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Contemporary Topics
Volume 43, Issue 4 (July 2004)
Vendor Apparent Source of Mouse Parvovirus in Sentinel Mice (p. 8-11)
Summary: Mouse Parvovirus (MPV) is growing in prevalence in laboratory animal facilities. It tends to
be hardy in the environment and can be transmitted primarily by fecal oral contact and fomite spread.
After the detection of sporadic MPV in the sentinel population, the author developed a system to assess
vendor mice used as sentinels by group housing them in individually identified large cages (A-U) in a
isolation type location, deploying some as sentinels into the facility, while retaining some in the
isolation location. Two mice from each of these cages were tested serologically for murine viruses 3
weeks after arrival and before sentinels were deployed from them. All tested negative at the time.
After a month, the sentinels were eye bled under anesthesia or terminal collections were done
on mice euthanized with CO2. The blood was analyzed using at a major testing facility to look for
routine murine viruses including MPV. The results indicated that mice deployed from cage C in the
isolation area were positive for MPV. As a matter of of fact all the sentinels deployed from cage C were
positive for MPV. None of the other group housed cages in the isolation area had deployed sentinels that
tested positive. At this time the remaining mice in cage C which had been isolated and not exposed to
other mice were tested and they too were seropositive. This seems to indicate that there is a possibility
that these mice came in from the vendor infected with MPV, and subsequently seroconverted in the lab
animal facility.
While the vendor facility tested rigorously (.8% of the population monthly), they housed their
mice in open top cages with the prevailing thought being that if all of the mice would be exposed to
everything by aerosol. MPV has been documented to be transferred by direct contact (contact sentinels),
and by fomite (dirty bedding), but has not been documented as being transmitted by aerosol. This may
account for it not being detected at the vendor location.
Questions:
1. Parvoviruses are:
a. Hardy
b. Transmitted by fecal oral
c. Becoming more prevalent in research facilities
d. All of the above
Answer D
Title: Evaluation of Cage Micro-Environment of Mice Housed on Various Types of Bedding
Materials (pages 12-17)
Summary:
A variety of environmental factors can affect study outcomes. Bedding is one such factor. This study
addressed the cage micro-environment (ammonia levels, temperature, humidity) of mice on different
beddings and their combinations. The types of beddings studied were as follows: ALPHA-dri (Alpha
cellulose), Bed-O"-cobs (corncob), Beta Chip (Hardwoods maple, beech and poplar), Cell-Sorb Plus
(recycled newspaper), CareFRESH Ultra (long-fiber, high-grade bleached pulp), and pine shavings. The
study also compared results for bedding supplied as Nestpaks vs. loose bedding.
With the exception of one component of the study that compared static vs. ventilated cages, the
remaining mice in this study were housed in static duplex cages for a period of three weeks with no
change-out. Each side of the duplex cage housed 4 male mice or a litter with both parents. The results
showed little effect of bedding type on in-cage temperature and humidity but considerable variation in
ammonia concentrations.
Past studies have reported the effects of exposure to gaseous ammonia on laboratory rodents.
Such effects have included mortality in rats, immunosuppression in guinea pigs, decreased
concentration-dependent running (on a wheel) in Long-Evans rats and Swiss mice, and decreased ciliary
activity in rats. A previous study showed that, regarding rats undergoing continuous exposure to 262
ppm ammonia for 90 or more days, 25% had a mild nasal discharge. Continuous exposure to >400 ppm
ammonia resulted in the death of 32 of 51 rats by day 25 of exposure and 50 of 51 by day 65. These rats
had mild dyspnea with nasal irritation. However, the authors also pointed out that the early literature that
addressed the noxious effects of ammonia concentrations on rodents must be considered in the context
of their microbial status at the time. Infectious diseases of the respiratory tract, specifically M. pulmonis
infection in rats, have been reported as cofactors in ammonia toxicity. Therefore, contemporary rodents
may be able to tolerate higher in-cage ammonia concentrations in the absence of such exacerbating
cofactors.
Human exposure to ammonia concentrations as low as 20 ppm can cause discomfort and
conjunctival hyperemia. Standards put forth by NIOSH (National Institute of Occupational Safety and
Health) indicate that workplace exposure to ammonia should not exceed 25 ppm over 8 hours or 35 ppm
over a 15 minute period. There are no accepted standards for rodent exposure to ammonia. In the
absence of data indicating what ammonia concentrations might be noxious for mice, the authors suggest
we may need to defer to the OSHA standards established for people.
The ammonia levels in cages with loose bedding did not differ from those with Nestpaks
containing the same bedding. Beta-chips and a combination bedding (Bed-O-Cobs mixed with ALPHAdri) yielded the lowest intra-cage ammonia concentrations. CareFRESH Utlra bedding yielded the
greatest ammonia concentrations of all beddings studied. In light of cage micro-environment results,
especially ammonia levels, the authors concluded that all bedding types that evaluated, except for
CareFRESH Ultra, were acceptable choices for use in static cages and that the practice of changing
bedding every 2 weeks would likewise be acceptable. Ammonia concentrations in ventilated cages
housing mice on CareFRESH Ultra bedding were acceptable, however.
Questions:
1.
T/F Infectious diseases of the respiratory tract coupled with
ammonia levels have been reported as synergistic factors in regards to ammonia toxicity.
2.
What does the acronym NIOSH stand for?
Answers:
1.
True
2.
National Institute of Occupational Safety and Health
To Enrich or Not to Enrich: Providing Shelter Does Not Complicate handling of Laboratory Mice
(pages 18-21)
Summary
The authors describe Environmental Enrichment (EE) as a method used in laboratory animal
housing to provide stimuli exceeding those of barren cages and is intended to improve the welfare of
captive animals.
Providing EE is more effective in reducing abnormal behavior than is housing animals in
larger cages. The authors mentioned some of commonly used methods of EE for rodents as providing
access for conspecifics, shelters, nesting materials, climbing structure, and gnawing objects. Animals
when given access to EE, express a more species specific repertoire of behavior, have a better learning
abilities, neurologically more stable and don’t perform stereotypic behavior to the same degree as when
maintained without EE.
Specific aim of this study was to evaluate whether the use of EE would complicate catching
and handling mice by comparing the time needed to catch animals and by evaluating the response to
handling. Inbred FVB/Nhan Hsd and outbred NMRI/Hsd Win strains were used in the study.
The study was conducted by housing 20 FVB and 20 NMRI male mice in Standard cages and
another 20 FVB and 20 NMRI male mice in cages enriched with two PVC conduits. Measurement of
food, water consumption, weight, latency Of catching, and a behavior score in response to handling
during a sham subcutaneous injections were performed weekly for four consecutive weeks when mice
were 10 weeks old.
Food and water consumption and weight were influenced by strains, but The presence of EE in
the home cage did not affect these parameters as much.
Outbred mice ate, drank, and weighed more than the inbred mice, but didn’tsignificantly gain
weight during the course of the four testing weeks. Cage enrichment in the form of PVC conduits
decreased the time needed to catch outbred mice and didn’t increase the time needed to catch mice from
the inbred strain. No difference in resistance to being held during the sham injection between animals
from the enriched versus non-enriched group.
In conclusion, the authors, based on the result of the study suggest, EE in the form of
sheltering object doesn’t complicate catching or handling. Allowing access to enrichment in the
laboratory cage, which has been Shown to have positive effect on welfare, doesn’t interfere with the
management or cost of laboratory animals.
Endotracheal Tubes Versus Laryngeal Mask Airways in Rabbit Inhalation Anesthesia: Ease of
Use and Waste Gas Emissions (p. 22-25)
The rabbit is a standard animal model for biomedical research in which many procedures require
anesthetic procedures. The anatomical features of the rabbit makes it difficult to use inhalation gas
anesthesia. This purpose of this study was to compare ease of placement of cuffed endotracheal tube,
uncuffed endotracheal tube (Cole) and pediatric laryngeal mask airways (LMA) and to assess the extent
of anesthetic pollution. A second purpose was to produce a comprehensive procedure for the placement
of LMAs in the rabbit The hypothesis was that the LMAs would be a simpler method of gas delivery but
would permit greater leakage of anesthetic gas. The reason for the study was to develop training
procedures for animal technicians and researchers to deliver gas anesthesia with lecture and hands on
training.
Rabbits were anesthetized with ketamine and xylazine and maintained with isoflurane using a precision
vaporizer to two circuits, one to a two liter acrylic box and on with direct delivery to the animal. The
exhaust system was connected to gas-scavenging canisters. The system was calibrated before use and
evaluated with portable equipment during the study for leaks. Also, during the study waste gas was
measured at the rabbit's oral commissure and in the operator's breathing zone. Rabbits were recovered
on heating pads maintained at 37 degrees C.
Rabbits were placed in ventral recumbancy with the heads tilted up at a 90 degree angle to inset the
endotracheal tubes blindly into the trachea. The tubes were advanced with the convex side of the
tube against the pharynx down into the trachea. The rabbits were placed in right lateral recumbancy for
LMA placement and the head tilted upward 90 degrees. The LMAs were held laterally with the aperture
turned toward the tongue and the LMA were rotated 90 degrees in a counterclockwise so the cuff would
cover the edges of the larynx.
Rabbits were evaluated every 5 minutes for pain (toe pinch) and levels of waste anesthesia
were measured. The original hypothesis was proven to be true; LMAs are easier to place for animal
technicians and researchers with a minimal amount of training. Adequate levels of anesthesia were
established and maintained during the study. In addition, smaller amounts of anesthetic waste was
emitted from both the cuffed and non-cuffed endotracheal tubes as compared to the LMAs emissions
which was higher and statistically significant. However, the amounts of emitted isoflurane from all three
anesthesia delivery methods exceeded NIOSH levels of 2 ppm; the results of this study measured 6.3 to
8.4 ppm at the commissures of the rabbits' mouths. The waste anesthesia levels did not surpass most
common European 8 hr time-weighted average defined for stand-alone use of isoflurane (10 ppm
established by Germany, Sweden and Switzerland).
Questions:
1. What were the three methods of distributing isoflurane to the rabbits in the study?
2. (True or False) Did the waste anesthesia measured in this study meet HIOSH guidelines?
3. Which guideline did the waste anesthesia for this method meet?
Answers:
1. cuffed endotracheal tube, uncuffed endotracheal tube (Cole) and pediatric laryngeal mask airways
(LMA)
2. False
3. European 8 hr time-weighted average defined for stand-alone use of isoflurane (10 ppm established
by Germany, Sweden and Switzerland)
Vitamin A Toxicity and Vitamin E Deficiency in a Rabbit Colony (pages 26-30)
Summary: Clinical signs of both vitamin A toxicity and deficiency include absorption of fetuses,
abortions, stillborn kits, decreased litter size, low neonatal viability, and hydrocephalic kits.
Hypovitaminosis A can affect cartilage formation in rapidly growing rabbits, which present clinically
with droopy ears. Except in cases of severe deficiency or toxicity, rabbits maintain fairly constant blood
serum levels of vitamin A; therefore, serum levels may not accurately reflect an animal’s true vitamin A
status. Liver values provide a more faithful representation of vitamin A status and are thus considered
the “gold standard.”
Rabbits are the most sensitive laboratory animals to vitamin E deficiency. Accordingly, rabbits
have been used as models to determine the physiological role of vitamin E. Vitamin E functions as an
anti-oxidant that prevents peroxide damage to tissues. Clinical signs of vitamin E deficiency in kits
include muscular dystrophy, with paresis to paralysis of hind limbs. Breeding does experience problems
with infertility, abortions, stillbirths, and in vivo hemolysis. Vitamin E deficiency can be diagnosed by
the in vitro peroxide hemolytic test, serum Creatine Phosphokinase, or vitamin E levels in the serum or
liver.
Vitamin E has been shown to modulate the effect and levels of vitamin A. Whereas small doses
of vitamin E increase vitamin A utilization, larger doses markedly reduce the amount of vitamin A
formed and stored in the liver.
Vitamin A toxicities are frequently reported in rabbit literature, but no treatment regimens have
been proposed. Here the authors evaluate vitamin A and E levels in rabbits with clinical signs of vitamin
A toxicosis and vitamin E deficiency, and assess the impact of vitamin E-based therapy on clinical signs,
reproduction, and vitamin A and E levels.
Case presentation: A concerned commercial rabbitry owner contacted MURADIL regarding a 46 week history during which several does had reabsorbed fetuses or had spontaneous abortions or
hydrocephalic kits. The few kits that survived were unthrifty or became weak, with most dying by 27
days of age. The diet consisted of a commercial pelleted rabbit chow that the owner had used with
confidence for 4 years. Several rabbits were submitted to MURADIL for diagnostic necropsies. Vitamin
A toxicosis and vitamin E deficiency were diagnosed. Specifically, laboratory tests showed elevated
serum and liver vitamin A levels and low serum and liver vitamin E levels. The vitamin E deficiency
had produced paresis and muscular dystrophy in the juvenile rabbits.
It is important to note that clinical manisfestations of toxicity or deficiency consequent to
misformulated rations may remain silent for as long as 3 months in rabbits after eating the feed. In
addition, one should note that hypervitaminosis A may result in elevated vitamin A levels in the milk at
kindling and cause hypervitaminosis A in the offspring.
In addition, the owner donated several rabbits from the affected colony to MURADIL. The
donated rabbits were provided Laboratory Rabbit Diet 5321 (Purina) and supplemented with parenteral
vitamin E (SQ injections q 2 days X 14 days) to assess the effect of vitamin E therapy on clinical signs,
reproduction, and vitamin A and E serum and liver levels. Over a 60-day period, this diet had little effect
on serum vitamin E levels, and this finding may indicate that diet alone does not adequately correct
hypovitaminosis E. In this study, pregnancy also may have played a role in the high serum vitamin A
levels, as the females were close to kindling when samples were drawn. In dams, serum vitamin A levels
are known to increase significantly at kindling, then decrease within 1 week after kindling to preparturition levels.
Blood samples were taken before and after dietary changes and vitamin E therapy. The
parenteral administration of the vitamin E for 2 weeks lowered the serum vitamin A levels and increased
the vitamin E serum and liver levels. In conclusion, vitamin E therapy appears to be an effective
treatment for hypervitaminosis A. In addition, previous studies have shown that when high doses of
vitamin A were given to rats, rabbits, and guinea pigs, the animals rapidly became deficient in vitamin
E. The mechanism of interaction between vitamins A and E and its relevance to vitamin A toxicity are
not fully understood.
In agreement with other findings, rabbits in this study appeared to return to normal reproductive
levels 12 weeks after the RADIL diet change. The authors conclude that the administration of vitamin E
decreases the serum levels of vitamin A in rabbits with hypovitaminosis E. Vitamin E therapy appears to
be an effective treatment for hypervitaminosis A in rabbits, and it may decrease the teratogenic effects
of hypervitaminosis A during embryonic development.
Questions:
1. Why are rabbits are among the most susceptible laboratory animals to misformulated rations?
2. Where is vitamin A primarily stored in the body?
Answers:
1. Because of their rapid growth and development, heavy reproductive demands, and short
gestation period.
2. In the liver.
Vaginal and Cervical Atresia in a Cynomolgus Macaque (p 31-32)
Clinical Case Summary: A 2.5 year old, female, 2.3 kg, cynomolgus monkey ( Macaca fascicularis captive-bred, and imported from south centralChina) presented clinically with a firm, slightly mobile
mass in the caudal abdominal. Ultrasound exam revealed a fluid filled mass with a granular freefloating appearance that enlarged from ~2 cm in diameter on ultrasound to double the size within a
month. Exploratory laparotomy revealed a large caudal abdominal mass encompassing the middle of
the uterus, ovaries, rectum, and bladder. Due to extensive adhesions and severely decreased pelvic
outlet, animal was euthanized under general anesthesia. Mass was removed and fixed in formalin.
Histological evaluation identified the mass as an enlarged uterus with no neoplastic components. It
appeared grossly that the cervix and uterus had no external opening and resulted in dilation of the uterus
and fluid (blood, presumptive) build-up. There was also a lack of an opening between the cervix and
vagina. Diagnosis: vaginal and cervical atresia. Different surgical techniques have been described to
correct this anomaly in human patients, with some presented in the discussion section.
Normal epithelium histology for the cynomolgus female reproductive tract:
Vagina: stratified squamous epithelium
Cervix: columnar epithelium
Glandular portion of uterus: cuboidal to columnar epithelium
Luminal surface of uterus: pseudostratified columnar epithelium
Questions:
1.
What is the scientific name of the cynomolgus macaque?
2.
What is the CDC mandated quarantine period for NHP imported from a foreign country?
3.
What are some clinical signs that would be consistent with a diagnosis of vaginal and cervical
atresia?
4.
What would be some differential diagnoses for a fluid-filled caudal abdominal mass in a 2.5 year
old female cynomolgus macaque?
Answers:
1.
Macaca fascicularis
2.
31 days, beginningwith the importation date, whether or not the primates were held for part of
the period at another location, and this applies to all nonhuman members of the Order Primates.
3.
Amenorrhea, uterine distention, caudal abdominal mass on ultrasound and/or palpation.
4.
Congenital malformation, traumatic or inflammatory insult to the lower abdomen, neoplasia,
cyst, bacterial or protozoal infection, and parasitism.
Brine Shrimp Dispenser (page 33-34)
Over the past 15 years, the zebrafish (Brachydanio rerio) has emerged as an important animal model in
biomedical research. Areas of study using zebrafish include developmental biology, environmental
science, genetic research, and teratology. Brine shrimp are a staple food for zebrafish.
One animal facility used squeeze bottles to deliver the shrimp to the fish, but this method resulted in
physical problems (repetitive motion injuries) and physical hazards (climbing on a step stool) to
administer. The facility found that a hand-operated pressurized sprayer, like those that are used for
liquid fertilizer, could be used with a few minor modifications. The facility replayed the brass nozzle
that the sprayer came with and used a piece of PVC bubble tubing instead. The end of the plastic spout
on the sprayer was bent at approximately 8 inches from the nozzle to allow for easier placement of the
tip into the feeding hold of the fish tank lid. The facility used a plastic pippette placed in the valve
handle to hold the trigger in place, decreasing repetitive motion injuries.
The recommended feeding mixture is a liter of concentrated, newly hatched brine shrimp with 1 liter of
reverse-osmosis water. If an excessive amount of shrimp is loaded at one time, the shrimp settle, and
only the shrimp's sloughed shells are dispensed into the tanks. Gently swirling the sprayer through the
feeding assists in keeping the shrimp evenly distributed in the container.
Questions:
1. What is the genus and species of the zebrafish?
2. Name three main areas of research that zebrafish are used in.
3. Why would you want to avoid overfilling a feeding container with brine shrimp?
Answers:
1. Brachydanio rerio
2. Developmental biology, environmental science, teratology, genetics
3. Shrimp may settle to the bottom of the container and only the shrimp's sloughed shells will be
dispensed from the feeding container.
Operant Conditioning (pages 35-36)
Overview: Operant conditioning is a form of testing in which an animal learns that a response, such as
pressing a lever, results in a consequence, such as a pellet being delivered to the animal. The
consequence is also known as an unconditioned stimulus because the animal will react to it without
training. In most models, unconditioned stimuli are instinctive triggers like food or pain that can be
positive or negative, with reward (positive reinforcement) used most often. Animals may be trained to
associate consequences (rewards) with responses, and once an animal displays a learned behavior in the
absence of the reward, the behavior is termed a conditioned response. In some conditioning models,
discriminative stimuli (e.g., flashing lights) may be used to aid animals in learning. Most people are
familiar with classical conditioning, as performed by Pavlov, in which an animal produces an
unconditioned response (salivation) in response to an unconditioned stimulus (food) paired with a
conditioned stimulus (a ringing bell); eventually, the unconditioned response becomes linked with the
conditioned stimulus and the dog salivates with the ringing of the bell, despite the absence of food.
Because classical conditioning deals with a response formed between two stimuli and not between an
action and a stimulus, it is of limited use in testing memory and learning in rodents; operant
conditioning, which requires an animal to produce a response of some kind in order to receive the
stimulus, is used extensively. Operant conditioning paradigms exist for mice, rats, hamsters, gerbils and
guinea pigs, and batteries of tests built on operant conditioning tasks may be useful for the behavioral
phenotyping of genetically modified rodents. Rats appear to be more skilled than mice at complex
operant tasks.
Methods: Typical operant conditioning chambers are metal with a clear top and at least one clear
side so that animals may be observed while in the chamber; they usually have a wire-grid floor, which
may be electrified (negative stimulus), an automated food or water delivery system, and various levers
and cue lights positioned at the subject animal’s height. A typical rat-sized operant conditioning
chamber is 22 cm x 22 cm x 28 cm high cube (NOTE: look at the photo in article as it is likely to appear
on ACLAM practical exam); mouse chambers are smaller and may have smaller, more sensitive levers,
or holes for the mice to poke their noses into instead of levers. In most labs, chambers are connected to
computers with software that aids in training and the evaluation of responses. Although a lengthy
training period is required before animals may be tested for learning or memory deficits caused by brain
lesions, genetic manipulations, or the administration of drugs, one of the advantages of operant
conditioning is that once trained, each animal may serve as its own control. Operant conditioning
training commonly features use of fixed ratio (FR) or fixed interval (FI) schedules of reinforcement; FR
schedules reward the animal for every nth behavior, while FI schedules reward the animal for
performing the behavior at a certain time interval (e.g., one reward delivered per minute, no matter how
many times the lever is pressed during each minute). Animals usually exhibit the greatest number of
instances of the desired behavior when both the ratio and interval of reward is variable, and treatment
effects may be revealed by randomly varying the ratio of behaviors performed to rewards given.
Animal Welfare Considerations: Parameters for food and water restriction should be set before
the project begins, given that animals may be food- or water-restricted in order to perform adequately
for the reward presented in the operant conditioning task. Negative stimuli, if negative reinforcement is
used, should be as mild as possible to still achieve the desired result. The potential for the spread of
pathogens between subsequent animals should be kept in mind given that operant conditioning devices
are difficult/impossible to disassemble and thoroughly disinfect due to the complexity of the materials
used.
Questions:
1. What is the difference between operant conditioning and classical conditioning?
2. Despite the lengthy training period involved, what is one of the primary advantages of using operant
conditioning?
3. Under which of the following reward schedule combinations do animals usually exhibit the greatest
number of instances of the desired behavior?
a) fixed ratio and fixed interval of reward
b) fixed ratio and variable interval of reward
c) variable ratio and fixed interval of reward
d) variable ratio and variable interval of reward
4. List 3 animal welfare concerns associated with use of operant conditioning:
a)
b)
c)
Answers:
1. Classical conditioning deals with a response formed between two stimuli and not between an action
and a stimulus; operant conditioning requires an animal to produce a response of some kind in order to
receive the stimulus (reward).
2. Once trained, each operant conditioned animal may serve as its own control.
3. d) variable ratio and variable interval of reward
4. a) food- or water-restriction
b) severity of negative stimuli, if negative reinforcement is used
c) potential pathogen spread between subsequent animals using operant conditioning devices