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.

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.

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.

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.

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.

PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using "nested primers" increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

The estimated long-term impact of tsetse control on the size of the population of cattle in the Didessa Valley, western Ethiopia .

The long-term impact of tsetse control on cattle population size in the Didessa Valley, western Ethiopia, was analysed using an age-structured population model. A prior analytical assessment revealed that the risk of cattle dying in the tsetse-unprotected villages ranged from 4 to 9 times higher than in the tsetse-protected village. Model results show that during a period of 10 years the cattle population in the tsetse-protected village of Meti is likely to increase from 167 to 583 animals, while that in the adjacent tsetse-unprotected village of Gale remains almost constant. Model simulations also predict that improving the survival rate of calves in the tsetse-unprotected villages of Taikiltu and Temoloko (which presently have calf mortality rates of up to 35% would bring a substantial increase in their cattle population.

National survey of clinical and subclinical mastitis in Jamaican dairy herds, 1985-1986.

Between April 1985 and August 1986, 89 Jamaican dairy herds with 10 or more cows were visited, 1,645 lactating cows were examined using the CMT test and 254 composite milk samples collected for bacteriological examination. Widespread management faults were noted, especially of milking machine usage and maintenance and the abuse of antibiotics. Fifty-six per cent of all quarters were found to have CMT scores of one or higher, 0.8% showed clinical mastitis and 3.2% were blind. The most common bacterial pathogen Staphylococcus aureus, was recovered from 31% of sampled cows. The resultant milk loss from clinical and subclinical mastitis was estimated to be 20% of the potential national production.

Serological survey for leptospiral antibodies in goats in St Elizabeth Parish, Jamaica, 1985-1986.

A serological survey of goats in St Elizabeth Parish, Jamaica, comprising the breeding herd at the Agricultural Development Corporation (ADC) facility at Hounslow, five commercial herds and 21 family herds was carried out in 1985-1986. The Leptospira microscopic agglutination test indicated that one or more serovars in the Canicola serogroup were the most prevalent. Repeated samplings of goats at ADC, Hounslow demonstrated an increase in portlandvere and canicola seropositive animals following the onset of warmer weather and of icterohaemorrhagiae seropositive animals following the autumn rains and cooler weather. Apart from a cluster of family goats seropositive to pomona there was no geographical separation of reactors to the other serovars tested in the parish.

The financial and production impacts of bovine dermatophilosis in Zambia.

During October to December 1986, 365 traditional cattle herds in four provinces in Zambia were inspected and the owners interviewed. Information was collected on the treatment, management and disposal of cases of bovine dermatophilosis and on the effects of this disease on productivity for 1985. The contemporary financial cost per case of treatment and/or premature disposal, slaughter or death to the owners was K. 202 (US$91). The cost due to draft oxen being affected was estimated at K. 428 (US$193) per affected ox. The cost of reduced milk production, replacing affected cows and calf deaths, directly or indirectly from bovine dermatophilosis, was estimated at K. 132 (US$78) per affected milking cow. The total annual national cost of bovine dermatophilosis in 1985 was conservatively estimated to be some K. 6.9 million, (US$3 million). There were indications that the true financial cost in 1985 may have been up to 1.8 times the estimated cost.

The epidemiology of bovine dermatophilosis in Zambia.

Bovine dermatophilosis (Senkobo disease) has been reported annually in Zambia for many years. However, its epidemiology under Zambian conditions had never been adequately studied. Officially the disease has never been recognized as being of any economic consequence. A field study was designed and conducted from August to December 1986, to provide estimates of epidemiological statistics and other factors for the period January 1985 to December 1986 in four districts. These districts supported approximately 28% of the national cattle herd. The study was conducted in communally grazed herds as the disease was reportedly of little significance in commercial herds. A total of 365 herds containing 22,344 head of cattle were inspected and the owners interviewed; 286 herds (78.4%) and 1114 cattle (5.0%) were found to be affected. Rainy weather, vegetation type such as grass savannah, woodland savannah and thorny bush, ticks and biting flies were indicated as important factors in the appearance and course of dermatophilosis in Zambia.

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana.

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.

The cost of anaplasmosis in the Red River Plains and south-east areas of Louisiana.

Losses from morbidity and mortality due to anaplasmosis were assessed in beef and dairy cattle of the Red River Plains and south-east areas of Louisiana. The cost of treatment and prevention of anaplasmosis and losses in milk production and those due to deaths or culling were valued at $0.5 million for the year 1983 in these two areas. The cost of prevention included the use of tetracyclines in feed and salt-mineral supplements, injectable tetracycline, vaccination and an estimated 10% of the cost of insecticidal ear tags, sprays, pour-on applications and dust bags. The remainder of these costs and the total cost of vector control in dairy cattle was attributed to the control of fly annoyance. Treatment costs included veterinary fees and the owner's treatment costs, including labour costs.