Artículos de revista (Inst. Salud Tropical)
Permanent URI for this collectionhttps://hdl.handle.net/10171/52056
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11 results
Results
- Immunomodulatory properties of Brucella melitensis lipopolysaccharide determinants on mouse dendritic cells in vitro and in vivo(Taylor & Francis, 2018) Moriyon, I. (Ignacio); Mémet, S. (Sylvie); Arce-Gorvel, V. (Vilma); Zhao, Y. (Yun); Gorvel, J.P. (Jean Pierre); Hanniffy, S. (Sean); Oh, S. (SangKon); Conde-Alvarez, R. (Raquel)The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligandderived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (BmwadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.
- Development and evaluation of the Galleria mellonella (greater wax moth) infection model to study Brucella host-pathogen interaction(2023) Loperena-Barber, M. (Maite); Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Elizalde-Bielsa, A. (Aitor); Conde-Alvarez, R. (Raquel)Brucellosis is a zoonotic disease caused by Gram-negative bacteria of the genus Brucella. These pathogens cause long-lasting infections, a process in which Brucella modifications in the lipopolysaccharide (LPS) and envelope lipids reduce pathogen-associated molecular pattern (PAMP) recognition, thus hampering innate immunity activation. In vivo models are essential to investigate bacterial virulence, mice being the most used model. However, ethical and practical considerations impede their use in high-throughput screening studies. Although lacking the complexity of the mammalian immune system, insects share key-aspects of innate immunity with mammals, and Galleria mellonella has been used increasingly as a model. G. mellonella larvae have been shown useful in virulence analyses, including Gram-negative pathogens like Klebsiella pneumoniae and Legionella pneumophila. To assess its potential to study Brucella virulence, we first evaluated larva survival upon infection with representative Brucella species (i.e.B. abortus 2308W, B. microti CCM4915 and B. suis biovar 2) and mutants in the VirB type-IV secretion system (T4SS) or in the LPS-O-polysaccharide (O-PS). As compared to K.pneumoniae, the Brucella spp. tested induced a delayed and less severe mortality profile consistent with an escape of innate immunity detection. Brucella replication within larvae was affected by the lack of O-PS, which is reminiscent of their attenuation in natural hosts. On the contrary, replication was not affected by T4SS dysfunction and the mutant induced only slightly less mortality (not statistically significant) than its parental strain. We also evaluated G. mellonella to efficiently recognise Brucella and their LPS by quantification of the pro-phenoloxidase system and melanisation activation, using Pseudomonas LPS as a positive control. Among the brucellae, only B. microti LPS triggered an early-melanisation response consistent with the slightly increased endotoxicity of this species in mice. Therefore, G. mellonella represents a tool to screen for potential Brucella factors modulating innate immunity, but its usefulness to investigate other mechanisms relevant in Brucella intracellular life is limited.
- The prevalence of brucellosis and bovine tuberculosis in ruminants in Sidi Kacem Province, Morocco(NCBI, 2018) Hattendorf, J. (Jan); Moriyon, I. (Ignacio); Azami, H.Y. (Hind Yahyaoui); Zinsstag, J. (Jakob); Ducrotoy, M.J. (Marie J.); Welburn, S.C. (Susan C.); Zuñiga-Ripa, A. (Amaia); Muñoz, P. (Pilar); Thrusfield, M. (Mike); Bryssinckx, W. (Ward); Bouslikhane, M. (Mohammed); Mick, V. (Virginie); RaquelBovine tuberculosis (BTB) and brucellosis are major endemic zoonoses in ruminants in Morocco that impact on both animal and human health. This study presents an assessment of the epidemiological and socioeconomic burden of bacterial zoonoses in Sidi Kacem Province in Northern Morocco from a cross-sectional survey of 125 cattle and/or small ruminantowning households. In total, 1082 sheep and goats were examined from 81 households. The single intradermal comparative cervical test to screen for bovine tuberculosis was undertaken on 1194 cattle from 123 households and all cattle were blood sampled. Cattle and small ruminant sera were tested for brucellosis using the standard Rose Bengal Test (sRBT) and the modified Rose Bengal Test (mRBT). Bacteriology was performed on 21 milk samples obtained from cattle that were seropositive for brucellosis for isolation and phenotyping of circulating Brucella strains. Individual and herd prevalence for BTB in cattle of 20.4% (95% CI 18%-23%) and 57.7% (95% CI 48%-66%), respectively, were observed in this study. The prevalence of brucellosis in cattle at individual and herd level was 1.9% (95% CI 1.2%-2.8%) and 9% (95% CI 4.5%-1.5%), respectively. Brucella pathogens were isolated from three cattle milk samples and were identified as B. abortus using Bruceladder (R) multiplex PCR and B. abortus biovar 1 by classical phenotyping. All small ruminants were seronegative to sRBT, two were positive to mRBT. A higher risk of BTB and brucellosis was observed in cattle in intensive livestock systems, in imported and crossed breeds and in animals from larger herds (>15). The three risk factors were usually present in the same herds, leading to higher transmission risk and persistence of both zoonoses. These results highlight the importance of implementing control strategies for both BTB and brucellosis to reduce productivity losses and the risk of transmission to humans. Prioritising control for BTB and brucellosis in intensive livestock production systems is essential for human and animal health.
- The fast-growing Brucella suis Biovar 5 depends on phosphoenolpyruvatecCarboxykinase and pyruvate phosphate dikinase but not on Fbp and GlpX fructose-1,6-bisphosphatases or isocitrate lyase for full virulence in laboratory models(NCBI, 2018) Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Muñoz, P. (Pilar); Lázaro-Antón, L. (Leticia); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Raquel; Barbier, T. (Thibault)Bacteria of the genus Brucella infect a range of vertebrates causing a worldwide extended zoonosis. The best-characterized brucellae infect domestic livestock, behaving as stealthy facultative intracellular parasites. This stealthiness depends on envelope molecules with reduced pathogen-associated molecular patterns, as revealed by the low lethality and ability to persist in mice of these bacteria. Infected cells are often engorged with brucellae without signs of distress, suggesting that stealthiness could also reflect an adaptation of the parasite metabolism to use local nutrients without harming the cell. To investigate this, we compared key metabolic abilities of Brucella abortus 2308 Wisconsin (2308W), a cattle biovar 1 virulent strain, and B. suis 513, the reference strain of the ancestral biovar 5 found in wild rodents. B. suis 513 used a larger number of C substrates and showed faster growth rates in vitro, two features similar to those of B. microti, a species phylogenomically close to B. suis biovar 5 that infects voles. However, whereas B. microti shows enhanced lethality and reduced persistence in mice, B. suis 513 was similar to B. abortus 2308W in this regard. Mutant analyses showed that B. suis 513 and B. abortus 2308W were similar in that both depend on phosphoenolpyruvate synthesis for virulence but not on the classical gluconeogenic fructose-1,6-bisphosphatases Fbp-GlpX or on isocitrate lyase (AceA). However, B. suis 513 used pyruvate phosphate dikinase (PpdK) and phosphoenolpyruvate carboxykinase (PckA) for phosphoenolpyruvate synthesis in vitro while B. abortus 2308W used only PpdK. Moreover, whereas PpdK dysfunction causes attenuation of B. abortus 2308W in mice, in B. suis, 513 attenuation occurred only in the double PckA-PpdK mutant. Also contrary to what occurs in B. abortus 2308, a B. suis 513 malic enzyme (Mae) mutant was not attenuated, and this independence of Mae and the role of PpdK was confirmed by the lack of attenuation of a double Mae-PckA mutant. Altogether, these results decouple fast growth rates from enhanced mouse lethality in the brucellae and suggest that an Fbp-GlpX-independent gluconeogenic mechanism is ancestral in this group and show differences in central C metabolic steps that may reflect a progressive adaptation to intracellular growth.
- Genomic insertion of a heterologous acetyltransferase generates a new lipopolysaccharide antigenic structure in brucella abortus and brucella melitensis(NCBI, 2018) Moriyon, I. (Ignacio); Ståhle, J. (Jonas); Zaccheus, M.V. (Mona V.); Gil-Ramirez, Y. (Yolanda); Zuñiga-Ripa, A. (Amaia); Iriarte-Cilveti, M. (Maite); Martinez-Gomez, E. (Estrella); Raquel; Widmalm, G. (Göran)Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a per R mutant. In mice, the wbdR constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against Brucella.
- WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization(NCBI, 2018) Cloeckaert, A. (Axel); Miguel, M.J. (María Jesús) de; Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zygmunt, M. (Michel); Gil-Ramirez, Y. (Yolanda); Zuñiga-Ripa, A. (Amaia); Muñoz, P. (Pilar); Iriarte-Cilveti, M. (Maite); Martinez-Gomez, E. (Estrella); RaquelBrucellosis, an infectious disease caused by Brucella, is one of the most extended bacterial zoonosis in the world and an important cause of economic losses and human suffering. The lipopolysaccharide (LPS) of Brucella plays a major role in virulence as it impairs normal recognition by the innate immune system and delays the immune response. The LPS core is a branched structure involved in resistance to complement and polycationic peptides, and mutants in glycosyltransferases required for the synthesis of the lateral branch not linked to the O-polysaccharide (O-PS) are attenuated and have been proposed as vaccine candidates. For this reason, the complete understanding of the genes involved in the synthesis of this LPS section is of particular interest. The chemical structure of the Brucella LPS core suggests that, in addition to the already identified WadB and WadC glycosyltransferases, others could be implicated in the synthesis of this lateral branch. To clarify this point, we identified and constructed mutants in 11 ORFs encoding putative glycosyltransferases in B. abortus. Four of these ORFs, regulated by the virulence regulator MucR (involved in LPS synthesis) or the BvrR/BvrS system (implicated in the synthesis of surface components), were not required for the synthesis of a complete LPS neither for virulence or interaction with polycationic peptides and/or complement. Among the other seven ORFs, six seemed not to be required for the synthesis of the core LPS since the corresponding mutants kept the O-PS and reacted as the wild type with polyclonal sera. Interestingly, mutant in ORF BAB1_0953 (renamed wadD) lost reactivity against antibodies that recognize the core section while kept the O-PS. This suggests that WadD is a new glycosyltransferase adding one or more sugars to the core lateral branch. WadD mutants were more sensitive than the parental strain to components of the innate immune system and played a role in chronic stages of infection. These results corroborate and extend previous work indicating that the Brucella LPS core is a branched structure that constitutes a steric impairment preventing the elements of the innate immune system to fight against Brucella
- Comparative performance of lateral flow immunochromatography, iELISA and Rose Bengal tests for the diagnosis of cattle, sheep, goat and swine brucellosis(Public Library of Science (PLoS), 2019) Bertu, W.J. (Wilson J.); Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Smits, H.L. (Henk L.); Gusi, A.M. (Amahyel M.); Muñoz, P. (Pilar); Dieste-Pérez, L. (Lucía); Blasco, J.M. (J. M.); Ocholi, R.A. (Reuben A.)Background Brucellosis is a world-wide extended zoonosis that causes a grave problem in developing economies. Animal vaccination and diagnosis are essential to control brucellosis, and the need for accurate but also simple and low-cost tests that can be implemented in low-infrastructure laboratories has been emphasized. Methodology We evaluated bovine, sheep, goat and swine lateral flow immunochromatography assay kits (LFA), the Rose Bengal test (RBT) and a well-validated protein G indirect ELISA (iELISA) using sera of Brucella culture-positive and unvaccinated brucellosis free livestock. Sera from cattle vaccinated with S19 and RB51 brucellosis vaccines were also tested. Finally, we compared RBT and LFA using sera of white Fulani cattle of unknown bacteriological status from a brucellosis endemic area of Nigeria. Results and conclusions Although differences were not statistically significant, RBT showed the highest values for diagnostic sensitivity/specificity in cattle (LFA, 96.6/98.8; RBT, 98.9/100; and iELISA, 96.6/100) and the iELISA yielded highest values in sheep (LFA, 94.0/100; RBT, 92.0/100; iELISA, 100/100), goats (LFA, 95.7/96.2; RBT, 97.8/100; iELISA, 100/100) and pigs (LFA, 92.3/100; RBT, 92.3/100; iELISA, 100/100). Vaccine S19 administered subcutaneously interfered in all tests but conjunctival application minimized the problem. Although designed not to interfere in serodiagnosis, vaccine RB51 interfered in LFA and iELISA but not in the RBT. We found closely similar apparent prevalence results when testing the Nigerian Fulani cattle by RBT and LFA. Although both RBT and LFA (showing similar diagnostic performance) are suitable for small laboratories in resource-limited areas, RBT has the advantage that a single reagent is useful in all animal species. Considering these advantages, its low cost and that it is also useful for human brucellosis diagnosis, RBT might be a good choice for resource-limited laboratories.
- Corrigendum: genetic and phenotypic characterization of the etiological agent of canine orchiepididymitis smooth brucella sp. BCCN84.3(Frontiers Media SA, 2019) Suárez-Esquivel, M. (Marcela); Cloeckaert, A. (Axel); Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Gonnet, M. (Mathieu); Víquez-Ruiz, E. (Eunice); Campos, E. (Elena); Zygmunt, M. (Michel); Thomson, N.R. (Nicholas R.); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Blasco, J.M. (J. M.); Baker, K.S. (Kate S.); Moreno, E. (Edgardo); Ruíz-Villalobos, N. (Nazareth); Guzman-Verri, C. (Caterina); RaquelIn the original article, there was an error. In the Funding statement is written that MZ was granted with a fellowship from SEP, Universidad de Costa Rica. The correct Initials are MS-E.
- Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3(Frontiers Media SA, 2019) Suárez-Esquivel, M. (Marcela); Cloeckaert, A. (Axel); Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Gonnet, M. (Mathieu); Víquez-Ruiz, E. (Eunice); Campos, E. (Elena); Zygmunt, M. (Michel); Thomson, N.R. (Nicholas R.); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Blasco, J.M. (J. M.); Baker, K.S. (Kate S.); Moreno, E. (Edgardo); Ruíz-Villalobos, N. (Nazareth); Guzman-Verri, C. (Caterina); RaquelMembers of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as “Brucella melitensis biovar 2.” Further molecular typing, identified the strain as an atypical “Brucella suis.” Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella clade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution.
- Rev1 wbdR tagged vaccines against Brucella ovis(Springer Science and Business Media LLC, 2019) Miguel, M.J. (María Jesús) de; Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Martinez-Gomez, E. (Estrella); RaquelSheep brucellosis is a worldwide extended disease caused by B. melitensis and B. ovis, two species respectively carrying smooth or rough lipopolysaccharide. Vaccine B. melitensis Rev1 is used against B. melitensis and B. ovis but induces an anti-smooth-lipopolysaccharide response interfering with B. melitensis serodiagnosis, which precludes its use against B. ovis where B. melitensis is absent. In mice, Rev1 deleted in wbkC (Brucella lipopolysaccharide formyltransferase) and carrying wbdR (E. coli acetyl-transferase) triggered antibodies that could be diferentiated from those evoked by wild-type strains, was comparatively attenuated and protected against B. ovis, suggesting its potential as a B. ovis vaccine.