Spatial clusters of human and livestock anthrax define high-risk areas requiring intervention in Lao Cai Province, Vietnam 1991-2022.

Anthrax, a widespread zoonosis in low and middle-income countries with low disease awareness and insufficient livestock vaccination coverage, has been known in Lao Cai Province in northern Vietnam for years before its apparent absence in 2009, which requires investigation as this infection is frequently reported from neighbouring provinces and countries. We aimed to describe the seasonal patterns of anthrax (1991-2008), compare livestock anthrax vaccine coverage to disease occurrence (1991- 2022), and delineate the high-risk areas to inform local disease surveillance in the province. We illustrated the seasonal pattern of anthrax and provided a comparison between livestock vaccine coverage and disease occurrence by purely spatial SaTScan (Poisson model, 25% population at risk) to detect spatial clusters of human and livestock anthrax using population derived from zonal statistics routines. The number of cases, crude cumulative incidence, and spatial clusters of human and livestock anthrax were mapped in QGIS. Results indicate peak anthrax incidence from May to October. Buffalo, domestic cattle, and horses accounted for 75% of total animal cases. Horse anthrax was more common in Lao Cai than in its neighbours and often occurred in years with human mortality. Vaccination covered less than 30% of the livestock population. We found an apparent pattern where anthrax was controlled from 1998-2003 with higher vaccine coverage (>20%) and identified spatial clusters of human and livestock anthrax in Muong Khuong, Bao Thang, and Bac Ha districts of Lao Cai. The local public health and veterinary agencies are recommended to revisit the high-risk areas and communicate with neighbouring provinces for a regional approach to anthrax surveillance and control.

Spatial epidemiology of human anthrax in Son La province, Vietnam, 2003-2022.

AIMS: Anthrax is reported with frequency but poorly understood in Southeast Asian countries including Vietnam. In Vietnam, anthrax surveillance is national. However, case detection, prevention, and control are implemented locally at the provincial level. Here, we describe the epidemiological characteristics, identify spatial clusters of human anthrax, and compare the variation in livestock anthrax vaccine coverage to disease incidence in humans and livestock using historical data in Son La province, Vietnam (2003-2020). METHODS AND RESULTS: Most human cases occurred between April and September. Most of the patients were male, aged 15-54 years old. The human cases were mainly reported by public district hospitals. There was a delay between disease onset and hospitalization of ~5 days. We identified spatial clusters of high-high incidence communes in the northern communes of the province using the local Moran's I statistic. The vaccine coverage sharply decreased across the study period. The province reported sporadic human anthrax outbreaks, while animal cases were only reported in 2005 and 2022. CONCLUSIONS: These results suggest underreporting for human and livestock anthrax in the province. Intersectoral information sharing is needed to aid livestock vaccination planning, which currently relies on reported livestock cases. The spatial clusters identify areas for targeted surveillance and livestock vaccination, while the seasonal case data suggest prioritizing vaccination campaigns for February or early March ahead of the April peak. A regional approach for studying the role of livestock trading between Son La and neighbouring provinces in anthrax occurrence is recommended.

Spatial analysis of human and livestock anthrax in Lai Chau province, Vietnam (2004-2021).

Anthrax is reported globally with varying disease intensity and seasonality among countries. In Vietnam, anthrax epidemiology and ecology remain understudied. We used historical data of human and livestock anthrax from 2004 to 2021 in Lai Chau province, to identify spatial clusters of human and livestock anthrax, describe epidemiological characteristics, and compare livestock anthrax vaccine coverage to human and livestock disease incidence. Local Moran's I (LISA) using spatial Bayes smoothed commune-level cumulative incidence (per 10,000) for the study period, epidemiological descriptive statistics, livestock vaccine coverage data, and annual incidence rates (per 10,000) at provincial level were used. LISA identified a human anthrax hotspot (high-high) in the southeast which did not overlap spatially with livestock anthrax hotspots in southeastern and northeastern communes. Most human cases were male, aged 15-59 years, handled sick animals, and/or consumed contaminated meat. Almost all cases were reported by grassroot health facilities with a delay of 6.3 days between exposure and case notification to the national surveillance system. 80 % of human cases were reported from June-October. The increase in disease incidence occurred shortly after livestock anthrax vaccine coverage decreased. This study informs vaccination strategy and targeted surveillance and control measures in newly identified high-risk areas and seasons of anthrax.

The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Spatial and phylogenetic patterns reveal hidden infection sources of Bacillus anthracis in an anthrax outbreak in Son La province, Vietnam.

Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

Genomic and Phylogenetic Analysis of Bacillus cereus Biovar anthracis Isolated from Archival Bone Samples Reveals Earlier Natural History of the Pathogen.

(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.

Blood analytes of clinically normal and diseased neonatal and weaned farmed white-tailed deer (Odocoileus virginianus) fawns.

Recent research focused on farmed deer has exposed many knowledge gaps regarding health assessment protocols for white-tailed deer (WTD). The objectives of this study were to establish de novo blood analyte reference intervals for farmed WTD fawns at birth (1-2 days of age; n = 84) and again at weaning (76-125 days of age; n = 28), to compare data at birth and at weaning to understand how these analytes are affected by the intrinsic factors age and sex in clinically normal WTD fawns, and to compare between clinically normal and sick WTD weanlings (respiratory disease n = 12; orbivirus-infected n = 6). Reference intervals were established for WTD fawns at birth and weaning. Female WTD neonates had significantly higher red blood cell counts, hematocrit, and hemoglobin compared to males. Most blood analytes were significantly different in clinically normal WTD neonates compared to weanlings, suggesting an effect of age. The observed sex- and age-related variations in WTD highlight the need to establish reference intervals that account for intrinsic factors. The comparison of clinically normal and sick WTD weanlings in this study identified higher MCHC and absolute monocytes in sick weanlings but these findings were presumably not biologically relevant given the small sample size for sick fawns. While the reference interval data presented herein will be useful for the veterinary care of WTD fawns at critical time periods in a high-density farm setting, this study also demonstrates the need to identify more sensitive and specific biomarkers for the assessment of health status in farmed WTD with specific underlying diseases.

Spatio-temporal patterns of malaria in Nepal from 2005 to 2018: A country progressing towards malaria elimination.

Nepal aims to eliminate malaria by 2026. This study analyzed district-level spatio-temporal patterns of malaria in Nepal from 2005 to 2018, following the introduction of Long-Lasting Insecticidal Nets (LLINs) for vector control intervention. The spatial variation in a temporal trend (SVTT) method in SaTScan was used to detect significantly high or low temporal trends of five malaria indicators: Indigenous, Imported, PV (Plasmodium vivax), PF (Plasmodium falciparum), and Total Malaria; results were mapped as clusters with associated trends. Spatial clusters of increasing malaria were found for all five indicators. Indigenous Malaria increased 113.71% in a cluster of three previously non-endemic mountainous districts. The most prominent cluster of Imported Malaria increased by 156.22%, and included the capital, Kathmandu. While some clusters had decreasing malaria, the rate of decrease in clusters was lower than outside the clusters. Overall, malaria burden is decreasing in Nepal as the country progresses closer to the elimination deadline. However, spatial clusters of increasing malaria, and clusters of lower rates of decreasing malaria, point to a need to focus vector control interventions on these clusters.

Incidence and Spatial distribution of Human and Livestock Anthrax in Cao Bang Province, Vietnam (2004-2020).

Specific knowledge on the distribution of anthrax, a zoonosis caused by Bacillus anthracis, in Southeast Asia, including Vietnam, remains limited. In this study, we describe disease incidence and spatial distribution of human and livestock anthrax using spatially smoothed cumulative incidence from 2004 to 2020 in Cao Bang province, Vietnam. We employed the zonal statistics routine a geographic information system (GIS) using QGIS, and spatial rate smoothing using spatial Bayes smoothing in GeoDa. Results showed higher incidence of livestock anthrax compared with human anthrax. We also identified co-occurrence of anthrax in humans and livestock in northwestern districts and the province center. Livestock anthrax vaccine coverage was <6% and not equally distributed among the districts of Cao Bang province. We provide implications for future studies and recommend improving disease surveillance and response through data sharing between human and animal health sectors.

Analyzing Spatial and Temporal Patterns of Designated Malaria Risk Areas in Nepal from 2018 to 2021.

Background: Nepal is preparing to eliminate malaria by 2026. To evaluate the progress of vector control and prioritize areas for targeted intervention, understanding the recent changing distribution of high and moderate malaria risk areas is vital. Methods: Patterns of designated high and moderate malaria risk wards in Nepal between 2018 and 2021 were analyzed to identify stable and newly generated high- and moderate-risk (HaMR) wards, using the Spatial Temporal Analysis of Moving Polygons (STAMP) method. Results and Conclusions: High-risk and moderate-risk wards decreased by about 55% and the number of districts containing these wards also decreased from 20 to 14. However, several stable and new HaMR wards, mostly in the northwest and the southwest of the country, are apparent, despite intervention efforts. Public health officials should prioritize those wards for malaria surveillance and vector control, and future studies should explore the underlying reasons for persistent risk wards.

Beyond the spore, the exosporium sugar anthrose impacts vegetative Bacillus anthracis gene regulation in cis and trans.

The Bacillus anthracis exosporium nap is the outermost portion of spore that interacts with the environment and host systems. Changes to this layer have the potential to impact wide-ranging physiological and immunological processes. The unique sugar, anthrose, normally coats the exosporium nap at its most distal points. We previously identified additional mechanisms rendering B. anthracis anthrose negative. In this work, several new ant - B. anthracis strains are identified and the impact of anthrose negativity on spore physiology is investigated. We demonstrate that live-attenuated Sterne vaccines as well as culture filtrate anthrax vaccines generate antibodies targeting non-protein components of the spore. The role of anthrose as a vegetative B. anthracis Sterne signaling molecule is implicated by luminescent expression strain assays, RNA-seq experiments, and toxin secretion analysis by western blot. Pure anthrose and the sporulation-inducing nucleoside analogue decoyinine had similar effects on toxin expression. Co-culture experiments demonstrated gene expression changes in B. anthracis depend on intracellular anthrose status (cis) in addition to anthrose status of extracellular interactions (trans). These findings provide a mechanism for how a unique spore-specific sugar residue affects physiology, expression and genetics of vegetative B. anthracis with impacts on the ecology, pathogenesis, and vaccinology of anthrax.

Informing One Health Anthrax Surveillance and Vaccination Strategy from Spatial Analysis of Anthrax in Humans and Livestock in Ha Giang Province, Vietnam (1999-2020).

Anthrax, caused by Bacillus anthracis, has a nearly global distribution but is understudied in Southeast Asia, including Vietnam. Here, we used historical data from 1999 to 2020 in Ha Giang, a province in northern Vietnam. The objectives were to describe the spatiotemporal patterns and epidemiology of human and livestock anthrax in the province and compare livestock vaccine coverage with human and livestock anthrax incidence. Annual incidence rates (per 10,000) for humans, buffalo/cattle, and goats were used to explore anthrax patterns and for comparison with livestock annual vaccine variations. A data subset describes anthrax epidemiology in humans by gender, age, source of infection, type of anthrax, admission site, and season. Zonal statistics and SaTScan were used to identify spatial and space-time clusters of human anthrax. SaTScan revealed space-time clusters in 1999, 2004, and 2007-2008 in the province, including in the northeastern, eastern, and western areas. Most human anthrax was reported between July and October. Most patients were male, aged 15-59 years, who had handled sick animals and/or consumed contaminated meat. High case-fatality rates were reported with gastrointestinal or respiratory cases. Our data suggest that vaccination in buffalo and cattle reduces the disease burden in humans and vaccinated animals but does not reduce the incidence in unvaccinated animals (goats). This study identified spatial areas of high risk for anthrax and can inform One Health surveillance and livestock vaccination planning in contextual settings similar to Ha Giang province.

Spatial analysis of human and livestock anthrax in Dien Bien province, Vietnam (2010-2019) and the significance of anthrax vaccination in livestock.

Anthrax is a serious zoonosis caused by Bacillus anthracis, which primarily affects wild herbivorous animals with spillover into humans. The disease occurs nearly worldwide but is poorly reported in Southeast Asian countries. In Vietnam, anthrax is underreported, and little is known about its temporal and spatial distributions. This paper examines the spatio-temporal distribution and epidemiological characteristics of human and livestock anthrax from Dien Bien province, Vietnam from 2010 to 2019. We also aim to define the role of livestock vaccination in reducing human cases. Historical anthrax data were collected by local human and animal health sectors in the province. Spatial rate smoothing and spatial clustering analysis, using Local Moran's I in GeoDa and space-time scan statistic in SaTScan, were employed to address these objectives. We found temporal and spatial overlap of anthrax incidence in humans and livestock with hotspots of human anthrax in the east. We identified three significant space-time clusters of human anthrax persisting from 2010 to 2014 in the east and southeast, each with high relative risk. Most of the human cases were male (69%), aged 15-59 years (80%), involved in processing, slaughtering, or eating meat of sick or dead livestock (96.9%) but environmental and unknown exposure were reported. Animal reports were limited compared to humans and at coarser spatial scale, but in areas with human case clusters. In years when livestock vaccination was high (>~25%), human incidence was reduced, with the opposite effect when vaccine rates dropped. This indicates livestock vaccination campaigns reduce anthrax burden in both humans and livestock in Vietnam, though livestock surveillance needs immediate improvement. These findings suggest further investigation and measures to strengthen the surveillance of human and animal anthrax for other provinces of Vietnam, as well as in other countries with similar disease context.

Malaria transmission in Nepal under climate change: anticipated shifts in extent and season, and comparison with risk definitions for intervention.

BACKGROUND: Climate and climate change affect the spatial pattern and seasonality of malaria risk. Season lengths and spatial extents of mapped current and future malaria transmission suitability predictions for Nepal were assessed for a combination of malaria vector and parasites: Anopheles stephensi and Plasmodium falciparum (ASPF) and An. stephensi and Plasmodium vivax (ASPV) and compared with observed estimates of malaria risk in Nepal. METHODS: Thermal bounds of malaria transmission suitability for baseline (1960-1990) and future climate projections (RCP 4.5 and RCP 8.5 in 2030 and 2050) were extracted from global climate models and mapped for Nepal. Season length and spatial extent of suitability between baseline and future climate scenarios for ASPF and ASPV were compared using the Warren's I metric. Official 2010 DoHS risk districts (DRDs) and 2021 DoHS risk wards (DRWs), and spatiotemporal incidence trend clusters (ITCs) were overlaid on suitability season length and extent maps to assess agreement, and potential mismatches. RESULTS: Shifts in season length and extent of malaria transmission suitability in Nepal are anticipated under both RCP 4.5 and RCP 8.5 scenarios in 2030 and 2050, compared to baseline climate. The changes are broadly consistent across both future climate scenarios for ASPF and ASPV. There will be emergence of suitability and increasing length of season for both ASPF and ASPV and decreasing length of season for ASPV by 2050. The emergence of suitability will occur in low and no-risk DRDs and outside of high and moderate-risk DRWs, season length increase will occur across all DRD categories, and outside of high and moderate-risk DRWs. The high and moderate risk DRWs of 2021 fall into ITCs with decreasing trend. CONCLUSIONS: The study identified areas of Nepal where malaria transmission suitability will emerge, disappear, increase, and decrease in the future. However, most of these areas are anticipated outside of the government's current and previously designated high and moderate-risk areas, and thus outside the focus of vector control interventions. Public health officials could use these anticipated changing areas of malaria risk to inform vector control interventions for eliminating malaria from the country, and to prevent malaria resurgence.

An Investigation of Burkholderia pseudomallei Seroprevalence in Market Pigs Slaughtered at Selected Pig Abattoirs in Uganda.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a disease of humans and animals. It is primarily transmitted through direct contact with contaminated soil and surface water. The epidemiology of this pathogen in Africa, including Uganda, is largely unknown. The objectives of this study were to estimate the seroprevalence of B. pseudomallei in pigs slaughtered in central Uganda and to identify potential hotspots for this pathogen in the country. A total of 1035 pig sera were analyzed for serological responses to B. pseudomallei with type A and type B LPS using OPS type A and OPS type B ELISAs. Of the 1035 samples, 75 (7.25%, 95% CI: 5.8-9%) were seropositive to the OPS-A ELISA using a two standard deviations (SD) cutoff and 19 (1.84%, 95% CI: 1.2-2.9%) at 3 SD. For the OPS-B ELISA, 93/1035 (8.99%, 95% CI: 7.4-10.9%) were seropositive at the 2 SD cutoff, and 28/1035 (2.71%, 95% CI: 1.9-3.9%) at the 3 SD cutoff. Pigs slaughtered in central Uganda were exposed to B. pseudomallei, and there is a higher seroprevalence in the rainy months. Public health awareness campaigns about melioidosis may be needed.

Spatiotemporal Patterns of Anthrax, Vietnam, 1990-2015.

Anthrax is a priority zoonosis for control in Vietnam. The geographic distribution of anthrax remains to be defined, challenging our ability to target areas for control. We analyzed human anthrax cases in Vietnam to obtain anthrax incidence at the national and provincial level. Nationally, the trendline for cases remained at ≈61 cases/year throughout the 26 years of available data, indicating control efforts are not effectively reducing disease burden over time. Most anthrax cases occurred in the Northern Midlands and Mountainous regions, and the provinces of Lai Chau, Dien Bien, Lao Cai, Ha Giang, Cao Bang, and Son La experienced some of the highest incidence rates. Based on spatial Bayes smoothed maps, every region of Vietnam experienced human anthrax cases during the study period. Clarifying the distribution of anthrax in Vietnam will enable us to better identify risk areas for improved surveillance, rapid clinical care, and livestock vaccination campaigns.

Geographical distribution and space-time clustering of human illnesses with major Salmonella serotypes in Florida, USA, 2017-2018.

Nontyphoidal salmonellosis is the leading reported foodborne illness in Florida. Although the diversity of Salmonella serotypes circulating in Florida has been identified, the geographical characteristics of the major serotypes are poorly described. Here we examined the geospatial patterns of 803 whole-genome sequenced Salmonella isolates within seven major serotypes (Enteritidis, Newport, Javiana, Sandiego, Braenderup, Typhimurium and I 4,[5],12:i:-) with the metadata obtained from Florida Department of Health during 2017-2018. Geographically, the distribution of incidence rates varied distinctively between serotypes. Illnesses with Enteritidis and Newport serotypes were widespread in Florida. The incidence rate for Javiana was relatively higher in the north compared to the south. Typhimurium was concentrated in the northwest, while I 4,[5],12:i:-, the monophasic variant of Typhimurium was limited to the south. We also evaluated space-time clustering of isolates at the zip code level using scan statistic models. Space-time clusters were detected for each major serotype during 2017-2018. The multinomial scan statistic found the risk of illness with Javiana was higher in the north and southwest in the fall of 2017 compared to other major serotypes. This serotype-specific clustering analysis will assist in further unpacking the associations between distinct reservoirs and illnesses with major serotypes in Florida.

Characterization of Bacillus anthracis replication and persistence on environmental substrates associated with wildlife anthrax outbreaks.

Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 105 spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.

Assays for Identification and Differentiation of Brucella Species: A Review.

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.