Total Leishmania antigens with Poly(I:C) induce Th1 protective response.

Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries.

Yersinia-triggered arthritis in IL-12p40-deficient mice: relevant antigens and local expression of Toll-like receptor mRNA.

OBJECTIVES: To study the role of IL-12p40 at the onset of reactive arthritis (ReA) after Yersinia enterocolitica O:3 infection, and analyse relevant microbial antigens and articular expression of Toll-like receptor (TLR) mRNA. METHODS: Wild-type C57BL/6 and IL-12p40-deficient (IL-12p40-/-) mice were orogastrically infected with Y. enterocolitica O:3. Early (day 3) and late (day 21) after infection, the number of bacteria were determined in Peyer's patches (PP), mesenteric lymph nodes (MLN), the spleen, and joints. Histological studies of joints were performed. Collagen-specific and anti-Yersinia antibodies were measured by enzyme-linked immunosorbent assay (ELISA). The presence of Yersinia antigens was studied by dot blot. Induction of articular mRNA of TLR2, TLR4, and tumour necrosis factor (TNF)-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR). TNFalpha protein levels were measured by ELISA. RESULTS: At day 3, bacterial recovery in PP, MLN, and spleen was significantly increased in IL-12p40-/- mice. Histopathological changes were observed in IL-12p40-/- mice at day 21 after infection, and correlated with higher antibody response against type II collagen. Although live bacteria could not be isolated at day 21 after infection, articular microbial components, especially from the outer membrane (OM), were detected. Moreover, intra-articular immunoglobulins to Yersinia antigens were significantly higher in IL-12p40-/- mice. Furthermore, mRNA levels for TLR2, TLR4 and TNFalpha, and TNFalpha protein were increased in joints from IL-12p40-/- mice. CONCLUSIONS: We concluded that IL-12p40 influences the resistance against Yersinia-triggered ReA. Bacterial products such as Yersinia OM could contribute to the ReA by induction of articular TLR expression, which results in an inflammatory response in the joint.

Role of TNFRp55 in Yersinia enterocolitica O:3-induced arthritis: triggering bacterial antigens and articular immune response.

OBJECTIVES: The pathogenesis of reactive arthritis (ReA), an aseptic synovitis that follows an extra-articular infection, is incompletely known. We studied the impact of tumour necrosis factor receptor (TNFR) p55 deficiency on the progression to ReA after oral Yersinia enterocolitica O:3 infection, the Yersinia antigens triggering articular inflammation and a possible articular TNFRp55-mediated mechanism that protects against ReA. METHODS: Wild-type C57BL/6 and TNFRp55-/- mice were orogastrically infected with Y. enterocolitica O:3 and monitored for survival and arthritis development. The bacterial load was determined in mesenteric lymph nodes (MLNs), the spleen and joints. Interferon (IFN)-gamma, TNF-alpha and IL-10 mRNA expression in MLN and joints were analysed by reverse transcription-polymerase chain reaction (RT-PCR). Articular antibodies to Yersinia antigens, TNF-alpha protein and nitric oxide (NO) levels were assessed. Acute arthritis was evaluated after joint injection of Yersinia antigens. RESULTS: The survival rate was 60% in TNFRp55-/- mice. They showed impaired bacterial clearance in MLN, the spleen and joints, and excessive mRNA expression of pro-inflammatory cytokines in MLN. Clinical and histological examinations revealed that TNFRp55-/- mice developed severe arthritis. Moreover, augmented articular outer membrane protein (OMP)-specific antibodies and TNF-alpha but impaired NO levels were detected in TNFRp55-/- mice. Synovial inflammatory response was detected by joint OMP injection. CONCLUSIONS: TNFRp55-mediated immune mechanisms prevent ReA development after oral infection with Y. enterocolitica O:3. Yersinia OMPs are the relevant antigens triggering ReA. NO induction through TNFRp55 signalling could have a local antibacterial function to prevent ReA. This study could contribute to ReA-specific therapeutic studies.

Interleukin-12 and interleukin-18 are indispensable for protective immunity against enteropathogenic Yersinia.

Previous results have demonstrated an essential role of gamma interferon (IFN-gamma) in resistance against Yersinia enterocolitica. Hence, we investigated the course of Yersinia infection in mice deficient for the IFN-gamma-inducing cytokines interleukin-12 (IL-12 p40(-/-)) or interleukin-18 (IL-18(-/-)). The experiments described herein argue for a critical role of both cytokines in protective immune responses against this pathogen.

Yersinia enterocolitica O:8 and O:5 lipopolysaccharide arthritogenicity in hamsters.

OBJECTIVE: To assess the arthritogenicity of Yersinia enterocolitica O:8 and O:5 lipopolysaccharide (LPS) administered separately as single antigens in hamsters. METHODS: Male hamsters of the Syria strain were intramuscularly injected into each of the hind paws with two doses of Y. enterocolitica LPS O:8 or O:5. The measurement of swelling using a plethysmometer, the analysis of histological changes by routine techniques and the kinetics of LPS-specific antibodies and autoantibodies evaluated by enzyme-linked immunosorbent assay (ELISA) were performed. RESULTS: LPS O:8 was demonstrated to be more arthritogenic than LPS O:5, inducing acute arthritis on day 3 post-injection as well as more significant and longer lasting joint swelling after a second dose. LPS O:8 caused severe histopathological changes in the joints. Important LPS O:8-specific IgG responses and antibodies against type I and II collagen were observed. CONCLUSION: LPS O:8 administered alone has arthritogenic power and induces activation of autoreactive clones. This study supports the key role of LPS in the development of reactive arthritis.

Effectiveness of various disinfectants in the elimination of Yersinia enterocolitica on fresh lettuce.

The effectiveness of various disinfectants against two potentially pathogenic Yersinia enterocolitica strains (Y. enterocolitica W1024 O:9 [strain A] and Y. enterocolitica B O:5 Lis Xz [strain B]) on shredded lettuce was examined. Dip-wash treatments using 25, 100, and 300 ppm of chlorine at 4 and 22 degrees C, 0.2% Orenco Peel 40, 0.1% Tergitol, 0.5% acetic acid, and 0.5% lactic acid at 22 degrees C were performed. Surfactants and organic acids were also tested in combination with 100 ppm of chlorine. Reductions of Y. enterocolitica counts with 100 ppm (2.68 log10 for strain A and 2.36 log10 for strain B at 22 degrees C) and 300 ppm of chlorine (3.15 log10 for strain A and 2.55 log10 for strain B at 4 degrees C) were observed after 10 min. Inhibitory effect of different chlorine solutions was not significantly (P < 0.05) influenced by temperature. Surfactants in combination with chlorine were more effective than surfactants alone. Treatment with 0.2% Orenco Peel 40 plus 100 ppm of chlorine resulted in reductions of 2.69 log10 CFU/g for strain A and 3.18 1og10 CFU/g for strain B at 10 min. Dip solutions containing 0.1% Tergitol plus 100 ppm of chlorine produced a significant reduction of 2.73 log10 CFU/g in strain A (P < 0.05). With the 0.5% lactic acid plus 100 ppm of chlorine combination, inactivation of Y. enterocolitica was >6 log10. The bactericidal effect of disinfectants was related to the concentration, exposure time, combination with chlorine (surfactants and organic acids), and susceptibility of each strain. Since the presence of pathogenic Y. enterocolitica on ready-to-use vegetables represents a health hazard, treatments as effective as 0.5% lactic acid plus 100 ppm of chlorine are recommended for washing of fresh lettuce.

Arthritogenicity of Yersinia enterocolitica O:8 in hamsters: analysis of the immune response.

An animal model, hamster, was used for the study of Yersinia-induced arthritis. The development of arthritis, estimated by measuring the inflammation on hind paws after infection, was correlated with the kinetics of the immune response. Histological and immunofluorescence (IFI) studies and serum antibody measurements were performed. Two inflammatory peaks were observed: an acute one on day 11 post-infection (p.i.) and a chronic one on days 26-35 p.i. Joint cultures were positive until day 14 p.i. IFI was used to demonstrate the deposit of bacterial antigens in the joint. A persistent response of cellular extract-specific IgG antibodies was observed until day 94. Lipopolysaccharide-specific IgG was statistically significant on day 26 p.i. Antibodies against bands 66 and 54 were observed by immunoblotting. Polyclonal activation was detected during reactive arthritis. It is shown that Y. enterocolitica is arthritogenic in hamsters, immune mechanisms participating in the development of this disease.

Intranasal immunization with Yersinia enterocolitica O:8 cellular extract protects against local challenge infection.

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5 x 10(6) colony-forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.-specific IgA and IgG, and serum-specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.

Humoral immune response in Yersinia enterocolitica O:5 induced arthritis in hamsters.

Yersinia enterocolitica can cause extraintestinal sequelae such as reactive arthritis. The immunopathogenic mechanisms of this disease have not been completely clarified. Autoimmunity and persistent immune responses against bacterial antigens have been related to Yersinia-induced arthritis. The arthritogenic capacity of Y. enterocolitica O:5 and the kinetics of the development of autoantibodies and Yersinia antigen-specific antibodies were studied in hamsters. The results indicated that Y. enterocolitica O:5 was arthritogenic in the animal model studied. The animals developed septic arthritis on day 2 post-infection (p.i.) and reactive arthritis on day 65 p.i. An important IgG response to types I and II collagen and the persistence of antibodies against lipopolysaccharide and bacterial cellular extract were observed. By immunoblotting, it was obtained that IgG reacted against a large number of bacterial antigens, the strongest being the responses against 88, 76, 63 and 36-33 kDa peptides. From the results obtained, it can be concluded that serovar O:5 was experimentally arthritogenic, and that both autoimmune mechanisms and Yersinia-specific antibodies participated in the development of Yersinia-induced reactive arthritis in the animal model studied.

Protective immunity induced by a Yersinia enterocolitica serovar 0:8 cellular extract.

The purpose of this study was to investigate the protective power of a cellular extract (CE) from Y. enterocolitica 0:8 grown in condition of expression of chromosomal antigens. Mice were immunized by s.c. route and challenged with: 0 LD50 (1 x 10(4) CFU/ml). Immunoblotting showed that CE-specific serum reacted with several CE antigens. Prominent bands, of molecular weights 60 and 35.5, were present in cytoplasmic and membrane fraction, respectively. The lipopolysaccharide (LPS) was detected in CE. These findings suggest that chromosomally-encoded antigens present in CE may induce protection against Y. enterocolitica infection. Both humoral and cellular immune response contribute to protection in mice.

Yersinia ssp. in surface water in San Luis, Argentina.

Yersinia spp. was examined in three rivers and two lakes located in the Province of San Luis, Argentina, over a 1-year period. Water samples were concentrated either by Moore's gauze technique or by filtering through diatomaceous earth. Five enrichment media: yeast extract--Bengal rose broth (YER) with bile-oxalate-sorbose broth (BOS); 67 mmol/L phosphate-buffered saline (pH 7.6; PBS); PBS enriched with a 1% mannitol and 1% peptone (PBSMP); PBS with lyzed 0.5% sheep blood (PBSB); Wauters broth (W); and five plating media: Mac Conkey agar (MC); Salmonella--Shigella agar (SS); 5% sheep blood agar (BA); lactose-sucrose-urea agar (LSU) and irgasan-novobiocin agar (IN) were used. The following strains were isolated: Y. intermedia B1 O:4,32-4,33 Lis Xz (four strains), Y. intermedia B1 O:57 Lis Xo (one strain), Y. intermedia B2 O:57 Lis Xo (one strain), Y. enterocolitica B1 O:10-34 Lis Xz (one strain), and Y. frederiksenii undetermined biovar, O:16-16,29 Lis Xz (two strains). The incidence of isolation of Yersinia spp. was 7.14%. YER-BOS proved to be the best enrichment method since it allowed the highest recovering Yersinia spp. strains. Among plating media, the best results were obtained with MC. Apparently, the isolation of Yersinia spp. can be related to environmental variables such as temperature differences between cold and warm seasons. Negative results obtained during virulence assays suggest that isolated strains lack the pathogenic potential against man.