Environmental relationships and anthrax epidemiology: field experiences of host resistance as opposed to dose-dependent experiments.

Even though anthrax is a disease of antiquity that has been studied for centuries, serious concerns have been raised about our understanding of its epidemiology. Since the 1960s, we have based the epidemiology of anthrax on the results of dose-dependent experiments, especially those involving cattle at that time. In this species the experiments demonstrated that the severity of infection was dependent upon the numbers of Bacillus anthracis spores ingested. The opinion was that ingesting only a few spores would be insufficient to cause an apparent infection; any infection that resulted would be latent (i.e., unrecognized). Based on the results of these experiments, it was accepted that the ingestion of large numbers of spores was the source of infection for hundreds of anthrax outbreaks. However, many investigations of both human and animal anthrax outbreaks have failed to identify sources of large numbers of spores, suggesting that these outbreaks are only rarely a consequence of ingestion or inhalation of large quantities of spores. This opinion piece builds upon the indirect evidence previously presented in an article focused on the existence of latent infections. Much of the evidence for the existence of latent infections was predicated upon a reduction of host resistance, which revealed how latent infections could be a source of more severe forms of the infection. That is, a latent infection can be the source of a severe infection, but the cause of the severe infection is the reduced host resistance. That first article concentrated on the arguments for latent infections, while this article concentrates on the arguments for host resistance. Host resistance is virtually impossible to measure objectively in the field. To provide a subjective measure of host resistance during anthrax outbreaks, we suggest the use of the opinions of livestock owners and or their veterinary practitioners and or field workers during investigations of anthrax outbreaks. When veterinary personal work in the field they are much like field biologists. In some ways field biologists better appreciate environmental factors, population ecology and other perspectives that are of use to epidemiologists. The more diverse the information the better the epidemiology is understood. To this effect we present our personal anecdotal and theoretical ideas from our experiences as well as a collection of bibliographic observations from others'. Our conclusions are that a combination of latent infections and reduced host resistance based on the host's relationship with its environment would better explain the epidemiology of severe infections in anthrax outbreaks for which large quantities of spores have not been located. This applies especially if the area has a history of the disease and/or if necropsies have shown the presence of latent infections in otherwise normal animals in the area and/or if environmental conditions are considered stressful and include intense insect activity.

Investigation on Anthrax in Bangladesh during the Outbreaks of 2011 and Definition of the Epidemiological Correlations.

In 2011, in Bangladesh, 11 anthrax outbreaks occurred in six districts of the country. Different types of samples were collected from May to September in the six districts where anthrax had occurred in order to detect and type Bacillus anthracis (B. anthracis) strains. Anthrax was detected in 46.6% of the samples analysed, in particular in soils, but also in bone samples, water, animal feed, and rumen ingesta of dead animals. Canonical single nucleotide polymorphisms (CanSNPs) analysis showed that all the isolates belonged to the major lineage A, sublineage A.Br.001/002 of China and Southeast Asia while the multi-locus variable number of tandem repeats (VNTRs) analysis (MLVA) with 15 VNTRs demonstrated the presence of five genotypes, of which two resulted to be new genotypes. The single nucleotide repeats (SNRs) analysis showed 13 SNR types; nevertheless, due to its higher discriminatory power, the presence of two isolates with different SNR-type polymorphisms was detected within two MLVA genotypes. This study assumes that soil is not the only reason for the spread of the disease in Bangladesh; contaminated feed and water can also play an important role in the epidemiology of anthrax. Possible explanations for these epidemiological relationships are discussed.

A Review of Arguments for the Existence of Latent Infections of Bacillus anthracis, and Research Needed to Understand their Role in the Outbreaks of Anthrax.

Hugh-Jones and Blackburn and Turnbull's collective World Health Organization (WHO) report did literature reviews of the theories and the bases for causes of anthrax outbreaks. Both comment on an often-mentioned suspicion that, even though unproven, latent infections are likely involved. Hugh-Jones suggested Gainer do an updated review of our present-day knowledge of latent infections, which was the basis for Gainer's talk at the Biology of Anthrax Conference in Bari, Italy 2019. At the Conference Gainer met Vergnaud who presented anthrax genome studies that implied that the disease might have spread throughout Asia and from Europe to North America in a short time span of three or four centuries. Vergnaud wondered if latent infections might have played a role in the process. Several other presenters at the Conference also mentioned results that might suggest the existence of latent infections. Vergnaud subsequently looked into some of the old French literature about related observations, results, and discussions of early Pasteur vaccine usage (late 1800's) and found mentions of suspected latent infections. The first part of the paper is a focused summary and interpretation of Hugh-Jones and Blackburn's and Turnbull's reviews specifically looking for suggestions of latent infections, a few additional studies with slightly different approaches, and several mentions made of presentations and posters at the Conference in Italy. In general, many different investigators in different areas and aspects of the anthrax study at the Conference found reasons to suspect the existence of latent infections. The authors conclude that the affected species most studied, including Homo sapiens, provide circumstantial evidence of latent infections and modified host resistance. The last part of the review explores the research needed to prove or disprove the existence of latent infections.

Parkinson's Disease in Louisiana, 1999-2012: Based on Hospital Primary Discharge Diagnoses, Incidence, and Risk in Relation to Local Agricultural Crops, Pesticides, and Aquifer Recharge.

The two major causes of Parkinson's disease (PD) are genetic susceptibility and exposure to agricultural pesticides. Access to 23,224 individuals' hospital primary discharge diagnoses of PD allowed the mapping of cases against known crop distributions and pesticides. Our main objective was to map PD risks (cases per 10,000 people) against crops and their pesticides. The ZIP Code address locations, and the 2000 and 2010 census data, were used to map the risks of PD throughout Louisiana and in relation to United States Department of Agriculture (USDA)-recorded crops. The introduction of glyphosate-resistant crops appears to have initiated the PD disappearance from northeastern parishes on the west bank of the Mississippi river. Rice and sugar cane are seemingly unassociated with PD, as is the Mississippi itself, except for Jefferson and St. Charles Parishes, which are essentially urban or industrial. The present major PD-affected areas are associated with commercial forests, woodlands, and pastures, and thus with certain arbor-pastoral pesticides, 2,4-D, chlorpyrifos, and paraquat. Human populations at maximum risk are those living in areas of moderate and high aquifer-recharge potential. The levels of estimated pesticide exposure in these recharge areas we were able to access were of variable use, but significant amounts of 2,4-D were being used.

The global distribution of Bacillus anthracis and associated anthrax risk to humans, livestock and wildlife.

Bacillus anthracis is a spore-forming, Gram-positive bacterium responsible for anthrax, an acute infection that most significantly affects grazing livestock and wild ungulates, but also poses a threat to human health. The geographic extent of B. anthracis is poorly understood, despite multi-decade research on anthrax epizootic and epidemic dynamics; many countries have limited or inadequate surveillance systems, even within known endemic regions. Here, we compile a global occurrence dataset of human, livestock and wildlife anthrax outbreaks. With these records, we use boosted regression trees to produce a map of the global distribution of B. anthracis as a proxy for anthrax risk. We estimate that 1.83 billion people (95% credible interval (CI): 0.59-4.16 billion) live within regions of anthrax risk, but most of that population faces little occupational exposure. More informatively, a global total of 63.8 million poor livestock keepers (95% CI: 17.5-168.6 million) and 1.1 billion livestock (95% CI: 0.4-2.3 billion) live within vulnerable regions. Human and livestock vulnerability are both concentrated in rural rainfed systems throughout arid and temperate land across Eurasia, Africa and North America. We conclude by mapping where anthrax risk could disrupt sensitive conservation efforts for wild ungulates that coincide with anthrax-prone landscapes.

Epidemiology of Human Anthrax in China, 1955-2014.

Using national surveillance data for 120,111 human anthrax cases recorded during 1955-2014, we analyzed the temporal, seasonal, geographic, and demographic distribution of this disease in China. After 1978, incidence decreased until 2013, when it reached a low of 0.014 cases/100,000 population. The case-fatality rate, cumulatively 3.6% during the study period, has also decreased since 1990. Cases occurred throughout the year, peaking in August. Geographic distribution decreased overall from west to east, but the cumulative number of affected counties increased during 2005-2014. The disease has shifted from industrial to agricultural workers; 86.7% of cases occurred in farmers and herdsmen. Most (97.7%) reported cases were the cutaneous form. Although progress has been made in reducing incidence, this study highlights areas that need improvement. Adequate laboratory diagnosis is lacking; only 7.6% of cases received laboratory confirmation. Geographic expansion of the disease indicates that livestock control programs will be essential in eradicating anthrax.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Spatio-temporal patterns of an anthrax outbreak in white-tailed deer, Odocoileus virginanus, and associated genetic diversity of Bacillus anthracis.

BACKGROUND: Anthrax, a soil-borne zoonosis caused by the bacterium Bacillus anthracis, is enzootic in areas of North America with frequent outbreaks in west Texas. Despite a long history of study, pathogen transmission during natural outbreaks remains poorly understood. Here we combined case-level spatio-temporal analysis and high resolution genotyping to investigate anthrax transmission dynamics. Carcass locations from a single white-tailed deer, Odocoileus virginanus, outbreak were analyzed for spatial clustering using K-function analysis and directionality with trend surface analysis and the direction test. RESULTS: The directionalities were compared to results of high resolution genotyping. The results of the spatial clustering analyses, combined with deer movement data, suggest anthrax transmission events occur within limited spatial areas, with carcass locations occurring within the activity space of adjacent cases. The directionality of the outbreak paralleled adjacent dry river beds. Isolates from the outbreak were represented by a single genotype based on multiple locus variable number tandem repeat analysis (MLVA); four sub-genotypes were identified using single nucleotide repeat (SNR) analysis. CONCLUSIONS: Areas of high transmission agreed spatially with areas of higher SNR genetic diversity; however, SNRs did not provide clear evidence of linear transmission. Overlap of case home ranges provides spatial and temporal support for localized transmission, which may include the role of necrophagous or hematophagous flies in outbreaks in this region. These results emphasize the need for active surveillance and prompt cleanup of anthrax carcasses to control anthrax both during outbreaks and between seasons.

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

The necrophagous fly anthrax transmission pathway: empirical and genetic evidence from wildlife epizootics.

Early studies confirmed Bacillus anthracis in emesis and feces of flies under laboratory conditions, but there is little empirical field evidence supporting the roles of flies in anthrax transmission. We collected samples during outbreaks of anthrax affecting livestock and native and exotic wildlife on two ranches in West Texas (2009-2010). Sampling included animal carcasses, maggots, adult flies feeding on or within several meters of carcasses, and leaves from surrounding vegetation. Microbiology and PCR were used to detect B. anthracis in the samples. Viable B. anthracis and/or PCR-positive results were obtained from all represented sample types. Genetic analysis of B. anthracis samples using multilocus variable number tandem repeat analysis (MLVA) confirmed that each ranch represented a distinct genetic lineage. Within each ranch, we detected the same genotype of B. anthracis from carcasses, maggots, and adult flies. The results of this study provide evidence supporting a transmission cycle in which blowflies contaminate vegetation near carcasses that may then infect additional browsing animals during anthrax outbreaks in the shrubland environment of West Texas.

Classification and management of animal anthrax outbreaks based on the source of infection.

Anthrax is a non-contagious infectious disease; it primarily affects herbivores, but all mammals, including humans, can be affected. Humans may contract anthrax directly or indirectly from infected animals. Veterinary surveillance systems, providing information about animal and human cases, should increase the efficacy of the animal anthrax management in order to protect population. Any aspect of the disease should be carefully monitored to implement effective prevention and control strategies. In this paper we propose a new, detailed classification of anthrax outbreaks, based on the source of the infection and the risk level for humans. We describe three different types of animal outbreaks and suggest the most effective procedures for their management and prevention.

Ecological niche modeling of Bacillus anthracis on three continents: evidence for genetic-ecological divergence?

We modeled the ecological niche of a globally successful Bacillus anthracis sublineage in the United States, Italy and Kazakhstan to better understand the geographic distribution of anthrax and potential associations between regional populations and ecology. Country-specific ecological-niche models were developed and reciprocally transferred to the other countries to determine if pathogen presence could be accurately predicted on novel landscapes. Native models accurately predicted endemic areas within each country, but transferred models failed to predict known occurrences in the outside countries. While the effects of variable selection and limitations of the genetic data should be considered, results suggest differing ecological associations for the B. anthracis populations within each country and may reflect niche specialization within the sublineage. Our findings provide guidance for developing accurate ecological niche models for this pathogen; models should be developed regionally, on the native landscape, and with consideration to population genetics. Further genomic analysis will improve our understanding of the genetic-ecological dynamics of B. anthracis across these countries and may lead to more refined predictive models for surveillance and proactive vaccination programs. Further studies should evaluate the impact of variable selection of native and transferred models.

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

BACKGROUND: In 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. RESULTS: The 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. CONCLUSION: The 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.

Analysing the spatial patterns of livestock anthrax in Kazakhstan in relation to environmental factors: a comparison of local (Gi*) and morphology cluster statistics.

We compared a local clustering and a cluster morphology statistic using anthrax outbreaks in large (cattle) and small (sheep and goats) domestic ruminants across Kazakhstan. The Getis-Ord (Gi*) statistic and a multidirectional optimal ecotope algorithm (AMOEBA) were compared using 1st, 2nd and 3rd order Rook contiguity matrices. Multivariate statistical tests were used to evaluate the environmental signatures between clusters and non-clusters from the AMOEBA and Gi* tests. A logistic regression was used to define a risk surface for anthrax outbreaks and to compare agreement between clustering methodologies. Tests revealed differences in the spatial distribution of clusters as well as the total number of clusters in large ruminants for AMOEBA (n = 149) and for small ruminants (n = 9). In contrast, Gi* revealed fewer large ruminant clusters (n = 122) and more small ruminant clusters (n = 61). Significant environmental differences were found between groups using the Kruskall-Wallis and Mann-Whitney U tests. Logistic regression was used to model the presence/absence of anthrax outbreaks and define a risk surface for large ruminants to compare with cluster analyses. The model predicted 32.2% of the landscape as high risk. Approximately 75% of AMOEBA clusters corresponded to predicted high risk, compared with ~64% of Gi* clusters. In general, AMOEBA predicted more irregularly shaped clusters of outbreaks in both livestock groups, while Gi* tended to predict larger, circular clusters. Here we provide an evaluation of both tests and a discussion of the use of each to detect environmental conditions associated with anthrax outbreak clusters in domestic livestock. These findings illustrate important differences in spatial statistical methods for defining local clusters and highlight the importance of selecting appropriate levels of data aggregation.

Confirmation of Bacillus anthracis from flesh-eating flies collected during a West Texas anthrax season.

This case study confirms the interaction between necrophilic flies and white-tailed deer, Odocoileus virginianus, during an anthrax outbreak in West Texas (summer 2005). Bacillus anthracis was identified by culture and PCR from one of eight pooled fly collections from deer carcasses on a deer ranch with a well-documented history of anthrax. These results provide the first known isolation of B. anthracis from flesh-eating flies associated with a wildlife anthrax outbreak in North America and are discussed in the context of wildlife ecology and anthrax epizootics.

Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan.

To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937-2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region.

Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP) to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2) and 8 km(2) and a 6-variable BioClim data set at 8 km(2). Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2), approximately 34% loss at 8 km(2), and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2). Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B. anthracis.

The ecology of Bacillus anthracis.

The global distribution of anthrax is largely determined by soils with high calcium levels and a pH above 6.1, which foster spore survival. It is speculated that the spore exosporium probably plays a key part by restricting dispersal and thereby increasing the probability of a grazing animal acquiring a lethal dose. 'Anthrax Seasons' are characterized by hot-dry weather which stresses animals and reduces their innate resistance to infection allowing low doses of spores to be infective. Necrophagic flies act as case-multipliers and haemophagic flies as space-multipliers; the latter are aided by climatic factors which play a key part in whether epidemics occur. Host death is a function of species sensitivity to the toxins. The major function of scavengers is to open the carcass, spill fluids, and thereby aid bacilli dispersal and initiate sporulation. In the context of landscape ecology viable spore distribution is a function of mean annual temperature, annual precipitation, elevation, mean NDVI, annual NDVI amplitude, soil moisture content, and soil pH.

Inhalation anthrax: dose response and risk analysis.

The notion that inhalation of a single Bacillus anthracis spore is fatal has become entrenched nearly to the point of urban legend, in part because of incomplete articulation of the scientific basis for microbial risk assessment, particularly dose-response assessment. Risk analysis (ie, risk assessment, risk communication, risk management) necessitates transparency: distinguishing scientific facts, hypotheses, judgments, biases in interpretations, and potential misinformation. The difficulty in achieving transparency for biothreat risk is magnified by misinformation and poor characterization of both dose-response relationships and the driving mechanisms that cause susceptibility or resistance to disease progression. Regrettably, this entrenchment unnecessarily restricts preparedness planning to a single response scenario: decontaminate until no spores are detectable in air, water, or on surfaces-essentially forcing a zero-tolerance policy inconsistent with the biology of anthrax. We present evidence about inhalation anthrax dose-response relationships, including reports from multiple studies documenting exposures insufficient to cause inhalation anthrax in laboratory animals and humans. The emphasis of the article is clarification about what is known from objective scientific evidence for doses of anthrax spores associated with survival and mortality. From this knowledge base, we discuss the need for future applications of more formal risk analysis processes to guide development of alternative non-zero criteria or standards based on science to inform preparedness planning and other risk management activities.