Production of a Bacillus anthracis Secretome with Suitable Characteristics as Antigen in a Complement Fixation Test.

In this study, we cultured the Bacillus anthracis vaccine strain Sterne 34F2 in a medium containing EDTA, and we assessed the best conditions to inhibit the activity of zinc-dependent metalloproteases to obtain a secretome containing a high concentration of non-degraded PA (PA83), as evaluated by the SDS-PAGE analysis. Then, we used this secretome as the antigen in a Complement Fixation Test (CFT) to monitor the production of antibodies against PA83 in the sera of rabbits vaccinated with Sterne 34F2 and then infected with a B. anthracis virulent strain to evaluate the potency of the vaccine. The PAS-based CFT results were compared with those obtained by using a commercial ELISA kit. The two serological tests gave similar results in terms of specificity and sensitivity, as the kinetics of the antibodies production was very similar. The Sterne 34F2 vaccine induced an antibody response to PA83, whose titer was not inferior to 1:8 in PAS-based CFT and 42 kU/mL in PA83-based ELISA, respectively, in all vaccinated rabbits. Our opinion is that the PAS-based CFT can be successfully employed in humans and in animals for epidemiological retrospective studies or post-vaccination monitoring. We also suggest the use of our method to test the efficacy of veterinary anthrax vaccines.

Rapid and safe one-step extraction method for the identification of Brucella strains at genus and species level by MALDI-TOF mass spectrometry.

Brucellosis is essentially a disease of domesticated livestock; however, humans can also be infected via the consumption of contaminated meat or dairy products, underlying the need for rapid and accurate identification methods. Procedures for microbiological identification and typing of Brucella spp. are expensive, time-consuming, and must be conducted in biohazard containment facilities to minimize operator risk. The development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based assay has reduced the processing time while maintaining performance standards. In this study, to improve the identification accuracy and suitability of the MALDI-TOF-based assay for routine diagnosis, we developed a new protein extraction protocol and generated a custom reference database containing Brucella strains representative of the most widespread species. The reference library was then challenged with blind-coded field samples isolated from infected animals. The results indicated that the database could be used to correctly identify 99.5% and 97% of Brucella strains at the genus and species level, respectively, indicating that the performance of the assay was not affected by the different culture conditions used for microbial isolation. Moreover, the inactivated samples were stored and shipped to reference laboratories with no ill effect on protein stability, thus confirming the reliability of our method for routine diagnosis. Finally, we evaluated the epidemiological value of the protocol by comparing the clustering analysis results of Brucella melitensis strains obtained via multiple locus variable-number tandem repeat analysis or MALDI-TOF MS. The results showed that the MALDI-TOF assay could not decipher the true phylogenetic tree, suggesting that the protein profile did not correspond with the genetic evolution of Brucella.

Development of a Sterne-Based Complement Fixation Test to Monitor the Humoral Response Induced by Anthrax Vaccines.

Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals, and humans depend on the prevention in livestock, principally cattle, sheep, and goats. Most veterinary vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2-) B. anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test (CFT) to detect specific antibodies induced in animals vaccinated with Sterne 34F2. We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to correctly identify vaccinated animals. It proved to be a very sensitive and specific test. Moreover, the Sterne-based CFT offers many benefits with regard to costs, standardization and reproducibility of the assay procedure.

Evaluation of the immunogenicity and safety of Brucella melitensis B115 vaccination in pregnant sheep.

In spite of its limitations, Rev.1 is currently recognized as the most suitable vaccine against Brucella melitensis (the causative agent of ovine and caprine brucellosis). However, its use is limited to young animals when test-and-slaughter programs are in place because of the occurrence of false positive-reactions due to Rev.1 vaccination. The B. melitensis B115 rough strain has demonstrated its efficacy against B. melitensis virulent strains in the mouse model, but there is a lack of information regarding its potential use in small ruminants for brucellosis control. Here, the safety and immune response elicited by B115 strain inoculation were evaluated in pregnant ewes vaccinated at their midpregnancy. Vaccinated (n=8) and non-vaccinated (n=3) sheep were periodically sampled and analyzed for the 108 days following inoculations using tests designed for the detection of the response elicited by the B115 strain and routine serological tests for brucellosis [Rose Bengal Test (RBT), Complement Fixation Test (CFT) and blocking ELISA (ELISAb)]. Five out of the 8 vaccinated animals aborted, indicating a significant abortifacient effect of B115 inoculation at midpregnancy. In addition, a smooth strain was recovered from one vaccinated animal, suggesting the occurrence of an in vivo reversion phenomenon. Only one animal was positive in both RBT and CFT simultaneously (91 days after vaccination) confirming the lack of induction of cross-reacting antibody responses interfering with routine brucellosis diagnostic tests in most B115-vaccinated animals.

Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice.

It has been demonstrated that antibodies specific for O-PS antigen of Brucella smooth strains are involved in the protective immunity of brucellosis. Since the rough strain Brucella melitensis B115 was able to protect mice against wild Brucella strains brucellosis despite the lack of anti-OPS antibodies, in this study we evaluated the biological significance of antibodies induced by this strain, directed to antigens other than O-PS, passively tranferred to untreated mice prior to infection with Brucella abortus 2308 and B. melitensis 16M virulent strains. The protective ability of specific antisera collected from mice vaccinated with B. melitensis B115, B. abortus RB51 and B. abortus S19 strains was compared. The results indicated that antibodies induced by B115 were able to confer a satisfactory protection, especially against B. abortus 2308, similar to that conferred by the antiserum S19, while the RB51 antiserum was ineffective. These findings suggest that antibodies induced by B115 could act as opsonins as well as antibodies anti-O-PS, thus triggering more efficient internalization and degradation of bacteria within phagocytes. This is the first study assessing the efficacy of antibodies directed to antigens other than O-PS in the course of brucellosis infection.

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18.

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.

B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections.

Brucellosis is one of the most serious zoonoses all over the world, with B. melitensis, B. abortus and B. suis being the most pathogenic species for humans. Vaccination of domesticated livestock still represents the most efficient way to prevent human infection. However, the available Brucella vaccines retain an important residual virulence and induce antibodies interfering with surveillance programs. Moreover, each vaccine shows different protective effects versus different Brucella species and different animal hosts. Nowadays, while B. melitensis and B. suis infections in cattle are emerging as a significant problem, there are no available vaccines to overcome such issue. B. melitensis strain B115, a natural, attenuated rough strain in our previous studies proved to be highly protective against B. melitensis and B. ovis infections in mice, without inducing interfering antibodies. In this study, we tested the efficiency of B115 as vaccine against B. abortus and B. suis. Vaccination of mice with 10(8) CFU/mouse of B. melitensis B115 conferred a satisfactory protection against B. abortus 2308. On the contrary, mice vaccinated once with 10(8) or 10(9) CFU/mouse of B115 were weakly protected against B. suis infection. Conversely, when mice were vaccinated twice with 10(9) CFU B115/mouse, the protective activity significantly increased. Unlike its rough phenotype, B115 showed an adequate persistence in mice accompanied to a solid humoral and cell-mediated immunity. All together, these findings suggest the potential usefulness of B115 to control brucellosis in animal hosts due to heterologous challenges.