Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

An Adolescent With Neurobrucellosis Caused by Brucella abortus Cattle Vaccine Strain RB51.

We present the case of an 18-year-old female with a 1-month history of fever, headache, and double vision, whose examination revealed papilledema and cranial nerve VI palsy. Blood cultures grew Brucella abortus cattle vaccine strain RB51, which is inherently resistant to rifampin. We discuss the management of the first known case of neurobrucellosis by this strain.

Avaliação genética das vacinas contra a brucelose bovina comercializadas no Brasil / Genetic evaluation of vaccine against bovine brucellosis marketed in Brazil

A prevenção contra infecções causadas por Brucella abortus em bovinos é realizada por meio da administração das amostras vacinais B19 e RB51. Existem relatos de que estas vacinas podem causar aborto em fêmeas vacinadas. Portanto, toda a ocorrência de aborto em animais vacinados merece um estudo aprofundado sobre a causa. No Brasil, não há registro sobre a origem das amostras B19 e RB51 utilizadas na produção das vacinas comerciais. Assim, um estudo para verificar possíveis mutações em relação às amostras referência USDA B19 e USDA RB51 de B. abortus se faz necessário, devido às amostras vacinais poderem reverter a sua virulência. Objetivou-se com este estudo caracterizar genotipicamente as amostras vacinais B19 e RB51 comercializadas no Brasil. A metodologia utilizada foi a genotipagem de genes marcadores destas amostras vacinais, por meio da amplificação pela reação em cadeia da polimerase. Os resultados obtidos permitiram a identificação do genótipo das vacinas comerciais B19 e RB51 disponíveis e utilizadas em bovinos no Brasil. A ausência de mutações nas vacinas testadas corrobora com a qualidade genética das mesmas, quanto à estabilidade dos genes analisados.

Vaccine strains B19 and RB51 are administered to cattle for prevention against infection by Brucella abortus. However, there are reports that these vaccines can cause miscarriages. Thus, every miscarriage among vaccinated animals should be thoroughly studied to determine the cause. In Brazil, there are no records on the origin of B19 and RB51 samples used in the preparation of commercial vaccines. Therefore, a study is needed to determine possible mutations in relation to the USDA reference samples of B. abortus due to the fact that vaccine samples could revert to the virulence of the disease. The aim of the present study was to perform a genotype analysis of vaccine strains B19 and RB51 used in Brazil. The methodology was based on the genotyping of marker genes of these vaccine strains by amplification using polymerase chain reaction. The results allowed the identification of the genotype of the B19 and RB51 commercial vaccine available for use on cattle in Brazil. The absence of mutations in the samples tested confirmed the genetic quality of the vaccines and stability of genes analyzed.

Asociación de patógenos bacterianos a células eucariotas: el caso de parasitismo intracelular de Brucella abortus / Asociation of bacterian pathogens in eucariotas cells

Brucella abortus es un parásito intracelular capaz de infectar una gran variedad de mamíferos incluyendo al hombre. Esta bacteria provoca su internalización en células epiteliales induciendo rearreglos locales del esqueleto celular. Una vez en el interior de la célula hospedero, Brucella reside inicialmente en un comportamiento temprano de la cascada de endocitosis/fagocitosis; sin embargo, rápidamente el patógeno se desliga desde el mecanismo de transporte intracelular y se asocia a la cascada de autofagocitosis. En los estadíos tardíos de la infección Brucella prolifera en el retículo endoplasmático de las células infectadas. Las brucelas poseen un sistema regulador de la transcripción de genes de virulencia formado por una proteína sensora de membrana y una proteína reguladora citoplasmática: este sistema de dos componentes permite a las bacterias adaptarse a los diferentes microambientes por los cuales transita durante el proceso de infección intracelular. Estas propiedades biológicas podrían favorecer el uso de Brucella abortus como modelo útil para el diseño de vacunas recombinantes. (Rev Cost Cienc Méd 1999; 20(1-2): 85-102) PALABRAS CLAVE: Brucella abortus, Parásito, Tráfico intracelular, Fagosoma, Autofagocitosis, Retículo endoplasmático, Sistema regulador, Lipopolisacárido, Péptidos catiónicos, Vacuna recombinante

Vacunas en la brucelosis humana / Vaccines in human brucellosis

Hace casi 100 años la brucelosis humana fue considerada por Zammit y Horrocks como una zoonosis, sólo 18 años después que Bruce identificó el agente etiológico de la fiebre de Malta. A lo largo de los años, el avance científico se ha visto encaminado primordialmente al control de la brucelosis animal en los aspectos de diagnóstico y vacunación. En cuanto a la enfermedad en el humano, el progreso más importante ha sido la quimioterapia, aunque en la última década el diagnóstico correcto ha sido un asunto de preocupación general. Las estrategias de erradicación de la brucelosis se divide en tres categorías: 1) Erradicación de la brucelosis animal; 2) Mejoramiento de las medidas de higiene individual y saneamiento y 3) Inmunización. Las vacunas contra la brucelosis han sido desarrolladas a lo largo de tres líneas: 1) vacunas vivas preparadas con cepas atenuadas como B. abortus cepa 19 y B. melitensis cepa Rev.1; 2) células completas inactivadas, como B. abortus cepa 45/20 y B. melitensis H-38 que se administran con adyuvante oleoso y 3) vacunas preparadas con fracciones celulares. Todas ellas han sido usadas ampliamente en el control de la brucelosis en animales y sólo en algunos casos muy particulares en humanos

[Structural changes in brucella chemical vaccine after gamma-irradiation]. / Strukturnye izmeneniia brutselleznoi khimicheskoi vaktsiny posle gamma-oblucheniia.

It was shown that gamma-irradiation of Brucella strain chemical vaccine stimulated phospholipid peroxidation therein: the content of extractable total phospholipids in the exposed vaccine decreased mainly due to diminution of phosphatidylcholines and phosphatidyl-ethanolamines. A relative content of high- and low-molecular weight protein components increased in the gamma-irradiated vaccine.

Antiviral activity of Brucella abortus preparations; separation of active components.

Injection into mice of heat-killed Brucella abortus or aqueous ether-extracted B. abortus (Bru-pel) induced a "virus-type" interferon response, with peak titers at 6.5 h. The animals also were protected against challenge with otherwise lethal doses of Semliki forest virus. Extraction of either heated B. abortus or BRU-PEL with a mixture of chloroform-methanol (2:1, vol/vol) (C-M( yielded an insoluble residue (extracted cells) and a C-M extract. Neither extracted cells nor C-M extract alone induced interferon or afforded protection against Semliki forest virus infection in mice. Full interferon-inducing and protective activity was restored when extracted cells were recombined with C-M extract. C-M extract from heat-killed Escherichia coli also was effective in restoring activity to extracted Brucella cells. Neither heat-killed E. coli nor its C-M extract was active, nor was C-M estracted E. coli recombined with the C-M extract from B. abortus. These results suggest that the interferon-inducing and antiviral protective properties of B. abortus are constituted of a C-M-extractable component that is common to B. abortus and E. coli and an unextractable component that is unique to B. abortus.

Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances.

A peptidoglycan-containing fraction called fraction P.I. (phenol insoluble), extracted from Brucella melitensis and previously described by some of us, had immunogenic and protective properties and did not produce any allergic reactions. Since it is well known that bacterial peptidoglycans studied so far have immunoadjuvant properties, the isolation of the active factor(s) of Brucella was undertaken. By successive enzymatic and chemical treatments, a new, much more purified fraction, called "4A" (approximately 5% of fraction P.I.), is obtained, retaining the same properties as P.I. and giving better protection against infection by Brucella. Immunogenicity, immunoadjuvant activity, allergizing capacity, and specific and nonspecific protective effects of fractions P.I. and 4A are compared. Chemically, fraction 4A is constituted by a lipoprotein covalently linked to peptidoglycan and by a few (lipo)proteins that could be solubilized by hot sodium dodecyl sulfate. Intrinsic properties of peptidoglycan could not be studied, but it does not seem to be essential for the activity. In conclusion, fractions P.I. and 4A are not agglutinogenic and, since fraction 4A induces better protection against infection by Brucella, it could advantageously replace fraction P.I. as a vaccine for humans.

The immunological properties of Brucella ribosomal preparations.

Ribosomes were isolated from Brucella abortus strains 19 and 45/20 by disruption of the cells followed by differential ultracentrifugation. The ribosome preparations contained 2-3 components reacting in immunodiffusion tests but were free of detectable lipopolysaccharide-protein agglutinogen. They crossreacted with antisera to Br. abortus, Br. melitensis, Br. suis and Br. ovis and elicited intradermal delayed hypersensitivity reactions in animals infected with Br. abortus, Br. melitensis or Br. suis. The ribosomes were antigenic in rabbits, guinea pigs and mice. Those from Br. abortus S19 induced agglutinins reaction with smooth brucella strains whereas those from Br. abortus 45/20 induced agglutinins reacting with rough brucella strains. Cattle vaccinated with S19 or 45/20 vaccines or infected with Br. abortus developed pricipitins to ribosomal components at an early stage in the immune response.