Comparative Medicine
Volume 60, Number 1, February 2010
ORIGINAL RESEARCH
Mouse Models
Craig et al. 7,12-Dimethylbenz[A]Anthracene Induces Sertoli-Leydig-Cell Tumors
in the Follicle-Depleted Ovaries of Mice Treated with 4-Vinylcyclohexen Diepoxide,
pp. 10-17
Domain 3: Research (Animal Models)
Primary Species: Mouse (Mus musculus)
SUMMARY: This study provides evidence that there is an increased incidence of 7, 12Dimethylbenz[A]Anthracene (DMBA)-induced ovarian neoplasms in the ovaries of a
mouse model of ovary-intact menopause compared with that in age-matched cycling
controls.
An ovary-intact rodent model of menopause was developed by repeated daily dosing of
4-vinylcyclohexene diepoxide (VCD). This model of menopause was combined with the
7, 12-Dimethylbenz[A]Anthracene (DMBA) model of carcinogenesis to cause ovarian
cancer in F344 rats. This paper outlines a study that uses both DMBA and VCD to
determine:


Whether ovarian failure affects susceptibility to the development of ovarian
neoplasms in mice
And to characterize DMBA-induced ovarian neoplasms in VCD-treated mice.
Female B6C3F1 mice were given VCD IP injections daily for 20 days to cause ovarian
failure. Four months after, mice were then given a single injection of DMBA under the
bursa of the right ovary. Ovaries were collected 3 or 5 months after injection, and
processed for histological evaluation. At 3 months, only 12.5% of the VCD+DMBA+
developed neoplasms while at 5 months, neoplasms were detected in 57.1% of the
VCD+DMBA+ and 14.3% of VCD+DMBA- ovaries. Immunohistochemistry confirmed that
the lesions were classified as Sertoli-Leydig cell tumors.
QUESTIONS:
1. How do daily doses of VCD affect murine ovarian tissue?
2. T/F VCD is non-specific and fast-acting in its destruction of ovarian tissue.
3. How is the activity of DMBA different to that of VCD?
4. In the above article, what was keratin 7 and inhibin? used for?
ANSWERS:
1. VCD selectively destroys ovarian primordial and primary follicles by accelerating the
natural process of follicular atresia.
2. False. VCD does not target larger follicles, hence ovarian follicular depletion in
treated mice is gradual.
3. DMBA targets all types of ovarian follicles and causes premature ovarian failure.
4. Keratin 7 used as a marker for Ovarian Surface Epithelium (OSE)
Inhibin ? used to label sex-cord-stromal tumors.
Patten et al. Perturbations in Cytokine Gene Expression after Inoculation of
C67BL/6 Mice with Pasteurella pneumotropica, pp. 18-24
Domain 1
Primary Species: Mus musculus
SUMMARY: Pasteurella pneumotropica is a common bacterial organism found in many
species of laboratory animals and is generally considered to be an opportunist. The
object of this study was to determine whether this organism alters the immune response
in infected mice and whether the response could have an effect on research. To test
this, the authors performed a number of experiments involving inoculation of female
C57BL/6 mice (housed in sterile conditions) with P. pneumotropica.
To determine the kinetics of the immune response, mice were inoculated with P.
pneumotropica orally intranasally, and 2-10 days later, measured levels of inflammatory
cytokine mRNA in the nasal turbinates (IL1B, TNFα, CCL3, CXCL1, CXCL2) were
determined by qPCR. The cytokine mRNA levels in some (but not all) of the infected
mice were 5-11 fold higher than the controls, peaking between days 2-8 postinoculation. Microarray was also performed on the nasal turbinates to look for
expression of 113 inflammatory genes, and 11 genes associated with acute
inflammatory response.
To mimic a natural infection (likely a dose much lower than that used in the previous
experiment), they inoculated mice with log-fold dilutions of P. pneumotropica and
determined a dose sufficient for low-dose exposure models. They then inoculated mice
with this low dose and a high dose and measured mRNA levels of cytokines in the
cervical and mandibular lymph nodes. 7 days after inoculation, increases in these
cytokine mRNA levels were seen in both the low and high doses groups, compared to
controls. This response was no longer seen at 28 days.
To determine whether bacterial viability was required to stimulate an immune response,
mice were inoculated with heat-killed P. pneumotropica. To determine whether a
genetically related nonpathogenic bacterium could cause a similar response, mice were
inoculated with Actinobacillus muris, which is considered a commensal in mice. There
was no difference in cytokine mRNA levels or histology between heat-killed P.
pneumotropica, A. muris, and sham-inoculated control groups.
The authors concluded that C57BL/6 mice colonized with P. pneumotropica can
manifest a mild and transient elevation in inflammatory cytokines and has the potential to
interfere with experimental studies.
QUESTIONS:
1. Regarding Pasteurella pneumotropica: Is it gram-positive or -negative? Hemolytic or
non-hemolytic? Motile or non-motile? Bacillus, coccus, or coccobacillus?
2. What are the two biotypes of P. pneumotropica?
3. Name 3 inflammatory cytokines that the authors examined in this study.
ANSWERS:
1. Gram-negative, nonhemolytic, nonmotile, coccobacillus
2. Jawetz & Heyl
3. IL1B, TNFα, CCL3, CXCL1, CXCL2
Rat Model
Booth et al. Spontaneous Coagulopathy in Inbred WAG/RijYcb Rats, pp. 25-30
Primary Species: Rat
SUMMARY: The authors report on a series of cases of spontaneous coagulopathy in a
colony of inbred rats from Yale University. The breeding colony had been established at
Yale from rats obtained from the Netherlands in 1970. There were some unexplained
deaths in postpartum rats but bleeding was not associated in them. The index case was
a 10 week old that sustained a subcutaneous hematoma after an injection to deliver
tumor cells. There followed several reports of unexplained injuries from investigator
reports. Evaluation of the index case revealed coagulopathy and subsequently there
were ~ 24 rats reported between ages 3 and 46 weeks of age that were euthanized for
humane reasons associated with bleeding. The index case and one other rat were the
only 2/24 to have had experimental manipulations.
Hematology parameters measured indicated prolonged aPTT (50%-70%), normal or
decreased Hct, normal PT, normal or decreased WBC normal or decreased RBC,
normal or decreased hemoglobin, variable platelet count, normal fibrinogen and D-dimer
concentrations. MCV, MCH and MCHC did not differ between affected rats and adult
female SD rats. These findings were consistent with normocytic, normochromic
regenerative anemia secondary to blood loss. The hemorrhagic diathesis identified was
characterized by a prolonged aPTT in rats with or without clinical signs of bleeding.
Although the causes of acute and chronic bleeding are variable, most potential acquired
coagulopathies were excluded
due to the colonies controlled husbandry, and
environmental conditions, through histopathological exam and through clinicopathologic
assays. All causes for hemorrhage aside from primary hemostatic defect have been
ruled out. The author's goals are to develop the WAG/RijY rat substrain to carry the
inherited coagulopathy in to a model to utilize for understanding the corresponding
human disease. They are working to identify which component of the coagulation
pathway is defective and responsible for the spontaneous coagulopathy in these rats.
QUESTIONS:
1. According to figure 6, acute hemorrhage can be caused by all except for which of the
following?
a. GI ulcers
b. Hemostatic defects including toxins, hemophilia A and B, DIC
c. Neoplasia including splenic hemangioma or hemangiosarcoma
d. Surgery
e. Thrombocytopenia
f. Trauma
g. Parasites
2. According to figure 6, chronic hemorrhage can be caused by all except for which of
the following?
a. GI ulcers
b. Hematuria
c. Neoplasia, vascular or GI
d. Parasites - ectoparasites or endoparasites
e. Vitamin K deficiency
f. surgery
ANSWERS:
1. g
2. f
Rabbit Model
Panda et al. Escherichia coli O157:H7 Infection in Dutch Belted and New Zealand
White Rabbits, pp. 31-37
Domain 3: Research; K3: Animal Models
Primary Species: Rabbit
SUMMARY: Enterohemorrhagic E. coli O157:H7 is a food borne pathogen that produces
Shiga toxins and causes bloody diarrhea in humans. Mouse models of E. coli O517:H7
do not satisfactorily replicate the pathology and clinical signs as seen in humans, and
generally germ-free mice are required. This study evaluated a rabbit model of E.
coli O517:H7 infection. Eight Dutch belted and 8 New Zealand White rabbits were
divided by breed into a control group of 2 rabbits and an experimental group 6 rabbits.
Experimental rabbits were inoculated with 109 CFU E. coli O517:H7 strain 933 in 1ml
PBS by intragastric gavage while control rabbits received 1ml PBS only. Rabbits were
monitored for clinical signs such as diarrhea, hemorrhagic colitis, fever, weight loss or
lethargy. Fecal samples were collected on days 2,4 and 6 for bacterial quantification
and bacterial colonies were collected for the presence of toxin genes Stx1 and Stx2 by
PCR. CBCs and chemistries were performed at baseline and at time of euthanasia.
Animals were euthanized on day 13 and tissues submitted for histopathology. All
experimental rabbits became infected with E. coli, and recovered bacteria contained
both Shiga toxins. Control rabbits had no clinical signs whereas infected rabbits had
diarrhea and weight loss. None of the rabbits had hemorrhagic colitis. One Dutch
belted rabbit was euthanized on day 8 due to greater than 20% weight loss. On
histopathology , 100% of New Zealand rabbits and 80% of Dutch belted rabbits
displayed enteritis. Multiple fibrinous thrombi in the renal arteries and tubular dilation
were seen in the kidney of one Dutch belted rabbit. CBCs and chemistries were not
significantly different between experimental and control rabbits. This study showed that
Dutch belted and New Zealand white rabbits are susceptible to oral challenge with E.
coli O517:H7 strain 933 and develop diarrhea, enteritis, and weight loss. This model may
prove useful for studying the pathogenesis of and novel therapeutic agents against E.
coli O517:H7 infection.
QUESTIONS:
1. E. coli O517:H7 is associated with what syndrome in humans, especially children?
2. E. coli O517:H7 primarily targets what organ system?
3. Rabbits in this study were/were not susceptible to oral challenge with E.
coli O517:H7 strain 933?
ANSWERS:
1. Hemolytic uremic syndrome
2. Gastrointestinal system
3. Were
Swine Models
Harig et al. Refinement of Pig Retroperfusion Technique: Global Retroperfusion
with Ligation of the Azygos Connection Preserves Hemodynamic Function in an
Acute Infarction Model in Pigs (Sus scrofa domestica), pp. 38-44
SUMMARY: This study aimed to refine the technical and functional aspects of a pig
model of acute myocardial infarction and retroperfusion with respect to the azygos
connections. Sixteen pigs were allocated into 4 groups of 4 pigs each. In each group,
acute ischemia was induced by ligation of the ramus interventricularis paraconalis
(equivalent to left anterior descending (LAD) artery in humans), followed by global
retroperfusion. Coronary sinus perfusion was performed in 8 pigs, but not the other 8,
and the azygos vein was ligated in 4 of the 8 pigs in each of these groups but left open
in the remaining animals.
They found that mid-LAD occlusion reduced cardiac output and worsened cardiac and
circulatory parameters. In addition, all pigs manifested rhythm disturbances during the
20-min LAD occlusion period.
So in short, global retroperfusion prevented
hemodynamic deterioration only if it was used in combination with azygos ligation. A
potential limitation was that the anesthetic regimen used may have caused both negative
and positive effects on hemodynamic function.
In conclusion, the authors found that it is crucial to identify and eliminate an azygos vein
connection to the coronary sinus when using a porcine model of global retroperfusion.
QUESTIONS:
1. What is the current pig model for global retroperfusion?
a. Yorkshire-Duroc
b. German landrace
c. Pietrain
d. American Yorkshire
2. Why is the clinical use of retroperfusion not widespread?
a. Because, until now there have been no animal models
b. Due to the fact that this is not a clinically relevant issue
c. Due to the fact that findings in the current pig models have generated
controversy
d. None of the above, its use is clinically widespread
ANSWERS:
1. B
2. C
Wei et al. Infection of Cesarean-Derived Colostrum-Deprived Pigs with Porcine
Circovirus Type 2 and Swine Influenza Virus, pp. 45-50
SUMMARY: Porcine respiratory disease complex (PRDC) is economically significant for
the pig industry. It is characterised by slow growth, poor food utilisation, lethargy,
anorexia, fever, cough and dyspnea in pigs from 16 to 22 weeks of age. Its aetiology
appears to from multiple viral and bacterial respiratory pathogens picked up concurrently
or sequentially. Porcine Circovirus 2 (PCV2) has been implicated in the aetiology. Swine
Influenza virus is a common pathogen also associated with PRDC. SIV is frequently
found in pigs with clinical signs of PRDC.
This study focussed on identifying a challenge model for PCV2 to determine whether
SIV influences PCV2 replication and increases severity of PCV2 associated disease.
The model used caesarean-derived colostrum- deprived Large White pigs housed in a
biosafety level 2 housing facility. Groups of 8 pigs (from one week of age) were assigned
as negative control, PCV2 only; PCV2 followed a week later with SIV. Inoculation was
via intratracheal instillation of the viruses or cell culture medium only (negative control).
Measures taken included: clinical sign evaluation and scoring, serologic assays for
antibodies by immunofluoresecent assay and hemagglutination, virus isolation and real
time PCR assay, histopathology and in situ hybridisation for PCV2. Coinfection with SIV
did not increase the number of PCV2 genomic copies in serum or target tissues or the
severity of microscopic lesions in lung or lymph node associated with PCV2. Antibody
titre to PCV2 did not differ significantly between the two virally infected groups i.e. PCV
alone or in combination with SIV. It seems that in the model used in this study SIV did
not influence PCV2 replication.
QUESTIONS:
1. T/F. Porcine respiratory disease complex mainly affects mature pigs
2. T/F. The complex seems to involve a combination of different viruses acting
synergistically.
3. T/F. PCV2 is a member of the family of viruses Circoviridae.
4. T/F. Swine Influenza is commonly isolated from clinical cases of PRDC.
5. T/F. Animals co -infected with SIV and PCV2 had higher antibody titres to PCV2.
6. T/F. Concurrent SIV infection did not increase the severity of the microscopic lesions
associated with PCV2.
ANSWERS:
1. F- It mainly affects growers.
2. F- It does seem to involve a number of viruses but bacteria are also implicated.
3. T
4. T
5. F
Nonhuman Primate Models
Fujimoto et al. Simian Betaretrovirus Infection in a Colony of Cynomolgus
Monkeys (Macaca fasicularis), pp. 51-53
Domain 4: Animal Care; Task 2 - Manage or provide indirect management/oversight of
animal husbandry programs
Primary Species: Macaques
SUMMARY: Simian betaretrovirus (SRV) cause symptoms of immunodeficiency,
including anemia, tumors, and persistent refractory diarrhea, in some infected macaques
but most infected monkeys exhibit few or no clinical signs. Establishing an SRV-free
breeding colony is paramount for a steady supply of appropriate monkeys for
experiments. Here, the authors investigated the actual prevalence and transmission of
SRV in the closed cynomolgus colony through several generations to prevent the spread
of the virus and establish an SRV-free colony. Blood samples were collected and
analyzed by Western blotting and RT-PCR. The results were that more than 90% of the
laboratory-born breeders were positive for SRV-specific antibodies or virus (or both).
The results also showed that all monkeys exhibiting SRV–specific antibodies without
viremia produced infants without viremia. Results also suggest that cynomolgus infants
infected in utero with SRV and born from viremic mothers are immunologically tolerant to
the virus and that they then become the source of SRV infection in the colony. The
authors planned to redesign the caging in order to prevent the monkeys from touching
the feces and urine from other monkeys and eliminate viremic dams from the breeding
colony which is critical to the establishment of an SRV-free breeding colony.
QUESTIONS
1. T or F Most monkeys infected with Simian betaretrovirus exhibit few or no clinical
signs.
2. The results of this investigation revealed that all monkeys exhibiting SRV –specific
antibodies without viremia produced
a. Infants without viremia
b. Infants with viremia
c. No infants at all
ANSWERS
1. True
2. a. Infants without viremia
Shipley et al. A Challenge Model for Shigella dysenteriae 1 in Cynomolgus
Monkeys (Macaca fasicularis), pp. 54-61
Task 9: Collaborate on the Selection and Development of Animal Models
Primary Species: Macaque fasciularis
Introduction: Shigella is a category B priority pathogen in humans. Infectious dose in
human is ~10 cfu. Since there is no vaccine, antibiotics are the only method of
treatment. The purpose of this study was to develop a Shigella dysenteriae 1
challenge model in cynomolgus monkeys and to establish the minimal challenge dose
required to induce clinical signs of shigellosis.
Materials and Methods: 6 macaques were exposed to increasing doses (105 to 109) of
wild type S. dysenteriae (1 dose per animal). The sixth monkey received 109 cfu that
was passaged in another macaque. Stool samples were collected twice daily until 4
days PI then once daily until 14 days PI. Blood was collected at days ), 7,14, 28 and 56
to measure antiShigella LPS antibodies.
5 macaques were divided into two groups: 2 were inoculated intraduodenally
(endoscopically) with 109 cfu S. dysenteriae 1 and 3 animals were given 1011 cfu of S.
dysenteriae 1 intragastrically. Each monkey received 15 mEq sodium bicarbonate
intragastrically (via stomach tube) immediately prior to inoculation. Blood was collected
at multiple time points post challenge for immunologic assays blood chemistries, and
hematologic analysis.
Animals were monitored daily diarrhea, dysentery fever, signs of respiratory distress,
changes in food intake and any other abnormal behavior. Animals that became ill were
treated with antibiotics and fluids.
Results: None of the animals exposed to increasing doses of S. dysenteriae 1 developed
clinical sings despite having persistent shedding for up to 10 days (108 and 109 cfu).
Monkeys exposed to 109 cfu mounted robust serologic IgG and IgA S. dysenteriae 1
LPS antibody response whereas animals with low doses (105 to 108 cfu) exhibited only
background response. Only animals given 1011 cfu S. dysenteriae 1 developed clinical
signs. CBC and chem panels were not remarkable. In animals given (109 and 1011 cfu)
increases in IgG and IgA titers were seen as early as 7 days PI and peaked at 14 days,
declined and reached a plateau at day 28.
QUESTIONS:
1. What is the Sereny test?
2. Which dose of S. dysenteriae 1 was necessary to induce clinical signs in cynomolgus
monkeys/
3. What was the classification given to S. dysenteriae by the National Institute of Allergy
and Infectious Disease?
ANSWERS:
1. The keratoconjunctivitis assay which is considered to be the gold standard to assess
the degree of attenuation of novel Shigella vaccine strains because this assay
measures mucosal invasiveness and reactogenicity.
2. 1011 cfu
3. Category B priority pathogen
Burke et al.
Alterations in Cytokines and Effects of Dexamethasone
Immunosuppression during Subclinical Infections of Invasive Klebsiella
pneumoniae with Hypermucoviscosity Phenotype in Rhesus (Macaca mulatta) and
Cynomolgus (Macaca fascicularis) Macaques, pp. 62-70
Domain 3: Research; Task 3 – Advise and consult with investigators on matters related
to their research
Primary Species: Macaques
SUMMARY: Non-human primates (NHPs) may be naturally infected with various strains
of Klebsiella pneumoniae, including a strain of K. pneumoniae designated as having a
hypermucosoviscosity (HMV) phenotype.
After one cynomolgus macaque
experimentally infected with monkey pox subsequently died from apparent HMV K.
pneuomiae infection it was hypothesized that virus induced immunosuppression allowed
bacterial proliferation and septicemia. To test the hypothesis that immunosuppression
can allow enhanced K. pneumoniae infections, and to better characterize potentially
confounding effects of subclinical infection with HMV K. pneumoniae in NHPs an
experiment was conducted wherein cynomolgus macaques and rhesus macaques were
experimentally infected with HMV K. pneumoniae either alone or in combination with
dexamethasone induced immunosuppression.
After infections animals were monitored for disease progression and levels of various
inflammatory cytokines in circulating plasma were quantified. Non-immunosuppressed
NHPs infected with MHV K. pneumoniae did not display any clinical signs of disease.
Clinical signs of disease in immunosuppressed animals were mild and transitory.
Pathologic findings in infected animals receiving immunosuppression showed periodic
evidence of peritonitis, fat necrosis, and typhlocolitis. Other pathological findings were
suppression of adrenal and lymphoid tissues. Bacteria infected animals had significant
alterations in cytokine expression, with cynomolgus macaques displayed significant
increases in GM-CSF, IL-6, IL-8 and IL-10. Rhesus macaques had significantly elevated
serum concentrations of IL-6 and IL-8. IL-8 was still elevated even after glucocorticoid
immunosuppression.
Subclinical infection with HMV K. pneumoniae can significantly alter cytokine expression
and potentially alter the immunological response to experimental treatments. The
elevated IL-8, even in the face of dexamethasone treatment, may partially explain why
clinical signs of HMV K. pneumoniae infection were difficult to produce. However, any
significance associated with cytokine levels must be made carefully due to the highly
interrelated functions of the various cytokines.
QUESTIONS:
1. What is the proposed mechanism that allowed HMV K. pneumoniae to produce
death in a NHP?
2. Did glucocorticoid immunosuppression replicate disease syndrome associated with
K. pneumoniae infection?
3. What effect did glucocorticoid immunosuppression have on cytokine expression
levels?
ANSWERS:
1. Virus induced immunosuppression/immunodysfunction which would allow systemic
proliferation of K. pneumoniae.
2. No
3. Immunosuppression did not significantly reduce IL-8 expression, possibly preventing
clinical disease associated with K. pneuomoniae infection.
Escaron and Sherry. Orally Ingested 13C2-Retinol is Incorporated into Hepatic
Retinyl Esters in a Nonhuman Primate (Macaca mulatta) Model of
Hypervitaminosis A, pp. 71-76
Primary Species: Macaca mulatta
Task 1
SUMMARY: Tissue vitamin A is primarily derived from either retinyl esters or provitamin
A carotenoids. In the intestine, retinyl esters are cleaved into retinols that are then
absorbed by enterocytes, esterified, and packaged into chylomicrons. These
chylomicrons are released into circulation and taken up by the liver. Once in the liver the
retinyl esters are hydrolyzed to retinol that is either secreted back into the circulation or
re-esterified and stored in hepatic stellate cells.
Little is known about vitamin A metabolism and storage during hypervitaminosis A. This
study examined ¹³C₂-retinol uptake in hypervitaminic A rhesus macaques. Male rhesus
monkeys received a physiologic dose of 14,15 ¹³C₂ retinyl acetate injected in a banana
that was sealed with peanut butter. Baseline (overnight fasted) blood sample and serial
blood collections after dose administration were drawn. Random liver biopsies at set
intervals pre- and post-treatment were also collected. Isotope ratio mass spectrometry
(IRMS) and a gas chromatograph were used to assess ¹³C- retinol levels of both the
serum and liver samples. It was hypothesized that de novo retinyl esterification would
continue to occur in the liver during hypervitaminosis A.
The results of this study showed that hepatic retinyl ester quantities continue to
accumulate in the nonhuman primate model of hypervitaminosis after receiving a
physiologic dose of vitamin A. These accumulations occurred for two days post dosing
suggesting that there is not a complete down regulation of this pathway. This study also
demonstrated that the heavy carbon isotope can be used to trace orally ingested vitamin
A, and thereby avoid the deleterious effects of using radioisotopes. These study
techniques could be potentially applied to metabolic studies on humans.
QUESTIONS:
1. Dietary vitamin A is primarily available as retinyl esters and carotenoids. T or F
2. It has been previously reported that hypervitaminosis A can lead to what
dysfunction?
a. Neurologic
b. Hepatic
c. Renal
d. Splenic
3. The physiologic dose of vitamin A administered to the rhesus macaques in this study
was:
a. Less than their daily intake from feed
b. More than their daily intake from feed
c. Equal to their daily intake from feed
4. The process of converting retinol from retinyl ester is:
a. Hydrolysis
b. Esterification
c. Oxidation
ANSWERS:
1. T
2. b
3. a
4. a