Peter C. B. Turnbull

Anthrax in animals

Anthrax is the archetype zoonosis; no other infectious disease affects such a wide range of species, including humans, although most susceptible are herbivorous mammals. Although the disease appears to have been recognized for centuries, it has yet to be established scientifically how animals contract it. While primarily a disease of warmer regions, it has long been spread to cooler zones through the trade of infected animals or contaminated animal products. Today it is still endemic in many countries of Africa and Asia and non-endemic countries must remain alert to the possibility of imports from such endemic areas resulting in outbreaks in their own livestock. The epidemiology of anthrax is becoming understood better with new systems coming on stream for distinguishing different genotypes and this is covered in detail. Clinical signs and pathology in animals are described.

Distribution and Molecular Evolution of Bacillus anthracis Genotypes in Namibia

The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP) and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983–2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA) and, in part, by twelve single nucleotide polymorphism (SNP) markers and four single nucleotide repeat (SNR) markers. A total of 37 genotypes (GT) were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate epidemiological relationships.

Effects Of Experimental Exclusion Of Scavengers From Carcasses Of Anthrax-Infected Herbivores On Bacillus Anthracis Sporulation, Survival, And Distribution

ABSTRACT Scavenging of anthrax carcasses has long been hypothesized to play a critical role in the production of the infectious spore stage of Bacillus anthracis after host death, though empirical studies assessing this are lacking. We compared B. anthracis spore production, distribution, and survival at naturally occurring anthrax herbivore carcasses that were either experimentally caged to exclude vertebrate scavengers or left unmanipulated. We found no significant effect of scavengers on soil spore density (P > 0.05). Soil stained with terminally hemorrhaged blood and with nonhemorrhagic fluids exhibited high levels of B. anthracis spore contamination (ranging from 103 to 108 spores/g), even in the absence of vertebrate scavengers. At most of the carcass sites, we also found that spore density in samples taken from hemorrhagic-fluid-stained soil continued to increase for >4 days after host death. We conclude that scavenging by vertebrates is not a critical factor in the life cycle of B. anthracis and that anthrax control measures relying on deterrence or exclusion of vertebrate scavengers to prevent sporulation are unlikely to be effective.

The fate of Bacillus anthracis in unpasteurized and pasteurized milk

Events following the inoculation of vegetative and spore forms of Bacillus anthracis into unpasteurized and pasteurized milk were monitored at 37°C, room temperature (RT; 20 × 2°C) and in a refrigerator (5–9°C). Counts of vegetative B. anthracis decreased rapidly in both unpasteurized and pasteurized milk, becoming unde‐tectable by 7 h at 37°C and by 24 h at RT and in the refrigerator. Spores in unpasteurized milk showed no significant signs of germination after 48 h. In pasteurized milk, there was a slight overall increase (1 log) in total and spore counts at 37°C; at RT there was an overall fall (1.5‐2 logs) and in the refrigerator, total counts remained steady while spore counts fell 1 log over a 48 h period.

Evaluation of the Biolog system for the identification of Bacillus anthracis

The potential of the Biolog system for the identification of Bacillus anthracis was evaluated. In‐house generated databases allowed the correct identification of 19 of 20 isolates of B. anthracis within 24 h. Five strains of the closely related B. cereus/thuringiensis group were misidentified as B. anthracis. For this reason the test could only serve as a primary screen with further testing being required to confirm identity. In addition 20% of all the strains of bacilli examined during the study gave unreadable reaction profiles due to false‐positive reactions.

Lethal exposure: An integrated approach to pathogen transmission via environmental reservoirs

To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1–2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways.

Research letterDifferentiation of Bacillus anthracis and other ‘Bacillus cereus group’ bacteria using IS231-derived sequences

Sequences based on the conserved 20 bp inverted repeat of IS231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group (B. anthracis, B. cereus, B. thuringiensis and B. mycoides), because of their close association with transposons, principally Tn4430 in B. thuringiensis. Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of 1S231 variants A-F. The same reaction conditions gave one or two bands for both B. anthracis and B. cereus that differed by restriction endonuclease mapping from the B. thuringiensis PCR product and established IS231 restriction maps; this does not preclude some kind of relationship between these products and IS231.

Co-infection of an animal with more than one genotype can occur in anthrax.

During the routine fingerprinting of outbreak strains of Bacillus anthracis of European and African origin by means of a 31‐marker multi‐locus variable number of tandem repeats analysis (MLVA), four cultures, two from the Etosha National Park (ENP), Namibia, and two from an outbreak in the Pyrenees in 1997, were found to harbour different genotypes (GTs). To investigate this further, isolates from 10 samples of blood‐soaked soil from beneath anthrax carcasses and 18 clinical swabs taken from carcasses in the ENP were examined by a 31‐marker MLVA. While only a single GT was found in any one of the 10 soil samples, four of the 18 swabs (22%) yielded different GTs. Two GTs were isolated from each of a zebra and a springbok and three GTs from each of a second zebra and an elephant. These animals had died in a region of the ENP where deaths caused by anthrax regularly occur every year. The results confirm the indications noted previously that co‐infection with more than one GT is probably not especially uncommon. The results show that, for the purpose of analysing genotypes involved in an outbreak, it is important to examine more than a single colony from a clinical sample.

Failure of Sterne- and Pasteur-Like Strains of Bacillus anthracis to Replicate and Survive in the Urban Bluebottle Blow Fly Calliphora vicina under Laboratory Conditions

This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4) known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7–9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

Genome Sequence of Bacillus anthracis STI, a Sterne-Like Georgian/Soviet Vaccine Strain

ABSTRACT The Bacillus anthracis strain STI is a Soviet vaccine strain that lacks the pXO2 plasmid. Previous data indicate that this isolate forms a new branch within the B. anthracis sub-group originally identified as A. Br.008/009.