Metagenomics Flashcards
Overview
- DSTL
- Classification of biological agents
- US definition of bioterrorism agents
- History of biological warfare
- Case studies- anthrax
- Microbial forensics and metagenomics
The ACDP has classified biological agents (which could be viruses, bacteria, fungi and their toxins) into different groups. What are these groups and what does this classification mean?
* HG1
o Unlikely to cause human disease
* HG2
o Can cause human disease; unlikely to spread to the community, prophylaxis, or treatment available
* HG3
o Can cause sever human disease, may present a risk of spreading to the community, usually effective prophylaxis, or treatment available
* HG4
o Cause severe human disease, likely to spread to community, usually no effective prophylaxis or treatment available
o All viral pathogens
What are CL3 and CL4 laboratories and whats good about them?
Containment level 3 (CL 3) is used for work with high risk biological agents and hazards, genetically modified organisms, animals and plants.
This is the highest level of biosecurity which is currently available. CL4 labs and rooms are not as common as their CL3 counterparts, as they handle only the most hazardous and exotic microbes which pose a serious risk to human health, and for which no treatment may be available
- Designed and built to prevent or control exposure to laboratory workers, other persons, and the environment to the biological agent
* HEPA air filter in cabinet so clean air that avoids contamination
*** Negative pressure cascade **
o Keeps individual safe whilst working and no reagents released to environment
o Air from outside into CL3 or CL4 lab
o Air goes through locker room, interlock, and autoclave
o The lobby area has agar and consumables
o Then different microbiological safety cabinets in each lab
o This air flow means that’s the air flow is contained if an accident occurred
What is meant by biological weapons?
- Biological weapons are microorganisms such as viruses, bacteria, fungi, and their toxins that are produced and released deliberately to cause disease and death in humans, animals or plants
- Biological weapons is a subset of a larger class of weapons referred to as weapons of mass destruction, which also incudes chemical, nuclear and radiological weapons
CDC has classified the bioterrorism agents/disease into what categories. What does this mean and give examples of some diseases found within this category
* Category A
o Easily disseminated or transmitter from person to person; high mortality rates, potential for major public health impact; might cause public panic; require special action for public health preparedness
o E.g., anthrax, botulism, plague, smallpox, tularaemia, VHF- Ebola, Marburg, Lassa, Machupo
* Category B
o Moderately easy to disseminate; result in moderately morbidity rates and low mortality rates; require specific enhancements of diagnostic capacity and enhances disease surveillance
o E.g., Brucellosis, E. Coli, O157:H7, Gladers, Meliodosis, Q Fever, Typhus fever, EE virus, cholera
* Category C
o Emerging pathogens that could be engineering for mass dissemination in the future because of availability, ease of production & dissemination and potential for high morbidity and mortality rates and major health impact
o E.g., emerging diseases such as Nipah virus and Hantavirus
Who were the foundation to microbiology?
*** Louis Pasteur (1822-1895) **
o French scientists responsible for pasteurisation and vaccination for anthrax and rabie
*** Robert Koch (1843-1910) **
o German scientist who discovered the disease cycle of anthrax and the bacteria responsible for TB and cholera
in 1914-1918 during WWI, what were common diseases and what were they caused by?
- Glanders (Burkholderia mallei) primarily a disease of horses, donkeys, and mules
- Anthrax (Bacillus anthracis) highly resistant spores
What is the Geneva protocol?
- Protocol for the prohibition of the use of war or asphyxiation, poisonous, or other gases, and of bacteriological methods of warefare
- The 1925 Geneva Protocol prohibits the use of Chemical and biological weapons in war. The protocol was drawn up and signed at a conference in 1925 and out into force in 1928
What happened between 1932-1945 (WWII)?
- Shiro Ishii began biological weapons research in 1930 (Unit 731)
- Killed 600 prisoners a year
- Poisoned wells in Chinese villages to study cholera and typhus outbreaks
- Dropped fleas infected with Y.pestis from planes over Chinese cities
- Suggested tens of thousands died as consequence of research
- In 1942, the US formed the war research served and investigated B.anthracis and botulinum toxin as use as a weapon
- In 1942 and 1943 the British tested B. anthracis bonds on Gruinard island of the NW
Whats the biological weapons convention?
- Convention on the prohibition of the development, production and stockpiling of bacteriological (biological) and toxin weapons and on their destruction
- The first multilateral disarmament treaty banning the development, production and stockpiling of an entire category of weapons of mass destruction
- Into force in 1975
What are the different forms that bacillus of the bacillus anthracis (anthrax) cam come in and what is the form often associated with?
o Single (causes food poisoning)
o Streptobacilli (rat bite fever)
o Diplobacilli (Q fever)
o Palisades (diptheria)
What group is anthrax part of ?
Part of the B. cereus group (sensu lato)
What are the 3 types of bacillus which are most commonly studied?
B.thuringiensis, B.anthracis, B. cereus are the most studies as are the ones which cause disease
Tell me about the structure of the bacillus anthracis
- Gram +ve bacteria (3-5µm tall, 1-1.2 µm wide)
- Non-motile, non-flagellated
- Facultative anaerobe- can survive without oxygen
- Two plasmids which encode virulent factors
o Toxins (pX01)
o** Capsule (pX02) ** - Spore formed- survived for decades in environment
- Zoonotic- transmitted from animal’s host to humans
What are the virulence factors of the B. anthracis?
Tell me some properties of them
*** The spore **
o Highly resisant to harsh conditions
o Can survive for many years in environment
o Infectious particles
*** The capsule (pX02) **
o Surrounds vegetative cells
o Prevents phagocytic killing
*** The toxins (pX01) **
o Edema toxin and lethal toxin
o Cause swelling and lysis
Tell me the stages to endospore formation of the B. anthracis
- Asymmetric cell division inside mother cells
- Spore receives own copy of genetic material
- Layers of spore shown on side
- Features of spore: Protection from host immune system, flexibility….
- Sporulation begins at the onset of the stationary phase when the nutrients are depleted, it is triggered by the activation of histidine sensor kinases
- Asymmetric division occur withint he mother cell to form a sporangium which is composed of 2 compartments, the larger mother cell and the smaller forespore, which then goes on to become the spore
- The Forespore is then engulfed by the mother cell during phagocytosis
- After engulfment, the forespore is a double membrane bound cell within the mother cell
- Late sporulation occurs
- The mother cell lyses to release a mature spore into the environment
- then germination…
What are the stages to germination of the B. anrhracis?
Germination occurs when nutrients such as Amino acids, or purine nucleosides stimulate specific germinant receptors located in the sport inner membrane
The germination has two stages:
1) Biophysical
2) Biochemical
Outgrowth of the spore then occurs
then endospore formation occurs again
Tell me about the capsule (pX02)
- polyD glutamic acid capsule offers protection to vegetative cell
- elevated bicarbonate levels in blood cause increased expression
- non-stimulatory to dendritic cells
- -ve charge
- Deceives host immune surveillance
- M’Fadyean stain: this stain can be used to visualise blood or tissue smears from dead animal. it is a higly reliable and rapid diagnostic test for anthrax
Tell me about Exotoxins (pX01)
- Encoded by pX01 plasmid
- Vegetative cells –> Protective antigen (used in vaccines) and Edema factor/ Lethal factor
- Host cell receptors: (type I transmembrane proteins)
o CMG2/ANTXR2
o TEM8/ANTXR1 - Without protective antigen the edema/ lethal factor cannot enter the cell
- Edema toxin (increase CAMP) and lethal toxins (decrease MAPKs)
Increase in cAMP leads to edema
Decreasein MAPKs leads to necrosis and hypoxia
Why is confirmatory PCR used?
- Confirmatory PCR
- Compliment microbial tests
- Specific to B.anthracis detection only
- Confirms presence or absence:
**o Ba chromosome target
o Ba pX01 target
o Ba pX02 target
**
What order was placed in 1991
The anthrax order
How did anthrax used to kill humans and how has this changed today?
Anthrax is a serious infectious disease caused by gram-positive, rod-shaped bacteria known as Bacillus anthracis. It occurs naturally in soil and commonly affects domestic and wild animals around the world. People can get sick with anthrax if they come in contact with infected animals or contaminated animal products.
Now adays there is still death from animals but also injected heroin users have died from anthrax
What are the 4 routes for human disease by anthrax?
Cutaneous
Pulmonary
Gastro-intestinal
Injectional
Tell me the following about cutaneous anthrax:
How infected?
Incubation period?
Symptoms?
Fatality %?
Spores enter abrasions in skin
20% fatality
Incubation period: 1-7 days post exposure
Symptoms: Blisters which turn to black eschar swelling (oedema)
