20       Bacillus species

 

Most Bacillus species are large, Gram-positive, endospore-producing rods up to 10 μm in length. In smears from tissues or cultures, cells occur singly, in pairs or in long chains. The genus comprises more than 200 species with diverse characteristics. Bacillus species are catalase-positive, aerobic or facultatively anaerobic and, with the exception of Bacillus anthracis and B. mycoides, motile. Most species are saprophytes with no pathogenic potential. Bacillus anthracis is the most important pathogen in the group.

Because they produce highly resistant endospores, Bacillus species are widely distributed in the environment. In soil, endospores of B. anthracis can survive for more than 50 years. The ability to grow aerobically and to produce catalase distinguishes Bacillus species from clostridia, which are also Gram-positive, endospore-forming rods. Differentiation of Bacillus species is largely based on colonial characteristics and biochemical tests or on molecular methods.

The major disease conditions caused by bacteria in this group are listed in Table 20.1. Anthrax is the most important of these diseases. Bacillus licheniformis can be a significant cause of abortion in sheep and cattle and is associated with the feeding of poorly preserved silage or hay. Because they have some similar phenotypic characteristics, B. anthracis and B. cereus require careful differentiation (Table 20.2).

Table 20.1 Clinical manifestations of diseases caused by Bacillus anthracis and other Bacillus species.

Bacillus species	Susceptible species	Clinical manifestations
B. anthracis	Cattle, sheep	Fatal peracute or acute septicaemic anthrax
	Pigs	Subacute anthrax with oedematous swelling in pharyngeal region; an intestinal form with higher mortality is less common
	Horses	Subacute anthrax with localized oedema; septicaemia with colic and enteritis sometimes occurs
	Humans	Skin, pulmonary and intestinal forms of anthrax are recorded in humans periodically
B. cereus	Cattle	Mastitis (rare)
	Humans	Food poisoning, eye infections
B. licheniformis	Cattle, sheep	Sporadic abortion

Table 20.2 Differentiating features of Bacillus anthracis and B. cereus.

Feature	B. anthracis	B. cereus
Motility	Non-motile	Motile
Appearance on sheep blood agar	Non-haemolytic	Haemolytic
Susceptibility to penicillin (10 unit disc)	Susceptible	Resistant
Lecithinase activity on egg yolk agar	Weak and slow	Strong and rapid
Effect of gamma phage	Lysis	Lysis rare
Pathogenicity for animals (application to scarified area at tail base of mouse)	Death in 24–48 hours	No effect

Anthrax

Anthrax is a severe disease which affects virtually all mammalian species including humans and is an important agent of bioterrorism. The disease, which occurs worldwide, is endemic in some countries. Ruminants are highly susceptible, often developing a rapidly fatal septicaemic form of the disease. Pigs and horses are moderately susceptible to infection, while carnivores are comparatively resistant. Birds are almost totally resistant to infection.

Endospore formation is the most important factor in the persistence and spread of anthrax. Outbreaks of disease in herbivores can occur when pastures are contaminated by spores originating from buried carcasses. Spores may be brought to the surface by flooding, excavation, subsidence, or by the activity of earthworms. Concentration of spores may also occur in some geographical regions with alkaline soils and following repeated cycles of flooding and evaporation in low-lying areas. Sporadic outbreaks of disease in non-endemic areas have been associated with the importation of contaminated meat-and-bone meal, fertilizers of animal origin and hides. Infection in animals is usually acquired by ingestion of spores and, less commonly, by inhalation or through skin abrasions.

The virulence of B. anthracis derives from the presence of a capsule and the ability to produce a complex toxin. Both virulence factors are encoded by plasmids and are required for disease production. The capsule, composed of poly-D-glutamic acid, inhibits phagocytosis. The complex toxin consists of three antigenic components: protective antigen, oedema factor and lethal factor. Protective antigen functions as the host cell-binding moiety for both oedema factor and lethal factor. Oedema factor is an adenylate cyclase enzyme which increases cAMP levels in the cell with resultant disturbance of water homeostasis. Lethal factor is a zinc metalloprotease which targets macrophages, dendritic cells, neutrophils and some epithelial and endothelial cells. In naturally occurring disease, local effects of the complex toxin include swelling and darkening of tissues due to oedema and necrosis. When septicaemia occurs, increased vascular permeability and extensive haemorrhage lead to shock and death.

The incubation period of anthrax ranges from hours to days. In cattle and sheep, the disease is usually septicaemic and rapidly fatal. Although most animals are found dead without premonitory signs, pyrexia, with temperatures up to 42°C, depression, congested mucosae and petechiae may be observed antemortem. In cattle, postmortem findings include rapid bloating, incomplete rigor mortis, widespread ecchymotic haemorrhages and oedema, dark unclotted blood and blood-stained fluid in body cavities. An extremely large soft spleen is characteristic of the disease in cattle. Splenomegaly and oedema are less prominent postmortem features in affected sheep. In pigs, infection generally results in oedematous swelling of the throat and head along with regional lymphadenitis. Some affected pigs may survive.

Carcasses of animals which have died from anthrax are bloated, putrefy rapidly and do not exhibit rigor mortis. Dark unclotted blood may issue from the mouth, nostrils and anus. The carcasses of such animals should not be opened because this will facilitate sporulation with the risk of long-term environmental contamination. Peripheral blood from the tail vein of ruminants or peritoneal fluid from pigs should be collected into a sterile syringe. Thin smears of blood or fluid, stained with polychrome methylene blue, reveal chains of square-ended, blue-staining rods surrounded by a pink capsule (M'Fadyean reaction). Blood and MacConkey agars are inoculated with the suspect specimens and incubated aerobically at 37°C for 24–48 hours. Identification criteria for isolates include colonial morphology, microscopic appearance in Gram-stained smears, absence of growth on MacConkey agar and other test procedures (Table 20.2). New molecular diagnostic methods based on PCR to amplify specific virulence-related plasmid markers have been developed and include real-time PCR procedures which are highly sensitive. Bacillus anthracis isolates are highly clonal but can be differentiated into different phylogenetic groups based on techniques such as multi-locus variable number tandem repeat analysis, with lineage A being the most widely distributed worldwide. Further subdivision of groups using techniques based on single nucleotide polymorphisms is possible and is useful in epidemiological investigations.

If administered early in the course of the disease, high doses of penicillin G or oxytetracycline may prove effective. Suspect cases of anthrax must be reported immediately to appropriate regulatory authorities. In endemic regions, annual vaccination of sheep and cattle with the Sterne strain spore vaccine is advisable. In non-endemic regions, movement of animals, feed and bedding is prohibited following a disease outbreak. Personnel implementing control measures should wear protective clothing and footwear. Carcasses should be incinerated or buried deeply away from watercourses. Contaminated equipment should be disinfected with 10% formalin. In-contact animals should be isolated and kept under close observation for at least two weeks. Contaminated buildings should be sealed and fumigated with formaldehyde before removal of bedding. Following removal of fittings and bedding, the building should be sprayed with 5% formalin which should be left to act for 10 hours before final washing.

Three main forms of anthrax occur in humans. Cutaneous anthrax (malignant pustule) is the result of endospores entering abraded skin. If not treated promptly, this localized lesion can progress to septicaemia. Pulmonary anthrax (wool-sorters' disease) follows inhalation of spores, while intestinal anthrax results from the ingestion of infective material. In the absence of early treatment, the disease may prove fatal.