Laboratory Animal Sciences 47 (1)

Laboratory Animal Science 47 (1)
1997

Detection of Enterotropic Mouse Hepatitis Virus Fecal Excretion by Polymerase Chain Reaction. Laboratory Animal Science 47 (1): 006.
MHV consists of a groups of coronavirus strains that vary in tissue tropism and pathogenic potential. These strains a divided into enteroptropic and respiratory tropic groups based on where the primary infection occurs. The most common form of infection is the enterotropic form, which is restricted to the intestinal tract. Serologic testing is currently used to detect the presence of MHV. Due to the fact that it takes several weeks for seroconversion, the authors conducted this study to determine if reverse transcription(RT) and PCR could be used to detect MHV in the feces of mice infected with an enterotropic strain of MHV.
The MHV UAB enterotropic strain was used in this study to infect the animals and serve as a positive control. Postive control was a single fecal pellet from an uninfected animals added to serial dilutions of the virus. Animals were anesthetized and inoculated oronasally. At 3 day intervals postinoculation, animals were restrained and a single fecal pellet was collected from each animal. At 30 days postinoculation both blood and feces were collected and the animals were euthanized. PCR detected MHV nucleic acid on postinoculation days 318 in all animals, and no virus was detected after postinoculation day 24. The RTPCR method consistently detected lower concentrations of MHV that with the other assays. This study confirmed that there is no long term carrier state or viral excretion in immunocompetant animals. The results indicate that RTPCR of fecal pellets provides a sensitive detection assay for MHV. However, it is important to remember that organotropism must be determined for the strain to be tested, for example tissues other than intestine would not be useful for determining infections with enterotropic strains of MHV.
Questions

1.   T/F MHV is a coronavirus that frequently causes a carrier state in mice.
2.   List 2 advantages for using PCR to detect MHV.
Answers
1.   F MHV does not produce a carrier state in immunocompetant
animals.
2.   a.   A noninvasive method can be used to collect samples for
          testing.
     b.   Virus can be detected as early as 3 days post
          infection as compared to requiring a t least 21 days
          for sentinel animals.

Morphologic Response of Myocardium to Freeze-Thaw Injury in Mouse Strains with Dystrophic Cardiac Calcification. Laboratory Animal Science 47 (1): 011.
The purpose of this study was twofold: (1)to determine when mineralization occurs in freezethaw injured myocardium of DBA and C3H mice and (2) to determine the pathogenesis of myocardial calcification in these strains by studying the ultrastructure of the myocardium.
       Dystrophic cardiac calcification(DCC) occur in certain inbred strains of mice, such as DBA and C3H, and is an autosomal recessive train in DBA mice. The authors used both these strains as well as C57 mice in this prject. Briefly, mice were anesthetized, laparotomies performed and a rod cooled in liquid nitrogen was applied to the diaphragm. The mice were sacrificed at intervals and the hearts examined for microscopic and ultrastructure changes. By day 2 mineralization of the necrotic myocytes was observed in the DBA and C3H strains, but not in the C57. By day 28, experimental mineralization was indistinguishable from naturally occuring disease in these strains, but no mineralization was present in C57. Ultrastructure study showed that calcification was due to mineralization of the mitochondria with extension to include the entire sarcoplasm.
Questions
1.   List 2 strains of mice that commomly develop dystrophic cardiac calification(DCC).
2.   T/F Genetic studies have shown that DCC is an autosomal dominant trait in DBA/2 mice.
3.   DCC starts with mineralization of which cellular structure?
Answers
1.   C3H, DBA/2
2.   False it is an autosomal recessive trait.
3.   Mitochondria

Spontaneous Pathology of the Gray Short-Tailed Opossum (Monodelphis domesetica). Laboratory Animal Science 47 (1): 019.
The order Marsupialia contains the New World opossums or Didelphidae, which have been used in research with limited success. The woolly (Caluromys sp), mouse (Marmosa sp), and Virginia (Didelphis virginiana) opossums are some examples that have been used in research. The Monodelphis domestica (gray short-tailed opposum) is a newly established model. This paper, focusing on disease incidence in a breeding colony with an annual census of 1,500 during an 11 year period, was written to expand the information on spontaneous disease in this species. The most spontaneous disease problems occurred in the digestive system and most diagnosis were lesions of the liver. The most common cause of death related to this system was rectal prolapse. This was largely a female reproductive problem. Neoplasia was most commonly found in the digestive system also ( liver followed by pancreas). The second most common system in whichlesions were diagnosed was the urogential system where the kidney was most frequently affected.
questions:
1. The genus and species of the gray short-tailed opossum?
2. What is essentially a problem of females and a leading cause of premature death in this species.
answers:
1. Monodelphis domestica
2. rectal prolapse ( not related to pinworms as seen in other rodents)

Increased Expression of Vascular Cell Adhesion Molecule-1 by the Aortic Endothelium of Rabbits with Pasteurella multocida Pneumonia. Laboratory Animal Science 47 (1): 027.
Objective: To determine if respiratory infection with Pasteurella multocida in rabbits would cause alterations in aortic endothelial cells with specific reference to VCAM-1 expression.
    Vascular Cell Adhesion Molecule -1 (VCAM-1) is a receptor expressed on endothelial cells and its expression is conducive to leukocyte adhesion to the vascular wall. Previous in vivo studies have shown that endothelial cell VCAM-1 expression is elevated by:
1. endotoxins     2. cholestrol-feeding   3. regeneration after vascular injury 4. alloxon induced diabetes mellitus
   Experimental Groups: A) SPF Rabbits, B)SPF Rabbits experimentally infected, C) Conventional Rabbits (naturally infected)
   Results: VCAM-1 expression was significantly elevated in the aortic endothelial cells of the infected groups (B&C).      Additionally, significant morphological differences noted by electron microscopy of the endothelial cells of infected rabbits (B & C) include:
1) cells were more vacuolated and contained a higher proportion of dilated rough endoplasmic reticulum
2) cells had a higher percentage of leukocytes attached to them
   This paper provides another example of how infectious disease in a laboratory animal may affect research results, especially in studies of the vascular system. Several electron micrographs are included.
Questions:
1. Increased serum cholestrol can result in increased expression of VCAM-1 in the rabbit. Which inbred rabbit strain (animal model) might you also predict to have elevated vascular VCAM-1 expression?
2. Endotoxins also result in increased expression of VCAM-1. Which serotype of Pasteurella multocida produces an endotoxin (A,B, D, E, or F)?
3. In addition to rhinitis and pneumonia, what are some other clinical manifestations of Pasteurella multocida in the rabbit (5)?
4. Infection of the rabbit's respiratory tract with this bacteria alone does not usually present with any clinical manifestations or lesions, however, infection of guinea pigs with this organisms is more likely to result in an acute severe respiratory tract infection. Name that bacteria.
Answers:
1. Watanabe Heritable Hyperlipidemic Rabbit   (St. Thomas Rabbit and the KHC rabbit are also rabbit models which have high circulating cholestrol levels)
2. D
3. otitis media & interna, conjunctivitis, abcesses, genital tract infection, septicemia
4. Bordetella bronchiseptica

Evaluation of a Vaginal Moisturizer in Baboons with Decreasing Ovarian Function. Laboratory Animal Science 47 (1): 036.
Ovariectomized babbons were used in a study to determine the effect of bioadhesive moisturizer on vaginal dryness found in postmenopausal women. The test compound if applied daily for 5 days was able to increase vaginal secretion, mucus production, and vaginal elasticity. Smear evaluation of the percentage of vaginal epithelial cell types seems to be the most practical histological technique for this type of study.
QUESTIONS:
1. Which animal is best suited for the study of the female reproductive tract, and why?
2. Name two possible treatment of vaginal dryness in postmenopausal women?
ANSWERS:
1. baboon, similar menstrual cycle as humans
2. systemic estrogen, bioadhesive vaginal moisturizer

The Atopic Dog: A Model for Food Allergy. Laboratory Animal Science 47 (1): 040.
High IgE producing inbred dogs were used as a model for food allergy. The animals were sensitized s.c. with food antigen extracts in alum after immunization with a live virus vaccine. Allergy status was tested using skin test, by measuring specific IgE levels and by gastroscopic food sensitivity testing (GSFT). GSFT was performed by injecting food extracts into the gastric mucosa by endoscope after intravenous injection of Evans blue. Changes in gastric mucosa included erythema, edema and blue patching. Dogs reacted to ingestion of food antigens with diarrhea (after 12h) and occasionally vomiting (after 1h).
QUESTIONS:
1. Which tests are used to confirm food allergy?
2. Describe a model for food allergies.
3. What is GSFT and how is it performed?
ANSWERS:
1. IgE levels, skin tests, gastroscopic food sensitivity testing
2. high IgE producing inbred dogs, sensitized s.c. with food antigens after immunization with live virus vaccines
3. gastroscopic food sensitivity testing, food antigen extracts are injected into gastric mucosa using an endoscope after intravenous injection of Evans blue, injection site is visually monitored for 3 minutes

Acute Neurologic Decompression Illness in Pigs: Lesions of the Spinal Cord and Brain. Laboratory Animal Science 47 (1): 050.
-Of 120 pigs, 40 (33.3%) were functionally unaffected and 80 (66.6%) developed neurologic decompression illness; 16 died, 64 survived.
-Petechial hemorrhages were grossly visible in the spinal cord of 73% of the survivors, 63% of the fatalities, and 3% of the clinically unaffected pigs, the thoracic part of the cord was most commonly involved. [thoracic> lumbar >cervical>sacral]
-Histologic cord lesions were found in 75 (63%) pigs: 83% of decompression illness survivors, 81% of the fatalities, and 23% of those clinically unaffected.
-Morphologically, hemorrhagic lesions were the most common (54%). Other common findings included spongiosis (48%), axonal swelling and loss (39%), and myelin degeneration (35%). [This is the pattern seen in other animal models(goats, dogs) and unfortunate divers. However, goats had vessel necrosis or microthrombi which was NOT seen in these pig cases]
-White matter hemorrhages in the spinal cord were generally more numerous and extensive than those affecting the gray matter; however, gray matter hemorrhage was associated with increasing disease severity.
-Brain lesions were present in 2% of pigs and were most frequent in fatalities. Cerebellar and brain stem hemorrhages were the most common brain lesions; the molecular layer of the cerebellum appeared particularly susceptible.
-Rapid onset and predilection for the spinal cord were consistent with the presentation and clinical picture in severe human cases of neurologic DCI
QUESTIONS:
1. What heart lesion in humans and pigs is associated with increased risk of neurologic DCI?
2. What part of the spinal cord was most affected?
3. Which was more affected in the spinal cord gray or white matter? How about in the brain?
ANSWERS:
1. Patent foramen ovale
2. Thoracic
3. White matter in the spinal cord and gray matter in the brain.

In Vitro Fertilization of Follicular Oocytes by Frozen-Thawed Spermatozoa in Japanese Monkeys (Macac fuscata). Laboratory Animal Science 47 (1): 058.
Can frozen epididymal spermatozoa be used for in vitro fertilization?
-Japanese monkeys are seasonal breeders.
-Performed the collection with one male monkey.
-Epididymal spermatozoa stored in mineral oil as a transport media showed the best motility and fertility.
-Frozenthawed spermatozoa maintained good motility up to 2 h of incubation; motility continually decreased thereafter, and the spermatozoa showed poor motility at the end of 8 h of incubation.
-Sexually mature female monkeys were used for ovarian stimulation in July and August during the nonbreeding season. Used one cycle of eCG and one dose of hCG.
-Compared with untreated ovaries, the ovaries treated with eCG and hCG were larger, and follicles were developed over a larger area of each ovary.
-Collected oocytes far outnumbered the follicles counted on the ovary surfaces.
-Many collected oocytes were judged to be immature[were in germinal vesicle stage and had not extruded first polar body nor had loosened cumulus cells]
-43%(29/69) of the oocytes were successfully fertilized=20
-4/29 fertilized oocytes progressed to the 8cell stage
-Succeeded in ovarian stimulation, spermatozoa cryopreservation, and IVF in Japanese monkeys during the nonbreeding season.
QUESTIONS:
1. When was hyperactive motility seen in frozenthawed spermatozoa?
2. What media is best for transporting epididymides?
3. Did dose of eCG make a difference in the number of oocytes collected?
4. What was one of the problems with the oocytes collected in this study?
ANSWERS:
1. 30 minutes after thawing
2. mineral oil
3. No
4. Many were immature

Estrus, Fertility, Early Embryo Development, and Autologous Embryo Transfer in Laboratory Woodchucks (Marmota monax). Laboratory Animal Science 47 (1): 063.
Reproductive parameters were studied in Woodchucks. Findings include evidence for induced ovulation, greatest fertility for 24 yr old females, mean litter size of 2.9 pups, vulva diameter
is indicative of proestrus and willingness to accept mounting by males. Gestation was 31.9 days. Woodchucks are marmotine, sciurid rodents and are models of hibernation, control of food intake, endogenous circannual cycles, including cycles of thyroid hormone activity and teste development. Not included in the report but an important model to remember is Woodchuck Hepatitis Virus leading to hepatocellular carcinoma. (see Journal of Hepatology. 26(6):132430, 1997 Jun.)
Questions:
1) Woodchucks are an animal model of what human disease.
A) Hepatitis
B) Hepatocellular Carcinoma
C) Diabetes Mellitus type I
D) Diabetes Mellitus type II
E) A and B are correct
2) What is the gestation period for the laboratory woodchuck.
A) 16 days
B) 21 days
C) 32 days
D) 45 days
E) 63 days
3) What is the genus and species of the common laboratory woodchuck.
Answers:
1. E
2. C
3. Marmota monax

Survey of Dermatophytes Isolated from the Coats of Laboratory Animals in Italy. Laboratory Animal Science 47 (1): 075.
Dermatophytosis has been reported in most laboratory animals, including rats, mice, guinea pigs, cats, and rabbits. This study documents the spectrum of dermatophytes infecting laboratory animals in Italy. Clinically, guinea pigs and cats developed circular 12 cm crusty ring lesions. Only the guinea pig developed pruritus. Cultures were taken and found Microsporum canis in all species, but rabbits. Trichophyton mentagrophytes was found in guinea pigs and rats only. There were no organisms isolated from rabbits. Treatment was recommended for cats only with appropriate antimycotic drugs. Rodents and rabbits are better eliminated to remove the carrier state. These organisms have zoonotic potential so workers need to be protected.
Questions:
1) Microsporum canis is a dermatophyte commonly isolated from
rabbits.
A) True
B) True, but also an isolate from rats and mice.
C) False, but has been reported to be isolated from guinea pigs.
D) False, but Trichophyton mentagrophytes is commonly isolated from
rabbits.
2) T/F Dermatophytes from laboratory animals have zoonotic
potential.
Answers:
1. C
2. T

Placement of Colonic Manometric Catheters and Electrodes in Cats. Laboratory Animal Science 47 (1): 078.
Constipation and fecal impaction of the colon is the most common GI problem associated with spinal cord injury (SCI). The authors describe an animal model to assess the efficacy of direct electrical stimulation of the colon to manage constipation. The cat was chosen because it develops constipation subsequent to SCI and it can tolerate well the instrumentation and SCI. Two survival surgeries were done: Surgery 1 Instrumentation: Laparotomy and insertion of bladder and colonic catheters, and the "suture" electrodes. The bladder catheter was used (i) to drain urine after the SCI and (ii) to indirectly measure intraabdominal pressure. The two silastic intraluminal colonic catheters were used (i) for the insertion of radiopaque markers and (ii) for manometric recordings. The six suture electrodes were implanted in the serosa in (i) a semicircular fashion around the colon, and (ii) longitudinally, 5 and 20 cm proximal to the anus. All electrodes and catheters were advanced subcutaneously and exited the dorsum of the cat. An animal jacket was placed on the cats to protect the equipment. Cats were maintained for 8 weeks before the second surgery. Surgery 2 Spinalization: The spinal cord was clamped at T4 with a hemostat for 30 sec. Cats were maintained for an additional 8 weeks to assess colonic function after SCI. Conclusions: (1) Cats recovered quickly from both surgeries and exhibited normal grooming behavior; (2) cats wore the jackets without preconditioning or discomfort; (3) SCI resulted in complete and permanent paraplegia without motor recovery; (4) on postmortem, the authors found minimal response to the instrumentation.
No questions

Rapid Tumor Development with a Modified MCF-7 Xenograft Model. Laboratory Animal Science 47 (1): 082.
The authors describe the growth of MCF7 human mammary tumors in 57 weekold sexually intact female Crl:CD1nuBR athymic mice. All mice were implanted subcutaneously with estradiol17beta pellets (MCF7 xenograft tumors require exogenous estrogen for growth) and some were subsequently treated with tamoxifen (a nonsteroidal antiestrogenic compound). Number and growth rate of resulting tumors were also compared between different tumor cell inoculum sizes, and between tumor cells suspended in PBS vs. cells suspended in PBS and Matrigel (which accelerates the early onset of tumors in inoculated mice). Residual Matrigel in the tumors could affect the distribution of therapeutic test compounds and confound the interpretation of results. Rapidgrowth phase tumors were treated with tamoxifen vs. placebo and with or without Matrigel. Conclusions: (1) Rapidly growing tumors in mice treated with tamoxifen regressed in size by 27% but increased by 144% in control animals; (2) Matrigel decreased the time required to achieve rapidgrowth phase (33 days vs. 48 d without Matrigel); however, the subsequent growth rate and final numbers of rapidly growing tumors were similar in both groups; and (3) inoculation with larger tumor volumes than those previously reported and maintaining high in vivo concentrations of estradiol increased the number and growth rate of resulting tumors.
No questions

Characterization of mRNAs 4 and 5 of Enterotropic Mouse Hepatitis Virus. Laboratory Animal Science 47 (1): 086.
Mouse hepatitis virus (MHV) belongs to the Coronaviridae family. These cornaviruses can cause either respiratory tract or enteric disease. Initial replication of the virus occurs in the upper respiratory tract and then disseminates to multiple organs of the body. Recent data indicates that mouse infections have shifted from predominately polytropic to predominately enterotropic. In this study they reported the nucleotide sequence of three enterotropic strains of MHV; (OMHVRI, MHVY and DVIM) to corresponding sequence of three polytropic strains (MHVA59, MHVS, and MHVJHM). In summary, polytropic and enterotropic strains of MHV are structurally alike in which the authors assume that small structural changes in the virion may be responsible for the difference in pathogenesis.
Questions:
1. Name three polytropic MHVs
2. Name three enterotopic MHVs.
3. Name the family that MHV belongs too.
Answers:
1. Polytropic strains MHV; (MHVA59, MHVS, and MHVJHM)
2. Enterotropic strains of MHV;(OMHVRI, MHVY and DVIM)
3. Coronaviridae

A Survey of Blastocystis sp. in Rodents. Laboratory Animal Science 47 (1): 091.
Blastocystis sp. is an intestinal protozoan parasite of mammals (rats, humans, swine, monkeys, etc.), reptiles, birds and insects. Blastocystis sp. was found not to infect mice, hamsters, or rabbits. Positive animals were identified by examining fresh fecal samples by light microscopy and inoculation into Jones medium containing 10 % horse serum. Culture of fresh fecal specimens inoculated into Jones' medium is more sensitive than direct stool examination for the detection of Blastocystis sp. infections. Blastocystis sp. has four main morphologic forms (vacuolar, Fig. 2A, granular, Fig. 2H, amoeboid, Fig. 2D and 2E ,and cyst, Fig. 2I, p. 9293). Wistar and Sprague Dawley rats have a higher prevalence than other stocks of rats. The cockroach can carry the cyst of this parasite in its cecal contents. The relationship of Blastocystis sp. infections between different species is not known.
Questions
1. T or F Blastocystis sp. can infect all laboratory rodents?
2. T or F The best detection method for Blastocystis sp. is direct examination of fresh feces?
Answers
1. False, not detected in mice or hamsters
2. False, Jones medium culture.