RESUMEN
The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.
Asunto(s)
Bacterias/aislamiento & purificación , Bacterias/metabolismo , Camélidos del Nuevo Mundo/microbiología , Fibras de la Dieta , Estómago/microbiología , Animales , Digestión , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease. RESULTS: In this work we present structural information as well as insights into the in vivo function of YqiC, a 99-residue protein of S. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S. Typhimurium yqiC deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures in vitro, although it was still able to invade and replicate inside epithelial and macrophages cell lines. CONCLUSION: This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/patogenicidad , Factores de Virulencia/metabolismo , Animales , Membrana Celular/química , Citoplasma/química , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos BALB C , Enfermedades de los Roedores/microbiología , Enfermedades de los Roedores/mortalidad , Salmonelosis Animal/microbiología , Salmonelosis Animal/mortalidad , Salmonella typhimurium/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Análisis de Supervivencia , VirulenciaRESUMEN
BACKGROUND AND AIM: Nitrate (NO3 -) reduces enteric methane emissions and could be a source of non-protein nitrogen in ruminant feeds. Nonetheless, it has a potential toxic effect that could compromise animal health and production. The purpose of this study was to determine the effects of progressive inclusion of NO3 - in the diet on the hematological, biochemical, and blood gases parameters, in turn, the effects on feed intake and live weight gain (LWG) in Holstein calves. MATERIALS AND METHODS: Eighteen Holstein heifers and steers (nine animals/treatment) were maintained in individual pens for 45 days. Animals were randomly allocated to either a control or nitrate diet (ND) (containing 15 g of NO3 -/kg of dry matter [DM]). The biochemical parameters and blood gases were analyzed only in the NO3 - group on days: -1, 1, 7, 13, 19, and 25 corresponding to 0, 20, 40, 60, 80, and 100% of the total inclusion of NO3 - in the diet, respectively. In addition, DM intake (DMI) and LWG were evaluated among dietary treatments. RESULTS: Feeding the ND did not influence DMI or LWG (p>0.05). Methemoglobin (MetHb) and deoxyhemoglobin increased according to the NO3 - concentrations in the diet (p<0.05), while an opposite effect was observed for oxyhemoglobin and carboxyhemoglobin (p<0.05). Hematocrit levels decreased (p<0.05), while albumin, alanine aminotransferase, and gamma-glutamyl transpeptidase concentrations were not modified (p>0.05). However, glucose, urea, aspartate aminotransferase (AST), and retinol concentrations increased (p<0.05) according to the NO3 - concentrations in the diet. CONCLUSION: This study confirmed that the progressive inclusion of 123 g of NO3 -/animal/day in the diet could be safe without affecting DMI and LWG of Holstein calves. In turn, a dose-response effect of the MetHb, glucose, urea, AST, and retinol was observed, but these values did not exceed reference values. These results highlighted the importance of using a scheme of progressive inclusion of NO3 - in the diet of calves to reduce the risks of NO3 - toxicity.
RESUMEN
The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis DeltabepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the DeltabepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the DeltabepE DeltabepG double mutant showed a more severe phenotype than the DeltabepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella suis/efectos de los fármacos , Farmacorresistencia Bacteriana , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Brucella suis/patogenicidad , Brucella suis/fisiología , Ácido Desoxicólico/farmacología , Células Epiteliales/microbiología , Etidio/farmacología , Eliminación de Gen , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Violeta de Genciana/farmacología , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , VirulenciaRESUMEN
Phenotypic differences between Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogen sulfide production, for example, is a trait exclusive to C. fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.
RESUMEN
LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.
Asunto(s)
Brucella/genética , Mycobacterium avium subsp. paratuberculosis/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Brucella/aislamiento & purificación , Brucella/patogenicidad , Brucelosis/diagnóstico , Brucelosis/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Paratuberculosis/diagnóstico , Paratuberculosis/microbiología , Sensibilidad y EspecificidadRESUMEN
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Asunto(s)
Brucella/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Técnicas Bacteriológicas/métodos , Brucella/genética , Brucelosis/diagnóstico , Cartilla de ADN/genética , Humanos , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculosis/diagnóstico , Factores de TiempoRESUMEN
Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis.
Asunto(s)
Brucella abortus/genética , Brucella abortus/patogenicidad , Proteínas de la Membrana/genética , Animales , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/normas , Brucella abortus/inmunología , Brucelosis/diagnóstico , Brucelosis/inmunología , Brucelosis/prevención & control , Farmacorresistencia Bacteriana/genética , Femenino , Silenciador del Gen , Marcadores Genéticos , Inmunización , Luciferasas/análisis , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Plásmidos/genética , VirulenciaRESUMEN
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Asunto(s)
Animales , Humanos , Brucella/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Bacteriológicas/métodos , Brucella/genética , Brucelosis/diagnóstico , Cartilla de ADN/genética , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculosis/diagnóstico , Factores de TiempoRESUMEN
Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella abortus/enzimología , Brucelosis/inmunología , Transferasas de Hidroximetilo y Formilo/metabolismo , Macrófagos/inmunología , Animales , Células de la Médula Ósea/inmunología , Brucella abortus/genética , Brucella abortus/inmunología , Brucella abortus/patogenicidad , Brucelosis/microbiología , Transferasas de Hidroximetilo y Formilo/genética , Interleucina-12/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Factor de Necrosis Tumoral alfa/inmunología , Vacunas Atenuadas/inmunología , VirulenciaRESUMEN
Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other alpha-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.
Asunto(s)
Antiinfecciosos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella suis/efectos de los fármacos , Brucella suis/patogenicidad , Resistencia a Medicamentos/genética , Animales , Clonación Molecular , Femenino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Filogenia , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Alphaproteobacteria/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/farmacología , Secuencia de Bases , Brucella abortus/genética , Brucella abortus/inmunología , Brucelosis/inmunología , Brucelosis/prevención & control , ADN Bacteriano/genética , Femenino , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/genética , VirulenciaRESUMEN
The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.