A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule.

The simple polychrome methylene blue (PMB) staining procedure for blood or tissue smears from dead animals (M'Fadyean reaction) established in 1903 remained accepted as a highly reliable, rapid diagnostic test for anthrax for six decades while that disease was still common in livestock throughout the world. Improvements in disease control led to anthrax becoming rare in industrialized countries and less frequent in developing countries with the result that quality controlled, commercially produced PMB became hard to obtain by the 1980s. Mixed results with alternative methylene blue-based stains then led to diagnosis failures, confusion among practitioners and mistrust of this procedure as a reliable test for anthrax. We now report that, for laboratories needing a reliable M'Fadyean stain at short notice, the best approach is to have available commercially pure azure B ready to constitute into a solution of 0.03 g azure B in 3 ml of 95% ethanol or methanol to which is then added 10 ml of 0.01% KOH (0.23% final azure B concentration) and which can then be used immediately and through to the end of the tests. Stored in the dark at room temperature, the shelf life is at least 12 months. Smears should be fixed with ethanol or methanol (95-100%), not by heat, and the stain left for 5 min before washing off for optimum effect.

Bangladesh anthrax outbreaks are probably caused by contaminated livestock feed.

In Bangladesh from 1 July to 30 September 2010 there were 104 animal cases of anthrax and 607 associated human cases. This investigation was conducted in Sirajganj district in December 2010, on eight farms where animal cases had occurred. Bacillus anthracis was recovered from soil samples and turbinate bones on six farms. Canonical single nucleotide polymorphism (SNP) analysis showed that all the isolates belonged to the major lineage A, sublineage A.Br.001/002 of China and South East Asia while a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) with 15 VNTRs demonstrated three unique genotypes. The single nucleotide repeat (SNR) analyses showed two SNR types in 97 out of 99 isolates; nevertheless, due to its higher discriminatory power the presence of two isolates with different SNR-type polymorphisms were detected within two MLVA genotypes. The epidemic occurred during the monsoon season, a time of extensive flooding, suggesting that the source was contaminated feed, not grazing, which is supported by the genetic variance.

Accidental and intentional animal disease outbreaks: assessing the risk and preparing an effective response.

Intentional animal disease outbreaks are infrequent, if not rare, yet they carry the potential for disastrous consequences. Normal but accidental outbreaks are not uncommon and they must be handled efficiently, effectively and economically. And whatever its origin a disease will then follow its usual epidemiology. Therefore, the effectiveness in dealing with the normal, and the practice, experience and confidence gained, will significantly aid a country in how it minimises the cost of an intentional disease outbreak. The response is what determines the financial and economic costs of a disease outbreak. This paper provides an overview of the various threats, targets, and possible government responses, all of which is then expanded upon in detail in the other papers in this issue of the Review.

Distinguishing between natural and unnatural outbreaks of animal diseases.

An intentional outbreak of disease among livestock, or agricultural crops, will share a number of characteristics with those aimed at humans - unexpected pattern of disease in season or place, possible explosive incidence, unusual pathogen either in itself or of its genetic structure, difficult diagnosis - but there will also be notable differences: human cases, if they occur, will be coincidental and the major impacts will be delayed and of severe economic consequence. An investigative and analytical protocol is proposed for identifying such an event. Unless the nature of the event is self-declaring, such investigations necessitate a very thorough and careful investigation by a dedicated and experienced epidemiologic team. Atthe same time a country should take steps in advance of such an event to be prepared and to save time later, such as determining possible targets, identifying early warning indicators, establishing molecular biologic expertise and reference collections of possible pathogens, and preparing a tactical and forensic response.

Retrospective characterization of Newcastle Disease Virus Antrim '73 in relation to other epidemics, past and present.

In November 1973 Newcastle disease suddenly appeared in Northern Ireland, where the viscerotropic disease had not been seen in 3 1/2 years and the two Irelands had been regarded as largely disease free for 30 years. It was successfully controlled with only 36 confirmed affected layer flocks, plus 10 more slaughtered as 'dangerous contacts'. Contemporary investigations failed to reveal the source of the Irish epidemic. Using archival virus samples from most of the affected flocks, RT PCR was conducted with primers selected for all six NDV genes. Phylogenetic analyses of three genes, HN, M and F, confirmed vaccine as the cause of one of the outbreaks. The other six samples were identical and closely related to previous outbreaks in the United States and western Europe initiated by infected imported Latin American parrots. The probable cause of the epidemic followed from the importation from The Netherlands of bulk feed grains contaminated with infected pigeon faeces.

Emergency response planning for anthrax outbreaks in bison herds of northern Canada: a balance between policy and science.

Anthrax outbreaks in northern Canada have implications for ongoing recovery efforts for the threatened wood bison and may pose a health risk to humans, other wildlife, and domestic livestock. RWED and WBNP maintain Anthrax Emergency Response Plans (AERPs) for their respective jurisdictions. An AERP is a pre-planned logistical framework for responding effectively and rapidly to an outbreak so as to minimize spread of the disease, reduce environmental load of spores available for future outbreaks, and minimize risk to public health. In this paper, we describe the main components of an AERP and outline areas for future research.

Anthrax and wildlife.

Although livestock anthrax is declining in many parts of the world, with an increasing number of countries probably truly free of the disease, anthrax remains enzootic in many national parks and even in some game ranching areas. These infected areas can present a persistent risk to surrounding livestock, which may otherwise be free of the disease, as well as a public health risk. The authors use as examples the national parks in southern Africa, the Wood Buffalo National Park in northern Alberta, Canada, and the deer ranching counties in south-west Texas, United States of America, to present the range of problems, epidemiology, and control procedures. While many advances have been achieved in the understanding of this disease, research is required into the genotypic grouping of anthrax isolates, improved field diagnostic techniques, and oral vaccines, as well as to provide a better understanding of spore survival in soil and the ecology of the disease under natural conditions.

Geographical information systems as a tool in epidemiological assessment and wildlife disease management.

Geographical information systems (GIS) facilitate the incorporation of spatial relationships into epidemiological investigations of wildlife diseases. Consisting of data input, management, analysis and presentation components, GIS act as an integrative technology in that a range of very varied data sources can be combined which describe different aspect of the environment of wild animals. The analytical functionality of GIS is still evolving, and ranges from visual to exploratory and modelling methods. Output generated by GIS in map format has the particular advantage of allowing implicit representation of spatial dependence relationships in an intuitive manner. The technology is becoming an essential component of modern disease surveillance systems.

The statistical analysis of truncated data: application to the Sverdlovsk anthrax outbreak.

An outbreak of anthrax occurred in the city of Sverdlovsk in Russia in the spring of 1979. The outbreak was due to the inhalation of spores that were accidentally released from a military microbiology facility. In response to the outbreak a public health intervention was mounted that included distribution of antibiotics and vaccine. The objective of this paper is to develop and apply statistical methodology to analyse the Sverdlovsk outbreak, and in particular to estimate the incubation period of inhalational anthrax and the number of deaths that may have been prevented by the public health intervention. The data available for analysis from this common source epidemic are the incubation periods of reported deaths. The statistical problem is that incubation periods are truncated because some individuals may have had their deaths prevented by the public health interventions and thus are not included in the data. However, it is not known how many persons received the intervention or how efficacious was the intervention. A likelihood function is formulated that accounts for the effects of truncation. The likelihood is decomposed into a binomial likelihood with unknown sample size and a conditional likelihood for the incubation periods. The methods are extended to allow for a phase-in of the intervention over time. Assuming a lognormal model for the incubation period distribution, the median and mean incubation periods were estimated to be 11.0 and 14.2 days respectively. These estimates are longer than have been previously reported in the literature. The death toll from the Sverdlovsk anthrax outbreak could have been about 14% larger had there not been a public health intervention; however, the confidence intervals are wide (95% CI 0-61%). The sensitivity of the results to model assumptions and the parametric model for the incubation period distribution are investigated. The results are useful for determining how long antibiotic therapy should be continued in suspected anthrax cases and also for estimating the ultimate number of deaths in a new outbreak in the absence of any public health interventions.

Geographic information system-aided analysis of factors associated with the spatial distribution of Echinococcus multilocularis infections of foxes.

To investigate the influence of environmental factors on the spatial epidemiology of infections with Echinococcus multilocularis, foxes were sampled in a focal endemic region in the Northwest of Brandenburg, Germany, and examined for infection by the parasite. The locations where foxes were obtained were recorded in a geographic information system database. Positions of infected and uninfected foxes were analyzed on the background of geographic vector data of water, settlements, streets, forests, crop, and pasture. Fox positions were allocated to different land-use classes by use of a Landsat Thematic Mapper (TM) satellite image. Infected foxes were more frequently shot near water, in areas of high soil humidity, and on pastures, suggesting that dryness may limit the tenacity of E. multilocularis oncospheres. Thus open landscapes with humid soil seem to be favorable for the life cycle of the parasite. In contrast, infected foxes were significantly underrepresented in forest areas.

Bacillus anthracis diversity in Kruger National Park.

The Kruger National Park (KNP), South Africa, has a recorded history of periodic anthrax epidemics causing widespread disease among wild animals. Bacillus anthracis is the causative agent of anthrax, a disease primarily affecting ungulate herbivores. Worldwide there is little diversity among B. anthracis isolates, but examination of variable-number tandem repeat (VNTR) loci has identified six major clones, with the most dissimilar types split into the A and B branches. Both the A and B types are found in southern Africa, giving this region the greatest genetic diversity of B. anthracis worldwide. Consequently, southern Africa has been hypothesized to be the geographic origin of B. anthracis. In this study, we identify the genotypic types of 98 KNP B. anthracis isolates using multiple-locus VNTR analysis. Two major types are evident, the A branch and the B branch. The spatial and temporal distribution of the different genotypes indicates that anthrax epidemic foci are independent, though correlated through environmental cues. Kruger B isolates were found on significantly higher-calcium and higher-pH soils than were Kruger type A. This relationship between genotype and soil chemistry may be due to adaptive differences among divergent anthrax strains. While this association may be simply fortuitous, adaptation of A types to diverse environmental conditions is consistent with their greater geographic dispersal and genetic dissimilarity.

Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis.

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

What and how do we communicate?

The rapid, inexpensive transmission of words and data, brought about by the internet and cheap computers, is changing the world faster than anything mankind has ever experienced, reaching into nearly every aspect of our lives, public and private, commercial and governmental. We are in an Information Age with an essentially instantaneous availability of large volumes of information and data. Technological pressures have their own momentum. The changes and improvements in data handling and information sharing will take place, however much or little we do in veterinary medicine, whether at the international or national levels. In addition, there are new superpowers emerging in the world. These are the small and medium third world countries that are gradually gaining democracy through political restructuring and involving the civil society in decision making. There are also the marginal pressure groups, once too small and insignificant to be noticed. These are changing the rules by which the way the world is governed and they are doing it now by the internet and banding together to serve their common interests. The rapid provision of accurate information on animal diseases is desirable goal from a public health as well as an economic viewpoint.

Meso-scale ecology of anthrax in southern Africa: a pilot study of diversity and clustering.

It has only recently been possible to detect sufficient genetic diversity among anthrax isolates to allow genotype grouping (Keim et al. 1997). Early results of such grouping suggest that the southern African subcontinent may be the geographical origin of Bacillus anthracis. This report describes a pilot investigation of the genetic diversity of a study group of isolates from the Kruger National Park, South Africa, and efforts to detect spatio-temporal clustering within the study group. This study has also served as further validation for the newly developed Multi-Locus VNTR Analysis (MLVA), designed to simplify genotyping of B. anthracis isolates. The results reveal a diverse range of genotypes within the park allied with three genotype reference groups, and show that the MLVA procedure is robust for rapid analysis of B. anthracis genotypes. We also observed multiple genotype groups within epidemics and between geographically and temporally close epidemic episodes. This is in contrast to earlier characterizations of anthrax epidemics. The result of a Mantel test for time-space clustering indicates clustering of the anthrax isolates selected for the study.

Molecular diversity in Bacillus anthracis.

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.

World Health Organization activities on anthrax surveillance and control.

The achievements of a World Health Organization Anthrax Working Group, established in 1990, have been the production of two editions of guidelines on anthrax surveillance and control and the formulation of templates to assist countries in the construction of their surveillance and control programmes. The latter was made possible by the active participation of the Department of Animal Production and Health, Ministry of Agriculture, Food and Fisheries, Zambia and the Livestock Development Programme, Mongu, Western Province, Zambia in a model country programme designed by the Working Group for the purpose. This paper elaborates on these achievements, particularly the lessons learned from the experience of Western Province, Zambia.

1996-97 Global Anthrax Report.

While there is a general decrease in the number of anthrax outbreaks, and thus of human cases, worldwide this is still a disease that is extensively under-diagnosed and under-reported. However, it is now very infrequent to rare in Canada, the United States, and many countries in Europe. An increasing number of countries are now free. At the other extreme, it is a significant problem in West Africa, Spain, Greece, Turkey, Albania, Romania and in Central Asia. In spite of the textbooks, livestock and wildlife deaths do occur, sometimes commonly, without any 'diagnostic' extravasation of blood and, if not realised, infected carcasses get recycled into meat and bone meals for feed.

Genetic diversity in the protective antigen gene of Bacillus anthracis.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.

Evaluation of alternative strategies to prevent Newcastle disease in Cambodia.

Velogenic viscerotropic Newcastle disease (vvNCD), which is endemic in Cambodia, can be prevented in theory by a combination of biosecurity and immunization of broiler flocks. The relative contribution of appropriate biosecurity and effective vaccination was quantified at the farm level, applying realistic projections for capital investment, fixed and variable production costs and losses following infection. Non-protected broiler flocks generate a loss when the probability of vvNCD infection exceeds 0.4. Applying both biosecurity and effective vaccination would sustain profitability up to a probability of exposure of 1.0. The benefit to cost ratios for alternative strategies were evaluated for a range of probabilities of exposure to vvNCD extending from 0.1 to 1.0. The benefit-to-cost ratio for biosecurity exceeded unity at a risk of exposure exceeding 0.1, and 0.2 for vaccination and the combination of vaccination and biosecurity respectively. A sensitivity analysis demonstrated that the efficiency of protection, feed cost, and financial consequences of infection markedly affected the projected benefit-to-cost ratios associated with alternative methods of prevention.