Detection of Brucella abortus B19 strain DNA in seminal plasma by polymerase chain reaction in Brazil.

A total of 27 seminal plasma samples from cattle-breeding farms or semen centres located in Minas Gerais, Brazil, previously negative by serological and tested positive for Brucella spp. with primer specific for the amplification of the gene virb5 by polymerase chain reaction (PCR) were analysed for the detection of Brucella abortus DNA by PCR. It was found that nine samples (33.33%) contained B. abortus B19 strain DNA, two (7.40%) contained B. abortus DNA and five (18.51%) contained both DNA. The larger number of samples with B. abortus B19 strain DNA would explained by the environmental contamination by vaccinated females with persistent excretion or some illegal vaccination process. It is first reported of male bovines detected with both DNA.

Detection of Brucella spp. DNA in the semen of seronegative bulls by polymerase chain reaction.

Semen samples from 88 reproductively mature bulls were screened to detect the presence of Brucella spp. by polymerase chain reaction. Twenty-seven samples were found to be positive, underscoring the importance of researching brucellosis in males and the need for greater care in the selection of sperm-donating bulls for semen centres.

Método eletroforético rápido para detecção da adulteração do leite caprino com leite bovino / Fast electrophoretic detection method of adulteration of caprine milk by bovine milk

Avaliaram-se os métodos de eletroforese em gel de poliacrilamida (PAGE) em presença de uréia (uréia-PAGE) e dodecil sulfato de sódio (SDS-PAGE) para identificar a adulteração do leite de cabra pela adição do leite de vaca. Um método foi otimizado para preparação do caseinato de sódio em poucos minutos para análise eletroforética. Uréia-PAGE foi o método mais apropriado para identificação desse tipo de fraude, em decorrência da presença da caseína alfas1 com migração mais rápida no leite bovino. A presença da alfas1-caseína bovina foi detectada a partir da adição de 2,5 por cento de leite de vaca utilizando uréia-PAGE. O limite de detecção, a repetibilidade, o tempo para execução indicaram que esse método pode ser aplicado como rotina no controle de qualidade do leite de cabra recebido pelas indústrias de processamento.

Polyacrylamide gel electrophoresis (PAGE) in presence of urea (urea-PAGE) or sodium dodecyl sulfate (SDS-PAGE) was evaluated to detect the presence of cow milk added to goat milk. A method was optimized to prepare sodium caseinate from milk in few minutes. After that, the sodium caseinate was analyzed by PAGE. The urea-PAGE was the most appropriated method to identify adulteration as caprine and bovine alphas1-caseins displayed different migration rates. When cow milk was added to goat milk at different proportions, the presence of bovine alphas1-casein was detected in the mixture by urea-PAGE for a minimal proportion of 2.5 percent of cow milk added to goat milk. The good sensitivity, the repeatability and the short time for execution indicate that the described method will be able to be routinely applied for the quality control of goat milk in dairy industry.

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice.

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.

Immunological properties of gene vaccines delivered by different routes

Gene vaccines represent a new and promising approach to control infectious diseases, inducing a protective immune response in the appropriate host. Several routes and methods of genetic immunization have been shown to induce antibody production as well as T helper (Th) cell and cytotoxic T lymphocyte activation. However, few studies have compared the nature of the immune responses generated by different gene vaccination delivery systems. In the present study we reviewed some aspects of immunity induced by gene immunization and compared the immune responses produced by intramuscular (im) DNA injection to gene gun-mediated DNA transfer into the skin of BALB/c mice. Using a reporter gene coding for ß-galactosidase, we have demonstrated that im injection raised a predominantly Th1 response with mostly IgG2a anti-ßgal produced, while gene gun immunization induced a mixed Th1/Th2 profile with a balanced production of IgG2a and IgG1 subclasses. Distinct types of immune responses were generated by different methods of gene delivery. These findings have important implications for genetic vaccine design. Firstly, a combination between these two systems may create optimal conditions for the induction of a broad-based immune response. Alternatively, a particular gene vaccine delivery method might be used according to the immune response required for host protection. Here, we describe the characteristics of the immune response induced by gene vaccination and the properties of DNA involved in this process