Comparative Medicine
Volume 59, Number 1, February 2009
OVERVIEWS
Chichlowski and Hale. Effects of Helicobacter Infection on Research: The Case
for Eradication of Helicobacter from Rodent Research Colonies, pp. 10-17
Species: Rodents, primary, secondary and tertiary
Domains/Tasks: Domain 1, Task 1, Domain 3, Task 3
SUMMARY:
Biological characteristics: Helicobacter spp. are gram-negative, long, narrow, slightly
curved rods with bipolar sheathed flagella bacteria. Helicobacter spp. vary in their
morphology, growth requirements, biochemical profiles, antibiotic susceptibility, and
sequence of conserved 16S rRNA genes. Several Helicobacter spp. (H. pylori, H.
hepaticus, H. bilis, H. muridarum) are urease-positive, that is, capable of producing
ammonia to neutralize gastric acid, whereas others (for example H. ganmani, H.
rodentium, H. trogontum, H. typhlonius) are urease-negative. Production of urease
allows microorganisms to survive in the very acidic gastric environment. Most rodent
Helicobacter species are urease-negative and thus preferentially colonize the intestine.
PCR-based techniques are used for identification and detection of most Helicobacter
spp. Molecular detection of Helicobacter DNA by using PCR is rapid and sensitive to the
early phases of infection. Further enhanced sensitivity is achieved by using nested
primers. One of the most important features of the PCR assay is that it can be performed
noninvasively on fecal pellets.
Rodent host species and the site of infection: (Table 1, page 11)





Hamster: H. aurati (stomach, intestine), H. cinaedi (intestine), H. cholecystus,
(gallbladder), H. mesocricetorum (intestine)
Mastomys natalensis: H. mastomyrinus (liver)
Gerbil: H. hepaticus ( intestine)
Mouse: H. bilis ( Intestine), H. ganmani, ( intestine), H. hepaticus ( intestine), H.
muridarium ( intestine, stomach), H. pylori , (experimental infections, stomach), H.
rodentium (intestine), H. trogontum (intestine), H. typhlonius (intestine)
Rat: H. bilis (intestine), H. ganmani (intestine), H. hepaticus (intestine), H.
muridarium (intestine, stomach), H. rodentium (intestine), H. trogontum (intestine), H.
typhlonius (intestine).
Transmission: Fecal-oral spread is the primary route of natural infection. The infection
is persistent with long-term shedding of the bacterial in feces. H. bilis can result in
delayed and inconsistent transmission to sentinel mice.
Eradication: Restocking with helicobacter-free mice, embryo transfer, cross-fostering,
antibiotic therapy. The efficiency of these methods is variable depending on species,
strain of rodent or helicobacter and experimental conditions.
Infection: The first report of pathogenic intestinal Helicobacter infections was in 1994.
Pathology of the most helicobacter species is host-dependent and infection may be
subclinical. Overall, Helicobacter organisms in mice have been linked to inflammatory
bowel disease and breast, liver, gastric and colon cancers.
Colonic inflammation and neoplasia: Helicobacter hepaticus or H. bilis have been used
most frequently to model microbial triggers of intestinal inflammation (typhlitis, proctitis,
colitis and rectal prolapse), because these species were the first to be linked to the
development of inflammatory bowel disease and inflammation associated neoplasia. H.
rodentium has been described to be nonpathogenic in adult wild-type mice, but this
species did enhance cytokine production in mice also infected with H. hepaticus. The
signaling pathway involving IL10 and IL6 is essential in maintaining epithelial
homeostasis and modulating epithelial invasion during bacterially driven inflammatory
diseases.
Gastric inflammation and inflammation-associated cancer: H. pylori does not naturally
infect mice but has been used to generate experimental mouse model of gastric
inflammation and cancer. H. pylori-infected INS-GAS mice (overexpressing amidated
gastrin) develop gastrointestinal intraepithelial neoplasia or gastric cancer at 28 wk after
infection, accompanied by inflammation, loss of parietal and chief cells, and hypertrophy
of foveolar glands. When H. pylori antimicrobial eradication therapy was instituted at 8
wk after infection, the risk of gastrointestinal intraepithelial neoplasia was reduced to a
level comparable to that of uninfected mice. H. muridarum which naturally colonizes the
ileum and cecum has a potential to colonize the stomach and elicit gastritis of older
animals.
Hepatocarcinoma and hepatic inflammation: H. hepaticus originally was discovered as
the causative agent for the development of chronic hepatitis and hepatocellular cancer in
A/JCr mice. In addition, H. hepaticus reportedly produces hepatocarcinoma with a male
bias. Tumors usually arise after 18 months, whereas after 1 year, susceptible mice
exhibit chronic hepatitis. Chronic active hepatitis was prevalent in other strains of mice
as well, including C3H/HeNCr, SJL/NCr, BALB/cAnNCr, and SCID/NCr.
Other
Helicobacter spp. associated with the rodent models of liver cancer and hepatobiliary
system are H. bilis, H. mastomyrinus, and H. cholecystus. In another study, coinfection
with both H. hepaticus and H. rodentium and monoinfection with H. bilis promoted
cholesterol gallstone formation in C57BL mice.
Mouse models of Helicobacter infection associated with gastrointestinal and liver cancer
(Table 2, p12).
 Gastric adenocarcinoma: H. pylori, H. felis (I NS-GAS mice)
 Hepatocellular carcinoma: H. hepaticus, (A/JCr mice)
 Colon carcinoma: H. hepaticus (IL10−/− (C57BL, IL10−/− Balb/c, A/JCr, Rag2−/−,
Mdr1a−/−), H. typhlonius, H. rodentium (IL10−/− (C57BL), H. hepaticus, H. bilis
(Smad3 and Mdr1a−/−).
Breast cancer: Inflammation induced in the gut by proinflammatory microbial infection
possibly could have systemic effects, which would then influence carcinogenic events in
distant organs. The H. hepaticus infection of C57BL/6 ApcMin/+ andRag2−/−ApcMin/+
mice enhances mammary carcinoma by a TNF-dependent mechanism.
Effects on reproduction: Thus far, the information regarding effects of Helicobacter spp.
on reproduction is scarce. H. typhlonius has been detected by PCR in the sex organs 12 weeks post-exposure and then, it was cleared from non-target tissues. H. hepaticus
has been cultured from fetal viscera of 2 of 11 pups sampled late in gestation from
infected SCID/NCr females, suggesting transplacental infection of H. hepaticus.
Experimental infection with H. pylori influenced murine pregnancy by increasing the
number of fetal resorptions and producing decreased fetal weights when compared with
those of noninfected CD1 mice. The reproductive success of C57BL/6 IL10−/− female
mice experimentally infected with H. typhlonius or H. rodentium (or both organisms) was
decreased compared with that of noninfected mice. Pregnancy rates and the number of
pups surviving to weaning were decreased in infected dams. Treatment with a 4-drug
anti-Helicobacter therapy eliminated PCR-detectable excretion of Helicobacter DNA,
improved fecundity, and enhanced survival of pups born to previously infected dams.
Effect on Immunity: The persistent, chronic gastric infection due to Helicobacter can
inhibit oral tolerance and result in food allergy, as well as with other allergic diseases like
chronic urticaria, atopic dermatitis, and hereditary angioneurotic edema.
QUESTIONS:
1. Which of the following is INCORRECT about Helicobacter?
a. Gram negative
b. All are urease positive
c. Are motile
d. Rod shape
2. All of the following are hamster Helicobacter EXCEPT:
a. H. aurati
b. H. cinaedi
c. H. bilis
d. H. mesocricetorum
3. Which of the following methods can be used to eradicate Helicobacter?
a. Cross-fostering
b. Embryo transfer
c. Antibiotics therapy
d. All of the above
4. The Helicobacter infection can be seen:
a. Gastrointestinal system
b. Reproductive system
c. Immune system
d. All of the above
5. Which of the following mice is the H. hepaticus/hepatocarcinomal model?
a. A/JCr mice
b. Balb/c mice
c. C3H mice
d. C57BL/6
6. T/F. H. pylori naturally infect mice.
7. Which of the following is a rapid, sensitive, noninvasive method for Helicobacter
detection?
a. ELISA
b. Fecal PCR
c. IFA
d. Fecal culture
8. T/F. There is an evidence of trans-placental infection in Helicobacter- infected
SCID/NCr females.
9. T/F. The persistent, chronic gastric infection due to Helicobacter can inhibit oral
tolerance and result in food allergy
10. Which of the following cytokine null mice are prone to develop helicobacter related
colon carcinoma?
a. IL16-/b. Il10-/c. Il5-/d. Il35-/ANSWERS:
1. b
2. c
3. d
4. d
5. a
6. F (experimentally)
7. b
8. T
9. T
10. b
Novak and Meyer. Alopecia: Possible Causes and Treatments, Particularly in
Captive Nonhuman Primates, pp. 18-26
Domain: 1
Primary, secondary and tertiary non-human primates.
SUMMARY: This paper is a review of possible causes and treatments of alopecia in
nonhuman primates (NHP).
The hair cycle:
Stages of hair growth include anagen (growth) phase, catagen (degradation) phase, and
telogen (resting) phase.
 Telogen effluvium – a prolongation of the telogen phase.
 Alopecia areata universalis – an autoimmune disease caused by a peri- or
intrafollicular infiltration of T-lymphocytes and macrophages. Results in defective
anagen-stage hair shafts and miniaturization of hair follicles.
 Androgenetic alopecia (male pattern baldness) – dysfunctional anagen-stage and
miniaturization of the follicles presumably caused by the effects of
dihydrotestosterone (DHT) on genetically susceptible hair follicles.
Causes of alopecia:
1) Naturally occurring hair loss
Seasonal Variation: Many mammals undergo seasonal change in hair production
associated with variations in day length and alterations in certain hormone levels. In
temperate regions, long day length is associated with lower levels of melanin and
prolaction and shorter hair (summer coat) and the opposite is seen for short day length
(high levels of melanin, prolactin and winter coat). While little is known about seasonal
hair coat variation in the NHP, prolactin’s role in seasonal molting patterns has been well
established for Djungarian hamsters, goats, sheep and deer. Furthermore, the hair
regrowth cycle is delayed in mice injected with prolactin.
Seasonal changes have been reported for some species of monkeys. In free-ranging
vervet monkeys, seasonal hair loss peaks between November and January and is more
obvious in subordinate monkeys. Similar changes have been observed in Rhesus
monkeys even when maintained under artificial light and apparently affects females
more than males. Considerable individual variation (from no hair loss to extreme hair
loss) is unexplained, but it is thought that sex and dominance rank may explain some of
the variance.
Aging: Hair loss has been clearly associated with the aging process. As humans age
there is a reduction in overall hair density and decreased growth rate of anagen hairs. In
two studies, older Rhesus monkeys showed skin abnormalities and thinning hair. These
changes were not associated with circulating levels of estradiol, thyroid stimulating
hormone, triiodothyronine, thyroxine, or cortisol. In a colony of squirrel monkeys, hair
loss in female monkeys was associated with older age and increased parity. Hair loss
was unrelated to body weight, serum chemistry values (hemoglobin, serum glucose,
BUN), or free thyroxine. Monkeys with hair loss had more telogen hairs suggesting an
alteration of the hair cycle consistent with chronic telogen effluvium.
2) Nutritional and hormonal imbalance
Nutritional Factors: Much of the focus has been on zinc, vitamin D, and protein.
Moderate to severe zinc deficiencies have been documented to cause alopecia in
rhesus and bonnet macaques, marmosets, talapoin monkeys, and children. Addition of
zinc to the food or drinking water results in reversal of the effects of zinc deficiency on
hair and skin. Exposure to toxic levels of zinc has also been associated with alopecia,
often accompanied by anemia and achromatricia (white monkey syndrome). Removal
from the source of zinc leads to reversal of clinical signs in most cases.
Most of the evidence for vitamin D playing a role in hair loss comes from experiments
with mice lacking the vitamin D receptor (VDR) and humans with VDR mutations.
Knockout of VDR in mice results in a cluster of clinical signs (hypercalcemia,
hyperparathyroidism, and rickets) that includes alopecia. Alopecia in these animals is
apparently caused by inadequate response of hair follicles to anagen initiation. The
actual role of vitamin d in regulating hair growth remains elusive and little is known about
the role of the VDR in hair loss in the NHP.
In Western lowland gorillas, a syndrome of alopecia, weight loss, and hypoalbuminemia
was traced to protein deficiency. Likewise, young baboons experimentally exposed to a
protein-deficient diet experienced hair loss. Alopecia was also linked to folacin deficiency
in squirrel monkeys.
Two other nutritional factors, iron deficiency and vitamin A levels, have been associated
with alopecia, but have not been studied in the NHP. Iron deficiency can result in
alopecia in humans, but low iron stores can’t account for all forms of hair loss. There is a
significant association of iron levels and hair loss in cases of androgenetic alopecia and
alopecia areata, but not in cases of alopecia universalis or telogen effluvium. Rats fed a
diet deficient in vitamin A developed clinical signs including alopecia and anemia.
Hypervitaminosis A in calves causes emaciation and alopecia. Vitamin A levels
designated for Old World monkeys have recently come under scrutiny as possibly being
too high.
Hormonal Imbalances and Changes: The best known example would be androgenetic
alopecia (male pattern baldness) in humans which is caused by conversion of
androgens to DHT in genetically vulnerable hair follicles. There is apparently no NHP
homologue to this condition. However, the stumptailed macaque has been used as a
model for studying the effects of minoxidil on hair growth. However, levels of other
hormones can be linked to alopecia in humans and animals. The authors focused on
hypothyroidism, tumors of the pituitary or adrenals causing hyperadrenocorticism, and
pregnancy.
Primary hypothyroidism in dogs gives rise to alopecia in approx. 25% of cases and is
apparently the result of telogen effluvium. Treatment with thyroxine generally induces
hair regrowth and improved skin. Hypothyroid-related hair loss has been reported for a
gorilla, an orangutan, and a chimpanzee. Treatment with thyroid hormone reversed all
cases except the chimpanzee.
Hyperadrenocorticism (Cushing syndrome) has been studied extensively in humans and
dogs. Caused by excessive therapeutic glucocorticoids (iatrogenic) or by excessive
glucocorticoid production in response to excessive adrenocorticotrophic hormone
secretion by the pituitary (usually due to a tumor in the pars distalis region). Hair loss in
a Japanese macaque was attributed to hyperadrenocorticism on the basis of changes in
skin morphology and hematologic parameters.
Pregnancy is associated with temporary hair loss (telogen effluvium) in as many as 30 –
50% of postpartum women. Both pregnancy and lactation have been associated with
telogen effluvium in some breeds of dog. A recent report links hair loss to pregnancy in
female rhesus macaques. Ten females showed substantial hair loss unrelated to
seasonal influences. Hair growth resumed after parturition and full hair coats were
restored within two months.
3) Immunologic and genetic factors
Autoimmune Hair Disorders: Alopecia areata is an immunologic disorder that affects
men and women between 20 and 40 years of age. The disease first manifests as rapid
patchy hair loss possibly progressing to hair loss over the total scalp (alopecia areata
totalis). Rarely, hair loss over the entire body (alopecia areata universalis) is seen.
Alopecia areata is caused by a dysfunctional immune response, in which the hair follicle
is invaded by lymphocytes (primarily CD4-positive T lymphocytes) and macrophages
that target specific antigens of the anagen hair follicle. A similar immune mediated
disease has been reported in rhesus monkeys and chimpanzees.
Lichen planus is an inflammatory disorder affecting the hair follicles resulting in hair loss.
Lupus erythematosus is associated with non-scarring alopecia in 40% of cases, but in a
small percentage of cases (14%), a scarring alopecia can develop secondary to discoid
lesions. Lichen planus has not been reported in NHP’s. A systemic lupus erythamatosis-
like syndrome has been reported in a rhesus macaque and a similar syndrome was seen
in cynomolgus monkeys maintained on a 40% alfalfa sprout diet.
Mutations of the Hairless Gene: Hair loss has been linked to the hairless (hr) gene in
mice and humans. Atrichia with popular lesions is a genetic disorder in which the
individual is born with hair, but once shed it is not replaced. Mutations of hr can also
cause alopecia in rhesus macaques.
Inflammatory hair loss. A variety of etiologies including bacterial (primary or secondary),
parasitic infections, and allergic processes can induce hair loss.
Bacterial Infections: Staphylococcal bacteria can produce a scarring alopecia (folliculitis
decalvins) in middle-aged humans. Staphylococcal infections have also been shown to
cause skin lesions and alopecia in sheep, horses, and dogs. Induction of chronic
Salmonellosis in guinea pigs was associated with hair loss. There is no evidence that
bacterial infections play a leading role in hair loss in NHP’s.
Bacterial skin infections can develop as a secondary consequence of other diseases as
is the case with primates with diabetes. Skin infections were noted in mandrills with a
syndrome similar to type 2 diabetes.
Parasitic Infections: Fungal infections cause hair loss primarily associated with pruritus.
Tinea capitis is a disorder that causes scalp eruptions in children and adults and typically
is caused by Microsporum canis or Trichophyton tonsurans. Fungal infections (usually
M. canis) have been reported in rhesus monkeys, chimpanzees, and gibbons.
Sarcoptic mange is rare in NHP’s and is limited to those held in seminatural settings.
The few cases reported were effectively treated with Ivermectin.
Atopic Dermatitis: A chronic skin condition apparently precipitated by exposure to
allergens. Invariably associated with pruritus which sometimes results in oozing sores
and secondary bacterial infections. Allergen specific Ig-E tests were used to screen
potential allergens. Case one was identified as a possible latex allergy. A change to vinyl
gloves led to an improvement of the skin condition of this monkey. As is often the case in
other species, the allergen was not identified in the second monkey.
Despite diagnostic difficulties, several treatments have proven somewhat effective for
treating atopic dermatitis. Essential fatty acids have few side effects and effectively
reduce inflammation, skin lesions, and pruritus in humans and dogs. Administration of a
fatty acid (dihomo-gamma-linolenic acid) to NC/Nga mice that developed spontaneous
atopic dermatitis resulted in a dose-dependent reduction in the severity of the lesions.
Glucocorticoids may be the treatment of choice for some cases despite the possible side
effects. Cyclosporine may be as effective as glucocorticoids in eliminating atopic
dermatitis. Cyclosporine effectively reduces skin lesions and pruritus and produced hair
growth in dogs, cats, and the previously discussed rhesus monkey for which no specific
allergen could be identified.
4) Psychological factors and hair loss
Self-Induced Hair Loss: Monkeys and apes can develop the abnormal behavior of
pulling their own hair out. This disorder shares some similarities to the human disorder
trichotillomania. Hair pulling in NHP’s may be brought about by environmental stressors
or by impoverished captive environments. Other medical conditions can lead to hair
pulling. In a study of 21 cats previously diagnosed with psychogenic alopecia
(compulsive licking or grooming), 16 were found to have an underlying medical disorder
(primarily atopic dermatitis). This case underscores the possibility that psychogenic hair
pulling may be over diagnosed.
No treatment for hair pulling reliably reduces or eliminates this behavior. Little evidence
suggests that enrichment routinely alleviates this behavior, exercise cages or social
housing may provide some benefit.
Hair Pulling by Others: NHP’s living in social groups may be vulnerable to having their
hair pulled by others. Reduction was noted after monkeys were given a foraging task.
The behavior is seen most commonly in pair-housed NHP’s and is usually not alleviated
by changing partners. The only known successful treatment is separating of animals.
Stress: The notion that stress is associated with alopecia areata is in large part due to
conclusions reached from uncontrolled studies and anecdotal evidence. Alopecia in
captive macaques has been attributed to stress. However, little evidence supports this
conclusion. If alopecia is due to stress, pharmacotherapy might be of benefit. Cats with
verified cases of psychogenic alopecia responded favorably to antidepressants or
anxiolytic drug therapy. Such therapy has not been reported successful in NHP’s.
Managing alopecia in laboratory primates: The authors suggest a stepwise strategy for
evaluating hair loss in NHP’s. Step 1 is assessment and is best accomplished as part of
regular health exams. Careful evaluation of hair loss and skin surface including possible
photodocumentation for comparison from one health exam to the next. Step 2 is to
determine if immediate medical attention is required. Step 3 involves skin biopsy to
evaluate inflammation and to rule out bacterial, fungal, or parasitic infections. Biopsy
also provides info on hair follicle condition. All information acquired in this step should be
supplemented with behavioral observations. Step 4 includes obtaining blood samples for
routine screenings and evaluation of relevant hormone levels. Finally, all information is
used to narrow the possibilities and consider treatment options. The authors present
three scenarios involving the most common causes of hair loss in NHP’s.
Scenario 1. Scratching plus reddened skin with bacterial, fungal and parasitic infection
ruled out most probable diagnosis is atopic dermatitis. If allergen is not identifiable or
can’t be removed, oral steroids or cyclosporine should be considered.
Scenario 2. Self-induced alopecia (hair pulling) with bacterial, fungal and parasitic
infection ruled out most probable diagnosis is psychogenic or stress-induced hair loss. If
the stressor can’t be identified or removed then two strategies may be employed. First
option is an enrichment strategy in which the animal is given greater stimulation. The
second option is a pharmacotherapeutic strategy employing anxiolytic drugs such as
diazepam. There is no evidence that either strategy will be effective, and compliance
with experimental protocol may prohibit use of these strategies.
Scenario 3. If skin biopsies are normal and no behavioral reason can be found for hair
loss then three factors are the most common causes for hair loss. These are; seasonal
variation, reproduction, and age. If none of these factors are related to the hair loss or if
hair loss does not resolve, consider hormonal imbalance or nutritional deficiencies.
QUESTIONS:
1. Name the condition characterized by a prolongation of the telogen phase.
2. Name the condition characterized as an autoimmune disease caused by a peri- or
intrafollicular infiltration of T-lymphocytes and macrophages. Results in defective
anagen-stage hair shafts and miniaturization of hair follicles.
3. Name the condition characterized as a dysfunctional anagen-stage and
miniaturization of the follicles presumably caused by the effects of
dihydrotestosterone on genetically susceptible hair follicles.
ANSWERS:
1. Telogen effluvium
2. Alopecia areata.
3. Androgenetic alopecia (male pattern baldness).
ORIGINAL RESEARCH
Mouse Models
Kelmenson et al. Kinetics of Transmission, Infectivity, and Genome Stability of
Two Novel Mouse Norovirus Isolates in Breeding Mice, pp. 27-36
Species: mouse (primary)
Domain 1: Management of spontaneous and experimentally induced disease and
conditions ; Task 1- Prevent spontaneous Disease
Background and Methods: The purpose of the present study was to extend the current
knowledge of murine norovirus (MNV) by using 2 isolates of the virus in mouse strains
that have not been previously used as infection models for MNV. Two novel MNV wild
type isolates were used to examine the kinetics of transmission and tissue distribution in
breeding units of NOD.scid and backcrossed NOD.scid x NOD/ShiLtj (N1) mice (model
for type 1 diabetes). The authors examined the natural virus transmission from infected
breeders to offspring, kinetics of infection within litters of infected breeding mice, and the
pathogenesis of infection in breeding colonies of mice. In addition, they examined the
effect of virus passage from parents to offspring on genomic stability of these 2 viral
isolates. Exposure of offspring of immunodeficient mice and immunocompetent mice to
the 2 different isolates of MNV resulted in different patterns of virus transmission,
susceptibility to infection and kinetics of infection as shown by the progressive spread of
virus within litters and in intestinal and extraintestinal tissues. MNV was shed
persistently in the feces of all mice tested regardless of immune status, and viral
progeny isolated from offspring mice contained genome sequence differences from the
parent virus in the Orf2 gene, an area of the MNV genome known to be susceptible to
mutations.
Results/Discussion: Transmission of both the MNV5 and MNV6 wild type isolates used
in this study readily occurred from infected female breeders to cohoused uninfected
male breeders and the offspring of these matings. However, viral transmission was not
detected in cages of control animals, even when filter tops were not provided. The
finding suggests that appropriate husbandry practices are sufficient to prevent cage-tocage transmission of MNV. Among mice infected with MNV5, offspring were not positive
for fecal or tissue virus as determined by RT-PCR until approximately 3 weeks
postpartum, suggesting that the offspring become infected via coprophagy. Therefore,
ingesting virus-contaminated feces from infected parents may be a primary means of
spreading the virus within a cage of mice. There was a significant difference in
susceptibility to the virus occurred between the mouse strains used in this study,
suggesting that MNV transmission is dependent on the mouse strain exposed to the
virus. All of the fresh fecal pellets collected from pups exposed to MNV6 (and MNV5)
after detection of MNV-positive fecal pellets contained live virus. Thus, the pups were
continuously seeding their cage with live virus and perpetuating environmental exposure
to virus. The authors also found that the distribution of MNV5 and MNV6 in the tissues
of offspring mice from virus-exposed litters was similar between the mouse strains.
From the perspective of molecular diagnostic testing, the ileum, cecum, and colon were
the tissues infected most consistently in mice exposed to MNV5 or MNV6 and are
therefore most likely to provide a positive diagnosis when mouse colonies are infected.
QUESTIONS:
1. What tissues were used for molecular diagnostic testing and detection of MNV5 and
MNV6?
a. Liver, Kidney, and colon
b. Stomach, Lung, and Bladder
c. Ileum, cecum, and colon
d. Tongue, Lung, and Heart
2. T or F: The Fresh fecal collection from pups exposed to MNV6 was not positive for
live virus.
ANSWERS
1. C: Ileum, cecum, and colon
2. False: Pups exposed to MNV6 fecal pellets contained live virus
Stocking et al. Use of Low-Molecular-Weight Heparin to Decrease Mortality in
Mice after Intracardiac Injection of Tumor Cells, pp. 37-45
Domain 3 - Research Task 3: Design and Conduct Research
Species: Primary (Mice)
SUMMARY: The investigators sought to show that pre-treatment with low-molecularweight heparin (LMWH) blocks the hypercoagulable state that occurs following
intracardiac injection of tumor cells in nude mice. This model in nude mice is used to
mimic the metastasis of human tumors to bone. Tumor cells used in this study were
nonsmall cell lung cancer cell lines, which were transformed to express luciferase and
GFP by using a lentiviral vector. Female CrTac:NCr-Foxn1nu mice (4-8 wk of age) were
anesthetized with ketamine/xylazine IP and placed in dorsal recumbency. Twenty mice
received 10 mg/kg LMWH (enoxaparin) IV 10 minutes before receiving the intracardiac
injection of tumor cells. Another 20 mice received no LMWH treatment prior to receiving
intracardiac injection of tumor cells. To assess accuracy of intracardiac injection, mice
were injected IP with D-luciferin and then imaged 4 minutes later using a Xenogen
imager. Results: all 20 enoxaparin-treated mice survived to allow successful imaging
following intracardiac injections. All mice not treated with LMWH died within 5-10
minutes after intracardiac injection, and these mice had clear histological evidence of
hypercoagulability. The LMWH-treated group went on to develop bony metastasis as
expected.
QUESTIONS:
1. T or F. Enoxaparin is a form of low-molecular-weight heparin.
2. T or F. Intracardiac injection of tumor cells in nude mice is used as model of
metastasis of human tumors to bone.
3. T or F. Tumor cells have procoagulant activity.
4. T or F. Non-enoxaparin treated mice showed no histological evidence of
hypercoagulability.
5. T or F. Bioluminescent imaging was used in this study to confirm successful
intracardiac injection of tumor cells.
ANSWERS:
1. T
2. T
3. T
4. F
5. T
Rat Models
Muhammad et al. Effects of 4-Vinylcyclohexene Diepoxide on Peripubertal and
Adult Sprague-Dawley Rats: Ovarian, Clinical, and Pathologic Outcomes, pp. 4659
Primary Species - Rat
SUMMARY: Ovariectomized animals are poor models for human menopause because
they do not model the hormonal changes associated with the menopausal transition and
postmenopausal period. A better model is daily IP injections of VCD, which causes
selective destruction of primordial and primary follicles and gradual onset of ovarian
failure. Previous studies have used young mice and prepubertal Fisher 344 rats for this
model. These authors wanted to see if the VCD model could induce similar changes in
adult Sprague Dawley rats, since older animals are more appropriate for studying the
combined effects of aging and menopause, and SD rats are often used for age-related
studies.
Two preliminary studies were done. The first used the IM route to see if this might
provide an alternative to IP injection.
 When given IM at a dose of 160 mg/kg for 12-13 days, some rats became lethargic
and ataxic and were euthanized.
 Also, at 80 mg/kg, muscle inflammation was seen at the injection site
microscopically, even though no adverse clinical outcomes were noted
 Both 80 and 160 mg/kg caused destruction of primordial and primary follicles equally
A second preliminary study was done comparing dosages of 80 and 160 mg/kg IP for up
to 30 days.
 Some rats treated with 160 mg/kg became lethargic and dyspneic after 10 to 13 d
and were euthanized.
 Rats treated with 80 mg/kg completed the 30 day trial.
 IP injections resulted in similar pattern of decrease in follicle number as with IM
 80 mg/kg daily for 30 days reduced primordial and primary follicles by 80%, and a
reduction in number of secondary follicles was seen cf controls
 160 mg/kg daily for 10-13 days reduced primordial and primary follicles by 60%, but
there were more secondary follicles cf controls
The main study examined doses of 40 and 80 mg/kg IP for 30 days in both adult and
prepubertal rats.
 In both age groups, the effects of the treatments were significantly different from
each other
 For secondary follicles, only the 80 mg/kg dose differed from controls; both dosage
groups and the control showed a decrease in secondary follicles.
The authors determined that, when given similar doses, the magnitude of VCD effect on
primordial and primary follicles is not affected by age.
 After 30 daily injections of VCD at 80 mg/kg, the number of primordial follicles was
reduced by 85-90% in adults and 81% in prepubertals
 Untreated adults also had a reduction in follicles, with almost 2/3 fewer follicles
present than the young rats
 Also, adult rats treated with the lower dose had similar follicle numbers post
treatment to young rats given the high dose.
 It may be possible to use a lower dose of VCD in adult SD rats to achieve ovarian
failure
 Although 160 mg/kg is often used to cause ovarian failure in mice, the same dose in
adult SD rats caused almost 100% loss of primordial follicles in only 10 to 13 days,
however some rats became clinically affected.
 No persistent adverse effects have been seen on young rats treated with 80 mg/kg
for up to 30 days
 VCD did not affect the liver in any negative way other than to cause an increase in its
weight, likely due to induction of metabolic enzymes and increased hepatic proteins
 Adult rats given the higher dose developed a neutrophilia, likely due to inflammation
at the injection site.
 Increase in BUN and creatinine also occurred in 4 adult rats given the higher dose,
and these rats developed adverse clinical signs. VCD is a contact irritant to tissues
 The VCD did not cause abnormal pathology in any tissues examined, other than an
inflammatory reaction at the site of injection, which was greater in the adults than in
the young, and was most severe with the highest dose used
Conclusion: As with young mice and F344 rats, VCD effectively destroys primordial and
primary follicles in adult as well as prepubertal SD rats, suggesting that the adult VCDtreated SD rat is suitable for modeling menopause in women. Adult rats were more
sensitive than young to the irritant effects of VCD, and the use of a lower dose (i.e. 40 or
60 mg/kg) for longer periods, different vehicles or more dilute mixtures may be
warranted.
QUESTIONS:
1. Which one of the following represents the most common adverse effect of VCD given
IP in rats?
a. Hepatic necrosis
b. Thymic destruction
c. Peritonitis
d. Gastric ulceration
2. What is the explanation for adult rats treated with 40 mg/kg VCD having similar
follicle numbers after treatment to young rats given 80 mg/kg?
3. T/F: Adult rats were less sensitive than young to the irritant effects of VCD.
ANSWERS:
1. c. VCD is a contact irritant to tissues
2. Adult rats will undergo a normal age-related reduction in follicles, as was seen in the
adult controls in this study.
3. F. Adult rats were more sensitive than young rats to the irritant effects of VCD.
Curtin et al. Evaluation of Buprenorphine in a Postoperative Pain Model in Rats,
pp. 60-71
SUMMARY: Buprenorphine was evaluated as a postoperative pain model in rats. The
study had 2 goals. The first was to establish the minimum dose of buprenorphine
needed to relieve acute postoperative pain effectively in rats. The second goal was to
evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is,
rebound hyperalgesia and allodynia) or create long term changes (that is, sensitization
or tolerance to opiates). Rats received surgery (paw incision under isoflurane
anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively
with 1 of several doses of subcutaneous buprenorphine. Buprenorphine at 0.05mg/kg
SQ was determined to be the most effective; this dose induced isoalgesia during the
acute postoperative period and the longest period of pain relief, and it did not induce
long term changes in opioid sensitivity in 2 functional measures of the opioid system.
QUESTIONS:
1. What is the typical recommended dose range of buprenorphine in rats?
2. What are the side effects encountered with buprenorphine administration in rats?
3. What is central sensitization?
4. Define allodynia?
5. In this study what test was used to measure pain threshold at 1, 4, 24, and 72 hrs
after surgery?
a. Tail flick
b. Hot plate
c. Paw pressure algesiometric
d. Von Frey
6. What was used to measure primary and secondary allodynia?
7. Buprenorphine reduced allodynia in a dose-dependent manner at 1 and 4 hrs, but
had no effect at 24 and 72 hrs.
ANSWERS:
1. 0.02 to 0.05mg/kg
2. Respiratory depression, nausea, GI distress, pica
3. Central sensitization is a well studies neural adaptation expressed in the brain and
spinal cord and induced by nociceptive stimulation that manifests as hyperalgesia
and allodynia
4. Allodynia is the appearance of pain-like responses to nonnoxious tactile stimuli
5. c
6. Nylon filaments
7. T
Canine Models
Kang et al. Three-Dimensional Time-of-Flight Magnetic Resonance Angiography
of Intracranial Vessels in a Canine Model of Ischemic Stroke with Permanent
Occlusion of the Middle Cerebral Artery, pp. 72-77
Species: Primary (Dog)
Domain 3: Research, K2, research methods and equipment
SUMMARY: This study evaluated the efficacy of a type of magnetic resonance
angiography (MRA) to validate an ischemic stroke model used in the study of human
neurological disease. Nonrodent species are frequently the preferred model in certain
neurological studies as they can tolerate frequent blood collections and complex
physiologic monitoring, have brains structurally and functionally similar to humans, and
the larger brain size can provide for higher imaging resolution. Dogs were used in this
study and were imaged before the surgical occlusion, at 3 days and at 10 days after the
surgery. All dogs were euthanized at completion of the study, and necropsy performed
to confirm obstruction of the cerebral arterial blood supply.
This study used the middle cerebral artery occlusion (MCAO) to induce focal cerebral
ischemia. For the surgical procedure, a cervical incision and blunt dissection were done
to expose the carotid artery at the level of its bifurcation. An embolus consisting of 7mm silicone attached to the end of a silk suture was then flushed with physiologic saline
into the internal carotid artery to the level of the middle carotid artery.
The angiography used was the 3-dimensional time-of-flight magnetic resonance imaging
(TOF-MRA). This method of imaging is based on the motion of water protons to detect
contrast between stationary tissue and flowing blood, as opposed to contrast enhanced
MRA (CE-MRA) which images the IV injection of the contrast passage through the
arterial system. The interpretations of the angiograms were done by both a neurologist
and a neuroradiologist, both of whom had no knowledge of the dog’s condition.
In three of the dogs, complete occlusion of the distal internal and middle cerebral
arteries was achieved. Neurologic signs related to these lesions included altered
mentation, hemianopsia, hemiparesis, head turning, circling and perceptual deficits.
Some of these neurological deficits partially improved after the seventh day.
Angiograms were obtained at the third day post MCAO and showed an absence of blood
flow at the occluded MCA, ipsilateral circle of Willis, and the internal carotid artery.
Obstruction of the MCA was confirmed at necropsy.
TOF-MRA on the other two dogs showed a reduced flow signal in the proximal portion of
the MCA, opacification of the distal branches and a delay in flow signal. These results
pointed to an incomplete MCA occlusion.
The authors conclude that embolus
placement could not be confirmed immediately after surgery due to transportation
issues. For a more effective stroke model, the proper placement of the occlusive device
needs to be ascertained in time to afford possible correction. This was not possible in
this study.
The authors conclude that the TOF-MRA did not afford an accurate picture of the degree
of vascular obstruction and also was unable to detect collateral vasculature. Because
TOF-MRA can only accurately detect fast moving blood flow, it can give an
overestimation of the amount of vascular obstruction. Overall, the use of TOF-MRA
along with other MR sequences might further increase the utility of the ischemia model in
the dogs. And due to some disadvantages of the TOF-MRA, other imaging methods,
such as the CE-MRA, need to be assessed.
QUESTIONS:
1. For their study, cerebral ischemia was accomplished by occlusion of which artery?
a. Internal carotid
b. External carotid
c. Middle cerebral
2. Venous structures are difficult to detect using TOF-MRA due to their low blood flow
velocity. T or F
3. Which of the following is not true regarding the advantages of the TOF-MRA?
a. It is noninvasive
b. It can consistently and accurately estimate the degree of vascular obstruction
c. No contrast media is needed
d. It can be repeated many times
4. The animal stroke model used in this experimental model is what type of cerebral
ischemia?
a. Focal
b. Multifocal
c. Hemispheric
d. Global
ANSWERS:
1. c
2. T
3. b
4. a
Ruben et al. Refinement of Canine Pancreatitis Model: Inducing Pancreatitis by
Using Endoscopic Retrograde Cholangiopancreatography, pp. 78-82
Species: Dog
SUMMARY: Over the subsequent 150 y, multiple species and multiple techniques have
been used in the induction and treatment of pancreatitis.
Nonhuman primates are the ideal model but are expensive. Dogs are used more often,
for various reasons. The canine pancreas closely mimics the human pancreas in size,
facilitating manipulations. The canine pancreas is freely mobile, suspended in the
duodenal mesentery, and both the major and minor ducts enter the duodenum
separately from the bile duct. Other species that have been used include mice, rats,
rabbits, pigs, possums, and cats.
In addition to a variety of species, numerous techniques have been used to induce acute
pancreatitis. Some of the noninvasive methods include administration of caerulein,
alcohol, or L-arginine and feeding a choline-deficient diet. Invasive methods include
closed duodenal loop, biliopancreatic duct ligation, pancreatic duct infusion, and
pancreatic vascular ligation. Each method has its own advantages and disadvantages,
but all result in clinical signs of pancreatitis (abdominal pain, vomiting, lethargy, and
others). Authors in this paper describe a new method of inducing pancreatitis that does
not lead to overt clinical signs. In humans, endoscopic retrograde
cholangiopancreatography (ERCP) is a procedure that is performed to help diagnose
various pancreatic and biliary diseases. Postprocedural acute pancreatitis is 1
complication of ERCP. The exact cause of postERCP pancreatitis is unclear.
Given the prevalence of postERCP pancreatitis, clinical research efforts have focused
on various methods of prevention, such as types of contrast used and pharmacologic
agents used as prophylaxis before or during the procedure. To further investigate
postERCP pancreatitis, authors performed a pilot study to determine whether ERCP
could be performed in the dog and whether pancreatitis could be induced through
several different manipulations.
All 8 dogs used in this study developed pancreatitis, and 7 of the 8 had no clinical signs.
During the course of this project, all 8 dogs developed biochemical and histological
evidence of mild to severe pancreatitis, with only 1 dog showing any of the classic
clinical signs of severe abdominal pain, vomiting, inappetance, and lethargy.
The initial goal of this study was to determine whether ERCP could be performed in dogs
and whether pancreatitis could be induced by using this method. These goals were
achieved. Overall, ERCP-induced pancreatitis has the potential to be a reliable,
reproducible animal model of acute pancreatitis without potentially severe procedureassociated complications that can be difficult to manage. By reducing or eliminating pain
and distress associated with current pancreatitis models, ERCP can be a refinement of
current methods of inducing acute pancreatitis.
QUESTIONS:
1. What animal model is the ideal model of induction and treatment of pancreatitis?
a. NHP
b. Canine
c. Feline
d. Mice
2. Which one is the characteristic of pancreas in dogs?
a. The canine pancreas is freely mobile
b. It is suspended in the duodenal mesentery c. both the major and minor ducts
fuse together before entering the duodenum d. a&b is correct
3. Which one would be considered as a noninvasive method of inducing pancreatitis?
a. Administration of caerulein,
b. Administration of alcohol
c. Biliopancreatic duct ligation
d. Feeding a choline-deficient diet
e. a, b, d
ANSWERS:
1. a (NHP)
2. d (a & b is correct)
3. e (biliopancreatic duct ligation is an invasive method)
CASE STUDY
Artwohl et al. Extreme Susceptibility of African Naked Mole Rats (Heterocephalus
glaber) to Experimental Infection with Herpes Simplex Viruse Type 1, pp. 83-90
Task: 1, 2, 3 & 9
Species: Tertiary
SUMMARY: Herpes Simplex virus type 1 (HSV1) is a pathogen that when present in
immunocompetent humans, causes a lifelong latent infection that can be interrupted by
episodes of viral reactivation. Several species, such as mice, rabbits, rats and guinea
pigs, have been used to study the pathogenesis of the virus. In these species the
pathogenesis of HSV1 is similar to that of humans, and in immunocompetent animals,
rarely causes any disease. In several areas of research, HSV1 isolates are being used
as vectors for gene delivery due to the virus’s capacity to carry large amounts of
heterologous DNA and the ability to establish persistent, lifelong infections. The virus
can also infect a wide range of human cell lines and primary cultures.
The naked mole rat is often times used in pain research due to the fact that they do not
produce substance P and calcitonin gene-related peptide from the C-fibers in their skin.
These peptides play an important role in pain signaling in the spinal cord and initiating
local immune response. In this case report, naked mole rats were infected with HSV1
engineered to express a gene that encodes the pain-related neuropeptides substance P
and neurokinin A for pain research. 10 days after the infection, one of the naked mole
rats was found dead, and others extremely lethargic. Necropsy, histology, and
immunohistochemistry showed severe, systemic HSV1 infection. A follow-up, controlled,
comparative study, to assess the susceptibility of naked mole rats to HSV1, showed
extreme susceptibility of the rats to the virus. After being inoculated with replicationcompetent, replication-conditional, or replication-defective HSV1, all naked mole rats
developed systemic infections with notable histological lesions in the liver, spleen, and
lymphatic tissue. Mice used as a comparison in the study showed no gross lesions or
signs of disease.
The naked mole rat’s extreme susceptibility to HSV1 is speculative. The best theory
points to the animal’s isolation from other mammals due to their fully subterranean
lifestyle. This may have limited their ability to evolve protective mechanisms to other
mammalian pathogens.
QUESTIONS:
1. T/F – Herpes Simplex 1 is fatal in mice, rats, rabbits, and guinea pigs.
2. What is unique about the naked mole rat that makes it popular for use in pain
research?
3. T/F – Herpes Simplex 1 will cause overwhelming systemic infection in naked mole
rats.
ANSWERS:
1. False
2. Naked mole rats do not produce substance P and calcitonin gene-related peptide
from the C-fibers in their skin, and they lack C-fiber-related
responses to
Capsaicin. These peptides play an important role in pain signaling to the spinal cord
and initiating local immune responses.
3. True