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Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.

Brucella ovis is a non-zoonotic Brucella species lacking specific vaccine. It presents a narrow host range, a unique biology relative to other Brucella species, and important distinct surface properties. To increase our knowledge on its peculiar surface and virulence features, and seeking to develop a specific vaccine, multiple mutants for nine relevant cell-envelope-related genes were investigated. Mutants lacking Omp10 plus Omp19 could not be obtained, suggesting that at least one of these lipoproteins is required for viability. A similar result was obtained for the double deletion of omp31 and omp25 that encode two major surface proteins. Conversely, the absence of major Omp25c (proved essential for internalization in HeLa cells) together with Omp25 or Omp31 was tolerated by the bacterium. Although showing important in vitro and in vivo defects, the Δomp10Δomp31Δomp25c mutant was obtained, demonstrating that B. ovis PA survives to the simultaneous absence of Omp10 and four out seven proteins of the Omp25/Omp31 family (i.e., Omp31, Omp25c, Omp25b, and Omp31b, the two latter naturally absent in B. ovis). Three multiple mutants were selected for a detailed analysis of virulence in the mouse model. The Δomp31Δcgs and Δomp10Δomp31Δomp25c mutants were highly attenuated when inoculated at 106 colony forming units/mouse but they established a persistent infection when the infection dose was increased 100-fold. The Δomp10ΔugpBΔomp31 mutant showed a similar behavior until week 3 post-infection but was then totally cleared from spleen. Accordingly, it was retained as vaccine candidate for mice protection assays. When compared to classical B. melitensis Rev1 heterologous vaccine, the triple mutant induced limited splenomegaly, a significantly higher antibody response against whole B. ovis PA cells, an equivalent memory cellular response and, according to spleen colonization measurements, better protection against a challenge with virulent B. ovis PA. Therefore, it would be a good candidate to be evaluated in the natural host as a specific vaccine against B. ovis that would avoid the drawbacks of B. melitensis Rev1. In addition, the lack in this attenuated strain of Omp31, recognized as a highly immunogenic protein during B. ovis infection, would favor the differentiation between infected and vaccinated animals using Omp31 as diagnostic target.

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Brucella ovis is a facultative intracellular bacterium that causes a non-zoonotic ovine brucellosis mainly characterized by male genital lesions and is responsible for important economic losses in sheep farming areas. Studies about the virulence mechanisms of Brucella have been mostly performed with smooth (bearing O-polysaccharide in lipopolysaccharide) zoonotic species, and those performed with B. ovis have revealed similarities but also relevant differences. Except for few strains recently isolated from unconventional hosts, Brucella species are non-motile but contain the genes required to assemble a flagellum, which are organized in three main loci of about 18.5, 6.4, and 7.8 kb. Although these loci contain different pseudogenes depending on the non-motile Brucella species, smooth B. melitensis 16M builds a sheathed flagellum under particular culture conditions and requires flagellar genes for virulence. However, nothing is known in this respect regarding other Brucella strains. In this work, we have constructed a panel of B. ovis PA mutants defective in one, two or the three flagellar loci in order to assess their role in virulence of this rough (lacking O-polysaccharide) Brucella species. No relevant differences in growth, outer membrane-related properties or intracellular behavior in cellular models were observed between flagellar mutants and the parental strain, which is in accordance with previous results with B. melitensis 16M single-gene mutants. However, contrary to these B. melitensis mutants, unable to establish a chronic infection in mice, removal of the three flagellar loci in B. ovis did not affect virulence in the mouse model. These results evidence new relevant differences between B. ovis and B. melitensis, two species highly homologous at the DNA level and that cause ovine brucellosis, but that exhibit differences in the zoonotic potential, pathogenicity and tissue tropism.

Brucella ovis is a non-zoonotic bacterium causing contagious epididymitis and other genital lesions in rams and responsible for significant economic losses in sheep-breeding areas. It is a naturally rough (without O-chains in the lipopolysaccharide) Brucella species whose virulence mechanisms have been less explored than those of zoonotic smooth brucellae (bearing O-chains that mask other outer membrane molecules). Considering the rough nature of Brucella ovis, the influence of surface components other than O-chains on its biological properties may be greater than in smooth Brucella species. Here we describe the construction and characterization of the mucR deletion mutant of virulent B. ovis PA, which is defective in a transcriptional regulator, affecting surface properties and virulence in smooth brucellae. This mutant showed increased amounts of three proteins identified as HdeA (acid-activated chaperone), Omp25d (outer membrane protein undetectable in the parental strain), and BOV_A0299 (hypothetical protein of unknown function). This observation correlated with the enhanced transcription of the corresponding genes and constitutes the first report on this type of proteome alteration in Brucella ΔmucR mutants. The upstream regions of the three genes contained AT rich domains with T-A steps described as binding sites for MucR in the Brucella abortus 2308 babR promoter (gene also upregulated in B. ovis ΔmucR), which suggests that hdeA, omp25d, and BOV_A0299 expression could be repressed by MucR through a direct binding to their promoter regions. Relative quantification of transcripts of several other genes selected according to the transcriptome of smooth brucellae ΔmucR mutants revealed not only similarities but also relevant differences among strains, such as those detected in flagellar and virB genes. Periplasmic HdeA has been related to the resistance of B. abortus to acidic pH, conditions encountered by Brucella inside phagocytes, but the deletion of hdeA in B. ovis PA and the ΔmucR mutant did not modify any of the evaluated properties of these strains. The B. ovis PA ΔmucR and ΔmucRΔhdeA mutants had defective in vitro growth and altered surface properties and architecture, exemplified by detectable amounts of Omp25d. Moreover, they showed virulence attenuation but established persistent splenic infection in mice, which encourages their evaluation as specifical attenuated vaccines against B. ovis.

Background and Aims Renin-angiotensin system inhibitors, diuretics and non-steroidal anti-inflammatory drugs (NSAIDs) is known as Triple Whammy (TW), a common combination used in hypertensive patients suffering from pain or inflammation; this combination of drugs can cause in some cases acute kidney injury (AKI). The renal outcome of the TW therapy seems to be influenced by additional factors, as the incidence of TW-induced AKI ranges from <1% to up to 22% depending on the study. The incidence of TW-AKI is higher among older adults, therefore age and other clinical conditions such as dehydration could be associated with TW-AKI. Furthermore, loss of functional nephrons in chronic kidney disease is a risk or predisposing factor for new AKI episodes. Consequently, the aim of our study was to analyse in vivo the influence of dehydration, age, renal mass reduction and hypertension (HTA) in TW therapy-induced AKI. Method Five experimental groups were subjected to TW therapy: (1) control group (3 months old Wistar rats); (2) dehydrated rats (3 months old Wistar rats with 60-70% water restriction); (3) aged rats (16 month old Wistar rats); (4) renal mass reduction (RMR) rats (3 months old Wistar rats with a 5/6 reduction of renal mass one month before the experiment); and (5) 3 months old spontaneously hypertensive rats). All rats received antihypertensive double therapy with Trandolapril in water and Furosemide i.p. for 4 days, and then a triple therapy for 2 days with Trandolapril and Ibuprofen in drinking water and Furosemide i.p. Blood and urine samples were collected at baseline (B), after 4 days of double therapy (day 4) and two days after triple therapy (day 6). Renal outcomes of the TW therapy were analysed by plasma creatinine, creatinine clearance, plasma urea and proteinuria. Results Risk factors such as dehydration or the loss of nephrons significantly increase plasma creatinine and urea and reduce creatinine clearance at day 6 compared to 3 months old Wistar rats without risk factors. Creatinine clearance is significantly reduced at day 4 in RMR and dehydration groups. Also, the RMR group shows at baseline and day 4 decreased creatinine clearance and increased plasma creatinine and urea than 3 months old Wistar rats. Proteinuria values are within normal ranges (< 10mg/day) in all experimental groups. Conclusion Renal mass reduction and dehydration are important risk factors in the development of AKI after TW therapy, while hypertension and age do not seem to influence renal outcomes. The knowledge of the specific conditions and the different factors that increase the risk of developing AKI in patients undergoing TW therapy will help us to stratify these patients based on their individual risk and to apply in each case the appropriate individualized treatment. This research was funded by grants from Instituto de Salud Carlos III (ISCIII) PI21/00548 and PI21/01226 co-funded by the European Union and Red de Investigación Renal RICORS2040 (Kidney Disease), RD21/0005/0004 co-funded by the European Union – NextGenerationEU, Mecanismo para la Recuperación y la Resiliencia (MRR). Eva M. Baranda-Alonso is recipient of a predoctoral fellowship from the Junta de Castilla y Leon (Spain) and the European Social Fund from the European Commission.Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

Background and Aims Renal frailty (RF) is a premorbid and, at least partly, modifiable condition arising from diminished renal functional reserve and defective adaptive response capacity predisposing to acute kidney injury (AKI). RF ensues from subclinical wear or distortion of the renal haemodynamic and tubular homeostatic responses that defend the renal excretory function from supervening circumstances (i.e., stressors such as drugs). In this work, we aimed to study the impact of RF generated by the combined effect of the hydration state, hypertension and ageing on the nephrotoxicity developed by a low dose of cisplatin in the rat. Method Young Wistar rats, young spontaneously hypertensive rats (SHR), and aged Wistar rats were used. Rats in each group were randomly subject to four experimental conditions: - Control: rats with ad libitum water intake, receiving vehicle (0.9% NaCl, i.p). - Cisplatin: rats with ad libitum water intake, treated with 2.5 mg/kg, i.p. cisplatin. - Water deprivation: rats deprived of water for 48 h, receiving vehicle (0.9% NaCl, i.p.). - Water deprivation + cisplatin: rats deprived of water for the 48 h prior to 2.5 mg/kg i.p. cisplatin administration. Tail vein blood and 24-hour urine samples were collected at basal time (B), after water deprivation (D0), and 4 days after cisplatin/vehicle administration (D4, day of maximum kidney damage). Haematocrit was measured and plasma osmolality, plasma creatinine (Jaffe reaction) and urea (Jung method) concentrations were determined. Urine samples were analysed for volume (urinary flow), osmolality, and proteinuria (Bradford method). Body fluid composition was measured by bioimpedance spectroscopy with an ImpediVETTM VetBIS1 device. Results All rats showed clear signs of dehydration after 48 hours of water deprivation: weight loss, increases in plasma osmolality and haematocrit (more pronouncedly in young Wistar and SHR than in aged animals), and reduced flow of a highly concentrated urine (i.e., with elevated osmolality). Bioimpedance analysis revealed a parallel loss of intracellular and extracellular water and net loss of total body water (explaining most of weight loss), with no changes in the liquid distribution between the intracellular and extracellular compartments. Dehydration was reverted after 4 days of rehydration in vehicle-treated rats, and no alteration of renal function was observed due to water deprivation. Administration of cisplatin in young, normohydrated Wistar rats showed minimal loss of renal function that was not worsened by water deprivation. Hypertensive rats showed completely normal renal function when cisplatin was administered under normohydration. Interestingly, however, dehydrated SHR did suffer a cisplatin-induced AKI (significant increases in plasma creatinine, plasma urea and proteinuria). In aged Wistar rats, cisplatin compromised renal function (as supported by increases in plasma creatinine and urea concentration), and this effect was significantly amplified by water deprivation. Conclusion This study suggests that dehydration in fully competent young rats is not in itself a significant risk factor for AKI, while the combination of dehydration and hypertension in young rats significantly increases the risk of AKI. Furthermore, ageing poses animals in a state of frailty that renders their kidneys unable to respond to stressors (such as a low dose of cisplatin). Frailty is boldly worsened in dehydrated aged animals. In perspective, models reproducing conditions of increased vulnerability to AKI provide useful tools to search for biomarkers pre-emptively predicting undesired health outcomes before exposure to stressors, and for developing preventing strategies. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Consejería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.