Engineering Control of Airborne Disease Transmission in Animal Laboratories. Contemporary Topics 41 (3): 9.
The purpose of this paper was to review the problem of controlling airborne disease transmission in animal research facilities. Engineering design and air-treatment technologies were specifically addressed.

Diseases can be transmitted between animals and between human and animals through 2 general means direct contact and airborne transmission. Disease transmission through direct contact can be avoided by implementation of appropriate operating procedures (things that the worker can do). Airborne transmission is best avoided through construction of engineered systems (building/HVAC design, etc). The most common and reliable methods for decreasing/removing microbes from the air include dilution ventilation, filtration, and ultraviolet germicidal irradiation (UVGI). Also, air pressure differentials can be used to isolate areas.

There are three broad categories of microbes viruses, bacteria, and spores that can be grouped by physical size. Spores are usually large and easily removed by filters. They are uncommon contaminates of animal facilities, but are common in outdoor air. Poultry and swine houses are well known for excessive airborne particle concentrations.

There are no agreed upon upper limits for concentrations of indoor airborne microorganisms. The following publications provide some suggestions for designing and running facilities: Guide for the Care and Use of Laboratory Animals; American Society for Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE) Handbook of Applications; American Institute of Architects (AIA) Guidelines for Hospitals; and American National Standards Institute (ANSI/AIHA) Guidelines. Generally, it is suggested that there be 10-15 air changes per hour (ACH). Often (most of the time) 100% of the air supply is taken from the outside air; a minimum of 50% outside air is suggested. High Efficiency Particulate Air (HEPA) filtration as well as charcoal absorbers are mentioned. UVGI is not mentioned.

There are 4 categories of methods used to control the spread of airborne pathogens and allergens: 1. source control - local ventilation of the animal cage. 2. room/building ventilation system dilutes building air and exhausts contaminants 3. pressure differentials between rooms to isolate areas 4. air-treatment systems intercept and destroy contaminants

Source Control The source of most airborne contaminants of concern is usually the animals and their cages. People can also be sources of contamination although the highest priority should be on protecting the worker. Use of ventilated caging is one method of source control. Some racks use negative pressure to draw air out of the cages while others use positive pressure to blow air into the cages. Two potential problems with ventilated racks are that racks with fans mounted directly on the rack may be subject to unacceptable levels of noise and vibrations. Also, air filters may be operated beyond their design face velocity (the air may be moving too fast for the filter to remove air particulates).

Dilution Ventilation (a.k.a.purge ventilation) Air is constantly being mixed in the building. If 100% outside air is being used for the air supply, the displaced building air can be exhausted to the outside untreated (although many places do clean or filter the air in some way). If complete mixing of the air in a room is occurring, increasing the ACH beyond 10-12 ACH is of limited value. While increasing the ACH in a room may seem like a good way to improve air quality, one must consider related expenses such as the life-cycle cost of the system (including fan energy which is often the highest cost) and the cost of treating outside air (especially when outside air reaches high or low temperature extremes, or requires dehumidification).

Pressurization Control The idea is to ensure that airflow moves from areas of low contamination e kill rav bà-potential to areas of high contamination potential. (from clean to dirty areas). Contaminated exhaust air is either exhausted outside or treated prior to reuse. Areas must be sufficiently airtight to be able to maintain pressure differentials. The actual pressure differences are not important as long as the air is traveling in the correct direction.

Filtration The most popular filter type is the HEPA filter which is designed to remove at least 99.97% of particles 0.3mm or larger when operated at design air velocity. Other filters are available with ratings of 25% - 95%. In one study, 80% - 90% filters offered a considerable improvement over using no filters, but the HEPA filter offered little improvement over the 80% and 90% filters. Therefore, one should question if the use of HEPA filters are necessary in each HVAC design.

Ultraviolet Germicidal Irradiation - For this method, a UV lamp is placed transverse to the air stream in a duct. Airborne microorganisms are exposed to the UV (both from the light and reflected from the duct). Depending on the susceptibility of the organisms, the kill rate may be rapid and sufficient to sterilize the air, or it may only destroy a portion of the microbes. Viruses are the smallest and, in general, the easiest microbes to kill with UV. However, spores are most difficult to inactivate with UVGI, but are the easiest to remove with filters. Bacteria may be vulnerable or resistant based on the species. UVGI and air filtration are considered mutually complementary.

Carbon Absorption - Carbon absorbers can be used to remove odors and volatile organic compounds (which filters and UVGI cannot do). They have little to no effect on airborne microorganisms.

Alternate Technologies - Photocatalytic oxidation (PCO) uses UV lamps to irradiate filter-like materials made of titanium dioxide that then oxidizes anything that touches it. PCO can remove many volatile organic compounds, odors, and airborne microorganisms. However, there is little data on use of this technology.
Ionization involves the generation of negative ions that causes dust particles (on which microorganisms may rest) to agglomerate and settle out of the air. This can be used to improve efficiency of filter units. It is currently being used in poultry houses.

Electrostatic filtration has the limitation that performance is affected by changes in humidity.

Biocidal filters are filters with anti-microbial coatings. However, these have not been used much since there is a concern that biocidal materials may dislodge and be spread in the air.
1. What does ASHRAEW stand for?
2. What does HEPA stand for?
3. HEPA filters are rated to remove at least _____% of all particles ____mm in diameter when operated at design air velocity.
4. What is the most common reason that HEPA filters fail?
5. T/F Spores are not usually removed through use of air filters.
6. T/FUVGI and air filtration can be two complementary methods to remove/inactivate airborne microorganisms.
1. American Society for Heating, Refrigerating, and Air-Conditioning Engineers
2. High Efficiency Particulate Air
3. 99.97%;0.3mm
4. air filters may be operated beyond their design face velocity
5. False, spores are large and are usually removed by filters
6. True, of course, this will depend on the needs and abilities of each facility

Outbreak: Detection and Investigation. Contemporary Topics 41 (3): 18.
Many institutions are trying to convert from conventional to SPF colonies exclusively to decrease the potential for outbreak of infectious agents into the SPF colonies
Conventional housing--animal rooms have one or more agents recognized as pathogens
Specific Pathogen Free--(SPF) housing--animal rooms free of specific pathogens
Outbreak--detection of an infectious agent in rodents in a location where the agent is unwanted
due to it jeopardizing the health of the rodent or the jeopardizing the research.
Outbreaks are usually detected through a surveillance program involving sentinel animals.
Detection and Investigation must be used to deal with an outbreak.
Contamination--the event that caused the outbreak.
DETECTION
Surveillance is used to maximize the identification of the contamination
1) Direct Monitoring
--sampling rodents used by investigator
--may be indicated for breeding colonies housed in open caging
--cannot be used on immunodeficient rodents that lack antibody responses
2) Indirect Monitoring--sentinel mice used as indicators to monitor the health status
of the colony.
SENTINEL MICE--WHICH STRAIN TO USE
Rodent strains and stocks may have differing susceptibilities
to various groups of agents
1) Inbred vs Outbred
Consider susceptibility--choose animals that are most likely to become infected;
frequency of seroconversion can vary between strains
Consider variability--less physiological variability between animals of the same
inbred strain.
2) Sex and Age
Females are preferred to decrease in-cage fighting
Animals should be old enough to have lost all maternal antibodies that may interfere
(usually older than 6 weeks is safe for mice) and old enough to be immunocompetant
(usually 4-8 weeks).
3) Parasitology--youngest mice will develop the highest worm burdens
Overall recommendation is to begin with 6-8 week old mice.
4) Immune Status
In general, immunocompetant rodents develop acute subclinical infections (intermittent
shedding) and immunodeficient rodents develop persistent clinical infections (continuous
shedding), if morbidity does not intervene. Immunodeficient mice can be valuable
in monitoring for bacterial, viral and parasitological infections especially if it is important
to identify or recover the agent. There is also a time advantage because clinical signs
will occur prior to antibody response.
SENTINEL SYSTEM
Type of Exposure
1) Contact (animals cohoused)
Best exposure, but female sentinels will get pregnant and males tend to fight.
Neutering animals requires additional costs
Possibility of widespread contamination with open top cages
2) Soiled bedding
Some infections may not spread (respiratory tract pathogens)
Need frequent transfer of soiled bedding (some infections may not be shed for long)
SENTINEL CAGING
Open top
Better transmission of viruses
Use this system if investigator cages are opened
Combining open tops with transfer of soiled bedding will improve surveillance
Cages with open tops are placed low on the rack and rotated to maximize fomite
transmission through airborne particles
Rodents in cages with open tops may become a nidus of infection for the room and
allow more widespread dissemination of pathogens.
Sentinels in open cages may acquire pathogens from sources other than the investi-
gators' rodents.
SAMPLING OF INVESTIGATOR CAGES
Three options:
1) ALL: Sampling all cages at a specific time point--most complete coverage
2) SYSTEMATIC: All cages are sampled over a given time period in a consistent
fashion
3) RANDOM: Select cages to sample through a formal process of randomization
To improve detection, rank the risk of infection of cages (newly arrived rodents
may have an increased risk of infection vs resident rodents)
NUMBER OF SENTINEL CAGES PER RACK
What is the number of investigator cages that should contribute soiled bedding to a sentinel age for infections to be detected. Avoid contributing so many cages that the agent is diluted and undetectable in the sentinels. There is no clear rule to make this decision.
NUMBER OF RODENTS PER CAGE
1) The determining factor is the time period between introduction and removal.
2) All rodent strains will experience mortality unrelated to their use as a sentinel so
surplus rodents should be present to ensure availability at the time of evaluation.
3) Having more than one sentinel to sample may help in interpretation of results.
4) A single sentinel might suffice for a short period of time (6 weeks), but it is better
to use two animals for longer time periods.
NUMBER OF RODENTS TO TEST
A formula has been derived that predicts within 95% confidence limits, the number of animals required to detect a single case of disease assuming a known prevalence in a population. The formula has been used to create a table of the probability of detecting infection in a given sample of animals. This formula assumes randomization of diseased animals throughout the colony which implies a free flow of infectious agents by contact, aerosol, or airborne and has limited applicability. Historical data may be more helpful. A guideline based on historical data from a commercial barrier facility shows that a group of 4 sentinel mice should be tested every 4 weeks and a group of 8 rats should be tested every 6 weeks.
EVALUATION
Test intervals:
Sufficient time should be allowed for development of manifestations of infection. Antibody responses may take 2 weeks or more. Parasitology testing should be done after 4 weeks. Rooms with a history of recurrent infections will be high risk and more frequent testing is indicated(every 4-6 weeks) whereas rooms with few problems may need testing every 8-12 weeks.
Sampling vs Necropsy
Depends upon the sensitivity of the test
For most bacteria, viruses, protozoa sampling of rodents will suffice.
For parasitology, the definitive method is necropsy, but a combination of sampling and
necropsy can be used at different test intervals.
SURVEILLANCE
Monitor the agents that the colony is at most risk of acquiring. Rodents are at a high risk of acquiring agents in mice housed in a conventional facility. If no conventional housing exists, but animals are imported, monitor based on the prevalence of infections in other institutions.
EXAMPLE SURVEILLANCE
One sentinel cage of two rodents placed on each side of each rack quarterly (covering 35-70 cages per side). A tablespoon of soiled bedding is placed from each investigator cage into an empty cage that will house the sentinels on a weekly basis. 6 week CD-1 and ICR mice and S-D rats are used as sentinels. Every 6 weeks, one sentinel is bled [Sera are tested for MHV (mice) and RPV (rats)], and both sentinels are used for perianal tape tests and fecal samples (pinworms). Every 12 weeks sentinels are euthanized and bled (serum for MHV, MPV, EDIM in mice; serum for RPV, SDAV, mycoplasma in rats), pelts are examined for ectoparasites, and cecum contents are examined for endoparasites. Yearly mice are tested serologically for an extended panel of parasites.
INVESTIGATION
Steps in an outbreak investigation: 1) Verify the diagnosis 2)Determine the extent of contamination in colony 3) Describe the outbreak in terms of individual, place and time characteristics 4) Identify the mode of introduction of the agent 5) Implement measures for control and prevention
VERIFY THE DIAGNOSIS
Rule out the possibility of a false positive test
Repeat using the same serum with different dilutions to avoid non-specific reactivity
---A positive result at a high dilution is more likely to be a true positive
--The sample could be tested by another laboratory or by a different assay
--Rebleed the same sentinels later to compare paired sera (increas. titer = recent infect.)
--Previously untested cage mates should be bled and tested to clarify initial result
--If no surviving sentinels remain, test a new sentinel 10 days after exposure to soiled bedding
CONFIRM THE EXISTENCE AND DETERMINE THE EXTENT OF CONTAMINATION
--Confirmation needed because sentinels may have been contaminated independently of others (ie during shipping, during room placement, etc)
--Be suspicious if all sentinels are positive, especially in a room with multiple investigators
--May need to sample one animal from each investigator cage that contributed to soiled bedding to determine source of contamination. In case the cages have been moved around, random sampling from each investigator may suffice. Knowledge should be obtained in advance concerning the immune status of the animals. For immunocompromised animals, sentinels can be co-housed or small groups of animals can be set up with one sentinel. Alternatives to serology such as direct sampling of feces to detect MHV shedding by PCR can be considered, but keep in mind the false negatives associated with alternative testing.
DESCRIBE THE OUTBREAK AND IDENTIFY THE MODE OF INFECTION
--Individual characteristics include the investigator, strain, immune status, origin, use of biological materials, and movement in and out of the room
--A spot map should be constructed of the location and relationship of positive cages to determine
how the pathogen was spread (personnel vs feral rodents).
--A floor plan of the facility will help identify the risk associated with each room
--Time characteristics include the movement of animals in and out of the room and when the animals were added to experiments.
(Questionnaires are included in this article to assist in collecting facts about the outbreak)
CONTROL
Implement several new control measures
--Once detected, the goal is to prevent spread of the pathogen by using more stringent methods than normally in place for prevention of contamination.
1) quarantine the roomno movement of cages in or out; maintain cages in place; limit personnel access to room
2) prioritize cage changing schedule based on risk of infection
3) increase surveillance and testing during quarantine period
Depopulation
--May be the best option if the entire colony is infected, rodents are replaceable, and the infection
is chronic or untreatable.
--Should be followed by a thorough decontamination of the room
--Depopulation can mean relocating to conventional or quarantine facilities if available
Test and Removal
--more time and labor intensive
--used with smaller barrier rooms
--test weekly and remove positive rodents
--continue testing until 4 consecutive negative weekly tests
Infection/elimination (BURNOUT)
--allows infection to run its course and be eliminated naturally
--works for agents which do not persist in rodents (MHV, Sendai, and SDAV)
--may not work if immunocompromised animals are infected because they may be chronic
carriers; be cautious using this method with transgenic mice colonies
Burn out: Either open the tops to allow all cages in the room to be contaminated if prevalence approaches 100% or close the tops if the goal is to control the spread if there is a low prevalence. If the idea is to control the spread, cages must be changed in a laminar flow hood. No naïve rodents should be introduced (no breeding). Sentinel rodents should be introduced at the end of the time it takes for rodents to clear the infection. If the sentinels test positive, they should be replaced by new sentinels. Burn out may compromise detection of future outbreak investigations since the animals will be seropositive.
Chemotherapy
--Rodents with pinworms can be treated with fenbendazole in the feed or ivermectin
--Fecal float and tape tests are insensitive ways to measure presence of pinworms so necropsy of some sentinels should follow treatment.
--After treatment, sentinels should be tested weekly for at least one month beyond the prepatent period
OTHER CONSIDERATIONS
--Disposal of infected rodents and caging materials: Use containment methods such as decontamination and double bagging of cages when removing infected rodents and caging from the room
--Communication between investigators and veterinary staff should be goodfind a convenient and efficient way to communicate.
PREVENT RECURRENCE
--When the mode of outbreak is identified, make appropriate changes in procedure to prevent recurrence
--If more than one investigator has a break, examine the practices of the animal technicians as the spread from cage to cage should technically not occur.
SUMMARY
Collaborations between institutions requiring an exchange of rodents have emphasized the need to maintain SPF rodents. A good detection plan as well as a coordinated plan of action is necessary to maintain barrier conditions. Management strategies will vary with the type of housing available in each institution.
No questions

Fentanyl-fluanisone-midazolam combination results in more stable hemodynamics than does urethane-alpha-chloralose and 2.2.2-tribromoethanol in mice. Contemporary Topics 41 (3): 28.
The purpose of ths report was to compare the impact of two widely used anesthetic protocols in cardiovascular research (fentanyl-fluanisone-midazolam versus urethane-alpha chloralose-tribromoethanol) on two specific hemodynamic parameters (mean arterial blood pressure and heart rate). The authors concluded that the fentanyl combination combination provided more stable hemodynamics under anesthesia than the urethane combination. Materials and Methods: 13 male Swiss Webster mice were used for this study. They were divided into two separate groups. One group(six mice) received the fentanyl-fluanisone-midazolam (FFM)combination (conc .079mg/ml fentanyl citrate, 2.5mg/ml fluanisone, and 1.25 mg/ml midazolam) which was prepared by dissolving 1 ml Hypnorm (fentanyl and fluanisone), and 1 ml Dormicum (midazolam) in 2ml of sterile water. The other group received (seven mice)the Urethane-alpha chloralose-tribromoethanol mixture (U-alpha cl-TBE) mixture. This was prepared by mixing alpha chloralose in normal saline (to a concentration of 10 mg/ml), urethane, and a 2.5% solution of TBE. Induction: The FFM group received 7 ml of the combination IP, and the U-alpha ch-TBE group received 300mg/kg IP TBE first followed by 600mg/kg of Urethane, and 40mg/kg of alpha ch IP a few minutes later. The animals were then mainitaned under anesthesia by continuous rate infusion. Following that, the animals were instrumented surgically in order to measure mean arterial blood pressure and heart rate. Results: Induction time was greater for the U-alpha ch-TBE group (app. 25 minutes), than for the FFM group (app. 5 minutes). Additional drug (TBE only) was administered to the U-alpha ch-TBE group to some mice that reacted to painful stimuli during surgical preparation. None of the FFM treated mice required further drug during that period, and the initial dose given proved sufficient to complete the surgical process. The total time from the initiation of induction to the beginning of recording was longer for the U-alpha ch-TBE treated mice (87+/-22 min) than for the FFM mice (68+/-19 min). The U-alpha ch-TBE mice maintained a lower mean arterial pressure(49+/-4 mmHg) than the FFM mice 78+/-5 mmHg). There was marked variation in the heart rate of the U-alpha ch-TBE mice (308+/-34 bpm at the outset to 477+/-43 bpm at the end of the experiment), and there were marked changes in arterial blood pressure as well with this group during the experiment. The FFM group however maintained stable mean arterial pressures and heart rates (431+/-37 bpm) for the course of the experiment. Vasoconstriction in the U-alpha ch-TBE group reduced the volume of fluids that could be administered by tail vein compared to the FFM group. Discussion: The authors state that investigating the hemodynamics effects of cardiac interventions in mice require a stable anesthetic plane and maintenance of near physiologic blood pressure and heart rate. They concluded that FFM provided more stable and near physiological hemodynamics when compared to U-alpha ch-TBE. It was also decided that FFM was able to allow for consistent stable deep anesthetic plane while the U-alpha ch-TBE combination did not. They also stated that the FFM anesthetized mice had heart rates and mean arterial blood pressures similar to those found in awake unanesthetized mice. These findings along with the fact that FFM has no known carcinogenic or mutagenic properties in mice and no toxic implications for lab personnel make it a superior choice in experiments studying cardiac physiology in mice.
1. Which of these compounds is carcinogenic/mutgenic in mice
A. Tribromoethanol
B. Fluanisone
C. Urethane
D. Fentanyl
2. T of F FFM provides a more stable deeper plane of anesthesia with near physiological heart rates and blood pressures than does the combination of U-alpha ch-TBE?
Answers not provided

A technique for creating localized subcutaneous Blastomyces granulomas in rats. Contemporary Topics 41 (3): 33.
This article describes the development of a subcutaneous Blastomyces lesion model in rats. Blastomycosis is a systemic fungal infection caused by Blastomyces dermatitidis which is a natural soil inhabitant. Common route of infection is inhalation with dissemination from the lungs to other organs. Localized cutaneous infections through wounds or trauma are also noted. There are numerous animal models (mice, rabbits, guinea pigs, hamsters, dogs and rats) of experimentally induce pulmonary or disseminated blastomycosis however there are few models of cutaneous blastomycosis. The hamster has been the most commonly used species to study cutaneous blastomycosis. The authors wished to study cutaneous blastomycosis but needed to do tail vein injections to administer radioisotopes therefore making the hamster unsuitable. They investigated developing a model in rats as an alternative to the hamster.

They used 39 (250-400g) Sprague Dawley rats of which 34 were deemed successful models. They also used an isolate of B. dermatitidis from a clinical case of blastomycosis from a dog. The organism was injected subcutaneously over the distal right or left tibia while the rats were anesthetized. Initial doses of inoculum 10(5) and 10(6)CFU (colony-forming units) taken from hamster studies did not produce the nodules noted with cutaneous blastomycosis. They increased the dose to 10(9) and 10(10)CFU which did successfully develop palpable nodules by day 7 post-inoculation in all 34 rats. Rats were euthanized 7, 10, 14, 21 and 28 days after inoculation. Histopathology of the lesions can be described as well demarcated with a necrotic center surrounded by a high concentration of yeast organisms and with inflammatory cells (mostly neutrophils) noted in the periphery of the lesion. Histologically, the lesions were characterized as a focal granuloma with associated tissue reaction. There were no incidences of systemic disease.
1. Prior to this study, which species was historically the animal of choice to study subcutaneous blastomycosis?
a) Mus musculus
b) Meriones unguiculatus
c) Mesocricetus auratus
d) Marmota monax
e) Dasypus novemcinctus
2. Which of the following dose(s) of inoculum reportedly causes lesions or nodules in hamsters? in rats?
a) 10(5)CFU
b) 10(6)CFU
c) 10(8)CFU
d) 10(9)CFU
e) a and b
f) c and d
PART 2 of question 2: Why do rats require a higher dose?
3. T or F: The lesions noted with this model of experimental blastomycosis are the same as lesions with naturally occurring blastomycosis.
1. C, Mesocricetus auratus (Syrian or Golden hamster) is the likely answer however, I could not determine exactly which hamster was used so it may also be Cricetus griseus (Chinese hamster), Cricetus cricetus (European hamster) or Cricetulus migratorius (Armenian hamster). NOTE: the mouse (Mus musculus) is the common species used for study of experimental pulmonary or disseminated blastomycosis. Meriones unguiculatus (gerbil), Marmota monax (woodchuck), Dasypus novemcinctus (nine-banded armadillo).
2. In hamsters (E). In rats (definitely D, C has not been evaluated therefore is not reported to cause lesions). Rats require a larger dose of B. dermititidis basically because they are less sensitive to infection with B. dermititidis than hamsters.
3. False, the lesions are similar but there are differences. In the experimental model the lesions have a large central area of necrosis surrounded by clusters of yeast. In the natural disease this area of necrosis is not typical and there are neutrophils, macrophages and multinucleated giant cells with some yeast organisms dispersed throughout the inflammatory cells. These difference may be due to the age of lesions as the experimental lesion are days old verse the natural disease which is usually diagnosed weeks or months after initial infection.