1.01.03 Biosafety
1.01.03 Biosafety
1.01.03 Biosafety
CHAPTER 1.1.3.
INTRODUCTION
Laboratory work of the type described in this Terrestrial Manual should be carried out with a
minimum of risk to the health of the staff (biosafety) and the environment (biocontainment). This
requires careful consideration of the risks involved in a particular procedure, followed by
appropriate measures to minimise the risk of human disease and of possible release into the
environment. This is a complex subject that can only be considered in outline in an introductory
chapter. This chapter is concerned almost exclusively with risks from infectious agents, but physical
and chemical injuries in microbiology laboratories must also be prevented. Risks from infection are
reduced by good laboratory techniques and secure facilities, which aid in the containment of
pathogens. It is important to understand that containment of pathogens can be used for two
purposes. One is to prevent disease in humans in the laboratory; the other is to prevent the release
of the pathogen into the environment and causing disease in animals or humans. Often the same
methods of containment are used for both preventing laboratory-acquired infection in humans and
for preventing escape of pathogens that could cause an outbreak of animal diseases. Although the
methods, techniques and facilities required may be the same, the list of pathogens and
categorisation into levels of risk will differ depending on whether it is human or animal diseases
control that is the primary objective.
Existing national and international reference laboratories have considerable experience in the
operation of safe working practices and provision of appropriate facilities. When new laboratories
are being established, it would be prudent to seek advice from the relevant regulatory authorities
and the competent authorities at established institutes. It is important to comply with legislative
requirements.
16
Known occurrence of human and animal infection with the organism or related organisms with similar
characteristics, any history of laboratory-acquired infection, infective dose and disease severity; production
of toxins or allergens.
Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
2.
The volume of culture to be handled and the concentration of the organism likely to be present. (Procedures
such as antigen or vaccine production that require large quantities of organisms usually carry a higher risk
than attempted isolation procedures.)
3.
The origin of the sample, for example samples from wildlife species may contain human or animal
pathogens not normally encountered.
4.
The history of the isolate being handled. Pathogens on primary isolation or of low passage level are often
more dangerous than pathogens of high passage level. In some cases, pathogenicity may be enhanced by
passage or subculture using different media.
5.
The possibility of aerosol formation should be especially taken into consideration when handling fluid
samples or, for example, during grinding, homogenisation and centrifugation.
6.
The threat that the organism may pose to food-producing or companion animals or to wildlife, irrespective of
the threat to laboratory personnel. Additional precautions for handling and storage are required for animal
disease agents from foreign countries.
7.
The physical state of the employees. For example, in the case of pregnancy, immunodeficiency or allergy,
special precautions may be required. Sometimes certain individuals have to be excluded from particular
types of work that would be especially hazardous to them.
8.
A higher level of risk may arise when agents such as Brucella or Mycobacterium are inoculated into animals.
To evaluate the impact of animal inoculation, a risk assessment should be conducted and the following
factors should be considered:
i)
Host species versus inoculated species;
ii)
Strain/treatment and concentration of the inoculum;
iii) Route of inoculation;
iv) Animal housing;
v)
Types of sampling during the experiment.
9.
Some pathogens need to be transmitted by specific vectors or require intermediate hosts to complete their
life cycles before they can infect animals and cause disease. In countries where such vectors or
intermediate hosts do not occur, or where climatic or environmental factors mitigate against their survival,
the pathogen poses a lower risk to animal health than in countries where such vectors or intermediate hosts
occur naturally or could survive.
Group 1 Organisms that are unlikely to cause human or animal disease and are disease-producing
organisms in animals that are enzootic but not subject to official control.
Group 2 Organisms that may cause human or animal disease but are unlikely to be spread in the
community or animal population and for which effective prophylaxis and treatment are available; examples of
Group 2 animal pathogens:
i)
They do not depend on vectors or intermediate hosts for transmission.
ii)
There is very limited or no transmission between different animal species.
iii) Geographical spread if released from the laboratory is limited.
iv) Direct animal to animal transmission is relatively limited.
v)
Mode of transmission is primarily through ingestion, inoculation or mucus membrane route.
vi) The need to confine diseased or infected nondiseased animals is minimal.
vii) The disease is of limited economic and/or clinical significance.
viii) Short-term survival in the environment and effective treatment or prevention is available.
ix) May be either exotic or enzootic but are subject to official control and have a low risk of spread from
the laboratory.
Group 3 Organisms that can cause severe human or animal disease and may spread in the community
and/or animal population but for which there is usually effective prophylaxis and treatment; examples of
Group 3 animal pathogens:
i)
They may depend on vectors or intermediate hosts for transmission.
ii)
Transmission between different animal species may readily occur.
iii) Geographical spread if released from the laboratory is moderate.
iv) Direct animal to animal transmission occurs relatively easily.
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
v)
vi)
vii)
viii)
ix)
Group 4 Organisms that cause severe human or animal disease, may represent a high risk of spread in
the community or animal population and for which there is usually no effective prophylaxis or treatment.
i)
They may depend on vectors or intermediate hosts for transmission.
ii)
Transmission between different animal species may occur very readily.
iii) Geographical spread if released from the laboratory is widespread.
iv) Direct animal to animal transmission occurs very easily.
v)
Can be transmitted through casual contact or indirectly.
vi) The statutory confinement of diseased, infected and in-contact animals is necessary.
vii) The statutory control of animal movements over a wide area is necessary.
viii) The disease is of extremely severe economic and/or clinical significance.
ix) No satisfactory prophylactic and/or therapeutic treatments are available.
x)
Have a high risk of spread from the laboratory into the environment and the national animal population.
Infectious organisms that might be encountered in laboratory work have been assigned to Risk Groups 14 by
authorities in several countries (Advisory Committee on Dangerous Pathogens, 1995; Barbeito et al., 1995).
Some examples of pathogens that may cause disease in humans, and also may be found in a veterinary
laboratory, are listed in Table 1 Also, some very serious Group 4 agents, including Hendra and Nipah, have been
isolated from diagnostic specimens in veterinary laboratories.
Table 1. Examples of some of the microorganisms in Risk Groups 2 and 3 that
are capable of causing human disease and that may be present in a veterinary laboratory
Group 2
Viruses: Influenza viruses types A, B, C other than notifiable avian influenza (NAI); Newcastle disease virus; Orf
(parapox virus)
Bacteria: Alcaligenes spp.; Arizona spp.; Campylobacter spp.; Chlamydophila psittaci (nonavian); Clostridium tetani;
Clostridium botulinum; Corynebacterium spp.; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus spp.;
Leptospira spp.; Listeria monocytogenes; Moraxella spp.; Mycobacterium avium; Pasteurella spp.; Proteus spp.;
Pseudomonas spp.; Salmonella spp.; Staphylococcus spp.; Yersinia enterocolitica; Yersinia pseudotuberculosis
Fungi: Aspergillus fumigatus; Microsporum spp.; Trichophyton spp.
Group 3
Viruses: Rabies virus; Equine encephalomyelitis virus (Eastern, Western and Venezuelan); Japanese B encephalitis
virus; Louping ill virus
Bacteria: Bacillus anthracis; Burkholderia mallei (Pseudomonas mallei); Brucella spp.; Chlamydia psittaci (avian strains
only); Coxiella burnetti; Mycobacterium bovis
Known pathogens
Having decided the risk level of certain work, it is then possible to decide the appropriate containment level that
is needed to minimise the risk of human disease and the risk of spread of disease to animals and the
environment. The containment level is defined by a combination of the physical facilities and working practices
employed. Organisms of the four Risk Groups indicated above may be placed into containment levels appropriate
for safe working, see below. Laboratories usually appoint a Biological Safety Officer, responsible for ensuring that
microorganisms are handled at the appropriate containment level. They should have sufficient expertise and be of
sufficient seniority to oversee and advise on all safety matters. In large organisations with a network of
laboratories, it is appropriate to appoint a central Safety Officer to advise on and coordinate safety matters of a
corporate nature, which are implemented by local laboratory Safety Officers at each site. The working methods for
a particular procedure or work station should be written out and readily available. Staff must be fully trained and
fully aware of any health risks associated with their work and in procedures for reporting incidents or accidents.
Staff should also be given a medical card indicating pathogens to which they might be exposed. In some cases,
staff can be specially vaccinated to give additional protection, e.g. when working with the rabies virus; this should
also be recorded on the medical card. Such information is useful for a medical practitioner in the event of illness
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
occurring. Regular medical examinations of employees are recommended and, as appropriate, monitoring tests of
employees working with the organisms that cause certain serious human diseases, such as brucellosis and
tuberculosis.
Much information is available on containment of pathogens, and sophisticated apparatus and buildings may be
constructed for containment of the more hazardous organisms as required by the guidelines, standards and
regulations of each country. The requirements depend on the containment required, from the most basic to the
highest level.
Essential requirements for all laboratory work. The essential requirements for any work with infectious agents,
however innocuous they may seem, are as follows:
1.
The laboratory should be easy to clean, with surfaces that are impervious to water and resistant to
chemicals. There shall be a wash-hand basin and emergency shower, including an eye bath, in each
laboratory suite as appropriate for the chemicals and other hazards present. Procedures shall be established
for frequent cleaning and disinfection during and at the end of the work period;
2.
Personnel access to the work area should be restricted; appropriate security measures such as controlled
electronic access may be necessary with higher risk agents.
3.
Personal protective equipment such as long-sleeved lab coats or gowns, closed-toe footwear, disposable
gloves, masks, safety glasses, face shields, and oro-nasal respirators, as appropriate, shall be worn in the
laboratory and removed when leaving the laboratory
4.
The laboratory door should be closed when work is in progress and ventilation should be provided by
extracting air from the room. (Where biosafety cabinets are used, care shall be taken to balance ventilation
systems.);
5.
Food (including chewing gum, candy, throat lozenges and cough drops) and/or drink shall not be stored or
consumed in laboratories;
6.
Smoking and/or application of cosmetics shall not take place in the laboratory;
7.
8.
9.
Emergency response plans should be developed to deal with the biohazard of spills. Some of the items
addressed in the plans should include having effective disinfectant available for cleaning spills, removal of
and decontamination of contaminated protective clothing, washing of hands, and cleaning and disinfection of
bench tops;
10. Used laboratory glassware and other contaminated material shall be stored safely. Materials for disposal
shall be transported without spillage in strong containers. Waste material should be autoclaved, incinerated
or otherwise decontaminated before disposal. Reusable material shall be decontaminated by appropriate
means;
11. No infectious material shall be discarded down laboratory sinks or any other drain;
12. Any accidents or incidents shall be recorded and reported to the Safety Officer.
Containment level for Group 2 pathogens, in addition to the points given above, a Class I, II or III
microbiological safety cabinet should be used when there is potential for generating aerosols or when handling
large quantities of culture or where there is a real need to protect the biological product (see Section D).
Appropriate signs are required at all entry doors to indicate the hazard present and the name and telephone
number of the person(s) responsible. Emergency protocols should be posted within the laboratory to advise
personnel of procedures to follow in case of a pathogen spill or the need to evacuate the laboratory in the event of
a fire or other emergency.
Containment level for Group 3 pathogens, it is advisable that the laboratory be in an isolated location; access
should be limited to appropriately trained level 3 staff. Emergency protocols should be posted within the
laboratory to advise personnel of procedures to follow in case of a pathogen spill or the need to evacuate the
laboratory in the event of a fire. OIE containment level for Group 3 pathogens surpasses biosafety level-3 (BSL-3)
guidelines as outlined by the United States Department of Health and Human Services (DHHS) joint publication
with CDC and NIH (2009) and the United States Department of Agriculture (USDA, 2002).
In addition to the previous requirements, the laboratory shall be under negative pressure and the pressure
differentials should be monitored; a procedure should be developed to provide an alarm if there is a problem and
personnel to respond to the alarm. A ventilation system is required that removes air from the laboratory through a
high efficiency particulate air (HEPA) filter. HEPA filters shall be verified regularly (usually annually); this would
include HEPA filters in biosafety cabinets and on room and equipment exhausts. The laboratory should be
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
sealable for fumigation and contain an airlock entry. There is a requirement to treat effluent depending on the
pathogen. Biological safety cabinets of Class I, II or III shall be used whenever the process to be undertaken is
likely to generate an aerosol (DHHS/CDC/NIH, 2000). It may be necessary for staff to shower on exit from the
laboratory and they must wear dedicated laboratory clothing that is left in the laboratory before leaving the
building.
Note. Because of the link between bovine spongiform encephalopathy (BSE) and new variant Creutzfeldt-Jakob
disease in humans, BSE and related agents are now categorised with the human transmissible spongiform
encephalopathies in Risk Group 3. Consequently, veterinarians and laboratory workers conducting necropsies on
BSE-suspect animals or handling tissues derived from such animals must conduct the work under appropriately
strict containment conditions, sometimes with derogations allowed by the nature of the work and the results of
local risk assessment. It is important that appropriate protective clothing be worn and that a strict code of practice
be followed to prevent exposure to the agent. Laboratories conducting work on BSE must comply with national
biocontainment and biosafety regulations (Advisory Committee on Dangerous Pathogens, 1998).
Containment level for Group 4 pathogens, the most stringent precautions are required, including access to the
building through air locks, and the building being maintained under negative air pressure. Inlet air to the laboratory
shall be filtered through a single HEPA filter and extracted air through double HEPA filters in series. All work with
infective materials shall be conducted in a Class III cabinet or in a Class II cabinet in conjunction with the use of
one-piece positive-pressure suits. All sewage from the laboratory, laboratory effluent and autoclave drain effluent
shall be treated by appropriate means to ensure that all infectious material is destroyed before entering the
sewerage outside the laboratory. Staff shall shower and change their clothing before leaving the building. Other
precautions as described for Group 3 would also apply. The use of one-piece positive-pressure suits is now an
internationally accepted way of providing additional protection at level 4.
OIE guidelines for the containment level for Group 4 pathogens are generally equal to the USDAs biosafety
level 3 Ag guidelines (USDA, 2002). The primary difference between OIE level 4 and BSL-3 Ag is that the BSL-3
Ag guidelines specify that the laboratory will be airtight and shall pass a pressure decay test to confirm that it
does not surpass the prescribed maximum leak rate.
2.
Diagnostic specimens
Veterinary diagnostic centres readily receive specimens that are submitted because they are suspect for a variety of
diseases. The infectious nature of the specimens is usually unknown, but they have the potential to contain biological
agents that may cause disease in animals and humans. Practices and procedures need to be in place that will
minimise the risk of occupational exposure of employees to such pathogens. Unless suspected of containing a
pathogen requiring a higher containment level, it is advisable that initial processing of all unknown specimens should
be carried out as though the material contained a Group 2 pathogen. The most important aspects are to prevent
percutaneous, mucous membrane exposure, particularly inhalation and ingestion. Biological safety cabinets should
be used for all manipulations that may generate aerosols. Class I or II are appropriate depending on the need for
protection of the samples from contamination. Additionally, there should be no mouth pipetting, personal protective
clothing shall be worn with, in some cases, eye and respiratory protection, depending on the anticipated level of
exposure. Although initial diagnostic procedures may be carried out at level 2, once a Group 3 or 4 organism has
been isolated (or suspected) further work must be carried out at the higher containment level.
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
E. STORAGE OF PATHOGENS
Storage of live pathogens requires appropriate containment and security to avoid risks due to breakage or
unauthorised use of material. Storage facilities should be appropriately labelled to indicate the nature of the
pathogens (e.g. their Group) and the contact information for the person(s) responsible for them. A complete
inventory of the pathogens in storage should be kept up to date and available. Special care must be taken when
opening glass vials of freeze-dried pathogens, as these can sometimes shatter. Care must be taken when
working with liquid nitrogen or rooms where asphyxiating gasses may be produced.
Many of the considerations given above relate not only to human safety but also to prevention of the spread of
infection to animals. In a veterinary laboratory an important responsibility is to minimise any risk of escape of
pathogens to animals, either wild or domestic, in the outside community. Close communication must be
maintained with the veterinary authorities. There may be national requirements for special licences to work with
certain microorganisms.
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
H. EMERGENCY PROVISIONS
First-aid equipment should be readily available, but stored in a location that is unlikely to be contaminated by work
conducted in the laboratory (for example, in the air-lock or ante-room). This equipment shall be appropriate to the
work and properly maintained. It shall be kept ready to hand for immediate emergency use by trained first aid
personnel. Bandages and dressings should be available. Some staff shall receive training in safety and first aid
from recognised authorities and shall possess a valid certificate as evidence of competence. Personnel working in
Containment Level 4 facilities shall have advanced first aid competence. Their names and locations should be
known to everyone and posted on notice boards. All staff should be aware of the importance of safety. There
must be suitable procedures and equipment for dealing with spillages and decontamination. A record must be
kept of all incidents and in some countries there may be a legal obligation to report incidents to the enforcing
authority.
There must be written procedures for dealing with emergency failure of all safety and containment systems, for
example in biosafety cabinets or biocontainment rooms, which can lead to loss of containment.
Many laboratories have a staff safety committee to increase safety awareness and to discuss safety issues with
management. Personnel are responsible for their own safety and those around them. Managers are equally
responsible for safety in their area of command and should not allow consideration of speed or cost of work to
come before the safety of personnel or containment of animal disease agents.
There must be an emergency procedure for obtaining medical assistance if required, and for hospitalisation in
appropriate infectious disease facilities when needed. Fire alarms shall be fitted, and tested regularly. The
institute or laboratory must designate a warden to control and communicate in emergency situations and conduct
periodic drills to make staff aware of what to do and where to assemble in the event of an emergency. The
warden is responsible for checking that everyone is in a safe location. Procedures for natural disasters, such as
hurricanes and earthquakes, should be in place where they present a risk. All these procedures should be written
down and periodically reviewed.
a)
b)
c)
d)
J. CONTAINMENT GROUPS
1.
22
The principal purpose of containment is to prevent the escape of the pathogen from the laboratory into the
national animal population. Some animal pathogens can infect humans. In these instances the risk to human
Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
health may demand additional containment than would otherwise be considered necessary from purely
animal health considerations. The risk of human to animal transmission of disease must also be considered
and controlled. In addition, other animals being used for experimental work on the pathogen should be held
in the appropriate containment level.
2.
The level of physical containment and biosafety procedures and practices should be not less than the Group
into which the pathogen has been placed and the detailed requirements should be appropriate to the type of
organism (i.e. bacterium, virus, fungus or parasite). The lowest containment level will be required for
pathogens in Group 1 and the highest level for those in Group 4. Guidance on the containment requirements
for Groups 2, 3 and 4 is provided in Section K.
3.
Arthropods may be pathogens or vectors for pathogens. If they are a vector for a pathogen being used in the
laboratory, the appropriate containment level for the pathogen will be necessary in addition to the
containment facilities for the arthropod.
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Single on extract
Yes
Yes
Yes
Yes
Yes
Yes
Available
Yes
Yes on site
Yes
Yes
Yes
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
Containment Group
Requirements of the laboratory/animal facility
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes with
double doors
Yes with
double doors
Yes
Yes
Yes
Yes
Preferable
Yes
Yes
Yes
It may be necessary
for staff to shower on
exit the laboratory
and they must wear
dedicated laboratory
clothing that is left in
the laboratory before
leaving the building
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
C) Laboratory discipline
24
Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
Containment Group
Requirements of the laboratory/animal facility
Yes
Yes
Yes
Yes
Yes
Yes
Yes
D) Handling of specimens
42. Packaging requirements to be advised prior to submission
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
1.
1.
The animal facility should be easy to clean, with surfaces that are impervious to water and resistant to
chemicals used in the area.
2.
Personnel access to the work area should be restricted; appropriate security measures such as controlled
electronic access may be necessary with higher risk agents.
3.
Personal protective equipment such as coveralls, boots, disposable gloves, masks, safety glasses, face
shields, and oro-nasal respirators, as appropriate, shall be worn in the animal facility and removed when
leaving the animal facility.
4.
The animal facility door should be closed when work is in progress and ventilation should be provided by
extracting air from the room. (Where biosafety cabinets are used, care shall be taken to balance ventilation
systems.)
5.
Food (including chewing gum, candy, throat lozenges and cough drops) and/or drink shall not be stored or
consumed in animal facilities.
6.
Smoking and/or application of cosmetics shall not take place in the animal facility.
10. Used laboratory glassware and other materials shall be stored safely before disinfection. Materials for
disposal shall be transported without spillage in strong containers. Waste material should be autoclaved,
incinerated or otherwise made safe before disposal. Reusable material shall be decontaminated by
appropriate means.
11. No infectious material shall be discarded down animal facility drains without appropriate waste treatment in
place.
12. Any accidents or incidents shall be recorded and reported to the Safety Officer.
L. CONCLUSION
High standards of laboratory safety and containment that will ensure healthy working conditions for laboratory
staff and protection of the environment must be of the greatest priority. They can only be achieved by careful
study of the principles involved followed by practical application to premises, facilities, operating procedures and
hygiene. Training of all laboratory personnel must be a high priority and no personnel should be allowed to work
until appropriate training and competence has been demonstrated and documented. There is a large published
literature on all aspects of the subject, and further reading is recommended (Beran & Steele, 1994; European
Committee for Standardisation, 2000; Richmond, 19962002; Sewell, 1995; World Health Organization, 2004).
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Chapter 1.1.3. Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
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