Mapping high probability area for the Bacillus anthracis occurrence in wildlife protected area, South Omo, Ethiopia.

Anthrax is a zoonotic disease caused by a spore-forming gram-positive bacterium, Bacillus anthracis. Increased anthropogenic factors inside wildlife-protected areas may worsen the spillover of the disease at the interface. Consequently, environmental suitability prediction for B. anthracis spore survival to locate a high-risk area is urgent. Here, we identified a potentially suitable habitat and a high-risk area for appropriate control measures. Our result revealed that a relatively largest segment of Omo National Park, about 23.7% (1,218 square kilometers) of the total area; 36.6% (711 square kilometers) of Mago National Park, and 29.4% (489 square kilometers) of Tama wildlife Reserve predicted as a high-risk area for the anthrax occurrence in the current situation. Therefore, the findings of this study provide the priority area to focus on and allocate resources for effective surveillance, prevention, and control of anthrax before it causes devastating effects on wildlife.

Whole Genome-Sequencing and Phylogenetic Analysis of Bacillus anthracis from 2019-2023 in Ningxia Hui Autonomous Region, China.

BACKGROUND: Since 2019, the incidence of anthrax in the Ningxia Hui Autonomous Region has increased significantly compared with previous years, so in this situation the anthrax in the Ningxia region not only had a detrimental impact on public health, but also inflicted significant economic repercussions. Therefore, we conducted a molecular epidemiological study of 20 strains from 2019-2023 isolates. This study investigated the origin of Bacillus anthracis and its genetic diversity. METHODS: We conducted canonical single-nucleotide polymorphisms (CanSNPs) typing and whole genome sequencing based on the extracted nucleic acid of Bacillus anthracis. Based on the whole genome drafts, we studied the genomic characteristics of 20 isolates. Meanwhile, we performed phylogenetic studies based on genome-wide core single-nucleotide polymorphisms (SNPs) using MEGA's Maximum Likelihood (ML) method and core-genome-based multilocus sequence typing (cgMLST) of the core genomes of these strains using BioNumerics' minimum spanning tree (MST) model. RESULTS: The 20 isolates were categorized into sub-lineages A.Br.001/002, and comparative genomic analyses of these strains with other isolates from other parts of the world showed that the strains from Ningxia were correlated with isolates from Europe, Indonesia, Georgia (USA), and Beijing (China). For the 20 isolates in Ningxia, the genetic relationship of the isolates isolated from the same year or region was relatively close. CONCLUSION: The A.Br.001/002 subgroup was the dominant endemic strain in Ningxia. The genetic relationship and phylogenesis between isolates from Ningxia and strains from Europe and Indonesia suggest that anthrax spread around the globe through ancient trade routes.

Fate and transport of viable Bacillus anthracis simulant spores in ambient air during a large outdoor decontamination field exercise.

The Wide Area Demonstration (WAD) was a field exercise conducted under the U.S. EPA's Analysis of Coastal Operational Resiliency program, in conjunction with the U.S. Department of Homeland Security and the U.S. Coast Guard. The purpose of the WAD was to operationalize at field scale aspects of remediation activities that would occur following an outdoor release of Bacillus anthracis spores, including sampling and analysis, decontamination, data management, and waste management. The WAD was conducted in May 2022 at Fort Walker (formerly known as Fort A.P. Hill) and utilized Bacillus atrophaeus as a benign simulant for B. anthracis. B. atrophaeus spores were inoculated onto the study area at the beginning of the study, and air samples were collected daily during each of the different phases of the WAD using Dry Filter Units (DFUs). Ten DFU air samplers were placed at the perimeter of the study area to collect bioaerosols onto two parallel 47-mm diameter polyester felt filters, which were then subsequently analyzed in a microbiological laboratory for the quantification of B. atrophaeus. The study demonstrated the use of DFUs as a rugged and robust bioaerosol collection device. The results indicated that the highest B. atrophaeus spore air concentrations (up to ~ 5 colony forming units/m3) occurred at the beginning of the demonstration (e.g. during inoculation and characterization sampling phases) and generally downwind from the test site, suggesting transport of the spores was occurring from the study area. Very few B. atrophaeus spores were detected in the air after several weeks and following decontamination of exterior surfaces, thus providing an indication of the site decontamination procedures' effectiveness. No B. atrophaeus spores were detected in any of the blank or background samples.Implications: Following an incident involving a release of Bacillus anthracis spores or other biological threat agent into the outdoor environment, understanding the factors that may affect the bioagent's fate and transport can help predict viable contaminant spread via the ambient air. This paper provides scientific data for the first time on ambient air concentrations of bacterial spores over time and location during different phases of a field test in which Bacillus atrophaeus (surrogate for B. anthracis) spores were released outdoors as part of a full-scale study on sampling and decontamination in an urban environment. This study advances the knowledge related to the fate and transport of bacterial spores (such as those causing anthrax disease) as an aerosol in the outdoor environment over the course of three weeks in a mock urban environment and has exposure and health risk implications. The highest spore air concentrations occurred at the beginning of the study (e.g. during inoculation of surfaces and characterization sampling), and in the downwind direction, but diminished over time; few B. atrophaeus spores were detected in the air after several weeks and following decontamination. Therefore, in an actual incident, potential reaerosolization of the microorganism and subsequent transport in the air during surface sampling and remediation efforts should be considered for determining exclusion zone locations and estimating potential risk to neighboring communities. The data also provide evidence suggesting that the large-scale decontamination of outdoor surfaces may reduce air concentrations of the bioagent, which is important since exposure of B. anthracis via inhalation is a primary concern.

Smartphone-assisted mobile fluorescence sensor for self-calibrated detection of anthrax biomarker, Cu2+, and cysteine in food analysis.

Dipicolinic acid (DPA), as a biomarker for Bacillus anthracis, is highly toxic at trace levels. Rapid and on-site quantitative detection of DPA is essential for maintaining food safety and public health. This work develops a dual-channel self-calibrated fluorescence sensor constructed by the YVO4:Eu and Tb-ß-diketone complex for rapid visual detection of DPA. This sensor exhibits high selectivity, fast response time, excellent detection sensitivity, and the detection limit is as low as 4.5 nM in the linear range of 0-16 µM. A smartphone APP and portable ultraviolet lamp can assemble a mobile fluorescence sensor for on-site analysis. Interestingly, adding Cu2+ ions can quench the fluorescence intensity of Tb3+. In contrast, the addition of cysteine can restore the fluorescence, allowing the accurate detection of Cu2+ ions and cysteine in environmental water and food samples. This work provides a portable sensor that facilitates real-time analysis of multiple targets in food and the environment.

Outbreak of Anthrax in Livestock with Human Occupational Exposures - Minnesota, 2023.

In July 2023, the Minnesota Department of Health (MDH) was notified of possible occupational exposures to anthrax during an outbreak in animals. In consultation with the Centers for Disease Control and Prevention, MDH epidemiologists created a questionnaire that assessed exposure risks and helped determine individual illness monitoring and antibiotic post-exposure prophylaxis needs. This investigation and the resources developed for it could be useful in future scenarios where there are occupational exposures to naturally occurring anthrax.

Comparative evaluation of in-house developed latex agglutination test (LAT) with World Organisation for Animal Health (WOAH) -recommended methods for the detection of Bacillus anthracis spores from the soil.

In-house developed Bacillus anthracis-specific synthetic peptide-based latex agglutination test (LAT) assay was comparatively evaluated with World Organisation for Animal Health (WOAH)-recommended polymerase chain reaction (PCR)/real-time PCR (qPCR) methods for the screening of B. anthracis spores from the soil to provide a simple, rapid, and economical immunodiagnostic test for field application.

First PCR Confirmed anthrax outbreaks in Ethiopia-Amhara region, 2018-2019.

BACKGROUND: Anthrax is a disease that affects humans and animals. In Ethiopia, anthrax is a reportable disease and assumed to be endemic, although laboratory confirmation has not been routinely performed until recently. We describe the findings from the investigation of two outbreaks in Amhara region. METHODS: Following reports of suspected outbreaks in Wag Hamra zone (Outbreak 1) and South Gondar zone (Outbreak 2), multi-sectoral teams involving both animal and public health officials were deployed to investigate and establish control programs. A suspect case was defined as: sudden death with rapid bloating or bleeding from orifice(s) with unclotted blood (animals); and signs compatible with cutaneous, ingestion, or inhalation anthrax ≤7 days after exposure to a suspect animal (humans). Suspect human cases were interviewed using a standard questionnaire. Samples were collected from humans with suspected anthrax (Outbreak 1 and Outbreak 2) as well as dried meat of suspect animal cases (Outbreak 2). A case was confirmed if a positive test was returned using real-time polymerase chain reaction (qPCR). RESULTS: In Outbreak 1, a total of 49 cows died due to suspected anthrax and 22 humans developed symptoms consistent with cutaneous anthrax (40% attack rate), two of whom died due to suspected ingestion anthrax. Three people were confirmed to have anthrax by qPCR. In Outbreak 2, anthrax was suspected to have caused the deaths of two livestock animals and one human. Subsequent investigation revealed 18 suspected cases of cutaneous anthrax in humans (27% attack rate). None of the 12 human samples collected tested positive, however, a swab taken from the dried meat of one animal case (goat) was positive by qPCR. CONCLUSION: We report the first qPCR-confirmed outbreaks of anthrax in Ethiopia. Both outbreaks were controlled through active case finding, carcass management, ring vaccination of livestock, training of health professionals and outreach with livestock owners. Human and animal health authorities should work together using a One Health approach to improve case reporting and vaccine coverage.

A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis.

As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

Ratiometric fluorescent detection of dipicolinic acid as an anthrax biomarker based on a high-nuclearity Yb18 nanoring.

An 18-metal lanthanide nanoring [Yb18(L1)8(HL2)2(OAc)20(MeOH)8(EtOH)6(H2O)4] (1), which shows a ratiometric fluorescent response to DPA, was constructed through the strategy of using two types of polydentate organic ligands. The addition of DPA increases the visible ligand-centered emission, but decreases the NIR lanthanide luminescence of 1. The limit of luminescent detection of 1 for DPA is 1.5 µM. The high fluorescence sensitivity of 1 to DPA is not affected by the existence of interferents such as aromatic carboxylates and ions.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies.

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium's close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

Identification of Bacillus anthracis, Brucella spp., and Coxiella burnetii DNA signatures from bushmeat.

Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.

Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax.

Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 103 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.

Modeling the spatial distribution of anthrax in southern Kenya.

BACKGROUND: Anthrax is an important zoonotic disease in Kenya associated with high animal and public health burden and widespread socio-economic impacts. The disease occurs in sporadic outbreaks that involve livestock, wildlife, and humans, but knowledge on factors that affect the geographic distribution of these outbreaks is limited, challenging public health intervention planning. METHODS: Anthrax surveillance data reported in southern Kenya from 2011 to 2017 were modeled using a boosted regression trees (BRT) framework. An ensemble of 100 BRT experiments was developed using a variable set of 18 environmental covariates and 69 unique anthrax locations. Model performance was evaluated using AUC (area under the curve) ROC (receiver operating characteristics) curves. RESULTS: Cattle density, rainfall of wettest month, soil clay content, soil pH, soil organic carbon, length of longest dry season, vegetation index, temperature seasonality, in order, were identified as key variables for predicting environmental suitability for anthrax in the region. BRTs performed well with a mean AUC of 0.8. Areas highly suitable for anthrax were predicted predominantly in the southwestern region around the shared Kenya-Tanzania border and a belt through the regions and highlands in central Kenya. These suitable regions extend westwards to cover large areas in western highlands and the western regions around Lake Victoria and bordering Uganda. The entire eastern and lower-eastern regions towards the coastal region were predicted to have lower suitability for anthrax. CONCLUSION: These modeling efforts identified areas of anthrax suitability across southern Kenya, including high and medium agricultural potential regions and wildlife parks, important for tourism and foreign exchange. These predictions are useful for policy makers in designing targeted surveillance and/or control interventions in Kenya. We thank the staff of Directorate of Veterinary Services under the Ministry of Agriculture, Livestock and Fisheries, for collecting and providing the anthrax historical occurrence data.

Spatial clustering of livestock Anthrax events associated with agro-ecological zones in Kenya, 1957-2017.

BACKGROUND: Developing disease risk maps for priority endemic and episodic diseases is becoming increasingly important for more effective disease management, particularly in resource limited countries. For endemic and easily diagnosed diseases such as anthrax, using historical data to identify hotspots and start to define ecological risk factors of its occurrence is a plausible approach. Using 666 livestock anthrax events reported in Kenya over 60 years (1957-2017), we determined the temporal and spatial patterns of the disease as a step towards identifying and characterizing anthrax hotspots in the region. METHODS: Data were initially aggregated by administrative unit and later analyzed by agro-ecological zones (AEZ) to reveal anthrax spatio-temporal trends and patterns. Variations in the occurrence of anthrax events were estimated by fitting Poisson generalized linear mixed-effects models to the data with AEZs and calendar months as fixed effects and sub-counties as random effects. RESULTS: The country reported approximately 10 anthrax events annually, with the number increasing to as many as 50 annually by the year 2005. Spatial classification of the events in eight counties that reported the highest numbers revealed spatial clustering in certain administrative sub-counties, with 12% of the sub-counties responsible for over 30% of anthrax events, whereas 36% did not report any anthrax disease over the 60-year period. When segregated by AEZs, there was significantly greater risk of anthrax disease occurring in agro-alpine, high, and medium potential AEZs when compared to the agriculturally low potential arid and semi-arid AEZs of the country (p < 0.05). Interestingly, cattle were > 10 times more likely to be infected by B. anthracis than sheep, goats, or camels. There was lower risk of anthrax events in August (P = 0.034) and December (P = 0.061), months that follow long and short rain periods, respectively. CONCLUSION: Taken together, these findings suggest existence of certain geographic, ecological, and demographic risk factors that promote B. anthracis persistence and trasmission in the disease hotspots.

Molecular genotyping of 15 B. anthracis strains isolated in Eastern Siberia and Far East.

Bacillus anthracis is a pathogenic bacterium, which causes anthrax disease. The ability of this bacterium to form spores, which can be preserved in soil for decades and cause outbreaks later on, makes this pathogen a serious problem for veterinary and health services of many countries. Siberia is one of the most anthrax-influenced regions of Russia. In this research we report on the results of genotyping based on whole genome SNP analysis of 15 strains, isolated on the territory of Eastern Siberia and the Far East in 1956-2018. In this research, we sequenced 15 genomes of B. anthracis strains isolated from infected humans and animals, and from soil samples from the territory of Eastern Siberia and the Far East in the period from 1956 to 2018. We used genomic sequences obtained in this study and 219 B. anthracis genomes available in the international GenBank database to perform a comparative analysis. As a result we detected 6400 chromosomal SNPs which allowed to differentiate the studied strains. We built phylogenetic reconstruction of the global B. anthracis population based on the detected SNPs using the Maximum Likelihood Method and described genetic diversity of the strains isolated on the territory of Eastern Siberia and the Far East. Strains, isolated on this territory from 1956 to 2018 belong to 5 different genetic groups: "Ames", "STI", "Tsiankovskii", "Siberia" and "Asia". The greatest diversity of the strains is registered for two regions of the southern part of Eastern Siberia - Tyva and Buryatia. This research expands current understanding of genetic diversity of B. anthracis strains circulating on the territory of Russia.

A weakly luminescent Tb-MOF-based "turn-on" sensor for the highly selective and sensitive sensing of an anthrax biomarker.

Bacillus anthracis is an extremely dangerous bacterium that is associated with high morbidity and mortality. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis, and it is of great significance to be able to detect DPA in a rapid, efficient, and sensitive way. Herein, a 3D network metal-organic framework (Tb-MOF) with excellent thermal and water stability was synthesized. Tb-MOF could be used to selectively detect DPA via green fluorescence recovery with a fluorescence intensity enhancement factor of 103. In addition, due to the high detection sensitivity (a detection limit of 2.4 µM) and excellent anti-interference abilities, Tb-MOF was less affected by environmental factors when compared with a "turn-off"-response luminescence sensor; it can be employed as a promising "turn-on" luminescence sensor for DPA in the future. Finally, quantum calculations showed that a large energy difference appeared between the 5D4 level of Tb3+ and the first excited triplet energy level of H2-DHBDC2-, which was the reason that the complex did not show characteristic Tb3+ emission.

A serological survey of Bacillus anthracis reveals widespread exposure to the pathogen in free-range and captive lions in Zimbabwe.

Numerous unknown factors influence anthrax epidemiology in multi-host systems, especially at wildlife/livestock/human interfaces. Serology tests for anti-anthrax antibodies in carnivores are useful tools in identifying the presence or absence of Bacillus anthracis in a range. These were employed to ascertain whether the disease pattern followed the recognized high- and low-risk anthrax zonation in Zimbabwe and also to establish whether anthrax was absent from Hwange National Park in which there have been no reported outbreaks. African lions (Panthera leo) (n = 114) drawn from free-range protected areas and captive game parks located in recognized high- and low-risk zones across Zimbabwe were tested for antibodies to anthrax PA antigen using the ELISA immunoassay. A random selection of 27 lion sera samples comprising 17 seropositive and 10 seronegative sera was further tested in the species-independent toxin neutralization assay (TNA) in order to validate the former as a surveillance tool for anthrax in African lions. Using the ELISA-PA immunoassay, 21.9% (25/114) of the lions tested positive for antibodies to anthrax. Seropositivity was recorded in all study areas, and there was no significant difference (p = .852) in seropositivity between lions in high- and low-risk anthrax zones. Also, there was no significant difference (McNemar's chi-square test = 0.9, p = .343) in the proportion of lions testing positive to anti-PA anthrax antibodies on ELISA-PA immunoassay compared with the TNA, with fair agreement between the two tests [kappa (K) statistic = 0.30; 0.08 < K<0.613]. Results of this study indicate that anthrax could be more widespread than 42 currently realized in Zimbabwe, and present in recognized high- and low-risk zones, including 43 where it has not been reported in over 20 years such as Hwange National Park. This is also the 44 first report documenting the presence of anthrax lethal toxin-neutralizing antibodies in naturally 45 infected carnivores, further confirming exposure to B. anthracis. The research results point to a 46 need for revisiting the currently recognized anthrax risk zones in Zimbabwe. This should be based 47 on improved surveillance of the disease in both wild and domestic animals for better understanding and control of the disease.

Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis.

Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive, less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected B. anthracis cultures.