RESUMO
Babesia bovis is the causative agent of babesiosis, a tick-borne disease that is a major cause of loss to livestock production in Latin America. Vaccination against Babesia species represents a major challenge against cattle morbidity and mortality in enzootic areas. The aim of this study was to evaluate the capacity of Bacille Calmette-Guerin (BCG) to deliver the rhoptry associated protein (RAP-1) antigen of B. bovis and to stimulate specific cellular and humoral immune responses in mice. Two of five mycobacterial expression vectors efficiently expressed the antigen. These constructs were subsequently studied in vivo following three immunization protocols. The construct with the greatest in vivo stability proved to be the one that induced the strongest immune responses. Our data support the hypothesis that specific T lymphocyte priming by rBCG can be employed as a component of a combined vaccine strategy to induce long-lasting humoral and cellular immune responsiveness towards B. bovis and encourage further work on the application of rBCG to the development of Babesia vaccines.
Assuntos
Babesia bovis/genética , Vacinas Bacterianas/genética , Técnicas de Transferência de Genes , Mycobacterium bovis/genética , Proteínas de Protozoários/biossíntese , Animais , Babesia bovis/imunologia , Vacinas Bacterianas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Combinadas/genética , Vacinas Combinadas/imunologiaRESUMO
P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mutação/genética , Mycobacterium bovis/genética , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.
Assuntos
Bacteriófagos/classificação , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/virologia , Microbiologia do Solo , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , DNA Viral/análise , Engenharia Genética , Lisogenia , Microscopia Eletrônica , Dados de Sequência Molecular , Rhizobium leguminosarum/crescimento & desenvolvimento , Análise de Sequência de DNA , Transdução GenéticaRESUMO
Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.
Assuntos
Genes de RNAr , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Microbiologia do Solo , Genes Bacterianos , Dados de Sequência Molecular , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Filogenia , Análise de Sequência de DNARESUMO
This study examined the effects of NH(4)NO(3) fertilizer on the size and activity of nitrifying, autotrophic, ammonia-oxidizing populations of the beta subdivision of the class Proteobacteria in arable soils. Plots under different long-term fertilizer regimes were sampled before and after NH(4)NO(3) additions, and the rates of nitrification were determined by (15)N isotopic pool dilution assays. Ammonia-oxidizing populations in the plots were quantified by competitive PCR assays based on the amoA and ribosomal 16S genes. Prior to fertilizer addition, ammonium concentrations and nitrification rates in the plots were comparatively low; ammonia-oxidizing populations were present at 10(4) to 10(5) gene copies g of soil(-1). Three days after the application of fertilizer, nitrification rates had risen considerably but the size of the ammonia-oxidizing population was unchanged. Six weeks after fertilizer treatment, ammonium concentrations and nitrification rates had fallen while the ammonia-oxidizing populations in plots receiving fertilizer had increased. The rapidity of the rise in nitrification rates observed after 3 days suggests that it results from phenotypic changes in the ammonia-oxidizing bacterial population. Associated increases in population sizes were only observed after 6 weeks and did not correlate directly with nitrifying activity. Phylogenetic analyses of PCR products from one of the plots revealed a population dominated by Nitrosospira-type organisms, similar to those prevalent in other soils.
Assuntos
Betaproteobacteria/metabolismo , Fertilizantes , Compostos de Amônio Quaternário/metabolismo , Microbiologia do Solo , Betaproteobacteria/efeitos dos fármacos , Betaproteobacteria/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Genes de RNAr , Dados de Sequência Molecular , Radioisótopos de Nitrogênio/metabolismo , Oxirredução , Oxirredutases/genética , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genéticaRESUMO
Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.
Assuntos
Engenharia Genética , Reação em Cadeia da Polimerase/métodos , Rhizobium/genética , Microbiologia do Solo , Antibacterianos/farmacologia , Centrifugação com Gradiente de Concentração , Sondas de DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Genes Bacterianos/genética , Sensibilidade e EspecificidadeRESUMO
An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II (nptII) promoter of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.
