Contemporary Topics 38 (6)


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