Sheep model in orthopedic research: a literature review. Comparative Medicine 51(4), 292.
Abstract: The article was written to provide basic information on the suitability of the sheep model for in vivo orthopedic studies. The fundamental aspects addressed by the authors are: factors strictly related to bone anatomy and formation; and factors strictly affecting bone physiology, such as gastrointestinal mineral and vitamin absorption, and reproductive cycle. Awareness and proper application of these factors may result in a better knowledge of the animal used and the differences between the model and the human being. Therefore, more useful data can be derived from a well-planned orthopedic study.
<> The selected animal model for an orthopedic study should be carefully assessed to ensure analogy with humans
<> Animal species used for human orthopedic pathologic conditions include NHPs, dogs, cats, pigs, cattle, horses
<> In 21,500 orthopedic studies done over the past 30 years, rats were used more often (36%) than other species. Sheep accounted for 2% of the animals used
<> Among the large animals, dogs were used most often for fracture studies (42%) and osteoporosis (41%). Rabbits were used most often for osteoarthritis (44%), and rabbits and dogs were tied in bone lengthening studies (37%). Sheep use averaged between 9-12% for each of these.
<> Rodents are often used in basic orthopedic studies, toxicologic tests on biomaterials, and early stages of testing
<> Healing characteristics of animals should approximate those of humans during the late stages of testing
<> Studies involving fracture fixation, osteotomies, ligament repair, and cartilage defects need animals carefully selected for their size and anatomy
<> The evaluation of final orthopedic and prosthetic devices requires large animals such as NHPs, dogs, pigs, sheep, and goats
<> Sheep are becoming popular as animals models due to the cost, ethical, and zoonotic concerns with monkeys, ethical and legal problems with dogs, and the rapid growth and handling difficulties of pigs.
(i) General aspects
<> In the past decade, sheep were used to study fracture and articular ligament repair, limb lengthening, osteoarthrosis, osteoporosis, muscular disorders, osteomyelitis, spinal diseases, and biomaterial evaluation
<> Sheep are similar in body weight to humans, are large enough for serial sampling and multiple experimental procedures
<> Sheep reach sexual maturity between 7 and 12 months of age (breed dependent), yet skeletal growth still occurs for a long time post puberty. A complete table is included in the article which provides the closing times of the sheep ossification centers.
(ii) Bone anatomy
<> Accurate anatomic and biomechanical studies of the ovine spine have been published and establish which regions are used as a model for the human spine (Anatomically, the gross structure of the thoracic and lumbar parts of the spine are best used as models for the human spine Biomechanically, the distal portion of the cervical part of the spine and thoracolumbar junction are most similar to the human spine
<> The shoulder girdle of the sheep is often studied due to the similarity between its infraspinatus tendon and human supraspinatus tendon
<> With the exception of a few anatomical differences, the sheep stifle joint is quite similar to the human knee joint
(iii) Bone structure and pathophysiology
<> One group evaluated multiple species for bone compositions, density, and quality and concluded there is no ideal animal bone to be used for all orthopedic studies. However, it was decided that dog bone best approximated human bone based upon the above factors.
<> Bone mineral density is fairly similar between humans, cattle, and sheep
<> Bone regeneration is faster in animals than it is in humans
<> Interspecies differences may be overcome when the critical size defect (CSD) is used. The CSD is defined as "the smallest size intraosseous wounds in a particular bone and species of animal that will not heal spontaneously during the life time of the animal" or as "defects that have <10% bony regeneration during the lifetime of the animal."
<> The CSD depends on breed, age, skeletal site, presence or absence of fixation systems, kind of fixation. The most important factor is the relation between bone defect and size of the selected bone segment.
<> Using this knowledge, researchers create CSD or larger defects in cortical and trabecular bone of animals. Example: defects that are 1.5-2.0 times the length of the shaft diameter are created in cortical diaphyseal bone, which often result in nonunions.
<> The sheep has been used to study altered bone metabolism during hormone deficiency to evaluate effects of therapeutic drugs on bone tissue. Ovariectomized sheep have an increase in bone formation beginning at 10 weeks and persisting until 6 months after surgery. Bone calcium deposition peaks at about 1 year of age, then decreases until leveling off at 9 years.
<> When sheep are used as osteoporosis models, seasonal components or depression of animal bone turnover due to day length and hormonal changes may influence GI and endocrine physiology
(iv) Mineral metabolism
<> Plasma Ca++ is dependent upon the amount and efficiency of intestinal absorption, skeletal development and mobilization, concentrations of parathyroid hormone and calcitonin, and amounts of excretion in intestinal tract, urine, fetus, and milk.
<> When sheep are deprived of Vit D less than 20% of ingested Ca++ is absorbed.
<> Differences in Ca+ metabolism between sheep and humans depend upon the regulation of its absorption. In sheep the regulation is precise and Ca++ is absorbed as the body needs it. Absorption is variable, but excretion is constant. The opposite is true for humans: absorption is controlled by the availability of Ca++ in the diet and the Ca:P ratio.
<> The factors which interfere with the Ca:P ratio in sheep are geographic area of origin which is affected by light cycle and food quality; indoor housing; Vit D content of forage. Feed these animals higher Ca++ and P content and at the proper ratio
<> A table is included in the article providing the daily Ca++, P, and Vit D requirement in sheep
<> Pay attention to quality and composition of pasture or herbal composition of pelleted diet. Phytoestrogens and their metabolites should be carefully controlled because they adversely affect reproductive conditions, bone morphology, and bone growth.
(v) Influence of the reproductive system on bone
<> Sheep (like other animals) do not have a clear-cut menopause at mid-life (characterized by accelerated bone loss in women)
<> However, Dorset and Merino ewes have a reproductive cycle similar to women. Therefore, sheep may be more sensitive to estrogen deficiency than any other animal with less frequent estrous cycles (such as the dog).
<> Orthopedic studies require animals similar in body weight and size to humans
<> Attention should be paid to the quality and composition of the animal's diet in bone metabolism studies
<> Phytoestrogens and metabolites should be carefully controlled in ruminants as they affect reproduction, bone morphology, and growth.
Questions: 1. What animal species was used most often in orthopedic studies over the past three decades?
2. What are some characteristics which would make sheep good models for human orthopedic research?
3. What are three anatomical similarities sheep share with humans?
4. T or F Bone regenerates faster in humans than animals.
5. What is the most important factor in the cortical size defect (CSD)
c. Type of fixation device
d. The relation between bone defect and size of the bone segment
6. A sheep's Ca:P may be altered by the
a. Content of Vitamin D in forage
b. Indoor housing
c. The breed's geographic area of origin
d. All of the above
Answers: 1. Rat (36%)
2. Similar body weight, size and reproductive cycle to humans; fewer ethical concerns than with dogs and NHP, easier handling than with pigs, fairly rapid sexual maturity
3. Thoracic and lumbar spine, infraspinatus tendon, stifle joint
Primer for non-immunologists on immune-deficient mice and their applications in research. Comparative Medicine 51(4), 300.
Abstract: The nude (1966-68) and scid (1983) spontaneous recessive mouse mutations led to the widespread use of
immune-deficient rodents in many areas of research, and modification of the mouse genome by transgenesis, gene
ablation and crossbreeding to develop lines with multiple immune deficits has provided numerous other 'types' of
inherent immunologically impaired mice. Early investigators using mice to study immune function were limited to using
very young/old mice, or using mice with immune deficits induced externally by diet restriction during fetal
development, preconditioning with irradiation or antibody infusion, toxins and immunotoxins, pharmaceuticals,
hormones, stress, or surgery (e.g., thymectomy). Problems with externally induced immune deficits included high cost
(materials, labor, equipment), variability in the extent of the induced deficit within each animal (different organ
systems) and between animals, and variability in the duration of the induced deficit. This review article points out
key differences between some of the immunodeficient strains, illustrates the value of immunodeficient mouse-based
research and encourages expanded, creative use of these animals. In addition to emphasizing the vigilance required
to maintain and work with genetically immune-deficient mice, the authors admonish readers to be on the lookout for
unexpected outcomes that may arise and to take care when interpreting data gathered from research using
The Immune System
Pluripotent self-renewing stem cells from the bone marrow give rise to the erythroid cell line, the myeloid cell line
(neutrophils, mast cells, macrophages) and lymphoid stem cells that produce T lymphocytes, B lymphocytes, natural
killer [NK] cells. T cells are lymphocytes that mature in the thymus, while B cells originate in the bone marrow but
mature in Peyer's patches or bone marrow before migrating to the secondary lymph organs (lymph nodes, spleen); NK
cells are large granular lymphocytes that are neither T nor B cells. T cells further differentiated as helper or
cytolytic - are important in target cell destruction (e.g., virus-infected cells or cells from transplanted tissues that
are genetically different). B cells develop into antibody-producing cells in response to vaccination or spontaneous
infections, and antibody concentrations (titers) can be measured in blood plasma unless B-cell development or
function is blocked. NK cells are immediate-response lymphocytes that produce cytokines, perform lytic functions,
and perform special functions in the pregnant uterus, becoming the dominant immune cell type following implantation
in mice, rats and humans. A lymphocyte must have the appropriate cell surface receptors to detect specific growth
factors in the local environment before it can progress in its normal stepwise development (differentiation,
maturation, activation). Activated receptors must signal into the cell to affect the cell and alter the manufactured
product (additional surface molecules, antibodies, cytokines, etc.), and cell function can be blocked by a failure to
initiate product manufacturing, manufacturing defective products, or blocked product distribution. Immune
dysfunction can therefore manifest at a variety of levels in the development of the cell line, from the absence of
stem cells to the failure of differentiated, mature, activated cell subsets to produce one product. The immune
system also depends on interactions with other tissues in the body to carry out its normal function- intercellular
adhesion molecule (ICAM-1) is a glycoprotein found on different cell types that support leukocyte adhesion and
function, and ICAM-1 null mice develop altered immune responses because the immune cells, while competent, have
impaired movement and localization.
T-cell deficiency and the nude mouse: nude mutation (Foxn1nu/Foxn1nu) first described by Flanagan in 1966; immune
deficit noted in 1968, mutation fully defined in 1999. Autosomal recessive mutation located on chromosome 11;
homozygous nu/nu are athymic and hairless, with vibrissae lacking or crinkled at 24 hrs of age (Immunodeficient
Rodents, NRC, 1989). Do not develop thymic anlage as fetuses, are immunologically abnormal from gestation day 11.
Lymphocyte precursors are normal, as are all lymphocyte lineages that differentiate independent of the thymus,
including some T cell subsets in the GI tract and liver. Nude mice only a limited number of normal tissue allografts
and xenografts; major application and enduring usefulness in oncology research. Nude mice not immunologically inert,
with most graft failures attributed to unusually high NK cell activity. Nude mice crossbred to other known,
spontaneously mutant mice (beige, XID) to generate larger immune defects (nude-beige; beige/nude/XID) to allow
growth of broader range of normal xenogenic tissue; beige mutation reduces NK lytic activity, but not cytokine
production or overall NK numbers, while Xid mutation reduces B cell activity. BNX mice used extensively as hosts for
normal human hematopoietic stem cells.
T- and B-cell deficiency in SCID, RAG-1 null and RAG-2 null mice: scid mutation (Prkdcscid/Prkdcscid) identified in
1983 in SPF mice that failed to produce detectable antibodies in a breeding study examining antibody classes.
Autosomal recessive mutation, with scid locus mapped to centromeric end of chromosome 16; scid/scid mice are T-
and B-cell deficient, with little to no immunoglobulin in their serum; SCID mice cannot reject allogeneic grafts or
produce antibodies to common laboratory antigens, and their spleen cells do not proliferate in response to T- or B-cell
specific mitogens (NRC text). SCID mice advanced studies of B-cell lineage, showed NK cell lineage distinct from T
and B cells, provided a better "tissue culture" vessel for transplantation studies, supporting even normal human
immune system cells. SCID mice not perfect for graft studies due to presence of NK cells, a "leaky" phenotype that
produces immunoglobulins, a tendency (15%) to develop spontaneous thymomas, and high sensitivity to irradiation.
Similar T-cell, B-cell deficient phenotype created through deletion of recombinase activation genes Rag1 or Rag2;
Rag1 or Rag2 initiate rearrangements in antigen-specific receptors of T and B cells, acting earlier in T- and B-cell
differentiation than scid gene. Mice with defects in more than one lymphocyte lineage made simultaneous
co-engraftment of several types of tissue feasible and allowed development of more complex experiments, e.g., (1)
SCID mice grafted with human skin, allogeneic human leukocytes given to the graft, then angiogenesis studied in an
inflammatory context, (2) porcine skin grafted to RAG-1 null mouse, mouse reconstituted with human PBL and
xenograft destruction studied.
T-, B-, and NK-cell deficiencies combined in recently described strains: murine NK-cell deficient strains began
emerging in 1994; Strain tgÎ26 has multiple copies of a human transgene, with NK-cell deficit and T-cell deficit;
Ikaros null lacks all lymphocytes; cytokine receptor chain gamma (gc) deleted strain is a knockout strain with NK-, T-
cell deficiencies with deletion for cytokine receptor chain gamma; RAG-2 null/gc is a cross of gc knockout with RAG-2
null, with all lymphocytes deleted. The RAG-2 null/gc is commercially available and is gaining ground in murine and
xenogenic tissue graft studies; expected to be "the most important new strain since SCID" because it is a good
breeder, free from lymphomas, more immune deficient than nude or SCID, and could prove to be a universal recipient.
Other plans to develop lymphocyte-deficient strains include modifying severely immune deficient mice to make them
monocolonally reactive, making mice with functional T- or B-cell populations that are homogeneous in function, which
would (in theory) make them free from background immunoreactivity.
Applications of Immune-deficient Mice
Oncology: array technology tracking of gene statement in tumor-bearing mice suggests xenogenic transplantation
models may have accurate predictive value for tumor progression, tumor treatment.
Lymphohematopoietic stem cell research: SCIDS, RAG nulls and improved multiple strains critical to understanding
developmental and differentiation potentials of human cells; exploring feasibility of gene therapy in bone-marrow
derived cells, muscle or endothelium; allowing assessment of efficacy of construct and duration of therapy due
absence of antigen-specific immunity.
Infective agent research: challenges with bacteria, viruses, parasites, or multiple components feasible, with specifics
of resistance or susceptibility elucidated by comparing responses of mice with differing immune competencies.
Other applications: diabetes mellitus (study of pathophysiology and treatment); arthritis; ocular diseases;
cardiovascular diseases; dermatology; human antibody production biotechnology.
Complications and Unexpected Outcomes in Work with Immune-deficient Mice
Creating strains by pronuclear injection may induce unexpected immune deficits, and lines showing perinatal or early
post-weaning mortality patterns should be evaluated (phenotype, gross and histologic exam of immune tissues).
Immune deficiencies range in severity, but best to assume impaired health status and use SPF or germ-free
husbandry, with daily monitoring of body condition score, regular postmortem assessment of immune-compromised
stocks and immune-competent sentinels. Sources of potential infection include inocula of tumors, cell lines and sera;
cesarian derivation and blastocyst transfer remain the best strategies to interrupt/prevent disease spread among
immune-deficient animals. Beyond infective properties, graft tissue may have inductive properties, e.g., activation of
murine stromal cells by products from engrafted tumor cells.
Overall genetic background of a selected immune-deficient mouse strain is important as penetration and presenting
phenotype of genetic alteration spontaneous or induced- can vary among strains. Researchers using
immune-deficient mice should monitor for and explain any observed variability in experimental results, and
experimental validation and confirmation of results using multiple approaches may be necessary. Example: IL-10
cytokine-deleted mouse develops spontaneous inflammatory lesion in the small and large intestines, used to study
inflammatory bowel disease, including Chron's disease; use of other immune-deficient strains revealed that resident
enteric bacteria played a critical role in modulating the inflammatory response in the IL-10 null model.
New strains with gene deletions may unveil subtle (weaker) pathways for induction of responses and may yield
unexpected phenotypes, e.g., ablation of IL-6 gene altered temperature sensitivity and promoted aggressive behavior
in mice; deletion of membranous ICAM-1 revealed circulating forms of the molecule and acceleration of hair cycle
development. Gene ablation in a well-characterized mutant strain may alter the strain's known characteristics, e.g.,
male MRL-lpr/lpr mice have a known survival advantage over females that becomes lost with disruption of
5-lipoxygenase (enzyme involved with arachidonic acid metabolism).
Two Detailed Examples of Applying Immune-deficient Mice to a Research Problem
Uterine lymphocyte biology during pregnancy: the pregnant mouse uterus has a surge in large granulated cells that
appears shortly after implantation, decreases in the second half of gestation; implantation sites and cell lineage in
nude, SCID, beige, and SCID/beige mice identical to normal mice, while NK-cell deficient strains have
reduced/absent cell populations; bone marrow donation from SCID donors (T- and B-cell deficient) restores the
normal cell line, thus conclusion that pregnancy-associated large granulated lymphocytes differentiate via the
NK-cell lineage pathway. Further studies using RAG-2null/gc null mice suggest uterine NK-cells play a cytokine
production role (INF-g) during pregnancy rather than a 'normal' target lysis role, with the product (INF-g) playing an
autocrine role in effecting the senescence of uterine NK-cells after mid-gestation.
Demodectic mange in dogs: Demodex canis lives only in the hair follicles of dogs; it cannot be cultured in the lab, and
experimental infection of natural hosts requires severe immune suppression, which in turn complicates the study of
host-mite interactions and mite-induced immunity. Grafting normal dog skin onto immunodeficient mouse strains
(SCID/bg, ICR SCID, tgÎ26, RAG-2 null) gave a model for natural disease, and reconstituting the RAG-2 null
engrafted mice with cells from the dog's immune system allowed study of the role of lymphocytes and lymphocyte
products in ridding the engrafted skin of parasites. In creating the model, the authors noted that heavy infestation
in skin grafts did not induce hair loss or inflammation, suggesting that mites do not directly damage the skin and
likely do not induce the skin lesions characteristic of demodicosis. Authors conclude canine inflammatory responses
act as growth-promoting factors for D. canis, rather than growth-inhibitors.
Questions: 1. List methods used by early investigators to externally induce immune deficits in mice:
2. What problems were/are inherent with externally inducing immune deficits in mice?
3. When was the nude mouse developed? Strain designation? Immunologic hallmark? Enduring use?
4. Which of the following is not true:
- the actions of the nude mutation were not fully defined until 33 years after discovery
- nude mice have thymic dysgenesis due to a development failure of a thymic anlage
- nude mice are immunologically abnormal from gestation day 11
- nude mice have no T-cells
- lymphocyte precursors are normal in nude mice
5. T/F The nude mouse is an immunologically inert mouse.
6. Most graft failures in nude mice are attributed to "unusually high" activity of __ cells.
- T cells
- B cells
- NK cells
7. What does the X in BNX stand for? How do BNX mice differ from nude mice? Extensive use?
8. List presence (+) or absence (-) of T, B and NK cells for the following mutant loci combinations:
bg nu xid T B NK
+/+ +/+ +/+
bg/bg nu/nu +/+
+/+ nu/nu xid/xid
bg/bg nu/nu xid/xid
9. When was the scid mouse developed? Strain designation? Immunologic hallmark?
10. List four reasons why SCID mice were not "perfect" for graft studies:
11. What research options became possible using mice with defects in multiple lymphocyte lineages? Examples?
12. What new combined deficiency strain expected to be "the most important new strain since SCID" may supplant beige/nu/xid in xenogenic graft studies? List four reasons?
13. In a breeding colony of mice, what type of mortality pattern might suggest emergence of immunodeficiency?
14. Two "best" strategies to interrupt/prevent disease spread among immune-deficient animals?
15. In studying uterine lymphocyte biology in pregnant mice, what interesting NK-cell activity was revealed thanks to the use of RAG-2null/gc null mice?
16. What of four immunodeficient mouse strains were tested, and which proved to be the only successful model for studying natural infection of D. canis in dogs?
17. The author's experimental results in immunodeficient mice suggest that canine inflammatory responses act as growth- (promoting / inhibiting) factors for D. canis. (circle one)
Answers: 1. Use very young/old mice, or use mice with immune deficits induced externally by diet restriction during fetal development, preconditioning with irradiation or antibody infusion, toxins and immunotoxins, pharmaceuticals, hormones, stress, or surgery (e.g., thymectomy).
2. High cost (materials, labor, equipment), variability in extent of induced deficit within each animal (different organ systems) and between animals, and variability in duration of the induced deficit.
3. First described by Flanagan in 1966; immune deficit noted in 1968, fully defined in 1999; Foxn1nu/Foxn1nu; T-cell deficiency; major application enduring usefulness in oncology research
4. The statement "nude mice have no T-cells" is not true; nude mice have some T cell subsets that develop outside of the thymus in the GI tract and liver
5. False; the nude mouse is not an immunologically inert mouse
7. The "X" in BNX stands for Xid (x-linked immune deficiency); BNX have the T-cell deficiency of the nude mutation plus reduced NK lytic activity (beige mutation) plus reduced B-cell activity (Xid mutation); used extensively as hosts for normal human hematopoietic stem cells.
8. Presence (+) or absence (-) of T, B and NK cells for the following mutant loci combinations:
bg nu xid T B NK
+/+ +/+ +/+ + + +
bg/bg nu/nu +/+ - + -
+/+ nu/nu xid/xid - - +
bg/bg nu/nu xid/xid - - -
9. scid mutation identified in 1983; Prkdcscid/Prkdcscid; T- and B-cell deficient (hence SCID acronym for "severe combined immunodeficiency").
10. (1) presence of NK cells, (2) "leaky" phenotype that produces immunoglobulins, (3) tendency (15%) to develop spontaneous thymomas, and (4) high sensitivity to irradiation
11. Simultaneous co-engraftment of several types of tissue, and development of more complex experiments. Examples: (1) SCID mice grafted with human skin, allogeneic human leukocytes given to the graft, then angiogenesis studied in an inflammatory context; (2) porcine skin grafted to RAG-1 null mouse, mouse reconstituted with human PBL and xenograft destruction studied.
12. RAG-2 null/gc; (1) good breeder, (2) free from lymphomas, (3) more immune deficient than nude or SCID, and (4) could prove to be a universal recipient
13. perinatal or early post-weaning mortality patterns may signal emergence of immunodeficiency
14. cesarian derivation and blastocyst transfer
15. a cytokine production role (INF-g) rather than a 'normal' target lysis role, with the product (INF-g) playing an autocrine role in the senescence of uterine NK-cells after mid-gestation.
16. SCID/bg, ICR SCID, tgÎ26, RAG-2 null were tested, but only the RAG-2 null proved to be a successful model for studying natural infection of D. canis in dogs.
17. canine inflammatory responses act as growth-promoting factors for D. canis.
Establishment of a deer mouse (Peromyscus maniculatus rufinus) breeding colony from wild-caught founders: comparison of reproductive performance of wild-caught and laboratory-reared pairs. Comparative Medicine 51(4), 314.
Abstract: The purpose of this study was to establish a breeding colony of wild-caught Peromyscus maniculatus rufinus to study the pathogenesis of Sin Nombre (SN) virus in its reservoir host. There are currently 60 formally described subspecies and 49 species within the genus Peromyscus (family Muridae, subfamily Sigmodontinae). Deer mice occupy a wide variety of habitats through out North America. In New Mexico there are 2 subspecies of deer mice, P. maniculatus blandus that lives in the southern part of the state and P. maniculatus rufinus, found in the north. Peromyscus spp. have been used to study mammalian reproduction, development, endocrinology, behavior, thermoregulation, torpor, metabolism, alcohol catabolism, immunology and genetics. Members of the genus serve as natural reservoir for several human pathogens including the etiological agents that cause Lymes disease, granulocytic ehrlichiosis, babesiosis, bartonellosis, and hantavirus cardiopulmonary syndrome. In 1993, deer mice were found to be the predominant carrier of SN hantavirus
Material and Methods: Wild deer mice used to found the colony consisted of 2 geographically distinct populations from New Mexico: one trapped near Gallup and the other from the Manzano mountains. F2 generation mice served as sentinels for serologic testing for MHV, Sendai virus, PVM, reovirus, TMEV, ectromelia virus, Mad1 and 2, polyomavirus, Mycoplasma pulmonis, parvovirus, EDIM, LCMV, CAR-bacillus, and Clostridium piliforme. Parasite examinations, bacteriology tests, and histology screening were also performed.
Morphologic characteristics: Gallup, Manzano
Adult weight(g) 19.4, 20.1
tail length(mm)* 58.1, 65.3
hind foot length(mm)* 19.1, 20.2
Adult weight wild F2 28.6
Reproductive Performance: Wild-caught, Lab-reared
total pairs 26, 59
fertility incidence 0.85, 0.73
Days from pairing to litter 106, 71
Mean litter/pair 7.2, 5.5
Mean pups/litter 4.3, 4.5
Total pups born 721, 740
Incidence cannibalism* 0.05, 0.26
female/male pups 1/1.1 1.01/1.00 * = statistically significant
Questions: 1. T or F Peromyscus maniculatus rufinus are a natural reservoir for Sin Nombre virus.
2. T or F Wild-caught Peromyscus maniculatus rufinus are less likely to cannibalize their offspring than their laboratory-reared counterparts.
3. Members of the genus Peromyscus serve as natural reservoir for what human pathogens?
A. The etiological agents that cause Lymes disease.
B. granulocytic ehrlichiosis
E. All of the above
Answers: 1. T
Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapside gene. Comparative Medicine 51(4), 319.
Abstract: Mouse hepatitis virus (MHV), an enveloped corona virus with a large single-stranded RNA, is one of the most common murine viruses found in laboratory mice. Many strains of MHV have been identified, and are classified into two groups, polytropic (pulmonary) and enterotropic, on the basis of tissue distribution of the primary infection. Infection with the enterotropic strains of MHV is probably the most common form of natural infection. With the enterotropic strains, the virus is spread by the fecal-oral route. Infection with MHV is usually diagnosed by detection of serum antibody in an enzyme-linked immunosorbent assay (ELISA) or with immunofluorescent staining on tissue sections. This paper describes the use of RT-PCR of fecal specimens or viruses isolated from fecal specimens in DBT cell lines to amplify the coding region of the nucleocapsid (N) gene of MHV. Sequence analysis of the N gene was useful in distinguishing the MHV strains. A phylogenetic tree of 12 strains recently found in animal facilities in Japan was constructed in an effort to study the epidemiology of outbreaks in 11 institutions in Japan. Four strains from one location and one strain from each of 2 other institutions were closely related, suggesting that they may have originated from a common source. Other strains from other institutions, including 2 strains from one institution that have 2 outbreaks in 3 years, had low nucleotide sequence homology, suggesting different sources of infection in each case. This paper demonstrates the usefulness of PCR and phylogenetic analysis of the nucleocapsid gene in tracing source of infection with MHV.
Questions: 1. True/False Mouse Hepatitis virus is very contagious, consequently screening of sentinel animals is likely to identify the infection in a mouse colony.
2. Sources of infection with mouse hepatitis virus in a naive colony could come from:
A. New mouse introductions from another facility
BE. Undetected carrier animals in the current colony
C. Transplanted tumor or other tissues
D. All of the above
E. A &C
3. True/False To rid MHV from an mouse breeding colony, it is necessary to do cesarean rederivation of the breeding stock.
Answers: 1. False
Questions and answers were derived from "Companion Guide to Infectious Disease of Mice and Rats (National Research Council, 1991). Mouse hepatitis virus is very contagious, with an 80% prevalence rate in infected colonies, hence routine sentinel screening for antibodies should pick up an infection. The infection runs its course in 2-3 weeks and carrier states have not been identified, consequently, there is no need to do cesarean rederivation to rid a colony of MHV.
Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays. Comparative Medicine 51(4), 326.
Abstract: Rodent parvoviruses include minute virus of mice (MVM), mouse parvovirus (MPV), and hamster parvovirus (HaPV). These are
among the most prevalent infective agents in contemporary mouse colonies. MVM is a common contaminant of murine cell cultures and
tissues. Therefore, identification of infected animals and contaminated materials is critical.
Current diagnostics include serologic assays, MAP tests, and standard PCR. This paper describes the use of fluorogenic nuclease PCR
assays (aka real-time PCR or TaqMan PCR) and descibes its advantages over the current diagnostics methods.
Fluorogenic nuclease PCR amplifies DNA between two specific oligonucleotide primeres by thermocycling in the presence of Taq
polymerase. The reaction mix includes an internal fluorogenic hybridization probe with covalently linked fluorogenic and quencher dyes
in close proximately. The probe is cleaved during each round of amplification which releases the dye free of the quencher. The increase
in dye is measured optically at the end of each cycle and is transmitted to the computer in real time, thereby eliminating the post PCR
processing and carry over contamination.
The authors developed three fluorogenic nuclease PCR assays- one to specifically detect MPV(and HaPV since they are very similar
genetically), one to specifically detect MVM, and one to detect all rodent parvoviruses. Each proved to be more sensitative than the
standard PCR and serologic testing methods.
Questions: 1.The mouse antibody production test
a.offers increase sensitivity in immunocompromised rodents.
b.uses fewer animals that PCR
c.is used for the detection of MPV contamination of biologic materials.
2.Real time PCR offers these advantages over standard PCR.
a.Eliminates post PCR processing and carry over contamination.
b.Eliminates carryover contamination and sensitivity.
c.Eliminates post PCR processing and viral shedding.
Variation of serum alpha2-macroglobulin concentration in healthy rats and rats inoculated with Staphylococcus aureus or subjected to surgery. Comparative Medicine 51(4), 332.
Abstract: Purpose of the study was to investigate variations in the serum concentrations of alpha 2-Macroglobulin (alpha 2M) in healthy rats and in
rats inoculated with S. aureus or subjected to surgery. Also to measure changes associated with circadian rhythm (blood collected at 3
hour intervals), age (blood collected 9 times at one week intervals), or day to day (blood collected at 8 times at one day interval) variation
in alpha 2M over a nines week period. Concentrations were measured using an enzyme-linked immunosorbent assay.
alpha 2M is a proteotypic acute-phase reactant protein in rats. This is similar to C-reactive protein an acute phase reactant protein in
humans and canines. In humans and dogs the C-reactive protein has been shown to increase following bacterial exposure or surgical
Rats were subjected to either castration with scrotal suturing (males), or oophorohysterectomy via abdominal approach. Following surgery Orbifloxacin was administered for an undisclosed amount of time.
Serum concentrations of alpha 2M increased at one-day post treatment and peaked at two days for all three-treatment groups. The
highest increase was in rats exposed to surgery (8-33 times normal), followed by oophorohysterectomy (7-28 times normal), then castration (4-25 times normal).
No change was noted for alpha 2M for circadian, age, or day to day intervals. Previous studies reported inflammatory stimulation of
alpha 2M in rats inoculated with turpentine oil. The response of alpha 2M is greater in turpentine treated rats when compared to the
concentration of alpha 2M induced by surgery or S. aureus. About 3 million rats are used in experimental animals per year in Japan.
Questions: 1. Of the following treatments, which demonstrated the lowest increase of alpha 2M in the rat?
a. Turpentine treatment
d. S. aureus exposure
2. Baseline alpha 2M serum concentrations in the rat have been shown to vary with?
a. Circadium rhythm
b. Day to day interval
c. Week to week interval
d. None of the above
Answers: B, D
Similarity of bone ingrowth in rats and goats: a bone chamber study. Comparative Medicine 51(4), 336.
Abstract: Bone growth in Repeated Sampling Bone Chambers (RSBC) in goats was scant compared to bone growth in Bone Conduction Chambers (BCC) in rats. It was suspected, that this was due to the lower metabolic rate in goats compared to rats. However, when the same Bone Conduction Chambers were implanted using a similar technique and harvested 6 and 12 weeks later it was found that there was no difference between ingrown bone between goats and rats. In fact quantitation of ingrown tissue (not bone) was more in chambers from goats than from rats. It was concluded, that different metabolic rate was not limiting bone ingrowth.
Questions: 1) Osteons are comprised of a series of concentric rings of bone cells and bone matrix, surrounding a hole in the middle, which is filled by blood vessels. Is this anatomical feature the basic building block for all species? T or F?
2) Why is this study flawed?
Answers: 1) False, Osteons are seen in lager animals but not in rodents
2) In rats you drill away half the tibia compared to a tiny whole to implant the same chamber
The pig as a model for excisional skin wound healing: characterization of the molecular and cellular biology, and bacteriology of the healing process. Comparative Medicine 51(4), 341.
Abstract: 20 - 2cm diameter skin wounds were created on the backs of each of Yorkshire pigs to study the molecular events of wound healing in this model. Semi-quantitative RT-PCR was used to determine if the expression of specific genes of interest went up or down. Mice are not good models because keloids (a thick scar resulting from excessive growth of fibrous tissue) and intra-abdominal adhesions do not develop indicating that healing is not identical to humans. Primates also do not develop exuberant scars. Pigs are good in that the relative thickness of the dermis and epidermis is similar, and there is a lot of skin to work with. Results: Wounds had re-epithelialized by postinjury day 20 allowing enough time to study the progression of wound healing in this model. Cultures taken from the wounds indicate that bacterial counts were maximal between days 3-7 postinjury. Pseudomonas aeruginosa was largely isolated and Staphylococcus aureus was also present. Total RNA and DNA from samples taken from the wound site significantly increased by the first day, peaked at day 7 and remained high at day 49. RNA and DNA levels paralleled each other during healing. Apoptotic cells were observed at day 1, greatly increased by day 3, and peaked by day 14. (Programmed cell death is needed to clear cells no longer necessary in the healing process). Decorin, a proteoglycan that binds collagen and mediates fibril formation, was significantly high by day 7 and continued to increase to a peak at day 35. Tumor necrosis factor alpha, a cytokine, is released by monocytes/macrophages in response to inflamation and has angiogenic properties. TNF-alpha rose at day1, and started to decline day14-21. Connective tissue growth factor (CTGF) - stimulates cell proliferation, cell adhesion, chemotaxis, angiogenesis, and production of extracellular matrix components. Detected on day 7, peaked on day 21, and was still high on day 49. Insulin-like growth factor has a role in cell metabolism and growth and has been reported to have a role in tissue repair. Initially decreased, then increased by day14 and peaked at day 35. Unlike human tissue, the transcription factors c-fos and c-jun were detected in normal pig tissues and decreased during days 1-35 postinjury. In people these factor increase in wounded tissue. Possible species difference.
Questions: 1. A deficiency in mouse models of inflammation and healing include all of the following except:
A. small size limits sample collection
B. keloids do not occur
C. intra-abdominal adhesions do not develop
D. connective tissue growth factor is not altered in response to wounding
E. skin density is different
2. In a pig model of excisional skin wound healing which of the following were identified as contaminants of experimentally induced wounds.
A. Pseudomonas auruginosa and Staphylococcus aureus
B. Pseudomonas auruginosa and Streptococcus suis
C. Staphylococcus aureus and Streptocococcus suis
D. Staphylococcus aureus E. Streptocococcus suis
3. Selective agar used to grow primarily gram positive cocci:
A. Mueller Hinton Agar
B. Hayflicks Agar
C. Brilliant Green Agar
D. Mannitol Salt Agar
E. Triple Salt Iron Agar
4. If you wanted to find the sequence of the gene encoding porcine decorin (or any other gene) where is the best/most efficient place you would search:
E. DNA R Us
5. In this study, semi-quantitative RT-PCR was used to provide an estimate of mRNA expression. Provide another method that could be used to quantitate RNA:
A. Western Blot
B. Northern Blot
C. Southern Blot
D. Immuno Blot
E. Nested PCR
6. While looking at an H&E slide you cannot determine whether the cells are undergoing apoptosis or necrosis. What are some features that may help you delineate the two. What is a classical assay that you could use to determine if a group of cells were undergoing apoptosis?
7. In a pig model of excisional skin wound healing which of the following were identified as being different from previous studies in man or other animals:
A. DNA levels increased
B. Apoptosis levels increased
C. Connective tissue growth factor (CTGF) increased
D. Decorin levels increased
E. C-fos levels decreased
Answers: 1. D
6. Apoptosis - the cells tend to have more picnotic nuclei and inflamation is minimal. Necrosis - the nuclei tend to be more fragmented and inflamation may be involved. TUNEL assay. 7. E. In humans the c-fos and c-jun levels increased after wounding, whereas in the pig they decreased.
Development of a novel intestinal and vascular access port (IVAP) rabbit model to study regiospecific oral absorption pharmacokinetics. Comparative Medicine 51(4), 349.
Abstract: The liver is considered the primary site for drug metabolism/secretion when assessing drug bioavailability. There is also interest in evaluating intestinal mediated secretory transport and drug metabolism. Several in vitro models (excised intestinal tissues, everted gut sacs, single-pass intestinal perfusion, membrane vesicles, cell culture systems) have been used to study intestinal drug transport processes, but conscious animal models provide a more realistic evaluation of drug absorption/metabolism. However, these in vivo models are limited in that chronic portal vein sampling is not feasible, thus preventing quantitative assessment of hepatic drug absorption. Although an IVAP canine model has been described to overcome this limitation, cost and limited drug quantities are still constraints in this model. The authors developed and validated an IVAP rabbit model because rabbits: have a lower distribution volume, can tolerate serial blood sampling, have comparable general intestinal physiology and small intestinal permeability as humans, have somewhat homologous intestinal cytochrome P450 statement to humans, and express various secretory transporters (i.e. canalicular multispecific organic anion transporter (cMOAT/MRPs) and P-glycoprotein). 5-F Silastic catheters were placed in the proximal or distal small intestine or colon of animals, while a 5-F Heparin Coated Polyurethane (HCP) catheter was implanted in the portal vein. Catheters were tunneled out of the abdomen (modified Witzel technique) and attached to separate dorsal SQ access ports (Fig 1).
Initially during model development, unexpected surgical losses occurred due to the susceptibility of rabbits to lethal cardiac arrhythmias after prolonged (> 2 h) halothane exposure. No such losses occurred after the authors switched to isoflurane. Maintaining portal vein (PV) catheter patency proved to be critical. Rabbits had a localized fibrous reaction to Surgicel (thrombotic agent used for hemostasis), a reaction not seen in the canine model. This fibrous reaction caused PV catheter failure 2-4 weeks postoperatively. Discontinuation of Surgicel use resulted in > 6 months of PV catheter functionality. Maintenance of aseptic conditions was also found to be crucial to prolonged PV catheter patency. Occasionally, postoperative ileus and anorexia was seen and required dietary intervention (timothy hay) and abdominal massage. Fluoroscopic visualization of intestinal/portal venous catheters indicated no catheter interference with GI motility/hepatic blood flow and appropriate catheter placement/patency. Acute pH studies suggested that intestinal catheters had minimal effects on GI motility patterns. The IVAP rabbit model proved to be a dependable model of intestinal regiospecific drug absorption and hepatic drug extraction as well as for examination of drugs that are expensive and available in small quantities.
Questions: 1. Which of the following HAS NOT been used as an in vitro model to investigate intestinal drug transport processes?
A. Excised intestinal tissues
B. Everted gut sacs
C. Single-pass intestinal perfusion
D. IVAP canine model
E. Membrane vesicles
F. Caco-2, MDCK, CHO, NIH3T3 cell lines
G. All of the above have been used as in vitro models
2. Which of the following IS NOT an advantage of the IVAP rabbit model?
A. Chronic and prolonged portal vein sampling
B. Higher volume of distribution
C. Allows serial blood sampling
D. Comparable small intestinal physiology and permeability as humans
E. Somewhat homologous cytochrome P450 enzyme isoform statement to humans
3. What type of needle is used to sample from intestinal or vascular access ports? Why must this type of needle be used?
4. Which of the following reasons accounted for the unexpected surgical losses of rabbits during the initial development of an IVAP rabbit model?
B. Intestinal leakage/perforation
C. Lethal cardiac arrhythmias due to >2 h halothane exposure
D. GI ileus and bloat
5. Which of the following were differences in the IVAP surgery between rabbits and dogs?
A. Rabbits had pronounced localized fibrous reaction to thrombotic agents
B. Rabbits were more sensitive to prolonged halothane exposure
C. Rabbits occasionally needed abdominal massage to stimulate appetite and promote GI motility
D. All of the above.
Answers: Answers: 1) D is an in vivo model [CM 50(2):167-174, 2000].
2) B - rabbits have a lower volume of distribution.
3) Huber needle; to prevent coring of or damage to the access port.
4) C - Once the authors switched to isoflurane, there were no additional unexplained surgical losses.
Coxiella burnetii infection in C.B-17 Scid-bg mice xenotranplanted with feal bovine tissue. Comparative Medicine 51(4), 357.
Abstract: The SCID-bo mouse was developed by implanting fetal bovine tissues (liver, lymph node, and thymus) surgically in the peritoneum of female C.B-17 scid-bg mice. The result was a xeno-chimeric mouse in which bovine humoral immune responses to T-cell dependant antigens, and multi-lineage bovine hematopoiesis could be studied. The source of bovine tissue was aseptically collected fetal tissue from cows at a slaughter house. Tissue from one fetus was used in 50 mice. The mice were treated from one day before to 1 week after surgery with Trimeth-Sulfa in drinking water. Two cases are reported of mice becoming ill several months after the tissue implantation, with Coxiella burnetii eventually identified as the causative agent. Thus the importance is stresses of considering risk of xenoinfection when models such as this are developed. There were two cases reported, and each represent only a few of the mice in a transplant group. Clinical signs included dehydration, lethargy, and rough coat. On gross necropsy enlargement of liver and spleen were noted with irregular surfaces and depressed white foci on the liver. Microscopically there was chronic active hepatitis with inflammatory cells primarily in the portal regions, and some Kupffer cells containing cytoplasmic inclusions which were bright magenta when stained with Machiavello's stain. On transmission electron microscopy the inclusions contained pleomorphic organisms from spherical to rod shaped with bilaminar walls contained in spacious vacuoles in the cytoplasm of hepatocytes. Tissue from the affected mice was preserved by freezing and later used for an inoculation study. C.B-17 scid-bg mice were inoculated with either .5 ml of tissue homogenate from affected mice or .5 ml of PBS. All inoculated mice and none of the sham mice developed lesions similar to the original case mice. One sham developed peritonitis, one thymic lymphoma, and one was normal on necropsy. PCR sequencing first for 16s Bacterial ribosomal gene and then for specific sequence was done. The sequence was compared with GenBank and found to be Coxiella burnetii. Samples tested from the source bovine tissue were negative. Coxiella burnetii is an obligate intracellular bacterium, and the cause of Q-Fever in humans and domestic animals. A single organism is known to be infective. The disease is primarily one of ruminants but can be transmitted to other animals including humans by inhalation, or tick bites. The fact that no organisms were identified in the source tissue and that only a few mice were affected is indicative of a very low number of organisms in the source tissues. This is of importance when one considers the risk of infection in xeno-chimeric animals and in planning screening tests prior to such studies. Even with screening, such infections are a possibility. Diagnostic surveillance of xenografted animals must include screening for diseases of the donor species in the recipient animal, and when possible donor tissue should be taken from animals of known health status.
Questions: 1) Match the following:
a. Xenograft 1. Same species
b. Allograft 2. Self
c. Autograft 3. Different species
2) Name the disease caused by Coxiella burnetii. Can it be zoonotic?
3) What does "SCID" stand for?
Answers: 1. A-3, b-1, c-2
2. Q-Fever, Yes
3. Severe combined
High mortality in a large-scale zebrafish colony (Brachydanio rerio Hamilton & Buchannan, 1822) associated with Lecythophora mutabilis (van Beyma) W. Gams and McGinnis. Comparative Medicine 51(4), 361.
Abstract: Young zebrafish fry in an established research colony spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore. Housing some 27,000 fish in 680 tanks at the time of the outbreak, the research colony had operated for seven years without substantial disease problems. Morbidity in affected tanks ranged from 10-100% among fry mostly 5-24 days after hatching- with affected fish becoming lethargic, decreasing feeding behavior and dying. Workers in the laboratory described the infected fish as "bearded" and the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans. Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal infection. A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air. Hyphae and spores of the oomycete genus Aphanomyces were also occasionally isolated. Several genera of bacteria (Flavobacterium, Pseudomonas, Aeromonas, Micrococcus and Acinetobacter), considered normal flora for freshwater aquaria, were isolated from the water. The outbreak coincided with a major renovation of the building's reverse- osmosis-purified (RO) water system, and elimination of the epizootic correlated with reduction in the number of L. mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems. The fish-housing tanks ranged in size from 2-55 L and each held between 15 to 75 fish; water was supplied at a rate of ~ 3.5 L/h, with overflow water filtered and re-circulated to the tanks. Water quality parameters were evaluated (once fish started dying) and all findings were within normal parameters for tropical fish culture with the exception of low calcium values (CaCO3 = 0.1 ppm). Zebrafish are a hard water species that do best with calcium in the range of ~80-200 ppm, and low water hardness values increases susceptibility to other adverse water quality conditions (excess nitrogen, heavy metals, fluctuating temperature and pH, salinity, nutrition, stocking density, low alkalinity, and water hardness). The skin of fish is metabolically active and quickly responds to stress, and low water hardness values decreases survival rates, growth and disease resistance in developing fry. The identified "normal flora" freshwater bacteria can cause secondary or opportunistic infection in compromised fish due to trauma, environmental imbalance, or concurrent disease. Lecythophora mutabilis is a ubiquitous fungus in moist environments, found in soil, decaying vegetation, air and river water, and from wood of preservative treated utility poles. It is a potential opportunistic pathogen in various compromised species and has been implicated as a rare human pathogen causing peritonitis, eye infection and pericarditis. While Lecythophora mutabilis did not invade tissues in the affected young fry, it was concluded that the dense hyphal strands, along with mixed bacteria and protozoa, occluded the oral cavity and/or gills and lead to starvation and/or asphyxiation.
Questions: 1. What two fungal agents were identified in this epizootic?
2. What are some of the genera of "normal bacterial flora" for freshwater aquaria?
3. Name nine potential "environmental stressors" for fish? Which one of these likely played a role in this epizootic, and how?
4. True or False: Lecythophora mutabilis is a potential zoonotic agent.
5. What was the determined cause of mortality in the affected fry?
Answers: 1.Lecythophora mutabilis, Aphanomyces sp.
2. Flavobacterium, Pseudomonas, Aeromonas, Micrococcus and Acinetobacter
3. Excess nitrogen, heavy metals, fluctuating temperature, fluctuating pH, salinity, nutrition, stocking density, low alkalinity, water hardness
5. Starvation and/or asphyxiation secondary to occlusion of oral cavity and/or gills by masses of fungal hyphae mixed with bacteria and protozoa
Persistent transmission of mouse hepatitis virus by transgenic mice. Comparative Medicine 51(4), 369.
Abstract: The authors document a persisten transmission of MHV over a 2 year period by MHV seropositive transgenic mice. Transmission occured via both direct contact with the mice as well as by contaminated bedding. MHV was not detected at diagnostic labs using viral isolation or RT-PCR of tissues taken from MHV positive mice. The authors believe that this indicates that unknown or unexpected pathophysiologic presentation of murinhe viral diseases may present new challenges in trying to avoid these diseases in research animals. The transgenic strains in this report were CBA/CaJ- Tg(TcrBV8.1)Mab heterozygous males, B10.Br-H2-T18a/SgSnJ- Tg(TcrBV8.1)1Mab heterozygous males, BALB/c.BNZW congene homozygous males and female and C57BL/6.BNZW congene homozygous males and females. (PLEASE NOTE that I am unable to use superscript with this email program so you should check the article to make note of just how the nomenclature for these strains should be written). These newly arrived animals were quarantined for 7 weeks and tesed. Results were positive for MHV. The mice were considered to have cleared their infection and developed protective antibodies at the end of the quarantine period. However, for this study, sentinel mice and 1 non-transgenic female for the strains were tested weekly for MHV. The CBA/CaJ and B10.BRtg mice that were MHV positive caused seroconversion of sentinel mice but the BALB/cBNZW and C57BL/6.BNZW mice did not cause seroconversion. The authors found that the MHV seropositive mice from the original cohort were able to transmit MHV by direct contact sentinels for over 12 months.
Questions: 1.MHV strains most frequently seen in research colonies are:
d All of the above
2. MHV has numerous strains that differ by
b. tissue tropism
c. immune response
d All of the above
3. Viral isolation is an insensitive method of detecting enterotropic MHV because:
a. The virus is rapidly cleared by the GI tract so live virus is rarely obtained
b. the virus is difficult to grow in cell culture
c. Maternal antibodies interfere with the ability to obtain live virus from neonatal GI tracts
d All of the above
Answers: 1. b