Development and validation of a flow cytometry antibody test for Lawsonia intracellularis.

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (106 bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Characterisation of autophagy disruption in the ileum of pigs infected with Lawsonia intracellularis.

Lawsonia intracellularis is the aetiological agent of proliferative enteropathy, an enteric disease endemic in swine. Survival in its intracellular niche of the ileum epithelial lining requires the capacity to subvert, repress or exploit the host immune response to create an environment conducive to bacterial propagation. To better understand how L. intracellularis survives in its intracellular niche, we have performed an investigation into the dynamic relationship between infection and the host autophagy response by immunohistochemistry in experimentally infected porcine ileum samples.Beclin1, a protein required early in the autophagy pathway was observed to be distributed with a basal to apical concentration gradient in the crypts of healthy piglets, whilst infected piglets were observed to have no gradient of distribution and an increase in the presence of Beclin1 in crypts with histological characteristics of L. intracellularis residence. Detecting microtubule-associated protein light chain 3 (LC3) is used as a method for monitoring autophagy progression as it associates with mature autophagosomes. For LC3 there was no notable change in signal intensity between crypts with characteristic L. intracellularis infection and healthy crypts of uninfected pigs. Finally, as p62 is degraded with the internal substrate of an autophagosome it was used to measure autophagic flux. There was no observed reduction or redistribution of p62.These preliminary results of the autophagy response in the ileum suggest that L. intracellularis affects autophagy. This disruption to host ileum homeostasis may provide a mechanism that assists in bacterial propagation and contributes to pathogenesis.

Lawsonia intracellularis infected enterocytes lack sucrase-isomaltase which contributes to reduced pig digestive capacity.

Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol® Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.

PREVALENCE OF LAWSONIA INTRACELLULARIS INFECTION IN NONHUMAN PRIMATES AND PEST RODENTS IN A ZOOLOGICAL COLLECTION.

In 2016 and 2017, Lawsonia intracellularis was isolated from several pileated gibbons (Hylobates pileatus) presenting with diarrhea in Mulhouse Zoo (eastern France). To this day, infection with this bacterium has rarely been described in nonhuman primates (NHP) in captivity or in the wild and there are no data about the prevalence or transmission of the disease. This study focuses on finding the prevalence of this infection amongst Mulhouse Zoo's NHP collection and trying to identify a source of contamination responsible for this epizooty. Forty-eight real-time PCR were conducted on feces from all NHP species in the zoo and on small mammals trapped in the NHP housing structures. No NHP was experiencing symptoms at the time of the study, however test results showed that Lawsonia intracellularis can be found in 61.76% (21/34) of the group total (n = 34) and the prevalence even increases to 92.3% (12/13) in the Lemuriform infraorder (n = 13). In small mammals (n = 14), prevalence of the bacterium is 57.17% (8/14) including 77.78% in rodents (7/9). The results of this study show that several NHP species are healthy carriers and some species of small mammals can be considered as a potential source of contamination. Because of the difficulty encountered trying to isolate the bacterium, it is plausible that infections caused by Lawsonia intracellularis have been underdiagnosed to this day, and that it could be an emerging disease in Europe. Therefore, using real-time PCR to search for this bacterium seems essential in case of diarrhea occurring in nonhuman primates. Moreover, even though further studies on contamination sources need to be conducted, the issue of the presence of rodents in NHP housing structures has to be taken very seriously and tackled with the utmost care.

Review of methods for the detection of Lawsonia intracellularis infection in pigs.

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis-specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.

Uncommon isolation of Desulfovibrio vulgaris from a depressed fracture wound on the forehead.

Desulfovibrio spp. are gram negative, obligate anaerobes capable of reducing sulfate. They have caused infections in humans, but very rarely. They are slow growers and difficult to identify. Hence, they are often overlooked and their actual presence goes unnoticed. Here, we describe a case of a 15- year old boy who was involved in a road traffic accident and he presented with seropurulent discharge from a depressed fracture wound on the forehead. Desulfovibrio vulgaris (D.vulgaris), was isolated from the pus discharge, the first to be reported. The characteristic desulfoviridin pigment production in the organism aided in the identification. The infection was successfully managed with pain reliever and course of amoxicillin - clavulanic acid and linezolid.

Evaluation of swine enteroids as in vitro models for Lawsonia intracellularis infection1,2.

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However, these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs, specifically, the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study, we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Evaluation of mouse enteroids as a model for Lawsonia intracellularis infection.

Lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. The hallmark feature of L. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. A major limitation to the study of L. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. Here we investigated the suitability of mouse enteroids as a model to study L. intracellularis infection. Mouse enteroids were microinjected with L. intracellularis, filter-sterilized L. intracellularis culture supernatant, or sterile cell culture media (DMEM). L. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. There was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in L. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. L. intracellularis infection did not induce changes in gene expression of Ki-67 (proliferation marker), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells). These results indicate that although L. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. Nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine.

Mycoplasma hyopneumoniae-Lawsonia intracellularis dual challenge modulates intestinal integrity and function1.

Lawsonia intracellularis (LI) and Mycoplasma hyopneumoniae (Mh) are 2 globally distributed pathogens that cause significant morbidity and mortality in grow-finish pigs. However, mechanisms that reduce growth and feed efficiency during LI and Mh infection are poorly defined. We hypothesized that reductions in performance are partially due to declines in intestinal function and integrity; thus, this study aimed to evaluate intestinal function and integrity of pigs during a 21-d Mh and LI dual challenge (MhLI). Littermate pairs of barrows (48.1 ± 6.7 kg BW) were selected; 1 pig from each pair was assigned to either MhLI challenge or nonchallenge treatments (n = 12). Pigs were individually housed, fed a corn-soybean diet, and allowed to acclimate for 21 d prior to inoculation. On days postinoculation (dpi) 0, MhLI pigs were dual inoculated with LI and Mh. On dpi 21, all pigs were euthanized for ileal and colon tissue collection. Formalin-fixed tissues were clinically scored and morphology analyzed, frozen tissues assayed for digestive enzyme activities, and fresh tissues mounted into modified Ussing Chambers to assess active nutrient transport, barrier integrity, and bacterial translocation. Data were analyzed using the Mixed Procedure of SAS with treatment as a fixed effect, age and start BW as covariates, and litter as a random effect. Compared with controls, MhLI pigs had decreased ADG (38%, P < 0.001), ADFI (25%, P < 0.001), and G:F (19%, P = 0.012). The MhLI dual challenge did not alter ileum morphology or transepithelial resistance (P > 0.10); however, ex vivo mucosal to serosal translocation of S. Typhimurium in the colon was increased (60%, P = 0.003) in MhLI pigs compared with controls. Additionally, MhLI pigs had increased ileal glucose transport (30%, P = 0.05) and decreased sucrase activity (30%, P = 0.049) compared with controls. This MhLI challenge antagonized intestinal function and integrity, and this may be a contributing factor to reduced pig performance.

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

A Cell Proliferation and Inflammatory Signature Is Induced by Lawsonia intracellularis Infection in Swine.

Lawsonia intracellularis causes porcine proliferative enteropathy. This is an enteric disease characterized by thickening of the wall of the ileum that leads to decreased growth of animals and diarrhea. In this study, we investigated the host response to L. intracellularis infection by performing transcriptomic and pathway analysis of intestinal tissue samples from groups of infected and noninfected animals at 14, 21, and 28 days postchallenge. At the peak of infection, when animals developed the most severe lesions, infected animals had higher levels of several gene transcripts involved in cellular proliferation and inflammation, including matrix metalloproteinase-7 (MMP7), transglutaminase-2 (TGM2), and oncostatin M (OSM). Histomorphology also revealed general features of intestinal inflammation. This study identified important pathways associated with the host response in developing and resolving lesions due to L. intracellularis infection.IMPORTANCELawsonia intracellularis is among the most important enteric pathogens of swine, and it can also infect other mammalian species. Much is still unknown regarding its pathogenesis and the host response, especially at the site of infection. In this study, we uncovered several novel genes and pathways associated with infection. Differentially expressed transcripts, in addition to histological changes in infected tissue, revealed striking similarities between L. intracellularis infection and cellular proliferation mechanisms described in some cancers and inflammatory diseases of the gastrointestinal tract. This research sheds important light into the pathogenesis of L. intracellularis and the host response associated with the lesions caused by infection.

Effects of Lawsonia intracellularis infection in the proliferation of different mammalian cell lines.

Lawsonia intracellularis is an obligate intracellular bacterium that causes proliferative enteropathy in various animal species. While cellular proliferation of intestinal cells is recognized as the hallmark of L. intracellularis infection in vivo, it has not been demonstrated in in vitro models. In order to assay the effect of L. intracellularis, various cell lines were infected with pathogenic and non-pathogenic passages of the bacterium. Because of the high proliferative rate of these cell lines, serum deprivation, which is known to reduce proliferation, was applied to each of the cell lines to allow the observation of proliferation induced by L. intracellularis. Using antibodies for Ki-67 and L. intracellularis in dual immunofluorescence staining, we observed that L. intracellularis was more frequently observed in proliferating cells. Based on wound closure assays and on the amount of eukaryotic DNA content measured over time, we found no indication that cell lines infected with L. intracellularis increased proliferation and migration when compared to non-infected cells (p > 0.05). Cell arrest due to decreased serum in the culture media was cell-line dependent. Taken together, our findings provide data to support and expand previous subjective observations of the absence of in vitro proliferation caused by L. intracellularis in cell cultures and confirm that cell lines infected by L. intracellularis fail to serve as adequate models for understanding the cellular changes observed in proliferative enteropathy-affected intestines.

Effect of selected biocides on microbiologically influenced corrosion caused by Desulfovibrio ferrophilus IS5.

The marine bacterial strain Desulfovibrio ferrophilus IS5, known for its lithotrophic growth ability to use metallic iron as a sole electron donor and for causing corrosion of steel, was used in the current study. Four commonly used biocides in the oil and gas industry, namely tetrakis(hydroxymethyl) phosphonium sulfate (THPS), glutaraldehyde (GLUT), benzalkonium chloride (BAC), and GLUT/BAC were selected to study their efficacy in controlling carbon steel corrosion in the presence of this strain. Incubations containing strain IS5 and low carbon steel coupons were prepared in the presence and absence of the four biocides, and these were monitored using both electrochemical methods (electrochemical impedance spectroscopy, linear polarization resistance and potentiodynamic polarization) and surface analyses (scanning electron microscopy, confocal measurements, optical microscopy, and profilometry) to assess the biofilm/metal interactions. When THPS, BAC, and GLUT/BAC treatments were applied, minimal corrosion was measured by all methods. In contrast, severe pitting was observed in the presence of 50 ppm GLUT, similar to what was observed when D. ferrophilus IS5 was incubated in the absence of biocide, suggesting that GLUT alone may not be effective in controlling MIC in marine environments. This study also showed that the use of non-destructive electrochemical methods is effective for screening for real time biocide selection and monitoring of the impact of chemicals post-dosage in oil and gas operations.

Structural insights into the electron/proton transfer pathways in the quinol:fumarate reductase from Desulfovibrio gigas.

The membrane-embedded quinol:fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/proton-transfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6 Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structural insights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.

Bilophila wadsworthia aggravates high fat diet induced metabolic dysfunctions in mice.

Dietary lipids favor the growth of the pathobiont Bilophila wadsworthia, but the relevance of this expansion in metabolic syndrome pathogenesis is poorly understood. Here, we showed that B. wadsworthia synergizes with high fat diet (HFD) to promote higher inflammation, intestinal barrier dysfunction and bile acid dysmetabolism, leading to higher glucose dysmetabolism and hepatic steatosis. Host-microbiota transcriptomics analysis reveal pathways, particularly butanoate metabolism, which may underlie the metabolic effects mediated by B. wadsworthia. Pharmacological suppression of B. wadsworthia-associated inflammation demonstrate the bacterium's intrinsic capacity to induce a negative impact on glycemic control and hepatic function. Administration of the probiotic Lactobacillus rhamnosus CNCM I-3690 limits B. wadsworthia-induced immune and metabolic impairment by limiting its expansion, reducing inflammation and reinforcing intestinal barrier. Our results suggest a new avenue for interventions against western diet-driven inflammatory and metabolic diseases.

Potent O-antigen-deficient (rough) mutants of Salmonella Typhimurium secreting Lawsonia intracellularis antigens enhance immunogenicity and provide single-immunization protection against proliferative enteropathy and salmonellosis in a murine model.

The obligate intracellular pathogen Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), poses a substantial economic loss in the swine industry worldwide. In this study, we genetically engineered an O-antigen-deficient (rough) Salmonella strain secreting four selected immunogenic LI antigens, namely OptA, OptB, LfliC, and Lhly. The genes encoding these antigens were individually inserted in the expression vector plasmid pJHL65, and the resultant plasmids were transformed into the ∆asd ∆lon ∆cpxR ∆rfaL Salmonella Typhimurium (ST) strain JOL1800. The individual expression of the selected LI antigens in JOL1800 was validated by an immunoblotting assay. We observed significant (P < 0.05) induction of systemic IgG and mucosal IgA responses against each LI antigen or Salmonella outer membrane protein in mice immunized once orally with a mixture of four JOL1800-derived strains. Further, mRNA of IL-4 and IFN-γ were highly upregulated in splenic T cells re-stimulated in vitro with individual purified antigens. Subsequently, immunized mice showed significant protection against challenge with 106.9 TCID50 LI or 2 × 109 CFU of a virulent ST strain. At day 8 post-challenge, no mice in the immunized groups showed the presence of LI-specific genomic DNA (gDNA) in stool samples, while 50% of non-immunized mice were positive for LI-specific gDNA. Further, all the immunized mice survived the virulent ST challenge, compared to a 20% mortality rate observed in the control mice. Collectively, the constructed rough ST-based LI vaccine candidate efficiently elicited LI and ST-specific humoral and cell-mediated immunity and conferred proper dual protection against PE and salmonellosis.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

Experimental studies on effects of diet on Lawsonia intracellularis infections in fattening boars in a natural infection model.

BACKGROUND: Lawsonia intracellularis is one of the most economically important pathogens in swine production. This study tested the hypothesis that the composition of diets for pigs has an impact on the excretion of L. intracellularis in a natural infection model. RESULTS: Fifty boars (~ 90 kg BW) from a SPF-farm with a strict hygiene and management regime for reducing the spread of an L. intracellularis infection up to the beginning of the final fattening period were transported, regrouped and randomly allotted to groups of five animals each at the research facility. After a 1-week acclimatisation period groups were fed one of five diets 4 weeks before slaughter. These were either a finely ground pelleted diet (FP) or a coarsely ground meal diet (CM), both consisting of wheat (40.0%), barley (39.3%), soybean meal (16.0%), soybean oil (2.0%) and minor components. In the other meal diets parts of wheat, barley and soybean meal were substituted either with 22% cracked corn (CORN), 16.9% dried whey (WHEY) or 30% raw potato starch (RPS). The animals had a comparable serological status in a blocking-ELISA immediately before the start and at the end of the feeding experiment. Values increased significantly during the trial. In all subgroups (FP/CM/CORN/WHEY/RPS), shedding was detected in week 0 (genome equivalents = GE; log10 GE L. intracellularis/g faeces: 2.46 ± 2.64/3.58 ± 2.54/3.43 ± 2.37/2.30 ± 3.16/2.58 ± 2.73). The average number of L. intracellularis microbes in faeces during the trial period did not differ between the groups (log10 GE L. intracellularis/g faeces: 3.40 ± 1.53/3.01 ± 1.41/3.80 ± 1.71/3.98 ± 2.20/4.08 ± 2.13). In animals fed the WHEY-diet, significantly lower counts of L. intracellularis were found in the caecal content. The acetate content in the caecum was negatively correlated with the serological results at the end of the trial (r = - 0.36; P = 0.010). Butyrate concentrations in the caecal content were negatively correlated with the number of L. intracellularis in the caecum (r = - 0.32; P = 0.023). CONCLUSION: Therefore, this study provides preliminary evidence that there might be specific dietary effects on the course of a L. intracellularis infection.

Effect of a live attenuated vaccine against Lawsonia intracellularis in weaned and finishing pig settings in Finland.

BACKGROUND: The intracellular bacterium Lawsonia intracellularis is an important pathogen in modern swine production. The aim of this study was to investigate the effect of a live attenuated L. intracellularis vaccine (Enterisol Ileitis®) on the health and production parameters of weaned and finishing pigs in a commercial Finnish 850-sow farm with diagnosed L. intracellularis infection. The herd was free from enzootic pneumonia, swine dysentery, progressive atrophic rhinitis, sarcoptic mange and salmonellosis. Four weekly groups of approximately 500 piglets were included in the study for a total of approximately 2000 piglets. Half of these piglets were vaccinated at 3 weeks of age and the other half served as controls. The study piglets were ear-tagged with individual numbers and colour-coded and were individually weighed at weaning (4 weeks), delivery to the finishing farm (12-14 weeks) and at slaughter. Mortality, symptoms of diseases and medications of the study piglets were registered in the nursery and finishing unit. Feed conversion rate was calculated for the finishing period and lean meat percentage was measured at slaughter. RESULTS: Vaccinated piglets had a higher live weight than unvaccinated piglets at delivery to the finishing unit (+ 1.18 kg, P = 0.002) and at slaughter (+ 3.57 kg, P < 0.001). The daily weight gain of vaccinated piglets was better than unvaccinated piglets in the nursery (+ 14.8 g/d, P = 0.013) and in the finishing unit (+ 30.9 g/d, P < 0.001). Vaccination had no effect on feed conversion rate or lean meat percentage (P = 0.102). Altogether, 3.9 and 4.6% of the pigs were medicated for different reasons in the vaccinated and control groups, respectively. The return on investment for the vaccination was calculated to be 0.41. CONCLUSIONS: Immunisation of piglets with a live attenuated L. intracellularis vaccine resulted in higher meat yield in pig production via significantly higher live weight and average daily weight gain in a Finnish specific pathogen-free setting.