1. health and fitness
2. disease

LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
RABBITS:
Introduction to Use in Research
V-9001
G. L. Van Hoosier, Jr., DVM
R. F. DiGiacomo, DVM
Department of Comparative Medicine
School of Medicine
University of Washington
Seattle, Washington
The Laboratory Animal Medicine and Science - Series II has been developed by the following
committee for the American College of Laboratory Animal Medicine (ACLAM): C. W. McPherson,
D.V.M., Chair; J. E. Harkness, D.V.M.; J. F. Harwell, Jr., D.V.M.; J. M. Linn, D.V.M.; A. F.
Moreland, D.V.M. G. L. Van Hoosier, Jr., D.V.M.; L. Dahm, M.S.
Portions of the project have been funded by a grant from the National Agricultural Library.
Laboratory Animal Medicine and Science - Series II
is produced by the
Health Sciences Center for Educational Resources
University of Washington.
2
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
University of Washington
Health Sciences Center for Educational Resources
Box 357161, Seattle, WA 98195 -7161 206/685-1156
ISBN: 1-55910-039-7
Copyright © 2000
by the University of Washington Health Sciences Center for
Educational Resources and the American College of Laboratory
Animal Medicine
All rights reserved.
V-9001 RABBITS: Introduction to Use in Research
3
PRIMARY AUDIENCE
Investigators, research technicians, and biomedical science students.
GOALS
The program presents sources and scope of laws and guidelines
pertaining to use of rabbits, the historical uses of rabbits in research
and testing, development of alternatives, attributes of rabbits as
research animals, recognition of pain and disease, and signs and
significance of common diseases.
.
OBJECTIVES
When you complete this program on the use of hamsters in research,
you should be able to:
1. State three sources of laws and guidelines pertaining to the care
and use of rabbits for research, testing, or teaching.
2. List several noninfectious diseases that are studied using a rabbit
model.
3. List several infectious diseases that are studied using a rabbit
model.
4. Describe two or more traditional uses for rabbits in testing that are
being replaced by alternative methods.
5. Discuss unique characteristics of rabbits that may justify selection of
the rabbit rather than a rodent as an animal model.
6. List several behavioral and physiological signs of pain or distress in
rabbits.
7. State two methods of euthanatizing rabbits that are approved by the
AVMA Panel on Euthanasia.
8. List signs of disease in rabbits that should be reported to veterinary
services for assessment and/or care.
9. Discuss the relevance of subclinical infections to research
outcomes.
4
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
1. Series
Laboratory Animal Medicine and Science - Series II
2. Title
Rabbits: Introduction to Use in Research
3. Objectives
Upon completion of this program, you will know the major sources of
laws and guidelines controlling the use of rabbits in research, testing,
and teaching; the primary uses of rabbits in the past; several examples
of alternatives; and the attributes of rabbits that may be useful in
meeting research goals. You will also know how to recognize pain and
distress in rabbits, how to euthanatize them humanely, and how to
recognize signs of common diseases of rabbits maintained within a
laboratory setting.
4. Legislation and Guidelines
The use of rabbits for research, testing, or teaching is
covered under the Animal Welfare Act, as amended by the Improved
Standards for Laboratory Animals Act (Public Law 99-198) in 1985,
and by the Public Health Service Act, which was amended by the
Health Research Extension Act (Public Law 99-158) in 1985.
5. Scope
In general, these laws cover the responsibilities of institutions and
individuals using animals, such as training requirements and oversight
of research by an Institutional Animal Care and Use Committee
(IACUC); and many aspects of humane care, identification, shipping,
record keeping, caging and environment, surgical conditions and postsurgical care, requirements for use of anesthetics and analgesics, and
methods of euthanasia.
6. The Guide
A publication you should be very familiar with is the Guide to the Care
and Use of Laboratory Animals, 1985 revision. The Guide provides a
summary of principles and issues of humane use of laboratory animals
for research, testing, and teaching.
In addition, your institution may be controlled by State and local
regulations, or may have institutional policies that you should be
familiar with.
HISTORICAL USES
7. List of historical uses
The cause and treatment of many human diseases have been
studied using the rabbit as a model. Because of its response to
different diets, the rabbit was the first animal model used to study
atherosclerosis. Other noninfectious diseases studied in the rabbit
include osteoarthritis, pregnancy toxemia, endometrial
adenocarcinoma, drug teratogenesis, hydrocephalus, muscular
dystrophy, glomerulonephritis, and gallstones.
8. List (cont.)
Infectious diseases studied in the rabbit include staphylococcal
infection, bacterial endocarditis, and Reiter's polyarthritis syndrome.
Hereditary studies include familial hypercholesterolemia, dwarfism,
spina bifida, and glaucoma.
V-9001 RABBITS: Introduction to Use in Research
9.
5
Syphilis - darkfield
There are some diseases of man for which only the
rabbit can serve as a model. One of these is syphilis.
The rabbit is the only other mammal in which syphilis
occurs naturally. The causative agent of human
syphilis, Treponema pallidium, cannot be grown in
vitro.
10. Syphilis
But we can put the human organism into rabbits and
study its growth there. If and when a vaccine is
developed for human syphilis, trials will undoubtedly be
conducted using the rabbit first.
11. Pet vaccination
Animals have also benefited from research using
rabbits. Pasteur developed a vaccine to protect dogs
from rabies by using dried spinal cord from rabbits that
had been experimentally infected.
ALTERNATIVES
12. Alternatives
We do not advocate using rabbits, or any other animal, solely on the
basis of historical precedent. As science and technology have evolved,
new options have become available. The increasing costs of using
animals and societal attitudes toward their use have provided
incentives for industry to be very active in the development of
alternatives that reduce the numbers of animals used, use lower
species, and refine techniques to eliminate potential discomfort in
animals used. For example, ...
6
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
13. Example - Current pregnancy testing
...until recently the most common pregnancy test used
rabbits, and an animal was sacrificed for each test.
Now pregnancy testing can be done by adding
radioisotope-labeled antibodies to measure human
chronic gonadotropin (hCG) levels in samples taken
from the patient. Computers provide rapid and
accurate data analysis.
14. Example - chemilluminometric immunoassay
Another method of measuring hCG levels for
pregnancy testing is chemilluminometric immunoassay
procedure that uses monoclonal antibodies from a
mouse and a solid phase reagent containing polyclonal
antibodies from a goat. Although these procedures are
still dependent on the use of animals, substantially
fewer animals are needed.
15. Example - pyrogen testing
Similar progress has been made in pyrogen testing.
Traditionally, rabbits have been to monitor parenteral drugs and
solutions for the presence of contaminating endotoxins. In 1976,
Levine and Bang made a serendipitous observation that endotoxins
caused horseshoe crab blood (amebocytes) to coagulate -- an
observation that led to the development and commercialization of the
limulus amebocyte lysate assay (LAL). The LAL is now in wide use for
detection of endotoxins in a variety of settings, although the FDA still
views the rabbit pyrogen assay as the definitive assay for many
parenteral compounds. This may change, however, with refinement
and supplementation of the LAL.
16. Example - Draize test Perhaps the most controversial use of rabbits has been in vivo
cytotoxicity studies using the Draize test, which measures response to
potential irritants by instilling a test compound onto a rabbit's cornea.
The techniques used for the Draize test are being refined to reduce
both the severity of irritation and the number of animals used. In
addition, in vitro alternatives are at the stage of validation or early use.
V-9001 RABBITS: Introduction to Use in Research
7
17. Example - cytotoxicity
One of these is a visual morphological cytotoxicity assay
combined with a assay quantitative neutral red
spectrophotometric test, which shows good agreement
with in vivo data. The neutral red assay first used
cultured mammalian cells. Later it was adapted to also
use fish cells as bio-indicators of aquatic pollution.
18. Example - antibody production
The most frequent use of rabbits in experimental biology
and medicine probably for production of antibodies. Until quite recently
antibody production often involved repeated use of Freund's Complete
Adjuvant (FCA), which was associated with adverse reactions. Now if
Freund's is used, the complete adjuvant is given only for the initial
injection, with incomplete adjuvant given for subsequent inoculations.
Injections into the footpad are no longer accepted, with only
intramuscular or subcutaneous routes recommended. There are also
other alternative adjuvants that are commercially available.
ATTRIBUTES OF RABBITS AS RESEARCH ANIMALS
19. Justification
Considering our current socio-legal environment, it has become very
important for an investigator to be able to justify use of a particular
animal species; e.g., the species selected must correspond with the
research or teaching objectives and the research must produce
meaningful outcomes that cannot be obtained by using another
method or a lower species. The following are attributes of rabbits that
may justify their selection.
20. Size
Rabbits may be preferred over other common
laboratory animals because of their size. They have
sufficient blood volume to allow for large blood
samples to be taken either singly or serially. Because
of their size, they may be preferred over rodents for
studies that require temperature monitoring or surgical
procedures.
8
21. Inbred strains
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
When we think of inbred strains, rodents may come to mind first. But
the rabbit is highly desirable for studies that require larger animal and
also an inbred strain. It is probably the largest laboratory species in
which inbred strains are commonly available, and is often chosen for
genetic studies.
Multiple litters. The rabbit is one of the largest laboratory species to
produce multiple litters per year. A rabbit can produce 6 or more litters
a year, with a typical litter size of 7 or 8.
Availability. Rabbits are readily available and relatively inexpensive
when compared with species of similar size. Also, specific-pathogenfree (SPF) rabbits are available. This means that the breeder will
supply animals that are free of Pasteurella and other infectious agents.
22. Disposition / handling
Rabbits are generally docile animals that are easy to
handle. They are, however, capable of inflicting serious
injuries by scratching or kicking, and of seriously injuring
themselves if they are frightened. A rabbit should be
handled only by persons trained in proper methods of
handling and restraint.
23. Caging / care
Another attribute is that they are relatively easy to
maintain and have no special requirements. An adult
rabbit requires a smaller cage area than cats or dogs,
they produce less waste, and they have no special
requirements such as exercise or psychological
enrichment, making them easier to maintain than other
larger species.
24. Analogous systems Four other characteristics of rabbits make them desirable for specific
research tasks. First, by analogous systems, we mean that some of
the rabbit's organ systems are very similar to systems in man. The
rabbit is often the model of choice to study immune responses for this
reason.
V-9001 RABBITS: Introduction to Use in Research
9
25. Embryonic susceptibility
The placental barrier of the doe is hemochorial, similar to
the human placental barrier; and rabbit embryos are extremely
susceptible to both embryotoxic and teratogenic agents. Therefore, the
rabbit is the model of choice for testing any product that might be used
by pregnant women.
10
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
26. Reproductive anatomy
A doe has two uteri, each of which opens into the
vagina through a separate cervix. This unique
reproductive anatomy allows for an experimental group
and a control group within a single biologic host.
Rabbits are also useful for studies that require precise
timing during gestation, as a doe ovulates about ten
hours after mating, and gestation is well defined at 3032 days.
27. Susceptibility to disease
The last of these characteristics that make the rabbit
valuable for specialized studies is its susceptibility to spontaneous and
induced diseases; such as syphilis, herpetic conjunctivitis, tumors
associated with papilloma viruses, glaucoma, hyperlipidemia, and
nutritional muscular dystrophy. A more complete list of spontaneous
and artificially induced diseases of rabbits is provided in the Guide that
accompanies this program.
PAIN AND DISTRESS
28. Signs of distress
One of the earliest signs of pain or distress in a rabbit is often anorexia,
which may be accompanied by abnormal (hunched) posture or
unwillingness to move. Other behavior changes associated with pain or
distress are unexpected aggressions and, in extreme cases,
vocalizations. Physiological signs such as elevated temperature and
increased respiration rate confirm pain or distress.
29. Form for quantitative assessment
A quantitative assessment of pain can be generated by
assigning a point value to five considerations: body
weight, appearance, behaviors, clinical signs, and
response to stimuli. The form shown here is included in
the printed materials accompanying this program. A
high score in any individual category or a moderate
score in several categories is an indication of probable
pain or distress.
V-9001 RABBITS: Introduction to Use in Research
11
30. Euthanasia
Methods of euthanasia that comply with the AVMA
Panel on Euthanasia's recommendations for rabbits
include CO2 asphyxiation or intravenous pentobarbital
(120 mg/kg of body weight).
12
31.
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
SIGNS OF DISEASE
32. Rhinitis
In rabbits cold-like signs, or rhinitis, require immediate
veterinary assessment, as they often are a
manifestation of Pasteurellosis, commonly called
"snuffles." The rabbit may have yellowish discharge
around the nares, or matted hair on its forepaws from
wiping the nose.
33. Head tilt
Head tilt is another sign of Pasteurellosis. It results
when an ear infection extends to the inner ear.
34. Diarrhea
Acute diarrhea is a sign of several serious diseases of
rabbits. The attending veterinarian should be notified,
and isolation from the remainder of the colony may be
indicated. Often the etiology remains undetermined,
and a very high mortality rate occurs in affected
rabbits.
35. Patchy hair loss
V-9001 RABBITS: Introduction to Use in Research
13
Patchy areas of hair loss, sometimes with dry, crusty
skin lesions, may be a sign of ringworm. They
frequently occur on the head, as shown here. This is a
condition that should be diagnosed and treated, as
ringworm is transmissible to people who handle the
animal as well as to other animals.
36. Ear mite infestation Otoacariasis, or ear mite infestation, must not be ignored. The
condition causes significant discomfort and stress to the affected
animal, and may make access to the ear veins of the rabbit impossible.
37. Summary of signs
38. Other signs
So far, we have discussed the significance of reporting and treating
snuffles, head tilt, diarrhea, hair loss, and ear mites. Other signs of
disease or discomfort that should be reported to veterinary services
are...
Failure to move about freely
Failure to eat; weight loss
Cloudy or red eyes
Rough hair coat
Ulcerative lesions, swellings
Bloody discharge
39. Effects of disease
The effects of disease on research are complex. Treatment of rabbits
with antibiotics carries a high risk, as the alteration of gastrointestinal
microflora can precipitate life-threatening diarrhea. Even conservative
interventions such as isolation may generate stress and disturb the
homeostatic balance of the animals. In addition to the potential loss of
animals, the effects of disease include loss of time, increased costs,
and possible compromise of data.
40. Subclinical infections
Subclinical and persistent infections can also seriously distort
research outcomes. Here are two examples:
Assume you have set up a nutrition study in which the experimental
group is fed a diet that is deficient in a particular element while the
control group is fed a normal diet. You plan to measure the impact of
the deficient diet by recording weights and you anticipate that the
deficient diet will result in weight loss.
14
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
41. Example
Now, let's assume that many of the animals in both
groups have a subclinical infection and that you are not
aware of it because there are no apparent signs of
disease. The additional stress of the deficient diet may
be sufficient to trigger a clinical manifestation of the
disease in the experimental group. Your experimental
group may go off feed and lose weight, not as a direct
result of the variable you were studying, but because of
the combination of the subclinical disease plus the
stress of the research.
42. Subclinical infections
The second example shows how subclinical viral and
parasitic infections affect research outcomes by
modifying the immune responses in rabbits. In this
study, a natural infection of rabbits with
Encephalitozoon cuniculi produced varied immune
responses, with depressed immunoglobulin G values.
43. Investigator
When the rabbit is the model of choice, the investigator
should make efforts to acquire disease-free animals
from good stock, and the animals should be observed
daily for signs of disease.
44. Programs
This program has presented an introduction to the use of the rabbit for
research, testing, or teaching. Additional programs in this series cover
care and management; biology that is relevant to the use of rabbits in
research and testing; and diagnosis and management of common
diseases of rabbits.
V-9001 RABBITS: Introduction to Use in Research
15
45. ACLAM Credits
The Laboratory Animal Medicine and Science - Series II
has been developed under the following committee for the
American College of Laboratory Animal Medicine
C. W. McPherson, DVM, Chair
J. E. Harkness, DVM
J. F. Harwell, Jr., DVM
J. M. Linn, DVM
A. F. Moreland, DVM
G. L. Van Hoosier, Jr., DVM
L. Dahm, MS.
Portions of the project have been funded
by a grant from the National Agricultural Library.
46. HSCER Credits
Produced by the
Health Sciences Center for Educational Resources,
University of Washington,
Seattle WA 98195
2000
ACKNOWLEDGMENTS
The authors wish to thank the following for images contributed to the program:
E. Borenfreund, DVM
S. Lukehart, Ph.D.
16
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II
APPENDIX 1
Examples of Medical Conditions in Rabbits and Corresponding Human Diseases
Rabbit
Human
Hereditary:
achondroplasia
spina bifida
glaucoma
osteopetrosis
Pelger-Huet anomaly
hypogonadia
diaphragmatic hernia
lysozyme deficiency
von Willebrand's disease
C6-complement deficiency
hyperlipidemia
hypercholesterolemia
epilepsy
dwarfism
spina bifida
glaucoma
osteopetrosis
Pelger-Huet anomaly
hypogonadia
diaphragmatic hernia
biologic role of lysozyme
von Willebrand's disease
C6 deficiency
familial hyperlipidemia type III
familial hypercholesterolemia
epilepsy
Shope fibroma
Shope papilloma
treponemal infection
poxvirus-induced neoplasia
papovirus-induced neoplasia
syphilis
arteriosclerosis
atherosclerosis
hypocalcemia
toxemia of pregnancy
endometrial adenocarcinoma
arteriosclerosis
atherosclerosis
lactation hypocalcemia
toxemia of pregnancy
endometrial adenocarcinoma
Infectious:
Other:
Artificially-induced conditions
Rabbit
Human
Noninfectious conditions:
vitamin A deficiency hydrocephaly
hydrocephalus
nutritional muscular dystrophy
muscular dystrophy
thyroiditis
thyroiditis
nephrosclerosis
arteriolar nephrosclerosis
synovitis
synovitis
degenerative joint disease
osteoarthritis
membranous /proliferative pneumonitis
Interstitial pneumonitis
cholelithiasis
gallstones
endotoxin-induced DIC
DIC in endotoxemia
Infectious conditions:
staphylococcal infection
bacterial endocardititis
chlamydial arthritis
chronic staphylococcal infection
bacterial endocardititis
Reiter's syndrome
V-9001 RABBITS: Introduction to Use in Research
17
APPENDIX 2
QUANTITATIVE ASSESSMENT OF PAIN
INDEPENDENT VARIABLE
SCORE
BODY WEIGHT
0 Normal
1 < 10% weight loss
2 10 - 15% weight loss, eating
3 >20% weight loss, not eating
APPEARANCE
0 Normal
1 Lack of grooming
2 Coat rough, possible nasoculo discharge
3 Coat very rough, abnormal posture, pupils enlarged
CLINICAL SIGNS
0
1
2
3
Normal
Small change of potential significance
Temperature Rise 1-2 , 30% rise of respiratory/heart rates
Temperature change > 2 , 25% rise in respiratory/heart rates
(or markedly reduced/shallow)
UNPROVOKED BEHAVIOR
0 Normal
1 Minor changes
2 Abnormal behavior, less mobile, less alert, inactive when activity
expected
3 Unsolicited vocalization, extreme self-mutilation
BEHAVIORAL RESPONSE TO EXTERNAL STIMULI (PALPATION OF
INJECTION SITES)
0
1
2
3
Normal
Minor exaggerated response
Moderate abnormal response
Violent response
TOTAL SCORE
18
NOTES
LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II