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Bacillus cereus biovar anthracis (Bcbva) causes anthrax-like disease in animals, particularly in the non-human primates and great apes of West and Central Africa. Genomic analyses revealed Bcbva as a member of the B . cereus species that carries two plasmids, pBCXO1 and pBCXO2, which have high sequence homology to the B . anthracis toxin and polyglutamate capsule encoding plasmids pXO1 and pXO2, respectively. To date, only a few studies have investigated the effect of variations in Bcbva sporulation, toxin, and capsule synthesis on animal and macrophage pathogenicity compared to B . anthracis , therefore more research is needed to gain a better understanding of the pathogenesis of this emerging infection. Here, we report that Bcbva can multiply and vegetatively survive on nutrient-rich media for a minimum of six days while generating spores. Sporulation of Bcbva occurred faster and more extensively than B . anthracis Ames. Bcbva tended to secrete less protective antigen (PA) than B . anthracis Ames when cultured in growth medium. We found Bcbva produced a substantially higher amount of attached poly-ƴ-D-glutamic acid (PDGA) capsule than B . anthracis Ames when grown in medium supplemented with human serum and CO 2 . In a phagocytosis assay, Bcbva spores showed reduced internalization by mouse macrophages compared to B . anthracis Ames. Our research demonstrated that Bcbva is more virulent than B . anthracis Ames using two in vivo models, Galleria mellonella larvae and guinea pigs. Following that, the efficacy of the veterinary vaccine Sterne strain 34F2 against anthrax-like disease was assessed in guinea pigs. Sterne vaccinated guinea pigs had significantly increased anti-PA titers compared to the unvaccinated control group. Toxin neutralizing antibody titers in vaccinated guinea pigs correlated with anti-PA titers. This indicates the Sterne vaccine provides adequate protection against Bcbva infection in laboratory animals.

Brucellosis is one of the most important and widespread bacterial zoonoses worldwide. Cases are reported annually across the range of known infectious species of the genus Brucella. Globally, Brucella melitensis, primarily hosted by domestic sheep and goats, affects large proportions of livestock herds, and frequently spills over into humans. While some species, such as Brucella abortus, are well controlled in livestock in areas of North America, the Greater Yellowstone Ecosystem supports the species in native wild ungulates with occasional spillover to livestock. Elsewhere in North America, other Brucella species still infect domestic dogs and feral swine, with some associated human cases. Brucella spp. patterns vary across space globally with B. abortus and B. melitensis the most important for livestock control. A myriad of other species within the genus infect a wide range of marine mammals, wildlife, rodents, and even frogs. Infection in humans from these others varies with geography and bacterial species. Control in humans is primarily achieved through livestock vaccination and culling and requires accurate and rapid species confirmation; vaccination is Brucella spp.-specific and typically targets single livestock species for distribution. Traditional bacteriology methods are slow (some media can take up to 21 days for bacterial growth) and often lack the specificity of molecular techniques. Here, we summarize the molecular techniques for confirming and identifying specific Brucella species and provide recommendations for selecting the appropriate methods based on need, sensitivity, and laboratory capabilities/technology. As vaccination/culling approaches are costly and logistically challenging, proper diagnostics and species identification are critical tools for targeting surveillance and control.

Background The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis . Although originally thought to be ubiquitous in B. anthracis , previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. Results In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant − ) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. Conclusions Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators.

White-tailed deer, Odocoileus virginianus, anthrax epizootics have been frequently documented in Texas over the last two decades. Once outbreaks begin, there is evidence for the potential role of hematophagous flies as vectors for the disease. Hypotheses on the role of biting flies in the transmission of anthrax date back more than a century. Both laboratory experiments and field studies have provided evidence of a biting fly transmission pathway. In particular, several studies have implicated biting flies during severe wildlife outbreaks in North America. Despite these implications, there is a lack of spatial analysis relating flies and anthrax. Here we report on the spatial patterns of anthrax in white-tailed deer on a well-studied ranch with a documented anthrax history. These patterns were evaluated against the spatiotemporal patterns of biting flies during the anthrax risk period. Unbaited fly traps were used to collect flies across the study ranch from June through August 2005. Kernel density analysis confirmed biting fly hotspots concentrated in the areas with highest densities of deer carcasses. The average nearest neighbor index confirmed that deer carcasses were spatially clustered and density estimates suggest that these are in proximity to areas supporting high fly populations. Dual kernel density analysis of carcasses and deer population identified a large dry riverine habitat as a high anthrax risk. Fly catch rates across the period identified a similar pattern to the anthrax risk surface. The high overlap between areas of sustained high fly catch rates and anthrax cases does suggest a relationship warranting future research.

(1) Background: Bacillus cereus biovar anthracis (Bcbva) was the causative agent of an anthrax-like fatal disease among wild chimpanzees in 2001 in Côte d’Ivoire. Before this, there had not been any description of an anthrax-like disease caused by typically avirulent Bacillus cereus. Genetic analysis found that B. cereus had acquired two anthrax-like plasmids, one a pXO1-like toxin producing plasmid and the other a pXO2-like plasmid encoding capsule. Bcbva caused animal fatalities in Cameroon, Democratic Republic of Congo, and the Central African Republic between 2004 and 2012. (2) Methods: The pathogen had acquired plasmids in the wild and that was discovered as the cause of widespread animal fatalities in the early 2000s. Primate bones had been shipped out of the endemic zone for anthropological studies prior to the realized danger of contamination with Bcbva. Spores were isolated from the bone fragments and positively identified as Bcbva. Strains were characterized by classical microbiological methods and qPCR. Four new Bcbva isolates were whole-genome sequenced. Chromosomal and plasmid phylogenomic analysis was performed to provide temporal and spatial context to these new strains and previously sequenced Bcbva. Tau and principal component analyses were utilized to identify genetic and spatial case patterns in the Taï National Park anthrax zone. (3) Results: Preliminary studies positively identified Bcbva presence in several archival bone fragments. The animals in question died between 1994 and 2010. Previously, the earliest archival strains of Bcbva were identified in 1996. Though the pathogen has a homogeneous genome, spatial analyses of a subset of mappable isolates from Taï National Park revealed strains found closer together were generally more similar, with strains from chimpanzees and duikers having the widest distribution. Ancestral strains were located mostly in the west of the park and had lower spatial clustering compared to more recent isolates, indicating a local increase in genetic diversity of Bcbva in the park over space and time. Global clustering analysis indicates patterns of genetic diversity and distance are shared between the ancestral and more recently isolated type strains. (4) Conclusions: Our strains have the potential to unveil historical genomic information not available elsewhere. This information sheds light on the evolution and emergence of a dangerous anthrax-causing pathogen.

Diphtheria is an acute, communicable disease caused by Corynebacterium diphtheriae. The disease is generally characterized by local growth of the bacterium in the pharynx with pseudomembrane formation or, less commonly, in the stomach or lungs; systemic dissemination of toxin then invokes lesions in distant organs. Acute disease of the upper respiratory tract usually involves one or more of the following: tonsillar zones, larynx, soft palate, uvula, and nasal cavities. A recent epidemic in Russia emphasized the role of vaccination in reducing disease in children and adults.

Bacillus anthracis is the causative agent of anthrax in animals and humans. The organism lies in a dormant state in the soil until introduced into an animal via, ingestion, cutaneous inoculation or inhalation. Once in the host, spores germinate into rapidly growing vegetative cells elaborating toxins. When animals die of anthrax, vegetative bacteria sporulate upon nutrient limitation in the carcass or soil while in the presence of air. After release into the soil environment, spores form a localized infectious zone (LIZ) at and around the carcass. Laboratory strains of B. anthracis produce fewer proteins associated with growth and sporulation compared to wild strains isolated from recent zoonotic disease events. We verified wild strains grow more rapidly than lab strains demonstrating a greater responsiveness to nutrient availability. Sporulation was significantly more rapid in these wild strains compared to lab strains, indicating wild strains are able to sporulate faster due to nutrient limitation while laboratory strains have a decrease in the speed at which they utilize nutrients and an increase in time to sporulation. These findings have implications for disease control at the LIZ as well as on the infectious cycle of this dangerous zoonotic pathogen.

Histopathologic examination of lymph nodes from 39 patients with clinical and pathological criteria for cat scratch disease revealed delicate pleomorphic Gram-negative bacilli in 34 of the 39 nodes. They were within the walls of capillaries in or near areas of follicular hyperplasia and within microabscesses. They were best seen with the Warthin-Starry silver impregnation stain. Organisms in lymph node sections exposed to convalescent serum from three patients and to immunoperoxidase stained equally well with all three samples. The organisms did not react with hyperimmune sera to Legionella pneumophila nor to several species of Rickettsia. These bacilli appear to be the causative agents of cat scratch disease.

(1) Background: Bacillus cereus biovar anthracis (Bcbva) was the causative agent of an anthrax-like fatal disease among wild chimpanzees in 2001 in Côte d’Ivoire. Before this, there had not been any description of an anthrax-like disease caused by typically avirulent Bacillus cereus. Genetic analysis found that B. cereus had acquired two anthrax-like plasmids, one a pXO1-like toxin producing plasmid and the other a pXO2-like plasmid encoding capsule. Bcbva caused animal fatalities in Cameroon, Democratic Republic of Congo, and the Central African Republic between 2004 and 2012. (2) Methods: The pathogen had acquired plasmids in the wild and that was discovered as the cause of widespread animal fatalities in the early 2000s. Primate bones had been shipped out of the endemic zone for anthropological studies prior to the realized danger of contamination with Bcbva. Spores were isolated from the bone fragments and positively identified as Bcbva. Strains were characterized by classical microbiological methods and qPCR. Four new Bcbva isolates were whole-genome sequenced. Chromosomal and plasmid phylogenomic analysis was performed to provide temporal and spatial context to these new strains and previously sequenced Bcbva. Tau and principal component analyses were utilized to identify genetic and spatial case patterns in the Taï National Park anthrax zone. (3) Results: Preliminary studies positively identified Bcbva presence in several archival bone fragments. The animals in question died between 1994 and 2010. Previously, the earliest archival strains of Bcbva were identified in 1996. Though the pathogen has a homogeneous genome, spatial analyses of a subset of mappable isolates from Taï National Park revealed strains found closer together were generally more similar, with strains from chimpanzees and duikers having the widest distribution. Ancestral strains were located mostly in the west of the park and had lower spatial clustering compared to more recent isolates, indicating a local increase in genetic diversity of Bcbva in the park over space and time. Global clustering analysis indicates patterns of genetic diversity and distance are shared between the ancestral and more recently isolated type strains. (4) Conclusions: Our strains have the potential to unveil historical genomic information not available elsewhere. This information sheds light on the evolution and emergence of a dangerous anthrax-causing pathogen.