Comparative Medicine
Volume 59, Number 6, December 2009
OVERVIEWS
Cray et al. Acute Phase Response in Animals: A Review, pp. 517-526
Domain 1, K1 - diagnostic procedures (clinical pathology)
SUMMARY: Acute phase proteins have been recognized and used in human and
veterinary diagnostics and as prognostic indicators, particularly for cardiovascular
disease, autoimmune diseases, organ transplant, and cancer therapy. Several acute
phase proteins (APP) are discussed: C reactive protein, Serum amyloid A, Haptoglobin,
alpha1-acid glycoprotein, alpha2-macroglobulin, pig MAP, ceruloplasmin, fibrinogen,
transferrin, and albumin.
Acute phase proteins are being proposed as markers for humane endpoints in laboratory
animals. Most major proteins are observed to increase markedly within 48 hours of the
triggering event, and then rapidly decline due to their short half life.
Assays that can be used to detect APP?s are currently limited. Total protein, albumin
and globulin levels can be obtained through routine serum chemistry analyzers. For
greater accuracy, protein electrophoresis is the method of choice. ELISA tests are
currently expensive, time and labor intensive, and require species-specific reagents.
Commercially available kits are for CRP, SAA, and haptoglobin. Other techniques for
APP assay described in the literature include radioimmunoassay, nephelometry,
immunoturbidometry, western blot, and mRNA analysis. For prolonged storage, APP
should be kept at -70 deg C.
The article includes a table of all documented veterinary use of APP analyses, and
species in which the analysis was conducted. The sensitivity and specificity of using
APP versus using WBC count to monitor disease progression or resolution was
determined to be superior using APP. However, it is recommended that an APP index
be used when monitoring a disease process, with multiple APP?s and sampling times.
Other uses of APP?s have included gauging stress in farm animals (pigs and calves),
comparing levels among laboratory and companion dogs, and monitoring herd health in
cattle. While it is not believed that APP would be useful as a rodent sentinel test
parameter, it may be useful in the evaluation of individual animals in a colony including
use as a prognostic indicator, animals in quarantine, or for screening animals prior to
entry into an experiment. There is much research potential with APP's in lab animal
medicine.
QUESTIONS:
1. Which of the following is a negative APP?
a. Albumin
b. Haptoglobin
c. C reactive protein
d. Serum amyloid A
2. What is a disadvantage of current methods available to measure APP in laboratory
animals?
a.
b.
c.
d.
Cost of testing equipment
Poor differentiation of proteins
Requirement of large sample volumes
All of the above
ANSWERS:
1. a
2. a
Zhang and Croy. Using Ultrasonography to Define Fetal-Maternal Relationships,
pp. 527-533
SUMMARY: Pregnancy is a very complex process. Disorders of pregnancy are
commonly not understood. Most of the pregnancy complications in human and mice are
associated with reduction on blood flow. Ultrasonography has been used longer than fifty
years to study human gestational health in humans. Nowadays, it is the preferred
diagnostic tool because its safety, precision and cost. Ultrasonography has allowed
investigating and understanding the human maternal–fetal relationships that previously
had been inaccessible. However, the majority of studies of physiology and pathology of
pregnancy in animal models are still relying on postmortem approaches. The
development of microultrasonography constitutes a major improvement. It does not
require termination of animals in study reducing the total number of animals used in
experimentation. It facilitates collection of parameters of embryo and placenta on living
mice and other laboratory animal species. A review of the microultrasonography
applications and limitations on embryonic and placental development in mice is
presented.
QUESTIONS:
1. Ultrasonography has become the diagnostic tool of choice to study human
gestational health because:
a. Safety
b. Precision
c. Cost
d. A and B
e. All of the above
2. Ultrasonography has enabled:
a. Exploration of human maternal fetal relationship
b. Understanding of human maternal fetal relationship
c. Understanding of female reproductive behavior
d. Learning about male reproductive organs
e. All of the above
3. Complications for pregnancy in humans and mice are commonly associated to:
a. Decreased placental vascular development
b. Obstruction to placental blood flow
c. Obstruction to fetal blood flow
d. Decreased ovarian vascular development
e. A and B
ANSWERS:
1. e
2. e
3. e
ORIGINAL RESEARCH
Mouse Models
Gulani et al. The Effect of Helicobacter hepaticus Infection on Immune-Responses
Specific to Herpes Simplex Virus Type 1 and Characteristics of Dendritic Cells, pp.
534-544
Task 3 – Control spontaneous or unintended disease or condition
Primary Species – Mouse
SUMMARY: This study compared levels of HSV1-specific antibody and T cell-mediated
immune responses between H. hepaticus-infected and noninfected mice.
Materials and Methods: SPF C57BL/6 male mice, aged 3-4 weeks were used. Mice
received either H. hepaticus in PBS or PBS alone via gavage. Four weeks after
infection, mice were either infected IN with HSV1 (see paragraph below) or euthanized
for collection of spleens and colic lymph nodes (LN), during which the LN were enriched
for dendritic cells (DC). Splenocytes and colic LN cells were stimulated with LPS, and
DC were analyzed for maturation-associated surface markers. DC were then stained for
proinflammatory cytokines.
One week after HSV1 infection, mice were euthanized and HSV1-specific CD8+ T cells
were quantified.
-producing HSV1-specific CD8+ T cells were detected. Cytotoxic
CD107+CD8+ T cells were detected and quantified. HSV1 infection was confirmed via
virus neutralization assay.
Statistical significance was determined with the 2-tailed, unequal variance, unpaired t
test.
Results: H. hepaticus infection had no statistically significant effect on HSV1-specific
antibody and T cell-mediated immune responses in the superficial cervical and
tracheobronchal LN of HSV1-infected mice. Nonsignificant trends toward reduced HSV1specific T cell-mediated immune responses and increased HSV1-specific cytotoxic T
cells were observed in tracheobronchal LN and superficial cervical LN, respectively.
Surface expression of CD40, CD86, and MHC II was significantly lower on DC derived
from H. hepaticus-infected mice than noninfected mice. The percentages of IL12p40-producing DC in the colic LN were lower in H. hepaticus-infected mice than in
noninfected mice after exposure to both LPS and medium only. Surface expression of
CD40, CD80, CD86, and MHC II reached significantly higher levels on LPS-exposed
splenic DC from H. hepaticus-infected mice than noninfected mice, and expression of
CD86 was higher in H. hepaticus-infected mice after exposure to media.
Discussion: In conclusion, a variety of assays were used to show that H. hepaticus
infection of mice changes several immunologic parameters. These findings emphasize
the importance of using H. hepaticus-free mice in studies in which immunologic function
is a factor in the results.
QUESTIONS:
1. In this study, what type of cell showed surface expression changes in response to H.
hepaticus infection?
2. True or false: Significant difference were seen in HSV1-antibody and T cellmediated immune responses in the superficial cervical and tracheobronchal lymph
nodes in response to H. hepaticus infection.
ANSWERS:
1. Dendritic cells
2. False
Walters et al. Mutational Insertion of a ROSA25-EGFP Transgene Leads to Defects
in Spermiogenesis and Male Infertility in Mice, pp. 545-552
Primary Species: mouse
Domain 3: Research - TT3.5 - genetic modification/engineering technology including
application of molecular biology techniques
SUMMARY: Characterized is the male infertility phenotype of the FVB/NTacTg(Gt(ROSA)26Sor-EGFP)130910Eps/Mmmh strain. The animals of this strain were
generated by random insertion of a transgene construct containing EGFP controlled by
the mouse ROSA 26 promoter via pronuclear injection of FVB/NTac embryos.
Chromosome walking revealed a single integration site on chromosome 3 within a gene
that coded for a novel nucleoporin which is speculated by the authors to be involved in
germ cell differentiation. Natural mating of 8-10 week old homozygous male with
homozygous females or wild-type FVB females produces no offspring after 10 wk of cohousing.
However, homozygous females mated with wild-type FVB males, or
hemizygous males mated with hemizygous females or wild-type females, produced
viable offspring in normal sized litters. Of the FVB female mice mated to homozygous
males, 6 of 12 had evidence of copulatory plugs. However, of the 169 embryos
collected, none were fertilized.
Sperm samples collected manually revealed spermatozoa of normal concentration with
no motility, and 80% showing head defects and head-to-tail fusions. Males at 8-10 wks
of age had more motile sperm with less abnormalities when compared to males at 8-10
mo of age. Histological examination of the testes and epididymides of the homozygous
mice showed somniferous tubular epithelium with large vacuoles, abundant cellular
debris and multinucleated giant cells, as well as decreased numbers of all germ cell
stages indicating severe testicular degeneration. Staining of GATA4, a transcription
factor thought to be important in gonadal steroidogenesis and sex determination,
showed that the vacuolation of the seminiferous tubule was localized in the Sertoli cells,
but not in the germ cells. Similar spermatogenesis defects were noted in a nectin 2 KO,
a mouse model with Sertoli cell vacuolation and abnormal germ cell adhesion.
QUESTIONS:
1. T or F. GATA4 is localized in the nuclei of Leydig and Sertoli cells, but not germ
cells, in the testes of pre and postnatal mice.
2. T or F. The transgene inserted into a gene that encodes a mitochondrial porin.
3. Which strain of mice were used as wild-type controls?
a.
b.
c.
d.
ICR
C57Bl/6
FVB
SJL
ANSWERS:
1. T
2. The transgene inserted into a gene that encodes a nucleoporin.
3. c. FVB
Nadeau et al. A Mouse Surgical Model for Metastatic Ovarian Granulosa Cell
Tumor, pp. 553-556
Domains 1: Management of spontaneous and experimentally induced diseases and
conditions
Domain 3: Research
Primary Species: Mus musculus
SUMMARY: The authors described a genetically engineered mouse model that develops
ovarian granulosa cell tumors (GCTs) that mimic the advanced human disease, but the
primary tumors killed the hosts before metastases formation so the mice were unable to
be used to study metastatic disease. The authors describe a novel protocol for tumor
removal which led to postoperative survival of more than 90% (19/21) of the mice and
subsequent development of metastasis. The protocol included multimodal anesthesia, a
ventral midline celiotomy, perioperative fluid therapy, and analgesia. Tumors as large as
10% of the animal’s body weight were successfully removed. The animals were
euthanized 12 weeks after surgery and histopathology identified pulmonary (9 mice) and
adrenal metastases (13 mice). The authors conclude that this mouse model will be a
useful preclinical model for the development of novel treatment modalities and
diagnostic techniques for GCTs and also has applicability in other human cancer
models.
QUESTIONS:
1. Which of the following is the most prevalent sex cord tumor in women?
A. Theca cell tumors
B. Ovarian granulosa cell tumors
C. Teratomas
2. T or F: In the genetically engineered mouse model (Ptentm1Hwu/tm1Hwu.; Ctnnb1tm1Mmt/+;
Amhr2tm3(cre)Bhr/+) , an aggressive form of GCT develops only in females.
3. Which of the following was NOT included in the multimodal anesthesia regimen used
is this study?
A. Medetomidine
B. Ketamine
C. Xylazine
D. Isoflurane
E. Atipamezole
ANSWERS: (Note there are nice pictures in this article)
1. B. Ovarian granulosa cell tumors
2. False. Develops in both sexes
3. C. Xylazine
Rat Model
Fu et al. Paeoniflorin Prevents Diabetic Nephropathy in Rats, pp. 557-566
Domain 1, Task 4
Primary Species: Rat
SUMMARY: Peony plants have been used in traditional Chinese medicines and herbal
medicines in China and Japan. Paeoniflorin (isolated from P. lactifllora) is a predominant
component to peony plants. Peony extracts and their constituents have various biologic
and biomodulating activities, including antioxidant activity, improvement of memory,
antiepileptic activity, antimutagenic properties and antihyperglycemic effects. This study
was performed to in order to elucidate the effect, if any, paeoniflorin has on the kidney
during diabetic nephropathy.
To accomplish this the group evaluated the effect of paeoniflorin on urinary albumin
excretion in diabetic rats. Reductions in urinary albumin in diabetic nephropathy are
reportedly associated with renal protection. In addition, the group also looked at
glomerular hypertrophy, blood glucose, the expression of transforming growth factor β,
type IV collagen and intercellular adhesion molecule 1; and renal infiltration of
macrophages compared with levels in untreated diabetic mice.
Treatment with paeoniflorin ameliorated albuminuria and glomerular hypertrophy.
Paeoniflorin suppressed the diabetes-induced increases in the urinary
albumin:creatinine ratio. It markedly and dose-dependently decreased TGFβ, type IV
collagen, and ICAM1 mRNA and protein expression in diabetic kidneys and macrophage
infiltration in diabetic glomeruli.. It also inhibited serum MCP1 in diabetic nephropathy
rats and suppressed the activity of NFκB, a key transcription factor of many
proinflammatory genes. These finding suggest that paeoniflorin has an anti-inflammatory
effect in diabetic kidneys and prevents the development of nephropathy.
QUESTIONS:
1. What is considered the key measure of renal function?
2. What is suggested to be a predictive indicator for prognosis of diabetic nephropathy
and its elevated excretions is said to impair renal function?
ANSWERS:
1. Urinary albumin:creatinine ratio
2. Urinary albumin
Rabbit Model
Gallagher et al. Ribonucleotide Reductase Inhibitors Reduce Atherosclerosis in a
Double-Injury Rabbit Model, pp. 567-572
Domain 3: Research
Primary Species: Rabbit (Oryctolagus cuniculus)
SUMMARY: Obstructive coronary artery disease is currently treated with percutaneous
transluminal coronary angioplasty with stent deployment. However, this procedure often
has a high incidence of restenosis because of a molecular cascade that initiates
vascular smooth muscle cells. These cells proliferate in the tunica media and migrate,
causing neointimal formation. Ribonucleotide reductase inhibitors, such as hydroxyurea
and didox, have anti-inflammatory and antioxidant properties that may mitigate vascular
reactivity.
Rabbits are commonly used in atherosclerosis studies because they can develop
atheromatous foci that share many characteristics with human lesions. These are
achieved with a high cholesterol diet with or without vascular endothelial denudation by
balloon injury.
In this study, endothelial injury was induced in the right common carotid artery of NZW
rabbits. After surgery, the rabbits were placed on a high cholesterol diet of 2%
cholesterol and 1% peanut oil. After injury, rabbits were given either didox or
hydroxyurea (there were also injured/untreated and uninjured/untreated groups). 4
weeks after surgery, balloon angioplasty of the developing atherosclerotic lesion was
performed. The rabbits were recovered and the high cholesterol continued for an
additional 4 weeks. The carotid arteries were then removed for histologic examination.
Results showed that treatment with didox and hydroxyurea significantly reduced lesion
area and did not decrease lumen loss compared with the untreated injured group. The
RR-inhibitor treated groups also had lower numbers of circulating leukocytes, though
they did not have cholesterol lowering effects.
QUESTIONS:
1. Didox and hydroxyurea are what?
a. Antibiotics
b. Endothelial growth factors
c. Ribonucleotide reductase inhibitors
2. How do human atherosclerotic lesions differ from those in rabbits?
a. They have areas of fibrocalcification and necrosis
b. They have fatty streaks
c. They have accumulated foam cells
d. They have fibrous plaque formation
3. T/F: RR inhibitors have anti-inflammatory and antioxidant properties
ANSWERS:
1. c
2. a (both humans and rabbits have the lesions noted in b-d)
3. T
Swine Model
Clarkson et al. Using Multidetector Computed Tomography in a Swine Model to
Assess the Effects of Sublingual Nitroglycerin and Intravenous Adenosine on
Epicardial Coronary Arteries, pp. 573-579
Domain 3 (Research), Task 2
Primary Species: Swine (Sus scrofa)
SUMMARY: Multidetector computed tomography (MDCT) has the potential for early
diagnosis and functional assessment of coronary artery disease. This is a repeated
measures study to evaluate pre- and post- vasodialator epicardial vessel visualization
and cross-sectional area assessment.
Cross-sectional diameter was decreased
significantly (approx. 9.6%) by adenosine and increased significantly (approx. 11 %) by
nitroglycerine. This suggests that nitroglycerine my aid in enhanced visualization of
coronary arteries. Adenosine is evaluated as new technology in MDCT will allow the
evaluation coronary perfusion. Adenosine is commonly used to induce hyperemic
responses in perfusion studies. Adenosine reduced vessel diameter by reduced
perfusion pressures. This suggests that MDCT protocols evaluating perfusion may
impact the ability to visualize the epicardial coronary arteries.
QUESTIONS:
1. Swine were chosen as the experimental model because:
a. The coronary artery anatomy is analogous to humans
b. The heart rate does not need to be manipulated to achieve baseline heart rate
comparable to clinical protocols.
c. The animals are inexpensive
d. None of the above
2. True or False: Nitroglycerine acts as a nitric oxide inhibitor to induce vascular
smooth muscle relaxation.
3. True or False: The effects of adenosine are dependent on the endothelium and
secondary to increased flow through the microvasculature.
ANSWERS:
1. A
2. False, nitroglycerine is a nitric oxide generator
3. True
Nonhuman Primate Models
Gray et al. Comparative Analyses of Single-Nucleotide Polymorphisms in the TNF
Promoter Region Provide Further Validation for the Vervet Monkey Model of
Obesity, pp. 580-588
Tertiary Species: Other Nonhuman Primates
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions
Domain 3: Research.
SUMMARY: Tumor necrosis factor is a cytokine that plays a role in inflammation, the
innate immune response, and metabolic dysregulation associated with obesity. This
project is part of an ongoing initiative to further characterize vervet monkeys originating
from St Kitts as an animal model of obesity and inflammation, the authors sequenced
and genotyped the human ortholog vervet TNF gene and approximately 1 kb of the
flanking 3’ and 5’ regions from 265 monkeys in a closed, pedigreed colony. The
process revealed 11 single-nucleotide polymorphisms (SNPs). Using sequences from
humans, chimps, vervets, baboons, and rhesus monkeys for comparison, showed that
the SNPs were clustered nonrandomly and nonuniformly around conserved transcription
factor binding sites. This study adds to prior characterization of obesity-related
phenotypic measures and heritability of these traits.
QUESTIONS:
1. What is the scientific name for the vervet?
2. What are SNPs?
3. What is tumor necrosis factor?
ANSWERS:
1. Chlorocebus aethiops sabaeus
2. SNPs - single-nucleotide polymorphism is a DNA sequence variation occurring when
a single nucleotide — A, T, C, or G — in the genome (or other shared sequence)
differs between members of a species (or between paired chromosomes in an
individual).
3. Tumor necrosis factor is a cytokine that plays a role in inflammation, lymphocyte
biology, immune responses, and metabolic dysregulation associated with obesity.
Burke et al.
Epidemiology of Invasive Klebsiella pneumoniae with
Hypermucoviscosity Phenotype in a Research Colony of Nonhuman Primates, pp.
589-597
Domain 1:- Management of Spontaneous and Experimentally Induced Diseases and
Conditions
Domain 4: Animal Care
Primary Species: NHP (Primary Species)
SUMMARY: Environmental contamination was not determined to result in transference
of Klebsiella pneumoniae with Hypermucoviscosity Phenotype.
Klebsiella pneumoniae is an enteric, gram-negative bacillus. This bacterium is ubiquitous
found in water, soil and in biofilms. It is an opportunistic pathogen has been associated
with peritonitis, septicemia, pneumonia, and meningitis in both Old and New World
primates. Over several months, 7 African green monkeys (AGM; Chlorocebus aethiops)
developed abscesses in multiple locations and either died or were euthanized.
Hypermucoviscous phenotype HMV K. pneumoniae (as determined by positive string
test) of the K2 serotype and carrying rmpA was determined to be the cause of the
infection. March 2008 one clinically normal rhesus tested positive for HMK from an
oropharyngeal sample. 35 more rhesus macaques were tested resulting in 3 more
positive by Oropharyngeal or rectal swab.
Program of surveillance was instituted. OP and rectal swabs were taken from 307
macaques and AGM. Total samples = 2297. Swabs were cultured on MacConkey agar
plates. String test was used to check any suspected colony of HMV Klebsiella. 78 HMV
samples were tested by rapid real time PCR for magA and rmpA genes. Bacterial
genomic DNA was tested for Cy isolates using “Ready to Go” RAPD beads. RAPD is
used to determine genetic variability in a population. The gold standard is pulsed field
gel electrophoresis but it is technically demanding, requires specialized equipment and
is time consuming.
Environmental testing: 252 samples were cultured over 6 wks from various locations
NHP rooms. 28 MacConkey plates were attached in a grid pattern in rooms during
spray cleaning. 40 culture plates were exposed after high pressure spray cleaning.
195 of 2297 swabs were positive for K. pneumonia with 81 positive for HMV phenotype.
Of the HMV positive samples, 49 were magA+/ rmpA-, 17 were magA-/ rmpA+, and 12
were magA-/ rmpA-. Of the 18 samples for RAPD, no pattern was found to be
associated with a specific species. Of the 252 environmental samples none cultured
positive. The wall grid samples only yielded 1 positive. None of the exposed plates
showed positive contamination after high pressure spray.
Transmission by fecal-oral or droplet/aerosol is possible but environmental testing did
not support a widespread contamination even after high pressure spraying.
QUESTIONS:
1: Which of the following, easily performed, tests is used to determine genetic isolate
variability in a Klebsiella pneumoniae population?
a. PCR
b. MacConkey differential culture
c. Ready to Go” RAPD beads
d. pulsed field gel electrophoresis
2: What test is being demonstrated in the picture to differentiate Hypermucoviscous
phenotype HMV K. pneumoniae?
a. Positive string test
b. Optochin Test
c. MacConkey differential culture
d. Quellung reaction
ANSWERS:
1. C
2. A