Quarantine

Quarantine of rodents from non-commercial sources — Due to the increase in transgenic and knockout mouse strains over the past few years, a growing number of rodents (mostly mice) will be introduced from non-commercial sources, including other universities, research institutions or small biotechnology companies. While most of these sources maintain a health-monitoring program, they usually do not maintain closed colonies, and they do not have a financial interest in providing only high quality animals. Quarantining animal groups is necessary to determine their health status to assure that no unwanted organisms are introduced to the Vivarium. Quarantine length and testing strategy will depend on:

1. Level of barrier security and level of surveillance testing in the originating colony,
2. Length of stay here,
3. Destination of the rodents.

The program at the originating colony will be verified. The PI acquiring the animals should ensure the clinical veterinarian at the originating institution contacts the attending veterinarian here. The PI must provide a copy of the latest pathogen screening results done at the originating colony. Some common questions that should be answered include:

1. What is the contamination history of the colony?
2. What is being surveyed? How often?
3. Are husbandry and access methods secure?
4. Are there contaminated colonies in the facility?
5. What pinworm detection method is being used?

General approach to quarantine — Health reports from the originating colony will be obtained to determine which adventitious agents are present. Most facilities use serology to test for all viral agents and some bacterial pathogens. Pinworm detection presents more of a problem since some use a tape test to detect Syphacia, but fail to perform postmortem cecal exams to detect Aspicularis.

The quickest approach to screening new imports is to request several extra cohorts from the exporting facility for sacrifice. The imported group can be surveyed by screening representative animals from the group. If none are available, animals will typically be held in quarantine and sentinels placed. Sentinels will be screened after a minimum of 3 - 5 weeks exposure to potential pathogens through dirty bedding transfer or direct contact.

Another method that significantly expedites release of new animals is to automatically treat incoming animals for pinworms through commercially available feeding (e.g., fenbendazole-medicated feed). Ivermectin may be used to treat both ectoparasites and pinworms. Once treatment is completed, serology can be performed on a cohort of rodents to detect viral pathogens. Most bacterial pathogens can only be detected by culturing at necropsy.

Note: Several transgenic strains show increased hypersensitivity to Ivermectin. If unsure as to safety, it is strongly recommended to dose a few trial rodents before treating the entire line.

Factors affecting decisions about which agents to screen for include risk tolerance, research use of the imported rodents, length of time to be housed at the facility, and financial considerations. Ultimately, a balance must be reached between these factors.

Rodents destined to be euthanized within a few days or weeks — These rodents are usually moved directly to a quarantine cubicle or conventional room and used for research. Additionally, access to quarantine will be limited and special arrangements made between the investigators and the staff. It is all too common for investigators to serve as inadvertent vectors, transferring pathogens between the laboratory and the animal facility. Generally, a startup meeting between the veterinary staff and the investigator will be arranged for discussion of the intended procedure in the context of protecting the facility colonies from pathogens. If the investigator decides later to keep these animals, the quarantine process should start at that time.

Rodents destined for conventional rooms — These rodents can be moved directly to a conventional room for housing if there are no concerns of contaminating the existing colonies. However, a simplified examination will be performed to minimize the risk associated with importing novel pathogens.

Typical screening protocol for conventional rodents:

• Gross examination of tissue (if suspicious lesions are detected, tissues are processed for histology)
• Examination of pelage for ectoparasites:
  • Myobia sp.
  • Myocoptes sp.
  • Radfordia sp.
• Examination of cecal contents for internal parasites:
  • Giardia muris
  • Spironucleus muris
  • Aspicularis tetraptera
  • Syphacia obvelata
• Serology for common pathogens:

Mouse:
Mouse hepatitis virus
Mouse parvovirus
Mycoplasma pulmonis
Sendai virus

Rat:
Rat coronavirus/Sialodacryoadenitis
Mycoplasma pulmonis
Sendai virus

Rodents destined for SPF/barrier housing [“clean” animals with a history of extensive health testing with negative results, and housed in a tight barrier at the originating colony] —

Generally, these animals are assumed to be free from major pathogens and are commonly screened using two methods:

1. Examination of group cohorts
2. Placement of sentinel rodents for 3-5 weeks.

Typical screening protocol for SPF rodents:

• Gross examination of tissue
  (if suspicious lesions are detected, tissues are processed for histology)
• Examination of pelage for ectoparasites (see above)
• Examination of cecal contents for internal parasites (see above)
• Serology for common and uncommon pathogens: (these are subject to change)

Mouse:
Cilia-Associated Respiratory Bacillus (CAR Bacillus) 
Ectromelia virus 
Encephalitozoon cuniculi
Epizootic Diarrhea of Infant Mice Virus 
Hantaan virus 
Lactate dehydrogenase-elevating virus 
Lymphocytic choriomeningitis virus 
Minute virus of mice 
Mouse adenovirus FL/K87 
Mouse cytomegalovirus 
Mouse hepatitis virus 
Mouse Parvovirus 
Mouse pneumonitis virus 
Mouse Thymic Virus 
Mycoplasma pulmonis
Pneumonia virus of mice 
Polyoma virus 
Reovirus type 3 
Sendai virus 
Theiler's mouse encephalomyelitis virus (GDVII)

Rat:
CAR bacillus 
Encephalitozoon cuniculi
Hantaan virus
Kilham rat virus
Lymphocytic choriomeningitis virus
Mouse adenovirus FL/K87
Mycoplasma pulmonis
Pneumonia virus of mice
Rat coronavirus / Sialodacryoadenitis
Rat cytomegalovirus
Rat Parvovirus
Reovirus type 3
Sendai virus
Theiler's Murine Encephalomyelitis Virus
Toolan's H-1 virus

• Bacterial culture of nasopharynx and cecum
  • Nasopharangeal wash
    • Bordetella bronchiseptica
    • Corynebacterium kutscheri
    • Klebsiella pneumoniae/oxytoca
    • Pasteurella pnemotropica
    • Pseudomonas aeruginosa
    • Streptococcus (beta hemolytic)
    • Streptococcus pneumoniae
  • Cecum culture
    • Klebsiella pneumoniae/oxytoca
    • Pasteurella pneumotropica
    • Pseudomonas aeruginosa
    • Salmonella sp.
• Fecal PCR
  • • Helicobacter hepaticus/bilis/ sp.

The above comprehensive lists will be modified as needed dependent on institutional goals and the ever-changing list of pathogens.

For rodents with marginal (not equivalent to accepted assessment panel) health reports, missing or incomplete serology, known infection with adventitious agents, or a weak barrier at the originating colony —

Generally, these animals are assumed to be contaminated until proven otherwise and are processed through several methods:

• Examination of group cohorts
• Placement of sentinel rodents for 3-5 weeks
• Automatic rederivation (not performed here at this time)

If no extra animals are available, sentinel animals may be placed in the same cage to maximize exposure on arrival. The animals will be housed in individually ventilated cages under BSL 2 conditions to exclude transmission of infectious agents between the different quarantine groups. After 3-5 weeks, the sentinels are removed, necropsied and samples are submitted for pathology, microbiology/parasitology and serology for all relevant rodent pathogens. Total duration of quarantine for a clean group is approximately 6-10 weeks.

Sentinel Program

One of the most common ways to assess the health status of laboratory rodent colonies is to use sentinel animals. These sentinel animals are independent of the research colonies and are maintained for the expressed purpose of testing for the presence of pathogens (either directly or via antibodies to those pathogens). At most institutions, sentinel animals are euthanized to facilitate sample collection, to enable collection of sufficient quantities of serum, and to perform direct cecal examinations (for Aspiculuris pinworms); however, sentinel animals can be maintained for multiple sampling periods to provide additional historical evidence for circumstances associated with seroconversion.

Sentinels will be housed in the same method as the colony animals in the room. For rodents in cages with open tops (no microisolation filters), the sentinels will be exposed to pathogens that are spread by aerosol as well as those spread by fomites. For rooms featuring microisolation cages, housing the sentinels in these cages creates an obstacle to their exposure to any pathogens present in the room. Rodents in microisolation cages will receive soiled bedding from the colony animals’ cages. Sentinel animals may also be housed in cages with open tops, even though the colony animals are in microisolation cages, to allow surveying of the room environment, but not necessarily the status of the colony (which could be effectively protected from environmental pathogens by the filtered cage).

The source of sentinel animals can be either unused colony members, or rodents purchased (or bred in-house) with the expressed purpose of serving as sentinel animals. The advantage of using colony members is that it eliminates the risk of introducing a pathogen via the housing of extramural sentinels. The disadvantage of their use is that the strain chosen may respond differently to a pathogen than other colony members, especially in a colony of genetically modified animals. Purchase or breeding of sentinel animals can assure that an appropriately immunoresponsive strain is chosen, but adds the risk of possibly causing a contamination, in addition to detecting it. In either case, animals used for sentinels must be old enough to have functional immune systems without interference from passively transferred maternally derived antibody. In general, rodents 8 - 12 weeks are used. As younger animals are less expensive, it may be cost-efficient to purchase slightly younger animals (e.g., 5 weeks) as sentinels, knowing that they will be 11 - 13 weeks old at the time of sample collection.

The numbers of sentinels per cage, and the number of colony cages per sentinel cage, are commonly debated parameters of a rodent health surveillance system. Ultimately, the number of sentinels per cage and the number of colony cages per sentinel cage are dictated by cost efficiency and risk tolerance. Each animal, and each cage, bears a cost to the institution in purchase costs, per diem rates, charges for sample testing, and additional technical labor. This cost must then be balanced against the need for sufficient samples to generate meaningful information that can be used to appropriately guide colony management.