1.

Introduction to Cell & Tissue Culture

P. Mather, Penelope E Roberts

2. Culture of Animal Cells

R. Ian Freshney

1. The Merck Veterinary Manual

Merck

2. A Text Book of the Diseases of Cattle, Sheep, Pigs, Goats and Horses
Radostits, Gay, Blood, Hinchcliffe

3. Common Disease of Companion Animals

Alleice Summers
 Anthrax

Malignant Pustule, Malignant Edema, Woolsorters’

Disease, Ragpickers’ Disease, Splenic Fever
 Overview
 Organism

 History

 Epidemiology

 Transmission

 Disease in Animals

 Prevention and Control

 The Organism
 Bacillus anthracis

 Large, gram-positive, non-motile

rod

 Two forms
e.g. Vegetative & Spore

 Over 1,200 strains

Center for Food Security and Public Health
 ANTHRAX: History

 The anthrax bacillus, Bacillus anthracis, was the first

bacterium shown to be the cause of a disease- Koch’s Postulate

 In 1877, Robert Koch grew the organism in pure culture

 Anthrax is a disease of domesticated and wild animals

 Men suffer from anthrax occasionally due to close contact with

infected animal or animal products
Robert Koch's original micrographs of the Anthrax bacillus

textbookofbacteriology
 Spore
 Sporulation requires
Poor nutrient conditions
Presence of oxygen
1 spore per cell

 Spores
Very resistant and survive for decades
Protoplast carries the material for future vegetative
form
Cortex provides heat and radiation resistance cell

microscopresblog
 Epidemiology

 Distribution worldwide

 Not common in West. Common in Africa ( Zimbabwe),

S.E. Asia, China, South America, Turkey, Pakistan, India

 Human to human or animal to animal transmission is rare

( not contagious)

Grazing animals become infected through ingestion of

spores in the soil (Carcasses become the source)
Anthrax Distribution

Center for Food Security and Public Health

 Animal Transmission
Ingestion
– Most common
– Herbivores
• Contaminated soil
• Heavy rainfall, drought
– Carnivores Center for Food Security and Public Health
• Contaminated meat
Inhalation
Mechanical (insects)
 Pathogenesis

Bacillusanthracis.org Access.health.qld.gov.au
 Mechanism of Infection
• Anthrax spores enter body

• Germinate & multiple in lymph nodes

• PA, EF, LF excreted from bacteria

• PA binds to TEM8/CMG2

• PA nicked by protease furin

– 20-kDa segment off leaving 63-kDa
peptide
– Heptamer forms

• EF and/or LF binds

• Complex internalized by endocytosis

• Acidification of endosome

• LF or EF crosses into cytosol via PA

mediated ion-conductive channels

• LF cleaves MAPKK 1 & 2

• EF stimulates cAMP http://kugi.kribb.re.kr/KUGI/Pathways/BioCarta/anthraxPathway/

 Outcome
 Do not understand exactly how symptoms occur

 EF converts ATP to cAMP

– Increases cAMP levels over 1,000 fold
– Alters water homeostasis
– Edema
– Impairs neutrophil function

 LF cleaves MAPKK at its N terminus

– Disrupts pathways involved in cell growth & maturation
– Increased synthesis of tumor necrosis factor-α & interleukin-1β
– Macrophage lysis
– More cells infected with bacteria & toxin
– Septic shock & death

 Death probably results from high levels of bacteria secreting LF toxins in blood
– At death, blood contains as many as 109 bacilli/ml (depending on the species)
 Clinical Signs
 Many species affected
– Ruminants at greatest risk

 Three forms
– Peracute
• Ruminants (cattle, sheep, goats, antelope)
– Acute
• Ruminants and equine
– Subacute-chronic
• Swine, dogs, cats

Center for Food Security and Public Health,

Iowa State University, 2011
 Ruminants
 Peracute
– Sudden death

 Acute
– Tremors, dyspnea
– Bloody discharge orifices from b

 Chronic (rare) Center for Food Security and Public Health

– Pharyngeal and lingual edema
– Death from asphyxiation
 Equine
Acute
– Fever, anorexia, colic, bloody
diarrhea
– Swelling in neck
• Dyspnea
• Death from asphyxiation
• Death in 1 to 3 days
Photo from WHO

Insect bite
– Hot, painful swelling at site
Differential Diagnosis (Ruminants)
 Blackleg

 Botulism

 Poisoning
Plants, heavy metal, snake bite

 Lightning strike

 Peracute babesiosis
 SAMPLES FROM ANIMAL

Carcasses 1 or 2 day old

Aspirate blood - MacFadyean stain for bacilli
Direct plating on blood agar

Putrefying carcasses
Blood, tissue and hide
Culture on selective medium
Soil sample from the areas where the carcass as lying

Serological assay
ELISA: based on anthrax toxin ( PA, LF and EF) for routine confirmation and vaccine
response)

Molecular techniques ( Only in the referral laboratories):

- RFLP
- PCR Fingerprinting

Animal Inoculation: Guinea pig and mice inoculation

Confirmation by Culture
 Diagnosis and Treatment
 Necropsy not advised!

 Do not open carcass!

 Samples of peripheral blood needed

Cover collection site with disinfectant soaked bandage to prevent
leakage

 Treatment
 Penicillin, tetracyclines

 Reportable disease
 Vaccination
 Livestock in endemic areas

 Sterne strain
Live uncapsulated spore vaccine

 No U.S. vaccine for pets

Used in other countries
Adjuvant (e.g. saponin) may cause reactions

 Working dogs may be at risk

Center for Food Security and Public Health,

Iowa State University, 2011
 Prevention and Control
 Report to authorities

 Quarantine the area

 Do not open carcass

 Minimize contact

 Wear protective clothing

Latex gloves, face mask
 Prevention and Control
 Local regulations determine carcass disposal
options
Incineration
Deep burial

 Decontaminate soil with 5%

quicklime

 Remove organic
material and disinfect structures
 Prevention and Control
 Isolate sick animals

 Discourage scavengers

 Use insect control or repellants

 Prophylactic antibiotics

 Vaccination
In endemic areas
Endangered animals
Etosha National Park in Namibia
Burning a carcass in a hole... > 2 feet deep
 Disinfection
 Spores resistant to heat, sunlight, drying and many
disinfectants

 Disinfectants
Formaldehyde (5%)
Glutaraldehyde (2%)
Sodium hydroxide (NaOH) (10%)
Bleach

 Gas or heat sterilization

 Gamma radiation
 Vaccine against Anthrax

Killed bacilli and/or capsular antigens produce no significant immunity.

A nonencapsulated toxigenic strain (Sterne Strain) has been used effectively in livestock.

Vaccine for humans: ( avirulent and nonencapsulated) sublethal amounts of the toxin
produced

Licensed in the U.S. is a preparation of the protective antigen (PA)

Dose: A. 3 doses subcutaneously at the interval of 2 wks

B. Followed by three additional doses at 6,12 and 18 months
C. Annual booster dose

Who are to be vaccinated

- Professionals ( Veternarians, butcher, Zoo keeper, Wild life workers, Forest guards)
- Military personnels