Rosanna Adone

Genetic Bases of the Rifampin Resistance Phenotype in Brucella spp.

ABSTRACT Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the β subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rifr) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rifr in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rifr vaccine strain Brucella abortus RB51 and of 20 Rifr clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rifs) parental strains and to the published B. melitensis strain 16M. All Rifr strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two “hot” regions were investigated in eight additional RifrBrucella laboratory mutants and in 20 reference RifsBrucella strains. rpoB mutations were found in all Rifr mutants. In contrast, no missense mutations were found in any analyzed Rifs strains. Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rifr phenotype in prokaryotes. In addition, because of the relationship between Rifr and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella.

PCR Assay To Detect Bacillus anthracis Spores in Heat-Treated Specimens

ABSTRACT Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable Bacillus anthracis spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121°C for 45 min. The results indicate that heat treatment ensures the complete inactivation of B. anthracis spores without significantly affecting the efficiency of PCR assays.

Mouse cytokine profiles associated with Brucella abortus RB51 vaccination or B. abortus 2308 infection

ABSTRACT This study indicated that mice immunized with Brucella abortus RB51 bacteria and subsequently challenged with B. abortus 2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.

Evaluation of the House Fly Musca domestica as a Mechanical Vector for an Anthrax

Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs.

Protective Properties of Rifampin-Resistant Rough Mutants of Brucella melitensis

ABSTRACT Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.

SNR analysis: molecular investigation of an anthrax epidemic

BackgroundIn Italy, anthrax is endemic but occurs sporadically. During the summer of 2004, in the Pollino National Park, Basilicata, Southern Italy, an anthrax epidemic consisting of 41 outbreaks occurred; it claimed the lives of 124 animals belonging to different mammal species. This study is a retrospective molecular epidemiological investigation carried out on 53 isolates collected during the epidemic. A 25-loci Multiple Locus VNTR Analysis (MLVA) MLVA was initially performed to define genetic relationships, followed by an investigation of genetic diversity between epidemic strains through Single Nucleotide Repeat (SNR) analysis.Results53 Bacillus anthracis strains were isolated. The 25-loci MLVA analysis identified all of them as belonging to a single genotype, while the SNR analysis was able to detect the existence of five subgenotypes (SGTs), allowing a detailed epidemic investigation. SGT-1 was the most frequent (46/53); SGTs 2 (4/53), 3 (1/53) 4 (1/53) and 5 (1/53) were detected in the remaining seven isolates.ConclusionsThe analysis revealed the prevalent spread, during this epidemic, of a single anthrax clone. SGT-1 - widely distributed across the epidemic area and present throughout the period in question - may, thus, be the ancestral form. SGTs 2, 3 and 4 differed from SGT-1 at only one locus, suggesting that they could have evolved directly from the latter during the course of this epidemic. SGT-5 differed from the other SGTs at 2-3 loci. This isolate, thus, appears to be more distantly related to SGT-1 and may not be a direct descendant of the lineage responsible for the majority of cases in this epidemic. These data confirm the importance of molecular typing and subtyping methods for in-depth epidemiological analyses of anthrax epidemics.

Evaluation of Brucella melitensis B115 as rough-phenotype vaccine against B. melitensis and B. ovis infections.

Brucella melitensis strain Rev1 is used as vaccine for the prophylaxis of brucellosis in sheep and goats. Because of its smooth phenotype, however, it induces antibodies directed to the O-polysaccharide (O-PS) of the lipopolysaccharide (LPS), thus unabling to distinguish between vaccinated and infected animals. It has been speculated that alternative vaccines could be live, attenuated Brucella rough strains, which are devoid of the O-PS. B. melitensis B115 is a natural, attenuated, rough strain. The O-PS is not exposed at the surface but is present in the cytoplasm. We tested the protective activity of B115 against B. melitensis and B. ovis infections in mice, in comparison with that of Rev1. The residual virulence and the humoral response following B115 vaccination were also evaluated. Vaccination with B115 conferred significant protective immunity against B. melitensis 16M and B. ovis challenge strains, equivalent to that provided by Rev1. No interfering antibodies to O-PS were detected, while the B115 vaccination was monitored by a specific B115-based complement fixation test. These promising features suggest further evaluation of B. melitensis B115 as vaccine for target animal hosts.

Effect of Exogenous Interleukin-18 (IL-18) and IL-12 in the Course of Brucella abortus 2308 Infection in Mice

ABSTRACT In this study we demonstrated that combined inoculation of interleukin-12 (IL-12) and IL-18 reduced the number of bacteria in the spleens of mice infected with Brucella abortus 2308 and that the effect of the treatment was mediated by an increased capability of spleen cells to produce gamma interferon at the early phase of infection.

Field Validation of the Use of RB51 as Antigen in a Complement Fixation Test To Identify Calves Vaccinated with Brucella abortus RB51

ABSTRACT In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated withBrucella abortus strain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 1010 CFU of RB51 commercial vaccine, while only six calves received 109 CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed withB. abortus smooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 109 CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.

Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillus anthracis contamination

BackgroundIn this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field investigation.ResultsThe results indicated that, in contrast to the classic method, the GABRI method was able to detect B.anthracis in all contaminated samples. The GABRI method produces a more sensitive measure of anthrax spore presence significantly different from the standard method. In particular, the latter is more sensitive to the presence of normal soil contaminants.ConclusionThe main feature of the GABRI method is its ability to strongly reduce the presence of the environmental contaminants, which being much more numerous than B. anthracis tend to inhibit their germination and growth making it extremely difficult to visualize any colonies. The reduction of the microbial environment also allows one to be able to culture and test a larger quantity of potentially contaminated soil and to isolate B. anthracis when the spores are present in very low concentrations in the soil.