Bacillus

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Bacillus:

Bacillus DR SMINA 1/21/2013 1

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Scientific classification Domain: Bacteria Division: Firmicutes Class: Bacilli Order: Bacillales Family: Bacillacea Genus :Bacillus 1/21/2013 2

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Numerous, including: B. alcalophilus B. alvei B. aminovorans B. amyloliquefaciens B. aneurinolyticus B. anthracis B. aquaemaris B. brevis 3

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B. caldolyticus B. centrosporus B. cereus B. circulans B. coagulans B. firmus B. flavothermus B. fusiform 1/21/2013 4

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B. globigii B. infernus B. larvae B. laterosporus B. lentus B. licheniformis B. megaterium B. mesentericus 1/21/2013 5

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B. mucilaginosus B. mycoides B. natto B. pantothenticus B. polymyxa B. pseudoanthracis B. pumilus 1/21/2013 6

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B. schlegelii B. sphaericus B. sporothermodurans B. stearothermophilus B. subtilis B. thermoglucosidasius B. thuringiensis B. vulgatis B. weihenstephanensis 1/21/2013 7

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Sporogenous , rod shaped aerobic bacteria Form heat resistant spores Gram positive-get decolourised easily Motile- peritrichous flagella 1/21/2013 8

Definition:

Definition The production of resistant endospores in the presence of oxygen remains the defining feature for Bacillus 1/21/2013 9

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The definition was undermined by the discoveries of Bacillus infernus and B. arseniciselenatis  strictly anaerobic Spores not observed in B. halodenitrificans , B. infernus , B. selenitireducens , B. subterraneus , and B. thermoamylovorans . 1/21/2013 10

Taxonomy:

Taxonomy Bacillus divided better-defined groups on the basis of 16S rRNA gene sequencing studies So far, ten new genera have been proposed 1/21/2013 11

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Alicyclobacillus  six species of thermoacidophiles Paenibacillus  49 species Brevibacillus  12 species Virgibacillus Gracilibacillus 1/21/2013 12

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Salibacillus  two species of halophiles Geobacillus  ten species of thermophiles including Bacillus stearothermophilus Ureibacillus with two round- spored , thermophilic species Single - membered Marinibacillus Salibacillus has subsequently been merged with Virgibacilius 1/21/2013 13

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Bacillus continues to accommodate the best-known species such as B. subtilis , B. anthracis B. cereus , B. licheniformis , B. megaterium , B. pumilus , B. sphaericus , and B. thuringiensis . It still remains a large genus, with over 90 species. 1/21/2013 14

Habitats:

Habitats Most aerobic endospore -formers  saprophytes in the natural environment Some species are opportunistic or obligate pathogens animals humans other mammals, and insects 1/21/2013 15

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Habitats Soils of all kinds, ranging from acid to alkaline, hot to cold, and fertile to desert Water columns and bottom deposits of fresh and marine waters. 1/21/2013 16

Importance of the aerobic endospore-formers:

Importance of the aerobic endospore -formers Most species of aerobic endospore -forming bacteria have little or no pathogenic potential rarely associated with disease in humans and other animals. 1/21/2013 17

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The main exceptions are B. anthracis (anthrax) B. cereus  food poisoning and opportunistic infections B. thuringiensis , P. larvae and its subspecies, P. lentimorbus and P. popilliae (all pathogenic to invertebrates) 1/21/2013 18

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spores resistant to Radiation Heat Disinfectants, and desiccation Bacillus species troublesome contaminants in the -operating room On surgical dressings In pharmaceutical products, and in foods. 1/21/2013 19

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several aerobic endosporeformers are of clinical or health importance in very positive ways In the production of antibiotics Bacitracin from B. licheniformis or B. subtilis Gramicidin from Brevibacillus brevis Polymyxin from Paenibacillus polymyxa 1/21/2013 20

Vitamins :

Vitamins Vitamins B 12 and B 2 from B. megaterium , Biotin and riboflavin from B. subtilis 1/21/2013 21

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As the bases of antibiotic assays B. cereus , B. circulans , B. megaterium , B. pumilis , B. subtilis , and G.stearothermophilus 1/21/2013 22

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In the validation of disinfectants ( B. cereus ) Monitoring of fumigation ( B. subtilis ) Heat sterilization ( G. stearothermophilus ) Radiation ( B. pumilus ) 1/21/2013 23

In various clinical tests:

In various clinical tests Uric acid assay using B. fastidiosus Chlamydia detection assay - B. subtilis Blood screening test for phenylketonuria utilizing B. subtilis Bacillus species are the active ingredients of probiotics for animals and humans 1/21/2013 24

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Bacillus species  commercial importance in the production of industrial enzymes The largest consumer is the detergent industry,  one third of the global market in such products Proteases and amylases it uses are all of Bacillus origin 1/21/2013 25

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Bacillus thuringiensis - biopesticide The genes for the δ- endotoxins of B thuringiensis - development of transgenic crop plants Strains of several species show promise as agents to control fungal diseases of plants 1/21/2013 26

Peanut leaves-Bt toxin:

Peanut leaves-Bt toxin 1/21/2013 27

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Spore formers -important deleterious agents and contaminants in the food industry B. coagulans , B.licheniformis , B. subtilis , G. stearothermophilus , and P.macerans -  spoilage of evaporated milk, in which fermentation occurs without gas production 1/21/2013 28

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P. macerans and P. polymyxa – produce both acid and gas- canned food swells and cheese defects. B. cereus causes problems in the dairy industry – it is a regular contaminant of milk, and it causes ‘bitty cream defect ’and ‘sweet curdling ’. B. subtilis causes ‘rope ’in bread, which appears as slimy stringiness when the loaf is cut 1/21/2013 29

The endospore:

The endospore It was independently discovered by Cohn -1876, Koch -1876 and Tyndall The ability to form endospores in aerobic conditions  a defining character of the genus Bacillus since the 1920s 1/21/2013 30

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Endospores are so named - formed intracellularly Optically refractile Highly resistant to chemical and physical stresses that are lethal to vegetative cells 1/21/2013 31

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It is the spores of Bacillus subtilis  most intensively studied Especially those of strain 168 The first Bacillus strain to have its genome sequenced 1/21/2013 32

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Endospores formed at the end of exponential growth phase Spores will not be formed under all cultural conditions Cultures may die if conditions are not conducive to sporulation 1/21/2013 33

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One trigger for sporulation is nutritional deprivation Sporulation may be initiated by transferring an actively growing culture from a rich to a poor growth medium 1/21/2013 34

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Man environmental factors affect endospore formation These include Growth temperature Environmental pH Aeration Presence of certain minerals, carbon, nitrogen and phosphorus sources and their concentrations 1/21/2013 35

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Second influence is population density Sporulation is poor at low cell densities, even though the cells may be starved 1/21/2013 36

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Mass of a culture increases Extracellular accumulation of a secreted peptide (competence and sporulation factor (CSF)) Acts as an auto-inducer for quorum sensing High intracellular levels of CSF Increase of the phosphorylated form of a response regulator ( SpoOA ) Leads to derepression of various stationary phase genes, some of which are needed for sporulation 1/21/2013 37

Bacillus anthracis:

Bacillus anthracis 1. first pathogenic bacterium to be seen under microscope-Pollender,1849 2.first communicable disease to be transmitted by inoculation of infected blood-Davaine,1850 3.first bacterium to be isolated in pure culture and shown to possess spores-Kosh,1876 4. first bacterium used for preparation of attenuated vaccine(Pasteur,1881) 1/21/2013 38

BACILLUS ANTHRACIS:

BACILLUS ANTHRACIS morphology large (4-8 × 1-1.5 μm ) non-motile, sporing bacillus. spores form readily - artificial media,or soil oval, refractile and central in position. The temperature range for growth is 12-45°C (optimum 35°C); 1/21/2013 39

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In tissues –singly, pairs or in short chains Entire chain surrounded by a capsule Capsule –polypeptide in nature-polymer d- glutamic acid Capsule formed- bicarbonate added or incubated with10-25 % co 2 serum, albumin, charcoal , starch 1/21/2013 40

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Blood films containing anthrax bacilli stained with polychrome methylene blue amorphous purplish material seen around –capsular material- M , Fadyeans reaction 1/21/2013 41

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In cultures, bacilli arranged end to end in long chains 1/21/2013 42

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Ends of bacilli –truncated or concave and swollen- bamboo stick appearance 1/21/2013 43

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Sporulation occurs under unfavourable conditions Distilled water,2% Nacl , oxalated agar Inhibited by calcium chloride 1/21/2013 44

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Spores do not stain by ordinary methods-need special differential stains Sudan black stain-fat globules seen inside bacilli 1/21/2013 45

Cultural charecteristics:

Cultural charecteristics Aerobe and facultative anaerobe Optimum temp-35-37 0 C Optimum temp –sporulation25-30 0 C Growth occurs on ordinary media 1/21/2013 46

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on agar plates-irregularly round colonies 2-3 mm diameter, raised dull opaque greyish white frosted glass appearance 1/21/2013 47

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Blood agar media-non haemolytic colonies 1/21/2013 48

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Under low power- edge of colony –long interlacing chains of bacilli-resembling locks of matted hair -Medusa head appearance 1/21/2013 49

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Virulent capsulated strains form rough cultures Avirulent strains form smooth colonies 1/21/2013 50

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On gelatin stab culture -inverted fir tree appearance with slow liquefaction commencing from the top 1/21/2013 51

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In broth occurs as floccular deposit with no turbidity When bacillus anthraxis grown on solid medium containing 0.05-.50 u of penicillin/ ml,in 3-6 hrs cells become large spherical and occur in chains – string of pearls reaction 1/21/2013 52

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A selective medium –PLET – Polymyxin,lysozyme,EDTA , Thallous acetate added heart infusion agar-isolate bacillus anthracis from mixtures of othrer spore bearing bacilli 1/21/2013 53

Biochemical reactions:

Biochemical reactions Glucose,maltose,sucrose are fermented producing acid, no gas Nitrate reduced to nitrite Catalase is formed 1/21/2013 54

resistance:

resistance Bacilli killed at 60 0 C in 30 mts In dead animals, bacilli remain viable in bone marrow for 1 wk,skin for 2 wks. Spores highly resistant 4% KMno 4 kills in 15 mts 1/21/2013 55

duckering:

duckering Destruction of spores in animal products when imported in to non endemic countries Formaldehyde 2%, 30-40 0 C,20 mts  wool 0.25%, 60 0 C , 6 hrs  hairs, bristles 1/21/2013 56

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The pathogenicity of B. anthracis depends primarily on two major virulence factors the poly- d - glutamic acid capsule the toxin complex comprising three proteins - the protective antigen , oedema factor and lethal factor . 1/21/2013 57

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The capsule enhance the virulence of B. anthracis by inhibiting the phagocytosis of vegetative cells in the extracellular environment of the lymphatic system and bloodstream. loss of plasmid, p x 02, which control capsule production leads to loss of virulence 1/21/2013 58

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It is mainly the action of the toxin that mediates damage to the host. The three components of the tripartite toxin combine to form two binary toxins, the oedema toxin and lethal toxin, formed by association of the protective antigen with the oedema factor and lethal factor, respectively. 1/21/2013 59

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1/21/2013 60

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the protective antigen binds to host cells facilitates the entry of the associated oedema or lethal factor 1/21/2013 61

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The oedema toxin -localized swelling associated with cutaneous anthrax. Oedema factor : calmodulin -dependent adenylate cyclase  catalyses the production of intracellular cAMPfrom host aATP Inducing IL-6 and inhibiting TNF-α in monocytes Oedema toxin also increase host susceptibility to infection by impairing neutrophil function. 1/21/2013 62

Lethal toxin:

Lethal toxin Lethal factor :zinc metalloprotease  inactivates mitogen -activated protein kinase , particularly in macrophages. The lethal toxin stimulates macrophages to produce IL-1β and TNF-α. IL-1β accumulates within macrophages and TNF-α is released. concentration of lethal toxin increases,  macrophage lysis  sudden release of IL-1β  shock and death. 1/21/2013 63

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Loss of plasmid p x 0 1 which encodes LF toxin renders the strain avirulent The avirulent Sterne vaccine strain is devoid of plasmid coding for capsular polysaccharide 1/21/2013 64

pathogenesis:

pathogenesis Anthrax - zoonosis - a disease of animals transmissible secondarily to man. Human beings relatively resistant to infection 1/21/2013 65

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usually a consequence of the exposure of a susceptible host to spores of the bacillus. arises by inoculation through the skin of material from infected animals or their products. Spores are not found in host tissues, but appear on exposure of the vegetative cells to oxygen in the air. 1/21/2013 66

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Local gelatinous hemorrhagic edema at site of inoculation Extensive subcutaneous congestion Enlarged dark red friable spleen Dark blood ,coagulates less firmly 1/21/2013 67

Anthrax:

Anthrax Animals infected by ingestion of spores present in soil Direct spread from animal to animal is rare Infected animals shed in the dischrages from the mouth, nose and rectum large numbers of bacilli sporulate in soil and remain the source of infection 1/21/2013 68

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spores  body by abrasion, inhalation or ingestion phagocytosed by macrophages transported from the site of infection regional lymph nodes the spores germinate and vegetative bacteria multiply. bacilli  bloodstream causing massive septicaemia, with up to 10 8 colony-forming units/ml of blood. 1/21/2013 69

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1/21/2013 70

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The disease may be Cutaneous Pulmonary Intestinal All types can lead to fatal septicemia or meningitis 1/21/2013 71

Cutaneous anthrax:

Cutaneous anthrax The face , neck ,hands arms and back -usual sites The primary lesion  malignant pustule. . Coagulation necrosis of the centre of the pustule results in the formation of a dark-coloured eschar 1/21/2013 72

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surrounded by a ring of vesicles containing serous fluid and an area of oedema and induration severe toxic signs and widespread oedema  prognosis is poor. 1/21/2013 73

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1/21/2013 74

Hide porters disease :

Hide porters disease The disease used to be common in dock workers carrying loads of hides and skin on their bare backs 1/21/2013 75

Inhalational anthrax :

Inhalational anthrax consequence of the inhalation of spores acute form of disease  high mortality rate. 1/21/2013 76 The infectious human dose by the air-borne route  range 25000 to 55000 spores sometimes incorrectly referred to as pneumonic anthrax, the disease does not develop as a bronchopneumonia

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1/21/2013 77 multiplication of organisms in bronchi spread  lungs, lymphatics and bloodstream

Inhalational anthrax:

Inhalational anthrax Intense inflammation, haemorrhage and septicaemia Toxins and the considerable bacterial load Increased vascular permeability and hypotension similar to endotoxic shock. 1/21/2013 78

Wool sorter , s disease:

Wool sorter , s disease cases of inhalational anthrax persons working in industries handling animal skins, hides and wool 1/21/2013 79

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accident at Soviet Union biological weapons factory in the 1980s  release of spores into the air. In the town of Sverdlosk 79 cases of human inhalation recorded - 6 weeks after exposure 68 deaths. 1/21/2013 80

Intestinal anthrax:

Intestinal anthrax Occurs among pastoralists  eat dead infected animals. 1/21/2013 81

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after a day or so from haemorrhagic diarrhoea, and dies rapidly from septicaemia. occur as small outbreaks in a family or village. some may suffer only cutaneous lesions cause of the outbreak is easily made clinically 1/21/2013 82

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1/21/2013 83

Naturally occurring infections of animals:

Naturally occurring infections of animals All animals are susceptible to anthrax Carnivores relatively resistant Herbivores highly susceptible Omnivores intermediate level of resistance. 1/21/2013 84

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Wild herbivores or domesticated animals usually septicaemic Ingestion of spores along with coarse vegetation. Trauma of the intestinal tract  entry of the spores into the host. infection after inhalation rare Skin abrasions -malignant pustules. 1/21/2013 85

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1/21/2013 86 Sudden death in any herbivore should be treated with suspicion A veterinary officer summoned - examine the carcass without a post-mortem examination

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A blood slide taken for Gram or methylene blue staining. Under the Anthrax Order the animal must remain on the farm and be incinerated on site if found to be positive 1/21/2013 87

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Deep burial in quicklime -alternative method of disposal but the spores remain viable for many years and may contaminate pasture and infect grazing animals. 1/21/2013 88

Lab diagonosis:

Lab diagonosis Specimens Swabs ,fluid or pus from pustules Sputum and blood 1/21/2013 89

Lab diagnosis:

Lab diagnosis Full developed malignant pustule difficult to swab Central necrotic area -poor yield. Fluid aspirated from the surrounding vesicles, more likely to yield anthrax bacilli. Specimens taken before antibiotic therapy Clinical diagnosis easy -characteristic appearance and occupational exposure 1/21/2013 90

microscopy:

microscopy Gram's stain may show typical large Gram-positive bacill Indian ink- capsule appears as clear halo 1/21/2013 91

culture:

culture blood agar -large, flat, greyish colonies with the characteristic 'medusa head' appearance. Staining -colonies shows long chains of Gram-positive bacilli, some containing spores. Demonstration of non-motility, gelatin liquefaction, growth in straight chains and enhanced growth aerobically, as seen in the characteristic inverted fir tree appearance in a gelatin stab, will generally identify B. anthracis completely. 1/21/2013 92

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1/21/2013 93

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Serological diagnosis - ELISA –done retrospectively-seldom used diagnostically. Polymerase chain reaction- rapid identification and diagnosis B. anthracis -a number of unique genes, the selection of suitable gene targets not problematic. Toxin production can be demonstrated by immunological or gene probe methods in reference laboratories. 1/21/2013 94

B.Anthracis colonies lysis by gamma phage:

B.Anthracis colonies lysis by gamma phage 1/21/2013 95

Environmental samples:

Environmental samples occasionally necessary to isolate B. anthracis from potentially contaminated material such as animal hair, hides or soil. The heat treatment of aqueous extracts of these materials at 60°C for 1 h kills all except spore-forming bacteria and fungi. For some soil types selective agars have been developed that allow preferential growth of B. anthracis 1/21/2013 96

TREATMENT:

TREATMENT . Penicillin remains the drug of choice, as β- lactamase -producing strains of B. anthracis are rare. .Most strains sensitive to macrolides , aminoglycosides , tetracyclines and chloramphenicol . .Ciprofloxacin  prophylaxis or early treatment for greatest risk of exposure 1/21/2013 97

prophylaxis:

prophylaxis General methods-improvement of factory hygeine and proper sterilisation of animal products Animals given single dose sterne vaccine-1 yr protection 1/21/2013 98

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The anthrax vaccine - culture filtrates of an avirulent , nonencapsulated strain known as V770-NP1-R. No living organisms are present in the vaccine December 2008, the new BioThrax IM Intramuscular injections in the deltoid 0 and 4 weeks and then at 6, 12, and 18 months, followed by annual boosters. Contains aluminium hydroxide as an adjuvant 1/21/2013 99

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Prolonged initialization sequence is required with annual booster shots Anthrax vaccine's primary ingredient Anthrax Protective Antigen Can impair the life-cycle of the human immune systems memory B-Cells Memory T-cells through inducing the production of immunoglobulin G ( IgG ) which sequesters furin 1/21/2013 100

Anthrax bioterrorism:

Anthrax bioterrorism The possibility of creating aerosols containing anthrax spores has made B. anthracis a chosen weapon of bioterrorism. Iraq, Russia and as many as ten nations have the capability to load spores of B. anthracis into weapons. . 1/21/2013 101

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1/21/2013 102

ANTHRACOID BACILLI:

ANTHRACOID BACILLI Aerobic spore bearing bacilli similar- B.anthracis Some are frequent lab contaminants B.subtilis  opportunistic pathogen Eye infections Septicemia 1/21/2013 103

BACILLUS CEREUS :

BACILLUS CEREUS Large Gram-positive bacillus Resembles B. Anthracis Motile and lacks the glutamic acid capsule Saprophyte Soil water vegetation 1/21/2013 104

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B. cereus closely resembles B. anthracis in culture Forming large, grey, irregular colonies described as anthracoid 1/21/2013 105

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Large inocula injected into laboratory animals may cause death Without the haemorrhagic appearance of anthrax Blood smears do not show the characteristic pink capsule with McFadyean's stain 1/21/2013 106

PATHOGENESIS:

PATHOGENESIS B. cereus is most commonly associated with food poisoning The organism can also cause post-traumatic ophthalmitis , which requires rapid, aggressive management locally 1/21/2013 107

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An atypical strain capable of causing a disease that resembled inhalation anthrax has been described This strain appears to have acquired the toxin-encoding pX01 plasmid, and a plasmid encoding a polysaccharide capsule 1/21/2013 108

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The capsule has the same role as the poly- d - glutamic acid capsule on the surface of B. anthracis Preliminary animal studies suggest that the strain is as virulent as B. Anthracis 1/21/2013 109

Bacillus cereus:

Bacillus cereus Cause of food poisoning Seen in Soil Vegetables Milk Cereals Spices meat poultry 1/21/2013 110

Food poisoning:

Food poisoning Spores of B. cereus are heat-resistant most strains produce toxins. The organism is widespread in the environment and is found in most raw foods, especially cereals such as rice. Enormous numbers of organisms (up to 10 10 organisms/g) may be found in contaminated food (commonly lightly cooked Chinese dishes), leading to two types of food poisoning: 1/21/2013 111

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vomiting, occurring within 6 h of ingestion rice 1,3,5 It is caused by preformed toxin- lowmolecular weight, heat- and acid-stable peptide withstand intestinal proteolytic enzymes. 1/21/2013 112

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A diarrhoeal form, occurring 8-24 h after ingestion 2,6,8,9,10,12 similar to enteritis caused by Escherichia coli or Salmonella enterica serotypes. caused by enterotoxins - like the Clostridium perfringens enterotoxin are heat labile formed in the intestine. 1/21/2013 113

LABORATORY DIAGNOSIS:

LABORATORY DIAGNOSIS food poisoning,-laboratory confirmation is easy if suspect food available for testing. 10 8 organisms/g -make the diagnosis in the absence of other food-poisoning bacteria. Anthracoid colonies on blood agar - B. cereus . Food reference laboratories are able to confirm identification and type if necessary. 1/21/2013 114

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Methods for the laboratory identification of B. cereus strains that cause an inhalation anthrax-like disease have yet to be devised. It is likely that genetic tests will be effective in identifying these atypical strains. 1/21/2013 115

TREATMENT:

TREATMENT Emetic and diarrhoeal syndromes  short lived No specific treatment is needed. Acute symptoms last less than 24 h and recovery on a reduced diet and fluids is rapid. antibiotic regimens for anthrax would be equally effective for the treatment of disease caused by atypical strains of B. cereus strains. 1/21/2013 116

CONTROL:

CONTROL Food poisoning caused by B. cereus is easily prevented by proper cooling and storage of food. Ideally, all dishes should be freshly prepared and eaten. Rice, in particular, should not be stored for long periods above 10°C. 1/21/2013 117

STERILIZATION TEST BACILLI:

STERILIZATION TEST BACILLI Bacillus stearothermophilus was, until the discovery of archaebacteria in hot springs, the most heat-resistant organism known. spores withstand 121°C for up to 12 min testing autoclaves  to ensure the destruction of spores 1/21/2013 118

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Strips containing B. stearothermophilus are included with the material being autoclaved Subsequently examined by culture for surviving spores The organism grows only at raised temperatures, between 50°C and 60°C; there is hardly any growth below 40°C 1/21/2013 119

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Bacillus globigi , a red-pigmented variant of B. subtilis , has been used to test ethylene oxide sterilizers B. pumilus has been used to test the efficacy of ionizing radiation 1/21/2013 120