Evaluation of a hypervariable octameric oligonucleotide fingerprints assay for identification of and discrimination between wild-type and vaccine strains of Brucella melitensis.

OBJECTIVE To evaluate a hypervariable octameric oligonucleotide fingerprints (HOOF-Prints) assay for identification of and discrimination between wild-type and vaccine strains of Brucella melitensis. SAMPLE Brucella melitensis vaccine strain M5 and wild-type strain M43. PROCEDURES 8 pairs of primers (alterable, octameric nucleotides) were designed on the basis of a biological analysis of 8 flanking sequences in the DNA of B melitensis. The HOOF-Prints technique was used to identify wild-type and vaccine strains of B melitensis. Phylogenetic analysis of short, polymorphic fragments of DNA from B melitensis strains M5 and M43 was performed. RESULTS Variable-number tandem repeat DNA segments of B melitensis vaccine strain M5 and wild-type strain M43 were successfully amplified by means of PCR assay. All target gene fragments ranged in size from 100 to 300 bp. Separate phylogenetic analysis of each Brucella strain revealed considerable differences between the vaccine and wild-type strains. CONCLUSIONS AND CLINICAL RELEVANCE The results of this study suggested the HOOF-Prints assay may be useful for discriminating vaccine strains of B melitensis from wild-type strains. This ability could allow discrimination between animals that are seropositive because of vaccination against B melitensis and those that are seropositive because of B melitensis infection and could decrease the likelihood of importing Brucella-infected animals.

Identification of the Brucella melitensis vaccine strain Rev.1 in animals and humans in Israel by PCR analysis of the PstI site polymorphism of its omp2 gene.

Adverse effects of strain persistence and secretion in milk have been encountered with the Brucella melitensis vaccine strain Rev.1. Field isolates obtained from vaccinated animals and from a human resembled the vaccine strain Rev.1 by conventional bacteriological tests. The lack of a specific molecular marker that could specifically characterize the commercial vaccine strain prevented confirmation of the homology of the Rev.1-like field isolates to the vaccine strain. The composition of the omp2 locus from two gene copies with differences in their PstI restriction endonuclease sites was used to establish an epidemiologic fingerprint for the omp2 gene in the Rev.1 vaccine strain. Primers designed to amplify DNA sequences that overlap the PstI site revealed a single 282-bp DNA band common to all Brucella spp. Agarose gel electrophoresis of the PstI digests of the PCR products from strains 16M and the vaccine strain Rev.1 revealed a distinctive profile that included three bands: one band for the intact 282-bp fragment amplified from omp2a and two bands resulting from the digestion of the amplified omp2b gene fragment, 238- and 44-bp DNA fragments, respectively. Amplified fragments of 37 Rev.1-like isolates, including 2 human isolates, also exhibited this pattern. In contrast, DNA digests of all other Israeli field isolates, including atypical B. melitensis biotype 1 and representatives of the biotype 2 and 3 isolates, produced two bands of 238 and 44 bp, respectively, corresponding with the digestion of both omp2a and omp2b genes. This method facilitates identification of the Rev.1 vaccine strain in both animals and humans in Israel.

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina.

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.

Application of pulsed-field gel electrophoresis for differentiation of vaccine strain RB51 from field isolates of Brucella abortus from cattle, bison, and elk.

Restriction endonuclease patterns of genomic fragments separated by use of pulsed-field gel electrophoresis were used to differentiate Brucella abortus strain RB51, a rifampin-resistant mutant of the standard virulent strain 2308, from other brucellae. Results were compared with results obtained by use of standard methods for characterizing brucellae. Electrophoretic patterns of the ATCC type strains allowed identification of the strains to the level of species. Genomic profiles of B abortus biovars 1, 2, and 4 were similar, as were those of biovars 5, 6, and of biovar 3 was similar to that of biovars 5, 6, and 9, except for a missing band at 93 kb and additional bands at 65 and 67 kb. A different fingerprint was detected in B abortus strain RB51, using the pulsed-field gel electrophoresis patterns of genomic DNA digested with restrictive endonuclease Xba I. The profile of B abortus strain RB51 contained a band at 104 kb, as opposed to a 109-kb fragment within profiles of B abortus isolates from naturally infected cattle, bison, and elk. Despite known biochemical and biological differences between RB51 and its parent strain (2308), restriction endonuclease analysis results were similar.

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