Moriyon, I. (Ignacio)

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    Brucellosis and one health: inherited and future challenges
    (2023) Moriyon, I. (Ignacio); De-Massis, F. (Frabizio); Letesson, J.J. (Jean Jacques); Blasco, J.M. (J. M.); Moreno, E. (Edgardo)
    One Health is the collaborative efforts of multiple disciplines to attain optimal health for people, animals and the environment, a concept that historically owes much to the study of brucellosis, including recent political and ethical considerations. Brucellosis One Health actors include Public Health and Veterinary Services, microbiologists, medical and veterinary practitioners and breeders. Brucellosis awareness, and the correct use of diagnostic, epidemiological and prophylactic tools is essential. In brucellosis, One Health implementation faces inherited and new challenges, some aggravated by global warming and the intensification of breeding to meet growing food demands. In endemic scenarios, disease awareness, stakeholder sensitization/engagement and the need to build breeder trust are unresolved issues, all made difficult by the protean characteristics of this zoonosis. Extended infrastructural weaknesses, often accentuated by geography and climate, are critically important. Capacity-building faces misconceptions derived from an uncritical adoption of control/eradication strategies applied in countries with suitable means, and requires additional reference laboratories in endemic areas. Challenges for One Health implementation include the lack of research in species other than cattle and small ruminants, the need for a safer small ruminant vaccine, the need to fill in the infrastructure gap, the need for realistic capacity-building, the creation of reference laboratories in critical areas, and the stepwise implementation of measures not directly transposed from the so-called developed countries.
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    Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp
    (Society for General Microbiology, 1998) Lopez-Goñi, I. (Ignacio); Moriyon, I. (Ignacio); Leiva, J. (José); Diaz, R. (Ramón); Romero, C. (Conchi); Velasco, J. (Julián)
    The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331T, a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster. The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).
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    New antiseptic peptides to protect against endotoxin-mediated shock
    (American Society for Microbiology, 2010) Howe, J. (Jörg); Bartels, R. (Rainer); Brandenburg, K. (Klaus); Kowalski, I. (Ina); Moriyon, I. (Ignacio); Kaconis, Y. (Yani); Gutsmann, T. (Thomas); Sánchez-Gómez, S. (Susana); Rossle, M. (Manfred); Martinez-de-Tejada, G. (Guillermo); Razquin-Olazaran, I. (Iosu); Schürholz, T. (Tobias); Hornef, M. (Mathias)
    Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.
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    Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo
    (American Society for Microbiology, 2011) Brandenburg, K. (Klaus); Moriyon, I. (Ignacio); Leiva, J. (José); Jerala, R. (Roman); Sánchez-Gómez, S. (Susana); Fernández-Alonso, M. (Miriam); Andrä, J. (Jörg); Martinez-de-Tejada, G. (Guillermo); Blondelle, S.E. (Sylvie E.); Lohner, K. (Karl); Japelj, B. (Bostjan)
    Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.
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    Spontaneous excision of the O-polysaccharide wbkA glycosyltranferase gene is a cause of dissociation of smooth to rough Brucella colonies
    (American Society for Microbiology, 2012) Lopez-Goñi, I. (Ignacio); Mancilla, M. (Marcos); Moriyon, I. (Ignacio); Marin, C.M. (C. M.); Zarraga, A.M. (Ana María); Blasco, J.M. (J. M.)
    The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.
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    Raising the political profile of the neglected zoonotic diseases: three complementary European commission-funded projects to streamline research, build capacity and advocate for control
    (Public Library of Science, 2015) Berkvens, D. (Dirk); Okello, A.L. (Anna L.); Beange, I. (Iona); Moriyon, I. (Ignacio); Integrated Control of Neglected Zoonoses, (ICONZ); Gabriël, S. (Sarah); Welburn, S.C. (Susan C.); Mukaratirwa, S. (Samson); Bardosh, K. (Kevin); Saarnak, C. (Christopher); Shaw, A. (Alexandra); Johansen, M.V. (Maria Vang)
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    Release of outer membrane fragments by exponentially growing Brucella melitensis cells
    (American Society for Microbiology, 1987) Moriyon, I. (Ignacio); Gamazo, C. (Carlos)
    Rough and smooth strains of Brucella melitensis released a membranous material that was devoid of detectable NADH oxidase and succinic dehydrogenase activity (cytoplasmic membrane markers) but that contained lipopolysaccharide, proteins, and phospholipids. This material was composed of two fractions that had similar chemical compositions but that were of different sizes which were separated by differential ultracentrifugation. Electron microscopy showed that both fractions are made of unit membrane structures. The membrane fragments were released during the exponential phase of growth, and no leakage of malic dehydrogenase activity (cytosol marker) was detected. Thus, the fragments were unlikely a result of cell lysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that, although group 2 Brucella outer membrane proteins and lipoprotein were not detected, the proteins in the membranous material were outer membrane proteins. Gas-liquid chromatography analysis showed a similar fatty acid profile for the cell envelope and the outer membrane fragments of the smooth strain B. melitensis 16M. In contrast, the outer membrane fragments from the rough 115 strain were enriched in palmitic and stearic acids. With respect to the unfractionated cell envelope, outer membrane fragments were enriched in phosphatidylcholine, a phospholipid that is unusual in bacterial membranes.
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    Enzyme-linked immunosorbent assay with Brucella native hapten polysaccharide and smooth lipopolysaccharide
    (American Society for Microbiology, 1988) Alonso-Urmeneta, B. (Begoña); Moriyon, I. (Ignacio); Diaz, R. (Ramón); Blasco, J.M. (J. M.)
    Brucella melitensis native haptens (NH) are polysaccharides identical to the O-side chain of the smooth lipopolysaccharide (S-LPS) (E. Moreno, H. Mayer, and I. Moriyón, Infect. Immun. 55:2850-2853, 1987) which precipitate with sera from infected cattle but not from strain 19-vaccinated cattle. In the present work, NH was extracted by the hot-water method (R. Díaz, J. Toyos, M.D. Salvo, and M.L. Pardo, Ann. Rech. Vet. 12:35-39, 1981) and purified free of S-LPS and protein. Purified NH lacked the ability to coat polystyrene and sheep erythrocytes. In contrast, NH acylated with stearoyl chloride bound to both polystyrene and erythrocytes. By hemagglutination and enzyme-linked immunosorbent assay (ELISA), S-LPS and acylated NH gave similar results with blood sera from brucellosis-free, strain 19-vaccinated, and infected cattle. Moreover, a significant correlation between the results of NH ELISA and S-LPS ELISA was demonstrated with milk sera. However, in a competitive ELISA with milk sera, S-LPS in the liquid phase abrogated the binding of antibodies to acylated NH adsorbed to polystyrene, while NH in the liquid phase did not influence the binding of antibodies to polystyrene-adsorbed S-LPS. It is hypothesized that the different precipitations of NH and S-LPS with sera from infected or strain 19-vaccinated cattle are due to differences in the affinity of the antibodies produced upon vaccination or infection and in the physical state of aggregation of NH and S-LPS in aqueous solutions.
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    Specificity dependence between serological tests for diagnosing bovine brucellosis in Brucella-free farms showing false positive serological reactions due to Yersinia enterocolitica O:9
    (Canadian Veterinary Medical Association, 2005) Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Marin, C.M. (C. M.); Muñoz, P. (Pilar); Blasco, J.M. (J. M.); Mainar, R. (Raúl); Grillo, M.J. (María Jesús)
    When brucellosis false positive serological reactions happen in cattle, the serial use of pairs of specificity-correlated serological tests (rose bengal, complement fixation, competitive ELISA) results in specificities lower than expected. In this situation, highly specific tests, such as the indirect ELISA used alone, may be more adequate than serial testing.
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    The epitopic and structural characterization of Brucella suis biovar 2 O-polysaccharide demonstrates the existence of a new M-negative C-negative smooth Brucella serovar
    (Public Library of Science, 2013) Cloeckaert, A. (Axel); Ali, T. (Tara); Moriyon, I. (Ignacio); Zaccheus, M.V. (Mona V.); Widmalm, G. (Göran); Zygmunt, M. (Michel); Iriarte-Cilveti, M. (Maite); Weintraub, A. (Andrej)
    The brucellae are Gram-negative bacteria that cause an important zoonosis. Studies with the main Brucella species have shown that the O-antigens of the Brucella smooth lipopolysaccharide are α-(1 → 2) and α-(1 → 3)-linked N-formyl-perosamine polysaccharides that carry M, A and C (A = M, A>M and AA) and M specificities. However, the biovar 2 O-antigen bound monoclonal antibodies to the Brucella A epitope, and to the C/Y epitope shared by brucellae and Yersinia enterocolitica O:9, a bacterium that carries an N-formyl-perosamine O-antigen in exclusively α-(1 → 2)-linkages. By (13)C NMR spectroscopy, B. suis biovar 1 but not B. suis biovar 2 or Y. enterocolitica O:9 polysaccharide showed the signal characteristic of α-(1 → 3)-linked N-formyl-perosamine, indicating that biovar 2 may altogether lack this linkage. Taken together, the NMR spectroscopy and monoclonal antibody analyses strongly suggest a role for α-(1 → 3)-linked N-formyl-perosamine in the C (A = M) and C (M>A) epitopes. Moreover, they indicate that B. suis biovar 2 O-antigen lacks some lipopolysaccharide epitopes previously thought to be present in all smooth brucellae, thus representing a new brucella serovar that is M-negative, C-negative. Serologically and structurally this new serovar is more similar to Y. enterocolitica O:9 than to other brucellae.