Journal of the American Association for Laboratory Animal Science
Volume 46, Number 6, November 2007

OVERVIEW
Villar et al.  Biologic Effects of Fenbendazole in Rats and Mice: A Review, pp. 8-15

Primary species: rat, mice
Tasks: Task 1 – Treatment of disease

SUMMARY: Fenbendazole is a broad-spectrum benzimidazole anthelminth with activity against nematodes (particularly pinworms Syphacia and Aspicularis spp.) tapeworms, flukes and protozoa.  Although pinworms are relatively nonpathogenic, their presence may alter the outcome of some experiments.  Specifically, myelopoiesis and erythropoiesis are increases in pinworm-infected mice.  Immune stimulation including auto-immune responses and increased production of some interleukins has been linked to infestation.  There are also changes associated with mild chronic stress including alteration in endoneurocrine responses, exploratory behavior and growth of young animals.  Heavy infestation can lead to overt clinical disease.  
	
The most frequently used treatments for pinworms are avermectins and benzimidazoles.  Both are 100% orally and are often given in feed or water; however avermectins can cause toxicosis in young animals or in transgenic mice with p-glycoprotein deficiency.  Fenbendazole is used widely because of its wide margin of safety and efficacy.  This paper reviews the physiologic effects of fenbendazole and their potential effects on laboratory research.  
	
General Safety Data:  FDA studies in rats showed that the Maximum Tolerated Dose (MTB) is 45mg/kg and is associated with hepatocellular hypertrophy and hyperplasia.  Mice appear to be less sensitive than rats.  A 2-y carcinogenicity study in mice showed no effects at 405mg/kg daily.  A no-observed adverse effect level for mice is set at 135mg.kg daily. 
	
Tumor Promoter Studies: Fenbendazole appears to have similar liver tumor-promoting activity to Phenobarbital.  The GST-P assay indicated that FBZ acts as a tumor promoter but only at doses close to the MTD and well above therapeutic doses. 
	
Effects on Cytochrome P450: FBZ does not alter the total content of cytochrome P450 but does induce certain hepatic cytochrome P450 isoforms, 1A1, 1A2 and 2B1.  These isoforms are known to bioactivate procarcinogens.  FBZ may act as a tumor promoter when given after certain tumor initiators.
	
Bone Marrow Effects:  There are no myelosuppressive effects in rodents associated with FBZ.  
	
Effects on the Immune System:  There are conflicting reports on whether FBZ is immunomodulatory.  Most studies show that FBZ has no effect on selected immune responses, whether FBZ suppresses lymphocyte proliferation remains controversial.  
	
Repro, Terotalogic and Behavioral Studies:  No effects have been documented.

QUESTIONS:
1.	T or F	Myelopoiesis and erythropoiesis can be increased in pinworm infection.
2.	T or F	Avermectins can be toxic in young or p-glycoprotein deficient mice.
3.	T or F 	FBZ may act as a tumor promoter when given after certain tumor initiators.

ANWERS:
1.	True
2.	True
3.	True

ORIGINAL RESEARCH
Biology
Williams and Wyatt.  Comparison of Subcutaneous and Intramuscular Ketamine-Medetomidine With and Without Reversal by Atipamezole in Dutch Belted Rabbits (Oryctolagus cuniculus), pp. 16-20

Task2: K6
Primary Species: Rabbit (Oryctolagus cuniculus)
 
SUMMARY: Forty Dutch Belted rabbits enrolled in a minimally invasive pharmacokinetics study were used to compare the efficacy of an anesthetic combination delivered through two injection routes. Rabbits were randomly assigned to 4 groups to determine the sedative and physiologic effects of ketamine-medetomidine given either intramuscularly (IM) or subcutaneously (SC). Also, the reversal effects of an intravenous dose of atipamezole were assessed by comparing the return of the righting reflex in rabbits given the reversal agent with those that recovered spontaneously. In comparing the IM route, the SC route of ketamine-medetomidine effectively induced chemical restraint with an insignificant difference in onset of anesthesia and markedly less resistance during the injection. In all groups the combination of ketamine-medetomidine, regardless of route of administration, provided an adequate level of anesthesia. Reversal with atipamezole improved arterial hemoglobin oxygen saturation for both the SC and IM groups; however, an advanced rate of recovery from anesthesia was clinically apparent only for animals given the combination by the IM route. 
 
QUESTIONS:
1. 	What is the class of sedative for xylazine and medetomidine?
2. 	What is the hypothesis for the hypoxia seen with ketamine-medetomidine combinations?
3. 	Of the following, loss of righting, ear pinch, pedal reflexes, and palpebral reflexes, which is unreliable for use as an indication of anesthetic depth in rabbits?
 
ANSWERS:
1. 	Alpha-2 adrenergic agonist
2. 	The hypoxia is thought to be a direct consequence of peripheral vasoconstriction associated with medetomidine. 
3. 	Palpebral reflexes 

Hotchkiss et al.  Effect of Prolonged Ketamine Exposure on Cardiovascular Physiology in Pregnant and Infact Rhesus Monkeys (Macaca mulatta), pp. 21-28

Task:  3 (research support) - K5 (anesthesia, analgesia, patient monitoring)
Primary species: Macaca mulatta,  

Age, restraint, and chemical sedation can affect physiologic measurements. 

Two groups were monitored for study. One group was anesthetized with Ketamine and the other remained conscious. 

Each group was further subdivided by age namely pregnant adults (120-123 days), infants of post natal day (PND) 5, and infants of PND 35.

The infant conscious animals were restrained manually and the pregnant conscious animals were restrained in chairs.

Heart rate:
Heart rate was generally higher in infants than adults.

Ketamine treated animals in each group had a lower heart rate than conscious restrained animals in each group. 

Blood pressure 
The pressure is generally lower in infants than adults; with infants at PND 5 displaying a lower blood pressure than infants at PND 35. 

Pregnant adults generally have a lower blood pressure than non pregnant adults.

Ketamine treated animals in each group had a lower blood pressure than conscious restrained animals in each group. 

Respiratory rate:
Ketamine Infants have a lower respiratory rate than their conscious counterparts. 

Ketamine adults did not have a significant decrease over their conscious counterparts.

Glucose and hematocrit:
Ketamine administration did not affect these values. 

QUESTIONS:
1.	Which is true?  
A. 	Heart rates are generally higher when an animal is anesthetized with Ketamine  
B. 	Age of an animal has an effect on heart rate               
C. 	Ketamine does not affect the heart rate
D. 	None are true
2.	What is true regarding blood pressure? 
A. 	Ketamine generally raises blood pressure  
B. 	Age of an animal has an effect on blood pressure               
C. 	Pregnancy raises blood pressure   
D. 	PND 5 infants have the same pressure as PND 35 animals.
3.	What is true regarding Ketamine effects on respiration?  
A. 	It significantly decreases the rate in adults   
B. 	Conscious infants have a higher rate than ketamine infants  
C. 	Both   
D. 	None
4.	What remains unaffected by Ketamine administration?  
A. 	Heart rate   
B. 	Glucose  
C. 	Blood pressure  
D. 	None

ANSWERS: 
1. 	B    
2. 	B    
3. 	B   
4. 	B

Dufour et al.  Complications of Gastric Catheters Implanted in Rhesus Macaques (Macaca mulatta), pp. 29-34

Primary Species: Macaca mulatta

SUMMARY: This paper was a retrospective look at the complications of gastric catheters implanted in rhesus macaques undergoing a chronic alcohol consumption and simian immunodeficiency virus study.  Thirty-one rhesus macaques were implanted with gastric catheters (7-French silastic catheters) used to deliver alcohol or isocaloric sucrose (control).  Once implanted, the animals wore jackets and were housed in specialized cages modified with swivels and tethers.  During the course of the study, 3 animals developed clinical signs indicating possible instability of the implanted gastric catheter.  All 3 animals were found to have a string foreign body wrapped around the distal end of the catheter, with 2 of the catheters perforating the intestinal wall.  Gastroscopy was used to screen remaining animals to determine catheter position and the presence of a foreign body attached to the end of the catheter.  

Results of the screening revealed that of the 28 remaining animals, 9 had malpositioned catheters; string foreign bodies were associated with 3 of the 9 malpositioned catheters.  Initially, the authors hypothesized that the peristaltic motion of the stomach, combined with the attachment of string, which was probably ingested by subjects after manipulating their jackets, led to eventual catheter displacement.  Later, the authors concluded that the string may have played a secondary role but was not the primary cause of catheter instability, because several malpositioned catheters had no string attached at the time of diagnosis. Subsequent modifications were instituted, including modifying the surgical techniques, altering the type of gastric catheter used, and increasing environmental enrichment for animals with known tendency to manipulate their jackets.  Specifically, the surgical adjustments that were made included: 1) a catheter with a larger diameter attachment disc (3 cm rather than 1.5 cm) was used, 2) the catheter disc was secured to the peritoneal surface of the lateral abdominal wall after securing it to the gastric serosa, and 3) the catheter is secured to the abdominal musculature on exit from the abdominal wall.

QUESTIONS:
1. 	T/F  The string ingested from manipulation of the jackets was the primary reason behind the ultimate instability of the gastric catheters.
2. 	T/F  Enlarging the attachment disc from 1.5cm to 3.0cm will help prevent the gastric wall invaginating around the catheter disc and subsequently pulled into the gastric lumen via peristaltic movement.

ANSWERS:
1. 	False, the string ingested by the monkeys after manipulating their jackets was thought to be a secondary problem.
2. 	True, by increasing the disc size as well as securing the disc and gastric serosa to the peritoneal wall, the chances of the disc being pulled into the gastric lumen will be lessened.

Non-Human Primate Enrichment
Novak et al.  Use of Animal-operated Folding Perches by Rhesus Macaques (Macaca mulatta), pp. 35-43

Tasks 4, 6 - Develop and Manage Animal Husbandry Programs and Design and Operate Animal Facilities
Primary species-Macaque
   
SUMMARY: In recent years, there has been significant work evaluating how to optimize laboratory animals’ housing environments.  However, we are often limited by cost and improvements within a preexisting facility.  This study focused on 2 issues involving how to improve housing for Rhesus macaques:  More efficient use of existing caging and how to provide animals with a measure of control over their environment.  This study evaluated the design of an animal-operated folding perch placed within the interior of the cages.  The goals of the research were to establish if this novel system was something macaques would interact with and whether it provides them with an aspect of control over their environment.  
   
The research was performed by the National Institute of Child Health and Human Development at the NIH Animal Center in Poolesville, MD.  The perch platform known as the ‘Ruperch’, was made of stainless steal and mounted on the side of the cage.  Hinge tension was adjusted to allow the foldable platform to be pulled down to a horizontal position or folded vertically against the side of the cage without snapping into place.    
   
Two different experiments were conducted:  Experiment 1 involved 11 rhesus macaques of various ages housed in single or pair-cage housing.  Experiment 2 involved 86 rhesus that were free ranging.  Behavioral data was then collected after an habituation period.  Behavioral data evaluated the role of focal subject in any interaction, the behavior of the focal subject, and the use or nonuse of the folding perch versus permanent perches.  The resulting data was then used to address the following:  Would macaques use the folding perches; how would they use them; to what degree would macaques use the folding perches versus the permanent perches; and would the folding perches influence behaviors other than perch use?
   
Results indicated that the folding perch was used by rhesus monkeys.  In experiment 1, the presence of the folding perches did not have a significant effect on the overall behavioral repertoires of the individuals, however the frequency of folding perch use and how it affected permanent perch use varied with individual animals.  Several variables may have contributed to this finding:  Some cages only had a folding perch and spent most of their time not using it.   Also, dominance among pair housed macaques may have affected individuals’ access to the folding perch.  Data from the field enclosure (experiment 2) is consistent with the argument that folding perches were preferred.  However young rhesus monkeys, including juveniles and infants of both sexes, used the folding perches the same amount as they used the permanent perches.  
   
In conclusion, there was concern that the novelty of the folding perch would decrease over time but data indicated that usage actually increased over a 5 week period.  This led the authors to place folding perches as continuous fixtures in some of their large social breeding groups.  They make a point to suggest that each folding perch device should be tailored to the size and tension requirements of the age and species being exposed.  Second, the authors suggest a for increased use of the folding perches is perhaps the environment was already lacking and therefore any new item would be preferred.  However, data from the field enclosure (experiment 2) does support folding perch preference since the animals had other indoor enclosures , a pond outdoors, and numerous other climbing and perching devices available yet they spent more time using the novel folding perches.  Last, there are 2 potential benefits to adding folding perches to nonhuman primate cages.  First, they increase the amount of floor space available in the cages and allow facility managers more variety and flexibility with how cages can be modified.  Second, they provide captive animals with an element of control over their environment and choice.  This is an important component of environmental enrichment for captive animals.  
   
Overall data demonstrated that adding folding perches enhancing environmental enrichment in rhesus macaques.  However, the study generated additional questions that need to be addressed.  First, the findings should be tested in other species and other facilities.  Second, a study needs to be conducted that will evaluate whether the devices affected abnormal behavior.  And last, it would be worthwhile to evaluate other ways the folding perches could be used such as steps for monkeys to move between adjacent areas of cages or how spacing between the perches can be used to affect spatial distribution of the animals.       
   
QUESTIONS:
1.  	What are examples of exterior environment enrichment that is available for primates?
2.  	What are some examples of interior environment enrichment for primates?
3.  	What are some examples of providing environmental control in a laboratory animal setting?
   
ANSWERS:
1.  	Mirrors, puzzle feeders, plastic balls, television, radios, and video
2.  Enhancing visual and social contact among species via cohousing, cage size and location, diet, climbing structures, next boxes, novelty and variability in diet, and rotating toys.
3.  Allow an animal an area where it can hide from observers; Provide objects that an animal can destroy or can alter/take apart its makeup; Have animal caretakers provide animals choices over food or socialization opportunities

Colony Management
Barbaric and Dear.  Optimizing Screening and Mating Strategies for Phenotype-driven Recessive N-Ethyl-N-nitrosourea Screens in Mice, pp. 44-49

SUMMARY: The aim of this study was to provide some guidelines and rationale for breeding schemes and animal numbers when planning recessive ENU mutagenesis screens. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screens in the mouse are being used to elucidate gene function and develop disease models.  Optimization will allow for minimization of the numbers of mice that must be bred and subjected to phenotypic screens while maximizing the number of mutant phenotypes that can be identified.  The results revealed that when the G3 screening costs are a key factor, then backcrossing a single G2 female to the G1 parent male is the optimal strategy.  When animal housing costs area a primary consideration, backcrossing 2 G2 females to the G1 parent male is the optimal strategy.   The authors feel that the calculations that they presented will assist scientist in planning and designing phenotype-driven recessive screens.  Although the screening strategies that had been devised using the mouse as a model system, they feel that they will be equally applicable to ENU recessive screens in other organisms, such as zebrafish.
   
QUESTIONS:
1. 	T or F.  ENU mutagenesis can be used to create an allelic series for any gene.
2. 	T or F.  ENU mutants may reveal drug targets more easily than other techniques.
   
ANSWERS:
1.	True, this information is useful in mapping functional domains.  
2.	True,  ENU mimics drug action more closely than do gene knockouts because many drugs do not inactivate proteins entirely.  

Experimental Technique
Kienstra et al.  Murine Neonatal Intravascular Injections: Modeling Newborn Disease, pp. 50-54

Task 3 
Primary Species:  Mouse

SUMMARY: This article described the establishment of two novel techniques for intravenous injections in birth to 6 day old C57 mouse pups.  The techniques filled a need to be able to induce certain disease states unique to newborns such as respiratory distress syndrome, retinopathy of prematurity, and necrotizing enterocolitis.  The typical intravenous injection access sites commonly used in adult mice, retro-orbital and tail vein, are not feasible in neonates due to persistent fusion of the eyelids and the tail vein being too small.

The first of the techniques involved intravenous injections into the external jugular vein.  This vein is easily observable in a neonate. It is large caliber, visible without magnification, tolerant of concentrated injectables, and remains visible throughout a reasonable portion of the animal's development for repeated injections.  The second technique, involving the superficial temporal vein, requires magnification and transillumination to visualize.  This vessel also offers the same advantages as the jugular, except for size.  Both vessels exist bilaterally.

Each technique involves a 4 step process, which requires two people.  The first step is the positioning of the neonate, done by person 1.  The second step, needle insertion, is also done by person 1. The third step, IV injection, is performed by person 2, as person 1 holds the animal and needle.  Finally, the fourth step, involves hemostasis and recovery.  Successful injections were measured by use of fluorescent dyes and immunohistochemistry.  Although these techniques require a steep learning curve, the authors demonstrated a 100% survival rate and highly successful intravascular delivery of injectables.  

QUESTIONS:
1.  	Name two example of disease states unique to neonates.
2.  	What two vessels were successfully accessed and used for IV injections in neonatal mice?

ANSWERS:
1.  	Respiratory distress syndrome
2.  	Retinopathy of prematurity

CASE REPORTS
Snyder et al.  Malignant Peripheral Nerve Sheath Tumor in a Hamster, pp. 55-57

Task Designation: 1K8:  Clinical Medicine
Species Designation: Secondary

SUMMARY: This case report describes a malignant peripheral nerve sheath tumor in a 3 year old female hamster.  This tumor is relatively common in dogs, but this is the first description in hamsters.

QUESTIONS:
1.	T/F  Malignant peripheral nerve sheath tumors are common in hamsters. 
2.	T/F  Malignant peripheral nerve sheath tumors are common in cats and humans.
3.	T/F  Malignant peripheral nerve sheath tumors are common in dogs and cattle.

ANSWERS:
1.	F 
2.	F 
3.	T 

Allison et al.  Iatrogenic Hemolytic Anemia and Endocarditis in New Zealand White Rabbits Secondary to Achromobacter xylosoxidans Infection, pp. 58-62

Species: Rabbits
Task 1. Prevent, Diagnose, Control, and Treat Disease 
Task 3. Provide Research Support, Information, and Services

SUMMARY: Nine of 15 rabbits in a heart failure model experiment developed hemolytic anemia.  The heart failure model was a two step surgical procedure. Aortic insufficiency was created by causing traumatic damage to the aortic valve and a perforative lesion in the left ventricle to create volume overload. The next surgery created aortic stenosis by placing a ligature around the abdominal aorta proximal to the kidneys to create a pressure change in the femoral artery of 10-15 mm Hg.  During a period of 3 months 9 of 15 rabbits presented with anorexia, lethargy and pallor.  CBC's revealed regenerative anemia (hct range 13%-31%).  None of the animals responded to medical treatment.  Achromobacter xylosoxidans was cultured in the blood of 7 animals and two heart valves. The authors presume that the Achromobacter xylosoxidans caused a endocarditis which led to a fragmentary intravascular hemolysis.  Achromobacter xylosoxidans is an environmentally resistant ubiquitous aerobic, catalase positive, oxidase positive, gram negative bacillus. The source of the infection was traced to a pressure transducer and bag of IV fluids. 

QUESTIONS:
1.	Achromobacter xylosoxidans is a ____________ organism which is found in aqueous environments.
a.	Positive rod
b.	Negative rod
c.	Positive cocci
d.	Negative cocci
2.	True or false Achromobacter xylosoxidans has been cultured in chlorhexidine solution?
3.	True or false because RBC's lyse in intravascular hemolytic anemia releasing hemoglobin, increased serum hemoglobin provides support for the diagnosis of IVHA?

ANSWERS:
1.	B
2.	True
3.	False the serum hemoglobin is immediately bound by haptoglobin and thus resulting in low serum hemoglobin, low serum haptoglobin and hemoglobinuria

Lefkov and Mussig.  Tiletamine-Zolazepam and Xylazine is a Potent Cardiodepressive Combination: a Case Report, pp. 63-64

ACLAM Task: Task 3 -- Provide Research Support, Information, and Services
ACLAM species designation: Primary -- pig (/Sus scrofa/)

INTRODUCTION: This is a case report on a single Yucatan minipig (/Sus scrofa domestica/), which was used as a rapid-pacing model of heart failure and developed cardiodepressive and hypothermic effects due to its Telazol-Xylazine (T-X) preanesthetics.

SUMMARY: The female pig received 4.4 mg/kg of Telazol (tiletamine-zolazepam) and 2.2 mg/kg of xylazine IM before isoflurane anesthesia for a 70 day pacemaker lead placement study. After recovery from the implant surgery, the pig received T-X once weekly for follow-up exams. These evaluations include data retrieval from the pacemaker, echocardiogram, and chest fluoroscopic exam.  Among the parameters that were assessed were Vmax, dP/dt and Tau.

Intra-paragraph Definitions:

Vmax: Maximal Upstroke Velocity -- A measure of the contractility of the heart that is unaffected by loading.

Tau: The ventricular relation time constant from minimum dP/dt to the point where the pressure has dropped 66% of the distance from systolic to diastolic pressure. A prolonged Tau is an indication of diastolic dysfunction.

dP/dt: The first derivative of the blood pressure.

The phases of the study started with stabilization after the pacemaker was implanted for 4 weeks. The next phase consisted of high-rate pacing back and forth between 60 and 240 paces to induce heart failure. At the end of 4 weeks, the high-rate pacing ceased and the cardiovascular system was evaluated at its intrinsic rate.

What was found during the high-rate pacing phase was prolongation of the cardiac recovery time. Coincident with the administration of T-X were signs of cardiodepression (decreased heart rate, Vmax, left ventricular systolic pressure, a Tau measurement increase) and hypothermia.

Once these findings were documented and discussed, the T-X regimen was changed for the remainder of the study to ketamine/acepromazine as a preanesthetic and midazolam for sedation. Afterwards, undesirable cardiac side effects were not seen.

The Telazol package insert warns of possible myocardial depression. The Xylazine package insert warns of bradycardia, partial atrioventricular block, and cautions against its use in the setting of severe pathologic heart disease.

QUESTIONS:
1. 	Vmax is the acronym for what?
2. 	True or false: Tau is an indication of systolic dysfunction?
3. 	Define dP/dt.
4. 	In this case, what was the better drug regimen for this pig once T-X was discontinued?
5. 	List the cardiac warnings on the package insert for Telazol and Xylazine.

ANSWERS:
1. 	Maximal Upstroke Velocity
2. 	False -- diastolic
3. 	dP/dt: The first derivative of the blood pressure.
4.	Ketamine/acepromazine, then midazolam.
5. 	Telazol - possible myocardial depression. Xylazine - bradycardia, partial atrioventricular block, and cautions against its use in the setting of severe pathologic heart disease.

Stevens et al.  Corynebacterial Necrohemorrhagic Cystitis in Two Female Macaques, pp. 65-69

Task 1: Prevent, Diagnose, Control, and Treat Disease
Primary Species: Macaques
   
SUMMARY: Here the authors present 2 case studies of Corynebacterial Necrohemorrhagic Cystitis in female macaques. In the first instance, an adult female rhesus presented with a distended abdomen and a blood-stained perineum. Bloody urine was obtained via cystocentesis. Surgical exploration revealed a severely distended and dark red to black bladder with a gritty and mucoid plug. The animal died several hours after recovery from anesthesia. Gross necropsy revealed a friable and necrotic bladder mucosa, bilateral hydroureter, serosanguinous ascites, serous pleural effusion and serosanguineous pericardial effusion. Aerobic microbial culture of the bladder yielded C. renale and Strep acidominimus. 
   
In the second case, an adult female cyno presented with lethargy, dehydration and a blood stained perineum. The animal died of shock and sepsis despite aggressive antibiotic, fluid and anti-inflammatory therapy. Gross necropsy revealed anemia, a necrotic bladder containing dark red to greenish black fluid which yielded growth of nondiptheritic Corynebacterium. 
   
A source of infection in these 2 cases remains unknown.
   
QUESTIONS:
1. 	What are some characteristics of C. renale which facilitates urogenital infection in a host?
2. 	Which host factors can predispose female animals to infection? 
   
ANSWERS:
1. 	Pili allow attachment to the urogenital mucosa and facilitate ascension of microbes into the bladder and kidneys. The bacteria produce urease and hydrolyze urea, allowing them to survive in urine containing high levels of urea.
2. 	Females have short urethras, which decrease the distance bacteria must travel to reach the urinary bladder and kidneys. High estrogen levels may affect the integrity of the bladder urethral epithelium.