RESUMEN

Borrelia (B.) mayonii sp. nov. has recently been reported as a novel human pathogenic spirochete causing Lyme disease (LD) in North America. Previous data reveal a higher spirochaetemia in the blood compared to patients infected by LD spirochetes belonging to the B. burgdorferi sensu lato complex, suggesting that this novel genospecies must exploit strategies to overcome innate immunity, in particular complement. To elucidate the molecular mechanisms of immune evasion, we utilized various methodologies to phenotypically characterize B. mayonii and to identify determinants involved in the interaction with complement. Employing serum bactericidal assays, we demonstrated that B. mayonii resists complement-mediated killing. To further elucidate the role of the key regulators of the alternative pathway (AP), factor H (FH), and FH-like protein 1 (FHL-1) in immune evasion of B. mayonii, serum adsorption experiments were conducted. The data revealed that viable spirochetes recruit both regulators from human serum and FH retained its factor I-mediated C3b-inactivating activity when bound to the bacterial cells. In addition, two prominent FH-binding proteins of approximately 30 and 18 kDa were detected in B. mayonii strain MN14-1420. Bioinformatics identified a gene, exhibiting 60% identity at the DNA level to the cspA encoding gene of B. burgdorferi. Following PCR amplification, the gene product was produced as a His-tagged protein. The CspA-orthologous protein of B. mayonii interacted with FH and FHL-1, and both bound regulators promoted inactivation of C3b in the presence of factor I. Additionally, the CspA ortholog counteracted complement activation by inhibiting the alternative and terminal but not the classical and Lectin pathways, respectively. Increasing concentrations of CspA of B. mayonii also strongly affected C9 polymerization, terminating the formation of the membrane attack complex. To assess the role of CspA of B. mayonii in facilitating serum resistance, a gain-of-function strain was generated, harboring a shuttle vector allowing expression of the CspA encoding gene under its native promotor. Spirochetes producing the native protein on the cell surface overcame complement-mediated killing, indicating that CspA facilitates serum resistance of B. mayonii. In conclusion, here we describe the molecular mechanism utilized by B. mayonii to resists complement-mediated killing by capturing human immune regulators.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas del Sistema Complemento/metabolismo , Evasión Inmune/genética , Enfermedad de Lyme/inmunología , Infecciones por Spirochaetales/inmunología , Spirochaetales/fisiología , Proteínas Bacterianas/metabolismo , Bacteriólisis , Activación de Complemento , Proteínas Inactivadoras del Complemento C3b/metabolismo , Factor H de Complemento/metabolismo , Biología Computacional , Humanos , Inmunidad Innata , Unión Proteica

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas/inmunología , Inmunohistoquímica/métodos , Spirochaetales/inmunología , Treponema/inmunología , Diagnóstico Diferencial , Errores Diagnósticos/prevención & control , Humanos , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/inmunología , Enfermedades Intestinales/microbiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Infecciones por Spirochaetales/diagnóstico , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/microbiología , Sífilis/diagnóstico , Sífilis/inmunología , Sífilis/microbiología , Treponema pallidum/inmunología , Treponema pallidum/fisiología

Asunto(s)

Infecciones Bacterianas del Sistema Nervioso Central , Sistema Nervioso Central/microbiología , Modelos Animales de Enfermedad , Infecciones por Spirochaetales , Spirochaetales , Animales , Infecciones por Borrelia/inmunología , Infecciones por Borrelia/microbiología , Infecciones por Borrelia/patología , Infecciones Bacterianas del Sistema Nervioso Central/inmunología , Infecciones Bacterianas del Sistema Nervioso Central/microbiología , Infecciones Bacterianas del Sistema Nervioso Central/patología , Humanos , Leptospirosis/inmunología , Leptospirosis/microbiología , Leptospirosis/patología , Ratones , Neurosífilis/inmunología , Neurosífilis/microbiología , Neurosífilis/patología , Conejos , Ratas , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/microbiología , Infecciones por Spirochaetales/patología

RESUMEN

Previously, a clpX gene encoding a predicted 67 kDa membrane-associated ATPase subunit of the Clp protease (ClpX) was identified in a porcine strain (95/1,000) of the intestinal spirochaete Brachyspira pilosicoli. In the current study, the distribution of this large clpX gene was investigated in a collection of strains representing all seven Brachyspira spp. Using PCR with internal primers, an 878 bp portion of the gene was detected in 29 of 35 strains (83 %) of B. pilosicoli, 6 of 24 strains (25 %) of Brachyspira hyodysenteriae, 14 of 16 strains (88 %) of Brachyspira intermedia, 6 of 17 strains (35 %) of Brachyspira innocens, 1 of 6 strains (17 %) of Brachyspira murdochii, 1 of 2 strains (50 %) of Brachyspira aalborgi and not in the single strain of Brachyspira alvinipulli. The whole gene was sequenced from 20 Brachyspira spp. strains and compared with the clpX gene from B. pilosicoli 95/1,000 (GenBank accession no. AY466377). The genes had 99.3-99.7 % nucleotide sequence similarity and the predicted products had 99.7-100 % amino acid sequence similarity. The clpX gene from WesB, a human strain of B. pilosicoli, was cloned and expressed as a histidine-tagged fusion protein in Escherichia coli BL21. The purified protein was used to vaccinate mice and their sera were found to recognize the expected approximately 67 kDa protein in whole-cell preparations of WesB. Sera from mice vaccinated with formalin-treated whole-cell proteins of WesB reacted with the recombinant protein. These results indicate that ClpX is both conserved and immunogenic and hence might be useful as a subunit vaccine component for Brachyspira spp. infections. Sera from humans with no known exposure to B. pilosicoli reacted with the recombinant ClpX protein, indicating that it is unlikely to be useful as a reagent for serological detection of Brachyspira spp. infections.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Endopeptidasa Clp/genética , Endopeptidasa Clp/inmunología , Sueros Inmunes/inmunología , Proteínas Recombinantes/genética , Spirochaetales/enzimología , Animales , Humanos , Inmunización , Masculino , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes/inmunología , Spirochaetales/clasificación , Spirochaetales/genética , Spirochaetales/inmunología , Infecciones por Spirochaetales/diagnóstico , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/prevención & control

RESUMEN

The development of intestinal lesions after inoculation with Brachyspira hyodysenteriae was followed by repeated endoscopy and biopsy sampling through a caecal cannula. Seven eight-week-old pigs were cannulated and inoculated, two were cannulated but not inoculated, and two pigs were inoculated but not cannulated. Endoscopy, biopsy, and blood sampling to determine SAA (serum amyloid A), haptoglobin, cortisol, and WBC counts were performed at scheduled time-points. At the third day of disease, endoscopy showed a hyperaemic, perturbed mucosa and excessive amount of mucus. Histologically, crypt hyperplasia, depletion of goblet cell mucus, and erosions were noted. Simultaneously, elevated acute phase proteins and circulating monocytes, and decreased number of intraepithelial CD3(+) cells were observed. After five days the pigs recovered. Intestinal lesions were demarcated and interspersed among apparently normal mucosa and blood parameters returned to initial values. Endoscopy through an intestinal cannula made it possible to follow the development of intestinal alterations in vivo and describe the sequential events during the course of swine dysentery. The number of animals used in a study could thus be minimised and the precision of the experiment increased.

Asunto(s)

Biopsia/veterinaria , Cateterismo/veterinaria , Disentería/veterinaria , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/patología , Animales , Biopsia/instrumentación , Biopsia/métodos , Cateterismo/instrumentación , Cateterismo/métodos , Colon/inmunología , Colon/patología , Disentería/inmunología , Disentería/patología , Femenino , Masculino , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/patología , Infecciones por Spirochaetales/veterinaria , Porcinos , Factores de Tiempo

Asunto(s)

Parasitosis Intestinales/diagnóstico , Mucosa Intestinal/patología , Infecciones por Spirochaetales/diagnóstico , Spirochaetales/aislamiento & purificación , Anticuerpos Antibacterianos/análisis , Antígenos Bacterianos/inmunología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Spirochaetales/clasificación , Spirochaetales/inmunología , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/microbiología

RESUMEN

Serum obtained from a patient histopathologically diagnosed as intestinal spirochetosis was investigated serodiagnostically by agglutination test. B. aalborgi which is a human intestinal spirochete reacted strongly with the human serum, while B. pilosicoli which has potential pathogenicity to humans reacted with the serum, but as strongly and its titer was different than the other three species. On the other hand, intestinal spirochetes (Matsumoto isolates) were isolated from the biopsy samples of the patient. The morphological, biochemical, and genetic characteristics of the isolates were very similar to those of B. aalborgi. Furthermore, the protein profiles of the Matsumoto isolates were also similar to those of B. aalborgi but were different than those of B. pilosicoli and B. hyodysenteriae. The reaction profiles of the Matsumoto isolates in immunoblotting were relatively similar to those of B. aalborgi except for a 74 kDa band but were different from those of B. pilosicoli and B. hyodysenteriae. Therefore, we identified the Matsumoto isolates as B. aalborgi and diagnosed the patient with a B. aalborgi infection.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Diarrea/microbiología , Enfermedades Intestinales/microbiología , Spirochaetaceae/crecimiento & desarrollo , Infecciones por Spirochaetales/microbiología , Pruebas de Aglutinación , Especificidad de Anticuerpos , ADN Bacteriano/química , ADN Bacteriano/genética , Diarrea/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Enfermedades Intestinales/inmunología , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Spirochaetaceae/genética , Infecciones por Spirochaetales/inmunología

RESUMEN

Spirochaetes are organisms that can infect the colon of people with normal or compromised immune systems. Infected patients can present with a variety of gastrointestinal symptoms, including diarrhoea and rectal bleeding. However, some report a lack of association between specific symptoms and the presence of spirochaetes. It is therefore unclear whether the spirochaetes colonising the colon are true pathogens. Diagnosis is typically made by histological examination, with the biopsy specimen showing a band-like growth of spirochaetes adherent to the colonic luminal surface, giving an accentuated brush-border appearance. A course of metronidazole can eliminate the spirochaetes, but treatment might not lead to improvement of symptoms. Owing to the lack of a definite association between symptoms and the presence of spirochaetes, observation without specific antibiotic treatment can be pursued in most patients.

Asunto(s)

Enfermedades del Colon/diagnóstico , Infecciones por Spirochaetales/diagnóstico , Adulto , Enfermedades del Colon/complicaciones , Enfermedades del Colon/inmunología , Diarrea/microbiología , Hemorragia Gastrointestinal/microbiología , Humanos , Inmunocompetencia , Masculino , Infecciones por Spirochaetales/complicaciones , Infecciones por Spirochaetales/inmunología

RESUMEN

The aim of this study was to examine changes in the systemic immune response during the incubation period and following the onset of clinical swine dysentery, including the recovery period. Ten healthy conventional pigs were inoculated with Brachyspira hyodysenteriae. Blood was sampled at pre-inoculation, at days 4 and 14 post-inoculation, during the first 4 days with clinical signs of dysentery and at days 1, 3, 7, 11 and 15 of the recovery period. Eight pigs developed haemorrhagic diarrhoea. Flow-cytometric analyses of lymphocyte subpopulations showed that all animals, including the two that remained healthy, had an increase in CD8alpha+ CD4- cells and gammadelta T cells at days 4 and 14 post-inoculation. In addition, an increase in CD4+ CD8alpha+ cells and CD8alpha+ CD8beta+ cells was observed at days 4 and 14 post-inoculation in animals that developed dysentery. During clinical signs of dysentery, the acute-phase protein serum amyloid A was increased. There was a two- to threefold increase in both neutrophils and monocytes during signs of dysentery and at the beginning of the recovery period. The numbers of CD8alpha+ CD8beta- CD4-, CD45RA- lymphocytes also increased during the dysentery period. Circulating CD21+ cells and CD21+ CD45RA- cells decreased at the end of the incubation period, during signs of dysentery and at the beginning of the recovery period. The dysentery-affected animals developed antibodies to B. hyodysenteriae-specific antigens (approximately 16 kDa and approximately 30 kDa) from the first day of recovery, and gammadelta T cells showed an increase during the recovery period. In comparison with pre-inoculation, increased numbers of monocytes, neutrophils, CD8alpha+ CD8beta- CD4- lymphocytes and CD45RA- lymphocytes were observed during clinical dysentery. Increased numbers of neutrophils, gammadelta T cells and specific antibodies were seen during the recovery period.

Asunto(s)

Disentería/inmunología , Disentería/veterinaria , Spirochaetaceae/inmunología , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/veterinaria , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Western Blotting/veterinaria , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Disentería/microbiología , Femenino , Citometría de Flujo/veterinaria , Recuento de Leucocitos/veterinaria , Linfocitos/inmunología , Linfocitos/microbiología , Masculino , Monocitos/inmunología , Monocitos/microbiología , Neutrófilos/inmunología , Neutrófilos/microbiología , Proteína Amiloide A Sérica/análisis , Infecciones por Spirochaetales/microbiología , Porcinos

RESUMEN

Aberrant host immune responses to bacterial components of the resident microflora may initiate and perpetuate gastrointestinal inflammation. To investigate how microbial perturbation promotes host immunological responsiveness to commensal bacteria and contributes to the development of typhlocolitis, we selectively colonized defined (altered Schaedler) flora C3H mice with either Helicobacter bilis or Brachyspira hyodysenteriae. Following selective colonization, tissues were analyzed for gross/histopathologic lesions and bacterial antigen-specific B- and T-cell responses. Gnotobiotic mice colonized with H. bilis or B. hyodysenteriae developed typhlocolitis of varying severity, with the most severe gross and histopathogical lesions observed in B. hyodysenteriae-colonized mice. Antigen-specific IgG1 and IgG2a responses to the resident microflora were increased in both H. bilis-and B. hyodysenteriae-colonized mice. The greater antibody responses were associated with less severe cecal inflammation in H. bilis-colonized mice. Altered Schaedler flora (ASF)-stimulated mesenteric lymphocytes from B. hyodysenteriae-colonized mice produced higher levels of interferon-gamma and interleukin (IL)-4 than did lymphocytes from H. bilis-colonized mice. However, ASF-stimulated mesenteric and splenic lymphocytes from both H. bilis and B. hyodysenteriae-colonized mice secreted higher amounts of IL-10 compared to similarly stimulated lymphocytes recovered from control mice. These results indicate that microbial perturbation may induce differential immune responses to nonpathogenic resident bacteria that can lead to intestinal inflammation.

Asunto(s)

Colitis/inmunología , Colitis/microbiología , Vida Libre de Gérmenes , Infecciones por Helicobacter/inmunología , Helicobacter/inmunología , Spirochaetaceae/inmunología , Infecciones por Spirochaetales/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Colitis/patología , Citocinas/análisis , Citocinas/biosíntesis , Citocinas/inmunología , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Linfocitos/inmunología , Linfocitos/microbiología , Ratones , Ratones Endogámicos C3H , Infecciones por Spirochaetales/sangre , Infecciones por Spirochaetales/microbiología , Infecciones por Spirochaetales/patología

RESUMEN

In this study, we used the epidermal suction blister technique, in conjunction with multiparameter flow cytometry, to analyze the cellular and cytokine responses elicited by intradermal injection of human volunteers with synthetic analogs for spirochetal lipoproteins and compared the responses to findings previously reported from patients with erythema migrans (EM). Compared with peripheral blood (PB), lipopeptides derived from the N termini of the Borrelia burgdorferi outer surface protein C and the 17-kDa lipoprotein of Treponema pallidum (OspC-L and 17-L, respectively) elicited infiltrates enriched in monocytes/macrophages and dendritic cells (DCs) but also containing substantial percentages of neutrophils and T cells. Monocytoid (CD11c(+)) and plasmacytoid (CD11c(-)) DCs were selectively recruited to the skin in ratios similar to those in PB, but only the former expressed the activation/maturation surface markers CD80, CD83, and DC-SIGN. Monocytes/macrophages and monocytoid DCs, but not plasmacytoid DCs, displayed significant increases in surface expression of Toll-like receptor 1 (TLR1), TLR2, and TLR4. Staining for CD45RO and CD27 revealed that lipopeptides preferentially recruited antigen-experienced T-cell subsets; despite their lack of antigenicity, these agonists induced marked T-cell activation, as evidenced by surface expression of CD69, CD25, and CD71. Lipopeptides also induced significant increases in interleukin 12 (IL-12), IL-10, gamma interferon, and most notably IL-6 without corresponding increases in serum levels of these cytokines. Although lipopeptides and EM lesional infiltrates shared many similarities, differences were noted in a number of immunologic parameters. These studies have provided in situ evidence for a prominent "lipoprotein effect" during human infection while at the same time helping to pinpoint aspects of the cutaneous response that are uniquely driven by spirochetal pathogens.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/farmacología , Lipoproteínas/inmunología , Lipoproteínas/farmacología , Infecciones por Spirochaetales/inmunología , Adolescente , Adulto , Anciano , Grupo Borrelia Burgdorferi/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Pruebas Cutáneas , Sífilis/inmunología , Regulación hacia Arriba

RESUMEN

OBJECTIVE: To review the aetiologies and preventative methods associated with Jarisch-Herxheimer reactions (JHR). DATA SOURCES: Ovid Medline (1966-June Week 1 2004) was utilized to assess biomedical literature; a review of the bibliographies of articles was also performed. DATA SYNTHESIS: JHR often occurs with the treatment of spirochete infections. However, the mechanism by which the reaction takes place is not clearly defined. CONCLUSION: Studies suggest with conflicting evidence that the JHR is caused by release of endotoxin-like material from the spirochete as well as cytokine elevation in the body. It appears the type of drug and the rate of spirochete clearance from the body have little effect on the incidence of the reaction. Many pretreatment options have been explored with limited efficacy with the exception of anti-tumour necrosis factor antibodies.

Asunto(s)

Antibacterianos/efectos adversos , Hipersensibilidad a las Drogas/etiología , Hipersensibilidad a las Drogas/prevención & control , Infecciones por Spirochaetales/tratamiento farmacológico , Spirochaetales/efectos de los fármacos , Antibacterianos/uso terapéutico , Citocinas/metabolismo , Endotoxinas/metabolismo , Humanos , Spirochaetales/metabolismo , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/metabolismo

RESUMEN

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0.047) than for the control groups, with a approximately 50% reduction. BmpB appears to have potential as an SD vaccine component.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/uso terapéutico , Disentería/veterinaria , Lipoproteínas/inmunología , Infecciones por Spirochaetales/veterinaria , Spirochaetales/inmunología , Enfermedades de los Porcinos/prevención & control , Vacunación/veterinaria , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Colon/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Disentería/inmunología , Disentería/microbiología , Disentería/prevención & control , Ensayo de Inmunoadsorción Enzimática/veterinaria , Heces/microbiología , Femenino , Lipoproteínas/genética , Proteínas de Unión a Maltosa , Reacción en Cadena de la Polimerasa/veterinaria , Distribución Aleatoria , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Spirochaetales/genética , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/microbiología , Infecciones por Spirochaetales/prevención & control , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Vacunación/métodos , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéutico

RESUMEN

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.

Asunto(s)

Brachyspira hyodysenteriae/inmunología , Disentería/veterinaria , Subgrupos Linfocitarios/inmunología , Infecciones por Spirochaetales/veterinaria , Enfermedades de los Porcinos/inmunología , Animales , Disentería/inmunología , Disentería/microbiología , Femenino , Citometría de Flujo/veterinaria , Recuento de Leucocitos/veterinaria , Subgrupos Linfocitarios/citología , Masculino , Organismos Libres de Patógenos Específicos , Infecciones por Spirochaetales/inmunología , Porcinos , Enfermedades de los Porcinos/microbiología

RESUMEN

Alphabeta+ and gammadelta+ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4+ and gammadelta+ T cells in mixed lymphocyte cultures. CD4+ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas gammadelta+ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-alpha (IL-2Ralpha). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking gammadelta+ than CD4+ T cell proliferation. Immunization induced interferon-gamma (IFN-gamma) production by CD4+ T cells, whereas only a small fraction of the antigen-stimulated gammadelta+ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4+ T cell functions are more tightly regulated when compared to gammadelta+ T cells. We conclude that these differences are due, in part, to the enhanced gammadelta+ T cell responsiveness to IL-2.

Asunto(s)

Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Spirochaetales/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/inmunología , Epítopos/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-2/inmunología , Interleucina-2/farmacología , Subunidad alfa del Receptor de Interleucina-2 , Activación de Linfocitos/efectos de los fármacos , Receptores de Antígenos de Linfocitos T gamma-delta/efectos de los fármacos , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/inmunología , Infecciones por Spirochaetales/inmunología , Sus scrofa

RESUMEN

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.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Brachyspira hyodysenteriae/inmunología , Disentería/veterinaria , Infecciones por Spirochaetales/veterinaria , Enfermedades de los Porcinos/diagnóstico , Pruebas de Aglutinación/veterinaria , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Brachyspira hyodysenteriae/crecimiento & desarrollo , Brachyspira hyodysenteriae/aislamiento & purificación , Pruebas de Fijación del Complemento/veterinaria , Reacciones Cruzadas , Disentería/diagnóstico , Disentería/inmunología , Disentería/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Pruebas de Hemaglutinación/veterinaria , Hemólisis , Lipopolisacáridos/inmunología , Infecciones por Spirochaetales/diagnóstico , Infecciones por Spirochaetales/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología

RESUMEN

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.

Asunto(s)

Brachyspira hyodysenteriae , Linfocitos T CD8-positivos/inmunología , Infecciones por Spirochaetales/veterinaria , Enfermedades de los Porcinos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Brachyspira hyodysenteriae/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/sangre , Infecciones por Spirochaetales/inmunología , Porcinos , Vacunación

RESUMEN

Colitis develops in mice infected with Brachyspira (Serpulina) hyodysenteriae. Numerous granulocytes (PMNs) are evident in cecal tissue sections 24-48 h post-infection. The role of PMNs was assessed by utilizing monoclonal antibodies specific for CD18 or CD29 to block PMN recruitment. Macroscopic lesions were less severe in mice treated with either monoclonal antibody compared to lesions observed in isotype control-treated mice. While these monoclonal antibodies may inhibit extravasation of other leukocytes, the central role of PMNs was further demonstrated in that colitis was reduced following neutrophil depletion. There was less edema and epithelial erosions in ceca of mice receiving anti-Ly6G, -CD18 or -CD29 monoclonal antibody compared to mice receiving the control. Moreover, there was a significant reduction in PMN infiltration in tissues of mice treated with anti-CD18. The reduction in infiltrating PMNs did not result from downregulation of neutrophil chemoattractant MIP-2 expression in anti-CD18-treated mice. In contrast, PMN recruitment into the cecum was apparently CD29-independent. It is noteworthy that the number of PMNs observed in anti-CD18-treated mice was significantly higher than observed in non-infected mice. The data provide evidence for a threshold number of PMNs necessary for lesion development and indicate that CD18, but not CD29, adhesive pathways are crucial for PMN recruitment in bacterial colitis.

Asunto(s)

Antígenos CD18/análisis , Colitis/inmunología , Integrina beta1/análisis , Infecciones por Spirochaetales/inmunología , Spirochaetales/patogenicidad , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Adhesión Bacteriana/efectos de los fármacos , Ciego/microbiología , Ciego/patología , Quimiocina CXCL2 , Colitis/patología , Colitis/terapia , Modelos Animales de Enfermedad , Granulocitos/fisiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C3H , Monocinas/genética , Monocinas/metabolismo , Neutrófilos/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Spirochaetales/patología , Infecciones por Spirochaetales/terapia

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/fisiología , Lipoproteínas/fisiología , Spirochaeta/patogenicidad , Adaptación Fisiológica , Animales , Variación Antigénica/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Humanos , Inmunidad Innata , Lipoproteínas/química , Lipoproteínas/metabolismo , Spirochaeta/química , Spirochaetaceae/patogenicidad , Infecciones por Spirochaetales/inmunología , Vacunas Sintéticas/inmunología

RESUMEN

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.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Brachyspira hyodysenteriae/inmunología , Disentería/veterinaria , Infecciones por Spirochaetales/veterinaria , Enfermedades de los Porcinos/inmunología , Pruebas de Aglutinación , Animales , Antibacterianos/administración & dosificación , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/química , Brachyspira hyodysenteriae/aislamiento & purificación , Brachyspira hyodysenteriae/metabolismo , Disentería/microbiología , Disentería/prevención & control , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Ratones , Espectinomicina/administración & dosificación , Infecciones por Spirochaetales/inmunología , Infecciones por Spirochaetales/microbiología , Infecciones por Spirochaetales/prevención & control , Porcinos , Enfermedades de los Porcinos/microbiología