Demodex musculi in the skin of transgenic mice. Contemporary
Topics 38 (6): 13.
In mammals, Demodex spp mites are host- and microhabitat-specific.
Demodex has been described in many species including humans, dogs, cats,
cattle, swine, horses, sheep, goats, gerbils, golden hamsters, and rabbits.
Four to five species have been identified in mice where they have been
found in the stratified epithelium of the oral cavity, tongue, and esophagus,
and from ears, skin, eyelids, and preputial and clitoral glands. It is
still unknown how Demodex organisms are transmitted between hosts and if
transmission involves adult or immature stages.
In this case report, D. musculi was found in the skin of B6,CBA-TgN(CD3E)26Cpt
mice (a homozygous transgenic mouse lacking mature T lymphocytes and NK
cells and B6,SENCARB-TgN(pk5prad1)7111Sprd mice (a transgenic mouse overexpressing
human cyclin D1 and manifesting a severe thymic hyperplasia). No mice were
exhibiting clinical signs of illness. Prior to this, D. musculi had only
been identified in one case report in Great Britain in 1917.
Diagnostics:
^Å Negative on cellophane tape test. This is likely due to the
superficial location of mites in hair follicles.
^Å Sometimes positive by examining (under a dissecting microscope)
plucked hairs from the dorsal aspect of the neck
^Å Sometimes positive by histological exam. Mites were seen only
in superficial hair follicles. Minimal to no inflammatory reactions were
noted around affected hair follicles. Therefore, difficulty exists in definitively
identifying animals as infected or not infected.
D. musculi was transmitted to other strains of mice when they were
housed with infected individuals.
No questions
The influence of euthanasia methods on rat liver metabolism. Contemporary
Topics 38 (6): 19.
8 week old SD rats were euthanatized by either isoflurane anesthesia
(1.5L/min O2 and 3% isoflurane for 3 minutes) and exsanguination, carbon
dioxide (100% for 2.5 minutes) asphyxiation; or 100% oxygen for 1 minute
followed by CO2 (100% for 2.5 minutes) asphyxiation. After euthanasia,
livers were rapidly removed and frozen in liquid nitrogen until assayed
for glycogen phosphorylase, glycogen synthase, pyruvate dehydrogenase,
branched chain amino acid dehydrogenase, phospho-1-fructokinase, fructose-6-phosphate,
ATP, pyruvate kinase, phosphoenolpyruvate, L-alanine, cAMP, inositol triphosphate,
glucose, glycogen, and fructose 2,6-bisphosphate. (There is a nice chart
on page 20 of how these all interrelate). The enzymes measured in this
study were chosen because they are susceptible to changes in extracellular
hormones and intracellular energy status. Values from animals euthanatized
by decapitation were used as baseline for comparisons.
Glycogen breakdown and glucose utilization enzymes were stimulated
by all euthanasia methods compared. Glycogen stores decreased by 50% in
animal euthanatized by carbon dioxide or O2/CO2 method but not isoflurane.
Enzymes utilized at the start of the glycolytic pathway were stimulated
after CO2 or O2/CO2 euthanasia but terminal glycolytic enzyme was stimulated
only by O2/CO2 euthanasia. Euthanasia by CO2 or O2/CO2 decreased the regulatory
enzyme of branched-chain amino acid degradation.
Questions
1. According to the 1993 AVMA Panel on Euthanasia, what is one disadvantage
of using CO2 as a euthanasia agent/method.
2. True/false. Carbon dioxide euthanasia can be accomplished humanely
by using compressed CO2 gas as can be obtained from a cylinder or fire
extinguisher, or by use of dry ice to generate CO2.
3. What is the recommended body weight limit for use of inhalant anesthetics
for euthanasia?
4. Arrange the following in order of preference for acceptable euthanasia
agent in small animals: enflurane, ether, isoflurane, methoxyflurane, halothane.
5. Name one disadvantage of inhalant anesthetics as euthanasia agents.
Answers:
1. CO2 is heavier than air, therefore incomplete filling of the chamber
may permit taller or climbing animals to avoid exposure and to survive;
This adds to the animal distress. Also, some species have extraordinary
tolerance to CO2.
2. False - fire extinguisher is unacceptable.
3. < about 7 kg
4. halothane, enflurane, isoflurane, methoxyflurane, ether
5. Struggling and anxiety may develop during induction because of,
for example, irritation to mucous membranes. Ether is flammable and explosive.
Personnel can be injured by exposure to these agents.
Influence of blood collection sites on plasma glucose and insulin
concentration in conscious C57BL/6 mice. Contemporary Topics 38 (6): 25.
This article compared the plasma glucose and insulin concentrations
in blood samples collected from two sites, retroorbital sinus versus tail
vein, and also determined if the two sites could be used alternately within
the same study. Blood collection from conscious, unanesthetized mice is
often performed during the drug development process. Since people tend
to use the tail vein alot for small quantities of blood collection the
tail may after awhile be rendered useless for blood collection, thereby
leaving investigators using other routes such as retroorbital sinus for
blood collection. The question then becomes can one compare data collected
from two different blood collection sites reliably. Two experiments were
performed: in the first experiment the retroorbital and tail bleeds were
performed a few minutes apart and in the second one they were performed
a week apart. The retroorbital yielded lower glucose levels and higher
insulin levels than did tail vein collection. For an individual mouse the
difference between measured glucose in a retroorbital collection versus
a tail vein collection under the same conditions is likely to be in the
interval (-97,98)mg/dl. The insulin level is likely to be in the interval
(-0.6, 7.3)ng/ml. The acceptability of switching blood collection sites
within a study depends on the amount of variablility that is tolerable
within a study. On average, the retroorbital glucose values for this experiment
were 11% lower than the tail vein glucose values and the retroorbital insulin
values were about 40% higher than the tail vein insulin values. Therefore,
for glucose levels this may be an acceptable technique but the insulin
values appear quite different for the two techniques.
Questions:
1) When comparing the two bleeding sites (retroorbital vs tail vein)
which statement is true?
A) glucose and insulin values were the same
B) glucose was lower for retroorbital vs tail vein
C) glucose and insulin were lower for retroorbital vs tail vein
D) insulin values were lower for retroorbital sinus vs tail vein
Answer B
A low-cost computerized system to monitor running performance and
circadian rhytms of twenty mice simultaneously. Contemporary Topics 38
(6): 29.
The purpose of this article was to describe a low-cost computerized
system for monitoring the voluntary activity of mice on running wheels.
The article lists the internet address for obtaining the software. The
software functions with IBM-compatible computers. A running wheel was attached
to the lid of each mouse cage. The article gives the details of the computer
requirements as well as the details of the system setup.
To demonstrate the system's performance, two applications of the system
were conducted. First, the circadian rhythms of wild type C57BL/6 mice
were followed as the light cycle of the room was altered. A readout produced
by this computerized monitoring system demonstrated that the mice on a
daylight-night schedule were active (ran on the exercise wheel) during
the night while they rested during the day. The mice were then exposed
to constant darkness; the mice maintained same circadian rhythm under these
conditions. Next, the mice were readjusted to a normal daylight-night schedule
but with a 12-hour phase difference compared with the initial daylight-night
schedule; these mice gradually shifted from daytime to nightly activity
due to the effect of light on the biological clock.
The second application of the system involved the running performance
of wild type vs. knockout mice having symptoms which mimic muscular abnormality
glycogen storage disease type II; this disease results in muscle weakness.
As mentioned above, these mice were most active during the night and rested
during the day. However, the knockout mice tended to perform much worse
(much less active on the exercise wheel) than the wild type mice. This
test was effective in discriminating knockout vs. wild type mice.
Questions
Q1: The application described is an effective, low-cost system to monitor
rodent activity applicable to many areas of research. T/F
Q2: The application described uses software designed to run on an IBM-compatible
computer to measure the voluntary activity of rodents on an exercise wheel.
T/F
Answers
A1: True
A2: True
Direct laryngoscopy in Mice. Contemporary Topics 38 (6): 33.
The authors describe a direct laryngoscopy technique suitable for use
in mice as small as 20g, utilizing a pediatric otoscope.
Key points to the method are:
1. non-invasive
2. must use deep level of anesthesia - authors used ketamine/xylazine/ace
cocktail
3. used 2mmspeculum on pediatric otoscope mounted on ringstand so operators
hands are free
4. mouse's tongue is rolled out with cotton swab, then extended by
grasping with fingers. mouse is positioned by pulling on the loose dorsal
skin.
5. speculum is guided past soft palate and base of tongue, then vocal
cords are visualized by adjusting head and tongue position. Experimental
substances (~50 microliters) are administered using a gel-loading pipet
tip.
Advantages:
1. minimal risk of trauma because of direct visualization
2. non-invasive - can be performed multiple times on same animal if
necessary
3. takes less time than tracheostomy
4. relatively easy to become proficient
No questions
An atypical case of Mycobacterium bovis in a cynomolgus macaque
(Macaca fascicularis) imported from the Philippines. Contemporary
Topics 38 (6): 36.
This article profiles an unusual case of Mycobacterium bovis. 100 male,
purpose-bred, cynomolgus macaques were recieved in a shipment. One juvenile
male was presented with lethargy, incoordination, and weakness one month
after arrival. From the history, physical exam, clinical chemistry and
hematology, a tentative diagnosis of melioidosis was made and the animal
was euthanized. This animal and all cohort animals from the shipment had
completed two negative intradermal skin tests for TB. Samples from the
necropsy were submitted for histopathology and culture according to routine
necropsy procedures for quarantine animals.
Cultures and histopathology results were conclusive for M. bovis. The
remaining cohort animals and the 15 native-born animals were then euthanized
and necropsied. Two animals were positive for M. avium. The authors conclude
that this disease presentation underscores the importance of follow-up
exams and cultures.
QUESTIONS:
1. Macaques are sussceptible to:
a. M. bovis
b. M. avium
c. M. tuberculosis
d. all of the above
2. The main concern about M. bovis is the fact that it is a _____________
disease.
ANSWERS:
1. d
2. zoonotic
Pyometra in a Siberian Pole Cat. Contemporary Topics 38 (6): 39.
Case report of pyometra in a siberian polecat (Mustela eversmanni)
the closest living relative to the black-footed ferret (Mustela putorius).
It is a research model for the endangered black-footed ferret. The Siberian
polecat is also used to study infectious disease susceptibility and prophylaxis
to canine distemper and plague, to assess the safety of anesthetic regimes,
for reproductive studies, and to explore reintroduction-related issues.
Pyometra is a reproductive disorder occurring in domestic carnivores, it
has not been previously reported in Siberian polecats, but has been documented
in black-footed ferrets. Clinical signs include lethargy, anorexia, polydipsia,
polyuria, abnormal vaginal discharge, abdominal pain or discomfort, hypothermia
or fever, and an elevated white blood cell count. If detected early enough
medical therapy may be attempted if an appropriate antibiotic therapy can
be identified. However, surgical intervention, with removal of the septic
uterus, is the treatment of choice. Gestion length for this species at
this facility was 38 +/- 0.5 days. The animal was part of a breeding colony.
Euthanasia was performed on the animal because it was nonreproductive and
might yield information relevant to the breeding colony as a whole. Necropsy
revealed a distended fluctuant uterus containing mildly odiferous, thick,
yellow-green, purulent material. Histopathology confirmed the diagnosis
of pyometra. A pure, heavy growth of Enterococcus fecalis was cultured
from the uterine contents, it was resistant to all antibiotic tested via
the MIC antibiotic sensitivity screening.
No questions
Rhabdomyosarcoma associated with incisor malocclusion in a laboratory
rat. Contemporary Topics 38 (6): 42.
Case report of a Long Evans rat noted for rapid weight loss. On examination
, the rat was found to have elongated incisors as a result of malocclusion.
Gingival erosion and hyperplasia were noted. The teeth were trimmed, but
over the subsequent 6 weeks, a raised circumscribed mass formed at the
midline of the mandible.
Following euthanasia and necropsy, the mass was found to be neoplastic
and composed of pleomorphic cells which were fusiform to polyhedral in
shape. The cytoplasm of some cells was eosinophilic with cross-striations
typical of skeletal muscle. These cells were often elongated with a strap-like
morphology. The striations were best seen with PTAH (phosphotungstic acid
hematoxylin) staining.
The diagnosis of rhabdomyosarcoma was based on the characteristic striated
cell morphology in the"strap cells" seen in this lesion which are classically
associated with rhabdomyosarcoma. The etiology of the neoplasia is theorized
to be associated with the constant trauma to the gingiva from the malocclusion.
Note: The gross and histological figures would be "classics" for the
practical - know them!
Questions:
1. What is the phylogeny of the rat?
2. What is the dental formula of a rat?
3. Which teeth are open-rooted?
4. What does PTAH stand for?
Answers:
1.Family: Rodentia
Order: Myomorpha
Suborder: Muridae
Genus: Rattus
Species: norvegicus
2. 1 0 0 3
1 0 0 3
3. incisors
4. Phosphotungstic acid hematoxylin
Diarrhea in neonatal rats. Contemporary Topics 38 (6): 44.
A litter of eight 10-12day old SPF Sprague Dawley rat pups presented
with runting, rough, yellow-discolored hair coats, perineal scalding and
soiling, and epidermal ulcerations on their rear legs. The dam also had
soft stools and perineal soiling. All animals were normally active. The
bedding was excessively wet and dirty and no formed fecal pellets were
evident.
One additional dam and litter in another cage in the same cubicle presented
with similar signs.
Infectious differentials included:
Tyzzer's disease (Clostridium piliforme)
Salmonellosis (S. enteritidis, S. typhimurium)
Streptococcal enteropathy (Enterococcus faecium durans II, Enterococcus
hirae,
Enterococcus-like agent
Infectious Diarrhea of Infant Rats (Rotavirus-like agent, Reovirus
3)
Endoparasites (Spironucleus muris, Giardia muris)
Diagnostic Tests & Results
CBC - nothing significant
Serology - nothing significant
Necropsy - pups had full stomachs, ileum, cecum, and colon were distended
with gas and contained yellowish fluid. Dam's stomach was empty and there
were no feces in the intestines. One gross pathology figure presented.
Histopathology - increased numbers of cocci adherent to villous tips
predominately in the duodenum and in other segments of the intestines of
the dam and pups. Villi were of normal length. One histopathology figure
presented.
Bacteriology - Aerobic cultures from small intestines and liver on
Blood, Chocolate, and MacConkey Agar. Smooth small grey alpha hemolytic
colonies grew on blood and chocolate agar within 18h.
Identicult - AE Kit - Group D Enterococcus identified as causative
agent
Treatment: Cubicle was depopulated
Discussion: Another study showed that pups became resistant to infection
with E. hirae after 5days. The present report differs in that the adult
dams were also affected. Enterococcus-like organisms are much more slower
growing (>24 h) than the case presented here. Identification can be performed
with an identification kit as performed here or by the ability of Enterococcus
to grow in the presence of 6.5% NaCl and produce bile esculin.
Questions:
1. Provide 3 bacterial differentials for diarrhea in neonatal rats.
2. Provide 1 viral differential for diarrhea in neonatal rats. Name
2 etiologies of this differential.
3. Provide 2 parasitic etiologies for diarrhea in neonatal rats.
Answers:
1. Tyzzer's disease, Salmonellosis, Streptococcal enteropathy
2. Infectious Diarrhea of Infant Rats
2 Rotavirus-like agent, Reovirus 3
3. Spironucleus muris, Giardia muris
Peritonitis in nude mice in a xenograft study. Contemporary Topics
38 (6): 47.
Thirty SIM: (NCR) nu/nu female mice were anesthetized IP with 2.5%
tribromoethanol (500 mg/kg) in preparation for surgical orthotopic implantation
of mammary tumour cells. Although animals recovered uneventfully in the
immediate post-op period, over the ensuing 6 days, 24/30 animals died or
were euthanized in moribund condition. Animals selected for necropsy demonstrated
fibrinous peritonitis characterized by abdominal organs with fibrinous
+/- edematous serosal/capsular thickening with neutrophilic infiltrates
and reactive spleens and mesenteric lymph nodes. Multiple species of opportunistic,
predominantly gram negative bacteria (eg, E coli, P mirabilis) were cultured
from the abdomens of affected animals. Epidemiologic considerations suggested
the tribromoethanol solution as the inciting agent, despite negative culture
of the solution itself. Intraperitoneal injections of the same tribromoethanol
solution into 3 additional immunocompetent animals resulted in similar
bacterial peritonitis developing in 2/3 animals, confirming this hypothesis.
Questions:
1) Tribromoethanol must be stored in the dark at ambient temperature
to ensure stability (T/F)?
2) If crystals are noted in the tribromoethanol solution, the solution
should be gently warmed to dissolve them, prior to injection (T/F)?
Answers:
1) F - stored in the dark at 4C.
2) F - crystals indicate presence of decomposition products that can
be irritating when injected IP.
The Polymerase Chain Reaction (PCR). Contemporary Topics 39 (6):
50.
This article is a one page illustrated summary of PCR, a technique
that amplifies a small amount of DNA into a large amount by repeating three
steps:
denaturation, annealing, and synthesis
1) denature by heating to 95 C
2) annealing oligonucleotide primers
3) synthesis - Taq DNA polymerase adds complementary bases to the single
strand template DNA
Repeat 20-30 cycles, visualize results with gel electrophoresis and
ethidium bromide staining
Fact 1 (instead of a question): Taq DNA polymerase is derived from
Thermus aquaticus, a heat stable bacterium
Fact 2: Southern blot analysis is an alternative technique
Advantages of PCR over Southern blot:
1. high sensitivity due to exponential amplification
2. high specificity due to specificity of primers
3. rapid results, in one working day
Disadvantages:
1. small amt of contamination can produce false positive results
2. expen$ive
3. inhibition of PCR reaction can lead to false negative results
No questions