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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.

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.

Nigeria is the most populous country in Africa, has a large proportion of the world’s poor livestock keepers, and is a hotspot for neglected zoonoses. A review of the 127 accessible publications on brucellosis in Nigeria reveals only scant and fragmented evidence on its spatial and temporal distribution in different epidemiological contexts. The few bacteriological studies conducted demonstrate the existence of Brucella abortus in cattle and sheep, but evidence for B. melitensis in small ruminants is dated and unclear. The bulk of the evidence consists of seroprevalence studies, but test standardization and validation are not always adequately described, and misinterpretations exist with regard to sensitivity and/or specificity and ability to identify the infecting Brucella species. Despite this, early studies suggest that although brucellosis was endemic in extensive nomadic systems, seroprevalence was low, and brucellosis was not perceived as a real burden; recent studies, however, may reflect a changing trend. Concerning human brucellosis, no studies have identified the Brucella species and most reports provide only serological evidence of contact with Brucella in the classical risk groups; some suggest brucellosis misdiagnoses as malaria or other febrile conditions. The investigation of a severe outbreak that occurred in the late 1970s describes the emergence of animal and human disease caused by the settling of previously nomadic populations during the Sahelian drought. There appears to be an increasing risk of reemergence of brucellosis in sub-Saharan Africa, as a result of the co-existence of pastoralist movements and the increase of intensive management resulting from growing urbanization and food demand. Highly contagious zoonoses like brucellosis pose a threat with far-reaching social and political consequences.

Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48 h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-gamma, TLR9, Myd88, T gamma delta and TNF-beta favor Brucella replication; whereas IL-1 beta, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-gamma and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (>= 10(8) bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella beta 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella beta 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella beta 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

La brucelosis es una de las zoonosis bacterianas más importantes y afecta a mamíferos terrestres y marinos. El lipopolisacárido de Brucella tiene un papel crucial en la virulencia y es considerado como un patrón asociado a patógeno alterado. En este trabajo, hemos investigado la función de dos hipotéticas glicosil transferasas de lipopolisacárido [BAB1_0351 (wadB) y BAB1_1620] y de una hipotética etanolaminafosfato transferasa (BMEI0118). El mutante wadB en B. abortus (BABwadB), B.melitensis y B. suis biovar 2 carece de parte del núcleo del lipopolisacárido, pero mantiene intacta la cadena O. BABwadB es más sensible al suero y a los péptidos bactericidas y está atenuado en células dendríticas y en ratones. Además, es capaz de inducir una respuesta inmune protectora. Por otro lado, BAB1_1620, una glicosil transferasa relacionada con otras implicadas en la síntesis del lipopolisacárido, parece no estar relacionada con este proceso en B. abortus. Así mismo, su función no es esencial para la virulencia en ratones. Por otra parte, hemos demostrado que la proteína codificada por BMEI0118 (BMElptA) actúa como una transferasa que incorpora etanolamina-fosfato al lípido A. Esta modificación contribuye al bajo reconocimiento del lipopolisacárido por parte de algunos efectores del sistema inmune innato, ya que el mutante en BMELptA es más sensible al efecto bactericida del suero normal y a los péptidos catiónicos. Esta ORF presenta ortólogos en otras -2 Proteobacteria como Ochrobactrum, Sinorhizobium y Agrobacterium. La interferencia de la vacunación en el diagnóstico es un aspecto crucial para el control de la brucelosis, ya que los anticuerpos generados, dirigidos principalmente frente al lipopolisacárido, son comunes a animales infectados y vacunados. La expresión en Brucella de wbdR (acetil transferasa de cadena O en E. coli O 157:H7) lleva a la incorporación de residuos acetilo a la perosamina, generando un nuevo epítopo inmunogénico. Esta modificación posibilitará la elaboración de un nuevo test diagnóstico. Brucellosis is a disease of terrestrial and marine mammals and an important zoonosis. Brucella lipopolysaccharide plays a major role in virulence and is considered as a molecule with a modified pathogen-associated molecular pattern. The role of two hypothetical lipopolysaccharide core glycosyltransferases [BAB1_0351 (wadB) and BAB1_1620] and a putative phosphoethanolamine transferase (BMEI0118) was investigated. A wadB mutant in B. abortus, B. melitensis and B. suis biovar 2 lacked part of the lipopolysaccharide core but kept the O-chain. The wadB B. abortus mutant was sensitive to normal serum and bactericidal peptides and attenuated in dendritic cells and mice. Moreover, it elicited a protective immunoresponse, confirming that core mutants are promising brucellosis vaccines. On the other hand, BAB1_1620, a glycosyltransferase related to others implicated in lipopolysaccharide synthesis, seems not to be related to this process in B. abortus and its function is not essential for full virulence in mice. It is demostrated that BMELptA acts as a transferase that incorporate phosphoethanolamine to the lipid A section. This modification contributes to low recognition of lipopolysaccharide by some innate immune effectors since BMELptA mutant was sensitive to normal serum and bactericidal peptides. BMELptA has orthologous in other -2 Proteobacteria such as Ochrobactrum, Sinorhizobium or Agrobacterium. A crucial aspect in the control of brucellosis is that the use of S-brucellae vaccines interferes in the diagnosis, since they generate antibodies against the O-chain of the lipopolysaccharide, which is also the dominant antigen in infected animals. In order to bypass this difficulty, we have followed a new approach. The expression of wbdR (E. coli O 157:H7 O chain acetyltransferase) in B. abortus 2308 leads to the incorporation of acetyl residues to the perosamine polysaccharide, creating a new immunogenic epitope that could be the base for the elaboration of new diagnostic tests.

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB(core) to distinguish it from the wbk manB(O-Ag). The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

Yersinia enterocolitica serotype 9 contained an antigenic component giving a reaction of total identity with Brucella native hapten and polysaccharide B. This component was present in a phenol-water extract (fraction 5; M. Redfearn, Ph.D. Thesis, University of Wisconsin, Madison, 1960) along with the smooth lipopolysaccharide. The native hapten could be purified free of lipopolysaccharide and proteins by gel filtration.

Los efectos del régimen hídrico y de distintos niveles de fertilidad sobre el rendimiento, calidad y tamaño del tomate de invierno. II. Características de las aguas salinas de Alicante destinadas al cultivo del tomate de invierno por V. Hernando y B. Orihuel Gasque.-- Estudio del suelo y relaciones con la vegetación en una ladera erosionada, por Estanislao Luis Calabuig y Angel Puerto Martín.-- Influencia de la simazina sobre microorganismos solubilizadores de fósforo en suelos calizos, por A. Ortuño. A. Hernansáez y J. Noguera.-- Efecto del ácido 2-hidroxifenilacético sobre la formación de raíces en esquejes de Phaseolus vulgaris L., por A. Vázquez, P. Castro y E. Vieitez.-- Estudio de la diversidad de la vegetación medida sobre un gradiente de profundidad del suelo, por .4. Puerto Martfn y J. M. Gómez Gutiérrez.-- Tierra negra andaluza (vertisol) sobre sedimentos de terrazas del Guadalquivir, por L. Clemente Salas .-- Estudios bioquímicos y fisiológicos en olivo. VI. Lípidos de hoja joven y cloroplastos aislados, por L. M. Daza y J. P. Donaire.-- Microbiological study of an andosol, by l. Moriyon, A. Martínez and A. Rodrígues-Burgos.-- Estudio de varios métodos radiométricos de determinación de fósforo en suelos, por T. Crisanto Herrero y J. Ovejero Escudero.-- Interacción del dipropilsulfóxido con montmorillonita. I. Estudio röntgenográfico y térmico, por G. Dios Cancela y S. González García.-- Relaciones entre la composición mineralógica de la fracción arcilla de algunos suelos característicos andaluces y su comportamiento frente al potasio, por L. Madrid, Sánchez del Villar, J. L. Pérez Rodríguez y R. Romero Díaz.-- Descripción automática de la vegetación. I. Análisis factorial de datos cuantitativos obtenidos en muestreos estratificado y sistemático, por F. Gonzáles Bernáldes y F. Díaz Pineda.-- Descripción automática de la vegetación. II. Análisis factorial de datos cualitativos obtenidos en muestreo estratificado, por F. Díaz Pineda y F. González Bernáldes.-- Supervivencia y conservación de la efectividad de Rhisobiwn meliloti en inóculos preparados a base de una turba neutro alcalina, por J. Romero, A. J. Palomares, F. Gallardo-Lara y J. Olivares.--Modificación del método de ataque para la determinación de C y N orgánico en suelos, por F. Guitián Rivera y G. Campillo Casais.-- Caracterización del K en los suelos españoles mediante electro-ultra-filtración, por J. A. Díez y V. Hernando.-- Suelo y vegetación de dos zonas salinas de la marisma de Lebrija (Sevilla). l. Características del substrato, por M. Chaves Sánchez y J. M. Murillo Carpio.—Notas. Federico Mayor Zaragoza, nombrado Director Adjunto de la UNESCO. Premio Osborne para la Defensa de la Naturaleza al proyecto presentado por el Dr. Arambarri.-- Nombramiento de Vicepresidentes y Secretario General del C. S. l. C.-- VII Reunión Nacional de Suelos.-- CIPAC Meeting.-- Reuniber-78.-- Actividades del Dr. Barrueco.-- Asamblea General del «Grupo Polifenoles».-- I Jornada Nacional Agrícola-- Bibliografía, 2019-08.- CopyBook.- Libnova.- Biblioteca ICA.

Trabajo presentado en el 9th European Biophysics Congress, celebrado en Lisboa del 13 al 17 de julio de 2013., Interactions between antimicrobial peptides (AMP) and bacterial membranes can be analysed by using reconstituted lipid membranes. Investigations with purified total lipids are limited and the activity of AMPs on Gram-negative membranes has been studied on few genus of the y-Proteobacteria (Proyeus, Salmonella, E, coli), that are quite sensitive.