Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii

BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7 )starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies

Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa—Chromosomal Monophyly and Broad Geographic Distribution

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d’Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Molecular and Biological Analysis of Eight Genetic Islands That Distinguish Neisseria meningitidis from the Closely Related Pathogen Neisseria gonorrhoeae

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis

Occurrence and Genetic Diversity of Bacillus anthracis Strains Isolated in an Active Wool-Cleaning Factory▿

Culturable microorganisms from various samples taken at an active factory performing wool and goat hair cleaning were isolated and analyzed. Bacillus anthracis was found in air filter dust, wastewater, and goat hairs, where it accounted for approximately 1% of the total counts of viable bacteria. Consistent with the countries of origin of the processed material (South Caucasian and Middle Eastern), all B. anthracis isolates belonged to the same phylogenetic cluster, as determined by variable-number tandem repeat (VNTR) typing at eight loci. Within this cluster, five closely related VNTR subtypes could be identified, of which two were previously unreported. Additional diversity was observed when more sensitive genetic markers were assayed, demonstrating the multifocal nature of goat hair contamination. Goat hair originating from areas where anthrax is endemic remains a material with high biological risk for modern woolworkers

Serological evidence for human exposure to Bacillus cereus biovar anthracis in the villages around Taï National Park, Côte d'Ivoire.

Bacillus cereus biovar anthracis (Bcbva) is an untypical anthrax-causing pathogen responsible for high wildlife mortality in Taï National Park (TNP), Côte d'Ivoire. However, nothing is known about its effect on the rural population living in the region bordering TNP. Contact to bushmeat is a known risk factor for exposure to a variety of zoonotic pathogens, but no human infections with Bcbva were noted so far. Therefore, we performed a retrospective seroprevalence analysis with sera from 1,386 study volunteers. We used assays which detect antibodies against the protective antigen PA, which is synthesized by both Bcbva and classic B. anthracis, and against the recently described antigen pXO2-60, a 35-kDa protein only produced by Bcbva. We found a high seroprevalence (22.37%) of antibodies against PA, and approximately half of those sera (10.46%) were also positive for the Bcbva-specific antigen pXO2-60. All sera negative for PA were also negative for antibodies against pXO2-60, confirming specificity and suitability of the PA/pXO2-60 combined serological assay. The fact that a large fraction of sera was positive for PA but negative for pXO2-60 can most likely be explained by lower immunogenicity of pXO2-60, but exposure to classic B. anthracis cannot be excluded. As only Bcbva has been detected in the TNP area so far, exposure to Bcbva can be suspected from the presence of antibodies against PA alone. In a questionnaire, most study participants reported contact to bushmeat and livestock carcasses. Unfortunately, risk factor analysis indicated that neither animal contacts, sex, age, nor country of origin were significant predictors of Bcbva seroprevalence. Nevertheless, our study added to an assessment of the distribution of Bcbva and its impact on the human population, and our data can serve to raise awareness of anthrax in the affected regions