Journal of the American Association for Laboratory Animal Science
Volume 47, Number 1, January 2008

ORIGINAL RESEARCH
Biology
Williams et al.  Ultrasonic Sound as an Indicator of Acute Pain in Laboratory Mice, pp. 8-10

Species:  Primary, mouse

SUMMARY: This study evaluated whether or not the measurement of either audible or ultrasonic vocalization was a reliable tool for assessing the level of pain or discomfort experienced by mice.  Mice frequently vocalize in the ultrasonic range (i.e. above the range of human hearing), thus raising questions that mice experiencing distress may be vocalizing to indicate their distress, which remains unrecognized by the people handling the animals.  The mice were divided into four groups:  those experiencing a tail snip, those experiencing an ear notch, or mice experiencing sham procedures for both of these groups.  None of the mice were anesthetized or given analgesics.  Mice were monitored for audible vocalizations, and also evaluated with an ultrasonic recording system.  65% of the mice undergoing presumably painful procedures did not produce either an audible or an ultrasonic sound during the procedure.  More mice did emit both audible and ultrasonic vocalizations during the tail snip or ear notch procedures than mice undergoing sham procedures, but at a level similar to mice just undergoing routine handling.  In all cases, ultrasonic vocalizations were accompanied by audible noises as well.  Previous studies with mice have shown that mice use ultrasonic vocalizations during social settings or as mouse pups during times of chill or distress, as well as during either anticipation or avoidance of a painful stimulus.  Based on this study, vocalization was not a useful tool for evaluating pain or distress in mice.  The authors had two theories for this:  either mice do not reliably vocalize in response to pain or distress, or these procedures were somehow not painful to mice.  

As a final point, the authors suggest that vocalization may be an indicator of pain or distress, but the absence of vocalizations does not necessarily indicate that the mice are pain or distress-free.

QUESTIONS:
1.	Ultrasonic hearing is above which frequency?
a.	10kHz
b.	20kHz
c.	40kHz
d.	80kHz
2.	T or F.  Female mice emit ultrasonic noises in the presence of males

ANSWERS:
1.	B
2.	F (but males do in the presence of females or their pheromones)

Buitrago et al.  Safety and Efficacy of Various Combinations of Injectable Anesthetics in BALB/c Mice, pp. 11-17

Task 2- Prevent, Alleviate, and Minimize Pain and Distress; K6-Practical applications of anesthesiology
Species: Mouse (Mus musculus)-Primary
 
Objective: To compare the safety and efficacy of three anesthetic+sedative/analgesic combination protocols in BALB/c mice to the commonly used Ketamine-xylazine anesthetic protocol.
 
Anesthetic protocols:
Ketamine-xylazine (KX)
Ketamine-xylazine-ace (KXA)
Ketamine-xylazine-buprenorphine (KXB)
Ketamine-xylazine-carprofen. (KXC)

Parameters:
Induction time
Anesthetic duration
Blood pressure
Pulse rate
Recovery time

Procedure: 4 random groups were selected and identified. All agents were diluted in sterile saline to provide a final dose of 0.1 ml/10g body weight and administered IP. *Carprofen was administered at 4mk/kg SC. The KX group served as a control.

Hemodynamic parameters: measured indirect blood pressure and pulse rate using a noninvasive blood-pressure analysis system.

Anesthetic induction and monitoring: variables recorded=induction time, time to loss of righting reflex, indirect systolic and diastolic arterial blood pressure, pulse rate, time to start of anesthesia (failure of response induction with tactile stimulus)

Assessment of anesthetic depth: vibrissae were touched with a sterile forceps. Response to was graded 1-4. Pedal withdrawal reflex assessed by grasping of the interdigital web with a modified forceps (standardized pressure).

Postanesthetic care and monitoring: 1 mL of 0.9% saline was administered SC once mice demonstrated spontaneous movement. Mice were monitored until fully ambulatory.

Results: KXA-produced stable physiologic parameters and longest duration (40+/-8)
All other combinations produced immobility; surgical plane of anesthesia not confirmed.
All produced significant hypotension without death.

KXA was found to be safe and reliable. Mice were ambulatory within 2 hours. KXA produced significant cardiovascular effects (low pulse rates and hypotension) Most likely due to acepromazine potentiating these effects.

KX being a commonly used anesthetic protocol, did not produce anesthesia alone or in combination with either analgesic. A 40% mortality rate was noted when attempts were made to increase the dose of KX to reach surgical plane of anesthesia. KX was found to provide inadequate anesthesia.

Neither KXC or KXB induced or prolonged anesthesia when compared to KX. Carprofen provided preemptive analgesia, but will not increase the depth of KX anesthesia.

Addition of Buprenorphine did not deepen or prolong the anesthesia induced but did increase cardiovascular depression.
 
Discussion: This study also provided an opportunity to use blood pressure analysis equipment not previously evaluated in anesthetized mice. (BP-2000, Visitech Systems, Apex, NC)

Techniques for measuring blood pressure in experimental animals can be divided into direct and indirect methods. In animals, the most common indirect method for monitoring blood pressure is the cuff technique.
 
Conclusions: The drug combinations tested suggests that KX, KXB, and KXC at the doses tested were not suitable anesthetics for major surgery in BALB/c mice. KXA was found to be efficacious in providing long, safe, and satisfactory anesthesia for performing surgical procedures in BALB/c mice.
 
QUESTIONS:
1.	Define balanced anesthesia:
2.	List a few advantages of balanced anesthesia:
3.	Classify the following drugs (i.e. carprofen=NSAID):
 	a.	Acepromazine
	b.	Xylazine
	c.	Ketamine
	d.	Buprenorphine
 
ANSWERS:
1.	Balanced anesthesia is the administration of a mixture of sedatives, analgesics, and anesthetics to produce anesthesia by using lower doses of each component than if each component were to be used by itself.
2.	Smooth induction, increased safety margin, and when analgesics are used, minimization of pain during the immediate postoperative period.
3.	a. 	Acepromazine = a phenothiazine tranquilizer
	b. 	Xylazine= Alpha2-adrenergic agonist
	c. 	Ketamine = a dissociative general anesthetic
	d. 	Buprenorphine= Partial opiate agonist

Suckow et al.  Field Trial of a Pasteurella multocida Extract Vaccine in Rabbits, pp. 18-21

Task 1 - Prevent, Diagnose, Control and Treat Disease; K9 - Preventative Medicine
Species: primary, Rabbit

SUMMARY: Pasteurella multocida is a primary bacterial pathogen that causes significant disease in rabbit colonies and which has a major impact on research activities.  Infection can be subclinical or latent and when disease occurs it can lead to disease severe enough to require euthanasia of affected rabbits.  Transmission occurs via direct contact of susceptible rabbits to carrier rabbits.  Airborne transmission is not efficient and requires an exposure period of greater than 3 weeks.  The likely site of initial infection is the upper respiratory tract.  P. multocida can be cultured on tryptic soy agar containing 5% sheep blood and can be identified as discrete, grayish, round mucoid colonies that contain small, gram-negative coccobacilli.

A potassium thiocyanate extract (PTE) produced from P. multocida serotype D: 3, 12, and 15 was used to vaccinate Pasteurella-free rabbits prior to their introduction into a rabbit colony having an endemic P. multocida serotype A: 3 infection.  PTE is a lysate that contains a mixture of soluble cellular components and is presumed to contain bacterial virulence factors.  The goal of the study was to determine if a heterologous P. multocida vaccine under field conditions could confer protection against P. multocida infection.  Two groups of 15 female New Zealand White rabbits were vaccinated either SC (subcutaneously) or IN (intranasally) with PTE once a week for 3 weeks.  A control group of 15 rabbits was sham-vaccinated IN with saline.  Serum samples were collected at 0, 5, and 24 weeks post-vaccination and evaluated by ELISA for anti-PTE IgG.  Anti-PTE serum IgG developed in the IN and SC PTE-vaccinated rabbits but not in the IN saline control rabbits.  Rabbits vaccinated SC with PTE developed significantly greater serum All rabbits were housed individually and did not have any direct physical contact.  Test rabbits were distributed throughout the colony with cages containing colony rabbits above, below, and on each side of them.  IgG activity than IN vaccinated or control groups.  Bacterial cultures of the nasopharynx at necropsy of the three groups of vaccinate was positive for 66% (10/15) of IN saline controls, 33% (5/15) of IN PTE vaccinates, and 6% (1/15) SC PTE vaccinates.

Conclusion:  PTE can stimulate protective immunity to a heterologous strain of P. multocida, with SC vaccination providing superior protection to IN vaccination.

QUESTIONS:
1. 	Which statement below correctly states the results of a study that vaccinated New Zealand White rabbits with a PTE based Pasteurella multocida vaccine:
a. 	Rabbits vaccinated with PTE either intranasally or subcutaneously developed significant levels of serum IgG and were equally protected from infection with P. multocida
b.	PTE vaccinated rabbits were not protected against a heterologous strain of P. multocida
c. 	Rabbits vaccinated with PTE vaccine subcutaneously developed significantly greater serum IgG antibody levels than either the IN PTE vaccinates or IN saline controls but were not protected from heterologous challenge with P. multocida
d. 	Subcutaneous vaccination of rabbits with PTE conferred the greatest level of protection against heterologous challenge with P. multocida when compared to rabbits vaccinated IN with PTE or sham-vaccinated IN with saline
2. 	PTE is an acronym for ____________  derived from   ___________ (name the bacteria).
3. 	T/F:  P. multocida is readily transmitted through direct contact or via airborne transmission.

ANSWERS:
1.  	d
2.  	Potassium thiocyanate extract; Pasteurella multocida
3.  	False, direct contact is efficient; however, airborne transmission requires exposure periods of greater than 3 weeks.

Sleeper et al.  Echocardiographic Reference Ranges for Sedated Healthy Cynomolgus Monkeys (Macaca fascicularis), pp. 22-25

Task 1: Prevent, control, diagnose and treat disease;   
Task 3: Provide Research Support, Information, and Services
Primary Species: Macaque

SUMMARY:  Echocardiographic evaluation was performed on 118 female and 119 male cynomolgus monkeys >2 years old under ketamine or tiletamine/zolazapam anesthesia.  Gender-specific reference ranges were developed for echocardiographic parameters (left ventricular size, left atrial diameter, aortic diameter, etc.).  After normalization for body weight, data was analyzed for gender- and sedation-associated differences.  Sedation-associated differences should be viewed with caution since animals anesthetized with ketamine were evaluated at a different facility with different personnel and equipment than animals anesthetized with telazol.

QUESTIONS:
1.   Which would be best evaluated with a Doppler echocardiogram?
a.	Movement of cardiac valves
b.	Direction and velocity of blood flow
c.	Thickness of the ventricular wall
d.	Both a and b
e.	All of the above
2.    Which are benefits of cardiac ultrasonography?
a.	It is non-invasive
b.	It allows cardiac functional evaluation 
c.	It is quick and easy for lab personnel to learn
d.	Both a and b
e.	All of the above

ANSWERS
1.  	b
2.  	d

Colony Management
Kastenmayer et al.  Eradication of Murine Norovirus from a Mouse Barrier Facility, pp. 26-30

Task 1 - Prevent , Diagnose, Control and Treat Disease
Species: primary, Mice

SUMMARY: Murine norovirus (MNV) is a non-enveloped single-stranded RNA virus of the Caliciviridae family and is related to human Norwalk-like virus. It was first described in mice that were deficient in Jak-STAT1 innate immunity pathway and they developed encephalitis, meningitis, cerebral vasculitis, focal interstitial pneumonia, peritonitis, pleuritis and hepatitis. It is spread by fecal-oral route. 

MNV is the most prevalent viral pathogen identified in lab animal facilities. The full effect of the infection on research is not known. This paper describes two eradication methods in a barrier facility where research in immunodeficient mice was being conducted. Discovery of the virus in the facility was made by sentinel testing of NIH Swiss Webster sentinels exposed weekly for 18 wks to dirty bedding from cages containing experimental mice. Each cage contained 3 mice and a new sentinel mouse was placed in the cage when the oldest was removed for testing. 
 
The source of the MNV infection was identified by mapping positives cage locations in two rooms. A common source was identified as a noncommercial contract facility that supplied limited numbers of mice to investigators. All shipments were halted from this facility. Room A was depopulated and decontaminated after initial quarantine procedures had been put in place. Room B, was quarantined, and the infection thought to be limited. Identification and removal of infected mice was performed in Room B. The method initially appeared effective but ultimately resulted in recurrence and was attributed to an environmental nidus of infection. This showed that this method of eradication was ineffective. It also required 6 months of quarantine plus expensive and extensive testing for the virus as well as proper coordination between the veterinarian and the technicians to identify positive mice. Testing procedures utilized include serology via microsphere-based serologic multiplexed fluorescent immunoassays and RT-PCR of fresh feces from mice. 

QUESTIONS:
1.	Murine Norovirus is an RNA virus and a member of which virus family?
a.	Caliciviridae
b.	Paramyxoviridae
c.	Parvoviridae
d.	Coronaviridae
2.	The best mechanism of eradication for MNV is to depopulate and decontaminate the infected room, T or F.
3. 	MNV is transmitted primarily by:
a. 	Aerosol
b.	Contact with blood or saliva of infected animals
c. 	Fecal-oral route.

ANSWERS: 
1. 	a
2. 	T
3. 	c

Syversen et al.  Temperature Variations Recorded During Interinstitutional Air Shipments of Laboratory Mice, pp. 31-36

Primary species, task 1

The NRC has recommended that mice be transported within a temperature range of 4 to 34C.  Couriers typically restrict shipment based on expected temperatures at enroute airports.  To determine the actual temperatures experienced by mice when shipped, the authors recorded temperature on standard domestic and international air shipments of mice from Johns Hopkins University.  

On 127 trips over the period of a year, temp was measured at 1 minute intervals by monitors taped to the outside of the shipping crates. Measurements during cooling and heating of crates were taken to establish whether or not exterior sensors appropriately represented internal temperature in the crates.  The authors concluded that outside and inside crate temperatures equilibrated within 10-15 minutes and that external sensors were appropriate for use.  Mice were shipped to the airport by TransporTech, then flew via freight aircraft or cargo holds of passenger airlines using 5 separate commercial couriers who also negotiated ground transport at destination.  

The authors obtained data on airline and ground shippers used, drop of and pickup times, flight arrival and departure times, and animal condition upon arrival.  24 shipments were excluded from analysis due to monitor dysfunction.  High temperatures were defined as exceeding 29.4C, low as lower than 7.2C, as this is the range defined by most couriers as acceptable for unacclimated rodents.  Mean duration of travel was 24 hours domestic, 52 hours international.  40% domestic, 77% international shipments reached or exceeded the high temperature range, 16% domestic and 12% international exceeded low temperature ranges.  61% of all shipments experienced temperature variations of over 11.1C during some portion of the shipment.  This was most frequent during stopovers, though airline was a variable.  The authors note that temperatures experienced by mice were likely to be as much as 5C above temperatures recorded due to mouse heat generation.

The authors conclude that times of loading and unloading were at high risk for temperature excursions outside the acceptable ranges. Passenger airline shipments, which was more likely to happen during the day, were more likely to experience high temperatures - mice in a shipment with temps that exceeded 65.6C died.  Low temperatures were more likely to occur during stopovers, and mice tolerated a low of -7.8C.  To decrease physiological stress of shipping, the authors recommend night shipments, that nesting material be used in shipping crates for insulation during cold weather, and that shippers specify use of cargo transport at temperatures suitable for live animals.  Authors noted data on duration and nature of mouse shipments that they didn't expect:  complicated coordination of airlines and couriers for even regional shipments, a surprising amount of 'holdover time' for international and domestic shipments, and complicated arrangements to hold animals in warehouses while awaiting flights. An admonition to remember that over 2 weeks post shipment may be required for glucocorticoid circadian rhythm to return to normal and more than a month for reproductive parameters is included.

QUESTIONS:
1. 	What temperature range does the NRC define as safe for shipment of adult mice?
a. 	5-25°C
b. 	4-29°C
c. 	0-30°C
d. 	7.2-29.4°C
2. 	Which type of flight is more likely to experience high temperatures, domestic or international?  Low temperatures?
3. 	When during flight are temperature excursions most likely to occur? Takeoff, in flight, stopover, or arrival.

ANSWERS:
1.	b
2.	High temps, international.  Low temps, domestic
3.	Stopover

Experimental Technique
Coleman et al.  Training Rhesus Macaques for Venipuncture Using Positive Reinforcement Techniques: A Comparison with Chimpanzees, pp. 37-41

Task 3:   Research support
Primary species: Macaca mulatta,  

SUMMARY: Macaques can be trained successfully to present for venipuncture using the same Positive Reinforcement Training (PRT) as performed on chimpanzees.

PRT has been used in chimps to eliminate the need for sedation during certain procedures and reduce aggression in group housing.

In general PRT may take months but once accomplished, can save time in the future. 

Negative Reinforcement Training requires a subject to perform a task to escape a negative stimulus. 

Positive reinforcement training (PRT) is a form of operant conditioning in which the trainer reinforces a desired behavior (i.e. sitting for a venipuncture) with a reward. Subject has control over its environment by exercising its choice to perform the task or not. 

PRT has the advantage of minimizing stress during study which in turn improves the quality of the data.

Terminology: 
a.	Primary reinforcer - usually a food item given as a reward.
b. 	Secondary reinforcer- a neutral stimulus (sound) used when the task is performed.
c.	Shaping- reinforcing successive progress towards a desired behavior. 

Methodology: 
1. 	Pair the primary reinforcer with the secondary reinforcer (i.e. food treat with a sound, 'clicker')
2. 	Shaping: Use of successive tasks which lead up to the desired behavior (i.e. approach sleeve -->put arm in sleeve -->remain in position --> venipuncture)
3. 	Each time a successive behavior has been completed; the animal hears a sound and is given a treat as a reward.

QUESTIONS:
1.	What is not true regarding PRT? 
a.	Subject has no choice in performing the task  
b.	It requires two types of reinforcement of behavior  
c.	It can help reduce stress d) all are not true
2.	Negative Reinforcement Training: 
a.	Inflicts a negative stimulus when the task is performed incorrectly  
b.	Rewards only the 'bad' behavior                                        
c.	Requires a subject to perform a task to escape a negative stimulus  
d.	There is no such term
3.	The secondary reinforcer is usually: 
a.	Painful stimulus   
b.	Food reward    
c.	A neutral stimulus  
d.	A punishment

ANSWERS:       
1.	a     
2.	c   
3.	c

CASE REPORTS
Clarke et al.  Peripheral Edema with Hypoalbuminemia in a Nonhuman Primate Infected with Simian-Human Immunodeficiency Virus: A Case Report, pp. 42-48

Task 1: Prevent, Diagnose, Control, and Treat Disease
Task 2: Prevent, Alleviate, and Minimize Pain and Distress

SUMMARY: The following is a case report on the development of peripheral edema with hypoalbuminemia in a non-human primate infected with Simian-Human Immunodeficiency virus (SHIV).  It is believed that a SHIV chimera virus is useful as a challenge organism for Human Immunodeficiency Virus (HIV) vaccine development. Furthermore chimera SHIV infection presents rapid progression of HIV like symptoms and is useful for HIV investigations precisely for this property. The animal presented in the following case report was a 7-year-old rhesus macaque (*Macaca mulatta)* infected with SHIV presented with edema around the abdomen and below the rib cage. The animal later developed edema in the scrotum. PCV, CBC and Chem, were performed revealing anemia, hypoalbuminema, hypoproteinemia, and azotemia in serial samples. Urine SG was 1.012. The authors arrived at a tentative diagnosis of renal impairment. Concurrent cardiovascular disease was ruled out on the basis of echocardiogram and the animal was medically managed and treated symptomatically for pain, dehydration, and other maladies associated with renal failure for approximately 4 months at which time it was determined the kidneys were unable to concentrate urine and clinical signs of edema became progressively more severe.

QUESTIONS:
1. 	In order to rule out concurrent cardiovascular disease rapidly in cases of peripheral edema, which of the following methods requires the least amount of complicated equipment and is inexpensive?
a. 	Measurement of central venous pressure
b. 	Echocardiogram
c.	Cardiac catheterization
d. 	Arterial blood pressure monitoring
2. 	In the aforementioned article, necropsy was performed. Histological diagnosis of lesions in the kidney revealed membranoproliferative glomerulonephritis. In addition to membranoproliferative glomerulonephritis, what are two other forms of histological lesions that may be seen in cases of immune complex glomerulonephritis?
3. 	The animal in this case had severe renal failure. What hormone produced in the kidney (that may now be deficient) might explain the patient's laboratory finding of anemia?

ANSWERS:
1.	a
2.	Membranous and proliferative
3.	Erythropoietin