Introduction

Good mental and physical health is integral to the success of a veterinarian in the workplace. One of the ways to ensure this is to take measures to ensure that work practices are safe.

Loss of life, serious injuries and infections that lead to severe debilitation affect us as individuals in our ability to earn an income not only for ourselves but also for our dependents. Students may find that they are delayed or disadvantaged in their studies. Employers experience losses in the form of: staff absenteeism; decreased production; over-burdening current staff; having to employ and up-skill new staff; and legal fees and compensation pay-outs. Countries find that the level of production and thus income is reduced and the number of persons requiring financial and other assistance is high.

How does biosafety fit into safe work practices?

• Decrease risks of occupational diseases in yourself and your staff, clients  i.e. infection with zoonotic agents
• Decrease the risk that you may act as a conduit of infectious agents to animals and people 
  

Definitions

Biosafety: The application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards. Biosafety is aimed at eliminating the risk of exposure of individuals (humans and animals) to potentially hazardous biological agents.

Biosecurity: Procedures or measures designed to protect a population against harmful biological or biochemical substances. It is part of biosafety but usually employs strategies to prevent the introduction of harmful pathogens i.e. at international borders, using fences to protect regions or farms. It also aims to spread the spread of harmful pathogens by containing them in an area i.e. quarantining the whole farm.

Zoonosis: An infectious or parasitic agent which can be transmitted to humans from animals.

Reverse zoonosis: An infectious or parasitic agent that is transmitted from humans to animals. SARS-CoV-2 is an example of a virus that transmits from people to felids (cats), canids (dogs) and mustelidae (ferrets and mink).

Occupational disease: A disease or disorder that is caused by the work or working conditions. Infectious diseases can also be occupational. Examples include infections due to dog or cat bites – the most common zoonosis in young vets. Hendravirus is also an occupational disease.

SAFE WORK PRACTICES

Scenario 1:

Consult the “Guidelines for veterinarians handling potential Hendra virus infection in horses” (2009) published by Queensland Primary Industries and Fisheries and the Department of Employment, Economic Development and Innovation.

Question 1:

Safe work practices are vital, but what does that mean in the light of biosafety

Question 2

HAZARD ANALYSIS

A hazard analysis is the process of deciding what might be a hazard, and what should be done if someone or something is exposed to this hazard.

This is not a strange concept as we are doing it all the time – just not writing it down. When you drive your car to university every day there is a slight risk that you will have a serious accident or the vehicle you are in breaks down and you, as a sitting duck, are attacked and robbed. However, you are a competent driver and you assume other drivers on the road are law-abiding, competent and polite.  Thus, there is a very low risk of the more serious consequences, and the benefits outweigh the risks – so you drive to uni.

Our activities at university and work are no different. So why undertake a formal risk (hazard) analysis?

• statutory requirement
• we are responsible for our own health and safety
• so we can remain healthy and have a good quality of life.

Five Basic Steps in Risk/Hazard Analysis

1. Identify the hazards

A hazard is any agent that can cause harm or damage to life, health, property or the environment. Thus any living agents such as infectious agents and dangerous and invasive animals and plants would be called a biohazard.

2. Assess the risks that may result from the hazard

Risk is the potential of gaining or losing something of value. Value can be life, health, property, income, social status etc..

In the light of biosafety, risk is the likelihood that death, injury or illness, or other loss of resources, might result because of the biohazard. This is all about consequences.

3. Decide on the most appropriate control measures that will prevent or minimise the level of the risks.

What should you do? i.e. wear gloves to protect your hands from per-cutaneous inoculation of agents. Wear protective clothing to contain spills.

4. Modify your work practices or work environment so that you can safely carry out the work

For example, work in a Class 2 Biosafety cabinet for infectious agents that easily form aerosols.

5. Monitor and review the effectiveness of measures 

Have the number of reported incidents decreased?

Examples of sharps hazards and how to assess the risks and how to reduce the risks are below

1. A needle stick injury.  Complete the short quiz below that provides an example of a hazard, the risks and management of the risks.

2. Glass, scalpel and needlestick injuries. See if you can answer the questions on the flash cards. Answers are provided when you turn the flashcards.

USING A RISK ANALYSIS TABLE TO ASSESS AND PRIORITISE BIOSAFETY RISKS

A simple way to decide how to deal with the risk associated with infectious agents is to assign a risk level based upon the seriousness of the disease they cause as well as the likelihood of it happening is to use the following Risk Analysis Table.

For more details to understand how to estimate risk level based on likelihood and consequences refer to:

Sensi, A., Brandenberg, O., Ghosh, K & Sonnino, A. (2011). Module C: Risk analysis. In Biosafety Resource Book (pp. 26-29). Food and Agriculture Organization of the United Nations (FAO).

Below are some questions that you can practice determining the risk level using the table above and information provided on some diseases.

Transmission route and laboratory-acquired infections

The transmission route of infectious agents is associated with different Biosafety Levels of Risk. For instance, the risk for systemic disease and consequently more serious disease is much higher in inhaled infectious agents compared to those who have entered via wounds. Below is a pie chart showing the transmission routes of pathogens associated with laboratory accidents.

The American Biological Safety Association has a database that is constantly updated with reported or published laboratory accidents. Below is a table showing the reported incidents of laboratory-associated infections worldwide since 2000.

The most common cause of laboratory-acquired zoonotic infections worldwide is brucellosis, caused by Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis and Brucella ceti.  There are more than 500,000 people infected annually with Brucella species with most of them being in China. However, only a small proportion are associated with laboratory-acquired infections. These are most often associated with the production of infected aerosols that are inhaled. Australia is in the fortunate position that it has eradicated Brucella abortus and never introduced Brucella melitensis and Brucella canis. i.e. the risks have been eliminated. This means that, in Australia, the hazards are Brucella suis infections in feral pigs and pigging dogs and Brucella ceti in marine mammals.

Below is a quiz that uses scenarios described in:

Zhou, C., Huang, W., Xiang, X., Qiu, J., Xiao, D., Yao, N., Shu, Q., & Zhou, S. (2022). Outbreak of occupational Brucella infection caused by live attenuated Brucella vaccine in a biological products company in Chongqing, China, 2020. Emerging Microbes & Infections, 11(1), 2544–2552.

Song, L., Gao, J., & Wu, Z. (2021). Laboratory-acquired infections with Brucella bacteria in China. Biosafety and Health 3(2), 101-104.

Pathogen Risk Group Classification

For a full explanation of the different biosafety levels see the YouTube video, Understanding Biosafety Levels.

Pathogens are classified into different risk classes (1 to 4) broadly based on their ability to cause serious life-threatening or debilitating diseases and whether the disease is treatable or not.

Other factors that are taken into account include the following:

• route of transmission
• host range
• exotic or endemic
• ability of the infectious agent to survive a long time either in the environment or the host/s
• effective control measures available.

They are contained into 4 levels with 4 different requirements with each higher level being more stringent regarding:

• safety equipment
• laboratory procedures
• infrastructure.

Below is a table showing the different categories of personal containment levels (the rest of the world calls them biosafety levels)

Examples of different pathogen risk agents

Write down the risk level of the listed pathogens. You will find all the answers in a web search.

At JCU you will be working in PC1  (BSL-1) and  PC2 (BSL-2) laboratories

Laboratory Rules

What to wear

Always dress professionally and make sure you have fully closed-in shoes that are easy to walk in. Joggers are good. In a microbiological laboratory, you will always be provided with a clean laboratory gown to put on over your normal clothes, gloves and safety glasses. You will also be provided in higher PC laboratories with full PPE which includes full face protection. This means that any microbes you work with will stay in the laboratory and not go home with you.

Some laboratory do nots, consequences and alternatives^[1]

Don’ts	Consequences	Alternatives

Biosafety Containment Laboratories

Personal containment 1 (PC1) Laboratory

• It usually deals with microorganisms that are not highly pathogenic to people
• It allows open bench working and is common in schools and physics and chemistry laboratories
• Standard laboratory practices i.e.: protective clothing (as needed), handwashing when dealing with infectious agents and when leaving laboratory, no eating, drinking, applying cosmetics or mouth pipetting, surfaces routinely cleaned and disinfected, easy human movement, closed shoes limited access and fire safety.

Personal containment 2 (PC2) laboratory

• It usually deals with micro-organisms that can infect animals and people but are easily treated. They are usually endemic to this region. Many clinical samples fall in this category as we don’t know what is in them. Once a risk 3 microorganism is identified – must move to a PC3 or be destroyed.
• Additional to PCl – No hand operated taps, no items on floor, access control, dedicated protective clothing including gloves, closed at front.
• The microbiology laboratories you work in at JCU are all classified as PC2.
• Air-conditioning is under negative pressure – allows organisms in, not out.
• It usually has additional containment i.e. Biosafety cabinet class 1 or 2 – Use when you expect splashes or aerosols or when dealing with microbes that easily become airborne i.e. some fungal spores.
• Proper decontamination of all surfaces. Usually, 70-80% alcohol is used.
• Proper disposal of Biological waste – autoclave or specialised removalist services.

Example of a laboratory-acquired infection of a laboratory worker with a Risk level 2 bacterium

Laboratory-acquired infections with MRSA are rarely reported, despite these infections being common in hospitalised patients. Below is a case where this has happened where a laboratory worker had his nasal cavity colonised with EMRSA (endemic methicillin-resistant Staphylococcus aureus) and only got a wound infected two months after the colonisation occurred.

Personal containment level  3 (PC3) laboratory

Serious, but treatable diseases both endemic and exotic, i.e. Q-fever ( Coxiella burnetii), bovine tuberculosis (Mycobacterium tuberculosis var. bovis), that are transmitted through inhalation and cause serious or lethal disease.

Same as PC2. In addition:

• Only trained and immunised person access
• Restricted access
• Airlock, sealed windows, negative pressure
• Separate air supply and removal – HEPA filters
• Biosafety cabinet class 2 must be used
• Everything, except for people, going out is autoclaved or disinfected

Personal containment level 4 (PC4) laboratory

Used for infectious agents that cause serious and often fatal disease and are not readily treatable and are highly transmissible i.e. Foot-and-mouth virus, Hendavirus, Ebola virus and Rabies virus. Only one animal PC4 facility in Australia at Geelong.

In addition to PC3:

• Shower in shower out – no personal clothes
• Proper decontamination of all substances leaving the laboratory
• Full protective clothing – no outside clothes
• All work within a Class 3 BSC
• Separate facility with dedicated air supply and exhaust and decontamination systems

What are Biosafety cabinets (BSC)?

Class 2 Biosafety cabinets allow a worker to work on microorganisms that may require more containment as they are more easily aerosolised. The inhalation of pathogens is one of the most hazardous means of transmission of infective agents within the laboratory. These are used in PC2 laboratories laboratories.

Class 3 Biosafety cabinets are used in PC3 and PC4 laboratories. They are fully sealed. Its features are discussed below.

Other safety equipment present in Biosafety laboratories

• emergency showers and eye wash stations
• biological waste disposal bags and sharps bins: These containers are often autoclaved at 121°C for 20 minutes before being collected for disposal. They can also be incinerated.
• a spills kit contains acid and alkali buffers, a towel to mop up, a dustpan and brush, warning signs
• handwash stations
• bunsen burner and electricity cut-off switches.
• fire extinguishers – flammable gas is an enormous fire risk. The number of microbiology laboratories using gas is decreasing with alternative methods being sought to reduce their need. For instance, dried Gram’s stains can be fixed with 100% methanol.

Disinfectants used in the laboratory (Click on the link to find out more about disinfectants)

In this section, I will only discuss disinfectants commonly used in laboratories. There is a wider range of disinfectants used in veterinary clinics and hospitals. You should become familiar with their properties before you use them.

1. In PC1 and PC2 laboratories, we generally use 70 to 80% ethanol as a disinfectant as it kills bacteria, fungi and most viruses but is not sporicidal. Ethanol works by denaturing proteins, which requires some water, thus 100% alcohol is not effective in killing microorganisms. Ethanol is not effective in the presence of organic material. It evaporates easily off surfaces without leaving a deposit and is not corrosive. Note that it requires a contact time of 10 to 15 seconds to be effective.
2. Other disinfectants commonly used in the laboratory of chlorhexidine (in hand soaps) and Virkon which contains potassium peroxymonosulfate (an oxidizing agent), sodium dodecylbenzenesulfonate (a detergent), sulfamic acid (a cleaning agent), and inorganic buffers. Both chlorhexidine and Virkon as well as killing bacteria, viruses, fungi and protozoa are also sporicidal. They also have residual activity which is not present for ethanol. They require a contact time of 10 minutes.
3. Although bleach, a chlorine compound, is a highly effective disinfectant within the laboratory, it is rarely used for general microbiology laboratories. Mainly because it is corrosive, irritant and bleaches everything. It is not effective in the presence of organic material. Bleach is, however, used in laboratories detecting DNA as it is able to destroy DNA and help avoid the severe DNA contamination some PCR laboratories have. It requires a contact time of 10 minutes. Note that bleach is also effective in removing biofilms and is rapidly produces chlorine gas which dissipates in the environment leaving no residue.
4. UV light. Whilst not essential, many biosafety cabinets usually have a source of UV light set to 254 nm wavelength. The UV light will denature any DNA coming into direct contact. To reach full bacteriocidal and virucidal activity, the UV light should be on for at least 12.5 minutes. Most cabinets are set to leave the light on for 20 minutes with an automatic shut-off and will only work when the sash cover is down. Nevertheless, one must avoid being near a cabinet when the light is on. UV light prevents cross-contamination in PCR work. Note that anything not in direct contact with the light waves i.e. in the light shadow will not be sterilised. The UV light produces ozone so is destructive to plastics and rubber. Reference: Meechan PJ., Wilson, C., (2006). Use of ultraviolet lights in biological safety cabinets: A contrarian view.  Applied Biosafety, 11(4) pp. 222-227.

Example of cutaneous anthrax in a laboratory worker

Below is an example of cutaneous anthrax reported to the CDC (Center for Disease Control, USA) in 2002. Anthrax cases in livestock are being reported annually in Australia in areas in and near the anthrax belt, the area of old stock routes in southern Queensland, New South Wales and northern Victoria.

control measures – hierarchy

Once the level of risk has been determined, it is important to decide on the control measures for the risk that should be implemented. Below is a table that lists control measures that can be taken to reduce risks. The higher the level of risk the more important it is to reduce the risk level.

RISK REDUCTION IN THE LABORATORY AND CLINIC

1. Implement biosafety control measures in a clinic or laboratory.  In a clinic, they might be called infection control policies and measures.
2. Safe transport procedures for samples
3. Safe laboratory practices in accordance with policies
4. Personal protective clothing and equipment (PPE)
5. Biosafety cabinets. i.e. BSC 2 in a BSL-2 laboratory (see earlier)
6. Correct labelling and storage of samples, reagents, growth media, cells, infectious agents etc.
7. Declutter
8. Reporting any accidents so that new controls can be put into place that reduce the risk of these accidents reoccurring

Below is a cluttered examination room. Help Dr Laurel identify the hazards and for each hazard suggest a control measure.

Implement control measures

1. Develop safe work procedures. These should be written down and accessible to all relevant persons.
2. Communication – inform personnel about the control measures.
3. Providing training and instruction to personnel and supervisors. Most laboratory accidents nowadays are associated with poor training.
4. Supervision – adequate supervision to ensure that control measures are being correctly implemented.
5. Maintenance – ongoing requirements to ensure the effectiveness of new controls.

Sample requirements for sending to a laboratory^[2]

Make sure of the following:    Correct sample that has been taken aseptically Place it in a leak-proof, non-breakable container that is properly labelled (BSL-3 & BSL-4 organisms require special packaging – this can be done by a certified courier for you) Fill in the submission forms fully Send via a specially registered courier to handle biological specimens – laboratories often contract couriers to do this.

Scenario: Suspected leptospirosis in a dog kennel

Below is a scenario of an outbreak of suspected leptospirosis in a dog kennel. You want to prove that the disease is present so will be collecting blood and urine samples from all the dogs.

Leptospirosis is a systemic bacterial disease causing fevers, liver and renal disease in animals and people. In the chronic stage, it may cause abortions, chronic kidney disease and even affect the brain and eyes. Animals can become subclinical (no clinical signs) carriers of the bacterium in their renal tubules and shed the leptospires in their urine. Infection can occur via any route as the bacterium can move across intact mucosae and through damaged skin. This bacterium is easily destroyed by disinfectants and drying out. It is treatable with antibiotics. This is to help you answer the quiz. Don’t learn.

Useful Readings

1. Sensi, F., Brandenburg, O., Ghosh, K and Sonnino A (2011) Biosafety Resource Book: Risk Analysis. Food and Agriculture Organisation.
2. O.I.E. (2015). Chapter 1.1. Biosafety and Biosecurity: Standard for managing biological risk in veterinary laboratory and animal facilities. OIE Terrestrial Manual 2015.
3. CDC Expert Panel (2009). Biosafety in Microbiological and Biomedical Laboratories. 5^th Edition. HHS Publication No. (CDC) 21-1112.
4. Queensland Primary Industries and Fisheries (2009). Guidelines for veterinarians handling potential Hendra virus infection in horses. Department of Agriculture, Fisheries and Forestry. Currently under review. New version will be available soon.