Journal of the American Association for Laboratory Animal Science
Volume 47, Number 5, September 2008

ORIGINAL RESEARCH
Biology
Purdue et al.  Suboptimal Ability of Dirt-bedding Sentinels to Detect Spironucleus muris in a Colony of Mice with Genetic Manipulations of the Adaptive Immune System, pp. 10-17

Task 1 & 3: Diagnose, Detect unwanted disease or condition
Species: Primary (Mouse)

SUMMARY: Spironucleus muris an unacceptable rodent pathogen is currently diagnosed by direct examination of sentinel intestinal smears. Clinical abnormalities associated with S. muris vary from no signs to severe lethal infection depending on other co-factors. One fresh cyst can remain infective for 14 days, and thus the inability to detect S. muris infection is confounding. The poor ability to detect infection is multifactorial: staff non-compliance to transfer dirty bedding, insufficient volume of dirty bedding, and number of fresh cysts in feces, sample collection and handling and experience of lab personnel in detecting S. muris. 

This study showed that the sensitivity for detecting S. muris infection using histology is 91% vs. 71% by direct smear examination. Also, the surveillance of sentinels to exposure period of 6 wk and 16 wk found to aid in more sensitive detection of S. muris infection. Thus the relative low incidence of S. muris infection in research colonies can be attributed to the pitfalls associated with dirty-bedding sentinel program. Further improvement in diagnostic and surveillance program will aid in detection and culling of animals infected with S. muris without affecting experimental data. 

QUESTIONS:
1.	The reported low incidence of S. muris infection in modern lab animal setting is due to 
a.	Improved husbandry
b.	Improper surveillance methodology used
c.	Early Detection and treatment 
2.	How many cysts are required to cause Spironucleus muris infection
d.	One fresh cyst
e.	10 
f.	25
g.	One old cyst
3.	The common sites of S. muris colonization is at 
h.	Large intestine
i.	Stomach
j.	Duodenum
k.	Ileum
4.	What is the characteristic motion seen flagellated trophozoites of S. muris? 
l.	Tumbling
m.	Circling
n.	Rolling

ANSWERS:
1. 	a
2. 	a
3. 	c 
4. 	c 

Freed et al.  Operant Task Performance and Corticosterone Concentrations in Rats Housed Directly on Bedding and on Wire, pp. 18-22

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions
Domain 2: Management of Pain and Distress
Domain 4: Animal Care

SUMMARY: This study examined the effect of housing conditions on task performance and corticosterone response.  One group of animals was housed on solid-bottom caging with corn-cob bedding.  A second group was housed on wire-bottom caging.  Both groups underwent training for an operant task.  After the learning the task, rats were exposed to an acute stress (60 min restraint) and a chronic stress (7d continuous light exposure).  Data from this study show that rats in both groups were able to perform equally well with learning the operant task.  Both sets of animals responded similarly to the constant light exposure.  However, rats on wire bottom cages had higher levels of corticosterone after1 hr of restraint and at 48 hrs after restraint.  This sustained elevation in corticosterone levels suggests that wire-bottomed cages exacerbate the physiologic effect of stress in rats.  The authors hope that these findings will be used to convince investigators that wire-bottomed caging is not necessary for all operant conditioning studies.
 
QUESTIONS:
1.	T or F – Various studies have shown that rats prefer solid bottom cages to wire bottom cages.
2.	The Guide and The Report of the Rodent Refinement Working Party both recommend _______ floored cages.
3.	How do you measure the hypothalamic-pituitary-adrenal axis response to stressors?
 
ANSWERS:
1.	True
2.	Solid
3.	Measuring corticosterone levels

Scorpio et al.  Retrospective Clinical and Molecular Analysis of Conditioned Laboratory Dogs (Canis familiaris) with Serologic Reactions to Ehrlichia canis, Borrelia burgdorferi, and Rickettsia rickettsii, pp. 23-28

ACLAM Task:  Tasks 1-3, prevent, control, and diagnose disease

SUMMARY:  Dogs are susceptible to a multitude of tick-borne infections such as Ehrlichia canis (canine monocytic ehrlichiosis), Borrrelia burgdorferi (Lyme disease), and Rickettsia rickettsii (Rocky Mountain spotted fever). These diseases can manifest with clinical signs to include fever, anorexia, malaise, lameness, rash, and bleeding episodes. However, these infections can also be subclinical. Hematologic abnormalities can include leukopenia, thrombocytopenia, hyperproteinemia, and hypergammaglobulinemia. 

Random-source dogs are susceptible to these diseases because of their increased risk of arthropod exposure.  In this study, 21 random-source (Class B) dogs were examined. Blood samples were taken for CBC, biochemistry profile, tick-borne pathogen serology, and PCR. Two (2) of these dogs presented with illness characterized by fever, malaise, lameness, or hemostatic abnormalities. Fifteen (15) had antibodies to one or more tick-borne pathogens. Specifically, 48% had antibodies to E. canis, 43% to B. burgdoferi, and 43% to R. rickettsii, with an overall prevalence of 71% of dogs positive for one or more pathogens. Further, 33% had antibodies to two pathogens, whereas 14% had antibodies to all 3 agents. Please note that Lyme disease vaccination can confound Lyme disease diagnosis because the vaccine induces antibodies detected by the standard serological assay. However, it is unlikely that the dogs at hand had received prior vaccination for Lyme disease.       

No specific hematologic or biochemical differences were apparent between seronegative and seropositive dogs with the exception of elevated globulin levels in the seropositive dogs. All dogs were negative for E. canis and B. burgdoferi PCR of tissues and blood (PCR used to assess if active infection). Serologic and molecular results indicate previous infection followed by clearance or persistence below that which could be detected by PCR. 

QUESTIONS:
1.	All 3 pathogens examined in this study are zoonotic, can cause human disease, and are the most frequent tick-borne agents in humans.  T/F      
2.	These agents can serve as a confounding variable due to causing blood and tissue alterations.   T/F
3.	 What are some common reasons for the continued use of Class B dogs even though they are much more likely to be disease laden as compared to Class A dogs?     

ANSWERS:
1.	T so long as one realizes that in humans monocytic ehrlichiosis is caused by E. chaffeensis.
2.	T
3.	lower cost, flexibility in the size and age of animals, and appropriateness for acute, terminal procedures.  

Tsuchida et al.  Maternal Behavior of Laboratory-born, Individually Reared Long-tailed Macaques (Macaca fascicularis), pp. 29-34

Species: Primary, Macaque spp. 

SUMMARY: The Tsukuba Primate Research Center (TPRC) employs individual rearing to maintain and supply SPF long-tailed macaque (Macaca fascicularis) monkeys for their research program. Monkeys are separated from their mothers at 5-6 months of age and pair-reared with an age-matched cage mate until 2 y of age. Thereafter, monkeys are maintained individually except for mating. Lack of social experience causes concern for interactive situations such as mating and mothering. This study analyzed data from a 20 y period from 3000 cases involving approximately 900 monkeys. 
  
Inadequate maternal behavior was defined as rejection, holding infant incorrectly, refusal to nurse, or violence towards the infant. Animal caretakers determined the adequacy of maternal behavior using records of daily observations of maternal behavior from birth to separation at approximately 6 months of age. The TPRC employed a strict scale in which even one instance of inadequate maternal behavior coded that dam as inadequate, even if it was a single incidence. The authors also examined the similarity of maternal behavior between mothers and their daughters and the effect of maternal care received as an infant on their maternal behavior as an adult. 
  
The results of the study indicated that only the level of parity had an effect on maternal behavior. The greatest improvement in maternal behavior was seen between the first and second delivery. No direct comparison was possible between TPRC individually reared maternal performance and that of group reared dams. 
  
QUESTIONS: 
1.	Besides the long-tailed macaque, what other common names are used for Macaca fascicularis? 
2.	What is the gestation period for Macaca fascicularis? 
  
ANSWERS: 
1.	Cynomolgus monkey and the crab-eating macaque
2.	167-193 days

Guenette et al.  Anesthetic Properties of Propofol in African Clawed Frogs (Xenopus laevis), pp. 35-38

SUMMARY: This study was performed in order to determine the level of anesthesia attained in African Clawed frogs, Xenopus laevis, using a propofol bath. Propofol is a general anesthetic that enhances the function of γ-aminobutyric acid (GABA) receptors. Binding to this receptor causes inhibitory activity of the central nervous system leading to immobility and unconsciousness. In this study, anesthetic depth was determined by performing the acetic acid test, assessing withdrawal reflex, righting reflex, heart rate, and respiratory frequency. Pharmacokinetics of propofol were calculated after blood concentration determination by tandem liquid chromatography-mass spectrometry analyses. Propofol produces brief, shallow anesthesia in African clawed frogs when administered by immersion at a concentration of 88 mg/l. High concentrations (175 and 350 mg/l) caused death in all animals. Behavioral tests revealed that the duration of analgesia was at least 30 min, but the depth of anesthesia was considered to be light. During the acetic acid test, some frogs showed muscle fasciculations at 15 min after induction, and some frogs reacted at 0 and 30 min after induction, respectively, during the withdrawal test. An appropriate anesthetic dose was determined to be 88 mg/l for 15 min. Short-duration anesthesia (less than 30 min) was obtained, and in many frogs, muscular fasciculation was seen during the acetic acid test. When administered as a single-bath immersion for 15 min, propofol does not appear to be a safe and effective anesthetic for Xenopus laevis frogs, due to a narrow dose-effect window, short duration, and shallow level of anesthesia obtained.

QUESTIONS:
1.	Name Genus & species of African clawed frog?
2.	What is the mechanism of action of propofol?
3.	According to this article, is a propofol bath an effective method for anesthesia for African Clawed frogs?
4.	List common anesthetics used in frogs.

 ANSWERS:
1.	Xenopus laevis
2.	Propofol is a general anesthetic that enhances the function of γ-aminobutyric acid (GABA) receptors. Binding to this receptor causes inhibitory activity of the central nervous system leading to immobility and unconsciousness.
3.	NO! propofol has a narrow dose effect window, short duration & a shallow level of anesthesia
4.	Anesthetics known to work well in frogs include MS222, eugenol & benzocaine

Fain et al.  Detection of Spironucleus muris in Unpreserved Mouse Tissue and Fecal Samples by Using a PCR Assay, pp. 39-43

Domain 1
Primary species: Mouse
 
SUMMARY: The purpose of this study was to develop a rapid DNA isolation method and a sensitive and specific PCR assay for detecting Spironucleus muris in mouse tissue and fecal samples.
 
Spironucleus muris is a flagellated protozoan found in the intestinal lumen of mice, rats, and hamsters.  It does not result in clinical signs.  Instead, the infection is identified histologically as accumulation of trophozoites in the intestinal lumen, between villi, and in the crypts of Luberkuhn.
 
Effects on research: (1) caused deaths among irradiated and cadmium-exposed mice, (2) altered macrophage activity and metabolism, and (3) depressed lymphocyte responsiveness to tetanus toxoid in the mouse.
 
Current diagnostic techniques: direct smear of intestinal contents, histological examination of intestinal samples, fecal smears, fecal smear immunochemical techniques, and light and electron microscope
Disadvantages: impractical for screening large numbers of animals due to time constraints, expense, or the need for highly trained personnel; require euthanized animals; poor sensitivity
 
Development of PCR assay: used experimental cull mice from a room naturally infected with S. muris.  The pyloric region of the stomach and proximal duodenum were recovered and the protozoa were identified along with cyst identified from fecal pellets from positive animals.
 
Direct examination, histology, and PCR assay comparison: In all cases the PCR assay identified as positive, samples found to be positive by either direct examination or histology.  
 
Fecal PCR sensitivity: rested on 2 assumptions (1) that any sample identified as positive for S. muris by either direct intestinal examination or histology were truly positive and (2) that samples testing positive by PCR in all 3 triplicate assays were true positives. 
Assay sensitivity= no. of true positives/ (no. of true positives + no. of false negatives) x 100%   
 
Conclusion: The PCR assay developed in this study is rapid, accurate, can easily be incorporated in to a health surveillance program, and the animal does not have to be euthanized.
 
QUESTIONS:
1. 	Name a flagellated protozoan found in the intestinal lumen of mice, rats, and hamsters.
2. 	Name 2 disadvantages of the current diagnostic techniques used to identify S. muris.
3. 	T/F Common clinical signs seen in animals infected with S. muris are dermatitis, excessive scratching, and hair loss.
 
ANSWERS:
1. 	Spironucleus muris 
2. 	Require euthanized animals, time constraints, expensive, poor sensitivity, need for highly trained personnel
3. 	F, does not result in clinical signs

Experimental Techniques
Ordodi et al.  Optimal Access to the Rat Heart by Transverse Bilateral Thoracotomy with Double Ligature of the Internal Thoracic Arteries, pp. 44-46

Primary species: Rattus norvegicus
Domain 3 : Research

SUMMARY: This paper describes a novel surgical approach to the thorax of a rat.  Current approaches include a median sternotomy and left thoracotomies.  Median sternotomies have bleeding from the sternum and internal thoracic arteries as a complication.  Left thoracotomies require exteriorization of the heart and has atelectasis in the left lung and perturbations in pulmonary gas exchange as possible complications.  Both of these approaches can have a mortality rates up to 35%.  An abdominal approach exits that accesses the thoracic cavity through the diaphragm but this approach gives poor visibility of the thorax.  The new techniques described in this paper gives good exposure of the heart and minimizes blood loss.

Description of the Technique: After surgical skin preparation a transverse incision is made at the level of the fifth intercostal space.  The pectoral muscles are dissected to expose the ribs and the 4th, 5th and 6th intercostal spaces.  The 5th intercostal space is incised 3mm from the edge of the sternum on both sides to allow for a blunt-ended surgical scissor to cross the thorax under the sternum.  The scissors are used to slightly raise the sternum while 3-0 vicryl is used to ligate the internal thoracic arteries at the 4th and 6th intercostal spaces.  The sternum can now be transected at the 5th intercostal space without hemorrhaging as a side effect.  The ends of the two ligatures are then pulled to provide excellent exposure of the heart.  The procedure is not technically demanding and can be performed in less then 10 minutes.  Blood oxygenation was compared between control animals, animals receiving a left thoracotomy and animals receiving surgery as described in this paper.  The partial pressure of oxygen in rats receiving a bilateral transverse thoracotomy was close to that of the controls and statistically higher then rats that had received a left thoracotomy.

QUESTIONS:
1. 	What are potential side effects of a) median sternotomy and b) left thoracotomy?
2. 	What is the novel addition of the surgical procedure described in this paper?
3. 	How does the partial pressure of oxygen compare in rats receiving a left thoracotomy versus a bilateral transverse thoracotomy as described in this paper?

ANSWERS:
1. 	a) Median sternotomies have bleeding from the sternum and internal thoracic arteries as a complication.
	b) Left thoracotomies require exteriorization of the heart and has atelectasis in the left lung and perturbations in pulmonary gas exchange as possible complications.
2. 	The novel addition is the ligation of the internal thoracic arteries.
3. 	The partial pressure of oxygen in rats receiving a bilateral transverse thoracotomy was close to that of the controls and statistically higher then rats that had received a left thoracotomy.

Singletary et al.  Modification of a Common BAL Technique to Enhance Sample Diagnostic Value, pp. 47-51

SUMMARY
1	2 different methods of BAL aspiration techniques were evaluated in 20 healthy, anesthetized rhesus macaques (Macaca mulatta), a conventional and a modified method
2	With both methods, a bronchosope was placed into a subsegmental bronchus and a seal maintained
3	The conventional method involved connecting the syringe directly to the biopsy channel of the scope
4	The modified method involved using a 70 cm section of sterile iv extension tubing with male and female ends to connect the syringe to the biopsy channel
5	2 aliquots of 25 ml saline were gently introduced and manually aspirated using the syringe
6	The modified method led to an 8.3% increase in fluid yield and a higher concentration of cells recovered without altering typical cellular ratios
7	No differences between heart rate, oxygen saturation or rectal temperatures were seen
8	In addition, more precise control of the syringe-generated suction, and easier syringe changing were noted, and increased operator comfort resulted
9	Advantages include
o	More fluid is recovered using the same amount of instillate
o	Enhance cell counts resulted without changes to differential cell count percentages
o	Increasing the cell concentration creates a more valuable diagnostic or research sample
o	Using less lavage fluid, decreasing turbulent flow and controlling suction more precisely most likely will reduce BAL associated tissue damage and morbidity
o	More control and improved operator comfort and better ergonomics resulted

QUESTIONS:
1. 	List 4 advantages to using the modified BAL technique described in this paper compared to conventional BAL.
2. 	Instillation of 50 cc of saline during modified BAL in rhesus macaques results in recovery of approximately how much fluid?
a.	5 to 15 cc
b.	15 to 25 cc
c.	25 to 35 cc
d.	35 to 45 cc

ANSWERS:
1. 	Advantages include:
1	Recovery of more fluid
2	Less residual fluid in lungs after aspiration
3	Higher concentration of cells in the fluid
4	More precise control over suction and syringe manipulations
5	Reduced hand, wrist and arm fatigue of the operator
6	Increased operator comfort
7	Possibly less suction-associated lung morbidity
2. 	d) 35 to 45 cc

Foster et al.  Whole-body Plethysmography in African Green Monkeys (Chlorocebus aethiops) with or without Jackets, pp. 52-55

Task 9 - Collaborate on the Selection and Development of Animal Models
Tertiary species: Other Non-Human Primate

SUMMARY: The authors of the study questioned the assumption that whole-body plesthymography (WBP) needs removal of the nonrestraining jacket to dose properly animals. The objective of the study was to determine whether results of WBP performed on a nonhuman primate wearing a nonrestraining jacket differed significantly from those from the same, but nonjacketed, animal.

Nonrestraining jackets are worn to avoid damage to indwelling central venous catheters used to facilitate frequent phlebotomy while minimizing stress and anesthetic effects on animals. However, nonhuman primates with central venous catheters must wear protective jackets. Removal of the jacket could dislodge the catheter making it unusable. The authors compared: minute volume, tidal volume, respiratory rate, and accumulated volume (cumulative sum of all tidal volumes measured during the collection period). The same animal acted as its own control. During week 1, the animals wore the jackets; the following week, unjacketed animals were analyzed.

The authors did not see any significant changes in measured respiratory parameters for anesthetized green monkeys wearing jackets, which indicate that appropriately sized protective jackets do not restrict the animals’ breathing and do not otherwise interfere with collection of respiratory data. The study standardized the anesthetic (sedation) regime using ketamine-acepromazine. Also, the authors discarded the use of an inhalant anesthetic regimen as this would interfere with the experimental endpoint because nonhuman primates cannot be exposed to an aerosol while breathing through an anesthesia circuit.

The authors conclude that WBP can be performed on African green monkeys wearing protective jackets, with no significant alterations in minute volume, tidal volume, respiratory rate, or accumulated volume.

QUESTIONS:
1. 	True or False. Appropriately sized protective jackets do not restrict the animals’ breathing and do not otherwise interfere with collection of respiratory data.
2. 	True or False. An inhalant anesthetic regimen would be ideal as nonhuman primates can be exposed to an aerosol while breathing through an anesthesia circuit.

ANSWERS:
1. 	True
2. 	False

CASE REPORTS
Lammey et al.  Diagnosis and Treatment of Pulmonary Arterial Hypertension and Atrial Fibrillation in an Adult Chimpanzee (Pan troglodytes), pp. 56-60

Task 1 - Prevent, Diagnose, Control, and Treat Disease
Species: Pan troglodytes (Tertiary-Other Nonhuman Primates)

SUMMARY: Cardiovascular disease is common in chimps, but the diagnosis and treatment has not been well described in the literature.  Pulmonary arterial hypertension (PAH) can be either primary (an inherited condition affecting small and medium arteries in humans) or secondary to a variety of conditions such as chronic obstructive pulmonary disease, pneumonia, pulmonary neoplasia, or heartworm in dogs.  This article describes a case of PAH in a 22-year-old male chimp who presented with multiform premature ventricular contractions and hepatomegaly on routine exam.  Echocardiography was performed 10 months later (he was mildly azotemic at that time), which showed severe right heart dilation and ventricular hypertrophy (right > left).  The pulmonary artery was dilated and there was regurgitation occurring at the tricuspid and pulmonic valves.  Pulmonary artery pressure was severely elevated.  A diagnosis of idiopathic pulmonary hypertension was made.  The animal was started on famotidine and aspirin.

Six months later, the animal presented with ascites, and physical exam revealed a heart murmur, tachycardia with an irregularly irregular rhythm consistent with atrial fibrillation, which was confirmed with ECG.  Echocardiography was mostly unchanged from previous, although pericardial and pleural effusions were seen.  The animal was started on furosemide, digoxin and enalapril, as well as sildenafil citrate, and the aspirin dose was increased.  In the next 48 hours, the animal began demonstrating signs of congestive heart failure, including coughing, increased respiratory effort, and cyanosis.  However, he was improved by 72 hours and by 96 hours was eupneic but with persistent ascites scrotal edema.  He was placed on a low-sodium diet with close monitoring.  

Two months later, the ascites and scrotal edema appeared worse, and hydrochlorothiazide and spironolactone therapies were initiated, which improved the animal’s condition within a week.  Three months later, the animal was found unconscious 5 minutes after distribution of enrichment, and attempts at resuscitation were unsuccessful.  Gross necropsy findings supported the echocardiographic and physical examination results.  Histopathology findings included interstitial myocardial fibrosis and myocytes hypertrophy; hemosiderin-laden alveolar macrophages; congestion and fibrosis of the liver.  This is the first case report of diagnosis and treatment of PAH and a-fib in a chimp.

QUESTIONS
1.	PAH leads to failure of ______-sided heart failure
2.	What is the common name for hemosiderin-laden alveolar macrophages?
3.	Name the type of drug for each of the medications described (i.e. diuretic, NSAID, etc):
a.	Furosemide
b.	Digoxin
c.	Enalapril
d.	Sildenafil (aka Viagra)
e.	Aspirin
f.	Hydrochlorothiazide
g.	Spironolactone 

ANSWERS
1.	Right
2.	Heart failure cells
3.	a) diuretic, b) cardiac glycoside (positive inotrope), c) ACE inhibitor, d) PDE5 inhibitor, e) NSAID, f) diuretic, g) diuretic

Fremont et al.  Acute Respiratory Distress Syndrome in Two Rhesus Macaques (Macaca mulatta), pp. 61-66

SUMMARY: ARDS can be initiated by indirect or direct pulmonary injury.  Indirect causes include: sepsis, severe trauma, blood transfusions, acute pancreatitis, drug overdose and shock.  Direct causes include: viral, bacterial, and fungal pneumonia, near drowning, toxin or gas inhalation, aspiration, pulmonary contusions and other lung injury.  ARDS is composed of 2 phases, “exudative phase” which occurs 24-48 hrs post-insult and “fibrinoproliferative phase” which occurs 48 hrs after insult.   The pathogenesis of ARDS is incompletely understood but involves excessive stimulation of inflammatory pathways leading to increased vascular permeability - > edema and emphysema, increased formation and deposition of fibrin causing microvasculature to clot, and inflammatory mediators neutralize surfactant leading to atelectasis.  Physiologically, ARDS is characterized by severe hypoxemia.  Treatment is supportive and can be unrewarding. 

This paper describes 2 ARDS in 2 rhesus monkeys that developed complications after experimental craniotomy.  The first was attributed to polymicrobial suppurative menigoencephalitis accompanied by marked neutrophilia with left shift.  The second monkey had focally severe neurodegenerative changes in the occipital lobe (site of craniotomy).  Both animals had progressive dyspnea and pulmonary edema and syndrome defining histopath criteria including edema with intra-alveolar neutrophils, fibrinohemorrhagic effusions with crescentic membranes and interstitial vascular degeneration. 

The paper discusses treatment options and discusses the need to aggressively address the underlying disease process in addition to providing supportive care.  Supportive treatment includes mechanical ventilation, IV fluid therapy, sedatives, diuretics, bronchodilators, respiratory stimulants, intra-bronchial surfactant administration, corticosteroids and prone positioning.   

This is the first case report of ARDS in rhesus macaque.  The authors assert that in case 2, ARDS was not the cause of death and was an incidental finding.  They suggest that ARDS may occur more frequently than is noted.

QUESTIONS:
1.  	Morphine is suggested as a good sedative in ARDS as it is reported to mobilize pulmonary fluid.  What is a potential side effect of opioids in non-human primates?
2. 	List the histopath characteristics of the exudative phase of ARDS.
3. 	List the modes of action of bronchodilators such as aminophylline that are beneficial in treating ARDS. 

ANSWERS:
1. 	Severe facial pruritus
2. Diffuse protein-rich pulmonary edema, variable numbers of neutrophils, alveolar hemorrhage with atelectasis.  Alveolar edema organizes with fibrin and hyaline membrane formation
3. 1) diuretic activity; 2) counteracts bronchospasm; 3) enhance mucocilliary function; 4) decrease diaphragmatic fatigue; and 5) decrease pulmonary edema