Bacillaceae Family Infections and Laboratory Diagnosis

Bacillaceae Family

Infections and Laboratory

Diagnosis

• Bacillaceae family, Bacillus genus

• B. subtilis

• B. anthracis

• B. cereus

• B. licheniformis

• B. megatorium

• B. thuringensis

• B. larvae (Paenibacillus larvae)

• More than 200 species

• Gram positive, large rod (10 μm)

• Endospores

• Catalase positive

• Aerobic or facultative anaerobic

• B. anthracis and B. mycoides

• Encapsulated

– In B. anthracis, the capsuleis not in polysaccharide structure but is in the polypeptide (poly-γ-D-glutamic acid) structure

General features-I

General features -II

• Colony forms are variable and B. anthracis always makes R type colony

• Spore forms are durable (live for 1 hour in 50%

ethanol)

• Can separated from Clostridium with aerobic

reproduction and catalase positive

• The most important disease that they do in humans and animals is anthrax caused by B.anthracis

• Apart from B. anthracis, other agents in Bacillaceae family are called «anthracoid»

(anthrax-like)

Bacilus Genus Sensitive Animals Clinical Course

B. anthracis Cattle, Sheep Fatal, peracute or acute septicemic Anthrax

Pig

Pharyngeal edema subacute Anthrax;

intestinal form (rare) high mortality

Horse

Local edematous subacute Anthrax;

septicemia and enteritis occasionally

Human Skin, respiratory and intestinal forms Anthrax

Bacillus Species Animals Clinical prognosis

B. cereus Cattle, Sheep, Goat Mastitis

Cow, Sheep Abortions

B. licheniformis Cattle, Sheep Sporadic abortions

B. subtilis Cattle, Sheep Mastitis

B. coagulans Cow Abortions

B. macerans Cow Abortions

B. pumilus Cow Mastitis

B. mycoides Catfish Ulcer and necrosis in the

muscle

Sheep Mastitis

B. thuringiensis Cow Mastitis

B. larvae (paenibacillus

larvae) Bee American foul brood

Anthrax

(Anthrax, Splenic Flank, Şirpençe)

• Anthrax (French): Anthrax (Greek): Kömür(Turkish)

• Increase in body temperature

• Swelling of the spleen

• Blood as dark as tar and not coagulate

• Formation of sero-hemorrhagic infiltrates in subcutaneous and sub- serous spaces

• Death

• Bacillus anthracis is an infection caused by a bacterium

Etiology

• 1876 Robert Koch first cultivated

• Gram positive rod

• Sports and capsule

• Aerobic and immotile

• It forms non-hemolytic R type (rough-edged, granular, gray colored) colony on blood agar (frosted glass-

medusa head)

• Adhesive at the bottom of broth culture

• Staining from tissues, the agents are either individually or in the form of chains of 2-8 bacilli

• In contrast, parallel filaments appear as hair in solid and

liquid cultures

Etiology

• Spores remain for 50-60 years in environment

• The dead animal is not sporing unless the body is opened

• Encapsulated in tissues (inside the body), spore form out of the body (contact with oxygen)

• Capsule staining (Mc Fadeyean painting, Giemsa)

• Spore staining

• The vegetative form of B. anthracis is not as durable as the spore form. They are destroyed in 3-6 days in unopened cadavers. It is not resistant to disinfectants. It dies in 10-15 minutes at 55-58 ° C

• On the other hand, spore form is very resistant to physical and

chemical agents. It becomes inactive in humid temperature at 121 °

C for 15 minutes in autoclave and at 160 ° C for 60 minutes at dry

temperature. Mercury chloride (1/1000) kills spores for 5 minutes

and formol (10%) for 15 minutes

• The spores are round or oval in structure and are

very resistant to physical and chemical agents

Anthrax Strains

• Stern strain (Weybridge strain)

• Max Sterne, 1930, vaccine strain

• Vollum strain

Weapon, 1937, United Kingdom

• Anthrax 836 strain

Weapon, 1953, Russia

• Ames strain(Amerithrax)

Weapons, 2001, envelopes sent to the America

Biological Weapon

• In 1943 the British tried anthrax bombs in

Gruinard island

• B. anthracis has two plasmids, pXO1 and pXO2

• plasmid pXO1

-Protective antigen -Lethal factor

-The edema factor is coded

• pXO2 carries the gene sequence that provides the capsule formation. If the bacteria lose one or both of these

plasmids, they become avirulent (loss of sickness)

Epidemiology

• Zoonotic (occupational disease, butcher, shepherd, veterinarian, wool worker)

• All mammals sensitive

• Cold-blooded animals (snakes, lizards, etc.) and poultry (except ostriches) are resistant to the disease

• Poultry body temperature 41-42 ° C, Ostrich 38 ° C

• Contagion is caused by digestion, respiration and skin

• Predators, worms, flood waters, stinging flies, meat-bone meal

CLINICAL SYMPTOMS

• Anthrax can be peracute, acute and subacute. The duration of incubation of the disease may vary from 1 to 14 days

• Peracute form

 It is mostly seen in cattle and sheep. It is characterized by sudden death

• In acute and subacute forms

 Body temperature increases up to 40-42°C and death usually takes

place shortly before the clinical semptoms develop

Necropsy

• The cadavers are quickly stinking. Rigor mortis is not complete or never seen

• The black blood come from natural holes and do not coagulate

• Cyanosis is seen in mucous membranes. Subcutaneous and serous membranes have septicemia

• The spleen is large, mud-like, and the section face is

charcoal

Anthrax lesions in humans

DIAGNOSIS

• Clinical Diagnosis: The disease may be confused with many acute and peracute diseases. Clinical symptoms are insufficient for diagnosis.

In cattle, Pyroplasmosis, Leptospirosis, Pastörellozis, Basiller hemoglobinuria In sheep Bradzot, Leptospirozis

Confused with stomach-intestinal diseases that are accompanied by pain in horses

• Necropsy: As a rule, due to spore contamination, anthrax suspicious animals are not autopsied !!!

Laboratory diagnosis

• Blood, spleen, bone marrow, ear pieces and parts taken from organs and edema fluid from anthrax suspicious animals can be sent for laboratory examination

• Bacterioscopy: From the materials sent to the laboratory,

the samples are stained with Gram and Giemsa staining

methods. Agents are seen in the form of individual chains

or chains of 2-8 bacilli. Agents with single or double red

painted capsules are typical in Giemsa staining

• Culture:

 Samples are sent to the laboratory cultured on appropriate medium and allowed incubated for 24-48 hours at 37°C under aerobic conditions. Typically, after 24 hours, 3- to 5-mm diameter R-shaped gray colonies are seen

 In gram staining, made from cultures, agents have long filamentous structure similar to the hair-like effect. Some sporadic Gram positive bacteria can ocur

 Differential identification should be done since samples taken after death may

contain many bacteria resembling B. anthracis. Especially, other Bacillus species (B.

subtilis, B. megaterium, etc.) and P. aeruginosa are very confused

• Animal Experiment: Inoculums prepared from samples are given to mice or guinea pigs subcutaneously or intraperitoneally. About 2-7 days later the animals die.

Autopsies of the dead animals are made. Smears are made from the other tissues, especially the blood and spleen. It is stained with Gram and Giemsa and a typical factor is sought. In addition, cultured from tissues such as spleen and liver. Colonies are examined for B. anthracis

• Serological Tests: Serologic tests include agar gel precipitation test, indirect microhemagglutination test and ELISA test, which are mainly Ascoli

thermoprecipitation test.

• Phage Typing: B. anthracis is sensitive to Gamma phage

Identification scheme for B. anthracis and other bacilli

Characteristics B. anthracis B. cereus

Motile immotile motile

Encapsulated in Tissue + -

Growth in broth The bottom sediment, the

upper part is clear Homogeneous turbidity View on Sheep Blood Agar Haemolysis (-) Haemolysis (+)

Gelatin hydrolysis V +

Penicillin Sensitivity (10 unit

disk) Sensitive Resistant

Gamma Sensitivity to phage Sensitive ineffective

PROTECTION AND CONTROL

• There is an effective vaccine

• The Turkish Universal Anthrax Vaccine produced by Professor Dr Süreyya Tahsin Aygün was used for the first in Turkey. Then the vaccine of Sterne started to be used in compliance with OIE standards

• The Max-Sterne vaccine, produced by the Etlik Central Veterinary Control and Research Institute applied to 2 to 6 months age animals by Ministry of Agriculture and Forestry, and is immunized for approximately 6-12 months

• Max Sterne vaccine lacks pXO2 plasmid. This is why the capsule is missing.

It is attenue and adjuvanted vaccine

• Anthrax is a compulsive and compensatory (3/4) disease

• In places where the disease has occurred, places with contamination

sources (pasture, barn, etc.) are closed or animals are kept away from them

• Required disinfection is done. 0.1% sublimation, 5% acid phenic, 5% cresol, 5% creolin and other disinfectants are used for the disinfection of tables and materials.

• The dead animals are buried in pits 2 m deep, autopsy is forbidden, and quicklime is thrown on them. Dishes and cadavers are burned.

• Cord and quarantine measures are applied to the area. 15 days after vaccination, vaccined animals can be grazed on contaminated pastures