Imported biologicals: unforeseen biosecurity risks in the laboratory

As imported biological products are so common place in our everyday lives and in laboratory settings it is easy to overlook that they are potential vectors for the introduction of significant biosecurity risks into Australia, such as pathogens of biosecurity concern as well as Security Sensitive Biological Agents (SSBA)¹. In addition to SSBAs, many of the most significant animal and environment biosecurity risks are listed in the national notifiable disease list of terrestrial animals² and the interim national priority list of exotic environmental pests, weeds and diseases³. Australia’s strong regulatory framework for imported biologicals considers these unforeseen risks and manages them according to Australia’s Appropriate Level of Protection (ALOP), which is a high level of sanitary and phytosanitary protection aimed at reducing biosecurity risks to a very low level, but not to zero⁴.

In this article, biologicals are considered to be products or goods produced using microorganisms, viruses or components derived from human, animal and plant tissues. These may be produced by biotechnology industries, or used by laboratories for diagnostics, reproductive services, and research and development. Commercial products such as vaccines and/or therapeutics containing materials of biological origin, and the myriad of specialised laboratory reagents may also be considered biologicals. Microorganisms, parasites and infectious agents used as tools for the development of products, imported as, or included within, the final product are also considered as biologicals for the purposes of this article.

Laboratory personnel and scientists frequently consider the risks posed to human health first and foremost when using biologicals in a laboratory. From a biosafety perspective, humans are most obviously at risk though laboratory-acquired infections⁵. Less obvious, but important, is the risk posed by biologicals to animals, plants and the environment. This lack of awareness can lead to inadvertent mishandling of imported biologicals contaminated with significant adventitious agents. A key role of the Department of Agriculture, Water and the Environment is to consider these unforeseen and unconsidered risks before biologicals are imported and to work with laboratory networks and importers to facilitate the safe and regulated importation of biologicals, allowing critical scientific research and development to be performed in Australia.

Adventitious or extraneous agents are microorganisms or infectious agents introduced into the manufacturing process of a biological product^6,7. Adventitious agents can be introduced into many stages of product manufacture from source material to final packaging. Although the likelihood of significant adventitious agents being in biologicals is relatively low, the consequences of their establishment in Australia can be extremely high⁸. Therefore, the department carefully assesses and manages these risks. There have been significant advances in quality control systems and regulation of the production of biologicals that ensure that the majority of biological products used present a managed biosecurity risk; however, there is still a small percentage of these products that may be contaminated with adventitious agents.

Eukaryotic cell culture is one of the most well established routine laboratory practices for introducing and maintaining biosecurity risks associated with biologicals. Contamination with Mycoplasma spp. is common in eukaryotic cell culture^9,10, and cross contamination incidents of cell lines, or contamination with other unknown cell lines is also extremely common¹¹. At least 1/3 of immortalised cell lines in cell culture collections are estimated or demonstrated to be either cross-contaminated, or misidentified^12,13. According to the register of misidentified cell lines¹² between 9% and 15% may be misidentified at the species level; misidentification at the species level introduces additional biosecurity risk as risk management such as adventitious agent testing or country of origin requirements may be incorrectly targeted. Moreover, continuous cell lines can be used for many years and the historical knowledge of the origin can be lost through inaccurate or absent record keeping further complicating an assessment of biosecurity risk. Numerous pathogenic viruses of animals can easily persist in cell lines as undetected adventitious agents, including significant agents of animal biosecurity concern such as bovine diarrhoea disease virus (BVDV)¹⁴, bluetongue virus (BTV)¹⁵ and porcine circovirus (PCV)¹⁶ among many others as well as agents identified as SSBAs, particularly classical swine fever virus (CSFV)¹⁷.

CSFV is the causative agent of classical swine fever (also known as hog cholera) and the last outbreak of CSFV in Australia was eradicated in 1961¹⁸. CSFV is notifiable to the World Organisation of Animal Health (OIE), nationally notifiable throughout Australia² and considered a Tier 2 SSBA as regulated by the Australian Government Department of Health¹. An outbreak in Australia would have significant animal health and economic consequences¹⁹. CSFV is known for persistent cell line contamination, specifically of porcine cell lines^17,20,21. The source of CSFV cell contamination is most likely through the use of infected source material²⁰. The routine use of porcine trypsin as a dissociative agent for the detachment of adhered cells in culture is also a potential route for the introduction of CSFV as it has been for other porcine viruses²².

An example of biological products of notable animal biosecurity risk is foetal bovine serum. Foetal bovine serum (FBS) is widely used as a cell culture supplement, as well as in the production of other biologicals and directly in reproductive technology. Between 15% and 49% of commercial FBS produced worldwide is estimated to be contaminated with BVDV^14,23–25, with the likely associated BVDV contamination in cell line collections between 24% and 33%^14,26,27. As a persistent adventitious agent of cell culture, detecting BVDV can be difficult due to the non-cytopathic nature of specific strains. Contamination with exotic BVDV is a significant biosecurity risk to Australia. A recent (2018) overseas example is the association of BVDV-1 outbreak in captive Rocky Mountain bighorn sheep after vaccination with a contaminated modified-live BTV-10 vaccine. In this example, there were significant herd mortalities that the authors hypothesise were directly linked to adventitious agents within the vaccine. Retrospective analysis determined that the vaccine was positive for BVDV-1²⁸, while the source of the BVDV in this case is unknown, cell lines, source material or sera are the most likely sources of infection.

The department utilises a range of regulatory tools and information sources as well as risk assessments to manage the animal and human biosecurity risks posed by imported biologicals. Import conditions, import permits and Approved Arrangements are the key regulatory tools available under the Biosecurity Act 2015. All imported biologicals used in the laboratory environment are managed using at least one of these regulatory tools. Most biological products used in Australia, present a well-managed biosecurity risk, however, there is still a possibility that these products can be contaminated with adventitious agents. Wherever possible, the department imposes the minimum regulatory requirements for each type of product. In most cases an import permit is required, involving an application, fees, provision of relevant information about the products and an assessment by the department of the risks posed and any conditions required to mitigate those risks. For most permit assessments for imported biologicals, the department is seeking to be assured the biologicals pose a very low biosecurity risk. When the biosecurity risk is not sufficiently low, wherever possible conditions will be imposed to reduce the risk, including sourcing, treatment, containment within an Approved Arrangement and end-use conditions (Figure 1), to reduce the risk below Australia’s established ALOP of very low.

Figure 1.  Importing biologicals into Australia.

An Approved Arrangement is a regulatory tool that provides a legally binding agreement between the department and an entity/institution for them to manage biosecurity risks in a defined way. While Approved Arrangements cover a wide variety of activities, including treatment facilities and containment laboratories. An Approved Arrangement sets the criteria that must be met and gives the department oversight of the activities and outcomes undertaken in the Approved Arrangement. The use of an Approved Arrangement allows the department to impose conditions under the Biosecurity Act 2015 and gives the department a high level of assurance that biosecurity risks will be appropriately managed. For importers, this regulatory assurance can broaden the scope for the use of biological materials in Australia. This may include flexibility in the experimental use of exotic infectious agents themselves where the biosecurity risks cannot otherwise be managed outside of an Approved Arrangement.

The Department of Agriculture, Water, and Environment regulates and performs risk assessments on imported biologicals to identify and manage biosecurity risks posed by adventitious agents in imported biologicals according to Australia’s ALOP. Many agents of biosecurity concern and threat are also established adventitious agents of biologicals. This presents challenges to the application of risk management strategies that can be both practical and feasible whilst minimising risk to an appropriate level. Laboratory managers, principal investigators, scientists and regulators have important roles to play in both risk assessment and management, ensuring that the ever-changing biologicals so critical for day-to-day research and development do not become the source of the next significant disease event.

The best source of information regarding biosecurity regulation of imported biologicals is the department’s BICON database: https://bicon.agriculture.gov.au/BiconWeb4.0. Further information about Approved Arrangements is available at: https://www.agriculture.gov.au/import/arrival/arrangements.

The authors declare no conflicts of interest.