Review: Occurrence and Distribution of Galanin in the Physiological and Inflammatory States in the Mammalian Gastrointestinal Tract.

Galanin (GAL) is a broad-spectrum peptide that was first identified 37 years ago. GAL, which acts through three specific receptor subtypes, is one of the most important molecules on an ever-growing list of neurotransmitters. Recent studies indicate that this peptide is commonly present in the gastrointestinal (GI) tract and GAL distribution can be seen in the enteric nervous system (ENS). The function of the GAL in the gastrointestinal tract is, inter alia, to regulate motility and secretion. It should be noted that the distribution of neuropeptides is largely dependent on the research model, as well as the part of the gastrointestinal tract under study. During the development of digestive disorders, fluctuations in GAL levels were observed. The occurrence of GAL largely depends on the stage of the disease, e.g., in porcine experimental colitis GAL secretion is caused by infection with Brachyspira hyodysenteriae. Many authors have suggested that increased GAL presence is related to the involvement of GAL in organ renewal. Additionally, it is tempting to speculate that GAL may be used in the treatment of gastroenteritis. This review aims to present the function of GAL in the mammalian gastrointestinal tract under physiological conditions. In addition, since GAL is undoubtedly involved in the regulation of inflammatory processes, and the aim of this publication is to provide up-to-date knowledge of the distribution of GAL in experimental models of gastrointestinal inflammation, which may help to accurately determine the role of this peptide in inflammatory diseases and its potential future use in the treatment of gastrointestinal disorders.

Adherence of Brachyspira hyodysenteriae to porcine intestinal epithelial cells is inhibited by antibodies against outer membrane proteins.

Attachment of Brachyspira hyodysenteriae to intestinal epithelial cell lines and its possible mediation by outer membrane proteins (OMPs) of the spirochete were examined. Different B. hyodysenteriae serotypes were shown to adhere to rat and swine intestinal epithelial cells (IEC-18 and IPEC-J2) in vitro but not to the human rectal tumor cell line (HRT-18). Adherence of strain B204 to IPEC-J2 cells was reduced by rOMP-specific antisera in amounts of 29 % (anti-rBhlp29.7), 59 % (anti-rBhlp16), 70 % (anti-rBhmp39h), and 74 % (anti-rBhmp39h), respectively. By use of pooled antisera against Bhlp16 and Bhmp39f inhibition rates of the other serotypes ranged from 53 to 91 %. In a western blot assay OMPs of all serotypes but one were detected by the respective rOMP antisera. Altogether the results indicated that OMPs of B. hyodysenteriae displayed a serotype overlapping antigenicity and mediated adherence of the spirochetes to animal cell cultures.

Evaluation of the use of recombinant Bhlp29.7 in immunoblotting with pig serum as a means to identify herds infected with Brachyspira hyodysenteriae.

AIMS: Aim of the study is to evaluate the use of recombinant Bhlp29.7 in immunoblotting with sera as a means to detect pig herds infected with Brachyspira hyodysenteriae. METHODS AND RESULTS: Sera samples from 789 sows and rectal swabs from 838 pigs of various categories on 22 farms of different size (median 450 animals), production type and history of swine dysentery (SD) were examined. Sera from 378 sows from farms with previous SD history were examined via immunoblotting. Specific antibodies were detected in 79 of these (20.9%). Examination of 411 serum samples from sows and gilts taken on 11 farms without previous history of SD detected specific antibodies in 13 sows and gilts (3.2%). These 13, however, had come from farms where the presence of B. hyodysenteriae was confirmed or SD status was not known. Seroprevalence in herds with previous SD history ranged from 2.5 to 35.7%. B. hyodysenteriae was confirmed on six (27.3%) of 22 monitored farms. CONCLUSIONS: Immunoblotting using recombinant antigen Bhlp29.7 in conjunction with culturing B. hyodysenteriae proved to be a valuable tool for detecting swine herds latently infected with B. hyodysenteriae. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of immunoblotting with recombinant Bhlp29.7 should prove to be a useful adjunct to detecting herds with SD, and hence, it will assist in controlling this important disease.

A reverse vaccinology approach to swine dysentery vaccine development.

Swine dysentery (SD) is a mucohaemorrhagic colitis of pigs resulting from infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Whole-cell bacterin vaccines are available to help control SD, but their performance has been inconsistent. This study aimed to use a reverse vaccinology approach to identify B. hyodysenteriae proteins for use as recombinant vaccine components. Nineteen open reading frames (ORFs) predicted to encode potential vaccine candidate molecules were identified from in silico analysis of partial genomic sequence data. The distribution of these ORFs among strains of B. hyodysenteriae was investigated by PCR, and widely distributed ORFs were cloned. The products were screened with a panel of immune pig sera, and from these a subset of conserved, immunogenic proteins was selected. Mice immunized intramuscularly with these recombinant proteins developed specific systemic antibody responses to them, and their sera agglutinated B. hyodysenteriae cells in vitro. In a pilot experiment, eight pigs were vaccinated twice intramuscularly with a combination of four of the proteins. The pigs developed antibodies to the proteins, and following experimental challenge only one developed SD compared to five of nine non-vaccinated control pigs. Although these differences in incidence were not significant, they indicated a trend towards protection using the recombinant proteins as immunogens. This study demonstrates that the reverse vaccinology approach has considerable potential for use in developing novel recombinant vaccines for SD.

Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery.

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.

An evaluation of the immunogenicity and protective responses to Brachyspira hyodysenteriae recombinant SmpB vaccination.

SmpB is an outer membrane protein of Brachyspira hyodysentariae that is present in some strains of the bacterium. It shares the same locus as SmpA, but all strains tested to date contain either one protein or the other, never both. In this study we have evaluated the efficacy of vaccination with SmpB to elicit immune responses in mice and to protect against a subsequent challenge. Immunised mice develop humoral and cellular responses to SmpB delivered as either a DNA vaccine or a recombinant protein, although the magnitude of the responses is greater after protein vaccination. The responses induced after protein vaccination offer moderate protection against disease and indicate that SmpB has potential as a component of a vaccine against B. hyodysentariae.

The selection of single-chain Fv antibody fragments specific to Bhlp 29.7 protein of Brachyspira hyodysenteriae.

Single-chain antibodies (scFv) specific to Brachyspira hyodysenteriae were isolated from a phagemid library. Recombinant Bhlp 29.7 protein was used for scFv selection and individual clones were tested by ELISA and immunofluorescent test; four unique clones were isolated. One of selected clones was able to bind specifically B. hyodysenteriae in ELISA and immunofluorescence test. This is the first report of species-specific recombinant antibodies against B. hyodysenteriae.

Differences in lymphocyte subpopulations and cell counts before and after experimentally induced swine dysentery.

The aim of this study was to examine the levels of circulating leukocytes and lymphocyte subpopulations before and immediately after experimentally induced swine dysentery. Twenty-one healthy crossbred pigs (approximately 22 kg) were orally inoculated with Brachyspira hyodysenteriae. Blood was sampled before inoculation and when clinical signs of swine dysentery occurred. Pigs that remained healthy were sampled when killed. Total and differential white blood cell counts were performed, and lymphocyte subpopulations were analysed using flow cytometry. Following a mean incubation period of 13 days, 12 pigs developed swine dysentery, whereas nine remained healthy throughout the study. Before inoculation, pigs that subsequently developed swine dysentery displayed higher levels of circulating gamma delta T cells (mean +/- se; 30.7 +/- 3.5 %) compared with pigs that remained healthy (14.9 +/- 1.4 %). Sick animals also displayed lower levels of CD8 cells (24.6 +/- 1.5 %), cytotoxic/suppressor T cells (10.9 +/- 1.3 %) and CD4 CD8 T cells (8.1 +/- 1.0 %) than the pigs that remained healthy (34.9 +/- 3.1 %; 17.6 +/- 2.0 %; 13.6 +/- 2.3 %). No difference was observed in leukocyte counts before inoculation. At onset of swine dysentery, there was an increase in monocytes (from 1.5 +/- 0.2 x 10 to 3.8 +/- 0.5 x 10 l) and CD4 CD8 T cells (from 5.8 +/- 0.9 to 8.9 +/- 0.7 %). In conclusion, gamma delta T cells and CD8 cells may be associated with susceptibility to experimentally induced swine dysentery, whereas monocytes and CD4 CD8 T cells appear to be the major responding leukocytes during the disease.

Immunomagnetic separation of the intestinal spirochaetes Brachyspira pilosicoli and Brachyspira hyodysenteriae from porcine faeces.

Porcine intestinal spirochaetes are fastidious anaerobic organisms and, as a consequence, it has been necessary to develop various protocols to enhance their isolation from or detection in faeces. Immunomagnetic separation (IMS) is a method developed recently to improve separation of target cells from mixed cell suspensions. The purpose of the present study was to compare the relative sensitivity of IMS for isolation of Brachyspira pilosicoli and Brachyspira hyodysenteriae with current routine diagnostic methods (culture on selective media and PCR) for detection of these micro-organisms in pig faeces. Neither direct nor indirect IMS methods enhanced the sensitivity of detection of either organism when performed with the recommended washings during sample processing. Performance of the IMS procedure without washing gave sensitivity at levels similar to direct culture onto selective medium. Further development of IMS techniques is required to improve isolation rates of Brachyspira species from faecal samples.

Serologic detection of Brachyspira (Serpulina) hyodysenteriae infections.

Swine dysentery (SD) caused by the intestinal spirochete Brachyspira hyodysenteriae is an economically important disease in pig-producing countries throughout the world. To date, no specific serologic assay is commercially available for the diagnosis of pigs with SD. Several serologic techniques have been identified in the past; however, these tests have all used either whole-cell proteins or lipopolysaccharide (LPS) as the antigen. Whole-cell antigens are plagued with false-positive reactions due to cross-reactivity with common proteins shared with other spirochetes. LPS antigens produce fewer false-positives; however, false-negatives may result due to LPS components being serogroup-specific. Generally, these techniques are useful for detecting infected herds, but are unreliable for the detection of individual infected pigs. In order to develop improved serologic tests it will be necessary to identify suitable diagnostic antigens, in particular immunogenic cell-surface structures which are specific to B. hyodysenteriae but common amongst different strains of the species. Recently, we identified and cloned a 30-kDa outer membrane lipoprotein (BmpB) which is specific to B. hyodysenteriae and is recognized by experimentally and naturally infected pigs. In this review we summarize the available serologic tests for SD, and speculate on the use of recombinant BmpB as an antigen for future development of an improved serologic test for SD diagnosis.

Antigen-specific proliferation of porcine CD8alphaalpha cells to an extracellular bacterial pathogen.

A vaccine inducing protective immunity to a spirochaete-induced colitis of pigs predominantly stimulates expansion of CD8+ cells in vivo and in antigen-stimulated lymphocyte cultures. CD8+ cells, however, are rarely considered necessary for protection against extracellular bacterial pathogens. In the present study, pigs recovering from colitis resulting from experimental infection with Brachyspira (Serpulina) hyodysenteriae had increased percentages of peripheral blood CD4- CD8+ (alphaalpha-expressing) cells compared with non-infected pigs. CD8alphaalpha+ cells proliferated in antigen-stimulated cultures of peripheral blood mononuclear cells from B. hyodysenteriae-vaccinated pigs. Proliferating CD8alphaalpha+ cells consisted of CD4-, CD4+ and gammadelta T-cell receptor-positive cells. CD4- CD8alphabeta+ cells from vaccinated or infected pigs did not proliferate upon in vitro antigen stimulation. Of the CD8alphaalpha cells that had proliferated, flow cytometric analysis indicated that the majority of the CD4+ CD8+ cells were large (i.e. lymphoblasts) whereas the CD4- CD8+ cells were predominantly small. Addition of monoclonal antibodies (mAb) specific for either porcine major histocompatibility complex (MHC) class I or class II antigens diminished B. hyodysenteriae-specific proliferative responses whereas addition of mAb to porcine MHC II, but not porcine MHC I, reduced the CD8alphaalpha response. In vitro depletion of CD4+ cells by flow cytometric cell sorting diminished, but did not completely abrogate, the proliferative response of cells from vaccinated pigs to B. hyodysenteriae antigen stimulation. These results suggest that CD8alphaalpha cells are involved in recovery and possibly protection from a spirochaete-induced colitis of pigs; yet, this response appears to be partially dependent upon CD4+ cells.

Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.

A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae.

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.

Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin.

Brachyspira (Serpulina) hyodysenteriae infection of pigs (swine dysentery) causes a mucohemorrhagic diarrhea resulting in significant economic losses for producers. A commercial vaccine consisting of a proteinase-digested bacterin has shown efficacy in the reduction of disease due to B. hyodysenteriae. Vaccines consisting of whole cell bacterins, however, generally fail to protect pigs from disease. In the present study, cellular immune responses induced by a proteinase-digested bacterin were compared to responses induced by a whole cell sonicate antigen preparation. In addition, usage of either squalene or Freund's incomplete adjuvants in combination with each antigen preparation was also compared. Both antigen preparations induced significant cellular immune responses as measured by in vitro (IFN-gamma production and T cell proliferation) and in vivo methods (DTH responses). No significant differences were detected in proliferative, interferon-gamma (IFN-gamma), or delayed type hypersensitivity (DTH) responses by pigs receiving either adjuvant or antigen preparation. T cells (CD3(+)) but not B cells from vaccinated animals proliferated in response to in vitro stimulation with B. hyodysenteriae antigen. CD8(+) (single positive and CD4/CD8 double positive) and gammadelta(+) T cells were particularly responsive. In addition, high percentages of both CD8 single positive and CD4/CD8 double positive cells were detected in antigen-stimulated cultures. These findings demonstrate the unique sensitivity of porcine CD8(+) T cells to priming for recall response by vaccination with a proteinase-digested B. hyodysenteriae bacterin.

Presence of 22- and 17-kDa proteins reacting with sera in mice experimentally infected with Brachyspira (Serpulina) hyodysenteriae.

The antibodies to B. (S.)hyodysenteriae in experimentally infected mice were detected by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). The reactions in MAT were serotype specific while those in ELISA were common to both strains. A further investigation with immunoblotting technique demonstrated that 22- and 17-kDa proteins reacted strongly with the sera. The proteins in ATCC 27164 strain strongly reacted with the serum from ATCC 31212 strain-infected mouse and vice versa. These proteins were sensitive to proteinase K.

Rapid identification of porcine Serpulina species by colony blot assay using a genus-specific monoclonal antibody.

An immunoglobulin G monoclonal antibody (mAb C9E8) recognising a genus-specific epitope on the 26 kDa protein of porcine Serpulina species organisms was used in a simple colony blot assay to detect Serpulina in cultures grown directly on blood agar plates from pig faeces and tissues. The mAb detected even a few colonies of the organism in the presence of an abundant growth of non-Serpulina organisms. The whole procedure was completed in less than three hours. A total of 123 strains of S hyodysenteriae and S innocens were correctly identified by the colony blot assay whereas all the 26 non-Serpulina Gram-negative organisms commonly isolated from faecal material or tissues of pigs remained negative. The assay was rapid, highly specific and sufficiently reliable to be used with confidence for identifying porcine Serpulina colonies directly on blood agar plates.

Identification of the swine pathogen Serpulina hyodysenteriae in rheas (Rhea americana).

Recently intestinal spirochetes were isolated from rheas in Ohio and Iowa with a necrotizing typhlocolitis. These intestinal spirochetes, strains R1 and NIV-1, were characterized and compared with other intestinal spirochetes, including strains of S. hyodysenteriae. Both rhea spirochetes were indole positive, strongly beta-hemolytic, grew under a 1% O2:99% N2 atmosphere, and were morphologically similar to spirochetes in the genus Serpulina. Analysis of rRNA gene restriction patterns (ribotypes), and immunoblots of whole cell proteins, indicated both spirochetes were similar to Serpulina hyodysenteriae strains from swine. Comparisons of nearly complete sequences (> 1458 bases) of the 16S rRNA gene of the two rhea spirochetes with S. hyodysenteriae strains confirmed that rhea spirochetes R1 and NIV-1 were strains of S. hyodysenteriae. These results indicate that S. hyodysenteriae has a broader host range than previously recognized.

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae.

A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae, the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non-S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae, and should prove to be a useful reagent for identification of S. hyodysenteriae.

Production and characterization of monoclonal antibodies against Serpulina hyodysenteriae and S. innocens and their use in serotyping.

Murine monoclonal antibodies (MAbs) directed against serotypes 1, 2, 8, and 9 of Serpulina hyodysenteriae and strain B256 of Serpulina innocens were produced and characterized. A serological classification of 96 field strains of S. hyodysenteriae and 28 field strains of S. innocens isolated from pigs showing clinical signs of swine dysentery was performed by rapid dot enzyme-linked immunosorbent assay (ELISA) with the MAbs. The results indicated that the majority of the field strains of S. hyodysenteriae (69%) belonged to serotypes 8, 1, and 9, whereas only 31% of the S. innocens strains were recognized by MAb 9H7, indicating the presence of antigenic heterogeneity among S. innocens isolates. Rapid dot ELISA with type-specific MAbs was found to be specific, sensitive, and easy to perform and thus to be suitable for routine serotyping of S. hyodysenteriae and S. innocens isolates. This is the first report of MAbs being used for serotyping clinical isolates of S. hyodysenteriae and S. innocens.

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.