Comparative Medicine
Volume 64, Number 1, February 2014
ORIGINAL RESEARCH
Mouse Models
March et al. Differences in Memory Development among C57BL/6NCrl, 129S2/SvPasCrl, and
FVB/NCrl Mice after Delay and Trace Fear Conditioning, pp. 4-12
Domain 3: Research
Primary Species: Mouse (Mus musculus)
SUMMARY: Fear conditioning (FC) is a form of Pavlovian associative-learning and used to study
differences in memory formation. Animals develop an association between an initially neutral explicit cue
(a tone or light), a training chamber and an aversive effect (foot shock). This fear usually manifests as
freezing and can be measured. This study used 3 inbred mouse strains: C57BL/6NCrl, 129SvPasCrl,
and FVB/NCrl obtained from Charles River to characterize conditioned fear memory. Two training
paradigms are used: (1) delayed conditioning in which an unconditional stimulus (foot shock)
coterminates with the presentation of a conditional stimulus (tone) and (2) trace conditioning in which the
conditional and unconditional stimuli are separated by a trace interval. For each paradigm, a recent
memory (3 d) and a remote memory (25 d) of the mice were carried out.
Results indicated that both C57BL/6NCrl and 129SvPasCrl mice developed strong and long-lasting
context and tone memories in both paradigms. FVB/NCrl mice showed a weaker but consistent tone
memory after the delay training paradigm. At 25d or the remote testing revealed that 129SvEPasCrl
mice tone memory diminished after delay training but was stable after trace training. In conclusion, the
C57BL/6NCrl and 129S2/SvPasCrl mice showed reliable and lasting fear memory after delay and trace
conditioning. The FVB strain showed reliable tone fear memory only after delay training.
QUESTIONS
1. Which strain has PDE68 that is implicated in retinal degeneration?
a. C57BL/b
b. 129/Sve
c. FVB
d. A
2. What is a common research for fear conditioning in mice?
a. Alzheimer disease
b. Blindness
c. Anxiety
d. Depression
ANSWERS
1. c
2. a
Trammell et al. Effects of Sleep Fragmentation on Sleep and Markers of Inflammation in Mice, pp.
13-24
Domain 3: Research; Task T3. Design and conduct research
Primary Species: Mouse (Mus musculus)
SUMMARY: The aim of the study was to develop and characterise an approach to create chronic
disruption of sleep in laboratory mice. People experience sleep disruption due to work, care giving or
lifestyle choices and there is increasing evidence that shows that many common disease conditions
develop in association with sleep disruption e.g. increased risk of obesity, glucose intolerance, type 2
diabetes.
In the experiment C57Bl6/6J mice were first housed in environmentally controlled chambers with a 12:12
hour light:dark cycle. Mice were surgically implanted with electrodes to monitor EEG and EMG. Sleep
fragmentation (SF) was induced by placing mice on a disc that rotated randomly; 3 different durations of
sleep fragmentation was induced. Sleep was measured by collecting EEG and EMG data; cytokine,
chemokine, insulin and adipokine measurements were made on serum and lung samples.
Sleep amount, depth and consolidation were significantly higher during the ad-libitum sleep phase that
occurred after each of the 3 SF regimens. In response to sleep fragmentation for 12 or 24 hours, mice
did not recover slow wave sleep (SWS) time during the subsequent 12 or 24 hour period leading to a
slow wave sleep debt. Mice recovered lost rapid eye movement sleep (REMS). Sleep fragmentation of 024 hours was associated with lower concentrations of some inflammatory mediators in the lung, whereas
effects on serum analytes were minimal. Therefore in this model acute SF can create a sustained sleep
debt and a modified inflammatory environment in the lung. The authors speculate that these changes
could contribute to a greater risk of lung disease secondary to prolonged sleep perturbation.
QUESTIONS
1. A sustained sleep debt may increase the risk of lung disease T or F
2. In humans, there is increasing evidence that sleep disruption is associated with
a. Obesity
b. Glucose intolerance
c. Type 2 diabetes
d. All of the above
ANSWERS
1. T
2. d
Walton et al. Comparison of 3 Real-Time, Quantitative Murine Models of Staphylococcal Biofilm
Infection by Using In Vivo Bioluminescent Imaging, pp. 25-33
Domain 3: Research
Primary Species: Mouse (Mus musculus)
SUMMARY: Staphylococcus aureus commonly causes difficult to treat hospital-acquired infections. One
of the resistance mechanisms for S. aureus is the formation of biofilms. These communities of microbes
in an extracellular polysaccharide matrix are protected from many host defenses and exogenously
administered antimicrobials. Since bacteria in a biofilm differ from their planktonic counterparts a valid
animal model of biofilm infection is important for developing therapeutics. The authors sought to use in
vivo bioluminescent imaging (BLI) to evaluate 3 different models of biofilm infection: infected tibial
intramedullary pin, subcutaneous catheter model, and subcutaneous mesh implant. Histopathology and
ex vivo bacterial quantification were also performed. All subcutaneous (SQ) catheter implants were lost
via abscessation and dehiscence of the implant site by day 20. Conversely both the IM pin and SQ mesh
maintained stable infections for at least 35 days. The average bioluminescence in the SQ mesh model
was slightly higher than the IM pin model. Bacterial counts and bioluminescent measurements were
compared to assess how well the bioluminescent signal correlated with bacterial load in each model. In
the IM pin model there was no significant correlation between bacterial count and BLI. In addition, the IM
pin had a relatively low bioluminescent signal compared to the SQ mesh despite a higher bacterial load
on ex vivo culture. The SQ mesh model had a strong correlation between the number of bacteria
recovered and the BLI data. They briefly discuss the morphologic characteristics of the model that can
potentially interfere with BLI such as pigmented skin, fur, and tissue density or depth. The authors
conclude that the SQ mesh model produces a stable infection with a superior correlation between
bioluminescence and bacterial counts as compared to the SQ catheter and IM pin models.
QUESTIONS
1. An investigator approaches you about incorporating in vivo bioluminescent imaging into his infectious
disease project. Which mouse model traits should provide the investigator with the most robust
bioluminescent signal?
a. Pigmented skin, nude or clipped hair, and a bioluminescent signal in superficial tissues (e.g.skin)
b. Albino, nude or clipped hair, and a bioluminescent signal in superficial tissues (e.g.- skin)
c. Pigmented skin, unclipped fur at the site of interest, and a bioluminescent signal in deep tissues
(e.g. kidney)
d. Albino, nude or clipped hair, and a bioluminescent signal in deep tissues (e.g. kidney)
2. Which of the following biofilm infection model created a stable infection for at least 35 days and a
strong correlation between bacterial quantification and bioluminescence?
a. Tibial intramedullary pin
b. Subcutaneous mesh implant
c. Subcutaneous catheter
d. All of the above biofilm infected implants were lost before 35 days post infection
3. Which common lab animal lesion are you the least likely to culture staphylococcal bacteria from?
a. Rabbit with mastitis
b. Rat with ulcerative dermatitis
c. Mouse with a preputial gland abscess
d. Guinea pig with suppurative cervical lymphadenitis
ANSWERS
1. b. Skin pigmentation, fur, and signal depth can result in decreased bioluminescent signals
2. b.
3. d. Think Strep zooepidemicus for the GP the others are classic manifestations of Staph infections
Rat Model
Inagaki et al. Spontaneous Intraocular Hemorrhage in Rats during Postnatal Ocular Development,
pp. 34-43
Primary Species: Rat (Rattus norvegicus)
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions
SUMMARY: This article describes an investigation into spontaneous intraocular hemorrhage in postnatal
Wistar Hannover (WH), Sprague-Dawley (SpD), and Long-Evans (LE) rat strains. Daily gross and
histologic examination of eyes at postnatal days 0 through 21 was performed. WH and SpD rats had
grossly identifiable intraocular hemorrhage at necropsy while the LE strain did not. All strains had
microscopic intraocular hemorrhage. WH and SpD rats had grossly identifiable red ring-shaped areas
that histologically appeared as hemorrhage of the tunica vasculosa lentis. WH and SpD rats also had
red spots in the eyes that histologically appeared as hemorrhage of the retina, choroid (WH only), or
hyaloid artery. LE rats only had histologically identifiable hemorrhage of the tunica vasculosa lentis and
hyaloid artery. WH had the highest incidence of intraocular hemorrhage. Hemorrhage was seen at
varying ages and durations for each strain but had all disappeared by time of weaning. The authors
posits that hemorrhage of the tunica vasculosa lentis is a normal process of rat ocular development and
indicates that no functional effect occurs due to this hemorrhage. Erythrocyte leakage from the tunica
vasculosa lentis and hyaloid artery may occur during regression. There are previous reports of retinal
and hyaloid artery hemorrhage in adult rats, but this article is the first known report in rat pups. No
reports of choroidal hemorrhage have been reported in adult or rat pups. Additional investigations
especially regarding retinal and choroidal hemorrhage should be undertaken to determine their
underlying mechanisms.
QUESTIONS
1. Which of the following anatomic structures are normally present only during ocular development?
a. Pupillary membrane
b. Tunica vasculosa lentis
c. Hyaloid artery
d. All of the above
e. None of the above
2. True or False. Rodent species are normally exophthalmic.
3. Which of the following statements regarding ocular pathology of rats is incorrect?
a. Albino rats housed on top shelves are more prone to more severe retinal degeneration
b. Rat coronavirus infection may lead to ocular pathology that is secondary to impairment of the
lacrimal glands
c. Conjunctivitis in nude rats may be alleviated by providing hardwood bedding
d. All of the above
e. None of the above
ANSWERS
1. d.
2. True
3. c. Hardwood bedding has been associated with CAUSING conjunctivitis/blepharitis in nude rats.
Swine Models
Newell-Fugate et al. Effects of Diet-Induced Obesity on Metabolic Parameters and Reproductive
Function in Female Ossabaw Minipigs, pp. 44-49
Primary Species: Pig (Sus scrofa)
Domain 3: Research; T3: Design and conduct research; K3: Animal models; K6: Characterization of
animal models
SUMMARY: This study characterizes the effect of an excess-calorie, high-fat, high-cholesterol, highfructose diet on metabolic parameters and reproductive function in female Ossabaw minipigs.
QUESTIONS
1. Obesity can cause?
a. Issues with the hypothalamic-pituitary axis
b. Issues with feedback from leptin and insulin
c. Issues with fertility
d. All of the above
2. Ossabaw minipigs are used as a model of?
a. Metabolic syndrome
b. Type II diabetes
c. Gut microbiota related studies
d. All of the above
3. Features of metabolic syndrome include?
a. Visceral obesity
b. Glucose intolerance
c. Dyslipidemia
d. Elevated fasting glucose
e. All of the above
ANSWERS
1. d. All of the above
2. d. All of the above
3. e. All of the above
Ploemen et al. Minipigs as an Animal Model for Dermal Vaccine Delivery, pp. 50-54
Domain 3
Primary Species: Pig (Sus scrofa)
SUMMARY: The authors evaluated the potential use of Gottingen miniature pigs as a model for
intradermal vaccine delivery. Pigs in general offer advantages over rodent models because their
physiology, anatomy, and biochemistry more closely resemble that of humans. Minipigs offer advantages
over regular pigs regarding husbandry and ease of handling due to their smaller size. Additionally, there
is a high similarity of skin anatomy between humans and minipigs compared to rodents and substantial
knowledge exists regarding the porcine immune system compared to other non-rodent species such as
dogs and Old World nonhuman primates.
Dermal delivery of vaccines is attractive due to the high prevalence of immunocompetent cells in the skin
such as Langerhans cells. There is increasing interest in developing alternative methods for traditional
dermal delivery of vaccines which require a needle and syringe (usually referred to as the ‘Mantoux
method’). One promising alternative method to needle delivery is the disposable syringe jet injector
(DSJI), which eliminates the need for needles (non-painful, no risk of needle stick injuries). DSJI also
facilitates the expression of nucleic acid vaccines without the need for subsequent electroporation. This
method is not feasible for testing in rodents due the mismatch of the size of the device compared to the
size of the rodents.
The authors used minpigs as a test model for DSJI delivery and compared it to standard intradermal and
intramuscular (IM) vaccine delivery, both using a needle and syringe (hepatitis B vaccine, HBsAg). Three
performed in awake animals for both vaccinations. Intradermal delivery by needle required animals to be
anesthetized during the second vaccination due to obvious distress of the animals and to ensure precise
delivery of vaccine. Blood was collected on day 0, 14, and 28 from the vena cava; ELISA was used to
quantify serum titers to the vaccine. Antibody titers on day 14 were significantly elevated for the DSJI and
IM vaccinated animals, but not the intradermal/needle vaccinated animals. By day 28 all groups had
significant increases in HBsAg specific antibodies. DSJI resulted in higher antibody titers compared to
conventional intradermal delivery and was not stressful to the animals. This study used HBsAg
vaccination as a proof of principle, demonstrating that minipigs can be effectively used for evaluation of
needle-free dermal vaccine delivery.
QUESTIONS
1. One advantage of disposable syringe jet injectors over traditional needle/syringe combinations is:
a. Use a significantly smaller needle
b. Lack a needle
c. Capable of injecting much larger volumes
d. Requires use of electroporation for nucleic acid vaccine
2. Which of the following IS NOT an advantage of working with pigs regarding dermal vaccine delivery?
a. High similarity of skin anatomy compared to humans
b. Substantial knowledge of the immune system exists compared to dogs and old world nonhuman
primates
c. High similarity of physiology and biochemistry compared to humans
d. Minipigs are used more frequently than regular pigs to evaluate vaccine efficiency
3. Conventional dermal delivery of vaccines using a needle is traditionally referred to as the:
a. Gebien method
b. Mantoux method
c. Burstein method
d. Baxter method
ANSWERS
1. b
2. d
3. b
Nonhuman Primate Model
Atkins et al. Characterization of Ovarian Aging and Reproductive Senescence in Vervet Monkeys
(Chlorocebus aethiops sabaeus), pp. 55-62
Domain 4; K6: Breeding colony management
Tertiary Species: Other Nonhuman Primates
SUMMARY: Reproductive senescence, or menopause, has not yet been c characterized in vervet
monkeys. Female vervets and macaques have been used as models for chronic diseases relevant to
women’s health, reproductive function and aging, as well as associated chronic diseases, such as bone
loss, atherosclerosis, obesity and diabetes. The vervet menstrual cycle is similar to macaques and
women with an average length of 29 days. Vervets are also well suited for reproductive studies because
that a seasonal anestrus, have a straight cervical canal, and do not act as host to B virus. Ovarian and
reproductive data for this study was collected from archived samples. Ovaries were collected from
females ranging from 6 days to 27.2 years old. Right ovaries (one per animal) were serially sectioned
and each 100th section was used for follicular counting. FSH, estradiol and Anti-Mullerian Hormone
(AMH) were measured using archived samples as well. Other parameters included longitudinal
assessment of menstrual cyclicity and evaluation of fecundity.
Results: Age group 0-9y had the highest number of primordial follicles, followed by 10-19 y. The 20-27y
group had very few follicles of any stage present compared to the other two groups. FSH was not
significantly associated with age. Serum AMH was negatively correlated with age. On histopathology,
vervets between 4-16y had normal signs of cyclicity. The 4 oldest animals (23-27y), showed remnants of
CLs, aggregates of adipocytes within the stroma, similar to changes seen in macaques (and postmenopausal women). Age group 4-10y had the highest percentage of females with offspring, followed
by 11-15y, 16-20y, with monkeys over 21 producing no offspring.
Conclusion: Vervets exhibit ovarian senescence similar to that observed in women and macaques,
reaching menopause around 20-23 years of age.
QUESTIONS
1. Macaques, vervets and apes have what type of placentation?
2. In older vervets approaching senescence, FSH will (increase/decrease) and AMH will
(increase/decrease)?
3. What age range of vervets are most fertile and capable of producing offspring?
ANSWERS
1. Interstitial, hemochorial villous
2. Increase; decrease
3. 4-10 years
CASE REPORT
Nonhuman Primate Models
Fong et al. Transmission of Chagas Disease via Blood Transfusions in 2 Immunosuppressed
Pigtailed Macaques (Macaca nemestrina), pp. 63-67
Domain 1: Management of Spontaneous an Experimentally Induced Diseases and Conditions
Primary Species: Macaques (Macaca spp.)
SUMMARY: These are the first reported cases of transmission of T. cruzi via blood transfusions in NHP.
The first animal was a 2.25 year old male pigtailed macaque (Macaca nemestrina) currently housed in
Seattle, Washington. The animal was on a protocol that included a bone marrow transplant after
irradiation. Due to experimental complications, the animal received multiple blood transfusions. Clinical
signs included pancytopenia and edema of the prepuce, scrotum, and legs. Included in the diagnostic
work-up was a blood smear that was positive for a trypomastigote consistent with Trypanosoma cruzi.
They determined from testing banked blood samples that the animal did not acquire Chagas disease in
Georgia where he had been originally housed outdoors. This finding lead to screening of the blood
donor animals (n=7). One of the donors tested positive. It was a 9 year old pigtail macaque that was
originally housed in Louisiana. A third animal on the same research protocol as the original case also
received transfusions from the infected donor and was antibody positive but did not have parasites
visible on the blood smear. This animal did not have any clinical signs associated with Chagas disease.
QUESTIONS
1. Chagas disease is described as all except what?
a. Infection with the hemoflagellate parasitic pathogen Trypanosoma cruzi, is the causative agent.
b. Chagas is the most serious parasitic disease in Southeast Asia, and despite control efforts, its
negative socioeconomic impact is rising
c. Most infected persons will die of heart disease (70% to 85%), digestive disorders (15% to 30%),
or neurologic disease (less than 5%)
d. It can be transmitted by a triatomine insect vector
2. How is Chagas disease transmitted? Which statement is not correct?
a. NHP’s are thought to contract the parasite by eating the insect vectors
b. Approximately 1% to 12% of offspring of infected mothers will acquire the parasite by congenital
transmission
c. Triatomine insects are commonly known as ‘kissing bugs’ (because as night feeders, they often
feed on the face) or ‘cone-nosed bugs
d. Most (80%) transmission occurs through insect vectors, specifically through contact of parasitecontaining saliva (which are deposited while the insect is taking a blood meal) with mammalian
mucous membranes or through a break in the skin.
3. Which rodent is not associated with the correct species of Trypanosoma?
a. White-footed mice and T. brucei rhodensiense
b. Dusty footed wood rat and T. neotomae
c. Eastern wood rat and T. kansasensis
d. Wild vole and T. microti
ANSWERS
1. b. Latin America
2. d. Feces
3. a. Multimammate Rat (Mastomys natalensis)
Radi and Morton. Lip Salivary-Gland Hamartoma in a Cynomolgus Macaque (Macaca
fascicularis), pp. 68-70
Domain 1; T3 : Diagnose disease or condition as appropriate
Primary Species: Macaques (Macaca spp.)
SUMMARY: An incidental, asymptomatic, well-circumscribed, solitary, submucosal nodular mass was
detected on the mucosal surface of the inner lower lip in a 2.4 y old female cynomolgus macaque during a
juvenile chronic toxicology study. The nodule (4mm in diameter, soft, not ulcerated with brown to tan
discoloration) was covered by normal stratified squamous epithelium and composed of well-circumscribed
irregular lobules containing hyperplastic and normal-appearing mucinous salivary gland acini and ducts,
which were separated by thick connective septae. A salivary gland hamartoma was diagnosed.
QUESTIONS
1. What is a hamartoma?
2. True or False
a. Hamartoma are frequent?
b. Intraoral salivary gland hamartoma have been describe in humans
c. Intraoral salivary gland hamartoma have been describe in veterinary species (laboratory animals)
3. This lesion resembles to what human clinical entity
ANSWERS
1. Focal, benign, nonneoplastic developmental malformation or inborn error manifesting as an
admixture of mature cells indigenous to the anatomic location of occurrence and that grows in a
disorganized mass.
2. a) F, b) T, c) F
3. Adenomatoid hyperplasia (or benign minor salivary hypertrophy or salivary gland hyperplasia)
Boedeker et al. Surgical Correction of an Arteriovenous Fistula in a Ring-Tailed Lemur (Lemur
catta), pp. 71-74
Domain 1, Task 4: Treat disease or condition
Tertiary Species: Other Nonhuman Primates
SUMMARY: A 10 yr old ring-tailed lemur presented for exacerbation of respiratory signs including
tachypnea and dyspnea during physical stimulation. Relevant clinical history included perineal dermatitis
and overgrooming, laceration repair, and an ovariohysterectomy performed due to persistent vulvar
swelling, ovarian cysts, follicular mineralization, cystic endometrial hyperplasia, and endometritis. A
hematoma formed at the femoral venipuncture site at the time of the ovariohysterectomy.
Physical exam revealed tachypnea, tachycardia, bilaterally muffled pulmonary sounds, a grade 2/6
systolic murmur detected over the left base with bilateral systolic ejection click, a doughy abdomen with
possible fluid distension, a right hind limb that was cool to the touch, a palpable thrill in the right inguinal
region, along with a bounding femoral pulse and auscultable bruit. Compression of the right inguinal
region resulted in a declined heart rate, consistent with a positive Nicoladani-Branham sign. Abdominal
ultrasound confirmed ascites. Thoracic radiographs revealed pleural effusion and right ventricular
enlargement. Echocardiography revealed mild enlargement of all 4 cardiac chambers, along with mild
tricuspid valve regurgitation. Ultrasound of the right inguinal region revealed a dilated femoral vein with
turbulent continuous flow. Exam findings were consistent with an arteriovenous fistula (AVF) of the right
femoral artery with congestive heart failure.
Treatment with furosemide was initiated (0.5mg/kg PO daily) resulting in rapid resolution of the
tachypnea, dyspnea, and exercise intolerance. Surgical repair 3 months later consisted of an oblique
incision over the area of palpable thrill, identification of the fistula between the common femoral artery
and corresponding deep vein, ligation of the fistula with 2 ligatures of 4-0 silk, and closure of the surgical
site in 2 layers. A topical paste of metronidazole tablets mixed with 0.9% saline was applied to deter
postoperative licking. Recovery and healing of the surgical site occurred without complication. At
recheck examination 2.5mths postoperatively, cardiomegaly and signs of congestive heart failure had
resolved with no palpable thrill in the right inguinal region. Other than occasional episodes of mild
alopecia and superficial erosions associated with overgrooming, the lemur remains clinically normal
4.5yrs after surgical repair.
AVF are not commonly reported in nondomestic species. The majority of cases are described in
macaques and suspected to be iatrogenic, as in this case. AVF should be considered a potential risk of
femoral venipuncture in lemurs, and probably all primates. The current case is the first AVF diagnosed
and successfully treated in a prosimian. AVF should be considered a treatable cause of congestive heart
failure in lemurs.
QUESTIONS
1. T/F – A congenital etiology is highly suspected as the cause of the AV fistula in the current case.
2. Select the most common clinical signs of AV fistulas
a. Exercise intolerance
b. Ascites
c. Palpable thrill at the site of the anomaly
d. All of the above
ANSWERS
1. False – An iatrogenic etiology is highly suspected as the cause of AV fistula in this case, as the lemur
had undergone repeated venipuncture during multiple health assessments, and there had been a
prior history of hematoma formation at the site of venipuncture.
2. d. All of the above. Congestive heart failure is a common development of patients with an
uncorrected AV fistula.