Contemporary Topics 39 (5)

Physiologic and behavioral assessment of rabbits immunized with Freund's complete adjuvant. Contemporary Topics 39(5), 08.
Abstract: This study was conducted to determine whether immunization at multiple sites with small volumes of Freund's Complete Adjuvant affects rabbit
well-being. Female NZW rabbits were immunized with antigen in FCA, FIA, or physiologic saline in 0.02-0.03 ml intradermal volumes in 30-40 sites, as well as 0.1 ml SC in 2 sites. Body weight, body temperature, blood samples, and behavior were used to evaluate for rabbit health and well being in response to the immunization procedure. Specifically, behavioral assessment testing assessed neurologic, muscular, sensory, autonomic, and motor functioning. After 10 weeks, rabbits were euthanized and all internal organs were examined grossly for the presence of lesions. Microscopic exam was conducted in lung, liver, and kidney tissue.
Rabbit well being was not compromised as a result of the immunization procedure. Body weight and temperature did not differ significantly
between groups. No significant trend in increased WBC counts was observed in rabbits that received FCA or FIA. Behavior of rabbits in all 3 groups did not differ significantly either. No granulomas were detected with gross exam of internal organs. The only significant difference was the degree of induration around injection sites in rabbits immunized with FCA vs. FIA. FCA immunized rabbits had a greater degree of induration.
Although this study suggests FCA can be used humanely for antibody production in rabbits, authors emphasized some recommendations to
follow to minimize development of complications with use of FCA:
1. Sterile preparation of immunogen and injection sites.
2. Limit volume of adjuvant injected into each site. (e.g. 0.03 ml/ site ID; 0.1 ml/site SC)
3. Use of multiple injection sites.
Questions: Questions:
1. What is the concern with use of FCA? Specifically, what can occur at injection sites?
2. List adjuvants that have been studied and used for polyclonal antibody production in rabbits.
3. T/F: In the literature there is great clinical evidence to support the conclusion that lesions from FCA injection are painful in rabbits.
4. Which is the current maximum concentration of mycobacteria in FCA recommended (by NIH) to minimize inflammatory reactions in the rabbit?
a. 0.5 mg/ml b. 2 mg/ml
c. 1 mg/ml d. none of the above
5. This study found degree of induration around injection sites differed significantly over time.
Which statements are TRUE.
a. Induration was greatest in the rabbits immunized with FCA.
b. Induration was almost nonexistant in rabbits immunized with saline.
c. None of the rabbits developed ulcerative dermatitis or abscessation at injection sites.
d. None of the rabbits exhibited any signs of discomfort upon palpation of injection sites.
e. All of the above.
6. What is the main difference between FCA and FIA?
7. T/F: The ID injection route has been shown to be an efficient method of antibody production leading to development of high titer and highly specific antibody.
Answers: Answers:
1. Granulomatous reactions may occur at injection sites. FCA can induce chronic inflammatory reactions.
2. FCA (Freund's Complete Adjuvant), FIA (Freund's Incomplete Adjuvant), RIBI, TiterMax
3. False
4. a (0.5 mg/ml)
5. e (all of the above)
6. FIA lacks the mycobacterial component.
7. True

Cardiovascular parameters telemeterically measured during pregnancy, parturition, and lactation in a common marmoset (Callithrix jacchus). Contemporary Topics 39(5), 14.
Abstract: The marmoset is an arboreal New World monkey from NE Brazil, in the family Callitrichidae. Mean weight for adult males is approx, 383 gms and non-pregnant females is approx. 420 gms. Marmosets typically give birth to twins, but can have triplets in captivity. Breeding occurs year-round, and gestation is 144 days. Females can become pregnant during lactation and therefore can give birth approx. every 5 months.
This paper describes the changes in heartrate (HR), systolic blood pressure (SBP), diastolic BP (DBP) and activity (ACT) during the last 2 weeks of gestation, through parturition, and during the first 2 weeks of lactation. Measurements were also taken during a "neutral" period 4 months after parturition. This marmoset had been instrumented for a behavior project and inadvertently became pregnant.
The authors divided the parturition process into 3 segments. Phase I (preparation for birth). Marmosets tend to be restless during this period. High ACT values were seen. Phase II(dilatation period) Varied behaviors during this period include "freezing" during contractions. Very low ACT values were seen during this time. Phase III (expulsive phase) Onset of expulsive contractions which continue till all neonates are expelled. Lasted approx. 40 minutes. HR fell back to baseline at the end of this period.
Results:
Gestation: SBP similiar to neutral period. HR were higher, especially at night. DBP was significantly higher.
Parturition: HR, SBP and DBP were all markedly increased
Lactation: SBP higher during the first 2 weeks than during gestation and neutral periods. DBP and HR decreased gradually during the first 2 weeks.
The findings for HR and SBP were in good agreement with human data. In women, BP does not vary significantly throughout pregnancy, but HR increases and remains elevated until delivery. HR and BP elevate in women during labor, but return to normal within 2 weeks after delivery.
Questions: Questions:
What is the genus and species of the common marmoset? What family does it belong to?
What is the gestation period of the common marmoset and how many offspring are usually produced?
What change in cardiac parameters is common to both women and the marmoset during pregnancy?
What change in cardiac parameters is common to both women and the marmoset after delivery?
Answers: Answers:
(Callithrix jacchus) ,Callitrichidae
144 days, twins. Can have triplets in captivity.
Increased HR
HR and BP return to normal within 2 weeks

Urolithiasis in a cynomolgus monkey (Macaca fascicularis): a case report. Contemporary Topics 39(5), 18.
Abstract: Case History: Captive-bred 4 yo male cynomolgus monkey was introduced to animal facility in September 1997. Animal was single-housed and quarantined for 6 weeks. Diet consisted of standard monkey chow (Monkey diet 5038, PMI Feed , Inc. Richmond, IN), daily fresh fruit, and ad lib water. Environmental conditions were standard. Animal was negative for herpes B, SIV, SRV, filovirus, CMV, STLV1, and tested negative for TB.
Clinical Findings: In December of 1997, animal presented with anorexia, lethargy, and distended urinary bladder. Clinician was unable to pass urinary catheter and plain film radiograph showed distended bladder with 2 urinary calculi, 1 in neck of bladder and the other in the penile urethra. Calulus in penile urethra was surgically removed, but bladder calculus was not. Animal urinated freely for 24 hours, but urine flow stopped by 48 hours. Animal was euthanized because of poor body condition. Calculus was sent for identification of chemical composition. Blood was drawn for hematology and chemistry profile. Liver, kidney, spleen, and urinary bladder sections were submitted for histopathology.
Analysis of stone was 100% calcium carbonate. BUN, creatinine, ALP, phosphorus, and potassium levels were elevated. Sodium and chloride levels were decreased.
Pathology: Thromboses were observed in the interlobular veins and cortical venules of both kidneys. Inflammatory cell aggregates and proliferation of spindle cells was observed in renal interstitium. Tubules exhibited slight abnormalities. Glomeruli, as well, exhibited minimal pathology. No evidence of proteinuria. Bladder wall was diffusely thickened, with slight epithelial hyperplasia and minimal to mild hemorrhage and edema.
Conclusion: Few cases of urolithiasis with calcium carbonate uroliths have been reported in cynomolgus monkeys. No cause of calculi was not determined.
Questions: Questions:
1.. Describe standard mechanism for urolith formation.
Answers: Answers:
1.. Supersaturation of urine leads to nucleation and crystal formation.

Reproducible echocardiography in juvenile sheep and its application in the evaluation of a pulmonary valve homograft implant. Contemporary Topics 39(5), 20.
Abstract: Increased use of the ovine animal model in cardiovascular surgical research has created a need for standardized ECG techniques. In this study they implanted 10 sheep with a cryopreserved pulmonary valve homograft and monitored them through weekly ECG exams for 20 weeks. The left cranial and left caudal transducer windows were used to get good images without prior sedation. The right apical window was used to get adequate views in the sedated animal. ECG data correlated with the results of the post explantation exams. The awake animal was placed in Left lateral recumbence, 2.5 or 3.5 MHz transducer. Left cranial window at left side of sternum at third intercostal space. Viewed: short axis view of the heart base showing the left atrium, right atrium, tricuspid valve, right ventricle, RVOT, pulmonary valve, aortic valve and ascending aorta Left caudal window at left side of sternum at the at the 5th intercostal space Viewed: long axis of heart - 4 chambered image, and short axis of the left ventricle. Right apical window immediately to the right of the sternum at 6th intercostal space Viewed: 2, 4, 5 chambered views RA, RV, LA, LV, aorta. Positioning was standardized and oriented the same direction for consistency. Color flow doppler was used to assess the degree of turbulent diastolic blood flow within the right ventricular outflow tract in order to semi-quantify valve regurgitation. thickening of the pulmonary valve homograft leaflets seen during diastole appeared thicker than the remnants of the native pulmonary leaflets. this may be indicative of calcification. Adhesions post surgically actually benefited the imaging since adheased the heart to the chest they decreased the interference of the air space of the lung.
Questions: Questions:
1. T/F Regarding the ultrasound probes the larger number of MHz, the better the resolution
2. Which of the "large" animals is most traditionally used for cardiology research? A sheep B cow C Goat D Pig
3. Why was the animal placed in left lateral recumbence if they are primarily viewing the left side ?
Answers: Answers:
1. True the higher the frequency, the better the resolution (greater detail) , but with high frequency come poor penetration.
2. Pigs are the "best" model for cardiology since they have hearts most similar to humans
3. The animal is placed in left lateral recumbancy because gravity draws the heart closer to the thoracic wall, pushing aside the lung lobes which interfere with the image (air interferes with the ultrasound image).

Method and complications of ileocutaeous anastomosis for collection of ileal digesta in neonatal pigs. Contemporary Topics 39(5), 26.
Abstract: This study describes a technique for collection of ileal digesta in neonatal pigs (<5 kg/ 10 day-old). This procedure has been performed in older pigs, but no method for collecting ileal digesta in neonatal pigs has been reported. The success of this procedure demonstrates an alternative to the "T-cannula" or ileorectal anastomosis for nutrition research in neonatal pigs.
The laparotomy incision was made in the right flank along the long axis of the body. The ileum was identified and transected 5-cm proximal to the ileocecal junction. The distal stump was sutured closed; the proximal stump was drawn to an incision 2-cm ventral to the initial surgical site where the ileocutaneous anastomosis was made.
Cannulas were placed initially, but all the pigs dislodged the device. The investigators had greater success collecting the digesta using the "free-catch" method by placing the animals in metabolic cages. Blood was collected from each pig before surgery and at day 7. All values remained in reference range. At the post-mortem exam, all pigs had adhesions surrounding the ileocutaneous anastomosis. This technique as demonstrated by these investigators was rapid, easy to learn, and led to few complications.
Questions: Questions:
1. What serum biochemistry changes are expected in pigs with ileorectal anastomosis (IRA)?
2. What post-operative medical considerations need to be addressed in this experiment?
3. What is a Thiry-Vella loop?
Answers: Answers:
1. Hypophosphatemia is common in animals with IRAs or extensive resection of the large colon.
2. Animals undergoing surgery on the digestive tract should receive peri-operative antibiotics and analgesia. The pigs in this study received flunixin meglumine and procaine penicillin G.
3. A Thiry-Vella loop is a technique used to create a small intestinal bypass. It is performed by isolating a segment of small intestine with the vascular and nerve supply intact, and exteriorizing both ends. This procedure is described and illustrated in: Surgery, Anesthesia, & Experimental Techniques in Swine by MM Swindle, Iowa State University Press, 1998.

Preparation of the inguinal fat pad for perfusion in situ in the rat: a surgical technique that preserves continuous blood flow. Contemporary Topics 39(5), 29.
Abstract: Development of a surgical technique using a tissue-isolated tumor model that preserves continuous blood flow to the organ (inguinal fat pad). May be useful for in vivo studies of dietary lipid intake and metabolism, hyperlipidemia, and cancer-associated cachexia.Methods: donor SD rat (fed and fasted) blood into Buffalo rats. Catheters into: epigastric artery, caudal superficial epigastric/femoral vein. Heat exchanger, artificial lung and chromatography to separate lipid extracts. Fat pad on left inguinal fossa exposed, butterfly ST infusion catheter into caudal epigastric/femoral vein draining the fat pad, arterial catheter into epigastric artery leading to the fat pad. Normal blood supply and drainage stopped (ligated). Perfusion system established and rat exanguinated. Fat pad removed for analysis of componentsHistorically, organ perfusion techniques involved prolonged interruption of host blood flow. Periods of anoxia and nutrient deprivation can cause long-lasting or irreversible tissue damage. In addition to describing the surgical technique, this study compares blood flow and fatty acid uptake & release in fed rats versus rats fasted 48 hours. There was little or no change in the rate of blood flow throughout the perfusions. No significant difference found in pH, pCO2 and pO2 between perfusions using blood from fed or fasted donors. However, rats lost 10-15% of total B.W.during a 48-hr fast, and the inguinal fat pad released fat in the form of FFA for energy expenditure. Concluded that the hemodynamics of the inguinal fat pad reached steady state within 15 to 20 min.
Questions: 1) Arterial blood for perfusion in this study was obtained from ______ rats that had free access to food or had been fasted (to increase / decrease plasma lipids) 48 hr prior to bleeding.
a) Sprague-Dawley
b) Buffalo
c) BB Wistar
2) Surgical procedures in this study to isolate the (pelvic / inguinal/dorsal) fat pad were performed in (fed / fasted/ both fed and fasted) ______ rats.
a) Sprague-Dawley
b) Buffalo
c) BB Wistar
3) The proportion of blood gas components in the arterial blood perfusate leading to the inguinal fat pad was adjusted to maintain pO2 at ___ mm Hg, pCO2 at ___ mm Hg, and pH at 7.4.
a) 130; 50
b) 30; 150
c) 150; 30
d) 50; 130
4) Measurement of lipid/ fatty acid transport in the inguinal fat pad is a "tissue-isolated" tumor model to study tumor growth and metabolism. Findings from this study (differed from / mimicked) that of tumor tissue which appears to ________ almost all lipid from arterial blood to varying degrees.
a) transport unchanged
b) remove
c) metabolize
d) convert to FFA
5) Lipids extracted from the fat pad were separated by using __________________ on silica gel G plates that were coated with rhodamine B in ethanol and activated at 100*C for 30 minutes.
a) High Performance Liquid Chromatography (HPLC)
b) Gas Chromatography (GC)
c) Agarose Gel Diffusion (AGD)
d) Thin Layer Chromatography (TLC)
e) method of Folch as modified by McDonald-Gibson
6) Which of the following are components of the total fatty acids (TFAs) in rat blood, (lipids that were extracted in this experiment)?
a) FFA (free fatty acids)
b) TGA (triglycerides)
c) PL (phospholipids)
d) CE (cholesterol esters)
e) All of the above
7) Which of the fatty acids listed in question #6 accounted for nearly 90% of fatty acid uptake and release, and was the only one significantly changed, by the fat pad?
8) After homogenizing fat pads from fed and fasted rats, the investigators found that >95% of the total fat is in the form of ____, with TFA content from fasted rats significantly (higher / lower).
a) FFA b) TGA c) PL d) CE
9) The inguinal fat pad released fat in the form of ____ for energy expenditure.
a) FFA b) TGA c) PL d) CE
10) The rats lost ______ % of their body weight during a 48-hour fast.
a) 5
b) 5-10
c) 10-15
d) 15-20
e) >20
11) What vein drains the inguinal fat pad into the femoral vein?
12) Which blood vessels are ligated in preparation for the fat pad perfusion procedure?
13) The BUF rat is a spontaneous animal model for fascicle # 316, which is:
a) IDDM
b) autoimmune thyroiditis
c) NIDDM
d) diabetes insipidus
14) Which is the principal lipid fraction that is transported by adipocytes in vitro?
a. cholesterol b. albumin
c. plasma free fatty acid d. glycerol
15) What are some disadvantages of the microdialysis technique as compared to the surgical perfusion technique?
Answers: 1) a;
2) inguinal, both, Buffalo;
3) c;
4) differed from, b;
5) d [Folch/McDonald-Gibson was the method used to extract all lipids];
6) e;
7) FFA;
8) TGA (fasted rats have lost TFAs in the form of FFA expended to produce energy), lower;
9) FFA;
10) c;
11) caudal superficial epigastric;
12) Femoral a., caudal superficial epigastric v. and femoral
v.;
13) b;
14) c;
15) Disruption of blood flow, excessive proteolysis, tissue destruction.

Congenital bilateral ureteral stenosis and hydronephosis in a neonatal puppy. Contemporary Topics 39(5), 34.
Abstract: This is a case report of a death of a 10 day old pup in a dog colony that carried a gene hereditary for canine spinal muscular atrophy. Parturition was uneventful. Three days after parturition, the Brittany spaniel/beagle puppy (Canis familiaris) was noted to be nursing but not gaining weight as rapidly as its litter mates. No other clinical signs were observed. Although its diet was supplemented, the puppy died 10 days after birth. Differential diagnoses for poor weight gain and sudden death in a neonatal puppy are numerous and include malnutrition / malabsorption, hypoglycemia, hypothermia, congenital defects and infectious disorders such as canine parvovirus and canine herpes virus. On necropsy, the renal pelves were found to be enlarged and filled with urine. Both ureters were thin throughout their length, and urine could not be expressed from either kidney into its respective ureter. The bladder contained no urine and was firmly embedded in the umbilicus. Histologically, both kidneys were hydronephrotic and contained hypoplastic collecting tubules. The diameter of the right (0.55 mm) and left (0.57 mm) ureters at the uropelvic junction were narrower than those of an age mated control of the same breed (1.03 mm and 1.02 mm) and were lined by hypoplastic urothelium. Trichrome staining of the ureters revealed disorganized smooth muscle fibers and excessive collagen between the smooth muscle fibers and in the adventitial layer; in contrast the control had predominantly circular smooth muscle fibers and less fibrous tissue.
The hypoplastic collecting tubules in this puppy are consitent with a developmental defect arising from abnormal differentiation of either ureter bud or metanephros. The abnormal differentiation of the ureters are most likely responsible for the intrinsic stenosis of the ureter. The hydronephrosis in this case was the result of intrinsic stenosis of the ureters. Although neither blood or aqueous humor could be evaluated for urea nitrogen, and the typical effects of uremia (e.g. pulmonary edema) were absent, we suspect the puppy died from uremia. Congenital anomalies of the ureter are rare in domestic animals and range from aplasia to complete duplication. This type of ureteral obstruction reported is both physical and functional, with the most common site being the uropelvic junction. This is the first report of bilateral stenosis of the ureters at the uropelvic junction and subsequent hydronephrosis in a puppy. The condition is similar to a form of uteropelvic obstruction in humans.
In humans 80% of cases of congenital hydronephrosis spontaneously resolve within the first 3 years of life. Persistent cases due to obstruction of the uropelvic junction are treated by performing pyeloplasty.
Questions: Questions:
1. What is the genus and species of the dog?
2. What would be the differential diagnosis for poor weight gain and sudden death in a puppy?
3. True or false? Congenital anomalies of the ureter are rare in domestic animals.
4. Was this type of obstruction functional or physical?
5. Is this a lethal congenital defect in dogs? In humans?
Answers: Answers:
1. Canis familiaris
2. Malnutrition / malabsorption, hypoglycemia, hypothermia, congenital defects and infectious disorders such as canine parvovirus and canine herpes virus
3. True
4. Both physical and functional.
5. Apparently in dogs (1st reported case). Not in humans since 80% resolve spontaneously in the first 3 years of life.

Novel use of the Elizabethan collar as a "hoop-skirt" for protecting wounds and catheters in nonhuman primates. Contemporary Topics 39(5), 37.
Abstract: A primate jacket and plastic Elizabethan collar (E-collar) were used to make a hoop-skirt device used in the management of proximal and
distal hindlimbs wounds in baboons and macaques. The device was developed for prostatectomized rhesus macaques with Foley catheters, to facilitate healing of the urethra.
The jacket was placed on the NHP and cable ties were used on the zipper pulls to prevent the animal from opening and removing the jacket.
After some modification of the E-collar (a minimum of 5 holes punched along the neck circumference 1 inch below the edge), it was placed around the NHP's waist, affixed to the jacket, and fastened with cable ties.
Within 24 hours of wearing the hoop-skirt the animal adapted to it. Animals were able to eat, perch, lay down and continue with their normal
behavioral repertoire, but were unable to remove the device or tamper with the bandages on the hindlimbs.
Questions: Questions:
1, Advantages of the hoop-skirt device include:
A. Durability
B. Minimal maintenance
C. Ease of implementation
D. Applicability to many species
E. All of the above
2. Disadvantages (inconveniences) of the hoop-skirt device include:
A. Inability to assess the animal's tumescence
B. difficulty in manuvering animals through cages entrance
C. Lack of access to lower limbs for IM injections
d. All of the above
3. The hoop-skirt device consist of:
4. Name 3 types of protective dressings used as methods to protect and maintain wounds and catheters in NHP.
Answers: Answers
1. E
2. C
3. Cable ties, Elizabethan collar, and primate jacket
4. Robert-Jones splints, thermoplastics, and fiberglass casting material

Corneal dermoid in a dwarf rabbit (Oryctolagus cuniculi). Contemporary Topics 39(5), 39.
Abstract: There are a few reports of occular dermoids in guinea pigs and rats, but no previous reports of corneal dermoids in rabbits. This case report describes a corneal dermoid in a 5 month old dwarf rabbit that was kept as a pet. The dermoid, in the right eye, was a small, light gray, elevated lesion with a few hairs on it. The left eye was normal. The abnormal tissue was removed and sent for histology. Histologically, the specimen consisted of a matrix of loose connective tissue, hair follicles and associated sebaceous glands, covered by a cornified squamous epithelium.
A hereditary disposition for corneal dermoids may be present in some species, including guinea pigs. Since this is a single report, genetic inheritance cannot be documented, but investigators should closely examine related animals if they discover a corneal dermoid in one of their research animals.

Dermoids are a type of choristoma, a hisologiaclly normal tissue in an anatomically abnormal location. They can also be classified as dysontogenetic tumors.
Questions: Questions:
1. What is a choristoma?
2.What is the histologic appearance of an ocular dermoid?
Answers: Answers:
1. A chorisotma is a histologically normal tissue in an abnormal anatomic location.
2. An ocular dermoid consists of cornified squamous epithelium overlying a stroma rich in connective tissue, hair follicles, sebaceous glands and fat.

Fluorogenic 5' nuclease PCR (Real time PCR). Contemporary Topics 39(5), 41.
Abstract: The fluorogenic 5' nuclease PCR assay (real time PCR) is a sensitive and specific assay capable of quantitating PCR products. Similar to other PCR assays it can also be modified to quantitate RNA. The authors assert that this method of PCR assay is gaining popularity in laboratory animal medicine diagnostics because of its ability to detect minute amounts of nucleic acid from adventitious agents. It is also useful for studying gene expression and to determine genetic alterations, such as allelic variation.
The steps of this PCR assay are:
Step 1 - DNA template. DNA is isolated from the sample to be tested. The template DNA is denatured resulting in two single strands of DNA from the one double stranded
Step 2 - Hybridization Probes. A non-extendable hybridization probe is designed to bind the SS DNA to the PCR product. The probe contains a reporter fluorescent dye on the 5' end and a quencher dye on the 3" end. During the annealing phase, the hybridization probe binds to the template DNA.
Step 3 - Annealing Phase: As the temperature continues to decline, the primers anneal to the template DNA containing the hybridized quenched probe.
Step 4 ? Synthesis of new DNA. Taq DNA polymerase synthesizes new DNA by adding complimentary bases to the denatured SS template DNA. Taq DNA polymerase has 5' to 3' exonuclease activity that allows it to cleave terminal nucleotides of DS DNA. As the hybridization probe is degraded, the quencher dye is separated from the reporter dye allowing detection of the fluorescent dye.
Step 5 - Product analysis: The amount of fluorescence is measured at the end of each thermal cycle and is proportional to the amount of PCR product. Relative quantitation (Rn) is expressed in terms of cycle threshold (Ct) the cycle at which fluorescence crosses the threshold value. Absolute quantitation can be expressed can also be expressed in terms of copy number.
Advantages: 1) Fluorogenic 5' nuclease PCR is very sensitive and
specific. 2) The assay is quantitative. 3) The assay has a rapid turn-around
time. 4) No post-PCR processing (e.g. gel electrophoresis) is required.
Disadvantages: 1) Cross contamination can easily lead to false positive results. 2) The technique is expensive and requires specialized instrumentation.
Alternative Techniques: Quantitative competitive (Nested) PCR, Southern blot hybridization, PCR-ELISA
Questions: 1. Real Time PCR is: A) sensitive B) specific C) quantitative D) all the above
2. T or F Using Real Time PCR can have false positive results from cross contamination?
3. T or F Real Time PCR requires specialized instrumentation and is expensive to perform?
Answers: 1) D 2) T 3) T