Laboratory Animal Science 49(1)

Laboratory Animal Science 49(1)

Meeting Report: Lovelace Respiratory Research Institute Annual Symposium validity of animal models of human respiratory diseases. Laboratory Animal Science 49(1), 007.
Abstract: This is a meeting report from the October 1998 meeting sponsored by Lovelace Respiratory Research Institute, the first in a new series of annual meetings. The complete set of abstracts submitted by the invited speakers as well as poster abstracts is available at the symposium website http://www.lovelace-symposium.org
The tone of the meeting was emphasis on strengths, weaknesses, and homology value of animal models of respiratory disease research with respect to the human disease. It was determined that reasonable homology exists for the evaluation of drugs to treat respiratory infections and for studies where the mechanism of the human disease process is known and can be shown to be similar in animal models. However, homology was determined to be more difficult in studies when the cause of the underlying disease in humans is not known or is potentially complex and in studies that assess risk where anatomy and metabolism play an important but poorly defined role. In these cases the extrapolation of data from rodents to humans may have little value.
Some important points that were made include:
1. There appears to be homology between mutations in tumor suppressor genes, but aberrant methylation patterns and effects on the expression of various oncogenes and suppressor genes.
2. Frequencies of the types of tumors induced in the usual rodent models differ from those observed in humans.
3. The use of rodent data for quantitative estimates of human cancer risk is difficult because current knowledge of differences in mechanistic basis for carcinogenesis among species is inaequate.
4. The importance of localized immune responses in human allergic asthma and the lack of such localization in rodents. Mice and rats instead have a lymphoid structure, the bronchus associated lymphoid tissue, that is not observed in humans, dogs and cats. This lymphoid tissue serves to drain the lung lymphatics to the systemic circulation. Thus antigen specific antibody and immune cells produced in rodent lungs do not remain in the lung.
5. One key response of human asthma is wheezing, a response that is not elicited in most animal models of the disease. However, asthmatic cats do display spontaneous wheezing, and appear to have similar histopathology to humans.
6. Differences among species and between strains of animals can play a complicating role. Differences in some parameters may be due to anatomic differences between the lungs of various animals, or between eosinophilic responses with airway hyper-responsiveness.
7. The power of transgenic and knockout technology has greatly improved the understanding of the roles of various cytokines and cellular responses in asthma. There is apparent potential for the use of genetically altered animals in many types of studies.
8. Microanatomy of the pulmonary vascular anatomy plays a more significant role than gravity in blood flow distribution in the lung.
Models of three specific infectious diseases (influenza, tuberculosis and Cryptococcus) were discussed. Responses of experimental animals (mice and guinea pigs) have led to important information that appears to be directly related to the management of human disease.
The overall consensus was the symposium provided a valuable venue for examining the relationship between studies performed in animals and the corresponding diseases and risk assessment in humans.
Questions: 1. What animal model appears to be the most homologus with human asthma by displaying wheezing characteristics and similar histopathology?
a. rat
b. guinea pig
c. cat
d. pig
2. What lymphoid structure dorats and mice have that is not observed in humans, dogs and cats and serves to drain the lung lymphatics to the systemic circulation?
3. Why is the use of rodent data for quantitative estimates of human cancer risk difficult?
a. current knowledge of differences in mechanistic basis for carcinogenesis among species is inadequate
b. microanatomy of the pulmonary vascular anatomy varies
c. rodents have bronchus associated lymphoid tissue
d. use of rodent data for quantitative estimates of human cancer risk is not difficult
Answers: 1. C.
2. bronchus associated lymphoid tissue
3. A.

LAS Journal Club: The existence of differing monkey B virus genotypes with possible implications for degree of virulence in humans. Laboratory Animal Science 49(1), 010.
Abstract: The purpose of the original study was to evaluate differences in B virus isolates from different species of macaque monkeys.
Isolates were examined from rhesus, Japanese, cynomolgus and pigtail macaques. At least three different genotypes exist. Phylogenetically the rhesus and Japanese macaque strains are more closely related, but the numbers in this study were low.
This information supports strain variation among B virus isolates and the suspicion that there are differences in pathogenecity in human beings.
Questions: Questions:
1. B-virus is a
a) alphaherpesvirus
b) betaherpesvirus
c) gammaherpesvirus
d) paramyxovirus
2. What does the RFLP stand for?
Answers: Answers
1. a
2. restriction fragment length polymorphism

Special Topic Overview: Spontaneous and engineered mutant mice as models for experimental and comparative pathology: history, comparison, and developmental technology. Laboratory Animal Science 49(1), 012.
Abstract: Development of Mouse Genetic Models of Disease
Spontaneous Model:
-polygenic models-The first inbred mouse stain DBA at the Harvard Bussey Institute in 1909. Research focused on cancer pathogenesis.
Examples of mice used for cancer research
--mammary and hepatic tumors in C3H
--testicular tumors in 129
--lung tumors in the strain A
--lymphocytic lymphoma in the AKR
--reticular cell neoplasms in the SJL/j
NON-NEOPLASIC
--amyloidosis A/J
--renal tubulointerstitial disease CBA/J
--dystrophic cardia calcinosis DBA/2
--systemic autoimmune disease NZB
Investigations of these characteristics in showed that several genes were involved.
Homozygouse-capable of producing progeny of identical genotype.
F1-hybrid mice produced by the mating of 2 inbred strains have genetic uniformity, albeit all loci differing between the 2 parental strains are present in the heterozygous state. the production fo congenic ingred strains of micese was pioneered in the 1940 by Snell.
Use of "marker" to develop fenetic linkage map.
The first genetic lankage between the recessive nutation a albino,c, and pink-eye dilution ,p, was described by Haldane. Other mutant markers genes dominant hammer -toe Hm and semi-dominant pintail Pt. The identification of polymorphisms provided a number of co-dominant marker gene for conventional mapping in constructed RI strains. RI strains were used for analyzing complex traits that were age-dependent or reflecting incidence-based datat, thus becoming an important experimental tool in studies such as the genetic determination of neoplasia.
Technologies which have advanced the pace of gene mapping RFLP restriction fragment length polymorphism PCR polymerase chain reaction. Efforts to dissect the gene map of inbred strains can be used in comparative mapping, developing models fo human disease, and cloning.
SINGLE GENE MODELS
Coisogenic mutant inbred strains to serve as controls to congenics. The former only differing by the gene of interest. The dwarf (dw) mouse developed by Snell was very important in the development of analogous or homologous pathogenic mechanisms of disease in humans. Now there was a defined background with a single mutation has the advantage of high reproducibility when compared to outbred or when there is linked or non-linked alleles.
SPONTANEOUS MUTATIONS
scid/scid a mutation of the C.B-17 inbred mouse discoved during routine radioimmuno immunoglobulin quantitation screening.
gld, generalized lymphoprliferative disease now, Fas-L gld, was the direct result of a search(phenodeviant search program) at Jackson labs. Were a tech found a lumpy bumpy mouse in a strain not noted for early onset lymphomas. The gld mutation and the lpr mutation are used in study of apoptosis and lymphocyte differentiation.
MURINE GENETIC DISEASES AND THE QUESTION OF HOMOLOGY
Of the 2,616 mapped loci int hemouse 917 have been mapped homolgues in humans, marking 101 segments of conserved linkage homology.
ENGNEERED MOUSE MODELS (NON-TRANSGENIC)
Mutations which are induced by whole-body, gonadal, or germ-cell exposure to chemicals or ionizing radiation. Used mostly to assess genetic risk of chemical and tye and dose of radiation a few muations of interest have resulted. The most frequently used chemical are ethylnitrosourea (ENU); urethane,7,12-dimethylbenz{a} anthracene; and 4-nitroquinoline 1-oxide. Radiation-induced mutations-the most common assessment of radiation risk is the specific-locus test (SLT) Examples of radiation induced mutations are the beige, bg mouse-lysosomal dysfunction and dominant cataract (cat-2t) and cleidocranial dysplasia. Allophenic mice-embryolagic manipulations to create tetraparental or mosaic mice. Enabled the tracking of genes involved in development. These models are used to study clonal pathways, differentiation commitments and neoplasia. These models have also help to identify gene interaction in mouse models of blood dyscrasias, immunologic aberrations and autoimmune daibetic insulinitis. Allophenic models are not generated with the expectationof perpetuation fo such genetic combinations in the germline.
ENGINEERED MOUSE MODELS
production of animals in which foreign DNA is introduced into the genome. Techniques: pronuclear injection, viral transgenic, and homologous recombination
Gain-of-function: developed by introducing either endogenous or exogenous gene copies with subsequent overexpression of the gene product or expression of noval gene products.
Loss-of-function: developed by disrupting and endogenous gene, with results ranging from complete loss of gene function to subtle mutation of a gene element.
Pronuclear injection: Most common method for gene transfer Direct injection of DNA of pronuclei of mouse zygotes.
RESULTS:
1) over expresion of a gene product
2) novel product from exogenous gene
3) disruptions of the host genome after insertional mutagenesis Transgenes are head to tail concatamer (two or more DNA molecules convalently joined end -to-end) 50 copies are usually injected. Transgenes are injected into ONE CELL embryo. FVB/N are used because: 1 respond well to superovulation, 2 many pronuclear stage embryos, large pronuclei.
RETRIEVABLE MUTATION SHUTTLE vectors-in vivo mutagenesis studies. A bacterial gene which is integrated into the mouse genome and is susceptible to mutation. Once exposed to the mutagen a high molecular weight DNA is isolated from the tissue samples. Using a bacteriophage packaging extract the bacterial genomic sequence is captured for analysis of non-mutated and mutated sequences.
EXAMPLES:
Big Blue Mouse and MutaMouse.
Viral transgenesis:
Moloney murine leukemia virus used on a pre-implantation stage embryo, a copy of the proviral DNA is inserted, there is no rearrangement of the host genome, and the host sequences on either end are duplicated thus allowing ease detection.
NOT USED BECAUSE:
1) extra steps involved in producing high titers of recombinant retroviral vectors
2) HIGH DEGREE OF MOSAICISM
3) low rate of germline transmission
4) multiple sites of insertion
THe adenoviral vector with a targeted lacZ gene has also been used. The advantageous because it does not result in a high degree of MOSAICISM.
HOMOLOGOUS RECOMBINATION (TARGETED MUTATIONS):
Technologies that allow the introduction of a defined genetic alteration at a specific site of the genome.
steps: 1) a vector is designed with delectable marker and flanking sequences which are homologous to the endogenous target gene. 2) the vector is introduced into the pluripotent stem cells by using transfection.
II. EXAMPLES OF TARGETED MOUSE MUTATIONS:
see chart on page 18>
Examples of gene knockout mutations affecting B ot T cell immunity:
A>uMT mutation: 1st model of complete loss of humoral immunity without concomitant lack of T cells. In this model B-cell differentiation is arrested at the pre-B-cell stage.
B>XLA agammaglobulinemi, mice homozygous for uMt used to study Bruton disease
C>knockout mutationBtk for patients with homologous XLA
D>C57Bl/6j-beta2m null mice lack Class-I dependent CD 8plus T cells can contain but not resolve listereria infections.
DEFICIENCY OF TRANSFORMING GROWTH FACTOR B1
Example where the targeted mutation was not achieved TGFB1 mice did not have pathogology associated with the heart but, with increased lymphocyte and immature neutrophils and expression of MHC molecules. Spectulation that mothers TGRB1 may protect the young and at 3wks animals died due to inflammatory disease of other organs.
TUMORIGENESIS ASSOCIATED WITH DISRUPTED p53
Homozygous animals P53-null mice develop tumors as early as 8wks the mean latent period is 20wks. Most commonly observed tumor is Malignant lymphoma. Because these animals die at less then 10 months they are considered good canididates for anticancer drug studies. In heterozygous animals the most common tumor is osteosarcoma. These animals are a good model for Li-Fraumeni syndrome. When the p53-null allele was place on a 129/Sc mouse the animal had an increase in testicular teratpcarcomas.
III. EXPRESSION OF INDIVIDUAL GENE AND INTERACTION BETWEEN MULTIPLE MUTANT GENES: CORRELATIONS BETWEEN SPONTANEOUS AND TARGETED MOUSE MUTATIONS.
PLEIOTROPISM:
example- the W locus on chromosome 5 the spotting mutation. there are three phenotypes for homozygous animals.1) mon-albinotic white spotting 2) macrocytic anemia and 3) oocyte depletion. Linked to the c-kit receptor example-the mutant steel Sl alleles, chromosom 10 also has a range of phenotypes. These changes are linked to the c-kit proto-oncogene ligand. other examples are the lpr (defect in the Fas- encoding gene) and gld( defect in the Fas ligand) mutations. Both share similar phenotypes but due to different defects.
PRIMARY GENE EXPRESSION DEPENDS UPON STRAIN BACKGROUND
Homozygous C57Bl/6J db/db-are obese and show insulin resistance but no permanent diabetes
Homozygous C57BL/6KsJ db/db are obese and have severe diabetes syndrome are a model for type II diabetes.
Massive lymphadenopathy and immune complex glomerulonephritis -lpr is expressed on the MRL/Mp strain. the expression is two-fold less on the C3H.He/J then the MRL/Mp and five time less on the C57BL/6J
EXPRESION OF THE PRIMARY GENE MAY BE ALTERED BY A SPECIFIC, DEFINED
SECONDARY GENE.
see TEXT:
AGE-DEPENDENT EXPRESSION:
example the bg mouse-homozygous had a late-onset syndrome characterized by tremor, ataxia, lethargy and death after 2 years of age. occures at 13 mounths in C3H/He bg/bg and 20 to 24 mounths in C57 bg/bg
Loss of greater than 50% Purkije cells.
MOLECULAR,EMBRYOLOGIC, and BREEDING TECHNIQUES FOR PRODUCING AND PROPAGATING GENE-TARGETED MOUSE MODELS
Gene targeting:
Embryonic stem (ES) line from pluripotent cells of the inner cell mass of the mouse preimplantation blastocyst-stage embryo. the cells are maintained in culture with either leukemia inhibitory factor or a feeder cell line. 129/Sv are the most commonly used mouse to dervive ES cell lines.
Selection strategies:
most frequently the complete ablation of gene fuction is used to create the null mutations.
1) These targeting vector conatins a positive-selection marker, the neomycin resistance gene. This gene also confers resitance to the aminoglycoside antibioric Geneticin which is used to select for ES cells that have incorporated the marker.
2)the use of hygromycin cassetts are also used to select of homologous recombination events.
3) Two positive-selection strategies:u
the use of a promotor or polyadenylation site
MOST COMMON
positive selection strategy uses a negative cassette to elimminate random intergration events. The thymidine kinase gene of herpes simplex virus. is the most common negative marker. This HSVtk is added to one or both end of the homologous DNA sequence. IF, a random event occurs the HSVtk are sensitive to gancyclovir and which will kill the cell SEE FIGURE 2 page 23.
HIT AND RUN:
this vector contains a mutation in the region of the homology plus a positive and negative marker. The first recombination requires the insertional event will duplicate the genomic sequence. The clones are i.d. through positive selection. In step 2, spontaneous intrachromosomal recombination removes the plasmid sequences and selction cassettes. AFter the removal of the neg. marker the clones revert to being resistant to the drugs used for negative selection and the clones selected.
DOUBLE HIT
the positive and negative markers are in the gene of interest. After the 1st recombination there is a positive selection. A second construct with genomic DNA of interest is transfected into the positive ES cell clones. the negative marker is lost and the clones revert to being resistant to the neg. selection agent.
CRe/loxP SYSTEM: uses bacteriophage p1 the enzume Cre recombinase aligns the phage DNA the loxP site and the intervening DNA. the vectror has the gene of interest flanked by loxP sites in the region of homology. Cre recombinase is used to exvise the selection markers, leaving a modified allele.
Once the ES cells are selcted clumps (clone) can be seen . A Southern plot is used or PCR to identify the successful targetin event. These are then injected into the donor blastocysts.
PRODUCTION OF GENE-TARGETED MICE:
ES cell chimeras:
THE MOST COMMON METHOD OF GENERATING ES CELL EMBRYOS CHIMERAS
see figure 4 page 24.
The use of micromanipulation system which is expensive and requires a great technical proficiency.
A new ES cell-Embryo chimeras are produced by aggregatin ES cells with eght celled embryos. This method does not require expensive equipment. 1) 8 cell embryos are collect 2) treated to remove the zona pellucida 3) then either the embryos are placed on bed of ES cells or the is plave in apposition to ES cell in s small depression in a tissue culture dish. The efficiency is similar to those of the injection technique.
Non- Chimeric embryo production: produced by aggregating tetraploid embryos with diploid ES cells. The tetraploid is the fussion of two-celled stage mouse embryos with a electric pulse. The Diploid/tetraploid chimeric embryos are formed by sandwiching ES cells between the 2 tetraploid embryos in a dish. They form a single, chimeric embryo.
EMBRYO TRANSFERES
the embryos are transfered to a psuedo pregnant fecmal. The coat colors are different thus one can i.d. the chimeria
SELECTION OF OFFSPRING:
The goal of producing offspring with the targeted allele is achieved by breeding chimeric male mice to females with a defined coat color. Thus selection is based on coat color. Example the male derived from strains 129/sv and C57BL/6 is breed to a C57BL/6 female. The mice with the agoutie color were dervived form the 129/Sv ES cell and have the target allele.
Males can be evaluated for the contribution of ES cells to the germline transmission by PCR of the spermatozoa.
Questions: None yet

Lymphoid organ alterations enhanced by sub-lethal doses of coronaviruses in experimentally induced Trypanosoma cruzi infection in mice. Laboratory Animal Science 49(1), 035.
Abstract: Trypanosoma cruzi is the causal agent of Chagas' disease. Infection with this parasite stimulates humoral and cell-mediated immunity, which can have adverse effects on the host. As shown in the literature, the microbial status of laboratory animals can affect outcomes of disease. This group has previously shown that contamination of their T. cruzi stocks with coronavirus resulted in increased parasitemia levels and decreased survival times.
Hypothesis: In this study, the group investigated the effect of a sublethal dose of coronaviruses (either MHV-3 or their contaminant "virus X") on the course of disease in mice infected with a mildly pathogenic strain (TC) of T. cruzi. Because coronaviruses are known to alter the immune response of the host, one would expect that resistance to T. cruzi would also be altered.
Experimental design: 5 groups of 25 SPF CBA mice were infected with either (1) parasites (2) virus X, (3) MHV-3, (4) parasites + virus X, or (5) parasites + MHV-3. Parasitemia and mortality were recorded; histology of lymphoid organs was done.
Results: 1) Mice that received parasites + coronavirus (either MHV-3 or virus X) were markedly parasitemic and died by day 15 after infection. There was no significant difference between the 2 coronaviruses tested. Other mice infected with either coronavirus OR parasites survived their respective infections.
2) Splenic weights and splenic and lymph node cell numbers were increased in mice inoculated with either strain TC or TC + coronavirus. Thymic weights and cell numbers were significantly reduced in mice inoculated with coronavirus alone or with TC + coronavirus.

Conclusions: Both pathogens depress the immune response of the host by acting on primary lyphoid organs. By inducing early depletion of T cells, coronavirus infection makes it difficult for the host to mount a T-cell response to the parasite and thus favors its multiplication. Research groups using mice from colonies with indigenous coronavirus infections should be aware that while the virus may be subclinical, it may still alter disease outcomes in infection studies.
Questions: Question:
1. What are the viral properties of coronaviruses? Name an example in laboratory mice and rats?
2. Trypanosoma cruzi is the causal agent of what disease?
Answers: Answers:
1. Coronaviruses are large enveloped single stranded RNA viruses.
Mice = mouse hepatitis virus (MHV)
Rats = sialodacryoadenitis virus (SDAV)
2. Chagas' disease

Management of a measles outbreak among old world nonhuman primates. Laboratory Animal Science 49(1), 042.
Abstract: This was an epidemiologic investigation of a measles outbreak in rhesus, cynomologus, and pig-tailed macaques housed at the National Institutes of Health. Two research buildings and one holding facility housing a total of 1735 NHPs were involved. Animal transfers between these facilities were common; initially monkeys went through a 60-d quarantine period, which involved five sequential TB tests and collection of sera for health-screening tests, including measles virus antibody testing. After that animals could be transferred to different facilities after a veterinary evaluation. The index case was discovered in the holding facility by a veterinarian while removing a cast from the arm of the monkey. A subtle maculopapular rash was detected that extended down the left side of the abdominal wall to the inguinal region, and mild hyperemia of the NHP's face was evident. After further investigation the monkey was returned to the room it had been housed in a strict facility quarantine was implemented and evaluation of the other NHPs was implemented. However, 7 monkeys from the holding facility were already in route to one of the research facilities, but upon arrival 3 monkeys were sent back to the holding facility. One monkey had maculopapular rashes and the other two monkeys had been housed in the same room. The remaining four monkeys were quarantined in the research facility, two received measles vaccination, as they had no documentation of previous measles seropositivity. In addition, 5 monkeys had also been sent to the other research facility. These monkeys were admitted to the facility before the suspected measles cases were identified at the holding facility or the other research building. Within 2 h of their admission they were pulled from their assigned rooms, evaluated, and moved into quarantine. During a routine surgical preparation another monkey, which had been transferred from the holding facility 11 days prior to the first identification of the measles case. During the outbreak investigation a probable
Questions: Questions:
1. What type of virus is measles?
2. What are the scientific names for rhesus monkeys, cynomologus monkeys, and pigtailed macaques?
Answers: Answers:
1. Single-stranded RNA virus member of the Paramyxovirus family.
2. Macaca mulatta, Macaca fascicularis, and Macaca nemestrina.

Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents. Laboratory Animal Science 49(1), 049.
Abstract: Purpose of this study was to examine the clinicopathologic features associated with ascites production of monoclonal antibodies because of the animal welfare concerns associated with this technique.
5 hybridoma cell lines, chosen to represent varied plasmacytoma fusion partners, varied isotypes of antibody, and different subclasses of antibody, were grown in mice. Each group consisted of 20 mice; On day 1, mice were given 0.5 ml pristane IP; day 14 - mice were injected with 10<6> cells IP each; Ascites fluid was collected very 1-3 days (when moderate distention was evident but before body weight increased approximately 20% over day-0 weight) for a maximum of 3 times. Clinical observations were made as well as body weights obtained before and after paracentesis; necropsies were performed on selected animals; pristane was cultured to detect bacterial contamination;
Overall survival was 98% (range, 90-100 %) to tap 1, 96% (85-100%) to tap 2 and 79% (35-100%) to tap 3; necropsy revealed disseminated intraabdominal seeding of tumors; differences were apparent between groups as well as within a group in terms of onset and rate of development of ascites. Clinical abnormalities observed were related to development of ascites fluid (rough haircoat, abdominal distention, weight gain, hunched posture - early on; thin appearance, dehydration, palpable abdominal masses, decrease activity - later signs). Immediately after paracentesis mice were observed to have rough haircoat, hunched posture, tachypnea, decreased activity and huddling, occ. dyspnea, and pallor to the ears and muzzle, signs which had significant impact on survivability. Authors interpreted signs to be compatible with those of circulatory shock. All mice that died did so within 30 minutes of paracentesis. Hemoperitoneum was observed in some mice during paracentesis.
Conclusions: significant clinicopathologic changes occur in mice during MAb production. Clinical condition worsens over time in association with increased tumor growth, ongoing ascites production and repeated paracentesis. Post-tap body weights decrease over time, suggesting a significant loss of mass in the face of increasing weight contribution by the tumor.
Questions: Questions:
1. What is the purpose of pristane?
2. What are the reported biologic effects of pristane?
Answers: Answers:
1. Pristane is a hydrocarbon derived from mineral oil. It functions as a ascitogenic agent to induce the production of ascites fluid (Studies have shown that mice given hybridomas without priming the abdomen produce solid tumors but no to little fluid).
2. Biologic effects include induction of granulomatous inflammation, induction of growth factors, lymphatic obstruction by oil-laden macrophages and neutrophils, and reduced clearance of particulate materials and cells from the peritoneal cavity. Transient (12 hours long) decreases in food consumption have been reported in mice receiving pristane IP.

Perizaeus-Merzbacher disease. Laboratory Animal Science 49(1), 054.
Abstract: A description of animal models for Pelizaeus-Merzbacher disease (PMD) and allelic forms of x-linked hereditary spastic paraplegia (HSP). These are rare inherited diseases that exhibit abnormal myelination of the CNS. Both PMD and the HSPs are among a group of mutations that affect the proteolipid protein (Plp) locus. In addition, the HSPs are heterogenous and may be linked to mutation of genes other than Plp. While Plp is transcribed and translated in the peripheral nervous system, it is not normally found in compact myelin there. The Plp gene is highly conserved at all levels.
PMD is a fatal demyelinating CNS disease that is either seen at birth or in the first few years of life (earlier onset form is more aggressive). The x-linked HSPs occur in 2 forms1) the pure spastic form and 2) a complicated form that resembles PMD. Mutations occur via missense, nonsense, silent, base deletion, base deletion/insertion, gene deletion, functional deletion (point mutation in the initiation codon), duplications (partial and complete), abnormal splice sites and rearrangements.
Due to the large number of mutation mechanisms, there are a number of naturally occurring and genetically engineered models. This paper describes 4 of them.
1. The 'jimpy' Plpjp mouse. One of the first models of PMD. Mouse experiences hypomyelination, tremors and early death (around 30 days) associated with seizures. An A to G mutation with resultant deletion of exon 5 and a frame shift. Histologically there are few mature oligodendrocytes, proliferation (but increased apoptosis) of immature oligodendrocytes and marked astrocytosis.
2. The 'rumpshaker' Plpjp-rsh mouse. Less severe phenotype; generalized tremor which peaks around 30 days. Tremor persists through life but becomes localized to hindquarters and observed only during movement then. No seizures, not fatal. A T to C mutation leading to an amino acid substitution in exon 4. Histologically see increased number of mature oligodendrocytes but hypomyelination still present. As mouse ages naked axons become ensheathed in some myelin.
3. #66 transgenic mouse. Homozygous mice experience dsymyelinatinon; hemizygous mice experience demyelination. Transgenes are autosomal, not x-linked. Severe hypomyelination, ataxic, intention tremor and death occurs early associated with seizures (40-60 days of age). Duplication mutation. Histologically oligodendrocyte Golgi bodies are abnormal, marked astrocytosis. Homozygous mice have decreased numbers of oligodendrocytes.
4. #72 transgenic mouse. Homozygotes develop ataxia and seizures between 50 and 150 days of age with demyelination and hypomyelination. Hemizygous have demyelination. #66 and #72 tg mice also exhibit subtle changes in the PNS.
Knock-outs appear to have mild defects with normal amounts of myelin. This is not similar to humans, in which gene deletion results in a progressive spastic paraplegia.
Questions: Questions
1. What cells are responsible for forming myelin?
2. Name 2 animal models for PMD and the x-linked HSPs.
Answers: Answers
1. The oligodendrocyte in the CNS and the Schwann cell in the peripheral nervous system.
2. Animal models include:
Mice - Plpjp (jimpy), Plpjp-rsh (rumpshaker), Plpjp-msd, Plpjp4, sh-pup,
#4e, #66, #72
md rat
Pt rabbit

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals. Laboratory Animal Science 49(1), 058.
Abstract: The purpose of the study was to evaluate whether early glycation products are increased in the vitreous humor in short-term diabetic animals (2-6 months duration). Chronic hyperglycemia leads to increased glycosylation (glycation) of proteins such as lens crystallin, collagen, and hemoglobin. Glucose reacts with the amino acids of the proteins and eventually yields to irreversible cross links called advanced glycosylated end products (AGE).
Diabetes Mellitus (DM) was induced in three cats by partial pancreatectomy. Three nondiabetic cats were used as controls. Blood glucose was maintained between 300-400mg/dl by administration of ultra-lente insulin. After 6 months of hyperglycemia, the cats were euthanized and eyes were removed.
Hyperglycemia (>200mg/dl preprandial blood glucose) was induced by IV administration of alloxan in 18 New Zealand White (NZW) rabbits. Seven of the 18 rabbits did not become hyperglycemic and were used as controls. After 2 months, the rabbits were euthanized and eyes were removed.
Fructosamine and glycated hemoglobin (GHb), 2 glycation products, were measured in serum and vitreous humor.
Blood glucose and serum fructosamine in diabetic cats were twice those of control cats. Fructosamine concentration was significantly higher in the vitreous humor of diabetic cats. Serum fructosamine and GHb values were significantly increased in hyperglycemic rabbits. Vitreous humor fructosamine values were also significantly higher in hyperglycemic rabbits.
Results indicate that there is an increase in glycation proteins in serum and vitreous humor when animals are hyperglycemic for as short a period as 2 months. Increased glycation may lead to increased superoxide (free radical) production causing cell damage (shown to occur in vitro). When the hyperglycemic state leads to increased glycation of capillary basement membrane proteins, endothelial cells and pericytes in the eyes may be damaged through cell membrane peroxidation. Lipid peroxidation has also been documented to injure retinal tissue.
Free radical production, cellular damage and eventually diabetic retinopathy are all sequelae of increased protein glycation products caused by chronic hyperglycemia. Pharmacologic inhibitors of non-enzymatic glycation (such as aminoguanidine) may be useful in reducing the severity of the complications of diabetes mellitus.
Questions: No questions

Bacterial lipopolysaccharide induces a conduction block in the sciatic nerves of rats. Laboratory Animal Science 49(1), 062.
Abstract: Gram negative bacteria and toxins have been implicated in the development of peripheral neuropathies in humans, so the authors were attempting to characterize an animal model of this eitology. They found that IP or IV injection of lipopolysaccharide from E. coli and Salmonella enteritidis, given to the male inbred Australian albino Wistar (AaW) rat would reliably create this lesion. Neurologic examinations were performed. Electrophysiologic analysis of the alpha-motor neurons of the sciatic nerve was also performed. Spinal cord evoked potentials (SCEP) were also assessed. Significant differences were noted in the results of the electrophysiologic studies (reduced compound muscle action potentials and elevated somatosensory evoked potentials). No significant differences were noted in the SCEP studies.
Interestingly, they noted that the effects of the lipopolysaccharide injections were transient. The degree of neurologic dysfunction was less on day 15 when compared to days 1 and 8. This suggests that this is not an ideal model for Guillian-Barre syndrome (GBS) and critical illness polyneuropathy (CIP), but it may model one of the important first steps in these disease properties.
Dexamethasone appeared to provide a protective effect (less severe neurologic sequelae in rats given dexamethasone prior to LPS injection). Gadolinium choloride also seemed to have protective capabilities.
The most common neurologic clinical signs included: gait abnormalities, hind limb proprioceptive loss, and abnormal righting reflex.
Questions: Questions:
1. Name two human syndromes that have peripheral neuropathies associated with them.
2. Which of the following affected the sign severity noted in these animals?
a. LPS dosage
b. Hydration
c. Sex
d. Genetic factors
e. All except B.
3. "Clinical and electrophysiologic results suggest that ____ diameter nerve fibers were preferentally affected in LPS-treated rats, compared with ____ diameter nerve fibers." (Put 'large' or 'small' in the sentence.)
4. List four inflammatory factors that glucocorticoids inhibit the production of.
5. What is gadolinium cholride (GAD)?
Answers: Answers:
1. Guillain-Barre syndrome (postinfectious polyneuritis) and critical illness polyneuropathy
2. E
3. Large; small
4. IL-1, IL-6, TNF-alpha, IL-8, prostaglandins, eicosanoids, nitric oxide synthase, and reactive oxygen intermediates.
5. It is a macrophage inhibitor

Monocolonal antibody production in murine ascites, I. Clinical and pathologic features. Laboratory Animal Science 49(1), 070.
Abstract: Purpose of this study was to examine the clinicopathologic features associated with ascites production of monoclonal antibodies because of the animal welfare concerns associated with this technique. 5 hybridoma cell lines, chosen to represent varied plasmacytoma fusion partners, varied isotypes of antibody, and different subclasses of antibody, were grown in mice. Each group consisted of 20 mice; On day 1, mice were given 0.5 ml pristane IP; day 14 - mice were injected with 10<6> cells IP each; Ascites fluid was collected very 1-3 days (when moderate distention was evident but before body weight increased approximately 20% over day-0 weight) for a maximum of 3 times. Clinical observations were made as well as body weights obtained before and after paracentesis; necropsies were performed on selected animals; pristane was cultured to detect bacterial contamination; Overall survival was 98% (range, 90-100 %) to tap 1, 96% (85-100%) to tap 2 and 79% (35-100%) to tap 3; necropsy revealed disseminated intraabdominal seeding of tumors; differences were apparent between groups as well as within a group in terms of onset and rate of development of ascites. Clinical abnormalities observed were related to development of ascites fluid (rough haircoat, abdominal distention, weight gain, hunched posture - early on; thin appearance, dehydration, palpable abdominal masses, decrease activity - later signs). Immediately after paracentesis mice were observed to have rough haircoat, hunched posture, tachypnea, decreased activity and huddling, occ. dyspnea, and pallor to the ears and muzzle, signs which had significant impact on survivability. Authors interpreted signs to be compatible with those of circulatory shock. All mice that died did so within 30 minutes of paracentesis. Hemoperitoneum was observed in some mice during paracentesis. Conclusions: significant clinicopathologic changes occur in mice during MAb production. Clinical condition worsens over time in association with increased tumor growth, ongoing ascites production and repeated paracentesis. Post-tap body weights decrease over time, suggesting a significant loss of mass in the face of increasing weight contribution by the tumor.
Questions: 1. What is the purpose of pristane?
2. What are the reported biologic effects of pristane?
1. What is the most commonly used adjuvant?
2. This adjuvant come in two flavors, what are they, how are they different, and how are they used?
3. What are Fab and Fc?
4. On what prominent cell type do you find CD4 antigens? CD8 antigens?
5. What mouse strain is most commonly used for hybridoma production? Why?
6. Antigens are usually injected in the mouse's body (SQ or IP). Name two other locations were antigens can be injected.
7. What two cell types are used to make a hybridoma?
8. What compound is used to fuse these cells together?
9. What compound is used to select for those cells forming hybridomas?
10. Name two agents that can be used to prime the peritoneal cavity of mice for ascites production?
11. Name at least two in vitro methods of monoclonal antibody production?
12. Compound that binds Fc region, used to purify IgGs?
13. Which two factors are most commonly sited by investigators for not wanting to use in vitro techniques for MAb production?
14. Why might SCID mice or nude mice be used for ascites production?
15. Commonly used experimental procedure that employs a polyacrylamide gel, nitrocellulose paper, electric current, and antibodies?
Answers: 1. Pristane is a hydrocarbon derived from mineral oil. It functions as a ascitogenic agent to induce the production of ascites fluid (Studies have shown that mice given hybridomas without priming the abdomen produce solid tumors but no to little fluid).
2. Biologic effects include induction of granulomatous inflammation, induction of growth factors, lymphatic obstruction by oil-laden macrophages and neutrophils, and reduced clearance of particulate materials and cells from the peritoneal cavity. Transient (12 hours long) decreases in food consumption have been reported in mice receiving pristane IP.
1. Freund's Adjuvant
2. Complete Freund's Adjuvant (CFA)- contains mycobacterium antigens, Incomplete Freund's Adjuvant (IFA) - does not contain mycobacterium Use CFA for first prime, following boosts use IFA. (2 CFA doses could result in serious reaction/anaphylaxis)
3. Fab = Fragment antibody - papain digested antibody fragment containing the variable region of the heavy and light chain joined by a disulfide bond, still binds antigen, monovalent, lacks effector function Fc - Fragment constant - papain digested antibody fragment that interacts with effector cells and complement. Ig's of the same class have highly homologous Fc regions
4. CD4 = T helper cells CD8 = cytotoxic T cells
5. BALB/c, first strain to demonstrate myelomas could be produced by injecting irritable oily substances IP. Myeloma cell lines were thus produced and are now commercially.
6. Intrasplenic and Popliteal lymph node (or footpad) - sometimes used because belief is that you might get an enhanced response to difficult antigen.
7. Immunized mouse splenocytes and myeloma cells
8. Polyethylene glycol (PEG)
9. HAT (hypoxanthine-aminopterin-thymidine)
10. Pristane and Incomplete Freund's Adjuvant
11. Gas permeable bags, batch tissue culture method, spinner culture flasks, semi-permeable membrane based systems (Integra system), hollow fiber bioreactors.
12. Protein A- sepharose
13. Cost and low concentration (and possibly less affinity/loss of function)
14. Hybridoma is nonsyngeneic to BALB/c (hamster, human splenocyte, or other used in original fusion). Need immunocompromised host to accept cells growing and producing ascites IP.
15. Western blot

Monoclonal antibody production in murine ascites, II. Production characteristics. Laboratory Animal Science 49(1), 081.
Abstract: Objective: To characterize monoclonal antibody production parameters of
five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice.
Methods: Five hybridoma cell lines were grown in-groups of 20 mice. Fourteen days prior to inoculation with 1 million hybridoma cells, mice were primed with 0.5 ml of pristane given IP. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample (i.e for each collection).
Results: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Total antibody production ranged from 422.9 to 966.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1,2, and 3 ranged from 2.5 to 15.03 mg/ml among cell lines.
Conclusions: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites. The findings in this paper stress that increased survival of mice positively impacts on total antibody production. Since the mice are basically growing cancer cells the balance must be reached where the animals can live with as little distress as possible and yet produce the maximal amount of ascites fluid. To address the distress issue the authors stress that careful clinical monitoring, assessment of the degree of abdominal distention, and timely performance of abdominal paracentesis are valuable in reducing mortality. The volume of inocula injected is also important in these regards. Higher inocula results in earlier onset of ascites production but earlier mortality; lower inocula result in fewer mice developing ascites and lower ascites fluid volumes. The authors also point out that the volume that the cells are injected in can affect production since when the same number of cells are given in 0.5 ml vs. 0.2 or 0.3 ml, the 0.5 ml volume yielded higher antibody production. In general, the more abdominal taps done, the higher the total antibody yields. If the number of abdominal taps is reduced, the amount of distress the animals experience will be reduced, but the total number of mice put in distress will be increased to achieve the same total level of antibody production. The decisions on limitations of abdominal taps should be based on (listed in the order of importance) the clinical condition of the mice, and antibody production parameters (i.e. ascites fluid volume and antibody concentration for taps over time). The authors suggest that vacuum aspiration as opposed to gravity flow may give higher yields of antibody. The authors found a great variability between the hybridoma clones in their ability to produce antibodies in vitro. This variability illustrates how bioreactor systems will not work for every hybridoma clone as efficiently. However, the authors suggest that in vitro. Alternatives for antibody production should always be considered.
Questions: Questions:
1. All mice used to produce ascites should be limited to one abdominal tap. T. or F?
2. Injecting mice with the maximal amount of hybridoma cells will always give the highest antibody production. T or F?
3. Irregardless of the hybridoma clone, bioreactor systems have been shown to work just as well as ascites fluid production in mice. T or F?
Answers: Answers:
1. F
2. F
3. F

Rib biopsy technique for cortical bone evaluation in rhesus monkeys (Macaca mulatta). Laboratory Animal Science 49(1), 087.
Abstract: Old World primates are often studied to model human skeletal physiology. An important advantage of monkeys over other animal models (i.e., rodents) is the presence of cortical bone Haversian remodeling. Seventy-five female rhesus monkeys (Macaca mulatta) were subjected to bone biopsy. With monkeys in lateral decubitus position, the tenth rib was surgically exposed and freed from periosteum by use of careful sharp and blunt dissection. The rib section was resected, using bone cutters, and the surgical wound was closed. This procedure was repeated for the contralateral rib at a later time point in 65 monkeys.
There was no mortality or appreciable morbidity. The bone specimens were (mean " SD) 2.50 " 0.25 cm long, with 5.5 " 1.0 mm2 total cross-sectional area. They were adequate for histologic, immunohistochemical, and quantitative histomorphometric examinations. Prevalence of pneumothorax was approximately 8.0% for the 140 procedures. This complication was immediately and successfully corrected by insertion of a small thoracic tube, evacuation of pneumothorax, and closure of the incision.
This well-tolerated, repeatable procedure yields excellent specimens for performance of cortical bone histologic examination without euthanasia, allowing longitudinal evaluation.
Questions: Questions:
1) What is the advantage of the monkey model over others (i.e. rodents) in studying osteoporosis and bone remodeling?
2) What is a limitation to using the rib when studying bone remodeling?
3) What does CITES stand for?
4) How many units of mammalian Old Tuberculin are injected ID when testing Old World NHP's?
5) What is the normal disease course of simian parvovirus in immunocompetent macaques?
6) Lung mite of Rhesus?
7) What cyclic changes do male squirrel monkeys undergo?
Answers: Answers:
1) An important advantage of the monkey model is the presence of cortical bone Haversian remodeling similar to that in human bone.
2) The rib is NOT a weight bearing bone compared with the tibia.
3) Convention on International Trade in Endangered Species
4) 1500 units
5) In immunocompetent macaques the infection results in a transient anemia that resolves in 2-3 weeks as immunity develops
6) Pneumonyssus simicola
7) They become heavier during breeding season (March - May in Northern Hemisphere) associated with increased testes size, spermatogenesis and increased plasma testosterone concentration

Embryo transfer in the rat as a tool to determine the genetic components of the gestational environment. Laboratory Animal Science 49(1), 090.
Abstract: A technique is described by which closely-related inbred rat strains, the hypertensive Dahl salt-sensitive SS/JrCtr (S) and normotensive Dahl salt-resistant SR/Jr (R) were examined to discriminate between factors responsible for their differing phenotypes which may be due to gestational effects as opposed to non-gestationally-related genetic differences. This was accomplished by performing reciprocal embryo transfers between the two strains. For control purposes, embryo transfers were also made into recipient females of each donor strain, and reciprocal transfers were made between the salt-sensitive SS/JrCtr strain and an unrelated nonresistant normotensive strain, the Dark Agouti (DA). Additional controls were rats gestated in a dam of their own parent strain, but taken less than six hours after birth and cross-fostered on dams of a different strain.
Embryos at the morula and blastocyst stages were harvested from donor rats at three or four days post-coitus (p.c.) and transferred to an avascular portion of the uterine horns (generally bilaterally) of recipient dams, which had been bred to a vasectomized male four days previously.
For a salt challenge, 0.9% NaCl saline solution was provided as a replacement for drinking water. Differences between the study groups were determined by measuring blood pressure (b.p.) of well-trained unheated progeny, twice weekly by tail-cuff plethysmography, commencing at an age of seven weeks.
Significant findings were as follows:
1. S embryos transferred to R dams (S et R), exhibited lowered blood pressure to normotensive levels from six to eleven weeks of age, rising to midrange between that of S controls and normotensives by 16 weeks of age. By 22 weeks of age, the blood pressure of female S et R rats had risen to the usual high levels of control S rats.
2. When these female S et R pups were subsequently bred to S males at 15 weeks of age (when their own b.p. was markedly lower than that of control S rats), the b.p. of their progeny (born when these S et R dams were 18 weeks
of age) was identical to that of control S rats (i.e. not low as was that of the S et R dams).
3. R rats embryo-transferred to S recipients (R et S) exhibited no change in b.p. from normal (normotensive) control R rats.
4. There was a small but significant elevation of b.p. in DA et S compared to DA control animals.
5. There was a significant drop in b.p. of S et DA rats versus S controls.
6. S rats typically weigh 10-12% more than R rats; this feature was not significantly altered by litter size, embryo transfer, or cross-fostering.
Many less unique findings corroborated observations of others in earlier work with embryo transfer in general, cross-fostering of pups in general, and general findings specific to the strains used. Examples are: 2 vasectomized rats produced viable progeny in their first mating at 12 days post-vasectomy, but subsequently became functionally infertile; about 27 to 53 percent of transferred embryos were viable, but if only those cases where at least one transferred embryo was successfully reared from a recipient dam are considered, the viability success rate was close to twice the overall average; factors noted to reduce embryo transfer success were inclusion of larger amounts of medium with transferred embryos, transfer of red blood cells with embryos, and trauma to the uterine lining during embryo transfer; exact timing of the donor dam was not critical, give or take a day or so, but departing by as much as a day from the four days p.c. timing of the recipient reduced the success rate of embryo transfer significantly; S rats are milder to handle than R; substituting 0.9% saline for drinking water did not affect b.p. of R rats, but elevated b.p. of both S and S et S.
DISCLAIMER: This and some other studies indicated no effect of cross-fostering of S and R rats; however, the authors pointed out that many other studies have noted b.p. of S (salt-responsive hypertensive) strains significantly lowered if cross-fostered on a normotensive strain. Environmental factors are cited as a significant factor in affecting b.p., even in similarly-aged animals of the same strain at the same institution on a different date (generally separated by a few months or more)---not all the causative factors were known, but this study, for example, was marred by a period of very low humidity (causing "ringtail" in some animals), annd frequent heavy construction noise around the facility.

The basic procedure of reciprocal embryo transfer described is postulated to
be a useful method of determining whether factors responsible for observed
differences in strains are purely "nature" (i.e. genetic) or somewhat due to
nurture" (gestational environmental factors).
Questions: QUESTIONS:
1. Are there factors in the gestational environment which can significantly alter characteristics normally attributed to a specific strain of animal? (If yes, give an example; if no, cite a specific reported negative experimental finding.)
2. Can embryo transfer between related strains of rats permanently change phenotype of second-generation offspring?
3. When Dahl salt-sensitive and Dahl salt-resistant rats are cross-fostered, what has been observed?
4. You are given two rats, one Dahl salt-sensitive, and the other an age-and-sex-matched Dahl salt-resistant one, and a device for measuring blood pressure by a tail-cuff method. With no additional items, describe how you would determine which animal is which.
Answers: ANSWERS:
1. Dahl Salt-Sensitive (S) rats embryo-transferred into Dahl salt-resistant (R) dam uteri develop with significantly reduced blood pressure relative to both unmanipulated S controls and S controls embryo-transferred into S dam uteri. This reduction continues through adulthood only, but then has been noted to taper back to the high levels of b.p. usually associated with the S strain.
2. Not demonstrated in this study (although at least one characteristic altered in embryo-transferred progeny reverted to the wild type in the next generation).
3. Reports have been variable as to the outcome of this manipulation; some have noted a lowering of the b.p. of S pups cross-fostered on the normotensive R strain; this study and others have not noted any effect of cross-fostering on b.p.
4. The hypertension in Dahl rats is salt-induced. Without first giving the animals 0.9% saline (not permitted in the terms of this question) in substitution for drinking water for an extended period of time (e.g. for six hours or longer) you should be unable to discriminate between the two by use of a tail-cuff plethysmograph. The 10-12% average greater weight of the SS/JrCtr (Dahl salt-sensitive) strain should be readily noticeable on picking up the two animals. The milder nature of the salt-sensitive strain can provide another valuable clue. Additionally (not pointed out in the summary, but fair game on an ACLAM question) the salt-sensitive strain has congenital cataracts due to an abnormal lens basement membrane, readily noted from the time of eye opening onwards.

Virulence Enhancement agents for Haemophilus influenzae Type B infection in mice. Laboratory Animal Science 49(1), 095.
Abstract: Most laboratory animal species are not susceptible to Haemophilus influenzae type B (Hib), a human pathogen. In order to study Hib pathogenicity, a number of experimental approaches have been developed to promote Hib infection in laboratory animals. Inracisternal inoculation, and concurrent administration of virulence enhancing agents (mucin, iron supplementation, and capsicum) are some of the experimental approaches used to promote Hib infection.
The goal of the study is to compare the ability of hemoglobin, iron dextran, trypsin, mineral oil, and bovine estrual mucous to cause Hib induced mortality in Balb/c mice.
Results:
1. The concurrent administration of saline and Hib did not induce mortality in Balb/c mice
2. Trypsin and iron dextran were the most effective virulence enhancing agents
3. Mineral oil had no effect on Hib virulence
4. Concurrent administration of estrual mucus and iron dextran decreased Hib virulence
5. The optimal time to administer virulence enhancing agents was the same day as bacterial inoculation
Questions: QUESTIONS
1. Species of the genus Haemophilus are
a. gram - rod/filament
b. gram + rod/filament
c. gram - cocci
d. gram + cocci
2. _____________ is the major B1-globulin that transports iron in the blood.
3. What is the function of the outer membrane proteins, Tbp1 and Tbp2, on Hib?
Answers: ANSWERS
1. A
2. TRANSFERRIN
3. Tbp1 and Tbp2 bind human transferrin - this allows the organism to acquire iron within the host

Transgenic mouse strain rescue by frozen ovaries. Laboratory Animal Science 49(1), 099.
Abstract: The authors of this study are from Jackson Laboratories, where FVB/N-TgN(MMTV Int 3)3Rnc transgenic mice were sent but became ill shortly after arrival due to rapidly growing mammary tumors. The immune compatible recipients FVB/NJ were not immediately available and so the ovaries were collected and sectioned in halves in M2 medium, then placed in cryoprotectant (CPA - 1.5M DMSO in M2 medium with 10% FCS). The cooling protocol was as follows: 23oC for 10 min, 0-5oC for 45 min, -6oC for 5 min, then "seeded on the surface of the medium by use of a Pasteur pipette cooled to -10oC"? Then cooled at -0.5oC/min to -80oC before being transferred into liquid nitrogen (-196oC) for 2 months. After the recipients became available, a second mouse of the same strain was received and was similarly ill. The ovaries were collected from this mouse, halved, washed in M2 medium, and were transferred to the recipients immediately. These served as a control for the frozen ovaries which were thawed at 23oC until the ice was melted, then washed in M2 medium. The recipient mice were anesthetized with tribromoethanol and ovariectomized, then one half of one ovary was transplanted into the right bursa of each recipient. The mice were mated to proven FVB/NJ males 10 days post-op.
Three of the 4 mice with fresh ovary transplants had a total of 8 litters in which only 2 of the 19 females born expressed the int3(+/-) transgene by PCR. (Males are infertile and so were not analyzed). Two of the 4 mice with frozen ovary transplants had a total of 3 litters in which 3 of the 8 females born expressed the transgene. There was no statistical analysis in this paper. The authors point out that the recipients were not affected by the mammary tumors of the donor strain, and so were able to reproduce normally. They caution the readers that the recipients may be exposed to pathogens by vertical transmission through the transplanted ovary. The recipients in this study remained sero-negative for transmissible disease, even though the donors were positive for MHV.
Questions: QUESTIONS:
1. Why was FVB/NJ strain used in this study?
A. They are a common background for micoinjection because the zygotes have large, easily identifiable pronuclei
B. They are immune-compatible with the transgenic strain
C. They tend to have large litters
D. All of the above
2. Tribromoethanol
A. Is a common anesthetic ingredient used in mouse embryo transfer
B. Has been implicated in a recent publication to cause peritonitis in mice
C. Was formerly available under the trade name Avertin
D. All of the above
Answers: ANSWERS: 1. D, 2. D

Plasma cytokine concentrations and splenic changes in cynomologus monkeys (Macac fascicularis) with malaria. Laboratory Animal Science 49(1), 101.
Abstract: A group wild caught cynomolgus monkeys, used on a 28 toxicity study, developed an uninduced malarial infection. Changes in cytokines and histopathology of spleen as a consequence of infection were discussed.
Pre treatment blood work- no sex or group (control or compound), differences noted or variation from reference ranges , no parasitized erythrocytes detected.
Day 14 one monkey in treated group had an increased reticulocyte count, and the lowest erythrocyte count. No differences between treated and control groups. IL-6 , TNF- alpha, IL-1 alpha were increased compared to pretreatment values. No monkeys found to have malaria via thin blood smears.
Day 27 No differences between treated and control groups. Erythrocyte numbers and hemoglobin concentration were significantly decreased, reticulocyte numbers increased. IL-6 , TNF- alpha, IL-1 alpha were increased compared to pretreatment values. 25/28 monkeys found to have malaria via thin blood smears, cytokines in remaining three were increased compared to pretreatment values
Day 28 Necropsy- hepatomegaly, splenomegaly with dark coloration. Parasite identified as Plasmodium knowlesi on the basis of morphologic features (ring formed trophozoites with a single large nucleus in the young stage, band formed trophozoites in the growing stage, oval and compact trophozoites in the mature stage, and the absence of applique or accole forms.
Splenic tissue findings- dilation of splenic sinus, deposition of malarial pigment, decreased lymphocyte numbers, increased numbers of large mononuclear cells in the white pulp.
Hepatic tissues increased numbers of kupfer cells and aggregation of macrophages in sinusoids which contained phagocytosed erythrocytes, malarial pigment and hemosiderin. Parasitized erythrocytes noted in glomerular capillaries Bone marrow ? normoblastic hyperplasia lungs parasitized erythrocytes in alveolar capillaries and deposits of malarial pigment in the capillary wall were present. No evidence of cerebral malaria
How did the monkeys get the infection? Speculation that monkey w/ high reticulocyte count had latent infection which was spread via use of a single needle used for i.v. administration of test substance, or through contamination of multiple dose vial used for i.m. injection of ketamine.
This study reports high reticulocyte count w/ malarial infection, not previously reported. IL-6 , TNF- alpha, IL-1 alpha are increase with malarial infection, and the TNF-alpha (which causes a decrease in lymphocyte numbers in vivo is the earliest responsive and most sensitive to malarial infection.
Questions: Questions
1. Splenic changes due to malaria (in this study).
a decreased lymphocyte numbers, decreased numbers of large mononuclear cells in the white pulp.
b decreased lymphocyte numbers, increased numbers of large mononuclear cells in the red pulp.
c increased lymphocyte numbers, decreased numbers of large mononuclear cells in the white pulp.
d decreased lymphocyte numbers, increased numbers of large mononuclear cells in the white pulp.
2. Which cytokine is considered to be the earliest and most sensitive indicator of malarial infection.
a. TNF- alpha
b. IL-6
c. IL-1 alpha
d IL-2
Answers: answers.
1.d
2.a

Bilateral prolapse of the deep gland of the third eyelid in a rabbit: Diagnosis and treatment. Laboratory Animal Science 49(1), 105.
Abstract: Protrusion of a large mass form the medial angle of the eye has been reported as an occasional finding of uncertain etiology in rabbits. The objective of this paper is to elucidate the cause of this clinical condition and to propose nomenclature for the orbital glands of the rabbit on behalf of the International Committee on Veterinary Gross Anatomical nomenclature.
Possible causes of swelling and protrusion of the third eyelid from the medial ocular angle of the rabbit include- infection and enlargement of orbital glands- prolapse of the ophthalmic venous sinus caused by periocular infection- prolapse of the deep gland of the third eyelid with unclear underlying cause. Clinical appearance is similar to the condition known as "cherry eye" in dogs, which is caused by the prolapse of an (enlarged) superficial gland of the third eyelid.
See article for figure and naming of the glands.
Case summary-
13 month lop eared male domestic rabbit with bilateral white conjuctival discharge, mild swelling and protrusion of the third eyelid for several weeks 3 month course of topical treatment w/ oxytetracycline hydrochloride ophthalmic ointment not helpful. Animal returned w/ increased swelling and protrusion of both third eyelids. Clinical exam indicated that both third eyelids contained a soft tissue mass and had congested blood vessels.
Presumptive diagnosis- prolapse of the third eyelid glandular tissue. Surgery (pocket technique described by Moore) was performed to correct the prolapse. Biopsy taken during surgery for histologic identification. Post surgery treatment 3 days w/SQ. chloramphenicol and topical chloramphenicol ophthalmic ointment. 1 and 3 months post sx- shrimer tear test WNL 16 months post surgery, swelling and protrusion of right eyelid, left eye normal. Surgical correction performed again- at 9 months post second sx both eyes normal.
3 3- months old male rabbits, without ocular disorders were used for histologic and anatomic identification of glandular structures.
Proper Lacrimal gland flat curved aggregation of small glandular lobules, situated dorsolateraly in the orbit and caudaldorsal to the eyeball. Composed of acinar secretory units and tubular ducts
Accessory lacrimal glands- extends about 4 cm along caudal and ventral orbital margins external part ids slender and protrudes medially into the orbit., dorsally a rounded retroorbital lobe extends into the temporal fossa. similar in histologic appearance to proper lacrimal gland
Superficial gland of the third eyelid few mm long, lies against convex surface of the slender curved cartilage of the third eyelid.
Deep gland of the third eyelid large solid pyramidal gland that lies rostromedially in the orbit. base is in apposition to the deep border of the cartilage of the third eyelid composed a dorsal white lobe and a bulky ventral pink lobe.
The histology of the prolapsed tissue from the case rabbit was similar to that of the pink ventral lobe of the deep gland of the third eyelid from the rabbits used for identification of the glands.
Questions: No questions

Surgical induction of cryptorchidism in rabbit pups. Laboratory Animal Science 49(1), 110.
Abstract: The purpose of this study was to surgically induce cryptorchidism in rabbit pups. There has not been a good model for evaluating endocrine-disrupting chemicals on adult reproductive function in humans as well as in animals. Specific abnormalities arising in males that may be related to environmental exposures to endocrine disruptions include cryptorchidism, hyospadias, and infertility. Rats were considered but the infantile period of rats last only a week which was considered too short. Rabbits were finally considered because the infantile period extends for about twelve weeks.
Twelve three-week old rabbit pups were studied. Three surgical procedures were used. The first procedure transected the gubernaculum and closed the inguinal ring. The second procedure excised a 1-cm section of the gubernaculum and closed the inguinal ring. The third procedure consisted of excising a section of the gubernaculum without manipulating the inguinal ring. These twelve animals were placed back with their dams for a period of four to twelve weeks.
All animals that underwent surgery remained bilateral cryptorchids. Two control and two bilateral cryptorchid pups were euthanized and were necropsied for unanticipated pathological changes, unusual adhesions, and other abnormalities which were not observed. The remaining animals were trained to ejaculate into an artificial vagina. All animals manifested normal sexual behavior and sexual capacity but were azoospermic throughout the collection. In contrast the animals exposed to environmental chemicals did not exhibit normal sexual behavior. There was a substantial difference in the weight of the testes in surgically treated animals compared to controls. Microscopically, germ-cell differentiation beyond the spermatogonial stage was not observed.
Conclusions: This study illustrates that surgical induction of cryptorchidism in prepubertal rabbits is a simple procedure with few complications. Also atypical cells observed in undescended testes after perinatal chemical exposure were not likely a sequel to the intra-abdominal per se; such cells were not observed in animals in which cryptorchidism was induce surgically. More studies are needed using the third surgical procedure with excision of the gubernaculum because it is sufficient to cause cryptorchidism, easy to perform and results in fewer post-operative adhesions.
Questions: Questions:
1. What is the primary cause of intraabdominal environmental disruption of normal progression of germ-differentiation in the seminiferous epithelium of the of cryptorchid individuals?
2. Which of the three surgical procedures were considered to be the easiest to use in the future?
Answers: Answers:
1. Higher temperature
2. Excision of the gubernaculun alone produced cryptorchidism individuals with fewer adhesions.

Detection of cilica-associated respiratory (CAR) bacillus in nasal-swab specimens from infected rats by use of polymerase chain reaction. Laboratory Animal Science 49(1), 114.
Abstract: Cilia-associated respiratory (CAR) bacillus is an unclassified, gram negative, motile bacteria that has caused respiratory tract disease in lab rodents. CAR bacillus infections are generally subclinical during the early stages of disese, and bacteria may be shed in nasal secretions before the disease is apparent.
Histo exam is a highly specific test because of the uinique colonization pattern of CAR bacillus: found lying parallel to the cilia of the nasopharyngeal, tracheal or bronciolar epithelium. The ELISA is sensitive in detecting subclinical infection, but cross reactivity with antibodies to other bacterail species may limit specificity of CAR bacillus ELISA's. Another disadvantage to serologic testing is that naturally infected rats may not develop high titer of antibody until 8-10 weeks of age. Culture is the most definitive diagnostic test, but cultivation has been limited to embryonating eggs, mammaliam cell cultures or modifications of the latter.
In this study the author evaluated the advantages of rDNA amplification and use of nasal swab specimens collected from CAR bacillus infected rats for the diagnosis of CAR baillus infection by PCR.
The animals were anesthetized and nasal swabs collected for PCR. Swabs containing calcium alginate were moistened with phosphate buffered saline prior to sample collection. After swabbing, 8 of the rats were euthanized and specimens were collected for PCR and histo exam.
To determine stability of CAR bacillus DNA collected by nasal swabs, specimens were collected from breeders, placed in sterile PBS & incubated at room temp for 6, 12, 24 & 48 hours. 4 swabs/time period were examined. The CAR baillus DNA was detected in 100% of samples from all time points, indicating that DNA was stable up to 48 hours at room temp.
To determine if nasal cavity secretions interfered with sensitivity of the CAR bacillus PCR, nasal swab specimens were collected from CAR bacillus free rats. The authors found that sensitivity of the assay was not affected by exposure to nasal secretions.
In this study, CAR bacillus infection by PCR of nasal swabs was detected in 94-100% of rats 5 weeks of age & older and in 54% of 3 week old rats.
Questions: Questions
1. List 2 advantages of using nasal swab PCR over other CAR bacillus testing methods.
2. List some of the clinical signs of CAR bacillus infection.
Answers: Answers:
1. a. PCR can be done antemortem. b. It can detect early infections.
2. Weight loss, rough coat, wheezing, cyanosis.