Zuñiga-Ripa, A. (Amaia)

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    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.
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    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); Raquel
    Bovine 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.
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    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); Raquel
    Sheep 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.
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    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); Raquel
    Brucellosis, 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
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    Correction: Rev1 wbdR tagged vaccines against Brucella ovis
    (Springer Nature, 2020) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Iriarte-Cilveti, M. (Maite); Martinez-Gomez, E. (Estrella); Conde-Alvarez, R. (Raquel)
    Correction to: Vet Res (2019) 50:95
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    The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus
    (2014) Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Arce-Gorvel, V. (Vilma); Zuñiga-Ripa, A. (Amaia); Hanniffy, S. (Sean); Iriarte-Cilveti, M. (Maite); Raquel; Grillo, M.J. (María Jesús)
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    The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species
    (2021) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Sholenkamp, C. (Christian); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Lázaro-Antón, L. (Leticia); Iriarte-Cilveti, M. (Maite); Vences-Guzmán, M.A. (Miguel Ángel); Conde-Alvarez, R. (Raquel)
    The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.
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    Prevalence and risk factors of brucellosis among febrile patients attending a community hospital in south western Uganda
    (NCBI, 2018) Bagenda, F. (Fred); Bonnet, M. (Maryline); Zuñiga-Ripa, A. (Amaia); Boum, Y. (Yap); Migisha, R. (Richard); Raquel; Nyehangane, D. (Dan); Page, A.L. (Anne Laure)
    Human brucellosis, a chronic disease contracted through contact with animals and consuption of unpasteurized dairy products is underreported in limited-resource countries. This cross-sectional study aimed to determine the prevalence and risk factors of brucellosis among febrile patients attending a community hospital in South western Uganda. A questionnaire that captured socio-demographic, occupational and clinical data was administered. Blood samples were tested for Brucella antibodies using Rose Bengal Plate Test (RBPT) and blood culture with standard aerobic BACTEC bottle was done. Of 235 patients enrolled, prevalence of brucellosis (RBPT or culture confirmed) was 14.9% (95%CI 10.6-20.1) with a culture confrmation in 4.3% of the participants. The factors independently associated with brucellosis were consumption of raw milk (aOR 406.15, 95% CI 47.67-3461.69); history of brucellosis in the family (aOR 9.19, 95% CI 1.98-42.54); and selling hides and skins (aOR 162.56, 95% CI 2.86-9256.31). Hepatomegaly (p < 0.001), splenomegaly (p = 0.018) and low body mass index (p = 0.032) were more common in patients with brucellosis compared to others. Our findings reveal a high prevalence of brucellosis among febrile patients and highlight a need for implementing appropiate tests, public awareness activities and vaccination of animals to control and eliminate the disease.
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    Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model
    (2020) Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Vizcaíno, N. (Nieves); Iriarte-Cilveti, M. (Maite); Pérez-Etayo, L. (Lara); Conde-Alvarez, R. (Raquel)
    Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortal‑ ity in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specifc vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identifed, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate ⇌ phosphoenolpyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.
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    A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams
    (2022) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Vizcaíno, N. (Nieves); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Andrés-Barranco, S. (Sara); Barberan, M. (Montserrat); Iriarte-Cilveti, M. (Maite); Blasco, J.M. (J. M.); Martinez-Gomez, E. (Estrella); Conde-Alvarez, R. (Raquel)
    Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.