RESUMO
The possibility of expressing a homologous antigen and a heterologous antigen simultaneously in an attenuated Brucella melitensis strain was investigated. The Brucella wboA gene encoding a mannosyltransferase involved in biosynthesis of lipopolysaccharide O-antigen, and the Bacillus anthracis pag gene encoding the protective antigen (PA) were cloned into plasmid pBBR4MCS. The resulting plasmid was introduced into O-antigen deficient B. melitensis strain WRRP1 to produce strain WRSPA. Strain WRSPA produced O-antigen and a series of PA products, induced protection in BALB/c mice against challenge with B. melitensis strain 16M, but failed to protect A/J mice against challenge with B. anthracis Sterne strain.
Assuntos
Antígenos de Bactérias/biossíntese , Bacillus anthracis/imunologia , Toxinas Bacterianas/biossíntese , Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Animais , Antraz/prevenção & controle , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Vacina contra Brucelose/genética , Brucella melitensis/genética , Brucelose/prevenção & controle , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-alpha subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Deltaure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Deltaure2K (ureB and ureC in ure2 operon), strain 1330Deltaure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Deltaure1KDeltaure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Deltaure2K and 1330Deltaure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Deltaure1KDeltaure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Deltaure1K was completely killed, strain 1330Deltaure2C was partially killed, but strains 1330 and 1330Deltaure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro.
Assuntos
Brucella suis/enzimologia , Brucella suis/patogenicidade , Brucelose/microbiologia , Intestinos/microbiologia , Urease/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Brucella suis/genética , Brucella suis/crescimento & desenvolvimento , Linhagem Celular , Modelos Animais de Doenças , Deleção de Genes , Concentração de Íons de Hidrogênio , Fígado/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Baço/microbiologia , Urease/biossíntese , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
This article reports epidemiological investigations on the occurrence of brucellosis in Romania. Like in other former communist countries, data concerning epidemiology of brucellosis and published articles are very few. The epidemiology and control of brucellosis in Romania was analyzed using data made available by the Office International des Epizooties and Veterinary Service of Romania. Romania, like many other developed countries, has eradicated Brucella abortus from cattle since 1969. Brucellosis caused by Brucella melitensis has never been reported. The incidence of brucellosis in swine and sheep is very rare but still there are a few outbreaks in some regions. In 2000, the number of cases was 47 in swine and 270 cases in sheep. Vaccination against brucellosis is prohibited in Romania.
