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Abstract

This article describes the refinement strategies that can be used in research requiring non-human primates to study infections of highly dangerous pathogens (biocontainment level 3 [BCL3] infectious agents). Successful attempts have been made to develop containment systems that balance the need for staff safety at all stages of the experimental process, whilst addressing the requirements of the animals for space, environmental enrichment and social interaction.

A state-of-the-art containment system has now been designed, built, validated and used in a series of non-human primate based studies. The system allows group housing of macaques that have been given BCL3 agents, such as Mycobacterium tuberculosis, by the most hazardous route (aerosol challenge) whilst offering quantifiable operator protection during infection, husbandry, experimental sampling and necropsy.

Keywords: biocontainment, infectious agents, macaques, refinement

Introduction

There are still some circumstances under which it is necessary to use non-human primate models for research. In the case of research on infectious diseases, the UK Government has accepted the conclusions of the 2006 Weatherall report on the use of non-human primates in research (1): "Given the vital role of non-human primates in some aspects of infectious disease research, particularly with regard to vaccine development, we therefore consider there to be a strong case for maintaining the use of non-human primates in this field. Furthermore, as evidenced by the recent appearance of SARS, there is a constant risk of devastating epidemics of infection by other newly emerging organisms. It is vital that expertise in the use of non-human primates is maintained, if rapid responses to these new infectious agents are to be effective."

Figure 1. Containment system for group housing macaques.

The UK Health Protection Agency (HPA) has, as part of its remit, a requirement to develop and evaluate vaccines and therapies for infectious diseases of humans in order to retain a capability to respond to emerging infectious agents that threaten human health.

A number of macaque models have been developed for highly infectious BCL3 agents in order to evaluate vaccines or therapies that are near to clinical use. For example, macaque models (Macaca mulatta and Macaca fascicularis) are currently used in research programmes, such as Tuberculosis (TB) vaccine evaluation, for the following reasons: 

• They have close similarities to humans in their anatomy, physiology and endocrinology
• Host molecules implicated in TB infections are present in macaques and humans but are not found, or are fundamentally different, in small animals
• They are susceptible to TB via the natural (respiratory) route of infection, resulting in disease that mimics human tuberculosis
• They are a valuable pre-clinical indicator of success in humans in terms of safety and immunogenicity and are considered to generate relevant efficacy data that can be used for regulatory submission
• The relative efficacy of different vaccine candidates can be assessed if a standardised model is used. Only a calibrated model can predict the relative efficacy in humans, achieved through the comparison of the immune responses that are induced in non-human primates and man when given the same vaccine
• Useful sequential volumes of blood, saliva and broncheo-alveolar lavage can be obtained that allow researchers to quantify and qualify the developing immune response to vaccination or challenge.

The natural route of infection for many BCL3 agents is by inhalation and the experimental models used by the HPA take advantage of the aerobiology (see Glossary) expertise that has been established over a number of decades. Administering dangerous pathogens to macaques by this route and the subsequent maintenance, sampling and necropsy (see Glossary) of the animals present a high degree of risk of infection to personnel. Very few research establishments are capable of dealing safely with the issues presented by such a combination of hazards.

Safety, welfare and science

Staff safety is the primary consideration for all experimental work and is assessed by a hierarchy of options:

• Removing the hazard: is the work necessary? (e.g. could a less dangerous species, a surrogate, or a less dangerous pathogen be used?)
• Isolating the hazard: by primary containment (e.g. totally enclosed cabinets, plastic film isolators, individually ventilated cages [IVCs])
• Partially isolating the hazard (e.g. by a combination of strategies, such as a directional flow system, transfer boxes, downdraught tables, personal protective equipment [PPE] and respiratory protective equipment [RPE] (see Glossary)
• Isolating the individual operator from the hazard (e.g. full airsuits or ventilated two-piece suits)
• Staff vaccination.

Animal welfare is also an important consideration. Primary containment systems that can house macaques are not commercially available and in many research establishments outside the UK there is a greater reliance on PPE and RPE than would be considered good practice under current Health and Safety regulations in this country (2). Furthermore it is common practice in some countries to minimise the risk to operators by housing infected animals singly in relatively small cages that simplify the process of feeding, husbandry and capture for scientific procedures. Many scientists will also argue from a scientific viewpoint that single housing is necessary to prevent cross-infection between experimental subjects that might confound the results and that this issue outweighs the needs of the animals for an environment in which to exhibit natural behaviours.

In the HPA there is a strong belief that, wherever possible, every effort should be made to improve animal welfare and to house infected animals in well-established social groups in cages of sufficient size and complexity that will allow interaction with conspecifics (see Glossary) and natural behaviours such as foraging and play. 

One particular HPA research establishment has developed a containment system for macaques that has achieved quantifiable levels of operator protection whilst optimising animal welfare. The approach taken has been based on many years of experience with containment strategies, not only for laboratory work but also with a wide range of animal models. During the design phase of this particular containment system considerable effort was made to gain the views of animal care staff and experts in the field of non-human primate behaviour and training.

Risk management when working with infected macaques

There are many risks as a consequence of working with experimentally infected macaques. There are physical risks of contamination from the animals coughing, biting, grabbing, urinating or throwing faeces. There is also a risk of aerosol infection from organisms shed by the animals and this risk is magnified if the animals are given enough space to move freely, forage, play and interact with other members of the group. Another area of risk is that associated with husbandry and procedural activities, where food, water and bedding need to be changed regularly and experimental procedures such as dosing, blood sampling and X-ray need to be conducted.

Addressing the hazards

Pre-training and acclimatisation

When working with dangerous pathogens, physically restraining macaques to administer substances by gavage (see Glossary) is not a safe option, and where significant volumes of blood are required it would be difficult to sample safely from an unsedated individual. However, the animals can be trained to take an oral dose of, for example, an antibiotic (Figure 2). Typically macaques in the HPA are trained using positive reinforcement techniques to i) station (move to a specific location in their home cage) so that all individuals can be effectively dosed without interference (Figure 3), and ii) co-operate by presenting themselves for intramuscular injection of a sedative, so that procedures such as X-ray, weighing and temperature measurement, which are vital to clinical assessment, can be conducted safely and with minimal stress caused to the animals.

Staff members, including researchers, are actively encouraged to interact with macaques before the infection phase of an experiment in order to familiarise the animals with the presence and behaviour of humans. Training the animals to receive an oral dose, to remain stationed whilst others receive theirs, and to come forward for injection is now an integral part of the experimental protocol and we anticipate that this will lead to scientific benefits in terms of the reproducibility of results.

Figure 2. Training for oral dosing.

Figure 3. Training for stationing.  

Due consideration is also given to the changes to the animals' environment that will occur once infectious challenge has taken place. For example, for short periods before challenge, the animals can be acclimatised to changes in the appearance of staff wearing protective clothing. Similarly, the animals can be introduced to containment housing prior to challenge so that they acclimatise to this novel environment. Some establishments do this by placing open cages within a larger gang pen set up, so that animals grow used to the cages, can enter and leave them at will and can use them as a refuge or feeding station.

Biocontainment

A containment system has been developed that utilises the principle of directional airflow away from the operator towards the back of the cage system in order to protect staff. A series of interconnected cages are placed inside modular booths that have a clear rigid plastic screen at the front of the system (Figure 1). This screen allows good observation of the macaques within the cages whilst providing protection from physical contamination by urination, defecation, cough, thrown objects or animals reaching through from the cage. Each screen is fitted with flap valves that control the velocity of the air into the system and each has a number of small access doors placed in strategic alignment with the cage front to allow animal care staff access to replace food and water, change bedding and sedate the occupants for subsequent procedures.

The number of airborne organisms is reduced by using this directional airflow away from the operator towards the rear of  the cage. With correct operation this flow is maintained at a minimum of 0.7m/second and the air removed from the rear of the cage is extracted by a total-loss room air handling system via HEPA filtration (see Glossary). Further operator protection is provided by using a transfer box for sedated animals and a downdraught table for more risky procedures, such as blood sampling, broncheoalveolar lavage, X-rays, close clinical assessment and necropsy.

Animal welfare needs are provided for by incorporating group housing and pre-training into the study design, utilising full height cages that meet the standards of the revised Council of Europe Convention ETS 123 (3), and providing foraging enrichment and toys such as balls, wooden dumbbells, chains and mirrors. Cages used for studies involving infectious agents need to be able to withstand robust decontamination processes, such as autoclaving and fumigation, that are required at the end of a study to provide for staff safety and for the scientific integrity of the following study. Whilst stainless steel is the obvious choice as a material for such cages, other materials can be used to provide a softer and less noisy environment, such as Trespa for cage partitions and polypropylene for platforms, shelves and perches (Figure 4).

Figure 4. A single component of three joined units. Note the Trespa side panels and polypropylene shelves.

When using some pathogens, it is not good containment practice to allow animals to have direct contact with the floor substrate. However, a grid floor can be provided above a tray containing substrate such as enviro-dry or sizzlenest to allow foraging behaviour. Alternatively, or additionally, puzzle feeders (Figure 5) and forage boxes (Figure 6) can be placed within the cage where they do not obstruct any dividing or restraining mechanisms. Forage boxes can be made of cheap, simple disposable materials, such as cardboard.

Figure 5. Puzzle feeder.

Figure 6. Forage box.

Control

When using highly infectious agents, the risks associated with the husbandry and experimental handling of non-human primates must be controlled by strict discipline with regard to feeding, watering and waste removal regimes. For these activities staff members are trained to adhere to a protocol described in a Code of Practice.

Setting robust, early experimental end points, using a range of clinical and behavioural parameters or microbiological indicators, minimises animal suffering (refinement) and reduces risk to staff. For example, a read-out end point for protection or efficacy could be virus isolation or viral load by polymerase chain reaction (PCR) in untreated controls before onset of disease (pilot experiments may be required to verify that this, combined with other clinical parameters, correlates with progression to disease). This approach is better from both an animal welfare and staff safety point of view rather than allowing progression to severe disease. This is an area where there is a strong case for data sharing on national and international levels, so that research groups can learn from previous work and not repeat experiments to establish such end-points.

Clinical monitoring can be carried out by taking X-rays (Figure 7), and by using telemetry implants to monitor heart rate, blood pressure, temperature, activity and respiration rate (Figure 8). Unfortunately, the telemetry system currently in use requires the macaques to be separated during data capture to avoid cross talk interference that may render the data meaningless, although they can be housed with non-implanted companions. However, telemetry systems are being developed that allow simultaneous data capture from  group-housed telemetered animals.

Figure 7. Taking an X-ray under BCL3 containment.

Figure 8. The telemetry receiver.

In order to observe important clinical signs there needs to be a commitment to round-the-clock monitoring for infected animals. This may mean that staff members need to enter the animal room on a regular basis throughout the night or, alternatively, observation can be made using closed circuit television (CCTV; Figure 9). It has been found that this is not only more convenient for staff but also is less disruptive for the animals. Furthermore, clinical assessment of animals that have been disturbed by the entry of an observer can be far less informative than remote observation of animals which are unaware of, and not stimulated by, the presence of humans.

Figure 9. Closed Circuit Television (CCTV) observation.

Scientific value

Group housing has been used without problems in terms of cross-infection, provided that all animals housed in each group receive the same treatment, such as the infectious dose and/or vaccination regime. Vaccinated and naïve animals are not housed together where a live vaccine such as BCG or a vaccine using a live viral vector is being used, as the close association of cage mates could bring about immune stimulation of the unvaccinated animals and compromise the results of the experiment. Compatible groups of up to six macaques have been housed together successfully with no confounding effect on the experimental outcomes.

Figure 10 shows MRI scans of the lungs of unvaccinated animals given two different doses (two animals per dose level) of the bacterium Mycobacterium tuberculosis which causes TB. It can be seen that the two animals housed together show the same level of pathology and that this is dependent upon the initial dose given rather than any cross-infection.

Figure 10.  MRI scans of the lungs of two animals given TB at dose levels, of either 30 CFU (colony forming units) or 75 CFU. The disease pathology is consistent with the dose received.

Conclusions

In order to allow work with macaques given highly infectious human pathogens, a containment system has been developed in the UK that can provide for good animal welfare whilst ensuring staff safety to a quantifiable level. The system allows group housing for macaques and has sufficient space and complexity to allow the animals to behave and interact naturally. Activities such as foraging and play can be supported whilst at the same time providing ready access to the animals so that procedures can be carried out safely. The system has been used successfully for a number of research programmes and a range of infectious agents. Pre-training the animals prior to infection enhances the environmental conditions, the reproducibility of scientific procedures and staff safety.

Glossary

Aerobiology: The science of the behaviour and survival of microbial aerosols and their delivery to experimental subjects.
BCG vaccine: A vaccine based upon a live but weakened form of Mycobacterium bovis (Bacillus Calmette-Guerin) that has been used for many years to prevent tuberculosis.
BCL3 agents: Biological agents that can cause severe human disease and a risk of spread to the community, where there is usually prophylaxis or treatment available.
Broncheo-alveolar lavage: Introduction of a physiologically compatible solution into the lungs via the trachea, and immediate removal of the recoverable fluid for immunological analysis.
Conspecifics: Animals of the same species.
Gavage: Administering a dose of fluid via the oesophagus using a flexible tube.
HEPA filtration: High efficiency particulate air filtration used to prevent the entry or exit of microbes from a room or cabinet.
Immunogenicity: The ability to stimulate an immune response.
Necropsy: The removal of tissues after death for analysis.
PPE: Personal protective equipment.
RPE: Respiratory protective equipment.
SARS: Severe Acute Respiratory Syndrome.
Tuberculosis (TB): A disease caused by the bacterium Mycobacterium tuberculosis .

References

1. Weatherall D, Goodfellow P, Harris J, Hinde R, Johnson L, Morris R, Ross N, Skehel J, Tickell C (2006) The Use of Non-human Primates in Research. Academy of Medical Sciences: London, p136.  http://www.acmedsci.ac.uk/p48prid6.html
2. Advisory Committee on Dangerous Pathogens: Categorisation of biological agents according to hazard and categories of containment (1995), 4th Edition, HSE Publications, Sudbury.
3. Council of Europe (2006) Appendix A of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123). Guidelines for Accommodation and Care of Animals (Article 5 of the Convention). Approved by the Multilateral Consultation. Cons 123 (2006) 3. Strasbourg: Council of Europe, 2006. http://www.coe.int/t/e/legal_affairs/legal_co-operation/biological_safety%2C_use_of_animals/laboratory_animals/2006/Cons123(2006)3AppendixA_en.pdf

All views and opinions expressed in this article are those of the author and do not necessarily reflect the views and opinions of the NC3Rs.