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.

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Use of oral fluids to detect anti Lawsonia intracellularis antibodies in experimentally infected pigs / Utilização de fluidos orais para detecção de anticorpos anti Lawsonia intracellularis em suínos experimentalmente infectados

Several pathogens and antibodies derived from serum or produced in tissues associated with the oral cavity are present in the oral fluid (OF). Considering the applicability of this alternative sample, recent studies in veterinary medicine have tested OF as a replacement for serum in diagnostic assays. The aim of this study was to standardize the immunoperoxidase monolayer assay (IPMA) to detect anti-Lawsonia intracellularis immunoglobulin A (IgA) and immunoglobulin G (IgG) in OF samples from experimentally infected pigs. Sixty-two pigs were divided into two groups: control (T1, n=30) and inoculated with L. intracellularis (T2, n=32). Blood, OF and fecal samples were collected at 0, 7, 14, 21, 28 and 42 days post-inoculation (dpi). Some adaptations of the standard technique for serum were made to IPMA for the detection of IgA and IgG in OF. The IPMA showed high specificity and sensitivity for serum samples and high specificity and moderate sensitivity for the detection of IgA and IgG in OF. There was high agreement between the results of serum IgG and OF IgA and IgG. Based on our results, oral fluid samples may be used for the evaluation and determination of anti-L. intracellularis antibodies in pigs, but not for individual diagnosis of swine proliferative enteropathy.(AU)

Vários patógenos e anticorpos derivados do soro ou produzidos em tecidos associados a cavidade oral estão presentes no fluido oral (FO). Considerando a aplicabilidade dessa amostra alternativa, estudos recentes em medicina veterinária têm testado o FO como substituto do soro para testes diagnósticos. O objetivo desse estudo foi padronizar a imunoperoxidase em monocamada de célula (IPMC) para a detecção de imunoglobulina A e imunoglobulina G anti-Lawsonia intracellularis em amostras de FO de suínos experimentalmente infectados. Um total de 62 suínos foram divididos em dois grupos: controle (T1, n=30) e inoculados com L. intracellularis (T2, n=32). Sangue, FO e amostras de fezes foram coletados aos 0, 7,14, 21, 28 e 42 dias após a inoculação (dpi). Algumas adaptações da técnica foram realizadas na técnica padrão da IPMC para a detecção de IgA e IgG. A IPMC demostrou alta especificidade e sensibilidade para amostras de soro e alta especificidade de moderada sensibilidade para a detecção de IgA e IgG em FO. Houve alta concordância entre resultados de detecção de IgG em soro com a IgA e IgG em amostras de FO. Baseado em nossos resultados, amostras de fluido oral podem ser usadas em avaliações e detecção de anticorpos anti-L. intracellularis em suínos, porém não de forma individual.(AU)

The effects of Lawsonia intracellularis, Salmonella enterica serovar Typhimurium and co-infection on IL-8 and TNFα expression in IPEC-J2 cells.

Lawsonia intracellularis is among the most important enteric pathogens of swine and has been shown to be a risk factor for increased Salmonella enterica shedding. S. enterica serovar Typhimurium, in addition to being a significant pathogen of swine, also remains one of the most common causes of foodborne illness worldwide. Inflammation and the expression of IL8 and TNFα are an important process in the establishment of S. Typhimurium infection. Yet the effect of L. intracellularis on the expression of these cytokines by enterocytes, the niche both pathogens occupy during infection, is poorly understood. In this study we compared cytokine gene expression between singly and dually infected IPEC-J2 cells, a non-transformed porcine enterocyte cell line. Our results show that L. intracellularis leads to increased expression of IL8 and TNFα and has an additive effect on their expression in co-infection. The increase in expression of inflammatory cytokines may be one mechanism by which L. intracellularis favors S. Typhimurium infection.

Efficacy of a novel inactivated Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions.

The efficacy of a novel inactivated Lawsonia intracellularis vaccine, Porcilis® Lawsonia, was compared to that of a commercially available live attenuated vaccine in three experimental vaccination-challenge studies in pigs. The efficacy of the new vaccine was further tested under field conditions on a farm with a history of acute ileitis. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV M Hyo; an existing combination vaccine against porcine circovirus type 2 and Mycoplasma hyopneumoniae. The three experimental vaccination-challenge trials had a similar design and for each trial 75 piglets were used, randomly allotted to three groups of 25 piglets. The pigs were vaccinated at 4 or 5 weeks of age with either Porcilis® Lawsonia in adjuvant or in associated mixed use with Porcilis® PCV M Hyo (group 1), with the live vaccine (group 2), or left as unvaccinated controls (group 3). The pigs were challenged with virulent Lawsonia intracellularis 3, 4 or 17 weeks after vaccination. Post-challenge the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months. The results of all three experimental vaccination-challenge trials showed that Porcilis® Lawsonia induced statistically significant protection against experimental Lawsonia intracellularis infection. This was demonstrated by lower clinical scores, improved weight gain, reduction of Lawsonia intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study, Porcilis® Lawsonia proved to be highly efficacious; reducing Lawsonia associated mortality to zero and improving key production parameters.

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.

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.

Effect of the route of administration on the mucosal and systemic immune responses to Lawsonia intracellularis vaccine in pigs.

In an on-farm study, 40 weaned piglets aged 3 weeks were vaccinated with Lawsonia intracellularis vaccine orally, IM or IP while a fourth group remained unvaccinated. All vaccinated animals showed increased serum levels of L. intracellularis-specific IgG antibodies, but significantly elevated concentrations of specific IgG, IgA and cytokines were generated in ileal mucosal secretions from the orally and IP vaccinated pigs when examined at 17 days after vaccination.

Immunomodulatory factors and infectious agents associated with the hepatic gene expression of the IGF system in nursery pigs.

Recent findings suggest there is a complex interaction between the IGF system and the inflammatory immune response. The objective of this study was to determine whether gene expression of growth factors (IGF-1, IGF-binding protein-3 (IGFBP-3) and growth hormone receptors (GHR)) in the liver is associated with gene expression of immunomodulators in the liver, including C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), interferon-α (IFN-α), IFN-γ, tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-10 and IL-18, as well as with the presence of Salmonella spp., Lawsonia intracellularis, Brachyspira spp., enterotoxigenic Escherichia coli, methicillin-resistant Staphylococcus aureus, swine influenza virus, and porcine reproductive and respiratory syndrome virus (PRRSV) in nursery pigs (n=74) from commercial farms (n=4). Gene expression was quantified using reverse transcription quantitative-PCR (RT-qPCR) and the data were modelled using logistic regression methods. Pigs with elevated IGF-1 expression were less likely to have increased expression of TNF-α (odds ratio (OR)=0.14, P<0.01) and IL-18 (OR=0.19, P<0.05), and less likely to be colonized with PRRSV (OR=0.03, P<0.01). Pigs with increased expression of IGFBP-3 were more likely to have elevated IL-6 expression (OR=8.5, P<0.05). It was also observed that IGFBP-3 and IGF-1 were significantly associated when Hp expression was low (OR=30, P<0.05), but this association was not significant when Hp expression was high (P=0.54). Pigs with increased expression of GHR were less likely to have elevated expression of SAA (OR=0.01, P<0.05) and IL-1ß (OR=0.03, P<0.05), but more likely to have increased expression of CRP (OR=290, P<0.01). Overall, there appears to be an inverse association between the hepatic expression of the IGF system (IGF-1, IGFBP-3, GHR) and certain cytokines (IL-1ß, IL-18, TNF-α) and acute-phase proteins (SAA, Hp).

Immunological responses to vaccination following experimental Lawsonia intracellularis virulent challenge in pigs.

Although a live attenuated vaccine has been used extensively to provide immunity against porcine proliferative enteropathy (PE) caused by Lawsonia intracellularis, the nature of the protective response is an area of considerable interest for the control of PE. Two trials investigated immune responses in pigs after oral and intramuscular (IM) vaccination followed by virulent L. intracellularis challenge. After an oral vaccination with 10(5.9) TCID50 organisms, significantly increased serum and mucosal secretions of IgM, IgG and higher mucosal TNF-α and TGF-ß1 were detected by day 17, together with a trend towards higher levels of IFN-γ and IL-6. Pigs vaccinated IM produced elevated serum antibody titres but mucosal immune responses were not detected. After challenge with virulent L. intracellularis, non-vaccinated control pigs had higher PE lesion scores and excreted significantly higher numbers of L. intracellularis in faeces than the vaccinated pigs. Reduced intestinal pathology and faecal L. intracellularis shedding were evident in the vaccinated groups. The results indicated that protection was associated with mucosal cytokine and specific IgG and IgA responses after vaccination and that systemic antibody responses were boosted following challenge. However in the search for an immune correlate with protection, a causal association was not evident from a kinetic analysis of immune parameters in serum, ileal pathology and faecal shedding.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model.

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

Microarray and cytokine analyses of field cases of pigs with diarrhoea.

This field study explored the cytokine expression in intestinal tissue and serum from 19 diarrhoeic and 9 healthy pigs in herds with a long-time history of Lawsonia intracellularis-infection. The disease, proliferative enteropathy (PE), is associated with diarrhoea and poor performance in growers and haemorrhagic diarrhoea and sudden death in finisher pigs, but the immunopathology is poorly understood. Histopathology, demonstration of L. intracellularis and porcine circovirus type 2 (PCV2) in intestinal tissue by PCR, and detection of serum antibodies to L. intracellularis, were performed. The presence of IL-1ß, IL-6, IL-10, IFN-α, IFN-γ, TNF-α and TGF-ß in sera was determined by immunoassays, and intestinal mRNA expression of these cytokines plus IL-12p40 was determined by qPCR. Intestinal specimens from pigs with intestinal adenomatosis (n=2), proliferative haemorrhagic enteropathy or swine dysentery (n=2), and controls (n=2) were analysed by a genome wide porcine microarray. The clinical signs of PE were not always supported by the subsequent analyses, and the presence of PCV2 may have contributed to an increased mRNA expression for IFN-γ in intestinal specimens from some pigs. The limited gene expression in the microarray analyses and the limited expression of cytokines in both sera and intestines, indicate that the immune response is poorly activated in the initial course of an infection with L. intracellularis. However, the gene encoding for insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated in two pigs with prominent mucosal proliferation.

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.

Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen.

Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.

Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis.

Little is known about the humoral and, especially, cell-mediated immune response in pigs exposed to Lawsonia intracellularis. The objectives of this study were to investigate the onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or a commercial live vaccine strain of L. intracellularis. Twenty-four 5-week-old pigs were exposed to 4.4x10(9) organisms of a pathogenic L. intracellularis isolate PHE/MN1-00 (10 pigs), a L. intracellularis live attenuated vaccine strain (10 pigs) or sham inoculum (4 pigs). Fecal, serum and whole blood samples were collected from all animals before exposure and weekly up to 13 weeks post inoculation and tested by PCR, immunoperoxidase monolayer assay serology and an interferon-gamma assay, respectively. One animal from each group was euthanized on day 22 post exposure to confirm infection. Humoral and cell-mediated immune responses were initially detected 2 weeks after exposure in pigs challenged with the pathogenic isolate, and 5 and 4 weeks, respectively, in pigs exposed to the modified-live vaccine group. Humoral and cell-mediated immune responses were still detected in some pigs from both L. intracellularis exposed groups 13 weeks after exposure. Fecal shedding was initially detected 1 week and lasted, intermittently, 12 weeks post exposure in pigs challenged with the pathogenic isolate, while fecal shedding was first detected 2 weeks and lasted, also intermittently, 9 weeks after exposure to the vaccine. In summary, both pathogenic isolate challenged and vaccine exposed pigs demonstrated long-term shedding of and immune responses to L. intracellularis.

A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy.

The currently used indirect fluorescent antibody test (IFAT) for the detection of antibodies against porcine proliferative enteropathy (PPE) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used in this comparison were collected from 5-week-old pigs on day 0 (pre-experimental challenge) and on days 7, 14, 21, and 28 after oral inoculation with intestinal homogenate from pigs affected by PPE (28 challenged pigs) and sucrose phosphate glutamate solution (2 control pigs). All animals were euthanized 4 weeks after inoculation. Immunohistochemistry staining was applied to formalin-fixed, paraffin-embedded sections of ileum for the detection of Lawsonia intracellularis antigen. The serology results with each method agreed in all samples, except on days 0 and 7 in 1 control animal, which was positive by IPMA, but negative by IFAT. The percentage of agreement between IFAT and IPMA was 98.6%.