Trichinella Infection in Culled Wild Boar (Sus scrofa) from El Palmar National Park, Argentina, and Exposure Risk in Humans and Dogs Consuming Wild Boar Meat.

Trichinellosis is a foodborne disease caused by ingestion of raw or undercooked meat containing Trichinella spp. larvae. Consumption of wild boar (Sus scrofa) meat represents an important source of human trichinellosis worldwide. In El Palmar National Park (EPNP), Argentina, invasive alien wild boars are controlled and meat from culled animals is released for public consumption following on-site artificial digestion (AD) testing. Meat trimmings and offal from the control program are often used as food for dogs (Canis familiaris). We evaluated infection and exposure to Trichinella spp. in wild boars from EPNP, as well as exposure to Trichinella spp. and associated risk factors in dogs and human consumers of wild boar meat. Trichinella spp. larvae were detected in muscle samples from 5/49 wild boars by AD (10.2%; 95% confidence interval [CI], 3.8%-23%), with a mean burden of 0.24 larvae per gram (lpg; range, 0.06-0.95 lpg). Anti-Trichinella antibodies were not detected in wild boar serum samples (n=42). In dogs, 12/34 were seropositive to Trichinella spp. (35.29%; 95%, CI, 20.3%-53.5%). Immunoglobulin (Ig) G antibodies were not detected in human serum samples (n=63). Our results reveal the presence, albeit at low prevalence, of Trichinella spp. in wild boars and exposure in dogs fed game offal. These findings suggest that the low prevalence and parasitic load in wild boars, together with the best practices applied by EPNP culling program personnel, contribute to keeping the risk of infection in people low. The dog results highlight that the parasite is circulating in the area, and therefore the risk of infection is not negligible. We recommend the implementation of an animal surveillance strategy in order to monitor the evolution of this zoonosis in the study area.

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Polymeric antigen BLSOmp31 in aluminium hydroxide induces serum bactericidal and opsonic antibodies against Brucella canis in dogs.

Polymeric antigen BLSOmp31 is an immunogenic vaccine candidate that confers protection against Brucella canis in mice. In this preliminary study, the immunogenicity and safety of BLSOmp31 adsorbed to aluminum hydroxide gel (BLSOmp31-AH) were evaluated in Beagle dogs. In addition, the potential to elicit serum antibodies with complement-dependent bactericidal activity and/or to enhance phagocytosis by neutrophils were analyzed. Dogs were immunized three times with BLSOmp31-AH by subcutaneous route, followed by an annual booster. The vaccine elicited specific antibodies 3 weeks after the first immunization. Annual booster induced comparable antibody response as the primary series. Humoral immune response stimulated by BLSOmp31-AH did not interfere with routine agglutination test for canine brucellosis. Antibodies demonstrated a high complement-dependent bactericidal activity against B. canis. Moreover, opsonization by immune serum not only stimulated binding and uptake of the bacteria by neutrophils but effectively enhanced the destruction of B. canis. Specific IgG was detected in 3/4 immunized dogs in preputial secretions. The antibody profile corresponded to a marked Th2 response, since IgG1 prevailed over IgG2 and cellular immune response was not detected in vitro or in vivo. These results require further evaluation in larger field studies to establish the full prophylactic activity of BLSOmp31 against canine brucellosis.

Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.

Evaluation of the efficacy of outer membrane protein 31 vaccine formulations for protection against Brucella canis in BALB/c mice.

Canine brucellosis is an infectious disease caused by the Gram-negative bacterium Brucella canis. Unlike conventional control programs for other species of the genus Brucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinant Brucella Omp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy against B. canis infection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) after in vitro stimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection against B. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine against B. canis infection.

The BtaF trimeric autotransporter of Brucella suis is involved in attachment to various surfaces, resistance to serum and virulence.

The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.

The vaccine candidate BLSOmp31 protects mice against Brucella canis infection.

Canine brucellosis represents a major reproductive problem worldwide and it is considered a zoonotic disease. New approaches are therefore urgently needed to develop an effective and safe immunization strategy against Brucella canis. In the present study, BALB/c mice were subcutaneously immunized with the recombinant chimera rBLSOmp31 formulated in different adjuvants. The different strategies induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. Besides, spleen cells from rBLSOmp31-immunized mice produced gamma interferon and IL-4, suggesting the induction of a mixed Th1-Th2. Vaccination with rBLSOmp31-IFA formulation provided the best protection levels comparable with that given by control vaccines. None of the immunization strategies induced serological interference in diagnosis. Hitherto, this is the first report that a recombinant vaccine confers protection against B. canis in mice.

Immune response and serum bactericidal activity against Brucella ovis elicited using a short immunization schedule with the polymeric antigen BLSOmp31 in rams.

Brucella ovis is the etiologic agent of ovine brucellosis. The control measures for this disease are periodical diagnosis by serological tests and/or bacteriological culture and culling of positive animals. Vaccination with Brucella melitensis Rev 1 is recommended when prevalence is high. This attenuated strain vaccine gives protection against B. ovis but it has important disadvantages associated with the development of antibodies interfering with serodiagnosis, virulence for humans and the prohibition of its use in countries considered free of B. melitensis. Consequently, there is a need for new safe and effective brucellosis vaccines to be developed. We have previously reported that the polymeric subcellular vaccine BLSOmp31 confers protection against experimental challenge with B. ovis when rams are immunized three times. In the present work we evaluated and characterized, along 56 weeks after the first immunization of adult rams, the evolution of the immune response elicited by BLSOmp31 using a short immunization schedule.

[Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy]. / Respuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación.

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsG1 (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. g1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

Respuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación / Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy

La brucelosis ovina por Brucella ovis es una enfermedad de prevalencia alta en Argentina. Para evaluar la patogenicidad de B. ovis y la respuesta serológica durante el último mes de gestación, 6 ovejas se distribuyeron en dos grupos: G1, ovejas preñadas, n = 4 y G2, ovejas no preñadas, n = 2. Tres ovejas del G1 (15 días preparto) y una del G2 fueron inoculadas con B. ovis. Se analizaron muestras de suero mediante diferentes pruebas serológicas. Se realizó aislamiento y PCR a partir de mucus cérvico-vaginal (mcv), placenta y leche. En las muestras de placenta se realizó histopatología. Las hembras del G1 parieron corderos vivos; se detectaron anticuerpos en las ovejas desafiadas del G1 a partir de los 5 días posinoculación. El mcv de las ovejas desafiadas resultó negativo al aislamiento en ambos grupos. Las muestras de leche del G1 fueron positivas por cultivo y PCR a B. ovis. La técnica de PCR resultó positiva en las placentas de las ovejas desafiadas del G1. La histopatología reveló una placentitis necrótica supurativa en una de las ovejas desafiadas. El desafío con B. ovis preparto resultó en la invasión de la placenta y de la glándula mamaria, con la consecuente excreción de la bacteria por leche. La infección con B. ovis indujo una respuesta humoral temprana en las ovejas. La colonización de la placenta por B. ovis y la excreción de la bacteria por la leche sugieren un potencial riesgo de infección activa para los corderos y la posibilidad de que estos se comporten como portadores latentes de la infección.

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsGI (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. G1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. Sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella suis.

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

[Development of an ELISA test to detect antibodies in vaccinated sheep or infected Corynebacterium pseudotuberculosis]. / Desarrollo de una prueba de ELISA para detectar anticuerpos en carneros vacunados o infectados con Corynebacterium pseudotuberculosis.

The aim of this study was to evaluate an indirect specific ELISA developed for the detection of humoral immune response in vaccinated sheep and/or challenged with a Corynebacterium pseudotuberculosis strain. Healthy 4 month-old lambs were distributed into 4 groups: Group 1 immunized (G1, n = 5), Group 2 vaccinated/inoculated (G2, n = 8), Group 3 inoculated (G3, n = 2) and Group 4 control (G4, n = 2). Groups G1 and G2 received two doses of an experimental bacterin. Four weeks postvaccination, G2 and G3 groups were challenged with a C. pseudotuberculosis strain. Serological titers were studied by ELISA for 7 months and pathological studies were performed in groups G2, G3 and G4 by taking lung and lymph node samples for bacteriology and histopathology. The inoculated strain in G2 and G3 animals reproduced the macroscopic and microscopic lesions typical of caseous lymphadenitis (CL) and was isolated from the inoculation site, lymph nodes and/or lung in 7/8 animals from G2, and 2/2 animals of G3. The developed ELISA test had sensitivity and specificity of 98% and 100% respectively, detected significant differences between serological reactors of different experimental groups and allowed to establish a relationship with the type of treatment. We conclude that the developed ELISA may be a useful tool to identify infected animals with positive clinical CL.

Desarrollo de una prueba de ELISA para detectar anticuerpos en carneros vacunados o infectados con Corynebacterium pseudotuberculosis / Development of an ELISA test to detect antibodies in vaccinated sheep or infected Corynebacterium pseudotuberculosis

El objetivo de este trabajo fue evaluar un ELISA indirecto desarrollado para medir la respuesta inmune humoral en carneros vacunados contra la linfoadenitis caseosa (LC) y/o desafiados con una cepa de Corynebacterium pseudotuberculosis homóloga. Se distribuyeron corderos de 4 meses clínicamente sanos en 4 grupos: grupo 1, corderos vacunados (G1, n = 5); grupo 2, corderos vacunados e inoculados (G2, n = 8); grupo 3, corderos inoculados (G3, n = 2); y grupo 4, control (G4, n = 2). Los animales del G1 y del G2 recibieron dos dosis de una bacterina experimental; los del G2 y del G3 fueron desafiados con una cepa de C. pseudotuberculosis cuatro semanas posvacunación. Se estudiaron por ELISA los títulos serológicos durante 7 meses y se efectuaron las necropsias en los grupos G2, G3 y G4. Se tomaron muestras de pulmón y linfonódulos para efectuar estudios bacteriológicos e histopatológicos. La cepa inoculada en los animales del G2 y del G3 reprodujo las lesiones macroscópicas y microscópicas típicas de la LC; ésta fue aislada del sitio de inoculación, de linfonódulos o de pulmón en 7/8 animales del G2 y en 2/2 animales del G3. La prueba de ELISA, con una sensibilidad del 98% y una especificidad del 100%, detectó diferencias significativas entre los serorreactores de los diferentes grupos experimentales y permitió establecer una relación con el tipo de tratamiento aplicado. Se concluye que el ELISA desarrollado puede ser una herramienta útil para identificar animales infectados y con clínica positiva a la LC.

The aim of this study was to evaluate an indirect specific ELISA developed for the detection of humoral immune response in vaccinated sheep and/or challenged with a Corynebacterium pseudotuberculosis strain. Healthy 4 month-old lambs were distributed into 4 groups: Group 1 immunized (G1, n = 5), Group 2 vaccinated/inoculated (G2, n = 8), Group 3 inoculated (G3, n = 2) and Group 4 control (G4, n = 2). Groups G1 and G2 received two doses of an experimental bacterin. Four weeks postvaccination, G2 and G3 groups were challenged with a C. pseudotuberculosis strain. Serological titers were studied by ELISA for 7 months and pathological studies were performed in groups G2, G3 and G4 by taking lung and lymph node samples for bacteriology and histopathology. The inoculated strain in G2 and G3 animals reproduced the macroscopic and microscopic lesions typical of caseous lymphadenitis (CL) and was isolated from the inoculation site, lymph nodes and/or lung in 7/8 animals from G2, and 2/2 animals of G3. The developed ELISA test had sensitivity and specificity of 98% and 100% respectively, detected significant differences between serological reactors of different experimental groups and allowed to establish a relationship with the type of treatment. We conclude that the developed ELISA may be a useful tool to identify infected animals with positive clinical CL.

An oral vaccine based on U-Omp19 induces protection against B. abortus mucosal challenge by inducing an adaptive IL-17 immune response in mice.

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4(+) T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity.

The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis.

Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease.

The polymeric antigen BLSOmp31 confers protection against Brucella ovis infection in rams.

We have engineered the polymeric vaccine BLSOmp31 by decorating the highly immunogenic and decameric Brucella lumazine synthase with an exposed loop of the Brucella outer membrane protein Omp31. In the present study, we have immunized different groups of rams with the recombinant chimera rBLSOmp31 in two different adjuvants (Incomplete Freund Adjuvant-IFA and QUIL A) and with the plasmid pCIBLSOmp31 administered either by i.m. injection alone or by using electroporation. In addition, we have used a heterologous prime-boost strategy consisting of repeated pCIBLSOmp31 electroporation priming followed by a single protein boost. Both, chimera rBLSOmp31 in IFA and the prime-boost strategy induced the highest IgG specific antibodies with bacteriolytic activity. While electroporation-enhanced humoral immune responses as compared to pCIBLSOmp31 injection alone, the highest levels of specific IFN-gamma and protection against bacterial challenge were achieved with prime-boost (76%) and chimera rBLSOmp31 in IFA (63%). Taken together these results strongly support the usefulness of the chimera BLSOmp31 as a vaccine against Brucella ovis in ovine brucellosis.

Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection.

Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4(+) as well as CD8(+) T cells producing gamma interferon. In vivo depletion of CD4(+) or CD8(+) T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis.

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria.

Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.

A DNA vaccine coding for the chimera BLSOmp31 induced a better degree of protection against B. ovis and a similar degree of protection against B. melitensis than Rev.1 vaccination.

In the present study, we reported an attempt to improve the immunogenicity and protective capacity of the chimera BLSOmp31 using a different antigen delivery: DNA vaccination. Vaccination of BALB/c mice with the DNA vaccine coding for the chimera BLSOmp31 (pCIBLSOmp31) provided the best protection level against Brucella ovis, which was significantly higher than the given by the co-delivery of both plasmids coding for the whole proteins (pcDNABLS+pCIOmp31) and even higher than the control vaccine Rev.1. Moreover, pCIBLSOmp31 induced higher protection against Brucella melitensis than pcDNABLS+pCIOmp31 but similar protection than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific cytotoxic T responses. The insertion of this peptide on BLS induced stronger T helper 1 responses specific for the carrier (BLS), thus our results represent a case of synergic strengthening between two Brucella antigens. Hitherto, this is the first indication that a recombinant subunit vaccine elicits greater protection than whole Brucella.