Mouse Sperm Cryopreservation: A Legacy in the Making. Laboratory Animal Science 48 (3): 224.
An editorial. The explosion of transgenic technology in mice has led to an almost overwhelming number of mouse models, transgenes and knock outs on different genetic backgrounds. The new problem is how to preserve and maintain so many different genetic lines. While zygotes and later stage ova are routinely cryopreserved, this still represents a large committment of labor and resources. The author points out that mouse sperm cryopreservation is a logical complement to the solution.  While mouse sperm was first frozen during the 1970s, it was not until the 1990s that acceptable post-thaw motility and fertility was achieved.  Technical advances and refinements have been made to allow freezing (followed by successful thawing and fertilization) of mouse sperm - both ejaculated and epididymis-derived. Reference is made to an article by Songsasen & Leibo in the same issue wherein live offspring were achieved.  The author feels that this new techology will allow the plethora of mouse lines to be maintained on a large-scale yet cost-effective basis.
Questions:
1. Name 2 drawbacks to prior attempts to utilize mouse sperm
cryopreservation.
2. Name 2 factors previously identified as problems involved in poor
post-thaw fertility.
Answers:
1. Mice had to be killed in order to obtain sperm. Post-thaw fertility
was unacceptably low.
2. Strain specificities(physiologic or biologic), decreased motility,
cryoprotectant toxicity, improper timing of freeze and/or thaw cycles,
freezing induced trauma.

International Conference on Emerging Infectious Diseases. Laboratory Animal Science 48 (3): 225.
This article summarized the three day international conference on emerging infectious diseases. Manuscripts submitted by the speakers will be published in the Emerging Infectious Diseases journal, available as a hard copy from the National Center for Infectious diseases or electronically at http://www.cdc.gov/ncidod/EID/eid.htm.
Day 1
Vector-borne infectious diseases, malaria, Lyme boreliosis, plague, dengue fever and dengue hemorrhagic fever, and yellow fever, are a huge problem around the world. Insecticide resistance, therapeutic drug resistance, and declining economic, social, and political support for control programs are the main factors responsible for the presence and/or re-emergence of these diseases.
Day 2
The U.S. is not thoroughly prepared for incidents involving the use of biologic weapons.
There has been a recent emergence of monkeypox in the democratic Republic of the Congo.
Encephalitozoon hellum has been isolated from Psiticines (including love birds), parrots and budgerigars, and there was a concern that immunosuppressed humans may be at risk for infection.
Equine morbillivirus, lyssavirus (rabies-related virus), and a paramyxovirus have been identified in fruit bats of the genus Pteropus. Based on genetic and protein analysis, the CDC suggested that the equine morbillivirus be removed from this taxon, and be named Hendravirus (based on the location of the 1994 outbreak).
Day3
No evidence of transmission of Bovine spongiform encephalopathy (BSE) to dogs fed raw meat; however, domestic cats, cheetahs, pumas, ocelots, and tigers fed raw meat were identified as carriers. Since the BSE outbreak, several cases of the so-called new variant Creutzfeld-Jacob disease have been identified in humans. The amount of infected cow brain necessary for the transmission of BSE to sheep is .5g , cows require 1g.
Chickens infected during the Hong Kong H5N1 influenza sudden death. 100% mortality was not uncommon for most of the chicken farms. Hong Kong^Òs surveillance program and quick response time prevented a major catastrophe.
Useful Acronyms
CDC = Centers for Disease Control
ASM = American Society of Microbiology
NCID = National Center for Infectious Diseases
QUESTIONS:
1. Encephalitozoon hellum is a:
A. Fungus
B. Protozoa
C. Bacteria
D. Virus
2. Which of the following statements is false:
A. Monkeypox is an enveloped DNA virus
B. Macaca mulata is the most significant host and reservoir for monkeypox
C. Humans can be infected with monkeypox
D. Monkeypox belongs to the genus Orthpoxvirus
3. Bovine Spongiform Encephalopathy is caused by a:
A. Virus
B. Bacterium
C. Prion
D. Fungus
ANSWERS:
1. B
2. B
3. C

Xenotransplant-Associated Infections. Laboratory Animal Science 48 (3): 228.
    Candidates for allotransplantation procedures continue to grow with no concurrent increase in the number of donors. The insufficient supply of human donors has increased research in the use of animal organs (xenotransplantation)in humans for the treatment of Parkinson's disease, diabetes mellitus, and acquired immunodeficiency disease. The primary problem of using animal tissues besides immunologic barriers are infections. These infections can be from endogenous flora or they can be transmitted within the donated tissue.
    Many infections attributable to human organs are predictable microbial agents maintained in latent or intracellular state such as herpesvirus, retrovirus, and intracellular parasites. Human cytomegalovirus (HCMV) and Epstein Barr Virus (EBV) are the best described herpesviruses that produce donor associated infections; however, other herpesviruses such as herpes simplex (HSV) and varicella zoster virus (VZV) do not present a problem. Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and hepatitis C virus (HCV) have been unintentionally transmitted after allotransplantation of donor organ or blood. Other infective agents that have been transmitted are parasites such as Toxoplasma gondii that harbors the latent cyst form which has a tropism for cardiac muscle. To prevent these infections from occurring, better screening methods need to be used.
    Xenozoonosis may occur when a non-pathogenic organism reacts and becomes pathogenic. Raising animals in controlled environments may help prevent transmission of acute infections. Another problem is that "species specificity" may be able to be overcome by the closeness in the human body and the immunodepressed state of the recipient. One such example is the alpha-herpesviruses Herpes simiae which is more severe in a foreign host and is fatal to humans.
Baboons and swine have been the two major animal sources considered for xenotransplantation. Baboons do not harbor Herpes B virus but have an analogue virus simian agent 8 (SA8) which is not pathogenic in man.  Swine have an alpha herpesvirus, pseudorabies, which has produce pathogenic problems in three immunocompetent humans. Baboons and swine have their own herpesviruses similar to HCMV and EBV. Some strains of CMV are similar to those seen in African Green monkeys but have not produced a pathogenic condition.  Transmission of retroviruses such as Simian Immunodeficiency Virus (SIV) is similar to Human Immunodeficiency Virus-2. Another retrovirus, Simian T-Lymphotrophic Virus (STLV), as well as SIV are associated with lymphomas in infected primate populations.  Little is known about retroviruses of swine. Other microbial organsims such as encephalocarditis may cause disease after xenotransplantation.  Parasites such as E. histolytica, Schistoma, T. gondii, and Babesia could be harbored in transplanted tissue and produce problems in immunocompromised recipents.
    Prevention of disease conditions may be reduced in animals providing transplantable organs and tissue when animals are raised in a germ-free environment. Pigs have a predictable gestation period and large number of offspring making a viable animal for a limited time period before increased waste and poor growth become a problem. SPF conditions are difficult to produce in baboons due to a variable gestation period and a single offspring. Another factor which can be a problem are diseases introduced by human care givers. Strict monitoring programs should be followed including records of vaccinations, documented linages, disease monitoring and diet products due to the spread of "mad cow " disease.  The diseases which should be excluded from these animals should be determined and then classified. Absolute contradictions are diseases known to be zoonotic and hazardous to humans. Another category of disease is "organisms with a potential for producing secondary disease to others." These secondary organisms include SIV, STLV, T. gondii, M. tuberculosis, and herpes B. A third category of disease is unavoidable infections that include baboon endogenous virus and all organisms not yet recognized. Serologic testing for these diseases have taken advantage of cross-reactivity making interpretation of test results difficult. Also cross-reactivity may not be the same in all cases.
Questions:
1. What are two common herpesviruses that produce infection in allotransplants?
2. What are the two most common species used for xenotransplantation?
3. How are xenozoonosis conditions reduced?
4. What disease classification group could become the worst one of the three mentioned?
Answers
1. HCMV and EBV
2. Swine and Non-human primates - baboons
3. Prevention of disease conditions may be reduced in animals providing transplantable organs and tissue raised in a germ-free environment.  Strict monitoring programs should be followed including records of vaccinations, documented linages, disease monitoring and diet products due to the spread of "mad cow " disease.
4. The disease group that includes the baboon endogenous viruses and all organisms not yet recognized.

Antigenic Analyses of Cilia-Associated Respiratory (CAR) Bacillus Isolates by use of Monoclonal Antibodies. Laboratory Animal Science 48 (3): 234.
Car bacillus has been reported in healthy and diseased respiratory tract epithelium from a variety of animals and infection has been associated with increased morbidity and mortality of laboratory rats and mice.  Methods of diagnosing CAR bacillus have been, culture, histological exam, serology and PCR. Though PCR is rapid, specific and sensitive it is not ideal for routine testing. Therefore a serologic test using monoclonal antibodies was developed. This method is more specific and sensitive than the previous serologic tests.  CAR bacillus is antigenic diverse between different hosts and even within the same host species. With the the development of the monoclonal antibodies the investigators able to develop reagents which specifically reacted with rat CAR bacillus. The 16 S rRNA analyses shows that the rabbit and the rat CAR bacillus are markedly different.
Questions/Answers:
1. What is the reason(s) for the false positive results in the ELISA assay
to detect CAR bacillus infection?
Both the intact and the sonicated preparations of CAR bacillus propagated
on the 3T3 cells contain many non CAR bacillus epitopes and probably
contribute to false positives.
2. Which of the following peptides 41, 50, and 105 KDA represent antigens
suitable for development of rat CAR bacillus specific serologic assay.
All three (41,50, and 105 peptides).

Mucosotropic Papillomavirus Infections. Laboratory Animal Science 48 (3): 240.
ETIOLOGIC AGENT: Canine oral papillomavirus (COPV)
HOST RANGE: Dogs (Canis familiaris), coyotes (Canis latrans), and wolves (Canis lupus)
GROSS LESIONS: Single or multiple, white to pink in color, firm exophytic growths on the tongue, buccal mucosa, and larynx
HISTIOLOGIC LESIONS: Noninvasive, exophytic proliferation of epithelial cells, which progress through an orderly maturation sequence. Basophilic keratohyalin-like granules, and amphophilic intranuclear inclusions may be present in tissue sections. Papillomas may progress to squamous cell carcinoma.
DIAGNOSIS: Histology, and Immunohistochemistry
COMPARISON WITH HUMAN DISEASE: Except for the absence of intranuclear inclusions in human papillomavirus- induced lesions, the human mucosotropic papillomavirus (HPV) has similar gross and microscopic features when compared to the COPV. HPV and COPV induced papillomas may progress to Squamous cell carcinoma.
USEFULNESS OF COPV AS A MODEL: Development of antiviral and vaccine strategies
QUESTIONS:
1. What type of genome does the papillomavirus have:
A. + RNA
B. - RNA
C. ssDNA
D. dsDNA
2. What viral family does COPV belong to:
A. Poxviridae
B. Papovaviridae
C. Herpesviridae
D. Hepadnaviridae
3. T or F. A live COPV vaccine, developed from fresh homogenates of oral papillomas, may cause a squamous cell carcinoma at the injection site.
ANSWERS:
1. D
2. B
3. True

Familial Megacecum and Colon in the Rat: A New Model of Gastrointestinal Neuromuscular Dysfunction. Laboratory Animal Science 48 (3): 243.
The authors describe a new mutant stock of rats that develops severe megacecum and colon with pseudo-obstruction shortly after weaning. The syndrome has been designated "Familial Megacecum and Colon (FMC)" and the mutant gene Fmc. Familial megacecum and colon is inherited as an autosomal recessive trait. It is characterized by a markedly dilated cecum and proximal colon followed distally by a short funnel shaped segment and distal portion of the colon with normal or slightly reduced lumen. The funnel zone marks the transition from the normally functioning proximal portion of the colon to the dysfunctional distal colon which inhibits normal flow. If allowed to progress, abdominal distention, perineal straining and diarrhea were observed and ultimately, the pups died from cecal or proximal colonic rupture. The exact cause of MC is still unknown.
Hirschrung's disease is the most common developmental motility disorder of the colon and is generally detected in the neonatal period. It is characterized by an absence of ganglion cells and numerous hypertrophied nerve bundles. This results in a contracted distal aganglionic segment resulting in pseudo-obstruction and dilation of the proximal segments. Although the clinical features and gross anatomical changes of the colon resemble Hirschprung's Disease, aganglionsis is not a feature of FMC.
The article has two illustrative photographs; one shows the exposed abdominal organs of an affected rat side by side with a normal control. The other photograph shows the gastrointestinal tract of a FMC rat showing the dilated cecum and colon, the funnel, and the distal constricted colon.
Questions
1. What section of the gastrointestinal tract is diseased in FMC rats?
A. The proximal dilated cecum and proximal colon
B. The funnel shaped section
C. The distal colon
D. The ileocecal junction
2. Which one of the following statements is false?
A. Hirschprung's Disease in people and animals and FMC rats are grossly similar in that both have a dilated cecum and proximal colon, a funnel shaped section and a distal colon with normal or slightly reduced lumen.
B. Hirschprung's Disease and FMC are both characterized by aganglionosis.
C. The genotype of unaffected heterozygous carriers of FMC is (fmc/+).
D. The genotype of affected rats with FMC is (fmc/fmc).
Answers
1. C
2. B FMC does not have aganglionosis.

Effect of Feeding of a Cholesterol-Reducing Bacterium, Eubacterium coprostanoligenes, to Germ-Free Mice (B). Laboratory Animal Science 48 (3): 253.
Eubacterium coprostanoligenes is a newly isolated bacterium that has previously been shown to decrease plasma cholesterol levels in hypercholesterolemic rabbits following oral administration. The mechanism of the decreased plasma cholesterol is by its conversion to coprostanol, which is minimally absorbed by the gastrointestinal tract. This paper describes the investigation of germ-free mice (Swiss Webster) as an alternative animal model to investigate the effect of orally administered E. coprostanoligenes on serum cholesterol levels. In a preliminary study, the authors observed that the serum cholesterol levels of germ-free mice were normally higher than of conventionally raised mice fed the same diet (similar observations have been reported in rats). Following a 2 week acclimation period, germ-free mice were fed 0.5 ml (0.5mg bacterial protein) of either killed E. coprostanoligenes (control group) or active E. coprostanoligenes (test group) for 1 week. Serum cholesterol concentrations and fecal coprostanol-to-cholesterol ratios were determined for control and test mice. Results revealed that orally administered E. coprostanoligenes (before and after administration of the bacterium; intragroup comparison) significantly decreased (11%) serum cholesterol concentrations and significantly increased the fecal coprostanol-to-cholesterol ratio.  Following cessation of administering E. coprostanoligenes, both serum cholesterol and fecal coprostanol-to-cholesterol levels returned to baseline. No effects were observed in control mice.
Questions:
1. Eubacterium coprostanoligenes converts cholesterol to ___________, which is poorly absorbed from the gastrointestinal tract.
2. T or F: Germ-free mice and rats tend to have higher serum cholesterol levels than of conventionally raised animals fed the same diet.
Answers;
1. coprostanol
2. True

Age-Related Changes in Blood Pressure in the Senescence-Accelerated Mouse (SAM): Aged SAMP1 Mice Manifest Hypertensive Vascular Disease (B). Laboratory Animal Science 48 (3): 256.
The senescence-accelerated mouse (SAM) was developed through selective inbreeding of the AKR/J strain. Nine (9) senescence-prone (SAMP) and three (3) senescence-resistant (SAMR) strains have been developed. All SAMP mice have early onset of aging, but each strain differs in its overall phenotype.  Examples of different phenotypes include senile amyloidosis, impaired immune responses, senile osteoporosis, learning and memory deficits, and emotional disorders. This paper described age-related changes in systolic blood pressure in 2 senescence-prone (SAMP 1 and 8) and 1 senescence-resistant (SAMR 1) strains. SAMR1 mice did not exhibit any change in blood pressure up to 27 months of age. In contrast. SAMP1 mice exhibited significantly increased blood pressures at 11-14 months of age and some of the mice had cardiac hypertrophy and arteriolar fibrinoid necrosis.  Prior to the increased blood pressure in SAMP1 mice, progressive renal changes were evident, suggesting that the increased blood pressures were secondary to renal pathology. SAMP8 mice exhibited decreased blood pressures as they aged, which the authors attributed to observed spongy degenerative changes and astrogliosis in the medulla oblongata.
Questions:
1. Senescence-accelerated mice (SAM) were developed through selective inbreeding of the _____ strain.
2. For each SAM strain, state whether there is an increase, decrease or no effect on blood pressure as the animal ages.
i. SAMR1
ii. SAMP1
iii. SAMP8
Answers:
1. AKR/J
2. i. No effect
ii. Increase
iii. Decrease

Growth Variation in Common Marmoset Monkeys (Callithrix jacchus) Fed a Purified Diet: Relation to Care Giving and Weaning Behaviors (B). Laboratory Animal Science 48 (3): 264.
This study examines the relationship between infant care and weaning behaviors and growth in a population of captive marmosets.
Results:
1) Infants that were not being carried as fequently by an adult marmoset during the first three weeks of life tended to gain less weight in the following three weeks.
2) Marmosets with heavier birthweights tended to be carried more often.
3) At 3-6 weeks of age, the number of times infants suckled was positively correlated to weight gain during that time period.
4) The more time infants spent eating solid food during weeks 3-6, the lower their weight gain and age specific weight during the 6-10 week period.
5) There were no correlations between being carried or weaning behavior early in life and day 75 knee-to-heel length. Infants that where off carriers more (0-21 days) were leaner.
6) There was a growth catch up period. Animals that ate solid food more frequently at an earlier age put on more weight later (days 120-220).
7) Abused and nonabused infants did not significantly differ in any growth parameter. Abused infants tended to have lower birthweights than nonabused infants.
Questions:
1) Common Marmoset, Genus and Species:
2) Which of the following is/are true:
A: Infant marmosets with lower birthweights tend to be carried more.
B: Abused infant marmosets tend to have lower birthweights
C: Infant marmosets which spend more time eating solid food early (weeks 3-6) in life tend to have lower age specific weights at 6-10 weeks of age
D: Infant marmosets which spend more time eating solid food early in life (weeks 3-6) tend to gain more weight than their cohorts during days 120-220.
E. Marmosets reproduce by binary fission.
Answers:
1) Callithrix jacchus
2) B, C, D

Effects of Single and Multiple Injections of Ketamine Hydrochloride on Serum Hormone Concentrations in Male Cynomologous Monkeys. Laboratory Animal Science 48 (3): 270.
The purpose of this study was to determine if the use of ketamine was an appropriate method of restraint in the study of reproductive hormones in male cyno's. Serum cortisol, testosterone, bioactive luteinizing hormone (BioLH), and immunoreactive luteinizing hormone (ILH) were measured in two groups of monkeys. ( BioLH represents hormone-specific receptor binding while ILH represents hormone-specific antibody binding. In this study BioLH levels were greater than ILH levels.)  Group 1 was used to "determine short-term effects". Eight monkeys were given 4 successive doses of IM ketamine at 0 (10mg/kg), 30 (5mg/kg), 60 (5mg/kg), and 110 (5mg/kg) minutes. Venous blood samples were obtained at 0, 15, 30, 45, 60, and 120 minutes. The authors found significant differences between 0-time values (obtained prior to ketamine injection) and later time values in individual animals. However, values increased in some animals and decreased in others so that when the data were pooled no significant differences were seen.   Group 2 was used to "determine long-term hormonal variation ... after a single dose ..." . Actually 10 monkeys were given 10mg/kg ketamine IM once a week for 4 consecutive weeks and a blood sample taken 5-10 minutes post-injection. The authors found no significant difference in any hormone at any time for each individual monkey (intra-individual) but did find significant differences for all hormones among individuals (inter-individual).  The authors note that some but not all previous studies agree with their results. Still they conclude that unbiased data on these hormones can be obtained from ketamine-anesthetized monkeys.
No questions.

Live Mice from Cryopreserved Embryos Derived In Vitro with Cryopreserved Ejaculated Spermatozoa (B). Laboratory Animal Science 48 (3): 275.
This is a very interesting paper which deals with one technique of cryopreserving (mouse) germ plasm from genetically valuable animals as an alternative to maintaining breeding colonies. Males were mated to females which were then killed and the ejaculated sperm collected by flushing the uterine horns. After mating, the males were also killed and spermatozoa were collected from the minced caudae epididymides. The ejaculated spermatozoa were then compared to the epididymal spermatozoa of the same male with respect to viability, motility, and kinetics of fertilization. Samples of each were also cryopreserved, later thawed and reevaluated for viability, motility, and in vitro fertilization ability. In the freshly obtained specimens, no significant differences seen in the concentration, viability, or motility between the two collection methods. Since the spermatozoa must undergo the process of capacitation before they are capable of fertilizing the oocyte, the investigators sought to determine if there would be some difference in the time required for sperm from the two sites to fertilize oocytes in an in vitro fertilization assay. Although differences in fertility were clearly seen between individual mice, the kinetics of fertilization were not different for the two types of spermatozoa. The percentage of viable sperm in frozen samples dropped approximately 50% for both after thawing. The % of motile spermatazoa was higher in the thawed epididymal sample but when incubated with oocytes there was no difference in the in vitro fertilization rate. Data was presented, but not analyzed for significance, on the number of live pups produced by transfer of embryos obtained from in vitro fertilization using both types of cryopreserved sperm. The authors conclude that the collection and cryopreservation of ejaculated sperm is an efficient and practicable method for the preservation of mouse germ plasm.
QUESTIONS: (1) What are some methods that have been used to collect sperm from the mouse?
ANSWERS: (1) a. Flushing from the uterus of recently mated mice. (This can be a delicate surgical procedure if you wish to preserve the female or a terminal tissue harvest if not.) b. Harvest from the cauda epididymus (also a terminal procedure). c. Certain drug combinations (Yohimbine, pilocarpine, steroids) can be used to induce ejaculation. d. electroejaculation (Deaths can occur using this technique from a variety of causes including the development of uremia due to the blockage of the urethra by coagulated ejaculate. Some of the old papers referenced in the current article make for facinating reading.)

Hypothermia Reduced Neurologic Deficits Associated with Placement of a Vascular Prothesis in the Abdominal Aorta of Rabbits. Laboratory Animal Science 48 (3): 282.
Original study of vascular prothesis in the infrarenal abdominal aorta in rabbits had had high incidence of hind limb neurologic deficits, secondary to focal ischemic damage to the spinal cord. These investigators induced mild hypothermia to block this ischemia.
Endothelial cells from each rabbit were cultured and sodded onto the prothesis. Hypothermia was induced in surgery by not using a heating pad and administering slightly chilled saline.
Mild hypothermia results in decreased blood pressure, heart rate, and cardiac output. Moderate hypothermia results in additional decreases in blood pressure and induces cardiac arrhythmias.
Mild hypothermia is beneficial in preventing ischemic damage to the spinal cord of rabbits.
Questions:
1) What causes vascular protheses to fail?
2) True or False: Mild hypothermia is useful in prventing ischemic injury to the spinal cord during placement of a vascular prothesis.
1. What is the theoretical mechanism of hypothermia in protecting ischemic
tissue?
2. What is the maximum time one can safely clamp the aorta in the rabbit?
a. 5 minutes
b. 15 minutes
c. 40 minutes
d. 90 minutes
Answers:
1) Acute thrombus formation at the biomaterial surface or microembolus formation (which results in decreased downstream tissue perfusion).
2) True
1. Mild hypothermia (defined as body temperature between 32 and 35°C) reduces the metabolic rate of the tissues, thereby reducing the oxygen requirements.
2. 40 minutes

Erythrocytosis in an Aged Beagle Dog. Laboratory Animal Science 48 (3): 288.
Diagnosed absolute secondary inappropriate idiopathic erythocytosis. Clinical signs were hyperemic mucous membranes and injected sclera with erythryoctosis and no evidence of hyperadrenocorticism, hyperthyroidism, renal masses, neoplasms, or polycystic renal disease.
Questions:
None

Transmission of Helicobacter hepaticus infection to sentinel mice by contaminated bedding. (B). Laboratory Animal Science 48 (3): 291.
Helicobacter hepaticus is a newly recognized, gram-negative, helical, microaerobic, pathogenic bacterium of mice that is transmitted by the fecal-oral route. It has been associated with chronic active hepatitis in a variety of mouse strains, with hepatocellular tumor in A/JCr mice, and IBD in immunodeficient and germ-free mice. H. hepaticus can confound or invalidate research data obtained from infected mice.
      Study was designed to see if sentinel mice could be used to detect H. hepaticus infection in mice by bedding transfer to the sentinels. 4 wk. old female C57BL/6NCr were screened to be free of H. hepaticus by fecal PCR. H. hepaticus-contaminated bedding was collected from a cage of 8 DBA/2 mice of an infected colony. Fecal shedding of the DBA/2's was confirmed by fecal PCR. Soiled bedding was transferred twice a week for 8 wks. from infected to sentinel mice. Every 2 wks. feces was tested for H. hepaticus by PCR and blood was collected to check titer levels by ELISA. 66% of sentinels were positive by PCR after 2 wks. exposure and all mice were positive by 4 wks. after exposure. None of the sentinels had a positive ELISA by 2 wks., 66% were positive by 4 wks. and all were seropositive by 6 wks. To date, they have not been successful in using sentinel mice to detect H. bilis-infected mouse colonies.
Conclusion: Sentinel mice exposed to H. hepaticus- contaminated bedding were rapidly colonized by the bacterium and that this infection could be detected by PCR and serologic testing so that fecal shedding appeared at 2-4 wks. and serum antibodies appeared at 4-6 wks. post exposure.
Ques:
1. Can bedding transfer be used to detect Helicobacter hepaticus infected mouse colonies and what diagnositic tests are used?
2. Can bedding transfer to sentinels be used to detect H. bilis infected mice?
Answers:
1. Yes, fecal PCR and serum ELISA
2. No, not so far at least.

Characterization of mouse parvovirus infection among BALB/c mice from an enzootically infected colony (B). Laboratory Animal Science 48 (3): 294.
Mouse parvovirus (MPV) is a newly recognized parvovirus of mice with nonstructural gene-coding sequences almost identical to those of the prototype parvovirus of mice, MVM; however there are major differences between the capsid- coding sequences of the 2 viruses. Both viruses target the small intestine and lymphoid tissue in mice, and MVM also replicates in the kidney. Infant mice are more permissive hosts for MVM infection whereas MPV replication is equally or more efficient in adult mice. MVM causes an acute, self-limiting infection, MPV infection persists well after seroconversion. Previous studies have indicated that BALB/c mice are susceptible to MPV infection so they were used in the study. 8 BALB/c mice from an enzootically infected colony were obtained at 1,2,3 and 6 months of age. 2 groups of 2 BALB/c mice in each age category were then set up, each with 2 adult contact sentinel ICR mice. 4 mice from each group were necropsied on arrival. All but 1 of the 16 mice were MPV seropositive. 8/12 mice in the age groups of 2-6 mos. old had MPV DNA in at least one tissue. MAP testing of intestinal and splenic homogenates from these mice indicated that tissues containing viral DNA also induced seroconversion in ICR mice.  Tissues from 4 of the 1mo. old mice did not contain viral DNA but intestinal homogenate induced seroconversion and only the 1 mo. old mice provoked seroconversion in the contact sentinels. Mice in the 3 older age groups had virus and viral DNA present in multiple tissues as determined by MAP testing and in situ hybridization, but these mice did not transmit to cage contacts. MAP test positive homogenates contain infectious virus. Because of the effects that rodent parvoviruses have on immunologic research, accurate results are sought. Serologic testing is the approach of choice because of its high, sensitivity, fast turn-around time, and low cost. A positive IFA test or EIA does not reveal when an animal was infected because MPV persists in tissues of infected mice. PCR, in situ hybridization, immunohistochemical analysis and MAP testing (requires 3-4 wks. for results) can also be used.
Questions: Can MPV be transferred by any age mouse to contact sentinels?
Answer: This study showed that enzootically infected BALB/c mice that were 1 month old had low antibody titers and had MPV detectable by MAP testing in the small intestine, despite the fact that MPV DNA was not visualized. This was the only age group that transmitted the infection to cage contact sentinels.

Hyaluronidase treatment and preimplantqtion development of mouse embryos (B). Laboratory Animal Science 48 (3): 298.
Hyaluronidase is an enzyme used to remove cumulus cells from around oocytes. Hyaluronidase dervived from the acrosomes of sperm is used in transgenic techniques such as zona drilling, sperm injection, and DNA microinjection. It has been recommended in the literature that oocytes not be left in the hyaluronidase for more than a few minutes after the cumulus cells are shed, for fear that it might harm the oocyte. However, this has never been determined empirically.
    The authors examined the effects of varying levels of hyalurionidase left on oocytes for varying amounts of time, in order to determine the developmental ability of fertilized oocytes treated with hyaluronidase.  They found that exposure of oocytes to hyaluronidase concentrations from 3-300 micrograms/ml from 30 minutes to 24 hours did not affect pre-implantation development of the embryos in vitro. (300 micrograms/ml removes cumulus cells in about 10 minutes, and is the concnetration recommended in the literature). However, exposure to 3000 micrograms/ml for more than 1 hour did affect development.
Questions:
1. What is the source of hyaluronidase used for making transgenic mice?
2. List some transgenic procedures for which hyaluronidase is used
3. T/F Hyaluronidase (300 micrograms/ml) will affect development of oocytes if they are exposed to it for more than 1 hour.
Answers:
1. acrosomes of sperm
2. DNA microinjection, zona drilling, sperm injection.
3. False. Exposure to this concentration (the "usual" one used for transgenic work) for up to 24 hours didn't affect development. However, exposure to 3000 micrograms/ml for more than 1 hour did affect development.

Evaluation of mutant mouse production by mice that are heterogeneous for the Mcfs^op gene (B). Laboratory Animal Science 48 (3): 300.
-Osteopetrotic mice (Mcsf^op) (aka op/op) have a point mutation in the M-CSF (macrophage colony stimulating factor) gene.
-op/op mice are used for research in pregnancy (M-CSF is required for fertility and mammary gland development), cellular development, bone remodeling, cellular immunuty (M-CSF is critical for microglial cell proliferation, osteoclast formation, and monocytopoeisis), and certain diseases including osteopetrosis and osteolysis.
-op/op phenotype: osteopetrosis (which leads to absence of marrow space and subsequent extramedulluary hematopoeisis with organomegaly, and anemia), defective monocytopoeisis (immunocompromised) and NO TEETH!!!!
-these mice don't survive well, and are maintained by breeding heterozygous pairs. The authors examined several breeding variables in order to maximize production of op/op mice that lived past weaning; variables were: housing (conventionsl vs SPF), litter size, parity, month of parturition, and interval between litters. Parameters measured were: litter size, neonatal deaths, preweaning mortality, nubmer and sex of op/op mice per litter, %op/op mice per litter.
Methods: 31 breeding pairs produced 212 litters/1527 pups; op/op pups were identified by the absence of incisors at 9-12 days, and were fed powdered food and fostered til weaning at 6 wks.
Results: type of housing didn't influence parameters; litter size increased with parity up to litter 4, then fell off; % mutant mice /litter decreased with parity, op/op pre-weaning mortality was high (~40%).
Recommendations: Parity was the only important variable; breeders should retire after 6 litters
Questions:
1. How can op/op mice be differentiated from +/op or +/+ mice?
2. What gene is defective in op/op mice?
3. what is the phenotype of op/op mice?
4. What is the most important variable in breeding op/op mice:
5. # of mutant pups/litter (increases or decreases) with parity
Answers:
1.macrophage-colony-stimulating factor
2. they have no teeth!!!
3.osteopetrosis (which leads to absence of marrow space and subsequent extramedulluary hematopoeisis with organomegaly, and anemia), defective monocytopoeisis (immunocompromised) and NO TEETH!!!!
4. parity
5. decreases

Experimentally Induced Infection with Helicobacter pylori in Squirrel Monkeys (Saimiri spp.): Clinical, Microbiological, and Histopathological Findings. Laboratory Animal Science 48 (3): 303.
Helicobacter pylori has been associated with gastritis, peptic ulcer disease, and gastric cancer. They are microaerophilic, motile and urease producing. The two latter are described as virulence factors. To study the pathogenesis of H. pylori the squirrel monkey was used as a model. Many animals have shown to be susceptible to experimental infection with H. pylori. Evaluation prior to inoculation showed animals to be in good health, however it was determined histologically, all monkeys has minimal to mild gastritis. Gastric biopsies were taken from the fundus and antrum. Tissue was stained for histology using H & E and a modified Steiner protocol. Scanning and transmission electron microscopy was also performed. Toluidine blue was the stain used for the TEM. A modified [13C]urea breath test was also used to determine if an infection was present. A lymphocytic gastritis was detected one animal otherwise it was not easy to detect presence of infection in the inoculated monkeys. Two animals did also demonstrate H. pylori resembling bacteria. Based on this study and others it seems as though H. pylori does not naturally colonize the squirrel monkey. Transient infection may occur and the presence of mild gastritis naturally, not a good model for induced gastritis. This model can be used for immune response against H. pylori vaccine and safety trials.
Questions
1. Name a stain used for transmission electron microscopy?
2. Minimal to mild gastritis is a sure sign of H. pylori infection in the squirrel monkey. T or F
3. List 2 virulence factors of H. pylori?
Answers:
1. Toluidine Blue
2. F
3. Urease and motility

Spontaneous Pre-Descemet's Membrane Corneal Opacities in Rabbits. Laboratory Animal Science 48 (3): 310.
This article describes discrete corneal opacities seen in NZW rabbits from a commercial SPF supplier in France. The lesion was present throughout the life of the rabbit, with no clinical progression. The condition appears to be inherited but the exact pattern of inheritance was not determined in this study. The corneal opacities were determined to be minute pre-Descemet's membrane linear cellular aggregates. The cells were ectopic endothelial cells actively secreting matrix material.
The authors conclude that this lesion should be considered a spontaneous ocular change when performing safety assessments in rabbits. Spontaneous ocular changes in laboratory rabbits can complicate the interpretation of ocular lesions in toxicity testing.
Questions:
1. What is the difference between the pre-Descemet's membrane corneal opacities seen in these NZW rabbits and those seen in humans?
a) bilateral and symmetrical in rabbits only
b) bilateral and symmetrical in humans only
c) late onset in humans
d) late onset in rabbits
e) a and c
f) b and c
Answers:
1. f - Usually does not occur in humans until 30-40 years of age. Bilateral and symmetric corneal opacities in humans. It is degeneration associated with aging in humans. Pathology in humans is limited to posterior stromal
keratonocyts with vaculoation and enlargement of affected cells with lipid-like material.