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PART I
Principles of Rodent Disease
Prevention
SCIENTIFIC OBJECTIVES
Animal experiments are essential to progress in the biomedical sciences (NRC,
1985~. Like investigations in any field of science, the merit of animal experiments
ultimately depends on rigid adherence to the principles of scientific method. Proper
practice of these principles yields data that are both reliable and reproducible, key
objectives of all good experiments (Bernard, 1865~.
INFECTION VERSUS DISEASE
A common misconception is that infection is synonymous with disease. Bacte-
rial opportunists and commensals, which are constitutents of the normal flora on
mucosal and body surfaces, are ubiquitous infections that usually cause disease only
when their hosts are immunosuppressed (Dubos et al., 1965; Savage, 1971~. The
viral and parasite pathogens of rodents vary considerably in pathogenicity. Some
cause severe disease, while others rarely do. It is also important to distinguish
between subclinical (inapparent, covert, or silent) and clinically apparent infec-
tions. Most natural infections with pathogenic organisms in mice and rats are
subclinical, and infection-induced aberrations in research results often occur in the
absence of clinical disease. Thus, it is important to prevent infection, not merely to
prevent clinical disease.
1
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2 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE AND RATS
TERMINOLOGY OF MICROBIAL AND PATHOGEN STATUS
Terms used in defining rodent microbial status vary greatly in precision of
meaning. Four terms (germfree, gnotobiotic, defined flora, and conventional),
representing the extremes of microbial status, have clear definitions that are
generally accepted and understood by scientists, as well as by technical personnel
(NRC, 1991~. However, there is major confusion about the definition and use of
terms representing the middle ground of pathogen status. Pathogen free, specific
pathogen free, virus antibody free, and clean conventional are relative terms that
require explicit definition every time they are used. The definition should include
the background of the rodent subpopulation in question (e.g., cesarean derived,
isolator maintained, barrier maintained), details of current housing (e.g., isolator,
barrier), and data from laboratory tests for pathogens (the specific tests done, the
number of tests, the frequency of testing, and the results) (Lindsey et al., 1986~.
COMMITMENT TO MAINTAINING
PATHOGEN-FREE STATUS OF RODENTS
Past experience demonstrates that maintaining rodents in the pathogen-free state
requires adherence to breeding, transportation, and maintenance programs spe-
cifically designed for the exclusion of pathogens. This means a strong commit-
mentby investigators, research staff, and animal care staff. Some essential elements
of that commitment are as follows:*
a. The investigator and the support personnel must understand the terminology
and principles involved.
b. Appropriate facilities and equipment must be available.
c. Housing practices must ensure physical separation and avoidance of cross-
contamination between different animal subpopulations throughout their lives.
d. Reliable health monitoring should be maintained to identify breeding popu-
lations free of pathogens and to redefine the microbiologic status of the animals at
regular intervals from the time they are received in the user facility until completion
of each study.
e. Written standard operating practices must be developed and followed without
interruption; clear objectives must be defined in advance, along with detailed
procedures for reaching those objectives.
*From a consensus developed during a seminar entitled "B. arrier Maintenance of Rodents in Multipurpose
Facilities," held at the Thirty-Sixth Annual Session of the American Association for Laboratory Animal
Science on November 3-8, 1985, in Baltimore, Md. Participants were J. R. Lindsey (leader); G. L. Van
Hoosier, Jr.; D. B. Casebolt; J. G. Fox; R. O. Jacoby; and T. E. Hamm, Jr.
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PRINCIPLES OF RODENT DISEASE PREVENTION 3
HEALTH SURVEILLANCE PROGRAMS
Health surveillance (or monitoring) is the term usually applied to the testing of
laboratory animals to determine their pathogen status and general state of health.
Health surveillance programs are systematic laboratory investigations that employ
batteries of diagnostic tests for the purpose of defining the pathogen and health
status of an animal population. These programs are crucially important in rodent
disease prevention because they provide data, which are the only reliable basis for
determining rodent pathogen status or providing health quality assurance.
Although the need for health surveillance programs is generally accepted, there
is a great diversity of opinion about the design of individual programs (Hsu et al.,
1980; Iwai et al., 1980; Thigpen and Tortorich, 1980; Jacoby and Barthold, 1981;
Hamm, 1983; Loew and Fox, 1983; Small, 19841. No two programs are identical.
Some are limited in scope; others are very comprehensive. Numerous factors
should be considered in designing individual programs, and special emphasis
should be placed on objectivity in testing rather than on the adoption of customary
practices. Some of those factors are listed in the following sections.
Scientific Objectives
Health surveillance efforts should, to the fullest extent possible, be matched
qualitatively and quantitatively with the specific scientific objectives of individual
research programs to ensure that the quality of the animal will meet these objectives.
For practical reasons, it is impossible to test for all known infectious agents of
rodents, or even all infectious agents that theoretically could interfere with a
particular study. In designing health surveillance programs, decisions must be
made about which agents should be covered in the test battery. Inclusion of a
pathogen in the test battery should be based on the likelihood that it will interfere
with the research being conducted. Such information is given in Part II of this
volume.
Test Procedures
The procedures used in health surveillance generally include serologic tests,
bacterial cultures, parasitologic examinations, and histopathology. Each category
can include a few or many procedures to detect different infectious agents or disease
processes. Some health surveillance programs are limited to only one of these types
of procedures, e.g., serologic testing.
Serologic tests are the main procedures used for detecting virus infections in
rodents, but they also have been found useful for some bacterial and protozoan
infections. The enzyme-linked immunosorbent assay (ELISA) and the indirect
immunofluorescent antibody (IFA) test have largely replaced the complement
fixation (CF) test and the hemagglutination inhibition (HAI) test. They are much
more sensitive than either the CF or HAI test and give fewer false positives than the
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4 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE AND RATS
HAI test. Serologic testing should rely on a primary test for each agent and one or
more additional tests to confirm the positive results of any primary test (Kraft and
Meyer, 1986; Smith, 1986b; Van Der Logt, 1986~.
One of the most useful applications of serologic testing in rodent health
surveillance is the mouse antibody production (MAP) test (Rowe et al., 1959, 1962~.
Although originally developed as a method for broadly screening mouse tissues for
viruses, it can be used to test transplantable tumors, hybridomas, cell lines, and other
biologic materials for contamination by infectious agents. An equivalent test, the
rat antibody production (RAP) test, is useful for screening biologic materials taken
from rats. Both these tests are generally considered more sensitive than virus
isolation (de Souza and Smith, 19891.
The isolation of bacteria using cultural methods and the demonstration and
identification of parasites using a microscope are the standard procedures for
detection of these agents. However, these methods also have limitations, depending
on the agent. In general, causative agents are more difficult to isolate or demonstrate
in subclinical infections than in clinically apparent infections. Some bacterial
infections of mice and rats, e.g., Corynebacterium kutscheri or Mycoplasma
arthritidis, commonly occur as subclinical infections in which cultural isolation is
extremely difficult. With each of the bacteria and parasites it is imperative that
specimens be collected from the most appropriate siteks) and processed expedi-
tiously using methods known to maximize the chances of successful recovery or
demonstration of the agent. Failure to collect specimens from the site that is most
appropriate for that microbe can result in false-negative tests.
Gross and microscopic evaluations of tissues for lesions are also invaluable in
health surveillance. In more comprehensive health surveillance programs, histo-
pathologic examination of all major organs by a qualified pathologist is standard
practice. Lesions caused by viral pathogens can occur before seroconversion. Some
histopathologic changes are diagnostic; others provide only clues to disease
processes.
Diagnostic methodology is in transition. Refinements continue to be made in
existing methods, and newer methods employing molecular biologic techniques,
e.g., nucleic acid hybridization and specific gene product detection, are being
developed at a rapid pace (Sklar, 1985; Edberg, 1986; Smith, 1986a,c; Delellis and
Wolfe, 1987; Howanitz, 1988).
Sampling Strategies
The purpose of health surveillance is to detect at least one animal with each of
the infections or diseases present in the population. The purpose is not to determine
prevalence of infection or disease.
The number of animals (sample size) to be tested is of critical importance and can
be determined mathematically by making important assumptions about the rates of
infection and the randomness in sampling (ILAR, 1976; Hsu et al., 1980; Small,
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PRINCIPLES OF RODENT DISEASE PREVENTION 5
TABLE 1 Confidence Limits for Detecting Infection Using Different Sample Sizes
and Assumed Rates of Infectiona
... . .
Sample Assumed Infection Rate (%)
Size (N)b 1 2 3 4 5 10 15 20 25 30 40 50
5 0.05 0.10 0.14 0.18 0.23 0.41 0.56 0.67 0.76 0.83 0.92 0.97
10 0.10 0.18 0.26 0.34 0.40 0.65 0.80 0.89 0.94 0.97 0.99
15 0.14 0.26 0.37 0.46 0.54 0.79 0.91 0.95 0.99
20 0.18 0.33 0.46 0.56 0.64 0.88 0.95 0.99
25 0.22 0.40 0.53 0.64 0.72 0.93 0.98
30 0.25 0.45 0.60 0.71 0.79 0.96 0.99
35 0.30 0.51 0.66 0.76 0.83 0.97
40 0.33 0.55 0.70 0.80 0.87 0.99
45 0.36 0.69 0.75 0.84 0.90 0.99
50 0.39 0.64 0.78 0.87 0.92 0.99
60 0.45 0.70 0.84 0.91 0.95
70 0.51 0.76 0.88 0.94 0.97
80 0.55 0.80 0.91 0.96 0.98
90 0.60 0.84 0.94 0.97 0.99
100 0.63 0.87 0.95 0.98 0.99
120 0.70 0.91 0.97 0.99
140 0.76 0.94 0.99
160 0.80 0.96 0.99
180 0.84 0.97
200 0.87 0.98
aFrom ILAR (1976a), Hsu et al. (1980), and Small (1984).
bat _ log(1 - probability of detecting infection)
log(1 - assumed infection rate)
1984; DiGiacomo and Koepsell, 1986~. As shown in Table 1, if one assumes that
40% of the animals in a population are infected with an agent, there is a 99%
probability that 1 infected animal will be detected in a randomly selected sample of
10 animals. At a 50% infection rate, a sample size of only 5 is required for a 97%
probability of detecting infection in at least 1 animal.
Although the sample size required to detect a single agent can be determined with
reasonable precision, it is virtually impossible to maintain the same degree of
precision for all agents to be included in a large test battery. Different agents
typically have very different infection rates within rodent colonies. For example,
typical rates for established infections in mouse colonies are greater than 90% for
Sendai virus, approximately 25% for pneumonia virus of mice, and less than 5% for
Salmonella enteritidis. In determining the number of animals to be used in a health
surveillance test battery for these three agents, the lowest assumed infection rate
should be used (i.e., 5%), and a 95% confidence limit would require a sample size
of at least 60 animals. This is entirely appropriate in instances where subclinical S.
enteritidis infection is suspected. However, for routine health surveillance, sample
sizes are usually based on assumed infection rates of 40-50% in order to keep sample
sizes reasonable.
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6 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE kD RATS
Proper sampling also requires that animals be taken from different cages,
shelves, and racks so that the sample is representative of the entire population.
Animals of both sexes and of two age groups should be sampled. For serologic
testing, sampling of young adults (approximately 90 days old) and retired breeders
is recommended. Young adults are best for detecting recent viral infections
(without interference from passive antibody), and retired breeders give an indica-
tion of the colony's infection history (Jacoby and Barthold, 1981~.
Test Frequency
One of the most difficult decisions to be made in designing health surveillance
programs is how frequently a given rodent population should be tested. There are
no established guidelines, but the problem seems to revolve around four central
issues: the specific purpose of the population in question, the potential or real
importance of a pathogen or other contamination to use of the population, the level
of risk of pathogen contamination from other nearby rodent populations, and
economic considerations. After evaluating these issues, one should have a basis for
deciding whether testing should be monthly, quarterly, biannually, or annually.
However, the frequency of testing may be different for different agents. For
example, if the greatest risks are deemed to be from mouse hepatitis virus and Sendai
virus, tests for these agents could be performed monthly, and a larger battery could
be done biannually.
Sentinel Animals
Sentinel rodents are sometimes introduced into a rodent population, housed in
open cages placed systematically throughout the colony, and used periodically for
testing. Pathogen transmission from the principal population to the sentinels can be
increased by transferring the sentinels into dirty cages from the principal population
at each cage change. Sentinel animals preferably should be of the same population
as the principal population and should be subjected to any experimental treatments
given to the principal population. The introduction of a second population as
sentinels, even if it is tested and found to be free of pathogens, poses an unnecessary
risk for contaminating the principal population.
RODENT DIAGNOSTIC LABORATORIES
Rodent diagnostic laboratories are indispensable to the production and mainte-
nance of mice and rats for high-quality research. Such laboratories specialize in
health surveillance testing, investigations of clinical diseases, and other quality
control methods specifically designed for laboratory rodents. Depending on the
breadth of their activities, these laboratories most often include competence in
serology, bacteriology, parasitology, and pathology. Virology and hematology
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PRINCIP f ES OF RODENT DISEASE PREVENTION 7
expertise may also be required in some instances. Many larger research institutions
have well-equipped and well-staffed institutional diagnostic laboratories. Testing
services also can be obtained through commercial laboratories.
Traditionally, rodent diagnostic laboratories have tended to give highest Snooty
to the investigation of clinical illnesses and necropsy evaluations of dead animals.
That approach is no longer acceptable. While those services certainly are necessary,
the needs of modern research and the principles of scientific method demand that
diagnostic laboratories give greater priorly to disease prevention. Most of the
pathogen infections and pathogen-induced diseases of laboratory rodents are
preventable.
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Representative terms from entire chapter:
laboratory animal
