Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

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.

The effects of zinc amino acid complex supplementation on the porcine host response to Lawsonia intracellularis infection.

Lawsonia intracellularis is among the most important enteric pathogens of swine and antibiotic alternatives are needed to help mitigate the negative effects of infection. Zinc is an essential trace mineral known to be crucial for maintaining intestinal barrier function and proper immune response. In this study, we investigated the porcine host response to L. intracellularis infection when supplemented with a zinc-amino acid complex, a form of zinc that can lead to greater bioavailability when compared to traditional inorganic forms of zinc. Our results show that a zinc-amino acid complex supplementation with a final concentration of 125 ppm of zinc in feed significantly (p < 0.05) decreased the number of animals with lesions and severity of lesions caused by L. intracellularis. Animals supplemented with the zinc-amino acid complex also exhibited a significantly (p < 0.05) earlier onset of seroconversion as well as an increased number of T cells in infected and non-infected intestinal tissue. This study demonstrated that this zinc-amino acid complex aids the host in responding to L. intracellularis infection and may be a new approach to help minimize negative effects of disease.

Seroprevalence of porcine proliferative enteropathy among wild boars in the Republic of Korea.

BACKGROUND: The importance of the wild boar as a reservoir of Lawsonia intracellularis was assessed by investigating the seroprevalence of this pathogen among wild boars in the Republic of Korea. The extent of exposure to L. intracellularis among wild boars (Sus scrofa coreanus) was monitored by a country-wide serological survey using an immunoperoxidase monolayer assay. RESULTS: In this study, antibodies to L. intracellularis were observed in 165 of 716 clinically healthy wild boars tested. The overall apparent prevalence calculated directly from the sample and the true prevalence calculated based on the accuracy of the test method were 23.0% (95% confidence interval: 20.0-26.3%) and 25.6% (95% confidence interval: 23.9-27.2%), respectively. Serologically positive animals were found in all the tested provinces. CONCLUSIONS: Our results confirm that L. intracellularis is present in the wild boar population worldwide, even in Far East Asia. Despite the high seroprevalence shown in wild boars, further studies are warranted to evaluate their potential as a reservoir species because seroprevalence does not prove ongoing infection nor shedding of the bacteria in amounts sufficient to infect other animals. It should also be determined whether the wild boar, like the domestic pig, is a natural host of L. intracellularis.

Seroprevalence of Lawsonia intracellularis antibodies in intensive pig farms in China.

BACKGROUND: Porcine proliferative enteropathy caused by Lawsonia intracellularis (L. intracellularis) is a major concern to the pig industry worldwide. Although 8.3 billion pigs are produced each year in China, few reports on the prevalence of L.intracellularis infection are available. The aim of the current study was to estimate the seroprevalence of L. intracellularis antibodies in intensive pig farms in China. RESULTS: A total of 1060 serum samples were collected from 14 commercial pig farms located throughout China. Animals from all age groups were sampled including pre-weaning piglets, weaners, fattening pigs, adult sows and boars. Antibodies against L. intracellularis were detected using a specific blocking ELISA. Of the 1060 serum samples, 602 were identified as positive using the ELISA test. The apparent seroprevalence of L. intracellularis seropositivity was 57% (95% CI 50 to 64%). The true prevalence (that is, prevalence corrected for the imperfect sensitivity and specificity of the testing method) was 77% (95% CI 70 to 83%). CONCLUSIONS: The highest true prevalence was observed in sows and boars, suggesting that within a herd these stock classes are a reservoir for infection. The prevalence of L. intracellularis seropositivity in local breed pigs was significantly less than that in imported breeds. A higher seroprevalence was found in pigs in herds in Central and Northern China, which may correspond to the greater use of the intensive production systems in these areas. We conclude that L. intracellularis is widely prevalent in commercial pigs in China.

Recent advances in understanding the pathogenesis of Lawsonia intracellularis infections.

Proliferative enteropathy is an infectious disease caused by an obligate intracellular bacterium, Lawsonia intracellularis, and characterized by thickening of the intestinal epithelium due to enterocyte proliferation. The disease is endemic in swine herds and has been occasionally reported in various other species. Furthermore, outbreaks among foals began to be reported on breeding farms worldwide within the past 5 years. Cell proliferation is directly associated with bacterial infection and replication in the intestinal epithelium. As a result, mild to severe diarrhea is the major clinical sign described in infected animals. The dynamics of L. intracellularis infection in vitro and in vivo have been well characterized, but little is known about the genetic basis for the pathogenesis or ecology of this organism. The present review focuses on the recent advances regarding the pathogenesis and host-pathogen interaction of L. intracellularis infections.

Vaccination with a Lawsonia intracellularis subunit water in oil emulsion vaccine mitigated some disease parameters but failed to affect shedding.

Lawsonia intracellularis is the causative agent of ileitis in swine that manifests as slower weight gain, mild or hemorrhagic diarrhea and/or death in severe cases. As an economically important swine pathogen, development of effective vaccines is important to the swine industry. In developing a subunit vaccine with three recombinant antigens - FliC, GroEL and YopN - we wanted to identify a formulation that would produce robust immune responses that reduce disease parameters associated with Lawsonia intracellularis infection. We formulated these three antigens with four adjuvants: Montanide ISA 660 VG, Montanide Gel 02 PR, Montanide IMS 1313 VG NST, and Montanide ISA 61 VG in an immunogenicity study. Groups vaccinated with formulations including Montanide ISA 660 VG or Montanide ISA 61 VG had significantly more robust immune responses than groups vaccinated with formulations including Montanide Gel 02 PR or Montanide IMS 1313 VG NST. In the challenge study, animals vaccinated with these antigens and Montanide ISA 61 VG had reduced lesion scores, reduced lesion lengths, and increased average daily gain, but no reduction in shedding relative to the control animals. This work shows that this vaccine formulation should be considered for future study in a field and performance trial.

Lawsonia intracellularis regulates nuclear factor-κB signalling pathway during infection.

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells.

Hybrid cluster proteins and flavodiiron proteins afford protection to Desulfovibrio vulgaris upon macrophage infection.

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.

Evidence of host adaptation in Lawsonia intracellularis infections.

BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate. MATERIALS AND METHODS: Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (109L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response. RESULTS: Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate. CONCLUSIONS: Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.

Cell-mediated and humoral immune responses in pigs following primary and challenge-exposure to Lawsonia intracellularis.

To investigate immune responses upon re-infection with Lawsonia intracellularis, local and peripheral humoral and cell-mediated immune responses to primary and challenge inoculations were studied in 22 pigs. Pigs were orally inoculated with virulent L. intracellularis at the age of 5-6 weeks, treated with antibiotics and challenged with a re-inoculation (RE) at the age of 12 weeks. Treatment control (TC) pigs received only the primary inoculation and challenge control (CC) pigs received only the secondary inoculation at 12 weeks of age. Following this regimen, all RE pigs were protected against the re-infection as defined by reduced colonisation and pathology of intestinal mucosa, absence of bacterial shedding and without increase in serum acute phase protein response. In the protected RE pigs, serum IgG responses were variable with both high and low responders. Serum IgA responses were not boosted by the re-inoculation, since identical intestinal IgA responses developed in response to the inoculation in both the susceptible CC pigs and the protected RE pigs. A memory recall cell-mediated immune response developed in RE pigs which was significantly stronger compared to the primary response in age-matched CC pigs as assessed by whole blood IFN-γ assay and by calculation of IFN-γ integrated median fluorescence intensity (iMFI) after flow cytometry. The major IFN-γ producing cells were identified as CD8+ and CD4+CD8+ double positive lymphocytes. The results indicate that cell-mediated immune responses are likely mediators of protective immunity against L. intracellularis, with CD8+ effector cells and CD4+CD8+ double positive memory T cells as main contributors to the antigen-specific IFN-γ production.

Evaluation of immune efficacy of Omp2 protein against Lawsonia intracellularis in mice.

Porcine proliferative enteropathy (PPE) is caused by the obligate intracellular bacterium Lawsonia intracellularis. Infection results in an enteric disease characterised by decreased growth performance of pigs, and presents a major economic burden for swine industries worldwide. Since vaccination is an effective technique for controlling PPE, novel effective vaccine platforms are need to be developed. In this study, five proteins of L. intracellularis were screened through animal experiments and the highly immunoprotective Omp2 protein was identified. Then, the immune efficacy of Omp2 was further evaluated based on humoral and cell mediated immune (CMI) responses, faecal bacterial shedding, histopathological lesions, immune barrier function of intestinal mucosa as well as digestive and absorptive capacity following challenge of mice with L. intracellularis. Mice immunised with Omp2 had reduced faecal shedding, fewer histopathological lesions and reduced bacteria colonisation of the ileum. Additionally, Omp2 immunised mice showed stronger serum IgG and IFN-γ levels, up-regulated Occludin and zonula occludens-1 (ZO-1) mRNA levels, as well as increased numbers of intestinal intraepithelial lymphocytes (IELs) and levels of sIgA. On the contrary, the activities of LPS, α-AMS and AKP were significantly increased. Our investigation indicated that immunization with Omp2 reduced the severity of clinical signs and provided efficacious immunoprotection for target animals against L. intracellularis infection in mouse model.

Evaluation of immunogenicity and protection mediated by Lawsonia intracellularis subunit vaccines.

Lawsonia intracellularis is an economically important bacterium that causes ileitis in pigs. Current vaccines for L. intracellularis do not allow for differentiation between infected and vaccinated animals (DIVA), which is beneficial for disease tracking and surveillance. Previously, we identified five putative surface L. intracellularis proteins that were targeted by antibodies from pigs infected with L. intracellularis which could serve as antigens in a subunit vaccine. We conducted two trials to determine whether these antigens were immunogenic and provided protection against infectious challenge and whether truncated glycoprotein D could be used as a DIVA antigen. For Trial 1, 5 week-old piglets were administered intramuscular monovalent vaccines comprised of a recombinant (r) flagella subunit protein (rFliC,) and DIVA antigen (truncated glycoprotein D (TgD), a herpes virus antigen) both formulated with a combination adjuvant consisting of polyinosinic:polycytidylic acid(poly I:C), host defense peptide 1002 and polyphosphazene, referred to as Triple Adjuvant (TriAdj). Relative to control animals, animals vaccinated with rFliC and rTgD had significantly elevated antigen-specific humoral immunity in sera suggesting that rFliC and TgD are immunogenic. Control animals had negligible anti-TgD titres suggesting that TgD may be a suitable DIVA antigen for pigs. For Trial 2, piglets were immunized with a trivalent vaccine (FOG vaccine consisting of rFLiC, rOppA protein (a ABC Type dipeptide transport system) and rGroEL (a stress response protein)) and a divalent vaccine (CM vaccine consisting of rClpP (an ATP-dependent Clp protease proteolytic subunit) and rMetK (a S-adenosyl methionine synthase)) formulated with Emulsigen®. Relative to the control pigs, pigs immunized with the FOG vaccine produced robust and significantly higher serum IgG antibodies against rFliC and rGroEL, and significantly higher anti-FliC and anti-GroEL IgA antibodies in jejunal (GroEL only) and ileal intestinal mucosa. Pigs immunized with CM vaccine produced significantly higher serum antibodies against rClpP and rMetK and significantly higher anti-rClpP IgA antibodies in the ileum relative to the control pigs. Quantitative polymerase chain reaction (qPCR) analysis showed that 18 days after challenge with infectious L. intracellularis, challenged/control pigs and pigs that received the CM vaccine, but not the pigs vaccinated with the FOG vaccine, shed significantly more bacteria in feces than the unchallenged controls pigs. These data suggest that the FOG vaccinated pigs showed limited protection. While promising, more work is needed to enhance the efficiency of the intramuscular vaccine to show significant disease protection.

Immune response and protection against Lawsonia intracellularis infections in pigs.

Lawsonia intracellularis are Gram-negative, obligate intracellular bacteria that cause proliferative enteropathy (PE), an economically important disease for the pig industry. Numerous reviews have been published on the characteristics and pathogenesis of this bacterium since its isolation and taxonomic characterization, with most reviews only partially covering how the host immune response develops during infection and the immune correlates of protection. With the development of increasingly more sophisticated immunological assays and tools for the pig, the immune response against L. intracellularis at distinct stages of pathogenesis has been published. In this review, we discuss current knowledge of the pig immune response against L. intracellularis and strategies to achieve immune protection. The immune response is presented in relation to chronological progression of pathological lesions and clinical symptoms, with emphasis on innate immunity and the adaptive humoral and cell-mediated immune response. The aim is to achieve a comprehensive understanding of the host immune response with respect to the stage-dependent cellular and biochemical processes important during PE development. Also, strategies for development of immune protection and new vaccination technologies are discussed in the light of new discoveries in the field.

Temporal detection of Lawsonia intracellularis using serology and real-time PCR in Thoroughbred horses residing on a farm endemic for equine proliferative enteropathy.

The goals of this study were to evaluate titers of antibodies against Lawsonia intracellularis in 68 resident broodmares from a farm known to be endemic for equine proliferative enteropathy (EPE) and to evaluate maternal antibodies, occurrence of seroconversion and fecal shedding in their foals. Serum samples collected from mares at delivery and from foals pre- and post-colostrum ingestion and monthly thereafter were tested for the presence of L. intracellularis antibodies by immunoperoxidase monolayer assay (IPMA). Further, feces collected from mares at delivery and foals post-partum and monthly thereafter were assayed for L. intracellularis using real-time PCR. Thirty-seven mares (54.4%) had detectable antibody titers (> or =60) against L. intracellularis by IPMA at the time of foaling. Passive transfer of colostral antibodies against L. intracellularis was documented in 37 foals (54.4%) and the colostral antibodies remained detectable in the serum of foals for 1-3 months. Overall, 22 foals (33.3%) showed evidence of natural exposure to L. intracellularis throughout the study period, however, none of the study foals developed signs compatible with EPE. The serological results showed that mares residing on a farm known to be endemic for EPE are routinely exposed to L. intracellularis and that antibodies against L. intracellularis are passively transferred to foals.

Immunoproteomic analysis of Lawsonia intracellularis identifies candidate neutralizing antibody targets for use in subunit vaccine development.

Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.

An attenuated Salmonella vaccine secreting Lawsonia intracellularis immunogenic antigens confers dual protection against porcine proliferative enteropathy and salmonellosis in a murine model.

Porcine proliferative enteropathy (PPE) caused by Lawsonia intracellularis (LI) is a global cause for substantial economic losses in the swine industry. Here, we constructed live attenuated Salmonella typhimurium (ST) mutant strains expressing and secreting 4 selected immunogenic LI antigens, namely, optA, optB, Lawsonia flagellin (LfliC), and Lawsonia hemolysin (Lhly); the resultant recombinant strains were designated Sal-optA, Sal-optB, Sal-LfliC, or Sal-Lhly, respectively. Using the BALB/c mouse model, we demonstrate that mice vaccinated once orally, either with a mixture of all 4 recombinant strains or with an individual recombinant strain, show significant (p < 0.05) production of LI-specific systemic immunoglobulin (Ig) G and mucosal IgA responses compared to the Salmonella alone group. Upon restimulation of vaccinated splenocytes with the LI-specific antigens, significant (p < 0.05) and comparable production of interferon-γ responses are found in all vaccinated groups, except the Sal-Lhly group, which shows non-significant levels. Challenge studies were performed in C57BL/6 vaccinated mice. On challenge with the LI (106.9 50% tissue culture infectious dose) 14 days post-vaccination, 20% (1/5) of mice in all vaccinated groups, except Sal-Lhly group, show the presence of the LI-specific genomic DNA (gDNA) in stool samples. In contrast, 40% (2/5) and 60% (3/5) of mice vaccinated with the Sal-Lhly strain and the attenuated Salmonella alone, respectively, were found positive for the LI-specific gDNA. Furthermore, 0% mortality was observed in mice vaccinated against the ST challenge compared to the 30% mortality observed in the unvaccinated control group. In conclusion, we demonstrate that the Salmonella-based LI-vaccines induce LI-specific humoral and cell-mediated immunities, and encompass the potential to offer dual protection against PPE and salmonellosis.

Systemic cytokine response in pigs infected orally with a Lawsonia intracellularis isolate of South Korean origin.

In the swine industry, Lawsonia intracellularis is one of the main enteric pathogens; it causes acute intestinal hemorrhage (proliferative hemorrhagic enteropathy) in naïve adult pigs and a wasting disease (proliferative enteropathy) in growing pigs. Among many kinds of cytokines, interferon-γ (IFN-γ) has previously been reported to play a significant role in limiting intracellular infection and increasing cellular proliferation associated with L. intracellularis. However, the levels of various circulating inflammatory cytokines, including IFN-γ, in animals infected with L. intracellularisis is still an area of considerable interest for understanding immunity against this bacterium. In addition, there has been no information on cytokine response in animals infected with any L. intracellularis isolate of South Korean origin or Asian origin. To determine the relationship between the changes in the systemic inflammatory cytokine response in the peripheral blood of the host after L. intracellularis infection, we measured the levels of some pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IFN-γ), anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-ß (TGF-ß)), and a chemokine (IL-8) in pigs infected with L. intracellularis isolated from South Korea. This study demonstrated that a L. intracellularis isolate of South Korean origin induced cytokine (TNF-α, IL-6, and IFN-γ) responses in infected animals within 15 days post-infection although the circulating levels of IL-4, IL-10, IL-8 and TGF-ß were induced relatively late.

Vaccination Against Lawsonia intracellularis Decreases Shedding of Salmonella enterica serovar Typhimurium in Co-Infected Pigs and Alters the Gut Microbiome.

Salmonella enterica serovar Typhimurium continues to be a major cause of foodborne illness worldwide and pork can serve as a source of infection. Co-infection of S. enterica with Lawsonia intracellularis, a common intestinal pathogen of swine, has been found as risk factor for increased S. enterica shedding. The objective of this study was to investigate if vaccination against L. intracellularis could lead to decreased S. Typhimurium shedding. To test this hypothesis, pigs were challenged with either S. Typhimurium or S. Typhimurium and L. intracellularis, with and without L. intracellularis vaccination (n = 9 per group). A non-challenged group served as a negative control. Vaccination decreased the shedding of S. Typhimurium in co-infected animals by 2.12 log10 organisms per gram of feces at 7 days post infection. Analysis of the microbiome showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum, in addition to other compositional changes that may explain the protection mediated against S. Typhimurium. These results indicate that vaccination against L. intracellularis in co-infected herds may provide a new tool to increase food safety by helping to prevent S. enterica without the need for antibiotics.

Autophagy counters LPS-mediated suppression of lysozyme.

Impaired Paneth cell expression of antimicrobial protein (AMP) lysozyme is found in patients with Crohn's disease with the autophagy gene ATG16L1 risk allele, in mice with mutations in autophagy genes Atg16L1, Atg5 and Atg7, and in Irgm1 knockout mice. Defective autophagy is also associated with expansion of resident Gram-negative bacteria in the intestinal lumen. These findings suggest that autophagy may control extracellular resident microbes by governing expression of lysozyme. To test the hypothesis that autophagy may have a defensive role in host response to resident extracellular microbes, we investigated the relationship between gut microbes, autophagy, and lysozyme. RAW 264.7 macrophages were treated with fecal slurry (FS), representing the resident microbial community; lipopolysaccharide (LPS); or butyrate, representing microbial products; or a representative resident Gram-negative bacterium Desulfovibrio vulgaris (DSV). FS, LPS, and DSV inhibited lysozyme expression, whereas butyrate had no effect. Induction of autophagy by rapamycin countered this inhibition, whereas silencing of the autophagy gene Irgm1 exacerbated the inhibitory effects of LPS on lysozyme expression. LPS also inhibited lysozyme activity against DSV and autophagy reversed this effect. Our results provide a novel insight into an interaction between gut bacteria, autophagy and AMP whereby autophagy may defend the host by countering the suppression of antimicrobial protein by Gram-negative bacteria.