Does witnessing experimental procedures produce stress in male rats? Contemporary Topics 41 (5): 8.
The Guide for the Care and Use of Laboratory Animals and The AVMA Panel on Euthanasia stipulate that animals should not be present when other animals are being euthanized. IACUCs concern that animals witnessing procedures such as euthanasia may be unduly stressed or if not stressed, then affected in such a way as to confound experimental results obtained from those animals. The authors hypothesized male rats are stressed by being in the same room in which euthanasia and experimental procedures are conducted on other rats and that the witnessing effect is influenced by housing density. To test the hypotheses behavior was monitored, and heart rate (HR) and mean arterial blood pressure (MAP) were obtained by radiotelemetry. Rats witnessed decapitation, decapitation and necropsy, cage change, restraint and subcutaneous injection, and restraint and tail-vein injection. Small increases in HR and MAP were observed in rats witnessing decapitation of other rats. Rats housed alone had significant greater responses than did rats housed two or four per cage. Witnessing decapitation and necropsy of other rats induced somewhat greater increases in HR and MAP than did witnessing decapitation only in animals housed alone but produced no significant changes in animals housed two or four per cage. There were no significant changes in HR or MAP in rats witnessing manual restraint and s.c. injection of other rats. Witnessing prolonged restraint in a rodent restrainer and tail-vein injection induced small, transient increases in HR and MAP in rats housed two per cage. Home cage sleeping behavior was reduced significantly only in animals witnessing decapitation and necropsy and then only in animals housed alone.
These results (small or no changes in HR and MAP and home-cage behavior in rats in the same room in which decapitation and other experimental procedures are conducted) do not support their hypothesis that witnessing these procedures stresses male rats. Further investigation is needed to examine responses in different rat strains and in other rodents.

Questions:
1. The Guide and AVMA stipulate that animals should not be present when euthanasia is performed. What is the reason for that?
2. Name 2 commonly used indices of stress.
3. In this study what methods were used to monitor stress?

Answers:
1. In some cases, vocalization and release of pheromone occur during induction of unconsciousness.
2. a) Heart Rate, b) Mean Arterial blood pressure
3. HR and MAP were determined by radiotelemetry, and home cage behaviors were scored.

Short-term effects of an environmental enrichment program for adult Cynomolgus monkeys. Contemporary Topics 41 (5): 13.
The purpose of this study was to examine the overall approach to nhp environmental enrichment (EE) stimulation used in a tox facility short-term housing adult cyno macaques, and to determine if the provision of enrichment opportunities affected the ease of training animals to perform a simple task (enter a transfer box). Adult Cynomolgus Monkeys (Macaca fascicularis), N=40, were placed into control and enriched study groups for a period of 4 weeks (10 Male or Female in both EE and Control groups). EE animals were exposed to various manipulanda, TV, and additional food foraging opportunities. Animals were observed directly for 10 minute periods each week, and the data was summarized. Additionally, each week the animals were made to enter a transfer box and weighed, and the time was recorded. Animals engaged in abnormal behaviors less than 11% of the time, regardless of their environment, even though EE animals were observed to interact with manipulanda and foraging devices. For both groups, animals spent the majority of their time inactive in their cages. For both groups, the behavior trend remained consistent across the 4 week observation period. EE did not have any effect on the training time required to perform a simple task (enter the transfer box). 25% of the control animals exhibited behavioral disorders at the end of the study, compared to 0% for the EE group. The author concludes that EE did have a positive effect on the overall health and well-being of these animals.

Questions:
1. What percentage of time did this study find the animals engaging in abnormal behavior?
2. What percentage of time did this study reference as being previously reported for similar animals, housed long-term, displaying abnormal behavior?
3. Did EE have a positive effect on the reduction in training time required to perform a simple task (enter the transfer box)?
4. T/F The author concludes that the provision of EE for adult cynomolgus macaques, housed short-term, is beneficial?

Answers:
1. 11%
2. 10-24%
3. No
4. True

Methods for predicting sexual maturity in male Cynomologus macaques on the basis of age, body weight, and histologic evaluation of the testes. Contemporary Topics 41 (5): 18.
This study evaluated the use of an animal's age, body weight and testicular size as simple prospective methods by which one could predict sexual maturity in male cynomolgus macaques. The most accurate measurement of sexual maturity, however, is via histologic evaluation of the testes accomplished by testicular biopsy or on postmortem examination. The data for this study was obtained retrospectively from 85 male cynos euthanized for other studies. Results of the study, using the animals' age and body weight with statistical methods predicting 90% probability of sexual maturity was that the animal should be at least 5 years 5 months of age and should weigh at least 5.3 kg, respectively. Testicular size, both left and right, was also measured by caliper, but the results were inconsistent with respect to volume calculations. Because of a large degree of variability in levels of maturation and inconsistency in testicular descent, testicular size is not considered a reliable prospective determinant of maturity and is not used as a determinant to select sexually mature animals for drug-safety purposes.

Questions:
1. Which of the following is not a primary characteristic defining maturity in macaques ?
A. body weight
B. age
C. testicular size
D. presence of ejaculate
2. Immature testes lack all of the following cellular components except
A. spermatogonia
B. spermatocytes
C. round spermatids
D. elongating spermatids
3. During which season are testosterone levels at their highest ? Name some seasonal breeding nonhuman primates.
A. winter
B. summer
C. fall
D. spring

Answers:
1. D; 2. A; 3. C

Review of growth plate closure compared with age at sexual maturity and lifespan in laboratory animals. Contemporary Topics 41 (5): 21.
In most animals, it has been assumed that growth plate closure occurs soon after sexual maturity. However, in rats, bone growth continues throughout their lifespan. This paper is an analysis of the literature with regards to growth plate closure times and age at sexual maturity in mice, rabbits, dogs, cat, sheep, cows, horses, non-human primates and humans. For all species, several ratios were calculated: age at growth plate closure to lifespan, age at growth plate closure to age at sexual maturity and age at sexual maturity to average lifespan.
In reviewing the literature, the focus was placed on data from the femur and tibia. Growth plate closure was not defined with respect to radiographic or histological criteria in most citations. No citation on the age at growth plate closure was found for any bone in the rats. Historical reports state the physis remained open for as long as 29 months in the rat tibia. For the ratio of age at growth plate closure to life expectancy, small NHP models had a small ratio meaning that growth plate closure occurs relatively early in the life of these animals. In NHP and humans, the ration was increased. However, the ratios in the mouse and the rat were the largest with rodents showing the presence of bone growth for more than 20% of their life expectancy.
In comparing ratios of growth plate closure to age at sexual maturity, only the dog and the rabbit showed growth plate closure times that occurred before or coincided with sexual maturity. Rodents showed markedly larger ratios indicating that sexual maturity preceded cessation of bone growth by a considerable time period. There was a gender-specific difference in the mouse due to the earlier onset of sexual maturity in the female mouse.
With ratios of age at sexual maturity to life expectancy, humans and NHP were the oldest at sexual maturity. All other species attained sexual maturity within 10% of their average lifespan.
The authors acknowledge that this study was limited by several factors including lack of criteria for growth plate closure in some citations, lack of documentation between the relationship between physis closure and longitudinal bone growth, breed/strain/gender variation and the lack of data on non-appendicular bones.
The take home message is that in selecting a research model for orthopedic studies, the investigator needs to consider species, age, and sexual maturity. Most laboratory animal species show synchrony between onset of sexual maturity and growth plate closure. Bone growth patterns in the mouse and rats stand out because of the rodents' later closure of growth plates and continued longitudinal bone growth after sexual maturity.

Questions:
1. T/F: In most mammals growth plate closure and cessation of bone growth occurs soon after sexual maturity.
2. T/F: Historical reports revealed that the physis of the rat remained open for 12 months in the rat.
3. T/F: Growth plate closure occurs early in the life of many small, nonprimate research models.
4. Rodents show the presence of bone growth for more than ________ of their life expectancy.
a. 10%
b. 20%
c. 50%
d. 75%
5. All species except ______ attained sexual maturity within 10% of their average lifespan.
a. rodents
b. dogs
c. rabbits
d. nonhuman primates.

Answers:
1. T
2. F Reports show it to be open for as long as 29 months.
3. T
4. b
5. d

Age- and gender-related changes in copper and zinc levels in the plasma of Mongolian gerbils. Contemporary Topics 41 (5): 27.
This study examines the plasma levels of copper and zinc in both male and female mongolian gerbils of three different ages. The authors chose to study these minerals because of their importance in various enzymes. Despite previous studies examining copper and zinc levels in bone, kidney and liver, not much is known regarding their levels in the plasma of gerbils. In other species, including humans, age and gender can affect the plasma levels of copper and zinc. For instance, in humans men have higher zinc levels than women but the reverse is true in dogs. Copper levels usually do not vary by gender except in the dog where males have consistently higher values until 5 to 6 years of age when they begin to decrease.

The study looked at 10 male and 10 female gerbils at 3 different ages (90, 180 and 360 days). The gerbils were fed ad lib pelleted rodent maintenance diet with 30 mg/kg copper and 95 mg/kg zinc. The gerbils had access to water with undetectable levels of zinc and copper levels of 0.45 mg/L. Blood was collected as a terminal procedure via cardiac puncture under pentobarbital anesthesia.

The results showed that for all age groups, males had lower plasma copper levels than females. The plasma copper levels did not vary with age for females whereas males at 180 days of age had lower levels than males at 90 and 360 days. Plasma zinc concentrations did not vary significantly between genders or age groups. A slight decrease in plasma zinc levels was noted with age.

Previously, mice, rabbits and most commonly rats, have been used to study copper and zinc metabolism. Without knowing the diets that were fed in those studies, it is difficult to determine how these gerbil values compare to data obtained in the rat. The male plasma copper levels were similar to values obtained in male and female rats in prior publications. The female plasma copper levels and male and female plasma zinc levels were higher in gerbils than those reported in rats in prior studies.

The authors conclude that the gerbil may prove to be an appropriate model for the study of trace element metabolism.

Questions:
1. What is the genus and species of the Mongolian gerbil?
2. Name 2 areas of research in which the gerbil is commonly used.
3. True or False, gerbils have a relatively long erythrocyte life span.
4. True or False, the plasma copper levels of male gerbils are similar to those reported in male and female rats

Answers:
1. Meriones unguiculates
2. Epileptiform seizures (gerbils are susceptible to spontaneous epileptiform seizures - this is believed to be a good model for human idiopathic epilepsy); cholesterol metabolism (gerbils develop high serum and hepatic cholesterol levels even when they are fed a diet low in fat); diabetes; hyperadrenocorticism, obesity and periodontal disease are other areas of research that utilize gerbils
3. False, gerbils have a relatively short erythrocyte life span. The half-life of gerbil erythrocytes is approximately 10 days, this is evidenced in blood smears as they contain large numbers of reticulocytes and basophilic erythrocytes.
4. True, however, it is difficult to compare the values reported in this article with those previously published for rats as the diet and water intake of the animals may not have been similar.

Cerebrospinal fluid centesis at the cerebellomedullary cistern of kittens. Contemporary Topics 41 (5): 30.
The purpose of this report is to describe a survival technique for obtaining cerebrospinal fluid from young kittens. The technique had been previously described in rats, and has been modified for use in kittens between 2-18 weeks of age.
Materials and Methods: 8 kittens (Felis catus or Felis domesticus) were anesthetized with isoflurane supplied by face mask. The face mask was adapted from a syringe case in order to allow for sufficient flexion of the neck when positioning the animals for CSF collection. A rodent-sized induction chamber was used as a positioning platform. Kittens were placed in sternal recumbency on the top of the chamber. The front limbs were pulled caudally and the head was lowered over the edge of the chamber to approximately a 90-degree angle to allow penetration of the collection needle into the cerebromedullary cistern. A micromanipulator with a 23-ga 3/4 inch butterfly needle (wings removed) was attached to a vacutainer blood collection set. The needle was positioned at the dorsal midline over the atlanto-occipital joint. The angle of entry was close to the angle of the longitudinal axis of the head. The manipulator was then lowered through tissue until CSF was visible in the collection tubing (approximately 1.0 to 1.5 cm in depth). CSF was allowed to fill the tubing under its own pressure. When the flow of CSF stopped, the needle was removed. After CSF collection, the isoflurane was stopped and kittens were maintained on 100% oxygen until they were responsive to noxious stimuli.
Results: Volumes of CSF collected ranged from 0.1-1.0 ml (average 0.4-0.5 ml). The entire procedure usually required 20-25 minutes. Blood contamination of CSF samples occurred in 4 of 33 samples. One kitten developed suppurative meningoencephalitis and was euthanized. A group G Streptococcus was cultured.
Discussion: The authors recommend no more than 3 attempts at repositioning the needle for centesis because of the likelihood of blood contamination. A surgical plane of anesthesia was required for the procedure in order to prevent even slight movement. Recovery from anesthesia was within 1 minute of cessation of isoflurane.
No questions

Pathology of a mouse model of X-linked chronic granulomatous disease. Contemporary Topics 41 (5): 33.
The purpose of this study was to document the spontaneous diseases present in these mice which are a murine model of X-linked Chronic Granulomatous Disease (CGD) and to compare these lesions to those of CGD in humans. CGD is characterized by the development of severe recurrent bacterial and fungal infections of the subcutaneous tissues, lymph nodes, lungs, liver, and bones. Because of their inability to generate hydrogen peroxide, hypochlorous acid, and chloramines and the lack of superoxide production by NADPH oxidase, phagocytic cells in patients with CGD are severely compromised in their ability to kill microorganisms. CGD patients are particularly susceptible to infections caused by catalase-containing organisms like S.aureus, A. fumigatus, and other fungi; gram-negative enteric bacilli like Seratia marcescens and Ps. cepacia; and Salmonella. Enterococci are the third most common organism isolated in nosocomial bacteremias in immunocompromised patients, in whom these bacteria cause endocarditis and urinary tract infections. Chronic granulomatous disease (CGD) in humans is caused by mutations in one of the five subunits of NADPH oxidase, which is a component of the superoxide generation system. NADPH oxidase contains two heme groups as well as flavin adenine nucleotide (FAD) and is responsible for transferringelectrons from molecular oxygen to superoxide in a process known as the respiratory burst. The a subunit (p22-phox) and subunit (gp91-phox) together form cytochrome b 558, which is an integral membrane component of the phagosome. This enzyme remains dormant until activated by the binding and engulfing of opsonized microorganisms. The other three components of NADPH are cytosolic proteins that translocate to the phagosome and bind the and components to activate the enzyme complex. The gp91-phox gene CYBB is found on the X chromosome, and defects in this gene account for the most common type of human CGD, whereas defects in the other NADPH subunit genes are transmitted as autosomal recessive traits.

Recently two knockout mouse models of CGD have been created. The p47-phox -/- mouse (gene designation, NCF-1) develops severe spontaneous bacterial and fungal infections similar to those developed by human CGD patients. The other model, the gp91-phox -/- mouse gene designation, Cybb tm1, is susceptible to experimentally induced Aspergillus pneumonia, but there is no report of spontaneous infections in these mice. This colony of knockout mice (Cybb tm1) has been maintained at the Medical University of South Carolina for 5 years. These mice are lacking the ß subunit of NADPH oxidase and are susceptible to experimental infection with Aspergillus fumigatus. In this study, all 72 necropsied mice had an acidophilic macrophage pneumonia, and 16 also had lobar suppurative and necrotizing pneumonias. Of the 72 animals, 36 had severe bacterial suppurative and necrotizing to pyogranulomatous pneumonias, 13 mice had a necrotizing and suppurative adenitis of the cervical lymph nodes, 30 had splenomegaly, and 11 had lymphadenopathy. The array of spontaneously occurring infectious diseases and lesions found in these mice is similar to that of human patients with CGD.

Questions:
1) CGD in humans is caused by?
a. A mutation of the NADPH oxidase enzyme of phagocytic cells.
b. Mycobacterial infections
c. Antigen stimulation
2) The CGD immune deficiency is caused by?
a. A failure of the phagocytic cells to produce superoxidase.
b. Is an X-Linked deficiency.
c. An inability to kill microorganisms.
d. All of the above.
3) True or False? CGD is characterized by sever bacterial and fungal infections?
4) True or False? CGD patients are very susceptible to catalase-containing organisms.
5) Spontaneous infections in X-Linked knockout mice (Cybb tm1) have?
a. Similar spontaneous infections as humans with CGD.
b. Are very susceptible to Aspergillus pneumonia.
c. Are immunocompromised.
d. All of the above.

Answers:
1) a 2) d 3) T 4) T 5) d

Multiple congenital genitourinary anomalies in a polled goat. Contemporary Topics 41 (5): 39.
The domestic goat (Capra Hircus) is mainly utilized in biomedical research for antisera production, reproductive studies, investigation of the pathogenesis of lentivirus infections, as a spontaneous animal model for congenital hypothyroidism, neuritic plaques, pulmonary adenomatosis, amelia, glomerulonephritis.
CASE HISTORY:
1. Signalment/History: - 1-day-old goat
- parents: polled Toggenburg dam and polled Nubian/Toggenburg sire
- doe completed a normal gestation period, surviving twin apparent normal male
2. Clinical Exam: - dysuria
- a large bladder-like mass extending from the ventral perineal midline
3. Differential Diagnosis: - cutaneous cyst
- ectopic urinary bladder
- urethral dilatation or diverticulum
4. Necropsy: - good nutritional status
- preputial ambiguity with opening located intermediately between that expected of a male or female
- bilateral cryptorchidism with scrotal absence - testicles found in a short inguinal canal confined within the subcutis
- segmental urethral hypoplasia - lack of urethral process, tiny external urethral orifice
- urethral dilatation/diverticula - one 5-cm, vascularized, bladder-like mass at ventral perineal midline and one 1-cm similar structure adjacent to prepuce, both separated by constricting band of fibrous tissue and confluent with pelvic urethra
5. Etiology: - presumptive etiology = intersex condition in association with polled breeds
- not confirmed, but least likely etiology = freemartinism
SUMMARY OF DISCUSSION:
Developmental anomalies result from genotypic, environmental influences, or a combination of both. Congenital or hereditary abnormalities associated with the distal urinary tract and/or reproductive tract occur infrequently in all animal species. These include in goats bipartite scrotum and cryptorchidism in Angora goats, testicular hypoplasia, sperm granulomas, and intersex. The term "intersex" refers to gender inconsistency, has been defined as "ambiguity in the structure of the gonads, reproductive tract, or external genitals", and is strongly associated with the polled condition in goat breeds of western European descent. The vast majority of reported cases of the intersex syndrome were genetically female (60.XX) with mixed male phenotype.
The interaction between hornlessness and intersex is termed Polled Intersex Syndrome (PIS) and believed to be caused by an interference with the development of female characteristics by aberrant _expression of male-promoting gene products. Phenotypically, the mutation (P) expresses polledness in an autosomal dominant fashion and results in sex-reversal in females as an autosomal recessive trait. Females that are homozygous for the polled gene (PP) will become infertile intersex.
Intersex secondary to freemartinism involves the sharing of cells and hormones between a male and female fetus following placental fusion and anastomosis, resulting in chimerism in the female twin. However, freemartinism in goats has a low prevalence.
Tests to differentiate between the two conditions include karyotype ananlysis performed from whole blood or cultured renal or pericardial fibroblasts, PCR, or Southern blot.
The relationship between the lack of horns and the PIS has to be considered in the reproductive management of goats for agricultural purposes. However, breeding schemes can be designed in biomedical research settings to produce PIS animals for the utilization as a model of the human XX-male intersex condition.

Questions:
1. Which of the following statements regarding Caprine Arthritis Encephalitis (CAE) is wrong?
a. The CAE virus is an enveloped, single stranded RNA lentivirus in the family Retroviridae.
b. Because of their natural affinity to the CAE virus, goats are suitable for the investigation of the pathogenesis of lentivirus infections in biomedical research.
c. Prevalence of infection decreases with age.
d. The CAE infection manifests clinically as polyarthritis in adult goats and less commonly as progressive paresis (leukoencephalomyelitis) in kids.
e. Clinical signs of CAE can also include subclinical or clinical interstitial pneumonia, indurative mastitis, and chronic wasting.
2. What is the diploid chromosome number in goats?
a. 60
b. 54
c. 58
d. 64
3. The gene for the polled condition in goats:
a. Is associated with high fertility when in the heterozygous state.
b. Expresses polledness in an autosomal dominant fashion and results in sex-reversal in females as an autosomal recessive trait.
c. Induces an infertile intersex condition in females that are heterozygous for the polled gene (Pp).
d. Is not associated with infertility.
4. Placental fusion, resulting in Freemartinism, is most prevalent in what species?
a. Goat
b. Sheep
c. Swine
d. Cattle
5. The following tests can be utilized to differentiate between PIS and Freemartinism, except:
a. Western blot
b. Southern blot
c. PCR
d. Karyotype analysis on lymphocytes or cultured renal or pericardial fibroblasts.
6. How do you discern if a goat is polled or disbudded as a kid?
a. There is no possibility to distinguish this in an older animal.
b. There is a central whorl of hair on the dorsal cranium.
c. There are at least two whorls on the dorsal cranium.
d. Polled goats are always black.

Answers:
1 c, 2 a, 3 b, 4 d, 5 a, 6 b (note: two whorls of hair overlying the area of horn growth present in disbudded kids)

Virucidal effects of rodent cage-cleaning practices on the viability of adenovirus vectors. Contemporary Topics 41 (5): 43.
Purpose - To determine whether available rodent cage-cleaning solutions produced virucidal effects for use in lieu of or prior to autoclaving the cages.
Materials and Methods
Experiment 1:The effects of the cleaning solutions alone on Human S8 cells was tested. S8 cells are derived from the human adenocarcinoma cell line A549 and express the Ad5 E1 genes and the E2a gene. Cleaning solutions used were CK-100 (Cage-Klenz 100), a liquid phosphate-free alkaline detergent; CK-220, a hydroxyacetic acid-based phosphate-free detergent solution; and Process NPD a phosphate-free cleaner/disinfectant consisting of four quaternary ammonium compounds in a compatible detergent solution. Cleaning solutions were diluted to the recommended use concentrations in Dulbecco's phosphate buffered saline (DPBS) and tested at the recommended use concentration and at 10-fold serial dilutions in DPBS to 10 -6 of the use concentration. The negative control was the S8 medium only. Results: No effect was seen on the S8 cells exposed to CK-100 or CK-220 at any dilution. NPD at use concentration and at a 10-fold lower concentration was toxic to the cells, as evidenced by a change in the medium color and loss of cells. For vector studies, NPD was used at a 100-fold dilution of the recommended use concentration was nontoxic (as determined by lack of CPE) to S8 cells for as long as 14 days in culture.
Experiment 2: Av3GFP vector was exposed to four different environmental conditions that simulate current rodent cage washing procedures: HEAT (74C for 6 min); HEAT +CK-100; HEAT +CK-220; and HEAT + NPD. The vector was diluted with S8 medium and diluted cleaning solutions to concentrations ranging from 1.24 × 10 3 to 1.24 × 10 8 vector particles/20 µL then placed into 96-well plates preseeded with S8 cells. The positive control was 1.24 × 10 3 vector particles of unheated vector/well and the negative control was S8 medium only. Cells were maintained at 37C with 5% CO 2 for 11 days and were observed for cytopathic effect (CPE), characterized by loss of cells from the confluent monolayer, and for GFP fluorescence. Results: HEAT-treated vector, no viral activity was seen at the concentration of 1.24 × 10 6 vector particles/well. For vector treated with HEAT +CK-100, no viral activity was seen at ~10 7 vector particles/well. For vector treated with HEAT +CK-220, no viral activity was detected at ~10 5 vector particles/well, and for vector treated with HEAT +NPD, no viral activity was seen at ~10 6 vector particles/well. Experiment 3: CK-100 was selected for further analysis at six conditions: MEDIUM alone, no heat (negative control); VIRUS alone, no heat (positive control); VIRUS +HEAT; CK-100 +HEAT; CK-100 +VIRUS, no heat; and CK-100 +VIRUS +HEAT. The Av3GFP vector was diluted in S8 medium and diluted CK 100 to concentrations of 10 8 to 10 -3 vector particles/100 µL,then placed in separate 96-well plates preseeded with S8 cells. Cells were maintained at 37C with 5% CO 2 for 11 days and were observed for CPE and GFP fluorescence.
Results: Infectivity of the Av3GFP vector was completely eliminated by heat alone, or by the combination of heat and CK-100 at all vector concentrations, resulting in greater than an 11-log reduction of viral infectivity. CK-100 alone possessed no virucidal activity.
Conclusion: Based on the results of this study, the authors recommend that cage-washing include dumping the contaminated bedding into a HEPA-filtered waste disposal system and autoclaving the bags of bedding before disposal, then cleaning the cages in the rack washer at wash temperatures of 74°C (165°F) and rinse temperatures of 82°C (180°F).

Questions:
1. Human adenoviruses and adenoviral vectors
a. are Risk Group 2 agents and require BSL2 containment and practices.
b. can be transmitted to other animals and/or personnel.
c. are shed in human and animal urine and feces.
d. All of the Above
e. None of the above
2. When handling BSL2 agents,
a. cage-wash staff are required to wear appropriate personnel protective equipment.
b. PPE is not required, but proper handwashing procedures should be followed.
c. Proper PPE includes street clothes covered by a Tyvek suit, hair covering, dust mask, shoes covers, and gloves.
d. Contaminated bedding should be bagged and autoclaved.
e. A and D are correct
f. B and C are correct
3. Autoclaving soiled polycarbonate rodent cages is not desirable because
a. Autoclaving is not necessary to eliminate adenoviral transmission.
b. Autoclaving softens and degrades the polycarbonate rodent cages and the ammonia present in the urine, bedding, and fecal material left in the dirty cages can adversely affect the cage.
c. The combination of heating and a liquid, phosphate-free alkaline detergent produced the same reduction in viral /vector infectivity.
d. All of the Above
e. None of the above
T or F
1. To determine the effectiveness of virucides, the Environmental Protection Agency will not accept data developed by virological techniques which only simulate the conditions under which the product is intended for use.
2. Viral shedding occurs in humans and laboratory animals in urine, feces, conjunctiva, and in bronchial secretion.
3. Virus shedding has been detected by qualitative polymerase-chain reaction (PCR)-based methods.
4. Heating an Av3GFP vector to a temperature of 74C (165F) for 6 min results in greater than an 11-log reduction in infectivity of the vector.
5. Autoclaving cages diminishes the stability and integrity of the polycarbonate cages without providing a further reduction in the risk of virus or vector transmission.
6. Av3GFP is a replication-defective adenoviral vector that expresses the green fluorescent protein (GFP) and is easily detected in culture within 24 to 48 h.

Answers:
Question 1-d; 2- e, 3-d. T/F - 1-false, 2 - 6 True

Nonhuman primate cage modifications for environmental enrichment. Contemporary Topics 41 (5): 47.
This article describes modifications to nonhuman primate caging to allow for group housing/socialization enrichment for NHP. Standard "two over two" cage design with four removable, stainless steel cages hanging from sides within a castor-mounted frame with squeeze back, side-opening door at the front, and a guillotine door within were modified to add perches and communication tunnels that could be opened/closed. Previous perches had to be removed in order to use the squeeze back, and previous tunnels were either too light (easily damaged) or too heavy (lifting problems). The described modifications were: semipermanent, did not require removal to use the squeeze back and could go through the cage washer. The perches, made of solid Delrin (acetyl homopolymer) rods were attached to a plate on the squeeze back. Slits were made in the ends of the perch rods corresponding to bars on the door to allow for complete closure of the squeeze back. Specific measurements and methods are listed. The Delrin polymer was chosen for its ability to withstand cagewash and durability; the authors state that other materials could be suitable. Connection tunnels between cages were created by cutting holes in the sides of adjacent cages (18% of the area of the cage side). These holes were placed at the front of the cage to allow easy open/close access for the staff and to allow less dominant animals to back out of the line of sight through the opening. The tunnel itself was a U-shaped piece of 14 gauge metal with a series of panels (one of which was removable upward sliding door, secured with a knurled set screw to be held open or secured closed from the outside) either bolted to the waste pan or "tack-welded" to the pan support. The sliding door can be replaced with a perforated sliding door to allow some access to temporarily separated animals. Animals in this housing (female, wild-caught, cynomologus monkeys) have been pair-housed for more than 18 months with no signs of aggression. This is speculated to be due to a combination of factors, including species, age, sex, and the request that the animals form housing pairs while still housed with the importer. The purpose of the article is the hope that principles outlined will allow others to make cost-effective changes to their caging which will enhance the well-being of the housed animals.

Questions:
Q1: What were previous problems with perches and external, hanging tunnels?
Q2: What was the rationale for the location & size of the tunnel hole?
Q3: What factors were speculated to contribute to the compatibility of paired animals?

Answers:
A1: required removal of perches to use squeeze back cage; tunnels were either lightweight making them prone to damage or too heavy to be lifted/manipulated easily.
A2: to allow the staff to easily open/close the cages and to allow the non-dominant animals to be able to remove themselves from the line of sight from the adjacent cage.
A3: species, sex, age and pre-pairing prior to introduction to the paired housing.

Flow cytometry. Contemporary Topics 41 (5): 50.
This article gives a brief schematic summary of the basics of flow cytometry as described in the referenced 1999 Radcliff and Jaroszeski article. I reviewed this article and outlined another brief reference from the LSU vetmed website (www.vetmed.lsu.edu/facs/fundamentals.htm). The LSU article is included.

Summary

Flow Cytometry uses laser technology and fluorescent labeled antibodies to examine the phenotypic and morphologic characteristics of individual cells. It can be used in nucleic acid analysis, cell function assays, and cell sorting and isolation. It can be performed on mammalian lymphocytes and other cellular elements, chromosomes, tumor cells, bacteria and fungi.

I. Basic Components of Flow Cytometry.
· Laser light source,
· Fluidic sample chamber and optical assembly
· Photodetectors and processors to convert light signals into digital signals
· Computer interface for the storage and analysis of digitized listmode data

II. Basic Flow Cytometry Process
· Individual cell membranes or intracellular constituents are label with fluorescent markers.
· Up to five or more fluorochrome conjugated antibodies are used simultaneously.
· Cells pass single file through a fluidic stream (isotonic saline).
· Individual cells intersect a laser beam.
· Scattered visible light is emitted, and fluorescence is detected.
· Two types of light scatter
· Forward angle light scatter- light scattered at very small angles (3-12<sup>o), a function of cell size, detected with a photodiode
· Right angle or side scatter -light scattered at larger angles (90o), proportional to the magnitude of cellular granularity or internal complexity, detected with a photomutliplier tube (PMT)
· Fluorescence emission - detected 90o to the incident laser beam with a PMT
· PMTs - allow conversion of photons to electrical impulses
· Subsequent signals are amplified, evaluated, digitized and fed to a pulse height analyzer (PHA) for storage.
· Resulting listmode data are histograms or dot plot displays with the computer interface.

III. Analysis of Physical and Chemical Properties of Cells
· Measurable Characteristics - cell size, cell shape, surface membrane receptors, DNA content, nuclear antigens, intracellular Ca2+, intracellular pH, and gene _expression.
· Cell populations - at rates of 1000 to 30,000 cells per second
· Cell Sorting - Identification and subsequent characterization of a subpopulation of cells within heterogeneous populations

IV. Advantages
· Id individual cell morphology and phenotype.
· Real time analysis and multiple, simultaneous analysis.
· Rapid - analysis of (10-6 cells in less than a minute
· FC vs fluorescence microscopy - large sampling statistics, precision, and sensitivity.
· FC vs RIA and ELIZA methodologies- CF can detect rare cells and select specific subsets within a mixed population.
.
V. Disadvantages
· Complex and expensive instrumentation
· Labor intensive.
· Requires trained personnel
· False positives due to overlapping emission wavelengths of fluorochromes.

VI. Alternative Methods
· Nucleic acid based techniques (Southernblot, PCR),
· Immunohistochemistry,
· Laser microdissection of individual cells,
· Western blot

VII. Three fluorochrome-conjugated antibodies
· fluorescine isothyocyanate (FITC)
· phycoerythrin (PE)
· allophycocyanin (APC)

Questions
Describe the two types of light scatter that occur in FC.
Name three fluorochrome antibodies used in FC.
What are the advantage of FC over fluorescence microscopy, RIA and ELIZA?
What physical and chemical properties of cells can be analyzed using FC?

Answers
1. Forward angle light scatter- light scattered at very small angles (3-12o), a function of cell size, detected with a photodiode
Right angle or side scatter -light scattered at larger angles (90o), proportional to the magnitude of cellular granularity or internal complexity, detected with a photomutliplier tube (PMT)

2. fluorescine isothyocyanate (FITC)
phycoerythrin (PE)
allophycocyanin (APC)

3. FC vs fluorescence microscopy - large sampling statistics, precision, and sensitivity
FC vs RIA and ELIZA methodologies- CF can detect rare cells and select specific subsets within a mixed population.

4. Measurable Characteristics - cell size, cell shape, surface membrane receptors, DNA content, nuclear antigens,intracellular Ca2+, intracellular pH, and gene _expression.
Cell populations - at rates of 1000 to 30,000 cells per second
Cell Sorting - Identification and subsequent characterization of a subpopulation of cells within heterogeneous populations

Fundamentals

What is Flow Cytometry?
Flow cytometry is a process in which individual cells or biological particles are labeled with fluorescent markers and pass in single file through a fluidic stream. While in the fluidic stream the cells are hit by a laser beam resulting in emitted scattered visible light and fluorescence detection. Physical and chemical properties of cells or particles are subsequently analyzed. Flow cytometric analysis has been performed on mammalian lymphocytes and other cellular elements, chromosomes, tumor cells, bacteria and fungi. What applications are performed using a flow cytometer?
Chemical and physical characteristics of cells which can be measured by flow cytometry include cell size, cell shape, surface membrane receptors, DNA content, nuclear antigens, intracellular Ca2+, intracellular pH, and gene _expression. Flow cytometers can analyze a population of cells, at rates of 1000 to 30,000 cells per second depending on the type of flow cytometer. One powerful application of flow cytometry requires identification and subsequent characterization of a subpopulation of cells within heterogeneous populations. This is referred to as cell sorting. In comparison to other technologies such as fluorescence microscopy, flow cytometry offers many advantages such as large sampling statistics, precision, and sensitivity. In comparison to RIA and ELIZA methodologies, flow cytometry has the ability to detect rare cells and select specific subsets within a mixed population.
What are the components of a flow cytometer?
The basic components of a flow cytometer/cell sorter are a laser light source, a fluidic sample chamber and optical assembly, and photodetectors and processors to convert light signals into subsequent digital signals. The instrument is interfaced with a computer system for the storage and analysis of digitized listmode data.
What parameters are measured with flow cytometry?
Cells pass in a single file into the center of a flow cell or nozzle where they are surrounded by sheath fluid which is typically isotonic saline. As the cells pass through the stream they are hit by the laser beam. Some light is scattered at very small angles (3-12o) and this is referred to as forward angle light scatter, which is a function of cell size and is detected with a photodiode. Light which is scattered at larger angles (90o), is proportional to the magnitude of cellular granularity or internal complexity, and this is referred to as right angle or side scatter and is detected with a photomutliplier tube (PMT). If the cells are stained with particular fluorescent marker(s), fluorescence emission is also detected 90o to the incident laser beam with a PMT. The PMTs allow conversion of photons to electrical impulses. Subsequently, signals are amplified and evaluated! , ! digitized and fed to a pulse height analyzer (PHA) for storage. The listmode data is illustrated in the form of histograms or dot plot displays with the computer interface.</sup>
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