Comparative Medicine
Volume 54, Issue 2 (April 2004)
Natural and Experimental Helicobacter Infections. pp. 128-158
The Helicobacter genus infects the stomach, cecum, colon, liver and genital
tract of mammals and birds. The host range for different species varies from limited to
wide, and genetic diversity of individual strains also ranges widely. The immune system
of the host is unable to clear infection and in some cases may worsen disease. Disease
may be seen in immunodeficient and some immunocompetent animals. Helicobacter are
gram negative, and molecular techniques are often required for species identification.
H. hepaticus has interfered with several carcinogenesis studies. It has also been
transmitted to SCID mice via injection of contaminated nonfrozen transplantable human
tumors. H. bilis accelerates, and H. hepaticus delays, development of spontaneous colitis
in multiple drug resistance-deficient mice. H. hepaticus can lessen severity of early MHV
lesions, and worsen hepatitis and meningitis later. Subclinical effects of helicobacters,
such as altered gene expression, may also confound research.
Eradicating infection in mice:
Commercial vendors shown to be free of infection should be chosen, and some
of the newly arrived animals should be confirmed infection-free based on PCR of cecal
scrapings or feces. Mice from sources not known to be free of infection should be
rederived, ideally with embryo transfer. C-section with fostering onto infection-free
dams, or fostering of neonates within 24 hours, has also been used successfully, but is not
as good. Note that H. hepaticus can cause transplacental infection in immunodeficient
mice.
Triple therapy (amoxi or tetracycline, plus metronidazole and bismuth) for 2
weeks may eradicate H. hepaticus in some immunocompetent mice, and may help
prevent disease in some immunodeficient mice.
Colony management to prevent fecal-oral spread is also important.
Use of sentinel mice:
Dirty bedding sentinel mice have been used to check for shedding of H.
hepaticus, H. rodentium and H. bilis. In one study PCR detected H. hepaticus as soon as 2
weeks, and all sentinels were positive by 4 weeks. The anti-helicobacter serum IgG
ELISA showed high sensitivity but low specificity, and in 2 studies the sentinels became
positive in 4-6 weeks.
Natural infection in mice:
H. hepaticus is found in the cecum, colon and liver, and has been a confounding
factor in long-term carcinogenesis studies. Some strains of mice develop typhlocolitis,
chronic hepatitis, and hepatocellular adenoma and carcinoma. A/JCr and SCID/NCr are
the most prone to hepatitis. SCID/NCr mice show many bacteria in the liver, with mild
monocytic and neutrophilic inflammation. Proliferative typhlitis in this strain shows
epithelial cell hyperplasia with mild inflammation. Immunocompetent A/JCr mice
develop substantial mononuclear inflammation and show only low numbers of bacteria in
the liver. Diagnosis can be pursued with culture (special requirements) of feces, tissues,
cecal scrapings; PCR of feces or cecal scrapings; and serology (not available
commercially). Warthin-Starry or Steiner silver stains can be used in liver and stomach
but not in cecum/colon as many normal bacteria will stain.
H. bilis is associated with proliferative typhlitis and chronic hepatitis in
immunocompromised mice. This species can also be found in dogs, rats, gerbils and
humans. PCR is considered the most sensitive test.
H. rodentium, along with H. hepaticus, has been commonly found in laboratory
mice. This species may cause typhlocolitis in SCID mice.
H. ganmani has been found in mice and is an anaerobic, as opposed to
microaerophilic, species. H. typhlonius can cause typhlocolitis in SCID mice and IL-10 /- mice. H. muridarum has been associated with typhlocolitis in some
immunocompromised mice. "H. muricola" and "H. rappini" have also been found in
mice.
Experimental infection in mice:
Mouse models have been especially important in looking at how chronic
infections and tissue inflammation may progress to dysplasia and cancer in the liver and
GI tract. Some studies have suggested a non-genotoxic tumor promotion mechanism,
which may be meditated by reactive oxygen species. Studies have also looked at how
host responses to inflammation are important in the pathogenesis of infection and disease.
Urease is a suspected virulence factor that may also be important in
carcinogenesis. All gastric helicobacters are urease positive, but only some enterohepatic
forms are. H. hepaticus and H. bilis are 2 enterohepatic species that are urease positive.
Mouse models have also been used to look at helicobacter-associated
inflammatory bowel disease-like conditions. H. hepaticus colonizes the cecum of
experimentally infected C57BL/6 mice to a greater extent than the cecum of A/JCr mice,
and C57BL/6 developed a mild hepatitis vs the more severe form found in A/JCr mice.
A/JCr immune response that limits cecal colonization may be involved in the
pathogenesis of liver disease.
H. trogontum in germ-free mice was associated with IBD-like inflammation.
The immune system's role in the inflammatory response to infection and
progression to cancer has been investigated in helicobacter-infected mice. A/JCr mice
produce mostly IgG2a antibodies to H. hepaticus infection, consistent with a Th1
response similar of that of humans to H. pylori. Cecal tissue gene expression profiles
show that some proinflammatory genes were up-regulated. IL-10 -/- mice develop a Th1mediated typhlocolitis when experimentally infected with H. hepaticus. The typhlocolitis
progresses to colorectal adenocarcinoma in 60% of IL-10-/- 129/SvEv mice by the age of
6 months. Different strains of mice infected with H. hepaticus have been used to help
define how innate immunity and adaptive immunity contribute to IBD.
Mice have also been used as experimental models of helicobacter-induced
gastritis. H. felis causes gastritis in germfree mice and gnotobiotic rats. C57BL/6 develop
severe gastritis. BALC-c mice devlop B-cell lymphoid follicles similar to MALT
lymphoma in humans. The Th1 response may also be important in carcinogenesis. The
SS1 strain of H. pylori can cause chronic gastritis and gastric atrophy in mice. This strain
has been used in looking at the link between a high-salt diet, H. pylori and preneoplastic
gastric lesions.
Infection in rats:
Natural infections occur in research rats, with a prevalence of 19% in one study.
H. bilis, H. typhlonius, H. hepaticus, H. rodentium, and unspeciated helicobacters have
been found. However, disease is not commonly seen in infected rats, with the exception
of H. bilis-associated proliferative, ulcerative typhlocolitis and proctitis in nude rats.
Experimental infections of rats have not been studied nearly as much as in mice.
H. felis and "H. heilmannii" can cause mild gastritis.
Infection in gerbils:
H. hepaticus and H. bilis can naturally infect gerbils, but disease is uncertain.
However, inbred Mongolian gerbils were the first successful model of gastric cancer due
to chronic H. pylori gastritis.
Infection in hamsters:
Hamsters are a reservoir host for H. cinaedi, which may infect and cause disease
in immunosuppressed humans. This helicobacter produces cytolethal distending toxin,
which is also found in certain enteropathogenic bacteria. H. mesocricetorium, H.
cholecystus and H. aurati have also been reported.
Infection in guinea pigs:
Although not studied extensively, guinea pigs and rabbits are not known to be
naturally infected with any helicobacter. The SS1 strain of H. pylori has experimentally
cause antral gastritis and gastric MALT in guinea pigs.
Infection in non-human primates:
Natural infections with H. pyloris and/or "H. heilmannii" are commonly
associated with subclinical chronic gastritis in both Old World and New World primates.
Clinical disease is uncertain.
H. cinaedi may naturally infect rhesus and may be associated with hepatitis and
typhlocolitis. A novel helicobacter species has been isolated from captive macaques with
chronic typhlocolitis. Cotton-top tamarins may also develop ulcerative colitis and colonic
adenocarcinoma due to a novel helicobacter.
H. pylori has been used experimentally to cause gastritis in primates.
Experimental infections with H. cinaedi and H. fennelliae have caused diarrhea and
septicemia in pig-tailed macaques.
Infection in ferrets:
Natural H. mustelae infection in ferrets was the first non-human gastric
helicobacter isolated. Kits acquire the infection from jills around the time of weaning.
Prevalence is about 100% in adult domestic ferrets, and natural infection is associated
with gastritis and ulceration. Experimental infection has shown carcinogenesis very
similar to H. pylori infection in humans. The ferret is the only domestic animal with
helicobacter-induced gastritis, and duodenal and gastric ulcers, as is seen in humans
infected with H. pylorus. As with H. pylori, flagellar motility of H. mustelae is important
for colonization and disease. H. pylori and H. mustelae may evade the host's immune
response partly by expressing Lewis blood group antigens that mimic the host's. Urease
activity is also necessary for colonization. The organism has been cleared from ferrets
with amoxicillin-based triple therapy for 28 days, and with clarithromycin plus ranitidine
bismuth citrate for 14 days.
Infection in cats and dogs:
"H. heilmannii" (H. bizzozeronii) is the bacteria most commonly found in the
stomachs of animals, including dogs, cats, nonhuman primates, cheetahs and swine. It's
occasionally found in humans also. It can cause gastritis in cats, but clinical signs are
uncertain.
Endemic H. pylori has been found in one commercial cat colony bred for
research. Chronic diffuse atrophic gastritis was seen, similar to infection in humans and
NHP's.
H. felis is found in cats and dogs. In cats it causes a lymphoid follicular
hyperplasia and mild gastritis, but gastric secretory function remains normal. This
organism can cause severe gastritis in mice with progression to cancer.
Several other helicobacters have been found naturally in some dogs and cats,
including those with enteritis and hepatitis.
In dogs and cats, experimental infection with H. pylori causes more severe
pathological changes than dose H. felis.
Infection in swine:
"H. suis" has been found in domestic swine and may be seen in farmers with
gastritis. It may be linied to ulceration of pars esophagea in swine. Experimental H.
pylori infections of swine have been used to study H. pylori-induced gastritis.
Infection in miscellaneous mammals:
H. rappini has been associated with hepatic necrosis in sheep fetuses and may
be zoonotic. H. bovis has been found in the cow abomasums.
H. marmotae may promote hepadnavirus-associated tumors in woodchucks.
H. cetorum has been found associated with gastritis and gastric ulcers in
dolphins and whales.
Infection in birds:
H. pullorum may be seen in the liver, duodenum and cecum of chickens, and
may cause gastroenteritis in humans. H. Canadensis is found in geese and may be
zoonotic. H. pametensis has been found in wild birds and domestic swine.
Questions:
1.)
What is the only domestic animal in which a helicobacter species may cause
gastritis with gastric and duodenal ulceration, similar to that found in humans infected
with H. pylori?
2.)
What are some methods to eradicate infection in mice?
3.)
Which species of helicobacter have been commonly found in lab mice?
4.)
T/F: Immunocompetent individuals are often able to clear helicobacter
infections, and may be resistant to reinfection.
5.)
Which mouse strains are most prone to hepatitis with H. hepaticus?
6.)
What type of disease is most commonly associated with H. hepaticus in mice?
Answers:
1.)
Ferrets infected with H. mustelae
2.) Repopulation with, and testing of, mice from commercial vendors free of the disease.
Rederivation via embryo transfer is recommended for other mice. Cross-fostering or
triple therapy may be successful in some immunocompetent mice.
3.)
H. hepaticus and H. rodentium
4.)
False.
5.)
A/JCr and SCID/NCr
6.) Typhlocolitis, chronic hepatitis, and hepatocellular adenoma and carcinoma
Minimally Invasive Surgery via Laparoscopy for Intra-Abdominal Biopsy in Obese
Rhesus Macaques (Macaca mulatto). pp. 159-164
SUMMARY: This paper demonstrates that laparoscopy in obese rhesus monkeys is a
minimally invasive surgical method to obtain repeated intra-abdominal liver and omental
adipose tissue biopsies. This approach greatly decreased operative time and stress,
provided generous tissue specimens in a time-efficient manner, and facilitated rapid and
full recovery in macaques.
The safety and effectiveness of laparoscopy for repeated intra-abdominal
biopsies was compared to liver wedge techniques in 9 obese rhesus monkeys performing
two procedures each, approximately six weeks apart. This technique was performed
under general anesthesia, used a three-port approach. A roticulating endoscopic
stapler/divider was used for obtaining tissue samples and a monopolar electrosurgery was
used for hemostasis. Liver biopsies of 3.8 g. and omental adipose biopsies of 16.6 g.
were obtained. Compared with previous studies of conventional laparotomy with liver
wedge resection, the monkeys experienced faster postoperative recovery via laparoscopy,
with rapid return to normal food intake and activity. Minimal to no adhesions were
observed by use of the repeat procedure in all monkeys, with no major complications. In
addition, a liver wedge obtained by laparotomy is considered a major surgical procedure
and only two major survival procedures should be performed on the same animal.
Questions:
1) T/F Laparoscopy is a safe and effective means for obtaining intra-abdominal
biopsies in Rhesus monkeys?
2) How many ports are typically used to perform abdominal laparoscopy?
3) How many major survival procedures should be performed on an animal?
Answers:
1) True, Rhesus recover more quickly to normal function, have fewer adhesions, and
fewer post-surgical
complications.
2) Three ports are normally used for abdominal laparoscopy: (insufflation, scope, and
biopsy)
3) Two
Metabolism of Diadzein by Intestinal Bacteria from Rhesus Monkeys (Macaca
mulatto). pp. 165-169
Summary: The main purpose of the paper was to identify the produced metabolites of an
isoflavanoid called Daidzein by colonic bacteria of rhesus monkeys. Isoflavanoids are
diphenol compounds found in some leguminous plants like soybeans. They have a role in
plant defense but their primary interest form a research perspective is that they may have
potentially beneficial effects in human disease in areas such as cardiovascular
(phytoestrogens have been demonstrated to impact LDL's by reducing them and HDL's
by increasing them) and endocrine related diseases. Daidzein, a weakly estrogenic
isoflavanoid, may be in diseases like hypercholesterolemia and osteoporosis. In this case
the Rhesus monkey's fecal bacteria were cultured with Daidzen to see if it was
metabolized, and what metabolites were produced. Metabolites were analyzed by using
(HPLC) and mass spectrometry.
Materials and Methods: Swab specimens were obtained from nine rhesus monkey
ranging from 2-12 years old. They were fed a diet that was 25% protein, 5% fiber, and
49.7% carbohydrate. The main ingredients of the diet were soybeans, wheat and corn.
Once the swabs were collected, they were placed in transport media and used to inoculate
tubes containing brain heart infusion media in an anaerobic container. Each tube then had
Daidzein added to it, and then incubated. Samples were then collected daily, prepped, and
subjected to HPLC analysis and cell density was measured by mass spectrometry.
Results: Three metabolites were identified: Dihydrodaidzein (all nine monkey swabs),
Equol (5 of 9 monkey swabs), and an unknown metabolite (3 of 9 monkey swabs) with a
molecular weight of 244.
Conclusion: The intestinal microflora of the NHP's produced similar Daidzein
metabolites, as did themicroflora in humans. There was variability in the production of
metabolites by the microflora of the NHP's as well, but not as great as in humans
(probably due to less dietary control of human subjects).
Questions:
1. Daidzein is a
a. Antibiotic
b. Isoflavanoid
c. Type of colonic bacteria
d. None of the above
2. Daidzein is a _________ compound that may have potential benefits in treating
________.
a. progestin and stress
b. estrogenic and diabetes
c. anti aging and hypercholesterolemia
d. estrogenic and hypercholesterolemia
3. The two principal metabolites of Daidzein produced by culturing Daidzein with NHP
microflora were:
a. Dihydrodaidzein and MW 243
b. Dihydrodaidzein and Equol
c. Equol and MW 243
d. None of the above.
Answers: 1, b, 2. d, and 3, b.
Effect of Water Hardness on Oocyte Quality and Embryo Development in the African
Clawed Frog (Xenopus laevis). pp. 170-175
Summary: The African clawed frog, Xenopus laevis, has been a common research animal
for over a century, and its embryos are especially valuable for studies of early
development.
Dechlorination and adequate salt concentration are known to affect oocyte
quality but water hardness has not been considered an important factor previously.
Females kept in soft water or water with marine salts alone result in a decrease in the
quality of oocytes. Increasing the water hardness results in increased survival and normal
development of embryos.
It is not surprising, therefore, that water from South African ponds that serve as
commercial sources of X. laevis is moderately to very hard.
By comparison, water hardness also affects the successful development of offspring
from fertilized eggs in various species of fish. Municipal water sources may vary
tremendously in hardness and conductivity on the basis of the geography and hydrology
of specific regions. Thus, water hardness should be an important consideration in
housing Xenopus females to be used for oocyte production.
Questions:
1.
What is the genus and species of the African Clawed Frog?
2.
What are common uses of the African Clawed Frog in biomedical research?
3.
Is Xenopus laevis a wholly aquatic species?
Answers:
1.
Xenopus laevis
2.
Used, among other things, in studies of development and differentiation at both
cellular and molecular levels
3.
Yes
Effect of Sex and Age on Serum Biochemical Reference Ranges in C57BL/6J Mice.
pp. 176-178
Summary: The C57BL/6J Mouse strain is widely used as a common genomic
background for many gene-modified murine models. However, few data on its clinical
biochemistry are available. Therefore, the investigators conducted a study to provide new
protocols for serum biochemical screening and developed the reference range for a set of
13 analytes that pertain to lipoprotein metabolism, electrolyte balance, and data reflecting
function of the heart, liver, kidneys, and pancreas. Male and female mice were studied,
and blood samples were obtained at six and 20 weeks of age. Of 13 parameters studied,
12 were affected by age and sex. Briefly, male mice had higher triglycerides, highdensity lipoprotein cholesterol, glucose, and amylase values. With age, mice of both
sexes developed higher triglycerides and glucose concentrations, as well as aspartate and
alanine transaminase activities. A significant difference between mice and humans was
noted for amylase activity, which is extremely high in this healthy mouse strain.
Therefore, the investigators suggest that caution should be taken when data are
interpreted to indicate gastrointestinal disease in murine models. The reference values
for murine clinical biochemical analytes obtained during the study reported here should
be useful for characterizing the biochemical phenotype of experimental mice.
Questions
True or False
1. Sex plays a role in mediating immune reactions and disease progression as a result of
effect of estrogens on cellular immune response.
2. Increased electrolyte and creatinine values may be due to hemoconcetration during
blood collection.
Answers
1. T
2. T
Differences in Spermatogenesis in Cryptorchid Testes among Various Strains of Mice.
pp. 179-184
Summary: Several strains of mice were studied to determine whether or not there was a
significant difference in spermatogenesis in crytorchid testes. The following strains were
studied: A/J, AKR/N (AKR), BALB/c (BALB), CBA/N (CBA), C3H/He (C3H),
C57BL/6 (B6), ddY, ICR, MRL/MpJ+/+(M+), MRL/MpJ lpr/lpr (lpr). Unilateral
cryptorchidism was created by relocation of the right testis from the scrotal sac and
suturing of the epididymal body to the abdominal wall. All mice were euthanized 14 days
post operatively.
The cryptorchid testis was weighed, and compared relative to the following
parameters: body weight, weight of intact testis, and the ratio of the weight cryptorchid
testis to that of intact testis. The tissue was submitted for histological examination
(quantitation of germ cells using the Sertoli cell index) and processed using the TUNEL
staining reaction (estimation of apoptosis) and PCNA immunostaining.
In A/J, BALB, C3H, CBA, B6, ddY and ICR mice, weight of the cryptorchid
testis was decreased to less than half of the non cryptorchid testis. These mice were
designated as “heat stress sensitive”, whereas the AKR, M+ and lpr mice in which the
weight of the cryptorchid testis was 70% of the intact testis comprised the “heat stress
resistant” group.
In both groups of mice, paralleling germ cell loss was the presence of nuclear
pyknosis, cellular atrophy and presence of tubular multinucleated giant cells within seven
to ten days post surgery. In heat stress sensitive strains the germ cell loss was still evident
by day 14 post operatively, and by day 21 resulted in presence of only spermatogonia and
Sertoli cells. Heat stress resistant strains demonstrated presence of pachytene spermatids
and round spermatids 14 days post procedure. The germ cell loss in the former group of
mice was attributed an increase in apoptosis.
Heat shock proteins act to protect somatic cells from thermal insults by preserving
the three dimensional structure of native proteins. Cells exposed to heat shock often
express highest levels of the hsp70i (inducible) of the hsp70 multigene family. Induction
levels of this gene in spermatocytes and spermatids is less than in somatic cells, and its
thermal protective effect in these germ cells yet to be decided.
Questions:
1. How many seminiferous tubule stages exist in the mouse ?
2. How many hours is a single spermatogenic cycle in the mouse ? How long does it take
for spermatogonia to mature ?
3. What is the origin of the MRL mouse ?
Answers:
1. 12
2. 233.6 hours, taking 4.5 cycles to achieve maturity.
3. LG (75 %), AKR (12.6%), C3H (12.1%) and B6(0.3%).
Antimicrobial Therapies for Pulmonary Klebsiella pneumoniae Infection in B6D2F1/J
Mice Immunocompromised by Use of Sublethal Irradiation. pp. 185-192
In this article the authors state that Klebsiella pneumoniae is a common cause of
nosocomially acquired pneumonia in immunocomprimised patients. The authors give an
excellent overview of the current epidemiology of K. pneumoniae infection. In brief, K.
pneumonia is the most common gram-negative bacteria that affect granulocytopenic
patients.
Klebsiella species cause 8 percent of all hospital acquired (nosocomial) infections and
second only to E. coli as the most common cause of gram-negative sepsis. Klebsiella
pneumoniae is responsible for infections of the soft tissue, urinary tract, bacterial
septicemia, and hospital acquired pneumonia.
In this study, the authors used their established Klebsiella pneumonia model using
B6D2F1/J mice sublethally irradiated with 7-Gy60Co
*-radiation and inoculating these mice intratracheally (i.t.) with the bacteria. This animal
model creates a pulmonary infection in which to evaluate different interventions to
combat K. pneumoniae infections.
Experimental design, mice were randomly distributed into seven groups of 20 using a
statistical program. Groups consisted of 4 groups given antimicrobial therapies of
ceftriaxone, gentamicin, ceftriaxone-gentamicin combination, or gatifloxacin. One group
did not receive any treatment, two groups were give sterile water as a vehicle control (one
group s.c., one group p.o.). All groups were given 7-Gy60Co *-radiation. The
experimental protocol is as follows:
Day 0 mice were irradiated. Day 4 mice were inoculated with 3.7 x 106 cfu of K.
pneumonia/0.1 ml. i.t. (370 LD 50/30). Day 5 mice started 10 day, single dose antibiotic
treatments.
Experiment 2: Mice followed the same experimental protocol as experiment 1 however;
mice in these experiment animals were inoculated with 3.7 x 106 cfu of K.
pneumonia/0.1 ml. i.t. (350 LD 50/30) on Day 4, then sub-sets from each group were
euthanized on days 5, 6, 8, 11, and 15 to evaluate the biological status of the mice after
treatment. Five untreated animals from this group were also evaluated.
Results: Experiment One Survival-Figure 1, page 188, 95% on mice treated with
ceftriaxone-gentamicin combo therapy survived, 80% with gatifloxacin, 75% given
ceftriaxone, and 10% given gentamicin. Survival of animals in treatment groups were
significantly greater that untreated and vehicle control groups (P<.05).
Experiment 2 Microbial Results-Figure 3, page 189, Clinical signs of disease (i.e.,
recumbency, dyspnea, kyphosis, ruffled hair coat, and /or reluctance to move when
stimulated) was noted only in untreated and vehicle control animals. Klebsiella
pneumonia was isolated from the lungs of 10 to 20% of antimicrobial-treated mice verses
73% in controls, in heart blood samples 0 to 10% of antimicrobial-treated mice verses
45% of control animals were positive for K. pneumoniae. These results showed the
therapeutic effect of the antimicrobial treatments throughout the treatment protocol.
Conclusion: This study suggest that ceftriaxone plus gentamicin, followed by ceftriaxone
alone or gatifloxacin as an alternative, is the most effective antimicrobial therapy for
treatment of acquired K. pneumonia in a mouse model of the human disease.
Antimicrobial resistance to treatments did not develop in this study. Future studies are
warranted to evaluate longer treatment periods and larger antimicrobial treatment doses.
Note on page 186 how the authors arrived at the antimicrobial doses used in this study.
Questions:
1.
Interpret the mouse nomenclature for the mice used in this experiment,
B6D2F1/J.
2.
Define the radiation unit Gray (Gy).
3.
Define what 370 LD 50/30 means in experiment 1.
Answers:
1.
A C57BL/6 female was crossed with a DBA2 male, and the first generation
offspring (F1) from these matings that were acquired from Jackson Labs (J) were used in
this experiment.
2.
A Gray (Gy) is the International System of Units base unit for absorbed energy
and equals one joule per kilogram. One joule is defined as the amount of energy exerted
when a force of one Newton is applied over a displacement of one meter.
3.
This is the amount of K. pneumoniae bacteria 3.7 x 106 (370) cfu of K.
pneumonia/0.1 ml. i.t., inoculated into mice that the lethal dose (LD) at which 50% of
the animals die in 30 days (50/30) on this study protocol.
Effects of Geographic Origin on Captive Macaca mulatto Mitochondrial DNA
Variation. pp. 193-201
Purpose of study: To help increase the mtDNA diversity database for rhesus
macaques using partial sequences of 12S and 16S rRNA genes to study the
influence from geographic origins on the genetic diversity among captive rhesus.
Background: There is a rising demand in biomedical research for genetically
well-characterized rhesus macaques. Strategies for genetic management of
domestic rhesus colonies (small and genetically heterogenous) are created to
monitor distribution of reproductive success, maintain genetic variability,
minimize inbreeding, and genetic subdivision. Even with careful planning to
maintain genetic diversity, colonies prone to genetic subdivision from colony
founder effects, genetic isolation, and genetic drift.
An example of differences in allele frequency distributions might occur at
quantitative trait loci is in viral disease resistance. Rhesus macaques originating
from China are more resistant to experimentally induced SIV infection than those
monkeys originating from India. At certain breeding centers, some Chinese and
Indian rhesus macaques have been indiscriminately cross-bred. In other centers,
the origin of the captive-bred animals is unknown. In such circumstances, mtDNA
haplotoypes should provide more sensitive and cost-efficient indicators of rhesus
geographic origin of non-admixed genealogy than do nuclear polymorphisms.
Monitoring the distribution of mtDNA haplotypes in captive rhesus colonies
should be an integral part of colony genetic management. This type of monitoring
will help to maintain the representation of all founding matrilines and patrilines in
approximately equal frequencies and maximize biogeographic representativeness
of the lineages in domestic captive rhesus colonies.
M&M: Blood and DNA samples were collected from 28 unrelated rhesus
macaques at 7 primate centers. These colonies represent three regional
populations: Northern India, SE Asia, and China.
Results: Sequence comparison of rhesus 12S and 16S rRNA genes reveals
significant and approximately equal degree of intraspecific diversity in both
genes. Results indicated marked divergence of rhesus macaques in China than in
India; the mtDNA sequences from Chinese macaques were more diverse than
Indian macaques. This outcome is consistent with China’s greater subspecies
diversity and with nuclear genotype distribution.
Conclusion: mtDNA haplotypes can be useful for genetically defining,
preserving maximal levels of genetic diversity and conforming the geographic
origin of captive breeding groups of rhesus macaques.
Questions:
1.
Which are more susceptible to experimentally induced SIV infection –
Indian or Chinese macaques?
2.
Which rhesus have greater subspecies diversity?
Answers:
1.
Indian macaques are more susceptible.
2. Chinese macaques have more subspecies diversity.
Neonatal Anesthesia for Studies of Hamster Parental Behavior when Infanticidal
Aggression is a Possibility. pp. 202-208
Infanticidal behavior directed toward unrelated neonatal pups is common in male rodent
species. Adult mice with (PPKO) do not show pup directed aggression. Male Djungarian
hamsters, Phodopus campbelli have an exceptional array of paternal behaviors and were
chosen to study these neuroendocrine mechanisms. Naive male and female Djungarian
hamsters were paired and checked after 18 days for birth and then were separated for
trials. In these trials their three-day old pups were anesthetized with xylazine and
ketamine administered IP. Pups were anesthetized to reduce pain from aggressive
behavior. Control groups received IP injections of saline. Parental behavior was tested
the last three hours of a 14:10 light phase. Testing was randomized with one adult in the
nidal cage and one in a separate cage but still in olfactory and auditory range. The pups
were placed in the cage after anesthesia had taken affect. Pups were evaluated for
movement, pedal reflex, audible vocalization and breaths per minute Then the parents
were evaluated for location and retrieval of unmanipulated pups.
100% of the females and 75% of the males picked up the saline injected pups and
no aggression was noted. Females picked up 93% of the anesthetized pups and retrieved
86% of the pups; however, there was a "testing effect" especially when the anesthetized
pup was presented first. Males picked up 70% and retrieved 60% of the anesthetized
pups; however, retrieval by males were more likely if the anesthetized pup was tested
second. Parental response to control pups (saline injection) was that 100% of the females
and 83% of the males picked up and retrieved them. No aggressive behavior was
exhibited by male and female parents. 100% of the naive males (nonparental) contacted
anesthetized pups, 8 picked up the pups, 45% of nonparental males that retrieved
pups attacked the pups while only 18% of nonparental males that retrieved pupd did not
attack them. In this study movement did not totally cease due to catalytic effect of
ketamine and spontaneous respiration was decreased by 50% while vocalization ceased
indicating anesthesia was reached. This method provides an efficient and effective means
of protecting pups wile allowing adults to express a wide range of parental behaviors. It
has an application in behavioral screening of transgenic strains toward unrelated young.
Questions:
1. What was the hamster used in this study?
2. What part of the female parental experiment had a "study effect"?
3. What are two useful aspects of this study?
Answers:
1. Djungarian, Phodupus campbelli
2. When females were presented the anesthetized pup first.
3. Effective way of protecting pups during these studies and screen transgenic strains
toward unrelated young.
Model of Angiogenesis in Mice with Severe Combined Immunodeficiency (SCID) and
Xenografted with Epstein-Barr Virus-Transformed B Cells. pp. 209-215
The SCID mouse model has been used extensively to evaluate the mechanisms of solidtumor angiogenesis. This study demonstrated the usefulness of this model to study
angiogenesis in B-cell lymphomas. Epstein-Barr Virus (EBV) is a gammaherpes virus
that causes B-cell lymphomas in immunosuppressed people; this mouse model would be
a valid system in which to study angiogenesis of these tumors.
The research group conducted two sets of experiments. The first was to
characterize the tumors. Lymphoblastoid cell lines (LCL) are cells that express EBV
latent proteins. These cells were injected intraperitoneally into SCID mice. Mice were
euthanized when clinical signs of illness were observed. The tumors excised from these
animals were analyzed by 1. immunohistochemistry for endothelial cells; 2. multi-probe
ribonucleic acid protection assay (RPA) for IL-8 and VEGF. In the other set of
experiments, IL-8 antibodies were administered to mice injected with LCL. This was to
determine if IL-8 inhibited lymphoma development.
Results:
1. The 2 angiogenic factors IL-8 and VEGF are present in LCL-SCID B cell
lymphomas.
2. 2. The research group confirmed that angiogenesis does occur during
lymphangiogenesis.
3. Reduction in IL-8 (through the anti-IL-8 antibody) did not cause a difference in
angiogenesis in tumors; it did cause a slight enhancement of tumor growth.
4. There was a strong positive correlation between tumor burden and circulating
serum Ig concentration. This would give researchers a good way of determining
tumor size in cases where tumors are not easily measured (like in the abdomen).
Questions:
1. Characterize the immune system of SCID mice.
2. What are two angiogenic factors in lymphomas?
3. What is a commonly used IHC monoclonal antibody used for detecting
endothelial cells?
Answers:
1. SCID mice display an absence of functional T cells and B cells, lymphopenia,
hypogammaglobulinemia, and a normal hematopoietic microenvironment.
Normal antigen-presenting cell, myeloid, and NK cell functions are strain
dependent. SCID mice carry a DNA repair defect and a defect in the
rearrangement of genes that code for antigen-specific receptors on lymphocytes.
Most homozygotes have no detectable IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA.
Thymus, lymph nodes, and splenic follicles are virtually devoid of lymphocytes.
2. IL-8 and VEGF
3. CD31
Barbering (Fur and Whisker Trimming) by Laboratory Mice as a Model of Human
Trichotillomania and Obsessive-Compulsive Spectrum Disorders. pp. 216-224.
Definitions, background Information, and interesting tid-bits:
·
Animal model validation types for human diseases:
o
Face validity = whether the behavior in animals has the same phenomenology,
demography, and etiology as the human disorder.
o
Construct validity = whether the same underlying processes are involved.
o
Predictive validity = whether treatments in humans can be predicted from the
model, and vice versa.
o
Discriminant validity = whether clinical manifestation show the unique
diagnostic criteria that discriminate behaviors in one disorder from symptoms in
other disorders.
·
Human trichotillomania sufferers repeatedly pluck hair from idiosyncratic body
locations, in particular the scalp, eyebrows, eyelashes, and the pubic region. Most is
self directed, but they will also pluck other individuals, upholstery, pets, and dolls.
·
Some models of human mental disorders ? most clinical signs of disease
manifested by the model are experimentally induced:
o
Models of schizophrenia and learned helplessness models of depression ?
isolation rearing and amphetamine
o
Model of autism ? use of Borna disease virus in neonatal rats
o
Models of obsessive-compulsive disorder (OCD) ? use of 8-hydroxy-2-(di-npropylamino)-tetralin hydrobromide (8-OH-DPAT), a serotonin 5hydroxytryptamine (5HT) 1A receptor agonist and D1CT (where dopamine D 1
receptors are potentiated with a cholera toxin transgene)
·
Other proposed spontaneous models of trichotillomania or OCSD: parrots, dogs,
and horses.
Summary:
This manuscript described a cross-sectional epidemiologic survey of a population of
laboratory mice to assess the face validity of barbering as a spontaneous model of
trichotillomania and obsessive-compulsive spectrum disorders (OCSD). Patterns of hair
loss and demographic and etiologic risk factors were recorded for each mouse then
analyzed via an array of statistical analyses. Of the 1979 mice included in the study,
7.2% are cagemate-barbers, and 0.7% are self-berbers. Whiskers were more severely
denuded by cagemate-barbers; the genitals, ventral surface of the body, and ventral views
of the limbs were more severely denuded by self-barbers. For details on statistical
analyses, please read the manuscript. Risk factors for barbering: 1) age ? onset during
puberty; prevalence increased with age; 2) genetics - background strain as an etiologic
factor ? C57BL/6J mice most likely to barber in the 7 inbred strains evaluated (but
there?s no statistical difference between the inbred strains and outbred stocks evaluated);
3) sex ? female biased; 4) breeding status ? an etiologic factor ? breeders are more likely
to barber. Authors concluded that barbering has many phenomenologic similarities to
those of trichotillomania, and may represent a refined, non-invasive spontaneous animal
model for the complex genetic/environmental etiologies of this disorder.
Questions:
1) True or False: The whiskers and the ventral surface of the neck and body and ventral
views of the limbs are strongly favored by self-barbers.
2) Name a few species proposed as animal models of trichotillomania or OCSD.
3) Name the risk factors for barbering suggested by authors in this manuscript.
Answers:
1)
False: barbering of the whiskers is a behavior seen in cagemate-barbers.
2)
Parrot (i.e. feather plucking), dogs (i.e. acral lick dermatitis), horses (i.e. cribbing).
3)
Age, sex, genetics, and breeding status.