with isolates from some STs detected only within a specific region in England, although others were more widespread, suggesting transmission between different regions. Also, some STs were more conserved in their core genome than others, despite these isolates being from different holdings, regions and years. Minimum inhibitory concentrations to commonly used antimicrobials (Tiamulin, Valnemulin, Doxycycline, Lincomycin, Tylosin, Tylvalosin) were determined for 82 of the genome-sequenced isolates; genomic analysis revealed mutations generally correlated well with the corresponding resistance phenotype. There was a major swine dysentery intervention program in 2009–2010, and antimicrobial survival curves showed a significant reduction in sensitivity to tiamulin and valnemulin in isolates collected in and after 2010, compared to earlier isolates. This correlated with a significant increase in post-2009 isolates harboring the pleuromutilin resistance gene tva(A), which if present, may facilitate higher levels of resistance. The reduction in susceptibility of Brachyspira from diagnostic submissions to pleuromutilins, emphasizes the need for prudent treatment, control and eradication strategies.

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.

Distribution of Flumequine in Intestinal Contents and Colon Tissue in Pigs after Its Therapeutic Use in the Drinking Water

Flumequine concentrations in plasma, colon tissue and intestinal contents were evaluated in 12 healthy pigs after oral administration (12 mg/kg every 24 h for 5 consecutive days in drinking water). Plasma, colon tissue and intestinal content samples were collected from animals sacrificed on days 3, 6 and 7. Concentrations were measured by high performance liquid chromatography after having validated the method, following the European Medicines Agency (EMA) requirements. The drug was not detected in any plasma sample. In colon tissue, concentrations were higher on day 3 (0.230 ± 0.033 µg/g, descending colon; 0.156 ± 0.093 µg/g, ascending colon) than on day 6 (0.187 ± 0.123 µg/g, descending colon; 0.107 ± 0.007 µg/g, ascending colon). Concentrations were considerably higher in intestinal contents, again on day 3 (1.349 ± 1.401 µg/g, descending colon; 0.591 ± 0.209 µg/g, ascending colon) than on days 6 (0.979 ± 0.346 µg/g, descending colon; 0.595 ± 0.075 µg/g, ascending colon) and 7 (0.247 ± 0.172 µg/g, descending colon; 0.172 ± 0.086 µg/g, ascending colon). Measured concentrations were lower than those effective against the most common intestinal pathogenic microorganisms in swine and, more specifically, Brachyspira hyodysenteriae.