The Health Protection Agency's (HPA) innovative "snout-only" aerosol exposure and biocontainment technique is driving significant research benefits, more efficient space use, and increased worker safety at the Centre for Emergency Preparedness and Response in Porton Down, U.K. HPA's highly accurate technique is based around the use of a modular aerosol generator and exposure system built inside a Class-III BSL cabinet. Nose-only delivery of aerosolized agents provides a controllable, accurate exposure system with a high degree of measurable safety that can be modified to accommodate different strategies for both rodent subjects and non-human primates.
“We have a variety of options for containment with the nose-only exposure system. Since it doesn’t require dedicated exposure facilities or cabinet lines we can be very flexible with our space use,” says Mike Dennis, a scientific leader at the HPA.
The Porton Down site has gone through a number of organizational changes since its initial development in the 1940s. Since 1994 the facility was known as the Microbiological Research Authority’s Centre for Applied Microbiology and Research. In 2003, it became the Health Protection Agency’s Centre for Emergency Preparedness and Response. Along with the name change came the mission of reducing the potential impact of infectious diseases, chemical hazards, poisons, and radiation on the public. One of the Centre’s primary roles is to conduct fundamental research for the testing and development of new vaccines and to coordinate responses to emerging diseases.
“One of our main projects at the moment involves the use of guinea pigs and primates in looking at the efficacy of TB vaccines. But we are also working with other inhalation models and that will include primate work as well,” says Dennis.
The primary component in the HPA’s exposure technique is a specialized piece of equipment known as the Henderson Apparatus. The device was originally developed by David Henderson, FRS, in the 1950s for testing the impact of particle size on the infectivity of anthrax spores. It evolved into a mobile unit in the 1960s that was used by the Ministry of Defence to gather field samples. At that time there was no containment associated with the device apart from safety suits. Eventually, components of the unit were incorporated into a modular biosafety cabinet.
“The controls and the elements that generated the aerosol were all integrated into one unit. It was mobile enough to move between labs, but it was a hefty piece of kit,” says Dennis.
The design was later modified so that the aerosol generating equipment remained in a BSL-3 cabinet but the equipment for controlling air flow and relative humidity was separated out, which greatly increased its flexibility and mobility. The modern Henderson Apparatus can deliver a highly accurate and controllable dose of aerosolized biological agents to a variety of species with no pipe work or permanent casing fixed to the room structure.
Aerosols are generated inside the safety cabinet with a Collison nebulizer and pulled by vacuum through an exposure tube called a sow, past the noses of the subjects, then circulated back into the control unit after passing through HEPA filters to remove the microorganisms. Primary containment is achieved using a downdraft table that keeps the flow of air moving down and away from research personnel.
“This system of nose-only exposure reduces the chance of contaminating the fur during the procedure so the system is considerably more precise. It is also safer for those handling the animals after they have been exposed,” says Dennis.
The nose-only system is adaptable to a wide range of species and biological agents. Small animals like mice and guinea pigs are placed in custom restrainers that align their snouts with the exposure tube. The aerosol is generated in the cabinet and channeled past their noses under negative pressure for a fixed amount of time. Due to the type of pathogens involved and the drive towards primary containment where possible, the HPA now encloses the process for guinea pigs and mice within a plastic film isolator while operators work through built-in half suits that have their own air supply and integrated communication systems so that researchers can talk to each other and to people outside the chamber.
“This system offers a high level of safety, but there is some compromise in dexterity. Obviously, the half suits have one-size-fits-all gloves so you don’t want to do a lot of fine technical work,” says Dennis.
Animals are transferred out of the system in sealable transport boxes that connect to the system so lab personnel can safely move them in or out of the caging system.
For aerosol delivery to primates they are sedated and encased from the neck down in a sealed plethysmography chamber that monitors lung volume and air exchange during the exposure procedure. The agent enters the mask and is extracted through an out-port all under negative pressure. There is also a sample port that allows researchers to monitor the numbers of organisms within the aerosol, enabling calculation of the exposure dose. Containment is still provided by a downdraft table with solid plastic screens.
Air sampling is essential to establishing baselines and thresholds for exposure. Air sampling, plethysmography, and estimated retention factors are all considered in the process of defining exposure and inhalated quantities of the agent.
Fewer Pipes, Better Research
One of the design considerations of the modular system is to minimize the length of pipe work from the aerosol-generating apparatus to the exposure area, reducing the decay of biological agents due to impaction on the internal surfaces of the pipes.
“In the old system, larger particles with high-humidity would impact on the pipe work and there would be a rapid drop off in aerosol concentration. With this new system that effect has leveled off considerably, which shows the advantage of not having too much pipe work when aerosols are circulating around,” says Dennis.
Maintaining the Balance
Under pre-exposure conditions the balance of procedures are weighted towards animal welfare and enrichment. Animals have access to gang pens with deep-litter foraging, swings, and other equipment so they can associate with each other socially within experimental groups. Modular cage systems work in conjunction with transfer boxes minimizing disruption.
After exposure to a respiratory pathogen the procedural balance shifts towards staff safety with no compromise on containment. To address the desired safety constraints and the need to group-house the animals, the HPA designed the system and its procedures in-house in order to assure quantifiable protection to staff.
Among the benefits of the HPA’s precise exposure technique is the ability to objectively measure the relative safety of various equipment and procedure combinations.
“One operating principle that is very important to us is being able to quantify the protection that these containment strategies provide,” says Dennis.
Aerosols of tracer organisms are generated within the system and measured by samplers placed within the working area. The operator protection factor is determined by looking at the ratio between airborne particle levels within the containment system and the highest levels recovered outside of the containment area. The system also goes through a regular validation process every six months, or whenever it is moved. The BSL cabinet, downdraft table, filters, and seals are all tested for pressure and performance according to British standards.
“We have a biosafety department that tests these virtually to destruction. They generate huge clouds of aerosol and sample the outside air. They enact accident scenario testing of what happens in a glove-off situation, or if there is a tear in the film of the isolator, and they have demonstrated quite convincingly that even if such an incident occurred, there is still a very high operator protection factor,” says Dennis.
By Johnathon Allen