Mcfarlane Burnet Centernary Symposium on Immunology and Virology. Laboratory Animal Science 49(5), 471.
Abstract: The following is a summary of topics discussed by various speakers:
Sir Frank Macfarlane won Nobel Prize in physiology and medicine in 1960 for his hypothesis of immunologic intolerance.
Gordon Ada: pioneered the use of embryonated eggs as substrates for vaccine production for influenza.
Frank Fenner: summarized the history of attempts to control rabbits introduced to Australia in 1859 for sport hunting. Myxoma virus was used to control rabbit population but did not eradicate rabbits.
Jacques Miller: MHC restriction is imposed intrathmically.
Peter Doherty: summarized how Type II Herpesvirus in voles (MHV-68) is similar (taxonomically) related to herpesvirus saimiri, human herpesvirus-8 and EBV.
Michael Oldstone: discussed how LCMV targets dendritic cells. The host cell receptor for LCMV has been identified as alpha-dystroglycan, a heavily glycosylated protein. The CD46 receptor is the host cell receptor for meales virus and mice transgenic for CD46 may be infected by MV intracerebrally, intravenously and intraperitoneally.
Peter Howley: noted that there are 500,000 new cases of human papillomavirus infections every year and HPV is the second most common killer among female cancers. Howley's talk emphasized the E6 and E7 genes of HPV, the genes most important to the transformation process.
G. Shellman: described murine CMV, a model for cytomegalovirus in humans. Cmv-1 gene is closely related to ectromelia virus resistance gene and to herpes simplex type 1 resistance gene.
Questions: No questions

Improved detection of Coxiella burnetii (Q fever) in livestock tissue through silica matrix DNA extraction and high-sensitivity PCR. Laboratory Animal Science 49(5), 474.
Abstract: This article describes a new diagnostic method to detect C. burnetti in clinical specimens, as well as gather information about shedding of rickettsial agents through bovine milk. The method described utilizes Trans PCR and a silica matrix for DNA extraction. Tested specimens included bovine blood, milk, placenta, liver, porcine heart valves, and sheep spleen. Further studies are needed to determine the true efficacy of this system in regards to diagnostic and epidemiological applications.
Questions: Questions
1. What are advantages of this method vs. others for C. burnetti detection?
2. How can Q fever be acquired?
3. What are disadvantages of direct culture techniques?
Answers: Answers
1. Fast, convenient, and inexpensive. Did not require a minimum specimen volume.
2. Exposure when utilizing sheep in environments with contaminated dust, urine, feces, amniotic fluid, placentas, and newborn lambs. Also, exposure from unpasteurized milk from infected livestock.
3. Time consuming, hazardous, expensive, and requires extensive laboratory support.

High Alveolar-Arterial Oxygen and End Tidal Arterial Carbon Dioxide Gradients in Mechanically Ventilated Rats. Laboratory Animal Science 49(5), 476.
Abstract: An unexpected inconsistency was noticed between the anticipated arterial oxygen partial pressure (PaO2) and measured PaO2 in anesthetized, mechanically ventilated rats. There was also a discrepancy between end tidal carbon dioxide concentration (EtCO2) and arterial CO2 partial pressure (PaCO2).
Outbred Sprague Dawley rats (Crl:CD[SD]BR) were anesthetized and mechanically ventilated (paralyzed with doxacurium) to investigate the difference between calculated (using the alveolar gas equation: See 49 (5): 476 if interested) and measured values for PaO2, PaCO2 and EtCO2. The calculated versus measured values for both PaO2 and EtCO2 were significantly different. PaO2 measured lower than expected. EtCO2 was higher than expected.

PaCO2 / EtCO2 discrepency:
The difference in PaCO2 values may be due to blood gas machine calibration error or failing to clear the sample line of air before obtaining the blood gas sample. Inaccurate calibration or internal algorithm errors in the micro-capnometer (measures CO2 and NO2 concentration in a gas sample by infrared absorption) were suggested as explanations for the discrepancy in EtCO2 values. A final, definitive explanation for these discrepancies was not given.
A second experiment was done to obtain blood gas analysis from arterial lines in rats which were either (1) spontaneously breathing room air, mechanically ventilated with (2) 99% O2 + 1% Halothane or (3) O2 / Halothane / N2O mixture. The investigator served as an additional group (!?). In all cases the measured PaO2 value was lower than predicted based on the alveolar gas equation, PaCO2 was lower than predicted based on EtCO2 measurement and pH values reflected a slight hyperventilation, consistent with measured PaCO2 values.

Alveolar / arterial discrepancy
Factors which may have impacted on the discrepancy between measured and calculated values include (1) Shunting: Systemic blood not contacting gas-exchange areas of the lung or Thebesian circulation (coronary venous blood into left ventricle) (2) Diffusion limitations: CO2 diffuses 20 times faster than O2 and is 24 times more soluble. A thickened alveolar membrane (pathology which may not be evident) may impact on O2 but not CO2 and (3) Ventilation (VA) / perfusion (Q) mismatch.
Commentary (provided in article): Measure versus calculate when PCO2, PO2, or pH might influence experimental interests.
Note: Two good summary algorithms (LAS 49 (5): 478)
Questions: Questions:
1. Which of the following is not likely to explain a discrepancy in measured versus calculated PaO2 levels?
a. ventilation / perfusion mismatch
b. thebesian circulation
c. shunting
d. thickened alveolar membrane
e. mechanical ventilation
2. The designation Crl:CD[SD]BR refers to:
a. an inbred strain
b. an outbred strain
c. an inbred stock
d. an outbred stock
e. none of the above
3. Doxacurium is :
a. an analgesic
b. an anti-inflammatory
c. a paralytic
d. a good alternative to this article
e. none of the above
Answers: Answers: (1) e (2) d (3) c ?

Animal models for neuron disease. Laboratory Animal Science 49(5), 480.
Abstract: Motor neuron disease is a general term applied to a broad class of neurodegenerative diseases that are characterized by fatally progressive muscular weakness, atrophy, and paralysis attributable to loss of motor neurons. At present, there is no cure for most motor neuron diseases, including amyotrophic lateral sclerosis (ALS), the most common human motor neuron disease-the cause of which remains largely unknown. Animal models of motor neuron disease (MND) have significantly contributed to the remarkable recent progress in understanding the cause, genetic factors, and pathologic mechanisms proposed for this class of human neurodegenerative disorders. Largely driven by ALS research, animal models of MND have proven their usefulness in elucidating potential causes and specific pathogenic mechanisms, and have helped to advance promising new treatments from "benchside to bedside." This review summarizes important features of selected established animal models of MND: genetically engineered mice and inherited or spontaneously occurring MND in the murine, canine, and equine species.
The term "motor neuron disease" (MND) is used to designate a variety of neurologic disorders, the principal features of which are attributable to dysfunction of upper and lower motor neurons. Patients with amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, manifest variable combinations of both upper and lower motor neuron signs, including spasticity, hyperreflexia, and extensive plantar signs (upper motor neuron signs); and progressive muscular weakness, fasciculations, and atrophy (lower motor neuron signs), leading to fatal paralysis .
The Greek derivation of the word amyotrophic means "muscles without nourishment" (a means without, myo means muscle, and trophic means nourishment). Lateral refers to the region of the spinal cord where the axons of diseased and dysfunctioning motor neurons have degenerated and are replaced by sclerosis, or scars.
The article summarizes the contributions and the limitations of selected experimentally induced and spontaneously occurring animal models for MND: murine transgenic motor neuron disease, and spontaneously occurring murine, canine, and equine motor neuron disease.
Genetically Engineered Animal Models of Motor Neuron Disease
To date, over 50 SOD1 mutations have been associated with familial amyotrophic lateral sclerosis (FALS). All these mutations have induced MND in mice that is clinically and pathologically similar to ALS. However, the gene copy number and level of gene expression significantly affects the pathologic changes and age at onset in the various transgenic lines. The combination of the results from experiments done with these transgenic mice suggests that motor neurons degenerate because mutant Cu/Zn SOD1 may itself be toxic or because it gains a toxic property or aberrant function.
In addition to the SOD1 transgenic mice, transgenic mice that express mutant neurofilaments (NF) or overexpress wild-type NF proteins have documented that primary NF alterations can cause MND that resembles ALS. However, the direct role of NFs in the etiopathogenesis is unclear, because NF gene mutations associated with FALS appear to be rare.
Targeted disruption of the genes that encode for brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and the tyrosine kinase B (trkB) receptor have enabled investigators to examine how these elements influence development and maintenance of motor neurons. Although mutations in genes encoding for these proteins have not been reported in association with ALS, experiments using knockout mice have yielded relevant information regarding the survival and maintenance of motor neurons in vivo.
Spontaneously Occurring Mouse Models of Motor Neuron Disease
Four spontaneously occurring, inherited mouse models of MND have received considerable attention: the motor neuron degenerative (mnd) mouse; the wobbler mouse; the wasted mutant; and the progressive motor neuronopathy (pmn) mouse. All are well-established models, characterized over the last decade or more, and all are autosomal recessive mutants.
The mnd mice have adult late onset motor neuron degeneration, and intraneuronal accumulation of NF and lipofuscin-like material. However, these mice do not have skeletal muscle denervation and motor neuron loss characteristic of ALS. The lipofuscin-like material contains a subunit of mitochondrial adenosine triphosphate synthase characteristic of neuronal ceroid lipofuscinosis . Thus the mnd mouse is a more appropriate animal model of Batten's disease, the most common human neuronal lipofuscinosis.
The wobbler mouse has anterior horn cell loss and muscular denervation atrophy. Wobbler mice develop generalized tremors and forelimb weakness. The term wobbler is used to describe the resulting gait abnormality. The predominant forelimb involvement differs from that of other animal models of ALS.
The wasted mutant is characterized by aggressive disease (e.g., onset at an early age and rapid progression to death). Wasted mutant mice are characterized by wasting and immunologic and neurologic abnormalities starting 21 days after birth, with death ensuing by 28 days. Selective vacuolar degeneration of the anterior horn cells and motor nuclei of the brainstem are prominent features of this model and are characteristic of ALS.
Muscular atrophy and paralysis are present in the pmn mouse. However, there is little loss of motor neurons in the spinal cord. Pathologically this model is characterized by a central-peripheral axonopathy in which proximal axons and cell bodies in the anterior horn are spared. It is not a primary disease of the cell soma, which is different from the lesion of ALS. The pmn mouse appears to be a model of dying- back axonal disease.
The role of oxidant stress, if any, in these murine models remains to be clarified. The lack of identification of the specific genetic mutations in the wobbler, mnd, and pmn mouse models is something of a disadvantage. It makes it difficult to define specific pathophysiologic mechanisms beyond descriptive characterizations, to detect affected progenies before the on-set of the disease, and to test specific treatments.
Inherited Canine MND
Inherited motor neuron disease in dogs, hereditary canine spinal muscular atrophy (HCSMA), was first reported in Brittany Spaniels in 1979. Affected dogs manifest clinical and neuro-pathologic features that are strikingly similar to those seen in ALS patients, with the exception that the motor cortex, which is affected in humans, is spared in dogs.
Pedigree analysis indicates that HCSMA is inherited as an autosomal dominant disease, similar to the mode of inheritance of FALS. Syntenic studies indicate that the markers linked to spinal muscular atrophy (SMA; the human MND that is clinically similar to HCSMA) are not linked to the HCSMA trait.
The rate of progression of clinical HCSMA is dependent on gene dose (e.g., homozygous pups develop an accelerated form of the disease by 6 to 8 weeks of age that rapidly leads to paralysis and necessitates euthanasia by 12 to 16 weeks of age). This is the only spontaneously occurring dominantly inherited animal MND in which onset and severity of the disease varies with the gene dose. Skeletal muscle degeneration develops in the accelerated and chronic forms of HCSMA, but spinal cord motor neuron loss is most apparent late in the disease in the heterozygotes.
Equine MND
Spontaneously occurring equine MND was first described in 1990. Since then, the epidemiologic, clinical, and neuropathologic data gathered from over 100 cases suggest commonalties with the sporadic form of ALS. Neuronal loss is less extensive than that associated with SALS, and there is no degeneration of the pyramidal pathway, which is rudimentary in the horse.
An important clinical feature in horses is the apparent tendency of the disease to "burn itself out" in some animals. These animals typically have sudden onset and rapid advancement of the clinical signs; the disease then appears to "stabilize" without further progression. These animals may survive for years, although they never completely recover. Some human ALS patients also experience this clinical scenario.
Equine MND is currently the only spontaneously occurring animal MND that could provide clues to the pathogenesis of sporadic ALS on the basis of the epidemiologic and clinical data for the naturally acquired disease.
Summary
None of the aforementioned animal species have a well-developed corticospinal tract, a system that is uniquely and specifically involved in ALS. A presymptomatic diagnosis is currently not possible in ALS patients, and clinical signs are usually readily apparent before treatment regimens are implemented.
Human autopsy material, the main source of tissue for the diagnosis and study of human MND, reflects end-stage disease processes. Thus, study of the presymptomatic period, the early disease process, and the temporal changes is important and possible with these experimental models. The composite information from the mouse, canine, and equine animal models for MND will continue to yield important insights into the pathogenesis of this disease.
Classification of selected animal models for MND
Experimentally induced
Genetically engineered mice
Cu/Zn SOD gene mutations
Neurofilament gene mutations and overexpression
CNTF, BDNF, and trkB gene knockouts
Neurotoxins
IDPN
Heavy metals-aluminum, lead, mercury
Cycad toxin: BMAA
Lathyrus sativus toxin: BOAA
Doxorubicin, vincristine, vinblastine
Nutritional deficiencies
Calcium and magnesium
Copper
Ascorbic acid
Viral
Poliomyelitis virus
Murine retrovirus
Lactate dehydrogenase-elevating virus
Autoimmune
Experimental allergic gray matter disease (EAGMD)
Experimental allergic MND (EAMND)
Axotomy induced
Spontaneously occurring
Heritable
Murine
Motor neuron degeneration (mnd)
Wasted mutant
Progressive motor neuronopathy (pmn)
Wobbler
Canine
Hereditary canine spinal muscular atrophy
Acquired
Equine MND
CNTF = ciliary neurotrophic factor; BDNF = brain-derived neurotrophic factor; trkB = tyrosine kinase B receptor; IDPN = _'_'-iminodipropionitrile; BMAA = _-methyl-amino-alanine; BOAA = _-N-oxalyl-amino-L-alanine
Questions: Questions:
1. Which of the following spontaneously occurring animal models of ALS is inherited as an autosomal dominant?
a. wobbler mouse
b. pmn mouse
c. wasted mutant mouse
d. hereditary canine spinal muscular atrophy
e. mnd mouse
2. The most common human motor neuron disease is:
a. Batten's disease
b. neuronal atrophy
c. amytotrophic lateral sclerosis
d. hyperparathyroidism
e. bulbospinal neuropathy
3. How does the disease expression in the wobbler mouse differ from the other animal models?
a. onset and severity of disease varies with gene dose
b. adult late onset motor neuron degeneration
c. aggressive disease with death ensuing by 28 days
d. basically a model of dying back neuronal disease
e. predominant forelimb involvement
Answers: Answers:
1. D - hereditary canine spinal muscular atrophy (HCSMA)
2. C - amytotrophic lateral sclerosis (ALS)
a. Batten's disease - most common human neuronal lipofuscinosis
3. E - predominant forelimb involvement
a. onset and severity of disease varies with gene dose - HCSMA
b. adult late onset motor neuron degeneration - mnd
c. aggressive disease with death ensuing by 28 days - wasted mutant
d. basically a model of dying back neuronal disease - pmn

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv. Laboratory Animal Science 49(5), 488.
Abstract: The investigators examined murine leukemia virus Akv (an ecotropic provirus found in the AKR/J strain) with regards to its effect on embryos (including pathological expression and embryonic lethality). Both provirus-carrying AKR/J mice and control SWR/J mice were used in a series of experiments to compare the effects of the virus. (AKR/J mice tend to spontaneously develop expression of ecotropic retrovirus, with titer increasing throughout their lives; SWR/J mice were selected as controls since they are one of comparatively few strains which are not known to harbor any endogenous ecotropic retrovirus.)
Several different paradigms were examined, involving presence or absence of the Akv provirus or virus in the dam, in the embryos being implanted in a recipient foster dam, in the recipient dams, or in the neonates themselves.
Significant findings were as follows:
1. Re: virus-free SWR/J embryos implanted into previously-infected SWR/J dams (these dams having been infected with Akv when they were newborns, and being viremic at the time they were subsequently pseudopregnant and used as recipients for the uninfected embryos): 13% of recovered embryos were dead, and new provirus integration was detected in 18% of recovered embryos. Nine percent of embryos developed normally at day 14 of gestation contained evidence of new provirus integration, demonstrable by Southern blot analysis; however, of five embryos dying between day 12 and day 14 of gestation, four (80%) contained evidence of both virus integration and viral protein expression.
2. Numbers of normally-developed embryos were significantly greater when healthy embryos were implanted in normal SWR/J recipient dams, than when the recipient dams were either Akv-infected SWR/J mice or Akv-provirus-containing AKR/J mice. The authors concluded that viremia of the dam during gestation impairs early embryonic development, and endogenous provirus expression is highly correlated with embryonic death..
3. Inoculation of previously-healthy SWR/J neonates with Akv retrovirus resulted in the development of nonthymic lymphoblastic lymphomas in 63% of the mice, during a 540-day observation period; only 8% of uninoculated controls developed lymphomas.
4. In the authors' experiments, higher levels of virus were seen in experimentally-infected SWR/J mice than occurred in AKR/J mice with spontaneously-expressed viremia of endogenous origin.
This work is considered of value as a model for transplacental transmission of retrovirus. The placental anatomy of humans and mice bear significant similarities. There are reports of transmission rates of human immunodeficiency virus (HIV) of up to 20% in infants born to HIV-positive mothers with AIDS. Similarly, in HIV-positive women, high levels of early embryonic death have been reported (e.g. 97% in the first eight weeks of pregnancy), reminiscent of observations noted in this mouse study. Additionally, the use of retroviral vectors for gene delivery in transgenic and knockout manipulations poses risks for unintentional transmission of retroviral infection and subsequent embryonic disease or death.
Questions: QUESTIONS:
1. Name one mouse strain which carries an endogenous retroviral provirus, name the virus, and name one mouse strain which for which no endogenous provirus has been detected.
2. State two possible sequelae of infection with murine Akv virus (acquired in utero or from endogenously-present provirus); give one possible sequela of post-natal infection with the same virus.
3. Name one possible model for transplacental transmission of human immunodeficiency virus (HIV).
Answers: ANSWERS:
1. The AKR/J strain of mouse carries the Akv retroviral provirus. The SWR/J strain carries no known endogenous provirus.
2. Infection in utero with Akv virus can cause viral integration in the embryo's genome, viral protein expression, developmental abnormalities and early embryonic death. Post-natal infection with Akv virus is associated with increased incidence of nonthymic lymphoblastic lymphoma.
3. Mouse dams infected with Akv retrovirus can transmit this agent transplacentally to embryos they are carrying.

Enteric lesion sin SCID mice infected with Helicobacter tryplonicus, a novel urease-negative Helicobacter species. Laboratory Animal Science 49(5), 496.
Abstract: Recently a novel urease negative helicobacter was isolated from IL-10-deficient mice with colitis and typhlitis. Another study had also indicated that this Helicobacter species led to typhlitis, colitis and proctitis in SCID/NCr mice. Because SCID mice are highly susceptible to enteric disease attributable to H. hepaticus and H. bilis, these mice are the most likely to be susceptible to enteric disease attributable to other helicobacters.
In this study, Helicobacter-free SCID mice were inoculated with the novel "H. typhlonicus" via gastric gavage. At 12, 24, and 36 weeks after inoculation, the animals were necropsied for histologic evaluation. A lesion-scoring system was used to assess the sections of liver and gastrointestinal tract isolated from the mice. The scoring was done blinded. In addition, a cohabitation study was performed using B6D2F1 mice infected with an uncharacterized urease-negative helicobacter. These mice were housed with SCID mice and the animals were necropsied after 11, 18, 34, and 45 weeks of cohabitation.
Results found that a 1.5 kb region of the 16S rRNA gene was 100% identical among three isolates of "H. typhlonicus". A 164-bp intervening sequence (IVS) was identified, yet it had less than 50% homology to the IVSs of H. bilis and H. canis, the only other helicobacters known to have an IVS. Sequencing comparisons showed that "H. typhlonicus" was most closely related to H. hepaticus, but was a distinct species having 97.64% identity and containing the aforementioned intervening sequence.
Phenotypic tests commonly used to characterize Helicobacters were performed on the isolates.
Clinical signs of disease were not evident at any time during the study. However, at all time points post-inoculation, histologic evidence of proliferative typhlitis was seen in 22 of 22 inoculated male and female mice. The findings were significant for only the 24 and 36 week groups. No liver abnormalities were found. Control mice did not have any evidence of cecal or colonic disease. Significant differences were not seen in proliferative typhlitis scores between inoculated male and female mice.
After 2 months of cohabitation, all SCID mice were culture positive for Helicobacter sp. Cecal lesions were detected in the 18 SCID mice. Relevant liver lesions were also found in these mice, consisting of some degree of necrosis or chronic active hepatitis. It is not totally clear why these mice had liver lesions that were not seen in the inoculated group. The paper speculated that it was possible that liver lesions attributable to H. typhlonicus develop only at late stages of the disease, or that the lesions were caused by a non-Helicobacter pathogen.
The findings highlight the need to screen rodent colonies, especially colonies of immunodeficient mice, for all Helicobacter species, not just those that have been speciated. The name of H. typhlonicus is only provisional at this time, official naming and recognition awaits identification and characterization of additional strains of this putative Helicobacter species.
Questions: Questions:
1) List identifying characteristics of Helicobacters.
2) T/F H. bilis has been associated with disease in both immunocompetent and immunodeficient rodents
3) Which 3 rodent Helicobacters contain an IVS (intervening sequence)?
Answers: Answers:
1) Microaerobic, curved to spiral rod morphology, motile by flagella that vary in number and location among various species, live in animal hosts where clonization is restricted to the gastrointestinal tract or, in some cases, the liver
2) FALSE. Only H. hepaticus has been documented to cause chronic active hepatitis and inflammatory bowel disease in both immunocompetent and immunodeficient mice. H. bilis has been convincingly associated only with disease in immunodeficient rodents, including mice and rats.
3) H. bilis, H. canis, and the putative H. typhlonicus

Reverse transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new rat coronavirus strain. Laboratory Animal Science 49(5), 506.
Abstract: The subject of this paper has been a topic of much Compmed discussion this winter. Rat coronavirus (RCV) is highly infectious and spreads rapidly through laboratory rat colonies, causing sneezing, nasal and ocular discharges, photophobia, and cervical swelling. Current diagnostic methods include serologic testing and histologic examination. The RT-PCR was used to detect RCV RNA in tissues from infected rats and on cages housing infected rats and to amplify portions of the RCV N, M, and S genes for molecular characterization. The RT-PCR detected RCV RNA on cages and in tissues from infected rats. The RCV-NJN gene is most closely related to the MHV-YN gene. The M proteins of RCV-NJ and RCV-SDA are 99% homologous and the six RCV S protein fragments are 97% to 100% homologous.
Use of RT-PCR with cage swab specimens was capable of diagnosing RCV infection in and viral excretion from rats. Also, molecular characterization of various genes can be used in performing further epidemiological studies.
Questions: Questions:1.Give three clinical signs of RAT-SDA.
2. T or F. RCV dried on plastic is still viable on rehydration 48 h later.
Answers: Answers: 1. sneezing, ventral cervical edema, and chromodacryorrhea.
2. T

Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups. Laboratory Animal Science 49(5), 514.
Abstract: To determine if different neutralization groups of rat coronaviruses (RCV) varied in their pathogenic potential, rats were innoculated with 3 different strains of RCV. Strains tested included RCV-SDA (sialodacryoadenitis--the prototype virus for this group of pathogens), RCV-BCMM, and RCV-W. Although clinical illness was not observed in any of the infected rats (go figure!), lesions were detected by histopathology 7 days post infection (PID) in the exorbital lacrimal glands (all strains); the Harderian glands, parotid salivary glands, nasal mucosa, and lungs (RCV-BCMM and RCV-W); and the submandibular salivary glands (RCV-W only). By PID 14, no histopathologic lesions were seen in rats infected with RCV-SDA. Rats infected with RCV-BCMM had lesions in Harderian glands, parotid salivary and exorbital lacrimal glands,lung, and nasal mucosa, although with the exception of the Harderian gland, lesions were mild. Rats infected with RCV-W had lesions in Harderian glands and parotid salivary glands, again with the Harderian glands more severely affected. Lesions in all tissues caused by all strains were typical, i.e. necrosis and inflammation. Good correlation was observed between tissues with high severity scores on histopath, and those that had high amounts of infectious virus and viral RNA (as detected by plaque assay and RT-PCR, respectively.) However, neutralization groups were not considered useful
for predicting pathogenicity.
Questions: Questions
1. Name two other major coronaviruses of veterinary interest.
2. List possible research confounders subsequent to RCV infection.
3. What are the clinical signs of RCV infection, and what other pathogens can cause similar signs?
Answers: Answers
1. ***mouse hepatitis virus***, bovine coronavirus, porcine transmissable gastroenteritis virus, turkey coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, canine coronavirus, porcine hemagglutinating encephalomyelitis virus, rabbit coronavirus (I'm sure I missed a couple <g>)
2. reduced food consumption with subsequent weight loss, chronic ocular lesions, reduced breeding performance, increased anesthetic risk, and the ever-popular secondary infection problem
3. Signs last about one week and include sneezing, sniffling, photophobia, cervical swelling, nasal and red-pigmented ocular discharges. Rule-outs include Sendai virus, M. pulmonis, CAR bacillus, various other respiratory bacterial infections (i.e. Strep pneumoniae)

Comparison of adjuvants with Leishmania antigens in a guinea pig model to induce delayed type hypersensitivity responses. Laboratory Animal Science 49(5), 519.
Abstract: Leishmaniasis is an arthropod-borne parasitic disease caused by a protozoan. Pathologic changes may develop in the skin, nasal mucosa, viscera (spleen, liver and lymph nodes), and bone marrow depending on the species of Leishmania contracted.
Diagnosis of Leishmania remains difficult. More rapid diagnostic tests are needed. One avenue of rapid diagnostic technology relies on use of antigens in immunologic assays. Guynea pigs were used to create a model to screen antigens for use in these assays, on the basis of their traditionnal role for delayed-type hypersensitivity (DTH) evaluations.
Complete Freund Adjuvant (CFA), TiterMax and liposomes were evaluated in combination with Leishmania antigen for their capacity to elicit DTH.
Liposomes and CFA were effective to elicit DTH. TiterMax was not. CFA may cause large ulcerative lesions, but liposomes can be used as a safe effective adjuvant.
Questions: Q1: Is Leishmania a zoonosis ?
Q2: What is the vector ?
Q3: For what reason were guinea pigs used ?
Answers: A1: Yes.
A2: Arthropod (sand fly)
A3: For their traditional role in delayed-type hypersensitivity evaluations.

Transdiaphragmatic lymphatic transport of intraperitoneally administered marker in hamsters. Laboratory Animal Science 49(5), 522.
Abstract: This study was developed to evaluate the movement of a marker substance (Monastral blue B) from the peritoneal cavity into the pleural cavity (transdiaphragmatic) in the Syrian hamsters (Mesocricetus auratus) compared to other animals in which similar studies have been done. Transdiaphragmatic transport of solutions is now considered the principal route of fluid drainage from the peritoneal cavity. On the peritoneal side of the diaphragm, particulate matter up to 22 microns and fluids are pulled into stomata (situated between mesothelial cells) centered on endothelial lined cisternae that connect with lymphatic channels leading into the pleural cavity. The negative intrathoracic pressure, especially during inspiration aided by the open stomata, "suck" fluids and particles into the cisternae. On expiration, the stomata close and fluid is propelled antegrade into the lymphatics.
Four hamsters were euthanitized at 7, 15 30 60,120,180 minutes and 24 hours after intraperitoneal injection of Monastral blue B. Light microscopy was used to detect presence of the marker substance in lymph nodes, liver, spleen, bone marrow, diaphragm, thymus, and testes. The order of marker appearance in the evaluated tissues are as follow:
thoracic lymph vessels and cranial mediastinal lymph nodes and testes (7 min); caudal mediastinal lymph nodes (30 min); liver, spleen, bone marrow , testes and diaphragm (60 min); mesenteric lymph nodes and mandibular lymph nodes (24 hours). Marker substance was considered to have entered the cranial and caudal mediastinal lymph nodes via lymph vessels and from these nodes disseminated hematogenously to the remainder of the tissues. The early appearance in the testes attests to the ease of migration of material in the lumen of the vaginal tunic and explains the fulminant orchitis, the Strauss phenomenon, seen in hamsters and guinea pigs after intraperitoneal administration of some bacteria. Marker was never found in the thymus or popliteal lymph nodes.
Conclusions: Authors found that their data were very consistent with previous studies in different species, using different or the same marker material or differences that existed could be explained.
Questions: Questions:
1. What is the Order and Suborder and Family of the species Mesocricetus auratus?:
2. What is the Strauss phenomenon?
Answers: Answers:
1. Order Rodentia; Suborder, Myomorpha, Family, Cricetidae
2. The Strauss phenomenon is an acute unilateral or bilateral orchitis and/or epididymitis, associated with intraperitoneal injection of bacteria, especially seen in hamsters and guinea pigs of the lab species.

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets. Laboratory Animal Science 49(5), 530.
Abstract: Pytoestrogens are plant compounds that exert estrogenic affects on the central nervous system, induce estrus, and (+) growth of the genital tract of female animals by binding to the estrogen receptor. Two main classes exist: lignans and isoflavonoids. Their potency is significantly less than steroidal estrogens, but wide variations in the concentration in plants and differences in species sensitivity make them potential confounders in rodent diets. Several rodent diets were assayed for phytoestrogen content using high performance liquid chromatography (HPLC). Brief review of terminology: Closed formula diet- ingredients are reported, but concentrations are not and can vary (ex: Rodent lab chow 5001). Open formula diet- all ingredients and their concentrations are reported (ex: NIH 31 diet). Soybean meal was the major source of these compounds, and their concentrations were directly correlated to the % of soybean meal in each diet. Variably high concentrations were found in different closed-formula diets as well as lot-to-lot variation of the same diet. For studies in which estrogenic compounds may influence results (such as for estrogenic, reproductive, and tumor development experiments) a standardized, open-formula diet in which estrogenic substances have been reduced and concentration reported are recommended.
Questions: Questions:
1. What is a phytoestrogen and why are they important to lab animals?
2. What is the difference between and open and closed formula diet?
3. Define HPLC
4. What of the following ingredient in rodent diets contains the highest amount of estrogenic compounds?
Corn meal
Alfalfa meal
Soybean meal
Wheat /Oats
Answers: Answers:
1. Pytoestrogens are plant compounds that exert estrogenic affects on the central nervous system, induce estrus, and (+) growth of the genital tract of female animals by binding to the estrogen receptor.
2. Open formula diet- all ingredients and their concentrations are reported; Closed formula diet- ingredients are reported, but concentrations are withheld and can vary significantly from lot to lot
3. high performance liquid chromatography
4. Soybean meal

Cardiovascular, respiratory, thermoregulatory, sedative, and analgesic effects of intravenous administration of medetomidine in Rhesus macaques (Macaca mulatta). Laboratory Animal Science 49(5), 537.
Abstract: The purpose of this study was to evaluate the effects of medetomidine in rhesus monkeys at therapeutic dosages. Medetomidine is an alpha 2 adrenergic receptor agonist. Historically, these agents have been used in a variety of species for their sedative/analgesic effects. The value in these agents is that they provide centrally mediated, dose-dependent sedation, analgesia, anxiolysis and muscular relaxation. They are rapidly reversible, act in synergy with other anesthetic agents and have predictable and manageable adverse effects (bradycardia, hypotension and hypothermia).
Four intravenously administered dosages of MED( 50, 100, 150 and 200 micrograms/Kg) were evaluated. Each animal (8 female, 7 male rhesus) received each dosage on a random basis with a 2 week rest period between trials. Blood pressure, heart rate and rhythm, temperature, respiratory rate, oxygen saturation and sedation/analgesia were evaluated. To determine the sedation/analgesia score, 6 parameters were evaluated: posture, mental state, ocular signs, bite reflex, response to noise/manipulation and response to aversive stimuli.
All 4 dosages tested were associated with reduction in heart rate, multiple arrhythmias, hypotension, hypothermia, sedation, anxiolysis and analgesia. There was an initial increase in respiratory rate, which then returned to baseline and continued to decrease. The sedation was interrupted by unexpected periods of arousal and aggressive behavior in response to manipulation or noise. The trials were terminated when values reached 70% of normal, the animals became agitated or continuation of the trial placed the animal's health in jeopardy. 58 of 60 trials were completed. 5 trials were terminated with the reversal agent, atipamezole. No adverse effects were noted with this treatment. It effectively reversed the hypotention and bradycardia, and also resulted in increased respiratory rate, diruesis, salivation, occasional bruxism and reddening of the skin. These authors conclude that due to the inconsistency of the anesthetic plane induced by medetomidine, it is unsatisfactory as a sole anesthetic agent. However, other reports describing the anesthetic properties of ketamine/medetomidine cocktails indicate this may be an acceptable regimen for primates.
Questions: Questions:
1. Medetomidine is classified as an __________________. Another commonly used agent in this group is ____________________.
2. Side effects noted with medetomidine included: ________________________.
3. Reversal of medetomidine can be accomplished using ___________________.
4. T/F The authors recommend the use of medetomidine as a sedative in NHP.
Answers: Answers:
1. alpha 2 adrenergic agonist
xylazine
2. Bradycardia, hypotension, hypothermia, multiple arrhythmias
3. atipamezole
4. F

Evaluation of surrogate markers of impending death in the galactosamine-sensitized murine model of bacterial endotoxemia. Laboratory Animal Science 49(5), 545.
Abstract: Refinement of animal experimentation as defined by Russell & Burch: to reduce to an absolute minimum the amount of distress imposed on those animals that are still used.
The purpose of this study was to define reliable markers of impending death in the galactosamine-sensitized murine model of endotoxemia to be used in vaccine trials. Endotoxins used were from: E. coli, K. pneumoniae & P. aeruginosa.
Several parameters reported as reliable endpoints in other studies include: serum biochemical markers in dogs for failing organ transplasnts, inability to ambulate in guinea pigs, sleep patterns in rabbits & weight loss in rats with CNS tumors. Hypothermia has been used a death alternative in mice for viral, bacterial & metal salt toxicity studies.
The parameters used in this study: temperature, body weight, coat condition, posture, inability to ambulate & loss of consciousness. To be useful for routine application, surrogate markers must be tailored to experimental protocol and species.
Galactosamine-sensitized mice were inoculated IP with endotoxin and montored for clinical disease signs. Wide flucuations in body temperautre were observed in survivors & nonsurvivors. Posture, coat & body weight were not accurate predictors of death. Only the inability to ambulate, with a positive predictive value of 100% accurately predicted death.
Questions: Questions
1. In addition to inability to ambulate, what other parameters have been used to predict death in research animals?
Answers: Answer
1. Serum biochemical markers, sleep patterns, temperature, weight loss.

Characterization of rabbit Pasteurella multocida isolates by use of whole-cell, outer-membrane, and polymerase chain reaction typing. Laboratory Animal Science 49(5), 551.
Abstract: Purpose: to characterize 50 isolates of Pasturella multocida from laboratory rabbits from 5 different geographic locations using serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP) and outer membrane proteins (OMP) and polymerase chain reaction (PCR) fingerprinting.
Serogrouping and serotyping: done using enzyme sensitivity to decapsulation determined by disk diffusion with hylauronidase, chondroitinase, and heparinase. Serotyping was done in agar gel precipitation with chicken antisera.
SDS-PAGE of WCP and OMP: Bacteria were sonicated and then run through the gel... Protein bands were stained and compared.
PCR: Primers to amplify the DNA were of M13 phage core sequences and microsatellite DNA sequences (4 different primer pairs)
Results: 45 were of serogroup A and 5 were serogroup D. Most were of serotype 2 or 3 only. There were one of each of group 4, 10, 11, and 15. Two were not typable.
SDS-PAGE of WCP identified 10 different groups, and 1 subgroup (one additional band). SDS-PAGE of OMP identified 10 different types. 4 of which were solely represented. PCR with the 4 different primer pairs reproducibly identified 7, 9, 5, and 9 different fingerprints each of which contained between 2 and 12 distinctive bands.
Conclusion: This study used statistical analysis to determine the discrimination index (D). This value is the probability that 2 unrelated strains randomly selected from the test population would fall into different typing groups.

Typing method Discrimination index
Serotype 0.80
WCP 0.88
OMP 0.81
PCR primers
M13 phage 0.62
M13 modified 0.79
(GTG)x5 (micro) 0.79
(GACA)x4 (micro) 0.84
All PCR's combined 0.90
WCP+OMP+ all PCR's 0.98
The findings support the use of PCR in the characterization of P.multocida isolates. However, PCR had no direct correlation with the other methods of characterization nor was any one primer was necessarily better than traditional typing methods.
Questions: Questions
1. Clinical sign/s of Pasturella multocida infection in laboratory rabbits include/s:
a) upper respiratory infection
b) fibrinopurulent bronchopneumonia
c) otitis media and meningitis
d) localized absessation
e) septicemia
f) all of the above
2. The most common serogroup to cause clinical signs in laboratory rabbits is:
a) type A
b) type B
c) type C
d) type D
e) type E
3. Pasturella multocida is a
a) gram negative rod
b) gram negative coccobacillus
c) gram positive coccus
d) gram positive rod
Answers: Answers

1. F
2. A. There are 5 capsular serogroups and 16 somatic serotypes of P. multocida. Type D is also common, however to a lesser extent.
3. B - they are bipolar staining

Rescue of (NZB x NZW) F1 mice from Oxygen-Derived Free Radical Injury by Use of Phosphatidylcholine-modified superoxide dismutase. Laboratory Animal Science 49(5), 560.
Abstract: Superoxide dismutase (SOD) is an enzyme that degrades active oxygen (ie. superoxide anion radicals). Overproduction of oxygen-derived free radicals may induce glomerular tissue damage. The use of SOD is limited by its' rapid metabolism. Biologically active proteins that have been coupled with lecithin (phosphatidylcholine, PC) increases the pharmacologic activity of these proteins by: 1) increasing cellular affinity, 2) decreasing adverse effects and 3) stimulating absorption.
This study investigated the use of PC-SOD in the treatment of lupus nephritis in (NZB x NZW) F1 mice. Mice were treated with various doses of PC-SOD via tail vein injection and euthanized after 23 weeks. Results (histology and electron microscopy) from this study indicated that activated PC-SOD scavenged oxygen-derived free radicals in mesangial and inflammatory cells. In addition, PC-SOD suppressed deposits in mitochondrial membranes of renal tissue sections.
Questions: Q1. The (NZB x NZW) F1 mouse is an animal model for what disease?
Q2. What type of inbred strain does (NZB x NZW) F1 represent?
Q3. The NZB refers to the male progenitor? (true or false)
Answers: A1. spontaneous lupus nephritis
A2. recombinant inbred strain
A3. False -female progenitor

Proliferative retinal changes in diabetic rats (WBN/Kob). Laboratory Animal Science 49(5), 565.
Abstract: 1. Human diabetes mellitus retinopathy has it origin in inappropriate neovascularization of the retina (proliferative retinopathy) leading to vitreous hemorrhage and/or traction retinal detachment, affecting a significant proportion of diabetics.
2. Current rodent models of diabetes mellitus (DM) have not been shown to exhibit the proliferative phase of the human disease, though there are vascular lesions in the rodent model.
3. The WBN/Kob strain exhibits a high incidence of spontaneous DM by 12 months of age, in males, though ocular angiopathy has not been documented to date. The onset of glucosuria (> 2 g/dL) begins ~ 14 months of age but returns to normal after 22-24 mos of age. Because of cataract formation in this model, there was speculation that retinal vascular damage may have been missed. This study was designed to evaluate late stage retinal damage. Silicone rubber injections into the aortic root with fixation, clearing and observation under stereomicroscopy and histopathology were used to document vascular lesions
4. Of 6 rats, all developed cataracts; all six became glucosuric and hyperglycemic (211- >500 g/dL) between 14 and 22 months of age with resolution to normal glucose values in 5/6 by 23 months and in all six by 24 months of age. Four of six demonstrated proliferative retinopathy but there was no correlation with the peak serum glucose; time of onset of hyperglycemia or duration of hyperglycemia. The lesions were seen even after the glucose levels in the serum returned to normal. The proliferating vessels grew into the vitreous and originated from the normal vasculature on the optic disc, with retinal detachment in 2/6
5. There is a model in dogs fed galactose for 84 months but the success rate of this long-term model is very poor (2/9) and there is no associated glucosuria, though the lesions are quite similar to that of this rodent model.
Questions: Questions:
1. In the WBN/Kob rat, diabetes _____________________
A. is a spontaneous phenomenon of both sexes
B. must be induced with streptozocin
C. does not resolve within the normal life span of the rat
D. is a spontaneous phenomenon of male WBN/Kob rats
2. In the dog model of diabetic retinopathy which exhibits proliferative neovascularization, ______________________.
A. the disease is induced by feeding lactose for long periods of time
B. the proliferative retinopathy is induced 100% of the time
C. the disease is induced by feeding galactose
D. glucosuria is a component of the ocular pathology
3. In the WBN/Kob rat model for proliferative retinopathy, _______________.
A. lesions resolve after 14 months of age
B. cataract formation was not an observed feature of the disease
C. lesions persisted with normal blood glucose and without glucosuria
D. retinal detachment is not an observed lesion
Answers: Answers:
1. D
2. C
3. C