Low-Cost Aerosol Exposure System for Guinea Pigs

Animals

Eight female, outbred Hartley guinea pigs, approximately 800–1200 g in weight (Charles River, Wilmington, MA), were housed in a biohazard level 3 facility, exposed to a 12-h light/dark cycle, and provided food and water ad libitum. After a 30-day acclimation period, animals were individually monitored through the insertion of glass encapsulated transponders (Bio-Medic Data Systems, Inc., Rockville, MD) into the back scruff. All procedures were reviewed and approved by an independent Institutional Animal Care and Use Committee (IACUC).

At the time of sacrifice, guinea pigs were euthanized by intraperitoneal injection (150 mg/kg) of sodium pentobarbital (Sleepaway; Fort Dodge Laboratories Inc., Fort Dodge, IA). The abdominal and thoracic cavities were opened aseptically. The lungs from each animal were weighed and homogenized in 50-mL tubes with 20-mL sterile saline using a hand-held tissue homogenizer (Kinematica Polytron PT1200E; Brinkmann Instruments, Westbury, NY). Serial dilutions (1:10) of homogenates were plated, in duplicate or triplicate.

P. aeruginosa, homogenate was plated on Brain heart Infusion agar and incubated at 37°C for 24-48 h. M. tuberculosis homgenate was plated on Middlebrook 7H11 agar and incubated at 37°C for 14–21 days. Colonies were averaged from the appropriate dilution quadrants (20 < quadrant count < 200) and expressed as mean colony forming units (CFU) per total right lung.

Design for four or eight animal exposure systems

The system is composed of a removable animal holder inserts (Fig. 1A) for four animals that fits inside of the four-animal chambers (Fig. 1B) or eight-animal chambers (Fig. 1C). As shown in Figure 1B, the complete four-animal aerosol exposure system is small (11″×11″×18.5″) so that one unit may fit within a conventional biosafety cabinet. The apparatus has a removable section where the animals are contained. The eight-animal system has two animal chambers for two inserts (Fig. 1C) and fits in large biosafety cabinets. A complete airflow diagram is shown in Figure 1D. Figure 2 shows the four-animal system complete and with guinea pigs.

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FIG. 1.

(A) Design of the animal holding insert and gasket. (B) Diagram of the four-animal system showing the dimensions and locations of the parts of the aerosol chambers. (C) Diagram of the eight-chamber system modifications. (D) Simplified diagram of the complete system showing the linear air flow and the arrangement of the components.

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FIG. 2.

Two views of guinea pigs in the four-chamber system. This illustrates the usage of the system for the third replicate of Pseudomonas infection.

Each tube was 12–14 inches long with 1/8th of an inch thickness. This was an appropriate size for the weight of the guinea pigs used in this study. This will have to be adjusted if other animal sizes are used. Because the animal holders are removable, construction of a variety of sizes to accommodate different needs is fairly simple. Some tubes had 4-inch interior diameters (800–1000 g, depending on obesity); others had 5-inch interior diameter for larger animals (1000–1200 g). Smaller animals, less than 800 g, will likely use a slightly smaller tube, and larger animals would use a bigger diameter. This can be tested with the same tubes commonly used for animal environmental enrichment. Guinea pigs naturally like the tubes, but be forewarned that dominant animals will try to assert control of the tubes in cages with multiple animals. The front of each animal-holding tube has a “smile”-shaped opening that follows most of the movement range of the animal's nose and mouth. The lid on the back of each individual animal holding tube can be held in place with tape or, preferentially, a lid with either a screw top or fastener. We often used tape and latches to ensure the animals would not accidentally back out of the tube. It is important to note that guinea pigs use similar tubes as enrichment in their standard caging.

Four of these go into the basic removable unit. The removable units fit into a larger gasket sealed box that contains the aerosol chamber and nebulizer chamber (Figs. 1 and 2). All openings are closed with clamp fittings and sealed by silicon or Goretex^™ gaskets (Figs. 1A and 2). The latches we used have a secondary safety catch to prevent accidental opening. We strongly recommend the use of polycarbonate plastic to build the system as it has excellent chemical and impact resistance.

The nebulizer is a conventional air jet nebulizer, driven by an air pump system that has both output and intake ports (Fig. 1D). For additional safety, the pump can also be placed within the biosafety cabinet to provide a backup for accidental failure of the inline HEPA filters. The nebulizer is a conventional air jet nebulizer that can hold about 5 cc of inoculum. The pump must have in-line HEPA filters, and we do recommend that this be placed in the cabinet. We have also used two-pump systems: one to provide pressure and one to provide vacuum. Airflow is controlled by an in-line regulator so that there is a net negative flow (∼−0.2 inch water pressure using the same low-pressure gauge, adjusted manually to start at 1-inch water pressure to be zero pressure) toward the intake ports and vaccum flask that is also separated by a HEPA filter. Flow rate with this system is 6 liters/min. Use of different nebulizers and pumps will require testing and potentially modification of the system to reach similar flow rates and pressures. Additional in-line dehumidifying filters are recommended for large volumes of inoculum.

A vacuum flask is used to collect the aerosol into chemical disinfectant and any air is filtered by in-line HEPA filters. We recommend labeling all filters and vacuum lines A–Z from the pressure (output) port of the pump to ensure that the correct orientation of all filters and correct attachment of lines is achieved at setup. It is also critical that each run and setup be tested before actual use to make sure the system is assembled correctly and fully functional. Run the system for at least 30 min during testing to make sure the pressures do not vary over time, especially if the filters could become saturated with inoculum, for example.

Parts list

Chemical resistance of all components for cleaning and disinfection

Another accomplishment is the minimized metallic content. In consultation with Cole-Parmer and Accurate Gasket about the best materials for the disinfection agents anticipated to be used (e.g., quaternary alkyl ammonium chlorides, hydrogen peroxide), the system components have been optimized to withstand any degradation caused by cleaning and disinfection.

“Quick connect” hosing connections

The intake and outtake hose connections are “quick connect” that seal when open to minimize any leakages. The ones recommended here were excellent for this task but there are many variations on this technology.

Port seals

The design also requires three “ports” each of which are potential leak points. Tapped, threaded ports are also glued so that the permanent connection points are sealed. These will then have quick-connect hose connections that also seal to minimize any potential leak points. The installation of the pressure meter is also attached by the same means and protected by an in-line HEPA filter.

Additional ports

The low-pressure airflow meter in this design is not exposed to infectious aerosol and is separated from the aerosol by a 0.2-micron HEPA filter. It is essential that a low-pressure gauge be used after adjustment to show zero at the 1-inch water mark to ensure that there is a slight negative pressure in the system. For ease of viewing, the pressure gauge is installed on the top of the aerosol chamber.

There is also a HEPA-filtered clean air vent on the aerosol chamber. This is simply a hose closed by a thumb clamp, which is opened prior to turning off the pump that drives the nebulizer. The most suitable location for this port is also on top of the aerosol chamber (Fig. 2). One modification that could also be used would be impinger sampling ports as well.⁽¹¹⁾

Disposability

Almost the entire design takes advantage of a certain amount of disposability, where any part with any question as to its safety or structural integrity can easily be replaced with minimal cost.

Leak testing

We prefer submersion in a large sink or tub (large storage tubs found at most stores, for example) that can be used to test the system after construction before testing with animals. When the apparatus is complete, use long hoses from the air pump(s) so they are not near any water and risk of electrocution. Seal the apparatus, turn on the pump(s), adjust the flow meter providing a slight positive pressure, and submerge the apparatus so that bubbles can be observed. This includes submerging the pressure gauge, vaccum flask, and flow meter.

Operation

We followed the protocol as described in the Supplementary Experimental Checklist (available at http:www.liebertonline.com/jamp). Briefly, the system is pretested using water or saline to make sure all tubing is assembled in the correct order, all seals are closed properly, and than the pressure gauge moves slightly negative when the pump is on. This is the stage to adjust the flow rate using the flow regulator. For consistency, we used the same nebulizer in our previous study where we performed a particle analysis.⁽¹⁰⁾

After the system is working as intended, the inoculum is prepared and loaded into the nebulizer. The animals are then loaded into the animal holder and placed in the system. Before starting the pump, we recommend double checking all seals and gaskets for proper closure. The pump is then turned on and the inoculum should fill the chamber. A close eye on the pressure gauge is essential to ensure no accidental leakage or discomfort to the guinea pigs.

Decontamination

Many chemicals can attack different parts of a system. Bleach attacks metals, peroxides destroy rubber, and alcohols and quaternary ammonium compounds can attack many different kinds of plastic. We found that the best combination of materials was polycarbonate plastic and Goretex for gaskets.

When the inoculation cycle is complete, the system is ventilated via the ventilation port and the nebulizer pump is turned off to allow the cloud to be cleared. We used 20 full aerosol chamber volumes (2 L) as our standard. As the system had a flow rate of 6 L/min, this corresponded to 20 sec per volume and approximately 7 min to get 20 volumes.

We had tuberculocidal disinfectants in spray bottles and sprayed every surface as the animal holders were removed, including into the now open aerosol chamber. We used Conflikt disinfectant (Fisher Scientific) for these studies. Additionally, we used 95% ethanol to wipe down the front feet and neck fur of the guinea pigs as it would be the least toxic to the guinea pigs but still kill mycobacteria.⁽¹²⁾ Finally, the entire system was dunked in chemical disinfectant for 15 min, rinsed in water, and dried. Each time the system was reassembled, we would again retest for leaks, cracks, broken pieces, and proper airflow rates (see Supplementary Experimental Checklist).

Fur contamination

Although the Madison Chamber and most other whole-body animal aerosol exposure systems do not address this issue, the tube design should minimize most fur contamination just to the very front of the animals, such as their front paws, nose, mouth, and neck. This is a hybrid between the whole-body exposures systems like the Madison Chamber and nose-only exposures systems such as those from CH Technologies (Westwood, NJ). It has the advantages of minimal restraint compared to the nose-only systems with minimal potential for fur contamination. We could see a small amount of aerosol reach the tubes interior just past the opening for the animal's mouth and nose. We were unable to culture bacteria from this aerosol during testing, however, and we added an ethanol or nontoxic disinfectant wipe of the animal's paws to minimize any contamination. The decontamination should be done within the biosafety cabinet as well. One other possibility is to place the animals within plastic bag cones (e.g., Decapicones, Braintree Scientific, Braintree, MA) so that only the nose and mouth are directly exposed.

Bacterial delivery

To test the ability of the system to deliver viable bacteria, the four-animal system was tested with luciferase expressing Pseudomonas aeruginosa and assessed by both homogenization of the lung and luminescent imaging (Fig. 3). The guinea pigs seemed to like the tubes and were reluctant to be taken out at the end of the exposures. The smooth plastic design helped, as there was no place for the animal to hold onto and accidentally get a nail caught. We did notice that the unique smell of Pseudomonas was unpleasant to the guinea pigs so we made the nose holes larger and “smile”-shaped so that no matter how the guinea pig moved, the nose was exposed to the aerosol. This helped make our results more consistent (Figs. 3–4).

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FIG. 3.

Results from high dose Pseudomonas infection in the four-chamber system. (A) Luciferase expressing Pseudomonas imaged in the lung 1 h after infection using a Xenogen imager showing wide distribution throughout the lung from experimental replicate 1. These images show the range of variation and the distribution of bacteria as detected by the color heat map (blue=fewer, red=higher bacterial concentration). As can be seen, the farthest right lung had the lowest bacterial load and is the spot around 100,000CFU in B. (B) Quantitation of bacterial cell counts 1 h after aerosol exposure for all three replicates. Error bars are standard error for each sample. No replicate was significantly different from the other replicates (Tukey-Kramer Honestly Significant Difference, p>0.05, JMP, SAS).

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FIG. 4.

Quantitation of low-dose Mycobacterium tuberculosis bacterial cell counts 1 h after aerosol exposure. The goal of the experiment was to get a very low dose (1–10 bacteria per animal) in all animals in the eight-tube system. All animals in both replicates were successfully inoculated with bacteria, even at a very low dose.

Typically 2–5 cc of inoculum took 5–10 min to exhaust completely, so approximately 15–30 liters of air were pumped through the system. Even though the animals were comfortable, they would usually urinate and defecate at some point so one might be aware of the liquid and fecal matter if this is a problem in the experiment. We found that using disinfectant soaked paper towels helped with cleanup.

High counts could be achieved in the lung, requiring a fairly high initial inoculum count. Consistently in pilot studies and in these tests, the system required approximately 1×10⁸ bacteria to ensure one would be deposited in the lung. So for a target of 1×10⁵/animal we used 1×10¹³ bacteria. This was consistent with both Pseudomonas at high doses and Mycobacterium tuberculosis at low doses in the eight-animal system where we were trying to get less than 10 bacteria in per animal (Fig. 4).

One important tip is that the inoculum, while it can be in sterile saline, must be relatively free of detergents, which cause foaming. Most Mycobacterial broths do contain detergents. We found that a single wash step by pelleting by centrifugation (1500 rpm, 5 min) in detergent free saline and resuspension in fresh sterile saline reduced the detergent to acceptable levels.