bronchoalveolar lavage analysis using urea dilution ......the respiratory system includes the mouth,...
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Bronchoalveolar lavage analysis using urea
dilution standardisation in diagnosis of respiratory
diseases in dogs
Amanda Elouise Helen Paul
BSc(Hons) BVSc(Hons) MVetSt
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I declare that this thesis is my own account of my
research and contains as its main content work which
has not previously been submitted for a degree at any
tertiary education institution.
………………………………………………….
Amanda E.H Paul
This thesis is presented for the degree of
Research Masters with Training, 2016
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I would like to thank my supervisors Dr Caroline Mansfield and Dr Peter Irwin for
their guidance and support, Dr Anthea Raisis in thesis preparation, and all the staff
of Murdoch University Clinical Pathology Laboratory for the assistance in
processing bronchoalveolar lavage samples.
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Abstract:
Differentiation of various chronic respiratory diseases may be made via analysis of
bronchoalveolar lavage (BAL) fluid using urea concentration of BAL relative to blood
urea concentration as a marker of dilution of pulmonary epithelial lining fluid (PELF).
Assessment of cell counts after adjusting for dilution may allow differentiation of the
primary disease process in dogs presenting with respiratory signs. Considerable
variation has been reported in total cell counts and concentration of biochemical
markers due to variable recovery of PELF in BAL fluid. A number of chronic
respiratory conditions can be difficult to diagnose definitively and accounting for
dilution of PELF may allow us to better differentiate respiratory disease.
Client-owned dogs presenting for investigation of respiratory disease were included.
All dogs had a BAL performed and BAL cell counts were corrected after using urea
as a marker for dilution and comparison of urea in blood to that of urea in BAL fluid.
A final diagnosis of respiratory disease was made after retrospective analysis of all
diagnostic investigations and response to treatment.
Seventy two BAL samples from a total of 48 dogs were analysed and thirteen
primary causes of respiratory disease identified based on diagnostic investigation
including BAL cell cytology and treatment response. Respiratory diseases were also
assigned to inflammatory, non-infectious, infectious, upper respiratory tract or
respiratory neoplasia categories based on the disease diagnosed. There was no
statistical difference in the adjusted total cell counts of BAL fluid (BALF) from dogs
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with different respiratory diseases or disease groups. Mycoplasma spp had no
effect on the total cell count in dogs with chronic bronchitis.
This study suggests total cell counts of BAL fluid corrected for dilution by urea
concentration cannot be used to distinguish between different respiratory diseases.
A larger number of cases and cross section of respiratory disease may further
identify significant differences in total and differential cell counts of various different
diseases.
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Contents
List of abbreviations ................................................................................................ 11
Chapter 1 Literature Review ................................................................................... 13
1.1 Introduction .................................................................................................... 13
1.2 Anatomy of the Respiratory System .............................................................. 13
1.3 Investigation of respiratory disease ............................................................... 17
1.3.1 History and Physical Examination ........................................................... 17
1.3.2 Initial diagnostic evaluation ..................................................................... 21
1.3.3 Radiology ................................................................................................ 22
1.3.4 Advanced Imaging .................................................................................. 23
1.3.5 Serum Natriuretic peptide concentration ............................................. 27
1.3.6 Serology in diagnosing respiratory disease ............................................ 28
1.3.7 Bronchoscopy and bronchoalveolar lavage ............................................ 30
1.3.8 Culture of bronchoalveolar lavage fluid ................................................... 34
1.3.9 Cytology of the bronchoalveoli ................................................................ 36
1.4 Respiratory cytology and characterisation of abnormal cytology ................... 40
1.4.1 Biochemical analysis of Bronchoalvolar lavage fluid ............................... 44
1.4.2 Standardisation of components of respiratory lavage fluid in animals .... 46
1.4.3 Exogenous markers used in the assessment of bronchoalveolar lavage
fluid .................................................................................................................. 47
1.4.3.1 Inulin 47
1.4.3.2 Methylene Blue 48
1.4.3.3 Technetium-99m diethlenetriaminepenta-acetic acid 48
1.4.4 Endogenous markers of PELF dilution.................................................... 49
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1.4.4.1 Albumin ............................................................................................. 49
1.4.4.2 Electrolytes 51
1.4.4.3 Urea 52
1.4.5 Urea standardisation of bronchoalveolar lavage fluid in dogs ................. 53
1.5 Conclusion ..................................................................................................... 56
Chapter 2 Aims and hypothesis of the study ........................................................... 57
2.1 Aims .............................................................................................................. 57
2.2 Hypothesis ..................................................................................................... 57
Chapter 3 Materials and Methods ........................................................................... 58
3.1 Case acquisition ............................................................................................ 58
3.2 Bronchoalveolar lavage and collection technique .......................................... 58
3.3 Blood urea measurement .............................................................................. 59
3.4 Bronchoalveolar lavage fluid processing ....................................................... 60
3.4.1 Urea concentration in Bronchoalveolar lavage fluid ................................ 60
3.4.2 Bronchoalveolar lavage fluid cell count ................................................... 61
3.4.3 Cytology of bronchoalveolar lavage fluid ................................................ 61
3.4.4 Culture of bronchoalveolar lavage fluid ................................................... 61
3.4.5 PCR of bronchoalveolar lavage fluid ....................................................... 62
3.5 Data Processing ................................................................................................ 62
3.5.1 Calculation of epithelial lining fluid recovery ........................................... 62
3.5.2 Standardisation of cell count ................................................................... 62
3.6 Diagnosis ....................................................................................................... 63
3.7 Characterisation of Respiratory Disease Groups .......................................... 65
3.8 Statistical Assessment................................................................................... 65
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Chapter 4 Results ................................................................................................... 66
4.1 Dogs .............................................................................................................. 66
4.2 Diseases Diagnosed ..................................................................................... 66
4.3 Epithelial Lining Fluid Recovery .................................................................... 68
4.4 Absolute and relative cell counts for each respiratory disease ...................... 69
4.4.1 Chronic bronchitis ................................................................................... 69
4.4.2 Aspiration Pneumonia ............................................................................. 72
4.4.3 Bronchointerstitial Pneumonia- non infectious ........................................ 74
4.4.4 Non- cardiogenic oedema ....................................................................... 76
4.4.5 Bacterial Pneumonia ............................................................................... 78
4.4.6 Pulmonary Fibrosis ................................................................................. 80
4.4.7 Neoplasia- metastatic and primary pulmonary carcinoma ...................... 82
4.4.8 Systemic Immune Mediated Disease ...................................................... 84
4.4.9 Laryngeal Paralysis................................................................................. 86
4.4.10 Laryngeal Collapse ............................................................................... 88
4.4.11 Sterile pyogranulomatous disease ........................................................ 90
4.4.12 Phaeochromocytoma ............................................................................ 91
4.4.13 Pulmonary carcinoma ........................................................................... 92
4.5 BALF cytology from dogs with airway collapse and chronic bronchitis -
tracheal or bronchial ............................................................................................ 93
4.6 Effect of Mycoplasma spp. on respiratory disease ........................................ 96
4.6.1 Chronic bronchitis ................................................................................... 97
4.6.2 Pulmonary Fibrosis ................................................................................. 99
4.6.3 Bacterial pneumonia ............................................................................. 100
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4.6.4 Non- cardiogenic oedema ..................................................................... 101
4.7 Respiratory Disease Groups ....................................................................... 103
4.7.1 Neoplasia .............................................................................................. 104
4.7.2 Upper Respiratory Tract Disease .......................................................... 105
4.7.3 Infectious disease ................................................................................. 106
4.7.4 Non-infectious disease .......................................................................... 107
4.7.5 Inflammatory disease category ............................................................. 108
4.8 Analysis Summary ....................................................................................... 109
4.8.1 Analysis between specific respiratory diseases .................................... 109
4.8.2 Assessment of broad respiratory groups .............................................. 114
Chapter 5 Discussion ............................................................................................ 118
5.1 Diagnosis of respiratory disease processes ................................................ 118
5.2 Pulmonary Epithelial Lining Fluid Recovery ................................................ 121
5.3 Total Cell Counts ......................................................................................... 122
5.4 Differential Cell Counts ................................................................................ 125
5.5 The effect of Mycoplasma spp on total and differential cell counts .............. 127
5.6 Effect of dynamic airway collapse on cell counts ......................................... 128
5.7 Conclusion ................................................................................................... 129
References............................................................................................................ 130
APPENDICES ....................................................................................................... 153
Appendix 1: Tests used for Respiratory Diagnosis ............................................ 154
Appendix 2: Raw and processed Bronchoalveolar lavage fluid data ................. 165
Appendix 3: Signalment of dogs included ......................................................... 181
Appendix 4: Mycoplasma spp. diagnosis in dogs included ................................ 186
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Appendix 5: Classification of diseases diagnosed and presence of Mycoplasma
spp. ................................................................................................................... 191
Appendix 6: Disease Group cell counts ............................................................. 198
Appendix 7: Statistical P values of respiratory disease when comparing all
respiratory diseases .......................................................................................... 204
Appendix 8: Statistical analysis of broad disease category groups ................... 205
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List of abbreviations
ANA
ARDS
Antinuclear Antibody
Acute respiratory distress syndrome
BAL Bronchoalveolar Lavage
BALF
BP
Bronchoalveolar Lavage Fluid
Blood Pressure
CAV Canine adenovirus
CBC
CFU
Complete Blood count
Colony forming units
CIRD Chronic Infectious Respiratory Disease
CIV Canine influenza virus
CNS Central Nervous System
CP Cytospin pellets
CPIV Canine parainfluenza virus
CRCoV
CSF
Canine Respiratory Coronavirus
Cerebrospinal fluid
CT
ECG
Computed Tomography
Electrocardiography
ELF Epithelial Lining Fluid
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FiO2 Fraction of inspired oxygen
ILD Interstitial Lung Disease
IQR Interquartile range
LDHp Pleural fluid lactate dehydrogenase
concentration
MRI Magnetic Resonance Imaging
MSP Manually smeared pellets
NaCl Sodium chloride
NT-proBNP Amino terminal-pro-B-type natriuretic
peptide
PELF Pulmonary Epithelial Lining Fluid
PTE Pulmonary thromboembolism
RT-PCR Reverse transcriptase-polymerase chain
reaction
SD Standard deviation
SLE Systemic Lupus Erythematosus
TPr
TCC
UPC
Pleural fluid/ serum total protein ratio
Transitional cell carcinoma
Urine protein concentration
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Chapter 1 Literature Review
1.1 Introduction
Respiratory disease can represent varied conditions such as inflammatory,
neoplastic, infectious and fibrotic airway disease (Silverstein and Drobatz, 2010).
Additionally, disease altering the haemodynamic status of dogs can result in signs
such as increased respiratory rate (tachypnoea) with no structural abnormality
present within the respiratory tract. Respiratory diseases can be present in the
upper airways including the nasal cavity, pharynx and larynx; the lower airways
including the trachea and bronchi; and the lung parenchyma. Respiratory disease
can be challenging to diagnose and manage in dogs as there are very few tests
available that diagnose specific respiratory disorders. A review of the approach
used to investigate respiratory diseases, and the uses and limitations of available
diagnostic tests is provided below.
1.2 Anatomy of the Respiratory System
When diagnosing respiratory disease, it is important to understand the anatomy of
the respiratory system as different diseases can be specific to different components
of the respiratory system. Furthermore when assessing BALF, it is important to
recognise that PELF is produced by the alveoli and thus may be an indicator of
disease affecting terminal airways (Creevy, 2009).
The respiratory system includes the mouth, nose, trachea, lungs and smaller
airways including the bronchi and bronchioles and terminal alveoli (West, 2008).
The left and right sides of the canine lungs are invaginated into a corresponding
pleural sac and are free floating, except at the dorsal root where they are attached
to the mediastinum and the caudal root of the lung where the pulmonary ligament
attaches to the diaphragm (Figure 1). Lungs are normally kept expanded by air
pressure within the respiratory tree. The airways consist of a series of branching
tubes, which become narrower, shorter and more numerous. The trachea extends
distally from the larynx and divides into main bronchi which then divide into lobar
then segmental bronchi, then terminal bronchioles. The terminal bronchioles divide
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into respiratory bronchioles which become alveolar ducts and have alveoli lining
their walls (Dyce et al, 1987).
Figure 1: A Medial Surface of the right lung of the dog;
B: Caudal view of the base of the lungs and heart of the dog
1. Trachea 2. Cranial lobe 3. Middle lobe 4. Caudal lobe 5. Accessory lobe
7. Oesophagus 8. Heart 9. Tracheobronchial lymph node 10. Area of
pulmonary ligament
(from Dyce et al, 1987)
Alveoli function in gas exchange where gas moves chiefly by diffusion (West, 2008).
The alveoli are lined by fluid which creates surface tension and subsequently
generates forces which tend to collapse the alveoli. Pneumocytes lining the alveoli
produce surfactant into the PELF which lowers the surface tension of the alveolar
lining layer and provides stability to the alveolus (West, 2008).The alveolar septum
separates two alveoli in lung tissue (Figure 2). On one side of the alveolar septum,
the epithelial and endothelial basement membranes are separated by a space of
variable thickness containing connective tissue fibrils, elastic fibers, fibroblasts, and
macrophages. This connective tissue is the backbone of the lung parenchyma. It
forms a continuum with the connective tissue sheaths around the conducting
airways and blood vessels. Thus, the pericapillary perialveolar interstitial space is
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continuous with the interstitial tissue space that surrounds terminal bronchioles and
vessels, and both spaces constitute the connective tissue space of the lung. There
are no lymphatics in the interstitial space of the alveolar septum. Instead, lymphatic
capillaries first appear in the interstitial space surrounding terminal bronchioles,
small arteries, and veins (Benumof, 2013).
The opposite side of the alveolar septum contains only fused epithelial and
endothelial basement membranes. The interstitial space is thus greatly restricted on
this side owing to fusion of the basement membranes. Interstitial fluid cannot
separate the endothelial and epithelial cells from one another, and as a result the
distance barrier to fluid movement from the capillary to alveolar compartment is
reduced and is composed only of the two cells and their associated basement
membranes (Benumof, 2013; West, 2008).
Between the individual endothelial and epithelial cells are junctions that provide a
potential pathway for fluid to move from the intravascular space to the interstitial
space and finally from the interstitial space to the alveolar space. The junctions
between endothelial cells are relatively large and termed loose; the junctions
between epithelial cells are relatively small and therefore termed tight. Pulmonary
capillary permeability is a direct function of the size of the holes in the endothelial
and epithelial linings. Fluid exchange occurs across the capillary endothelium and
obeys Starling’s law (Benumof, 2013; West, 2008). Fluid leaving the capillaries
leaks into the interstitium of the alveolar wall and tracks through the interstitial
space to the perivascular and peribronchial space within the lung. Interstitial fluid is
normally removed from the alveolar interstitial space into the lymphatics by a
pressure gradient mechanism. This is caused by the presence of the relatively
greater negative pressure surrounding the larger arteries and bronchi. The pressure
gradient is aided by the presence of valves in the lymph vessels. In addition,
because the lymphatics run in the same sheath as the pulmonary arteries, they are
exposed to the massaging action of arterial pulsations. The differential negative
pressure, the lymphatic valves, and the arterial pulsations all help to propel the
lymph proximally toward the hilum through the lymph nodes (Benumof, 2013).
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Lymphatics traverse in the perivascular spaces and transport fluid to the hilar lymph
nodes (Figure 2). Lymph then drains to the tracheobronchial and mediastinal lymph
nodes.
Figure 2: Possible paths for fluid that moves out of pulmonary capillaries in
the alveolar septum. 1. Fluid that enters the interstitium initially finds its way
into the perivascular and peribronchial spaces. 2. Fluid may cross the
alveolar wall filling alveolar spaces. (from West et al, 2008)
Most of the lung tissue is comprised of the bronchi, pulmonary vessels and
peribronchial and perivascular connective tissue (Dyce et al, 1987). Pulmonary
blood vessels form a series of branches from the pulmonary artery, to the capillaries
and back to pulmonary veins. There is significant anatomical variation of the origin
of the bronchial arteries in dogs. The bronchial artery could be branch of the right
5th, 6th or 7th intercostal artery which arises from the thoracic aorta. The course
followed by the bronchial artery is also subject to considerable variation. In the
majority of dogs, the bronchoesophageal artery crosses the left side of the
oesophagus and contributes an oesophageal branch before entering the hilum of
the lung (Evans and De Lahundra, 2013). In addition, small bronchial vessels that
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supply the hilum of the lung and arise from the pericardiophrenic or internal thoracic
arteries can occur. At the level of the respiratory bronchiole the bronchial artery
terminates in a capillary bed that is continuous with that of the pulmonary artery
(Kotoulas et al, 2014). The capillaries form a dense network in the walls of alveoli,
so that an almost continuous sheet of red blood cells in the alveolar wall exists for
efficient gas exchange (West, 2008). Capillary blood is separated from alveolar gas
by a series of anatomic layers: capillary endothelium, endothelial basement
membrane, interstitial space, epithelial basement membrane, and alveolar
epithelium (of the type I pneumocyte). The pulmonary arteries receive the whole
output from the right heart and generally follow the bronchi, while pulmonary veins
can run separately, alternating in position with the bronchoarterial associations.
Bronchial blood flow returns to the left side of the heart by pulmonary veins.True
bronchial veins are found only at the hilum of the lung. They empty into the azygos
vein or the intercostal vein at the level of the seventh thoracic vertebra (Dyce et al,
1987; West, 2008; Evans and De Lahundra, 2013).
1.3 Investigation of respiratory disease
1.3.1 History and Physical Examination
History
Accurate history taking in diagnosis of respiratory disease in dogs is often
problematic. Owners are often only vaguely aware of their pet’s illness and in many
cases, history can be incomplete and often missing the early clinical and historical
signs in chronic illness. However, indicators for investigation can be gained from the
signalment, environment, geographical region, travel and medical history of the dog
(Silverstein and Drobatz, 2010).
Signalment can also provide direction for investigation. Juvenile dogs are more
likely to contract infectious conditions (Jordan et al, 1993; Nolan and Smith, 1995).
Brachycephalic dogs are more likely to present with anatomical defects including
hypoplastic trachea, stenotic nares and everted laryngeal saccules (Ettinger, 2010).
Breed dispositions also have been described for respiratory disease such as
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idiopathic pulmonary fibrosis in West Highland White and Staffordshire Terriers
(Corcoran et al, 1999; Reinaro and Cohn, 2007; Syrä et al, 2013) and primary
ciliary dyskinesia in Dobermans, Bichon Frise and English Springer Spaniels for
example (Cohn, 2010; Crager, 1992; Edwards et al, 1992; Ettinger, 2010).
Travel history and geographical region are important components of a thorough
history. Some infectious agents have specific environmental niches and
geographical locations including blastomycosis in the Ohio River Valley or
coccidiomycosis in the southwest United States of America (Schmeidt et al, 2006;
Silverstein and Drobatz, 2010). Heartworm (Dirofilaria immitis) is common in
northern tropical –temperate regions of Australia and less common in cooler
southern climates of Australia (Carlisle and Atwell, 2008; Starr and Mulley, 1988).
As clinical signs of respiratory disease can be present with infection of Dirofilaria
immitis, this should also be considered in diagnosis of respiratory disease (Carlisle
and Atwell, 2008; Starr and Mulley, 1988).
Access to toxins including anticoagulants, smoke exposure and new environments
can also trigger airway reactions or pathology, and so knowledge of the dog’s
environment is helpful in determining respiratory disease (Silverstein and Drobatz,
2010).
Physical Examination
Physical examination may assist in location of lesions, but is a non- specific and
often a poorly sensitive diagnostic tool (Padrid, 2000; Silverstein and Drobatz,
2010). Respiratory rate and/or effort can often be increased in respiratory disease
however, hyperthermia, anxiety, cardiovascular disease, abdominal enlargement,
opioid administration, aspiration of respiratory irritants and light anaesthesia can
also cause these changes (Fine et al, 2008; Padrid, 2000).
Mucus membrane colour can identify cyanosis indicative of severe respiratory
compromise and hypoxemia which can occur in both respiratory and cardiac
disease; pallour can be associated with pain or reduced circulating blood volume; or
mucus membranes can have a dark red ‘injected’ appearance consistent with
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dehydration or septicemic shock, and are thus a poor localizing physical
examination finding (Silverstein and Drobatz, 2010).
Observation of inspiratory or expiratory difficulty can help to differentiate the location
of disease. Inspiratory dyspnoea can indicate an upper airway obstruction, pleural
space disease or a mediastinal mass (Sigrist et al, 2011). Expiratory dyspnoea is
often an indication of bronchoconstriction as occurs with allergic or non- infectious
inflammation (Silverstein and Drobatz, 2010). Disease of the lung parenchyma or
trachea can present with both inspiratory and expiratory dyspnoea (Silverstein and
Drobatz, 2010). Conversely, coughing is not specific for pulmonary disease and
may be a manifestation of either pulmonary or cardiovascular disease (Carlisle and
Atwell, 2008; Noble et al, 2011; Starr and Mulley, 1988).
Coughing is an important defensive reflex that enhances clearance of secretions
and particulates from the airways and protects from aspiration of foreign materials
occurring as a consequence of aspiration or inhalation of particulate matter,
pathogens, accumulated secretions, post-nasal drip, inflammation, and mediators
associated with inflammation (Polverino et al, 2012). Differences among several
sites from which cough stimuli can originate may result in variations in the sounds
and patterns of coughing. Sensory afferents are found in the upper airways to the
terminal bronchioles and lung parenchyma (Evans and De Lahundra, 2013). The
specific pattern of the cough depends on the site and type of stimulation (Polverino
et al, 2012; West, 2008). Mechanical laryngeal stimulation results in immediate
expiratory stimulation to protect the airway from aspiration. Stimulation distal to the
larynx causes a more prominent inspiratory phase, presumably to generate the
airflow necessary to remove the stimulus. Lesions that compress the upper airway,
including arteriovenous malformations and retrotracheal masses, may present with
chronic cough (Glumez et al, 1999; Park et al, 2004; West, 2008). Cough can also
be a symptom of tracheobronchomalacia, which results from loss of rigid support of
the large airways and inspiratory collapse, and can be seen in conjunction with
obstructive lung disease (Johnson and Pollard, 2010; Polverina et al, 2012; Tagner
and Hobson, 1982). Laryngeal stimulation produces a choking type of cough without
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a preceding inspiration. Inadequate mucociliary clearance mechanisms (as in
bronchiectasis or pulmonary fibrosis) may produce a pattern of coughing with less
violent acceleration of air and a sequence of interrupted expirations (Silverstein and
Drobatz, 2010; West, 2008)
Thoracic auscultation can detect crackles and wheezes indicative of respiratory
pathology, and percussion can detect changes in resonance indicative of respiratory
disease. However, an absence of abnormal respiratory sounds does not imply the
absence of respiratory disease (Silverstein and Drobatz, 2010).
Respiratory auscultation can also detect muffled or diminished breath sounds which
when auscultated in the ventral thorax can indicate presence of pleural fluid (Ludwig
et al, 2010). An asynchronous or inverse (restrictive) breathing pattern and
decreased lung auscultation was found to be significantly associated with pleural
space disease in dogs in a study by Sigrist et al (2011). The combination of
breathing pattern and lung auscultation was highly sensitive (99%) but not very
specific (45%) for diagnosis of pleural space disease (Sigrist et al, 2011).
When diminished breath sounds are auscultated in the dorsal lung fields, this is
more suggestive of accumulation of air in the pleural space (Silverstein and
Drobatz, 2010).
Increased adventitial sounds such as crackles or wheezes can sometimes be
auscultated. Localisation of these adventitial sounds can sometimes identify diffuse
or focal disease. Presence of adventitial sounds in the cranioventral or right middle
lung lobes is suggestive of aspiration pneumonia (Schulze and Rahilly, 2012).
Adventitial sounds auscultated in the perihilar region, is supportive of cardiogenic
pulmonary oedema and diffuse adventitial sounds throughout the thorax may
suggest a diffuse pulmonary parenchymal disease such as pneumonia,
inflammatory disease, pulmonary fibrosis or metastatic neoplasia (Corcoran et al,
1999; Heikkilä et al, 2011, Silverstein and Drobatz, 2011). Adventitial sounds that
are loudest in the caudodorsal thoracic fields are suggestive of non-cardiogenic
oedema or pneumonia (Silverstein and Drobatz, 2010).
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1.3.2 Initial diagnostic evaluation
Initial diagnostic evaluation generally consists of a complete blood count (CBC),
biochemistry panel, faecal flotation, urinalysis and arterial blood gas measurement
(McCullough and Brinson, 1999; Padrid, 2000). These tests can help localise a
respiratory disorder and determine the degree of respiratory compromise, and in
some cases identify parasitic disease (Crenosoma vulpis, Angiostrongylus vasorum,
Oslerus osleri) (Taubert et al, 2009; Tebb et al, 2007).
Haematological testing can identify changes to the leukon that reflects the aetiology
of respiratory disease. A leukocytosis with a left shift can indicate severe airway
inflammation or infection and leukopenia is common with sepsis (Schulze and
Rahilly, 2012; Silverstein and Drobatz, 2010). Eosinophilia may be present with
parasitic airway disease, allergic inflammation and eosinophilic
bronchopneumopathy (Clercx et al, 2000; Silverstein and Drobatz, 2010). An
elevated red cell count in dogs with respiratory disease can also be a result of
chronic hypoxia, which stimulates the kidney to produce erythropoietin and
subsequently stimulates erythrogenesis in the bone marrow (Graber and Krantz,
1978).
Serum biochemistry may demonstrate hypoalbuminemia associated with pleural
effusions which can assist in identifying a cause of pleural effusion (Eid et al, 1999).
Pulse oximeters are often used to measure oxygen saturation of haemoglobin which
can determine if animals require oxygen supplementation and give an indication of
respiratory compromise (Ortega, 2011; Silverstein and Drobatz, 2010). However,
pulse oximetry is not without issues to do with accuracy and the ability to obtain a
reading is affected by many factors such as probe placement, pigmented tissue,
tissue thickness, interference by ambient light, hypoperfusion, presence of
carboxyhaemoglobin, methaemoglobin or haemoglobin based oxygen carrier
molecules that have different light absorption properties (Ortega, 2011).
Before pulse oximetry was available to identify hypoxemia, clinicians relied on
invasive procedures, such as arterial puncture for blood gas analysis. The use of
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pulse oximetry reduces the need for arterial blood gas analyses and may allow
titration of the fraction of inspired oxygen (FiO2) in dogs requiring oxygen or
mechanical ventilation (Ortega, et al, 2011), however it does not identify the disease
aetiology.
Arterial blood gas analysis is the most accurate method for measuring the partial
pressure of oxygen in blood and can measure oxygenation, ventilation and acid
base balance (Kerl, 2010). The degree of respiratory impairment can be
determined. Specific diseases cannot be diagnosed, however assessment of pH
may assist in indicating if there is a primary respiratory or metabolic cause (Kerl,
2010; Silverstein and Drobatz, 2010). Respiratory alkalosis can suggest increased
alveolar ventilation with hypoxemia, anxiety, fear, central nervous system (CNS)
disease or primary metabolic acidosis (Kerl, 2010). Respiratory acidosis suggests
inadequate ventilation such as diseases of the pulmonary parenchyma, pleural
space, pulmonary vasculature, chest wall or CNS (Kerl, 2010).
However, advanced diagnostic testing is usually required to make a definitive
diagnosis of specific disease.
1.3.3 Radiology
Thoracic radiography can provide valuable information about lung patterns and
pleural changes in addition to cardiac size and contour, vascular patterns and
thoracic conformation in the dog with respiratory disease (Miller, 2007; Thrall,
1998). The difficulty in obtaining good quality radiographs often impedes
radiological interpretation of respiratory disease in clinical practice, as dogs with
respiratory disease may be poorly compliant and panting during radiography. Poorly
inflated lungs can appear as having increased opacity and structures can appear to
have abnormal size and shape when poorly positioned (Miller, 2007; Thrall, 1998).
Underexposure can lead clinicians to over-interpret lung field markings (Thrall,
1998). Similarly some diseases such as pulmonary thromboembolism (PTE) can
result in any roentgen pattern (Johnson et al, 1999). Three views of the thorax are
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often recommended to maximise lesion detection and to minimise superimposition
of thoracic structures (Miller, 2007; Thrall, 1998).
Radiological descriptors of the lung parenchyma are described as patterns in
relation to suspected histopathological changes. These patterns describe where the
pathologic change is located (bronchioles, interstitium, or alveoli) but do not provide
a definitive diagnosis and can be over-interpreted (Miller, 2007; Myer, 1980).
Many pulmonary diseases may appear as a mixture of the alveolar, interstitial,
vascular, and bronchial radiographic patterns (Johnson et al, 1999; Myer, 1980).
Radiographic appearances can also depend on the disease stage at which thoracic
radiographs are taken (Myer, 1980; Thrall, 1998). For example, in the early stages
of congestive left heart failure, there is an increase in interstitial pulmonary density
surrounding the left atrium (Myer, 1980). As this perivascular oedema progresses, a
subsequent alveolar or mixed alveolar and interstitial pattern is seen (Myer, 1980).
Radiographs of animals with diffuse lung disease are particularly difficult to interpret
as similar findings may be present in many diseases. For example, a nodular
interstitial pattern may represent neoplasia, parasitic infection, atypical bacteria or a
non- infectious inflammatory disease (Thrall, 1998). Conventional radiography can
also have false negative results when investigating pulmonary neoplasia (Hawkins
et al, 1990). Radiography will not detect approximately 25% of canine pulmonary
metastases and generally requires masses to be greater than 3-5mm (Suter et al,
1974; Hawkins et al, 1993; Hawkins et al, 1990).
1.3.4 Advanced Imaging
Computed Tomography (CT) and magnetic resonance imaging (MRI) can assess
lung parenchyma, the mediastinum, thoracic wall, lymph nodes and airways
(Johnson and Wisner, 2007). Computed Tomography can provide a three
dimensional image of the thoracic cavity and its contents, minimise artifact
summation and allow visualisation of images in saggital and axial planes
(Silverstein and Drobatz, 2010).
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Computed Tomography has increased sensitivity for detection of smaller masses in
the pulmonary structures not detected by radiography as well as pulmonary
thromboemboli (PTE) (Goggs et al, 2009; Johnson and Wisner, 2007).
Although conventional radiography is still the first diagnostic imaging approach to
respiratory disease, CT is invaluable as an adjunctive procedure in characterizing
nasal and thoracic pathologic changes. Computed Tomography eliminates
superimposition of overlying structures and offers superior contrast resolution as
compared with conventional radiography (Naidich et al, 1990). These advantages of
thoracic CT allow for more precise characterisation and localisation of lesions and
are invaluable for guiding rhinoscopic, bronchoscopic, and surgical procedures.
Thoracic CT may also be more accurate as an aid in the diagnosis of canine chronic
bronchitis as individual bronchi can be more easily seen and more accurately
characterized (Johnson and Wisner, 2007). Additionally, bronchial wall thickness
can be directly measured on a computer workstation and the peribronchial and
interstitial tissues can be more accurately assessed for pathologic change (Johnson
and Wisner, 2007). Bronchiectasis can also be detected earlier in the course of
disease than when conventional radiographs are used (Johnson and Wisner, 2007).
Recently high resolution CT has been used to evaluate pulmonary fibrosis in the
dog and this has increased the positive predictive value of achieving a diagnosis
(Johnson and Wisner, 2007).
Pneumothorax is readily diagnosed using conventional radiography, however the
inciting abnormality is not easily recognised in dogs, with the exception of traumatic
and iatrogenic pneumothorax (Au et al, 2006). Because the underlying cause of
pneumothorax has an impact on treatment and prognosis, CT is often indicated in
this subset to increase the sensitivity of identifying the underlying cause of
pneumothorax (Au et al, 2006).
Pleural effusions can also be assessed with CT. Imaging of pyothorax by CT that is
poorly responsive to conventional management techniques, and when a pleural or
pulmonary foreign body or abscess is suspected, can be useful due to the cross-
sectional nature of CT. Superimposition and silhouetting of structures that occurs on
25
thoracic radiographs caused by loculated or residual pleural fluid is eliminated
(Johnson and Wisner, 2007). Assessment of chylothorax with CT and direct
lymphangiography by ultrasound-guided mesenteric lymph node injection of
iodinated contrast material to achieve thoracic duct opacification provides excellent
contrast enhancement of the thoracic duct. Computed tomography also documents
dilated, tortuous, cranial mediastinal lymphatics which can be used for planning of
treatment modalities and techniques (Johnson et al, 2006).
Computed Tomography-angiography is used in veterinary medicine to detect PTE
(Goggs et al, 2009; Saunders and Keith 2004). Computed Tomography-
angiography may directly identify the pulmonary embolus as an intraluminal filling
defect and enables identification of other conditions that may cause similar
presenting signs (Mullins et al, 2000; Rathbun et al, 2000). Two recent studies in
people demonstrated consistently high sensitivity (87%) and specificity (91%) in the
diagnosis of PTE (Mullins et al, 2000; Rathbun et al, 2000). In experimentally
induced PTEs in dogs, the thrombus was detected in 64% to 76% of the dogs
(Woodward et al, 1995).
Magnetic Resonance Imaging (MRI) is based on nuclear magnetic resonance
principles that reflect chemical and physical characteristics of tissues examined
using absorption and emission of energy in the radiofrequency range of the
electromagnetic spectrum (Silverstin and Drobatz, 2010). In the thorax, MRI has
been used to assess tumors invading the chest wall and for mediastinal and cardiac
tumours (Fredericks et al, 2008). For pulmonary imaging, MRI is perceived as
inferior to CT imaging (Fredricks et al, 2008) however recent advancements in MRI
systems including improved pulse sequences, utilization of contrast media such as
hyperpolarized noble gases and new diffusion techniques have made MRI an
increasingly important tool for lung cancer staging in people (Erasmus et al, 2005;
Fredericks et al, 2008; Ohno et al, 2007). Reports have indicated the ability of MRI
to reveal invasion of the mediastinum by tumours and to help identify hilar and
mediastinal nodal metastases (Erasmus et al, 2005; Ohno et al, 2007).
A fluoroscopic study can be used to evaluate a coughing dog for the presence and
26
location of airway collapse (Maggiore, 2014). When radiography was compared
with fluoroscopy, radiographic evidence of collapse was at the incorrect location in
44% of dogs and it was not detected in 8% of dogs with radiographs alone.
(Macready et al, 2007). Fluoroscopic identification of lower airway collapse versus
tracheal collapse can be important in determining therapy (Maggiore, 2014).
27
1.3.5 Serum Natriuretic peptide concentration
Natriuretic peptides may help to differentiate congestive heart failure (CHF) from
primary pulmonary disease. This is useful as respiratory distress due to primary
pulmonary disease and congestive heart failure can be difficult to distinguish as the
clinical signs of CHF often are nonspecific. Cough and dyspnea, which are common
clinical signs associated with CHF, are also present in dogs with primary respiratory
disease or neoplasia (DeFrancesco et al, 2007). Natriuretic peptides are
structurally related hormones that include amino terminal-pro-B-type natriuretic
peptide (NT-proBNP) which is released into the circulation from cardiac myocytes in
response to ventricular dysfunction (Maack, 2006; MacDonald et al, 2003).
Assessment of NT-proBNP concentrations has been used as a tool to discriminate
between congestive heart failure and primary pulmonary disease in dogs as is
shown in Table One (Boswood et al, 2008; Fine et al, 2008; Oyama et al 2009).
Table 1: Circulating serum NT-proBNP in dogs with respiratory distress
attributable to congestive heart failure or primary pulmonary disease.
CHF: Congestive heart failure, IQR: Interquartile range 25-75%
Fine et al, 2007
Oyama et al,
2009
Boswood et al,
2008
CHF 2554 pmol/L
(IQR1651.5-3475.5)
2445 pmol/L
(IQR 449-3134)
1700 pmol/L
Range 186-9280
Primary Pulmonary
Disease
357 pmol/L
(IQR 192.5-565.5)
413 pmol/L
(IQR 245-857)
113 pmol/L
Range 42-362
CHF and Respiratory
disease
478pmol/L
(IQR 323-1158)
Heart disease with no
CHF
468 pmol/L
Range <42- 3980
28
The problems in studies to date show that the difference in median serum NT-
proBNP concentration between dogs with congestive heart failure that also have
concurrent respiratory disease, are not significantly different from those with
congestive heart failure and so differentiating these conditions remains difficult.
Furthermore, NT-proBNP can be elevated in dogs with heart disease but not failure,
which can also have respiratory disease and again, a definitive differentiation
between respiratory and cardiac disease is not present (Boswood et al, 2008;
Oyama et al, 2009). This test is also complicated in differentiating respiratory and
cardiac disease in dogs with azotemia as it results in NT-proBNP being increased to
concentrations reported as diagnostic of heart disease or heart failure in dogs
(Raffan et al, 2009). While the study by Fine et al (2008) suggests that a serum NT-
proBNP >1400pmol/L strongly suggests a dog will have CHF, an NT-proBNP
concentration <1400pmol/L is ambiguous and can be a result of both respiratory
and/or cardiac disease as is illustrated by Table One which presents the values of
different studies. Thus, serum NT-proBNP can be used in the establishment of a
diagnosis of respiratory disease, but in conjunction with other diagnostic tests and in
consideration of clinical signs.
1.3.6 Serology in diagnosing respiratory disease
Serology is often used as a diagnostic method for causes of respiratory disease in
both brochoalveolar lavage fluid (see below) and blood. A diagnosis of
Cryptococcus spp. or Mycoplasma spp. infection is often made using serology.
Mycoplasma spp are commonly found in airways of healthy dogs, with 20-25% of
healthy dogs harbouring mycoplasmas in the trachea or lungs (Randolph et al,
1993). Studies have shown that Mycoplasma spp can be related to clinical
respiratory disease (Chalker et al, 2004) and the lungs of dogs can be colonised by
Mycoplasma spp during pneumonia (Rosendal, 1982). Speculation as to the role of
Mycoplasma spp as primary or secondary pathogens in canine pulmonary disease
continues (Randolph et al, 1993; Chandler and Lappin, 2002). It is possible that
29
Mycoplasma spp infect the respiratory tract from the oro-pharynx following pre-
disposing immunosuppression or infection (Chalker, 2005).
A positive antibody response to M.cynos has been shown to be common in dogs
entering a re-homing kennel in one study, of which 80% had recorded clinical
respiratory disease (Rycroft et al, 2007). Mycoplasma spp. have been known for
some time to modulate host susceptibility to secondary infections (Kaklamanis and
Pavlatos, 1972; Thacker et al, 1999).
Serology is useful when Cryptococcus spp. are suspected such as when nodular
infiltrates, an interstitial pattern, pleural effusion and/or tracheobronchial
lymphadenopathy are seen on thoracic radiographs (Duncan et al, 2006).
Aspergillus spp. has a complex cytoplasmic organization, an impressive array of
morphological expression, and a remarkable diverse and adaptable metabolism.
Variation in serology diagnostic testing is a result of the large numbers of antigens
produced by the fungal cell, the widespread occurrence of cross-reactions between
related and at times unrelated pathogenic species, and the paucity of accepted
reference reagents and standard procedures. False negative antigen titres are rare
but can occur (Richardson and Page, 2017). False positive titres are uncommon
and usually associated with interfering substances (Kerl, 2003).
Heartworm disease in dogs can produce eosinophilic pneumonitis, pulmonary
hypertension, PTE, proliferative pulmonary endarteritis and inflammation (Simón et
al, 2012). Heartworm is diagnosed by the detection and specific identification of
microfilariae and by using tests for the detection of circulating adult worm antigens,
available only for D. immitis (Simón et al, 2012). Highly specific and sensitive
enzyme-linked immunosorbent assays (ELISAs) or immunochromatography-based
assays that detect circulating antigens of adult D. immitis females are commercially
available for the diagnosis of cardiopulmonary dirofilariasis. (Simón et al, 2001),
Serological tests allow the detection of amicrofilaremic infections, and a positive
microfilaria test followed by a positive antigen test conclusively confirms an infection
with D. immitis (Simón et al, 2012; Simón et al, 2001).
30
1.3.7 Bronchoscopy and bronchoalveolar lavage
Bronchoscopy is performed with the patient under general anaesthesia and is used
to see airways and additionally to collect samples from the respiratory tract (Amis
and McKiernan, 1986; Roudebush, 1990). The procedure is indicated for
assessment of chronic coughing, haemoptysis, pulmonary infiltrates on thoracic
radiography, or when an airway mass or foreign body is suspected (Silverstein and
Drobatz, 2010).
Systematic examination of the tracheobronchial tree is performed including patency,
colour and character of mucosa, presence and character of secretions, and
presence and location of masses or foreign bodies. A method of recording changes
by anatomical location in the respiratory tree was reported by Amis and McKiernan
(1986) which allows systematic examination and a means for subsequent
comparative re-evaluation as shown in Figure 3.
31
Figure 3: Bronchoscopic anatomy of the dog. Systematic identification of
endobronchial anatomy during bronchoscopy in the dog. (From Amis and
McKiernan, 1986)
32
Grade of tracheal collapse can also be recorded during bronchoscopy and the
scheme reported by Tagner and Hobson (1982) as demonstrated in Figure 4, is
most commonly used.
Figure 4: Classification scheme of collapsed trachea (from Tangner and
Hobson, 1982)
When observed using bronchoscopy, normal airway openings are round to ovoid in
appearance and demonstrate minimal (subjectively <20%) variation in luminal size
during phases of respiration. Airway collapse can be seen when there is >25%
static flattening of lobar airways, circumferential narrowing or distortion of the
normal round appearance of airway openings, or there are dynamic changes in
luminal diameter with respiration in sublobar and smaller airways (Johnson and
Pollard, 2010).
Ancillary procedures commonly performed during bronchoscopy include forceps
biopsy, brush biopsy, bronchoalveolar lavage (BAL), bronchial washings, and
transbronchial needle aspiration (Rha and Mahony, 1999; Roudebush, 1990).
Cytology brushes may be used to obtain samples of bronchial epithelial cells and
endobronchial masses (Rha and Mahony, 1999; Roudebush, 1990). Brushing
33
may be more sensitive than BAL for diagnosis of inflammatory states because
brushing detects white blood cells that are adherent to bronchial walls in addition to
those that are readily washed free (Hawkins et al, 2006).
Transbronchial biopsies are used to sample peribronchial tissue and lung
parenchyma but carry the risk of haemorrhage and pneumothorax and are
performed with caution (Rha and Mahony, 1999).
The process of bronchoalveolar lavage samples PELF on the luminal surface of the
respiratory tract (Ward et al, 1999). Cytology of PELF generally correlates well with
pulmonary histology (Hunninghake et al, 1981; Padrid et al, 1991). A BAL is ideally
performed at the site where radiographic or bronchoscopic changes have been
identified and if no lesions are identified, sampling from left and right lung lobes is
performed (Silverstein and Drobatz, 2010). This can be by blindly performed with a
catheter or guided endoscopically. Traditionally, approximately 10 to 25 mL of
warm, sterile saline (0.9% NaCl solution) is instilled through the port of the
bronchoscope to bathe dependant alveoli in that airway (Rha and Mahony, 1999). It
has been shown in one study, that when 2ml/kg BW fluid is infused during BAL, a
more uniform ELF recovery compared with that for a fixed-amount bronchoalveolar
lavage technique is achieved, thus facilitating more accurate comparisons of
cellular and noncellular constituents in BALF among dogs of various sizes
(Melamies et al, 2011). Other studies have used standardized volumes for
comparison of BALF between dogs, based on weight categories (Mills and Lister,
2005). This infused fluid, is then aspirated with gentle pressure (Rha and Mahony,
1999). Gentle suction using a suction pump to a maximum of 50 mmHg negative
pressure applied to a sterile plastic catheter using a suction trap connection, to
withdraw each flush has been show to have significant improved retrieval over
manual aspiration to recover BALF (Woods et al, 2014). Cytological, biochemical,
immunological and microbiologic evaluation of BAL fluid can then be used to gather
information on the cellular components and potential pathogens within the alveolar
space and further characterize pulmonary disease in the dog (Brownback and
34
Simpson, 2013; Hawkins et al, 1990; Rha and Mahony, 1999; Silverstein and
Drobatz, 2010).
As bronchoscopy and BAL requires general anaesthesia and many respiratory
patients have some degree of respiratory distress, it is important to gain as much
information as possible from each diagnostic test utilised and cytological,
serological, microbial and fungal analysis of the BALF is commonly performed
(Silverstein and Drobatz, 2010).
1.3.8 Culture of bronchoalveolar lavage fluid
Lower respiratory tract infections are a common clinical diagnosis in dogs (Epstein
et al, 2010). Knowledge of the infective agent causing lower respiratory tract
infection is imperative for directed treatment. Fluid, cells and tissue from the
respiratory tract can undergo microbiological testing and a variety of techniques are
used in order to obtain specimens (Silverstein and Drobatz, 2010).
Sampling from the upper respiratory tract can be difficult as there is a wide range of
bacteria in the nasal cavity of normal dogs (Bailie et al, 1978). Lower tracheal
cultures do not always correspond with the microbial cause of pneumonia and it has
been shown that in healthy dogs, up to 40% of tracheal cultures will be positive for
growth (McKiernan et al, 1984).
The main problems of diagnosis in lower respiratory tract infection are the
differentiation of infection from colonisation or contamination, and the isolation of a
reliable and true pathogen (Sparkes et al, 1997). Bacteria can often be found in fluid
obtained by BAL and may represent pulmonary infection (Peeters et al, 2000).
However, healthy dogs may harbour an aerobic bacterial population within their
mainstem bronchi at a concentration as high as 103 colony-forming units per
milliliter (CFU/mL) (Peeters et al, 2000).
One study found use of a threshold of 1.7 x 103 CFU/mL of BAL fluid produced a
diagnostic sensitivity and specificity of 86% and 100% respectively for infectious
lung disease (Peeters et al, 2000). The presence of intracellular bacteria on
cytologic examination as an additional diagnostic criterion changed these values
35
slightly, yielding a sensitivity of 87% and a specificity of 97% (Peeters et al, 2000).
This decrease in specificity can be explained by results of another study of 105
dogs with lower respiratory tract infection that found 2% of dogs had cytologically
detectable intracellular bacteria but no microbial growth on aerobic, anaerobic or
Mycoplasma culture (Johnson et al, 2013). The same study found that while 25% of
dogs did not have cytological evidence of infection, they subsequently cultured
miscellaneous bacteria. This illustrates the difficulty in confirming a definitive
diagnosis of respiratory tract infection (Johnson et al, 2013). As BAL also dilutes the
organisms present by a variable amount, even quantitative cultures need to be
interpreted in light of cytologic and clinical findings (Creevy, 2009).
Fungal culture may also be performed on BAL fluid and has increased importance
in areas of endemic fungal disease. Diagnosis of pulmonary aspergillosis is often
difficult given serology and PCR are often positive due to the ubiquitous nature of
the mould, with spores inhaled in large numbers daily (Chotirmall et al, 2013).
Positive PCR, serology using Aspergillus -galactomannan and fungal culture may
simply reflect colonisation rather than invasive disease (Avni et al, 2012; Chotirmall
et al, 2013). Aspergillus spp. in BAL may be seen with direct microscopy as
septated hyaline hyphae. Aspergillus spp. can also be cultured, however there are
many documented negative cultures (Neofytos et al, 2009). A recent study in people
may change the veterinary approach to BAL fungal culture with culture from
undiluted BAL having a much higher yield of Aspergillus spp. (Fraczek et al, 2013).
As M.cynos has been shown to be involved in the early stages of infectious disease,
and possibly acts as an initiating agent of chronic infectious respiratory disease
(CIRD), testing for Mycoplasma spp. has become routine. Mycoplasma spp. culture
and PCR, is performed on BAL samples. However, Mycoplasma spp. can be
isolated from the trachea and bronchial lavage in a proportion of healthy and
diseased dogs and so clinical judgement as to the significance and morbidity of the
organism as an opportunist, infecting organism or co-infective organism in
respiratory disease is ambiguous (Chalker et al, 2004; Rycroft et al, 2007).
36
Development of further tests may help identify in which dogs this organism is
contributing to the disease process present.
1.3.9 Cytology of the bronchoalveoli
Quantitative analysis of the type and number of inflammatory cells recovered from
the lung is useful in determining the aetiology of pulmonary disease (Foster et al,
2004; Hawkins et al, 2006; Johnson et al, 2013; McCullough and Brinson, 1999).
Important cytological criteria for evaluation of BALF, are the number and types of
cells recovered (Dehard et al, 2008). In particular, cytology of respiratory secretions
can be used to confirm infectious organisms and neoplastic disease (Silverstein and
Drobatz, 2010).
Qualitative examination of cells can reveal activation of macrophages and
phagocytosis of fungi, bacteria, red cells and other debris (Hawkins et al, 1995;
Hawkins, 2004; Hawkins et al, 2006). Increased numbers of reactive lymphocytes
and plasma cells can suggest immune stimulation (Hawkins et al, 1995; Hawkins,
2004; Hawkins et al, 2006). Neutrophils may demonstrate degenerative change or
intracellular bacteria and non- inflammatory cells can be examined for signs of
malignancy (Hawkins et al, 2006).
With a threshold for infection of >2 intracellular bacteria observed in any of 50 fields,
microscopic examination of gram stained BAL preparations had a sensitivity of
71% and a specificity of 97% in establishing lower respiratory tract infection
(Peeters et al, 2000). In another study of 105 dogs with lower respiratory tract
infection, suppurative septic inflammation was identified in 75% of BAL samples
examined (Johnson et al, 2013). Of the remaining 25% of dogs without cytological
evidence of intracellular bacteria, mixed inflammation with neutrophils, lymphocytes
or eosinophils was present (Johnson et al, 2013).
Diagnosis of neoplasia is possible if a tumour exfoliates, but often the diagnostic
yields for cytological examination of bronchoscopic sampling procedures on
neoplasms are dependent on location of bronchoscopically-visible tumours (Liam et
37
al, 2007). Peripheral lesions are usually poorly sampled during bronchoalveolar
lavage and hence yield little for cytological examination (Padrid, 2000).
Studies investigating use of bronchoscopic procedures for diagnosis of neoplasia in
people showed that sensitivity for diagnosis of endobronchial neoplastic disease
was highest for endobronchial biopsy (74%), followed by cytobrushing (59%) and
washing (48%). The sensitivity for all modalities combined was 88% (Schreiber and
McCrory, 2003).
Another medical study observed that BAL had a sensitivity of 43% in diagnosing
peripheral neoplastic lesions (Schreiber and McCrory, 2003). One of the main
reasons, the sensitivity of bronchoalveolar lavage is poor for neoplastic disease is
the variable exfoliation of cells from different neoplasms. Thus revised or newer
techniques are required to increase the diagnostic yield of procedures performed.
A retrospective review in people indicated that infiltrates that were predominantly
reticular or nodular in appearance on CT which are more commonly associated with
extra-alveolar disease, had the lowest diagnostic yield (36.5%) of BAL. In contrast
consolidated, ground-glass or tree-shaped infiltrates on CT, which are more
commonly associated with bronchiolar and alveolar disease, had highest (61.2%)
diagnostic yield. Diagnostic yield was also increased in patients with fever and
respiratory symptoms (Brownback and Simpson, 2013). No study in dogs has
examined this correlation, and although all dogs undergoing BAL usually have
clinical signs of respiratory disease, a similar study in dogs may be beneficial to
avoid morbidity in canine patients that are unlikely to benefit from the BAL
procedure.
Cytology of non-infectious inflammation can be problematic in interpretation of the
lower airway disease. Macrophages which reflect inflammation in other body fluids,
are the most abundant immune cell population in normal lung tissue (Byers and
Holtzman, 2011). Macrophages serve roles in innate and adaptive immune
response as well as development of inflammatory airway disease and can act as
confounders in interpretation of BAL samples (Byers and Holtzman, 2011; Hawkins
et al, 2006; Padrid, 2000).
38
Cytological responses in BAL of dogs with respiratory disease, has been
traditionally grouped into categories based on relative differential counts and
diagnosis of disease has been based around these descriptions (Hawkins et al,
2006; McCullough and Brinson; 1999). The characteristics of the broad categories
of cytological responses in respiratory disease and the likely disease aetiology are
listed in Table 2 (Hawkins, 2004; Hawkins et al, 2006; Derhard et al, 2008). There
are many categories that overlap, and not only can each category have multiple
differential diagnosis, but each cytological category can also have differential
diagnosis or disease aetiology that can have multiple cytological characteristics
(Hawkins, 2004; Hawkins et al, 2006; Derhard et al, 2008). These cytological
evaluations hence narrow the list of differential diagnosis but often cannot always
definitively diagnose respiratory disease.
39
Table 2: Cytological characteristics of abnormal bronchoalveolar lavage fluid
and differential diagnosis (Hawkins et al, 1990;McCullough and Brinson; 1999)
Classification Cytologic characteristics Aetiology
Acute Neutrophilic
Increased neutrophils
+/- Degenerate
Organism phagocytosis
Bacterial infection
Aspiration pneumonia
Mycotic infection
Neoplasia
Non-infectious inflammatory
disease
Rickettsial/ Protozoal
Iatrogenic
Chronic bronchitis
Chronic- active (mixed)
Increased neutrophils and
macrophages
+/- activated macrophages
+/- phagocytosis of cellular
debris
Infection
Neoplasia
Non-infectious inflammatory
disease
Resolving bacterial infections
Mycotic infection
(Blastomyces dermatitidis in
endemic areas)
Chronic (mixed)
Activated macrophages
Increased total cell count
Mild Increases of neutrophils,
reactive lymphocytes, plasma
cells
NON SPECIFIC
Infection
Neoplasia
Chronic bronchitis
Eosinophilic
High numbers eosinophils Hypersensitivity responses
Parasitic
Allergic pneumonitis
Allergic bronchitis
Haemorrhage
High numbers red cells
Erythrophagocytosis
Haemosiderin laden
macrophages
Chronic inflammatory changes
occur
(Blastomycosis in endemic
areas)
Neoplasia
Trauma
Foreign bodies
Thromboembolism
(Iatrogenic)
Neoplasia
Cells with multiple cytologic
criteria of malignancy
eg ↑ Mast Cells
eg ↑ Lymphoblasts
Neoplasia
(NB Differentiation between
hyperplastic and neoplastic
cells can be difficult)
40
1.4 Respiratory cytology and characterisation of abnormal cytology
Multiple reports in the literature describe that characterisation of normal respiratory
cell counts in dogs. Establishment of a normal reference interval has been
problematic due to variability and a lack of standardisation of collection and
processing techniques and lack of strict classification and universally accepted
guidelines for interpretation of BALF. Most of the variation probably reflects
differences in sampling technique, because a strict standardisation of the procedure
is lacking (Hawkins, 2004; Mills and Litster, 2005; Rennard et al, 1986). During
BAL, the results can be modified by the volume of fluid used, the time between
infusion of lavage fluid into a bronchus and aspiration of the fluid, the suction
pressure during aspiration of fluid and cytological slide preparation (Brown et al
1983; De Brauwer et al, 2000; Dehard et al, 2008; Hawkins, 2004; Mordelet-
Dambrine et al, 1984; Saltini et al, 1984).
The total volume of BALF instilled has been shown to influence the total cell count
and differential cell count, as well as the concentration of cells recovered in BALF
samples from horses (Sweeney et al, 1992). Large BALF volumes yielded lower
total cell counts and concentrations of neutrophils, lymphocytes, and macrophages
(Sweeney et al, 1992).
Immediate withdrawal of instilled fluid is necessary to obtain an adequate cell yield
and minimise loss of cells (Klech et al, 1989). Delay in BALF retrieval will result in
dissemination and absorption of the fluid by the lung parenchyma and loss of
volume and number of cells (Klech et al, 1989).
Variable BALF cell counts also occur if strict sample handling is not adhered to. It
has been demonstrated that BALF specimens need to be processed within 2 hours
of collection (Klech et al, 1989). Keeping samples refrigerated (at 4˚C) for up to 24
hours has been shown to decrease the number of cells, but not differential cell
counts in people and horses (Klech et al, 1989; Rennard et al, 1998). An additional
factor influencing BALF interpretation is cell lysis (Creevy, 2009). Cell lysis can
occur with a lack of rigidity of the suction catheter that links the endoscope to the
aspiration pump (Dehard et al, 2008). A flexible catheter can collapse under
41
aspiration and lead to sudden release of fluid and cells, which can induce cell
damage (Dehard et al, 2008).
When processing BALF, variations in the speed and duration of centrifugation also
influence differential cell count (Fleury-Feith et al, 1987; Mordelet-Dambrine et al,
1984). Lower cytocentrifugation speed can cause greater lymphocyte loss than
higher speeds, while the converse is true for macrophage cell counts (Fleury-Feith
et al, 1987; Mordelet-Dambrine et al, 1984). A short period of high-speed
cytocentrifugation (1200-2000 rpm) has been recommended to minimise
lymphocyte loss and standardise cell counts in BALF samples (Willcox et al, 1988).
Cytological slides can be prepared manually by streaking cell suspension or pellets
from centrifuged fluid onto a microscope slide, or by using a cytopsin that
automatically spreads cells onto a slide (Dehard et al, 2008; Hawkins, 2004;
Hawkins et al, 1995). Counting 300 cells in cytocentrifuged BALF samples in a
circular pattern around the centre of the cytocentrifuged spot has been shown to
lead to reliable differential cell counts (De Brauwer et al, 2002). Another study has
identified that it can be difficult to identify 300 intact cells which is why a differential
cell count based on the examination of 100 cells is generally performed (Dehard et
al, 2008; Hawkins, 2004).
A recent study in dogs and cats with inflammatory or infectious lower respiratory
disease evaluated the differences in the cytological interpretation of BALF after
cytospin preparation (CP) or manual smearing of pelleted cells preparation (MSP)
(Dehard et al, 2008). While the study only had small numbers of animals included (4
in each category), it demonstrated that CPs were considered of better quality than
MSP. When only MSPs were evaluated, differential neutrophil counts were found to
underestimate the counts found on CP slides (Dehard et al, 2008). Use of cytospin
preparation was thus recommended for the evaluation of BALF with low total cell
count.
Variability in interpretation of cell cytology is well illustrated when comparing studies
by Hawkins et al (1995) and Clerx et al (2000). In the study by Hawkins et al (1990),
neutrophilic, eosinophilic, or lymphocytic inflammation was diagnosed by relative
42
cell counts ≥12%, 14%, or 16% in BALF respectively. In diagnosing eosinophilic
bronchopneumopathy, Clerx et al (2000) used >50% eosinophils in BALF as a cut-
off for diagnosis, even though the disease can be diagnosed with significantly lower
eosinophil counts (Brandendistel et al, 1992; Clercx et al, 2002; Hawkins et al,
1995).
A poor correlation also exists between the pathologist’s prediction of the types of
cells likely to be present in BALF based on pulmonary histopathology, and the cells
that actually are present in BALF (Norris et al, 2001; Norris et al, 2002a).
Table 3 lists reported cell fluid populations in healthy dogs.
43
Table 3: Reported uncorrected reference range intervals for bronchoalveolar
lavage fluid cytology in healthy dogs not corrected for volume/dwell time (* %
reported for healthy dogs collected by bronchial wash, ‡ healthy beagles).
Reported as range of total cell numbers attained between brackets and mean
+/- standard deviation if reported.
Rebar 1980
Scott et al 1993
Mills and Litster 2005
King et al 1988
Rajamäki et al
1990‡
Hawkins et al
2006*
Kuehn 1987 &
Hawkins et al 1990
Total nucleated
cells/µl
516
(240-630)
163 +/- 100.3
(60-330) 104 +/- 64
56
(25-143)
200 +/-86 (54-454)
Differential Macrophages
% Range
83
69 (27-92)
102.8+/- 53.6
63 +/- 23 (28-186)
81
75 (68-72)
81 (67-89)
70 (49-93)
Lymphocytes %
Range 5.7
10
(1-52)
12.3+/-9.1 8 +/- 4 (1-28)
6
13 (7-19)
7
(1-19)
Neutrophils %
Range 5
6
(0-68)
14.2+/-9.6 7 +/- 4 (2-26)
5 5
(1-9)
7
(1-14)
5
(1-27)
Eosinophils %
Range 4.2
3
(0-35)
16.8+/-10.7 10 +/- 7 (4-29)
4 4
(0-9)
6
(1-11)
6
(0-19)
Mast Cells %
Range 2.3
1
(0-5)
1.1+/11.3 1 +/- 1 (0-4)
2
(0-4)
0
(0-1)
1
(0-5)
Epithelial Cells
% Range
0.6 (0-1.3)
5
(0-25)
1
(0-12)
Other recent studies used a reference interval of <5-8% eosinophils, neutrophils or
lymphocytes and 65-85% macrophages with an absence of intracellular bacteria as
normal BAL (Hawkins et al, 1995; Johnson et al, 2013). Samples with >8%
neutrophils were thus classified as suppurative and if neutrophils contained
intracellular bacteria, the BAL fluid was classified as septic (Hawkins et al, 1995;
Johnson et al, 2013).
Table 4 shows the results of a study on BAL cytology uncorrected for volume of
infusion, by Dehard et al (2008) in diseased dogs. While differences were significant
between dogs with infectious and non infectious disease there was a significant
44
overlap of cell counts and differential percentages which highlights that BALF
cytology cannot always be relied upon to diagnose a specific respiratory disease
aetiology.
Table 4: Total cell counts in BAL fluid reported from diseased dogs (from
Dehard et al, 2008) SEM: standard error of the mean. Range is found between
brackets
Total Cell
Count
Mean +/- SEM
(Range)
Macrophage
%
Neutrophil
%
Lymphocyte
%
Eosinophil
%
Bacterial
Infection
1775+/- 736
(500-3800) 20 +/- 4.6 77.4 +/- 4.4 1.8 +/- 6.5 0.75 +/-0.3
Non Bacterial
Inflammation
867 +/- 104
(700-1100) 55.4+/- 10.7 34.3 +/- 10.8 5.2 +/- 1.8 5.1 +/- 1.5
As discussed earlier, the cytologic responses found in BAL of dogs with respiratory
disease have been loosely grouped and based on relative differential cell counts
(Hawkins et al, 1995; Hawkins, 2004; Hawkins et al, 2006; Derhard et al, 2008).
Cytological responses have been grouped into acute neutrophilic inflammation,
chronic-active inflammation, chronic, eosinophilic inflammation, haemorrhage and
neoplasia (Hawkins et al, 1995). However, these groups are based on the greatest
percentage of a cell type and consideration should be given to the absolute
numbers of cells and differential leukocyte cell counts, similar to interpretation of
peripheral blood profiles.
1.4.1 Biochemical analysis of BALF
Determination of the biochemical profile of bronchoalveolar lavage fluid including
lactate dehydrogenase (LDH) and alkaline phosphatase, which reflect lung
45
parenchymal cell damage or cell death have also been investigated in humans to
determine whether there is practical value in distinguishing between infectious and
non-infectious pulmonary disorders n diagnosis (Cobben et al, 1999; Drent et al,
1996; Glick, 1969; Lott and Nemensanszky, 1986). The best discriminator between
infectious and non- infectious pulmonary disorders, appeared to be LDH isoenzyme
activities and LDH4/LDH5 had a sensitivity of 93.6% and specificity of 93.9%
(Cobben et al, 1999). In cats LDH has been measured in pleural fluid to distinguish
between transudate and exudate (Zoia et al, 2009). In the 20 cats examined in that
study, LDH of pleural fluid (LDHp) and pleural fluid/serum total protein ratio (TPr)
were found most reliable when distinguishing between transudates and exudates,
with sensitivity of 100% and 91% and specificity of 100%, respectively (Zoia et al,
2009). The range of activity of LDH of BAL in healthy cats has been reported as
110.2- 212.3 U/L compared to mean LDH of 145 U/L in BAL fluid from healthy
lambs (Pusch et al 1997) and mean LDH of 361 U/L in BALF of healthy humans
(Cobben et al, 1999).
A study reported the mean LDH activity from PELF in healthy dogs was 191.3 (SD
176.8) U/L (Mills and Litster, 2005). This was comparable to that of healthy lambs
(Pusch et al, 1997). The concentration of LDH of BAL fluid in dogs is much less
than that reported in healthy humans (Cobben et al, 1999). The LDH activity of
PELF from dogs is not routinely measured, and the potential practical value in the
veterinary setting remains undetermined.
Another problem associated with lavage of epithelial surfaces and using differential
cytology is the variable recovery of lavage fluid and cells lining the epithelium which
results in variation of cell counts in BALF as was seen in Table 1 (Mills and Litster,
2005; Vail et al, 1995). To overcome the variation, a reference substance or marker
can be used to determine the concentration of PELF in lavage fluid and more
accurately identify epithelial cell concentrations (McGorum et al, 1993; Mills and
Litster, 2005; Mills and Litster 2006; Rennard et al, 1986).
46
1.4.2 Standardisation of components of respiratory lavage fluid in animals
Bronchoalveolar lavage instills fluid into bronchi and alveoli which mixes with PELF
to form a composite fluid. The PELF from the alveoli and airways provides a cellular
profile for diagnosis of respiratory disease. However considerable variation has
been reported in cell counts and concentration of biomarkers in BALF from dogs
and other species (Cobben et al, 1999; Effros et al, 1990; Kirshvink et al, 2001;
Marcy et al, 1987; McGorum et al, 1993; Mills and Litster, 2005; Mills and Lister,
2006; Rennard et al, 1986; Ward et al, 1999). One of the problems associated with
interpreting a cell count which falls within previously reported reference values is the
uncertainty in the amount of PELF retrieved in the sample. Variable recovery of
PELF in lavage fluid can account for these differences in cell counts and is mainly
due to variable recovery of the instilled lavage fluid (Mills and Litster, 2005; Rennard
et al, 1986; Vail et al, 1995).
Furthermore, the concentration of a substance in BALF does not necessarily reflect
the concentration in the PELF (Rennard et al, 1986; Mills and Litster, 2005; Mills
and Lister 2006). Simply measuring a substance such as cells or cytokines (eg
TNFα) in BALF ignores the tremendous variability in dilution between samples.
Without standardising solute and cell concentrations in BALF, comparisons cannot
be made between different studies or disease processes.
There is no practical method for which the total PELF volume in lungs can be
directly measured (Rennard et al, 1986; Ward et al, 1999). To overcome the
problem of variable recovery of PELF in lavage fluid, use of exogenous and
endogenous biomarkers have been assessed to calculate the concentration of
PELF in BALF and estimate the concentrations of cellular and non-cellular
components of PELF (Rennard et al 1986, McGorum et al, 1993, Mills et al, 1996,
Kirschvink et al, 2001, Bayat et al, 2004, Cobben et al, 1999).
47
1.4.3 Exogenous markers used in the assessment of bronchoalveolar lavage
fluid
To adjust for dilution of PELF, an exogenous (introduced) marker can be added to
the BALF. The ideal exogenous marker must meet several conditions (Ward et al,
1999; Kirschvink et al, 2001) to be useful in interpretation of BALF;
1. The marker must not be lost from airspaces or cross the pulmonary epithelium
2. Precise methods for measuring concentrations of the macromolecule must be
met as dilution is expected to be <1% in normal lungs
3. No water enters BAL fluid from a source other than PELF
4. The only source of the marker is in the instilled lavage fluid
5. The marker is inert
6. The marker is not metabolised by cells
7. The marker mixes completely with the epithelial lining fluid in the lavaged
segment of lung
Inulin, methylene blue and radioactive tracers (99mTC-diethylenetriamine
pentaacetate, 51Cr-EDTA) have been studied as exogenous markers of BALF
dilution (Bayat et al, 1998; Kirschvink et al, 2001; Restick et al, 1995). These
exogenous markers are introduced with the BAL solution. Unfortunately the use of
these markers has not been successful with variable and artefactual results
reported (Ward et al, 1999; Kirschvink et al, 2001) due to loss of indicators from the
lung, imprecision of marker assays and complex fluid dynamics that occur during
BAL.
1.4.3.1 Inulin
Inulin is an inert polysaccharide of high molecular weight (5 kDa) that has been
tested as an exogenous marker of dilution in BAL fluid in healthy and horses with
chronic obstructive pulmonary disease (Kirshvink et al, 2001), foetal lambs
(Normand et al, 1971) and in people (Restrick et al, 1995). Inulin concentration in
48
the retrieved BALF is measured by a spectrophotometric assay (Kirshvink et al,
2001; Restrick et al, 1995). The benefit of using inulin as an exogenous marker of
BALF includes the need for minimal preparation and relatively simple processing.
One limitation is that dilution with inulin of instilled lavage fluid and mixing with PELF
is negligible and marker concentration changes are also nearly negligible in BALF
retrieved (Kirschvink et al, 2001). In addition, large lavage samples and long dwell
times appear to be required to allow adequate mixing of the lavage fluid with PELF
in horses. Furthermore, inulin did not demonstrate any advantages over an
endogenous marker, urea in estimating PELF when used in horses (Kirshvink et al,
2001). Given these findings, inulin is not a useful marker of PELF dilution in the dog.
1.4.3.2 Methylene blue
Methylene blue dilution in BALF has also been assessed. However, use of this
marker has not been found to be useful as it has been shown to bind to cells and to
diffuse into the pulmonary interstitium leading to overestimation of recovered PELF
(Rennard et al, 1986, Baughman et al, 1983). Discolouration caused by the dye can
also interfere with further analysis of BAL fluid (Ward et al, 1999). In the rat, there is
evidence that methylene blue is rapidly reduced to its colourless form
(leukomethylene blue) due to the presence of ascorbate in airspaces (Effros et al,
1994). This may also occur in the human lung via glutathione (Slade et al, 1993).
Reduction of methylene blue in the dog lung remains poorly characterised and thus
it is a poor candidate as an exogenous marker in this species.
1.4.3.3 Technetium-99m diethylenetriaminepenta-acetic acid (99mTc-DTPA)
Technetium-99m diethylenetriaminepenta-acetic acid is a radionuclide chelate
complex (Bayat et al, 1988). Equilibration of 99mTc-DTPA between blood and PELF
has been developed to account for the diffusion of the indicator as well as fluid
exchange during lavage (Bayat et al, 1988). The technique requires a gamma
counter and radio-isotope handling facilities generally not available in general
veterinary practice. Another limitation is the prolonged dwell times required.
Although prolonged dwell times of BAL fluid did not significantly alter PELF
49
estimation in healthy lungs, penetration from blood to PELF is greatly enhanced in
acutely inflamed lung (Bayat et al, 1988; Bayat et al, 2004). Blood/airspace
clearance of 99mTc-DTPA becomes elevated with an increase in intravascular lung
water and unchanged pulmonary wedge and capillary pressures with acute lung
injury (Bayat et al, 2004). It is likely the blood/air barrier becomes more permeable
with the BAL procedure itself particularly in inflamed lung and hence diffusion of the
indicator increases, making it unstable and not in equilibrium with blood (Ward et al,
1992).
The complexities in 99mTc-DTPA measurement, kinetics with changes in alveolar-
capillary permeability and use of a radioactive substance make it undesirable to use
as a marker of BALF dilution in clinical veterinary practice.
1.4.4 Endogenous markers of PELF dilution
Endogenous markers of PELF dilution are measured solutes that are not in the
instilled lavage fluid, but are normally present in circulation and therefore in PELF.
Ideally the concentration of the solute within PELF prior to lavage must be
equilibrated with plasma concentration or at a known constant fraction with plasma
and there must be no movement between the airspace and vascular compartment
at the time of lavage (Ward et al, 1999). The endogenous marker must also not be
affected by the airway disease itself (Kirschvink et al, 2001; Ward et al, 1999). Use
of an endogenous marker has an advantage in that it is not necessary to add a
tracer molecule to lavage fluid to estimate the dilution of the recovered PELF
(Cobben et al, 1999). Several endogenous markers have been investigated
including albumin, electrolytes and urea.
1.4.4.1 Albumin
Exact concentrations of albumin in PELF remain uncertain (Effros et al, 1990;
McGorum et al, 1993a). Proteins may be relatively excluded from the interstitial gel
near the epithelial membrane with which PELF is in equilibrium, and thus plasma
albumin exceeds albumin in PELF (Effros et al, 1990).
50
Studies of effects of disease on PELF albumin and protein have revealed varying
results. One study in people did not show any significant differences in albumin or
total protein in PELF between people with infectious or non-infectious respiratory
disease (Cobben et al, 1999). However, this study quantitated total albumin
concentration and did not use protein or albumin as a biomarker for PELF
collection. Another study found that albumin in BALF was increased
disproportionately relative to urea in people with a variety of interstitial lung
diseases which suggests that protein may increase when the integrity of the
epithelium is compromised and thus make it an unreliable marker for measuring
PELF dilution (Jones et al, 1990).
A study of PELF cell counts in healthy horses and horses with chronic obstructive
pulmonary disease also found a plasma: PELF ratio for albumin to be greater than
that of urea suggesting protein leakage in pulmonary disease making it an
unreliable marker in the equine species as well (McGorum et al, 1993). Studies in
rats estimate albumin concentration in PELF to be 60% of plasma level (Effros et al,
1990).
Several limitations to the use of albumin as a reference standard have been
identified (Rennard et al, 1986; McGorum et al, 1993a; Ward et al, 1997; Ward et al,
1999). These include;
a. only relative comparisons to plasma can be made and use of albumin as a
biomarker does not allow absolute concentrations of molecules in PELF to be
determined.
b. Albumin has an average molecular mass (69,000Da) and its validity
diminishes for molecules of greater or lesser mass.
c. lung diseases leading to altered alveolar membrane permeability are likely
to lead to altered albumin concentration in PELF
Thus as the alveolar membrane has reduced permeability to large albumin
molecules, plasma albumin concentrations exceed those of PELF in healthy lungs
making it a poor endogenous marker of pulmonary epithelial lining fluid due to its
51
variable concentration in PELF with respect to plasma (McGorum et al, 1993a;
Ward et al, 1997). The variable permeability of albumin with disease further
confirms it is a poor endogenous marker of BALF dilution (McGorum et al, 1993a).
1.4.4.2 Electrolytes eg Potassium, Calcium, Sodium, Chloride
The use of electrolytes has been investigated as endogenous markers.
Bronchoalveolar lavage with isotonic mannitol, saline or glucose yields high initial
and subsequent concentrations of potassium, and it has been found in both humans
and rats that potassium is secreted into the airspaces during first lavage (Effros et
al, 1990, Basset et al, 1988, Davis et al, 1982). As potassium is secreted during the
procedure and because any injury to lung cells might further increase potassium
concentration, use of potassium as a marker for PELF dilution is not recommended
(Effros et al, 1990, Basset et al, 1988, Davis et al, 1982).
Sodium and chloride concentration in PELF and plasma are very similar to each
other (Nielson, 1986). However this electrolyte is not suitable when saline is used
as the lavage fluid as it is impossible to distinguish between sodium and chloride in
the PELF from the quantities of ions infused into lungs. Experiments have
suggested isotonic mannitol may be a useful solution for lavage as it allows
measurement of sodium as an indicator of PELF, however safety studies have not
been undertaken (Effros et al, 1990).
Total calcium concentration cannot be used to quantitate PELF dilution when
isotonic saline or glucose is used to lavage the lungs, as calcium has been shown
to increase in instilled lavage fluid and calcium exchange may accelerate into air
spaces during BAL (Effros et al, 1999).
52
1.4.4.3 Urea
Urea is a potentially useful endogenous marker. Urea is ingested and absorbed
from the large intestine but the majority is synthesised in the hepatic urea cycle from
ammonia derived from protein catabolism (Duncan et al, 1994). Urea is an easily
measured component of plasma that diffuses freely throughout the body including
the alveolar wall (Taylor et al, 1965; Rennard et al, 1986). Urea transporters that
may potentially lead to variable urea in PELF and that are present in red blood cells,
kidney and liver cells do not appear to be found in pulmonary epithelial tissue
(Macey, 1984; Effros et al, 1992; Effros et al, 1993, Hediger et al, 1996). Urea does
not have a polarity at physiologic pH and is not used or produced by lung cells
(Rennard et al, 1986). Urea thus equilibrates between PELF and plasma. Direct
measurements in foetal sheep of PELF have demonstrated plasma and lung urea
concentrations are in equilibrium which suggests urea can be used as a marker of
PELF dilution when compared to serum urea concentration (Adams et al, 1963). A
study in rats where radiolabelled urea was administered one minute prior to BAL,
demonstrated 80% equilibration of urea with PELF (Effros et al, 1990). Conversely,
a study in people using radiolabelled 14C showed urea fully equilibrated between
plasma and BAL fluid within 5 minutes of injection of the radioisotopes (Ward et al,
1999). This supports use of urea as an endogenous biomarker of dilution of PELF.
Urea is used as an endogenous marker of PELF dilution because its free and rapid
diffusion across the alveolar capillary barrier from the plasmatic sector allows
equilibrium across the barrier at equal concentrations (Rennard et al, 1986).
However, this extreme diffusibility is also responsible for an increase in urea
diffusion from plasma to BALF over time, rendering its use as a stable marker of
PELF dilution problematic (Rennard et al, 1986; Ward et al, 1999). The underlying
mechanism proposed for urea diffusion has been specifically related to the BAL
procedure with local tissue distortion of tissue due to high hydrostatic pressures
opening non physiologic pores (Ward et al, 1999).
Lack of correlation between urea serum-to-BALF ratios and any other solute serum-
to-BALF ratio occurs when lavage volumes >20 mL are used, when sequential
53
instillations are used, and as lavage dwell time increases to >20 secs (Cheng et al,
1995).
When comparing urea to other endogenous biomarkers, there are important
differences between urea and albumin dilution of BALF. As the alveolar membrane
has reduced permeability to large albumin molecules, plasma albumin
concentrations exceed those of PELF, whereas urea is a small molecule that
diffuses throughout body fluids and is in equilibrium between PELF and plasma
(Rennard et al 1986). Thus, estimation of dilution with urea can be used to
determine the concentration of pulmonary PELF in BALF samples (McGorum et al,
1993a).
1.4.5 Urea standardisation of bronchoalveolar lavage fluid in dogs
Although urea is a potentially useful endogenous marker, there are some limitations
associated with its use that need to be recognised if this marker is to be used. Use
of urea as an endogenous marker can lead to underestimation of solute
concentrations in PELF due to diffusion of urea into BALF over time (Marcy et al,
1987; Effros et al, 1990). Some studies have used short dwell times (< 20 seconds)
to limit urea diffusion into BALF (Cheng et al, 1995; Peterson et al, 1993). This can
increase the error in determining disease from distal airways as it has been shown
the first aliquot of instilled fluid will only sample proximal airways and that peripheral
airways are only sampled with further lavage (Kelly et al, 1987). However, it
appears that using urea as an endogenous marker is less variable in injured lung
when compared to other markers (Bayat et al, 2004). Furthermore, in closed-chest
experiments on the dog, the haemodynamic effects of pulmonary lavage were
minimal as long as low positive and negative pressures were used to inject and
withdraw the lavage solution making diffusion of urea across the alveolar membrane
less likely during BAL thus maintaining urea equilibration between PELF and blood
(Cross et al, 1960). Diffusion of urea during BAL can thus be minimised by
procedural BAL protocols.
54
While urea does not seem suitable for estimating PELF recovered by a BAL
protocol using multiple aliquots of instilled saline into the same lung segment due to
diffusion of urea into the alveolar space during lavage (Marcy et al, 1987), single
lavage of affected lobes may provide a more accurate estimate of absolute volume
of PELF recovered by BAL (Cheng et al, 1995).
Only one study has assessed the usefulness of urea standardisation of BALF in
dogs. Both total nucleated cell count and differential percentage of cells from BALF
collected from nine healthy dogs was calculated in PELF, based on the relative
concentration of urea in plasma and BALF (Mills and Litster, 2005). Thus diagnosis
of ‘normal’ cell counts based on uncertain dilution of PELF without correcting for
dilution of PELF by instilled lavage fluid during investigation of pulmonary disease
may be misleading and lead to incorrect assumptions of health. The study clearly
demonstrated estimation of the PELF fraction in lavage fluid, using a standardised
lavage technique and correction for urea dilution, permitted estimation of cellular
and non-cellular components of PELF (Mills and Litster, 2005). Table 5 shows cell
counts found after correction for dilution of PELF in the studied dogs (Mills and
Litster, 2005).
55
Table 5: Cell counts of BAL fluid recovered from dogs. Mean +/- Standard
deviation reported. (from Mills and Litster, 2005) Range reported in
brackets().
Cells Unadjusted cell count
(cells/ µl) Urea adjusted cell
count (cells/µl)
Total Nucleated 163.2+/- 100.3
(60-330)
38,645 +/- 30, 347
(6,900-86,800)
Differential
Macrophages
%
Range
102.8 +/- 53.6
63+/-23
(28-186)
25, 272+/- 18,401
63 +/-23
4,485-50,400
Lymphocytes
%
Range
12.3 +/- 9.1
8+/-4
(1-28)
3,196+/- 3,168
8+/-4
(115-9,800)
Neutrophils
%
Range
14.2 +/- 9.6
7+/-4
(2-26)
3,364 +/- 4,221
7+/-4
(220-12,580)
Eosinophils
%
Range
15.8 +/- 10.7
10+/-7
(4-29)
2,573 +/- 1,832
10+/-7
(1,400-5,375)
Mast cells
%
Range
1.1 +/- 1.3
1+/-1
(0-4)
217 +/- 356
1+/-1
(0-1,120)
The most significant finding in this study was that cell counts in PELF of normal
dogs cannot be predicted from cell counts in BALF, however the clinical significance
of this in dogs with pulmonary disease was not evaluated, nor was the effect of
volume instilled based on bodyweight (Mills and Litster, 2005) Further
standardisation may have been possible using an instillation volume based on
56
bodyweight (ml/kg) rather than a set volume. At this time it is unknown if less
dilution in smaller dogs compared to larger dogs may reduce variability of cell
counts in BALF or PELF fluid.
1.5 Conclusion
The ability to quantify PELF volume and standardise cell counts obtained by BAL
has great potential to improve interpretation of BAL cytology. This is supported by
veterinary studies which have identified differences in inflammatory or epithelial cell
populations between different diseases such as infectious and non-infectious
inflammation and neoplasia in dogs (Clerxc et al, 2000; Hawkins et al, 2006). In
people studies have also shown differences in inflammatory or epithelial cell
populations in bronchial brushings between healthy people, patients with asthma,
cystic fibrosis and chronic bronchitis associated with smoking (Danel et al, 1996;
Gibson et al, 1993; Riise et al, 1992; Riise et al, 1996). Estimations of the
concentration of cells on the epithelial surface of the respiratory tract may prove
useful in understanding of the pathogenesis of pulmonary disorders in the dog as
well as staging animals with respiratory disease and providing prognostic indicators.
57
Chapter 2 Aims and Hypothesis of the Study
2.1 Aims
This study aims to; 1) measure and calculate a dilution factor of BALF from relative
concentration of urea in PELF and blood, and then to; 2) use this dilution factor to
accurately quantify cell counts in BALF, and thus to; 3) differentiate respiratory
diseases in dogs diagnosed with respiratory disease involving the bronchi and
parenchyma. The dogs presenting with respiratory disease were prospectively
recruited and diagnostic data analysed retrospectively to ensure a definitive
diagnosis was achieved. Cell counts of BALF were then assessed to account for
dilution of PELF and to determine if disease groups produced similar cell counts.
Diseases were assessed to see if BAL cell counts alone could distinguish between
specific respiratory diseases and broad disease processes including neoplasia,
inflammation, infectious and non-infectious disease and upper respiratory tract
disease.
2.2 Hypotheses
i. The use of urea concentration of bronchoalveolar lavage fluid (BALF) relative to
that of blood to estimate the volume of pulmonary epithelial lining fluid retrieved
during bronchoalveolar lavage, will allow the concentration of cellular components in
pulmonary epithelial lining fluid to be accurately calculated.
ii. Evaluation of the concentration of the cellular components of pulmonary epithelial
lining fluid described above can be used to differentiate between different
respiratory diseases or causes of respiratory clinical signs.
58
Chapter 3 Materials and Methods
3.1 Case acquisition
All dogs included in the study were client owned dogs presented to Murdoch
University Veterinary Hospital for investigation of respiratory disease. No animals
were actively recruited. Only animals requiring a BAL as part of their diagnostic
investigation were used. The cases were analysed retrospectively and classified
according to their final diagnosis. Statistical differences were assessed and
usefulness of standardised BAL fluid in respiratory disease analysed. The use of
animals in this study was approved by the Murdoch University Animal Ethics
Committee, Murdoch, Australia (Permit number R2024/06) and was performed in
accordance with the requirements of the Australian Code of Ethics for the Care and
Use of Animals for Scientific Purposes 7th Edition 2004.
3.2 Bronchoalveolar lavage and collection technique
Anaesthesia for bronchoscopy and BAL was performed using standardized
protocol. Induction of anaesthesia was performed using propofol (10mg/mL)
administered at 4-6 mg/kg by intravenous injection. Anaesthesia was then
maintained using an intravenous infusion of propofol at 0.2-0.6 mg/kg/minute. After
adequate depth of anaesthesia was obtained, a sterile plastic catheter was inserted
into the mid trachea and 100% oxygen was administered throughout the
bronchoscopic and BAL procedure.
Bronchoscopy was performed in a standardised manner. Grade of tracheal collapse
if present was recorded according to a previously defined scheme by Tangner and
Hobson (1982) based on the percent reduction in luminal size and as described in
section 1.3.7 (figure 4).
Bronchoalveolar lavage was performed via bronchoscopy to ensure a
representative sample of cells and secretions from the diseased lung was collected,
as it has been shown there can be marked clinical and statistical differences
between lung lobes in dogs with pulmonary disease (Hawkins et al, 2003). A 6mm
fibre-optic endoscope (Olympus flexible paediatric GI videoendoscope) was
59
advanced into the trachea and the bronchi and airways were examined. Aliquots of
10-20 mL warmed sterile saline were injected down a sterile plastic catheter in the
catheter port of the endoscope. Based on the study performed by Mills and Litster
(2005), standardised volumes of 10-20 mL of saline were used based on being a
small dog (<20kg) and large dog (>20kg). Small dogs had 10mL instilled and large
dogs had 20 mL saline infused. Gentle suction using a suction pump to a maximum
of 50 mmHg negative pressure was applied to the sterile plastic catheter using a
suction trap connection, to withdraw each flush before a subsequent aliquot was
instilled in an alternate pulmonary lobe if there was disease present in multiple
pulomonary lobes as described by Woods et al (2014). Only the first sample from
each site was included in the study to ensure there was not an artificial increase in
urea concentration in PELF subsequent to the first BAL procedure (Marcy et al,
1987; Cheng et al, 1995).
As the reliability for urea determination of PELF depends on the duration or ‘dwell
time’ (Cheng et al, 1995; Rennard et al, 1986), to avoid underestimation of the
dilution by BALF (overestimation of PELF collected), a set dwell time of 30 seconds
was selected for all samples collected, consistent with the experimental protocol of
Mills and Litster (2005). This protocol is supported by other studies that have shown
a dwell time of 30 seconds is unlikely to be associated with diffusion of urea into
PELF and loss of equilibrium of urea with that of plasma (Marcy et al, 1987;
McGorum et al, 1993; Ward et al, 1992).
Fluid withdrawn was collected directly into sterile sample containers, placed on ice
and transported directly for immediate laboratory cytological analysis. The first
aliquot was used for both urea and cytological analysis. Samples collected from the
same lung lobe after the first sample, were pooled for bacterial culture and
Mycoplasma spp PCR as indicated.
3.3 Blood urea measurement
Blood was withdrawn from the jugular vein of each dog with a 22 gauge needle and
5 mL syringe at the time of BAL in the anaesthetised patient. Blood was collected
into a 3 mL serum screw top collection tube (VACUETTE® Serum Clot Activator 4
60
mL; Greiner Bio-One, Germany). Serum blood urea concentration was measured
using a quantitative enzymatic kinematic method as shown in Figure 5 (Rx Daytona
auto analyzer, Randox Laboratories, UK)a.
FIGURE 5: Principle of urea measurement used by Rx Daytonaa
3.4 Bronchoalveolar lavage fluid processing
Samples of BALF were collected and analysed on the day of collection. Aliquots of
BALF were analysed for urea within 1 hour of collection. If there was any delay in
urea testing, samples were frozen and stored at -20°C and run as a batch no
greater than every 3 months. Urea has previously shown stability in rat serum at -
20°C to at least 90 days (Cray et al, 2009).
3.4.1 Urea concentration in BALF
Bronchoalveolar lavage fluid was centrifuged for 5 minutes at 1500 rpm and the
supernatant collected for measurement of urea concentration.
To account for differences in analyte concentrations between BALF and serum ,
higher volumes of lavage fluid were used than plasma (35 µL instead of 5 µL) and
urea in BAL fluid was measured using the quantitative urea enzymatic kinetic
method (Rx Daytona, Randox Laboratories, UK). A 1:7 dilution was used for test
analytes.
A precision analysis was performed by the laboratory before adopting this dilution
protocol prior to collection of study samples and standard laboratory control
Urease
Urea + H2O 2NH4+ + CO2
GLDH
2α-oxoglutarate + 2NH4+ + 2NADH 2L-glutamate + 2NAD+ + 2H2O
GLDH: Glutamate dehydrogenase
61
samples were run with each BALF urea processed to ensure accurate results.
Between -day –variation of urea measured in BALF was accounted for by the
laboratory, by running repeat BALF samples on pre-study samples subsequent
days prior to commencement of the study.
3.4.2 Bronchoalveolar lavage fluid cell count
A cell count was performed on the first aliquot of fluid collected from each
pulmonary lobe. The urea content of the BAL fluid was determined in the first aliquot
also.
New methylene blue stain was drawn into a blood capillary tube to coat the tube
surface. Fifty µL of BALF was then drawn into the capillary tube using a pipette. The
tube was then gently rocked to stain the cells. The sample was then pipetted into a
haemocytometer (improved neubauerchamber; depth 0.1 mm, 1/400 mm2 area) and
allowed to sit for 10 minutes. All nucleated cells where then counted.
3.4.3 Cytology of bronchoalveolar lavage fluid
Cytology was only performed using the first BAL sample from each pulmonary lobe.
Cell counts were performed on fluid samples that had been centrifuged at 500 rpm
for 8 minutes at slow acceleration in a cytospinner (Cytospin 2, Thermo Scientific).
The cytospinner concentrated and distributed the cells onto a slide for examination.
A 100-cell differential cell count was conducted on all smears of BAL fluid after
staining with Wright’s stain. Cells were also examined for signs of neoplasia. Cells
were reported as eosinophils, neutrophils, mast, epithelial and plasma cells and
macrophages. Macrophages were classified as not activated or activated if the
macrophage exhibited vacuolation or phagocytosis.
3.4.4 Culture of bronchoalveolar lavage fluid
Samples of BAL were cultured when indicated by cell cytology or upon request of
the case clinician. Fluid aliquots were pooled if specific organisms were not
identified on cytological smear examination, in one particular aliquot for culture.
62
Samples were cultured on sheep blood agar and MacConkey agar (Pathwest
media) and incubated at 35oC. Pure culture of any bacterium or fungus was
considered significant. Moderate to heavy growth of any microbe with minimal
growth of oral contaminants was also considered significant.
Positive microbial culture was only confirmed as the definitive aetiological agent if
history, cytology and response to appropriate treatment were supportive.
3.4.5 PCR of bronchoalveolar lavage fluid
Mycoplasma spp. nucleic acid detection PCR was performed on BAL fluid by
Pathwest laboratory. BAL samples were pooled after cell counts and cytological
analysis had been performed, for Mycoplasma spp. PCR.
3.5 Data Processing
The serum: BAL fluid urea ratio was calculated for each sample and an absolute
cell count was determined for the cell differential.
3.5.1 Calculation of epithelial lining fluid recovery
The percentage of PELF in BAL was calculated by;
([Urea BALF] x 100 ) / [Ureaserum] (after Rennard et al, 1996)
3.5.2 Standardisation of cell count
Serum:BAL fluid urea ratios were calculated for each sample. The urea-adjusted
cell count (cells/ µL) were calculated according to the following formula (McGorum
et al, 1993):-
[Ureaserum] (mmol/L) / [Urea BALF] (mmol/L) x cell countBALF (cells/µL)
An apparent differential cell count was calculated for each sample using the
following formula: Urea-adjusted cell count (cells/ul) x % recovered cell type.
For each disease diagnosed, adjusted cell counts and differential cell counts were
collated and mean cell counts and standard deviation for each cell type calculated.
These cell counts of BALF and PELF were also assessed in diseases with
63
concurrent presence of airway collapse and Mycoplasma spp. to determine if these
diseases led to a significant difference in absolute and differential cell counts.
3.6 Diagnosis
All diagnostic tests performed were dependent on the presenting clinical signs and
at the discretion of the attending veterinarian in order to achieve a diagnosis of
respiratory disease. The combined use of radiographs, CT, cytological analysis of
BAL fluid, microbiological analysis including Mycoplasma spp. PCR, biopsy or
necropsy and response to treatment were used to make a final diagnosis.
Respiratory diseases diagnosed were based upon diagnostic tests, necropsy when
applicable and treatment as listed in Appendix 1. The criteria used to make a
diagnosis of each respiratory disease is outlined in Table 6.
64
Table 6: Criteria used to establish definitive diagnosis of respiratory disease
Respiratory Disease Criteria for diagnosis
Chronic Bronchitis
Inflammatory BALF (neutrophilic), diagnosis of exclusion using all methodologies to rule out other disease eg poor or lack of response to treatment, negative culture, or no improvement after treatment for Mycoplasma spp, radiography
Pulmonary carcinoma
Cytology/ histopathology
Pulmonary metastatic neoplasia
Cytology/ histopathology
Sterile Pyogranulomatous
Pneumonia
Histopathology and necropsy with negative PCR and culture results, Inflammatory BALF.
Aspiration pneumonia
Alveolar pattern in right middle and cranial lung lobes, history of regurgitation and/or vomiting, response to treatment
Pulmonary fibrosis CT images, necropsy and histopathology and lack of response to therapy
Pneumonia- infectious- bacterial
Positive BAL culture and/or serology, alveolar infiltrates on imaging
Bronchointerstitial pneumonia- no
infection
Negative BAL culture and serology, bronchointerstitial imaging changes , one nosocomial contamination of BALF
Non-cardiogenic oedema
Negative BAL culture, Caudodorsal pulmonary imaging changes, response to therapy , supportive history
Laryngeal Paralysis Upper airway examination
Laryngeal collapse Upper airway examination
Systemic immune mediated disease
CBC, positive ANA, negative BAL culture, arthrocentesis cytology, proteinuria, lymphadenomegaly, splenomegaly
Phaeochromocytoma Hypertension, histopathology of adrenal mass
65
3.7 Characterisation of Respiratory Disease Groups
Respiratory disease was loosely categorised into respiratory groups based on the
aetiology of respiratory diseases to determine if broad categories of disease could
be identified by BALF corrected for dilution, rather than specific disease entities.
Diseases were assigned to the inflammatory, non-infectious, infectious, upper
respiratory tract or respiratory neoplasia categories (Appendix 6). This differs to the
broad groups of Hawkins et al (1990) in which groups were categorised according
to abnormal cytology. Presence of airway collapse and Mycoplasma spp. was also
recorded within these categories.
3.8 Statistical Assessment
Data in specific respiratory diseases and groups are presented as mean and range
of data to allow comparison with published literature. When more than 5 sample
numbers were available the 95 % confidence interval was also calculated. A paired
t-test, the Welch two sample t-test was used to compare unadjusted and urea
adjusted cell counts between each disease category. Differential cell counts
observed in different disases were assessed by ANOVA and the effect of
Mycoplasma spp. was assessed by linear regression. A p value of <0.05 was
selected for significance. Statistical analysis was performed by use of a command
driven statistical package, R version 2.15.1b.
66
Chapter 4 RESULTS
4.1 Dogs
A total of 48 dogs were included in this study. A complete list of dog breeds and
signalment included in this study is outlined in Appendix 3. A total of 72 BAL
samples were analysed. There were 58 samples from 23 pure breeds processed.
Fourteen samples were obtained from 8 crossbreed dogs. The greatest number of
BALF analysed, were collected from the Staffordshire Bull Terrier (8), Shih Tzu (7),
Labrador (6), West Highland White (5), Border Collie (5) and Jack Russell Terrier
(4).
The mean (Standard deviation (SD)) age of the dogs was 9 years (+/-20m). The
mean (SD) weight of the dogs was 17.7(+/-2.31kg).
4.2 Diseases Diagnosed
A complete list of diseases diagnosed with that had urea and cell counts measured
is listed in Appendix 2 and 5. Table 7 lists the respiratory diseases diagnosed.
Mycoplasma spp. was detected in 27 dogs by PCR (Appendices 2 and 4).
67
Table 7: List of Diseases Diagnosed in dogs with respiratory signs included
in this study.
Respiratory Disease Diagnosis
Number of
samples from
dogs assessed
Chronic Bronchitis 34
Aspiration pneumonia 4
Bronchointerstitial pneumonia- no infection 2
Non cardiogenic oedema 5
Pneumonia- infectious- bacterial 6
Pulmonary fibrosis 6
Pulmonary metastatic neoplasia 2
Systemic immune mediated disease 6
Laryngeal Paralysis 2
Laryngeal collapse 2
Sterile Pyogranulomatous Pneumonia 1
Phaeochromocytoma 1
Pulmonary carcinoma 1
68
4.3 Epithelial Lining Fluid Recovery
The calculated % recovery of PELF in BAL fluid was 25% (median 24.9%; range
0.53%-124%). Calculated PELF recovery is listed in Appendix 2 and Table 8. There
is marked variation of PELF recovery within each respiratory disease group
diagnosed.
Table 8: The median and (range) of the calculated % recovery of pulmonary
epithelial lining fluid (PELF) in each respiratory disease diagnosed. Where
n=2, both values are reported.
Respiratory Disease Diagnosis %PELF Recovery
Chronic Bronchitis (n= 34) 21.9
(0.05 – 94.9)
Pulmonary carcinoma (n=1) 11.3
Pulmonary metastatic neoplasia (n=2) 47.1
(7.87, 86.4)
Sterile Pyogranulomatous Pneumonia (n=1) 8.1
Aspiration pneumonia (n=4) 56.1
(10.8-124)
Pulmonary fibrosis (n=6) 13.7
(5-27.2)
Pneumonia- infectious- bacterial (n=6) 23.2
(13.7-37.9)
Bronchointerstitial pneumonia- no infection (n=2) 27.7
(19.4, 36.0)
Non cardiogenic oedema (n=5) 25.9
(2.50 – 81.9)
Laryngeal Paralysis (n=2) 19.7
(1.68, 37.7)
Laryngeal collapse (n= 2) 12.9
(10.2,15.6)
Systemic immune mediated disease (n=6) 36.5
(10.0-84.0)
Phaeochromocytoma (n=1) 39.1
69
4.4 Absolute and relative cell counts for each respiratory disease
The cell counts in BAL fluid and urea adjusted cell counts for each dog are listed in
Appendix 2. Cell counts for BAL fluid and PELF for each respiratory disease
diagnosed are presented in Tables 9 to 21.
4.4.1 Chronic bronchitis
There were 34 samples from 33 dogs diagnosed with chronic bronchitis (Table 9).
There were 22 males and 12 females. The distribution of unadjusted and urea
adjusted cell counts are shown in Figure 6. Concurrent disease was present in 23 of
the cases sampled and included bronchial collapse or dynamic airway disease (6),
tracheal collapse (4), bronchiectasis and bronchial collapse (3), laryngeal paralysis
(2), bacterial infection and tracheal collapse (2), tracheal and bronchial collapse (2),
and hypoplastic trachea and dynamic airway collapse (2). Bronchiectasis was
identified in one dog and bronchiectasis, bronchial and tracheal collapse was also
identified in one dog. Mycoplasma spp. was detected in 14 cases. In the cases in
which Mycoplasma spp. were detected, retrospective review demonstrated ongoing
respiratory clinical signs after treatment, hence the final diagnosis and inclusion into
the category of chronic bronchitis.
In one dog, Pseudomonas aeruginosa was identified by broth culture and not via
culture on sheep blood agar. This dog was treated with appropriate antibiotics for
P.aeruginosa and made no clinical improvement. The dog was alive 3 years post
diagnosis and bacterial infection was considered an unlikely cause of the dog’s
respiratory clinical signs.
70
Table 9: Cell counts in 34 BAL samples from 33 dogs with chronic bronchitis.
Cell counts are presented as mean (standard deviation) and [range].act =
activated macrophages; unact= unactivated macrophages
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 700 (318)
[10-11000]
2585(584)
[219- 20240]
Differential
Macrophages
Act: 190 (36.9)
[1.98-1079]
Unact: 34.4 (23.4)
[0-834]
Act: 1078 (166)
[62.2 – 5031]
Unact: 173 (107)
[0 – 3486]
Lymphocytes 43.8 (13.6)
[0 – 440]
191 (34.9)
[0 -810]
Neutrophils 416 (291)
[0 -10010]
1084 (545)
[0 -18418]
Eosinophils 15.8 (5.20)
[0 – 126]
90.1 (28.8)
[0 – 876]
Mast Cells 1.72 (0.77)
[0 - 25.8]
11.7 (4.34)
[0 -128]
Plasma Cells 0.98 (0.71)
[0 – 24.8]
3.21 (1.58)
[0 – 41.7]
71
Figure 6: Box and whisker plots showing urea adjusted and unadjusted cell
counts for each cell type in 34 dogs with chronic bronchitis (ooutlier, solid line
median, bars represent range). Eo= eosinophils, CellCt=cell count,
Lym=lymphocytes, Plas=plasma cells, Mph= macrophage
72
4.4.2 Aspiration Pneumonia
There were four BAL samples taken from 3 dogs diagnosed with aspiration
pneumonia (Table 10). The distribution of unadjusted and urea adjusted cell counts
are shown in Figure 7. Three of these dogs were male and one was female. There
was no Mycoplasma spp. detected in any of these samples. Concurrent disease
included megaoesophagus (2) and immune mediated polyarthritis (1).
Table 10: Cell counts in four bronchoalveolar fluid samples from three dogs
with aspiration pneumonia. Cell counts are presented as mean (standard
deviation) and [range]
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 2308 (616)
[470 – 3740]
7649 (3383)
[991– 18148]
Differential
Macrophages
822 (58.2)
[4.80 – 2745]
1418 (592)
[9.91- 2904]
Lymphocytes 98.0 (26.6)
[9.60- 153]
375(211)
[19.8 – 1089]
Neutrophils 1388(635)
[153 – 3403]
5855 (2862)
123 – 14156
Eosinophils 0 0
Mast Cells 0 0
Plasma Cells 0 0
73
Figure 7 : Box and Whisker plot showing urea adjusted and unadjusted cell
counts in dogs with aspiration pneumonia (line represents median, bars
represent range) Eo= eosinophils, CellCt=cell count, Lym=lymphocytes,
Plas=plasma cells, Mph= macrophage (act= activated, nonact= nonactivated)
74
4.4.3 Bronchointerstitial Pneumonia- non infectious
There were two samples taken, one from the right caudal and one sample from the
left caudal lobe in one male kelpie cross dog with a diagnosis of bronchointerstitial
pneumonia (Table 11). The distribution of unadjusted and urea adjusted cell counts
are shown in Figure 8. In these cases, a contaminant mucoid coliform was identified
on broth culture (samples 33 and 34, Appendix 1). It was unable to be cultured on
traditional sheep blood agar. Empirical antibiotic therapy also failed to be associated
with any clinical improvement. There was no other concurrent disease identified.
This led to the classification of non-infectious bronchointerstitial pneumonia.
Table 11: Two BAL cell counts from one dog with bronchointerstitial
pneumonia with no infectious cause identified. Cell count from both samples
[cell count ] and mean are presented.
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 235
[160 and 310]
1020
[444 and1596]
Differential
Macrophages
182
[110 and 254]
808
[307 and 1308]
Lymphocytes 19.7
[18.6 and20.8]
76.8
[57.8 and 95.7]
Neutrophils 15.8
[15.5 and 16.0]
62.1
[44.5 and 79.8]
Eosinophils 11.8
[12.8 and 21.7]
73.6
[35.6 and 112]
Mast Cells 0 0
Plasma Cells 0 0
75
Figure 8: Box and whisker plot showing urea adjusted and unadjusted cell
counts in dogs with bronchointerstitial pneumonia (ooutlier, bar represent
median and lines range) Eo= eosinophils, CellCt=cell count,
Lym=lymphocytes, Plas=plasma cells, Mph= macrophage (act= activated,
nonact= nonactivated)
76
4.4.4 Non- cardiogenic oedema
There were five BAL samples taken from two dogs with non-cardiogenic oedema
(Table 12). The distribution of unadjusted and urea adjusted cell counts are shown
in Figure Nine. Three samples were from one female and two from one male dog.
There was no other significant concurrent disease identified in any dog, however
Pseudomonas spp. was detected on broth culture only in samples from one dog
(case 54, 55 and 56; appendix 1). This sample was processed at an external
laboratory after a 48 hour time delay. No pure culture was grown on sheep blood
agar. Clinical history for that dog was consistent with a ‘near drowning’ episode.
Pseudomonas spp was considered a contaminant. Mycoplasma spp.was detected
in the same dog and given the dog clinically improved with no treatment for
Mycoplasma spp., it was considered this was a commensal bacteria.
Table 12: Cell counts from five broncho-alveolar lavage fluid from three dogs
with non- cardiogenic oedema. Cell counts presented as mean (standard
deviation) and [range].
Cells Unadjusted BAL fluid cell
count (cells/µL) Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 178 (42.9)
[90 – 330]
1880 (673)
[403 – 3778]
Differential
Macrophages
76.9 (17.4)
[44.1-153]
1010 (391)
[80.5- 2304]
Lymphocytes 18.5 (5.31)
[0.63-27.3]
187 (67.9)
[32.2- 453]
Neutrophils 78.9 (35.7)
[31.2- 238]
646 (238)
[101 – 869]
Eosinophils 3.75(1.28)
[0 - 7.50]
68.7 (19.6)
[24.0 – 113]
Mast Cells 1.70 (0.41)
[0-2.50]
24.9 (6.58)
[0 - 37.8]
Plasma Cells 0 0
77
Figure 9 : Box and Whisker plats of urea adjusted and unadjusted cell counts
in three dogs with non cardiogenic oedema (ooutlier, bar represents the
median , lines represent range) Eo= eosinophils, CellCt=cell count,
Lym=lymphocytes, Plas=plasma cells, Mph= macrophage (act= activated,
nonact= nonactivated)
78
4.4.5 Bacterial Pneumonia
There were six BAL samples taken from five dogs with bacterial pneumonia (Table
13). The distribution of unadjusted and urea adjusted cell counts are shown in
Figure 10. Three samples were from three females and three samples were from
two males. Concurrent disease was identified in one dog with bronchial collapse.
Mycoplasma spp. was detected in four of the six samples, including the dog with
concurrent bronchial collapse.
Table 13: Cell counts in 6 bronchoalveolar samples from 5 dogs with bacterial
pneumonia. Cell counts presented as mean (standard deviation) and [range]
with outlier removed ( * range includes single outlier).
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 736(320)
[160 – 15250]
14263(1602)*
[638 – 55953]
Differential
Macrophages
442 (8.21)
[19.7 – 686]
1870 (351)
[189 – 7992]
Lymphocytes 151(57.7)
[4.92-458]
600(251)
[47.2 -1679]
Neutrophils 84.7 (17.6)
[18.7-14106]
547 (197)
[70.1 – 51757]
Eosinophils
8.18(2.62)
[1.70-23.0]
449(17.6)
[6.38 – 100]
Mast Cells 1.02(0.51)
[0 – 3.40]
23.1(1.53)
[0 – 8.90]
Plasma Cells 0 0
79
Figure 10: Box and Whisker plot of urea adjusted and unadjusted cell counts
in dogs with bacterial pneumonia (ooutlier, bar represents median and lines
distribution) Eo= eosinophils, CellCt=cell count, Lym=lymphocytes,
Plas=plasma cells, Mph= macrophage (act= activated, nonact= nonactivated)
80
4.4.6 Pulmonary Fibrosis
There were six samples taken from six dogs with a diagnosis of pulmonary fibrosis
(Table 14). The distribution of unadjusted and urea adjusted cell counts are shown
in Figure 11. One sample was taken from one female and five from males. There
was concurrent disease in three cases. Two had concurrent bronchitis and one had
concurrent bronchiectasis. Five samples tested positive for Mycoplasma spp.
Table 14: Cell counts in 6 BAL samples from 6 dogs with pulmonary fibrosis.
Cell counts presented as mean (standard deviation) with outlier removed and
[range] including outlier.
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 555 (198)
[120-1340]
3954 (922)
[933– 5778]
Differential
Macrophages
27.3 (12.2)
[7 – 1057]
363(147)
[84.0 – 3891]
Lymphocytes 54.3 (16.7)
[0 – 201]
578 (256)
[0 – 1345]
Neutrophils 290(138)
[26.8 – 888]
2644(943)
[98.5 – 4622]
Eosinophils 20.0 (8.88)
[2.90-167]
375(189)
[39.6 – 1120]
Mast Cells 0
[0 – 13.4]
0
[0 – 49.3]
Plasma Cells 0
[0-20.3]
0
[0 – 277]
81
Figure 11: Box and Whisker plot of urea adjusted and unadjusted cell counts
in dogs with pulmonary fibrosis. Distribution of urea adjusted and unadjusted
cell counts in dogs with pulmonary fibrosis (ooutlier, bars represent range,
line represents mean). Eo= eosinophils, CellCt=cell count, Lym=lymphocytes,
Plas=plasma cells, Mph= macrophage (act= activated, nonact= nonactivated)
82
4.4.7 Neoplasia- metastatic and primary pulmonary carcinoma
There were three samples taken from two dogs with pulmonary metastatic
neoplasia (Table 15). One dog was a female crossbreed and the other dog, a male
crossbreed.The distribution of unadjusted and urea adjusted cell counts are shown
in Figure 12. There was no other concurrent disease identified. Mycoplasma spp.
was not tested for in either BAL sample. Both samples came from a male.
Table 15: Cell counts in 3 BAL samples collected from 2 dogs with neoplasia.
Cell counts presented as mean and [range]
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 162
[90.0- 300]
1324
[104- 2654]
Differential
Macrophages
102
[27.0- 123]
647
[31.6 – 1088]
Lymphocytes 9.21
[6.30 -12.4]
81.3
[7.29 -157]
Neutrophils 78.0
[15.2 – 162]
564
[65.6 – 1433]
Eosinophils 2.95
[0- 6.00]
29.7
[0 – 53.0]
Mast Cells 0 0
Plasma Cells 0 0
83
Figure 12: Box and Whisker plots demonstrating distribution of urea adjusted
and unadjusted cell counts in dogs with neoplasia (ooutlier, bar represents
mean and lines range) Eo= eosinophils, CellCt=cell count, Lym=lymphocytes,
Plas=plasma cells, Mph= macrophage (act= activated, nonact= nonactivated)
84
4.4.8 Systemic Immune Mediated Disease
There were six BAL samples taken from three dogs with systemic immune mediated
disease (Table 16). The diagnosis of systemic immune mediated disease was made
as per the guidelines outlined in table 6. The distribution of unadjusted and urea
adjusted cell counts are shown in Figure 13. Three samples were from one female
and three were from two males. There was no other disease detected in these
dogs. Mycoplasma spp. was not detected in any samples.
Table 16: Cell counts in 6 BAL sampels from 3 dogs with systemic immune
mediated disease. Cell counts presented as mean (standard deviation) and
[range] (*outlier removed)
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 651(196)
[8.00-1310]
2039 (703)
[28.6 – 5324]
Differential Macrophages
156 (56.1) [0.56 – 341]
500 (176) [1.99 – 1384]
Lymphocytes 36.4 (12.8)
[0.08 – 91.7] 123 (48.1)
[0.29 – 373]
Neutrophils 457(134)
[7.36 – 878] 1457 (491)
[26.3 – 3567]
Eosinophils 0 0
Mast Cells 0
[0 – 0.60]
0 [0 – 60]
*
Plasma Cells 0 0
85
Figure 13: Distribution of urea adjusted and unadjusted cell counts in dogs
with systemic immune mediated disease ( ooutlier, bars represent range, line
represents mean) Eo= eosinophils, CellCt=cell count, Lym=lymphocytes,
Plas=plasma cells, Mph= macrophage (act= activated, nonact= nonactivated)
86
4.4.9 Laryngeal Paralysis
There were two samples from two dogs with laryngeal paralysis (Table 17). The
distribution of unadjusted and urea adjusted cell counts are shown in Figure 14.
Both dogs were male. One dog had concurrent bronchial collapse. Mycoplasma
spp. was not detected in either sample. Both dogs underwent subsequent arytenoid
lateralisation to manage laryngeal paralysis. One dog improved and the other dog
with concurrent bronchial collapse was euthanased due to ongoing respiratory
compromise 1 week post operatively.
Table 17: Cell counts from 2 BAL samples from 2 dogs with laryngeal
paralysis. Cell count is reported as mean and [range]
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 238
[120 – 355]
3895
[683 – 7108]
Differential
Macrophages
155
[75.6-234]
2464
[451 – 4478]
Lymphocytes 40.3
2.40-78.1
146
142 – 150
Neutrophils 31.6
[28.4 – 34.8]
1058
[54.6 – 2061]
Eosinophils 6.55
[6.00-7.10]
185
[13.7 – 355]
Mast Cells .15
[1.20 – 7.10]
42.4
[13.7 – 71.1]
Plasma Cells 0 0
87
Figure 14: Box and whisker plots of urea adjusted and unadjusted cell counts
in dogs with laryngeal paralysis (bars represent range, line represents mean)
Eo= eosinophils, CellCt=cell count, Lym=lymphocytes, Plas=plasma cells,
Mph= macrophage (act= activated, nonact= nonactivated)
88
4.4.10 Laryngeal Collapse
Two samples were acquired from one male Cavalier King Charles spaniel dog with
laryngeal collapse (Table 18). The distribution of unadjusted and urea adjusted cell
counts are shown in Figure 15. Concurrent bronchial collapse was also present in
the dog samples were taken from. Mycoplasma spp. was not detected.
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 175
[140-210]
1355
[1340 – 1369]
Differential
Macrophages
83.7
[47.6-1120]
615
[466 – 764]
Lymphocytes 22.8
[18.2 - 27.3]
176
[174-178]
Neutrophils 67.6
[60.9 - 74.2]
57
[389 – 726]
Eosinophils 0 0
Mast Cells 0 0
Plasma Cells 0 0
Table 18: Cell counts in two samples from one dog with laryngeal collapse.
Cell count is reported as mean and [range]
89
Figure 15: Box and whisker plots of urea adjusted and unadjusted cell counts
in dogs with laryngeal collapse (ooutlier, bars represent range, line represents
mean)
90
4.4.11 Sterile pyogranulomatous disease
There was one male Staffordshire bull terrier dog diagnosed with sterile
pyogranulomatous pneumonia (Table 19). There was no other concurrent disease
in this dog. Mycoplasma spp. was detected. The dog had been recently imported
into Australia and extensive testing was performed. Testing for A. vasorum,
necropsy and serological testing for Borrelia sp, Bartonella spp. and rickettsial
organisms were negative for concurrent disease.
Table 19: Cell count in one BAL sample from one dog with sterile
pyogranulomatous disease
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 58.0 716
Differential
Macrophages 25.5 315
Lymphocytes 8.12 100
Neutrophils 24.4 301
Eosinophils 0 0
Mast Cells 0 0
Plasma Cells 0 0
91
4.4.12 Phaeochromocytoma
One female dog with phaeochromocytoma was assessed for respiratory signs. This
dog presented with tachypnea and exercise intolerance. The cell count is
presented in Table 20. Mycoplasma spp. was not detected in this sample.
Table 20: Cell count from one BAL sample from one dog with
phaeochromocytoma
Cells Unadjusted BAL fluid
cell count (cells/µL)
Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 100 256
Differential
Macrophages 88.0 2256
Lymphocytes 0 0
Neutrophils 10.0 25.6
Eosinophils 1.00 2.50
Mast Cells 1.00 2.50
Plasma Cells 0 0
92
4.4.13 Pulmonary carcinoma
One spayed female dog was assessed with pulmonary carcinoma. The cell count is
shown in Table 21. Mycoplasma spp. was not tested for in this dog.
Table 21: Cell counts in one BAL sample from one dog with pulmonary
carcinoma
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 300 2654
Differential
Macrophages 123 1088
Lymphocytes 9.00 79.6
Neutrophils 162 1433
Eosinophils 6.00 53.1
Mast Cells 0 0
Plasma Cells 0 0
93
4.5 BALF cytology from dogs with airway collapse and chronic bronchitis - tracheal or bronchial
Tracheal collapse was identified in six dogs with nine BAL samples reviewed. There
were two Jack Russell dogs, one West Highland White and one Pomeranian male
and one Shih Tzu and one Maltese female respectively. All these dogs had chronic
bronchitis as the primary diagnosis of respiratory disease.
There were eighteen dogs identified with dynamic airway disease and collapse of
primary bronchi. Seventeen of these dogs had chronic bronchitis as the primary
diagnosis of respiratory disease and one dog had laryngeal paralysis causing the
most significant component of respiratory disease. Cell counts were compared in
dogs with chronic bronchitis and airway collapse (Table 22).
Assessment of the effect of dynamic airway disease on total nucleated PELF counts
obtained produced no significant association (P=0.49). There was no significant
difference in total or differential cell counts in dogs diagnosed with chronic bronchitis
whether there was the presence of dynamic airway disease or not (Table 23).
94
Table 22: Cell counts in dogs with chronic bronchitis (CB) with and without
tracheal or bronchial airway collapse. All cell counts corrected for dilution.
Cell counts are reported mean (standard deviation) and [range] (*outlier
removed)
Cells Chronic Bronchitis (CB)
(no airway collapse)
CB and Tracheal Collapse
CB and Bronchial Collapse
Total Nucleated
(cells/µL)
1765(449)
[219-2320]
4961 (1917)
[691-1113]
2131(287)
[1200-4303]
Differential
Macrophages
(cells/ µL)
907(149)
[192-2171]
1427(522)
810-5032
1083(521)
[390-2170]
Lymphocytes
(cells/ µL)
188(41.5)
[45.1-617]
180(79.1)
[0-810]
205(71.9)
[78.0-791]
Neutrophils
(cells/µL)
262(55.1)
[75.3-5707]
3685(1296)
[0-18418]
460 (168)
[48.0-1785]
Eosinophils
(cells/µL)
59.9(29.0)
[0-386]
89.6(26.8)
[0-197]
136(27.3)
[0-875]
Mast Cells
(cells/µL)
7.87(3.88)
[0-57.9]
26.1(10.8)
[0-128]
10.9(4.85)
[0-57.8]
Plasma Cells
(cells/µL)
0* 0 0*
95
Table 23 : Statistical association of dogs with chronic bronchitis, with and
without dynamic airway disease.
Parameter P value P value with outliers
removed
Total Cell Count 0.7384 0.4955
Macrophages 0.9576 0.9422
Lymphocytes 0.1902 0.4608
Neutrophils 0.4878 0.401
Eosinophils 0.8924 0.8584
Mast Cells 0.3274 0.3363
Plasma Cells 0.7246 0.6735
96
4.6 Effect of Mycoplasma spp. on respiratory disease
For each disease, dogs were subdivided according to whether Mycoplasma spp.
was detected. The cell counts were compared to determine if Mycoplasma spp. had
any effect on differential and relative absolute cell counts.
A Mycoplasma spp. PCR was performed in fifty one BAL samples. Mycoplasma
spp. was detected in twenty seven BAL samples and in five respiratory disease
states. A summary of the number of dogs in each disease group, with and without
concurrent Mycoplasma spp. are presented in Table 24.
Table 24: The number of dogs with respiratory disease with and without
Mycoplasma spp. detected by PCR.
Respiratory Disease
Diagnosed
Mycoplasma spp.
detected
Mycoplasma spp. not
detected
Chronic bronchitis 15 18
Sterile Pyogranulomatous
Disease 1 0
Pulmonary fibrosis 5 1
Pneumonia- infectious-
bacterial 3 3
Non-cardiogenic oedema 3 2
97
4.6.1 Chronic bronchitis
Of the 33 BAL samples in dogs diagnosed with chronic bronchitis, 15 BAL samples
had no concurrent infection and 18 had concurrent Mycoplasma spp. detected.
Differential cell counts are resented in Table 25.
Table 25 Cell counts in dogs with chronic bronchitis with and without
detection of Mycoplasma spp. Cell counts reported as as mean (+/-standard
deviation) and [range](*outlier removed).
Cells Chronic bronchitis –
Mycoplasma ( n= 15)
Chronic bronchitis +
Mycoplasma ( n= 18)
Total Nucleated
(cells/ µL)
2059 (366)
[219 – 7711]
3169(1205)
[520 – 20,240]
Differential
Macrophages (cells/ µL)
1085 (172)
[89.9 – 3375]
1254 (333)
[62.2 – 5032]
Lymphocytes
(cells/ µL)
1612 (31.2)
[2.19 – 292]
222 (73.5)
[0 – 810]
Neutrophils
(cells/ µL)
649 (273)
[55.6 – 5706]
1602 (153)
[0 – 1876]
Eosinophils
(cells/ µL)
75.1 (25.5)
[0 – 386]
62.4 (19.5)
[0 – 250]
Mast Cells
(cells/ µL)
16.0 (7.04)
[0– 128]
4.98 (2.14)
[0 – 20.8]
Plasma Cells
(cells/ µL) 0*
[0 – 171]
Mean 0*
[0 – 41.7]
Mycoplasma spp. was detected in 18 of 34 BAL samples in dogs diagnosed with
chronic bronchitis. There was no significant difference in total cell counts in the BAL
of dogs with or without Mycoplasma spp., however there was a significant difference
98
in the lymphocyte count (p=0.01). Doxycycline was prescribed in all dogs that had
BAL samples in which Mycoplasma spp. was diagnosed and no single dog
responded to this medication as sole therapy.
99
4.6.2 Pulmonary Fibrosis
Of the six dogs with pulmonary fibrosis, five dogs had concurrent Mycoplasma spp.
infection and one dog had a negative PCR for Mycoplasma spp. (Table 26). The
total nucleated cell count between dogs with pulmonary fibrosis that did and did not
have Mycoplasma spp. correlated to each other (p=0.019).
Table 26: Pulmonary Fibrosis; Comparison of cell counts in dogs detected
with Mycoplasma spp and without detection of Mycoplasma spp. Data
presented as mean and 95% confidence interval and range (*outlier removed)
Cells Pulmonary Fibrosis – Mycoplasma (n= 1)
Pulmonary Fibrosis + Mycoplasma (n= 5)
Total Nucleated
(cells/µL)
5778 3954(1032)
[933-7000]
Differential
Macrophages (cells/ µL)
9289 943(183)*
[84.0-3891]
Lymphocytes
(cells/ µL)
0 193(229)
[0-1345]
Neutrophils
(cells/ µL)
4623 2402 (911)
[98.4-5740]
Eosinophils
(cells/ µL)
867 442(189)
[56.0-1120]
Mast Cells
(cells/ µL)
0 0
[0-49.3]*
Plasma Cells
(cells/ µL)
0 0
[0-277]*
100
4.6.3 Bacterial pneumonia
Of the five dogs with bacterial pneumonia, six BAL samples were assessed and five
of these for presence of Mycoplasma spp. There were two BAL samples in dogs
with bacterial pneumonia in which Mycoplasma spp. was not detected and three
BAL samples in dogs which tested positive for Mycoplasma spp. by PCR (Table
27).
Table 27: Cell counts in dogs with bacterial pneumonia with and without
detection of Mycoplasma spp. Cell counts presented as mean (standard
deviation) and [Range] (*outlier)
Cells Bacterial pneumonia – Mycoplasma spp
(n=2)
Bacterial pneumonia + Mycoplasma spp
(n=3)
Total Nucleated
(cells/µL)
1105
[638-and 1571]
4018 (245)
[897-55953]
Differential
Macrophages (cells/ µL)
263
[189 and 338]
2375 (581)
[210-7992]
Lymphocytes
(cells/ µL)
132
[47.1 and 217]
834 (379)
[69.98-1679]
Neutrophils
(cells/ µL)
664
[70.1 and 1257]
3878 (156)
[179-51757]
Eosinophils
(cells/ µL)
42.5
[6.38 and 78.5]
41.4(16.8)
[0-99.9]
Mast Cells
(cells/ µL) 0* 0*
Plasma Cells
(cells/ µL) 0 0
101
4.6.4 Non- cardiogenic oedema
Two dogs were diagnosed with non-cardiogenic oedema and five BAL samples
were assessed for Mycoplasma spp. Of the five BAL samples in dogs with
noncardiogenic oedema, two BAL samples did not have concurrent Mycoplasma
spp. detected and three BAL samples were from dogs which tested positive to
Mycoplasma spp. when PCR was performed (Table 28).
Table 28: Cell counts in dogs with non cardiogenic oedema with and and
without detection of Mycoplasma spp. Cell counts reported as mean
(standard deviation) and [Range] (*outlier removed)
Cells Non cardiogenic oedema - Mycoplasma
(n=2)
Non cardiogenic oedema + Mycoplasma
(n=3)
Total Nucleated
(cells/µL)
412
[403 -421]
2859 (679)
[1200-3778]
Differential
Macrophages (cells/ µL)
156
[80.5 - 232]
1580 (392)
[672 – 2304]
Lymphocytes
(cells/ µL)
60.3
[32.2 - 88.5]
271 (81.6)
[108-453]
Neutrophils
(cells/ µL)
195
[101-290]
946 (285)
[384-1584]
Eosinophils
(cells/ µL)
0
45.8 (28.2)
[0-113]
Mast Cells
(cells/ µL) 0
16.6 (4.24)*
[0-37.8]
Plasma Cells
(cells/ µL) 0 0
102
103
4.7 Respiratory Disease Groups
Broad respiratory groups were analysed for differences in cell counts as per the
methodology. Of the 47 dogs included in this study from which 71 BALF were
analysed and grouped into inflammatory, non-infectious, infectious, upper repiratory
tract disease and neoplasia (Appendix 6);
Two (three BALF) were categorised as having Neoplasia;
Three (four BALF) had Upper Respiratory Tract Disease;
Six (nine BALF) had Infectious Respiratory Disease;
Twenty eight (41 BALF) had Inflammatory Respiratory Disease
Eight (14 BALF) had Non-infectious Respiratory Disease.
The differential cell counts in dogs categorised accordingly are presented
separately below. In this study dogs were categorised into one generalised disease
category in retrospective analysis. Total and differential cell counts of both BALF
and PELF in different categories did overlap and in some instances and were
similar between disease processes as can be seen when comparing Tables 29 -33.
Thus cell counts were unable to differentiate broad respiratory groups.
104
4.7.1 Neoplasia
Three dogs included in the neoplasia category had a histopathological diagnosis of
neoplasia (Appendix 1). Cell counts are displayed in Table 29.
Table 29: Cell counts in three dogs with neoplasia. Total cell counts are
presented as mean (standard deviation) and [Range]. Differential cell counts
are reported as mean and [Range]
Cells Unadjusted BAL fluid
cell count (cells/µL)
Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 162 (56.5) [90.0- 300]
1322 (603) [104- 2654]
Differential Macrophages
102 [27.0- 123]
647 [31.6 – 1088]
Lymphocytes 9.21
[6.30 -12.4]
81.3 [7.29 -157]
Neutrophils 78.0
[15.2 – 162]
564 [65.6 – 1433]
Eosinophils 2.95
[0- 6.00] 29.7
[0 – 53.0]
Mast Cells 0 0
Plasma Cells 0 0
105
4.7.2 Upper Respiratory Tract Disease
There were four samples included from three dogs with upper respiratory tract
disease (Table 30). Two of these samples came from a dog with laryngeal paralysis
and two dogs had laryngeal collapse. None of these dogs had evidence of non-
cardiogenic oedema based on all tests performed.
Table 30: Cell counts in four BAL samples from three dogs with upper
respiratory tract disease. Cell counts presented as mean (standard deviation)
and [Range] interval.
Cells Unadjusted BAL fluid
cell count (cells/µL)
Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 206 (46.1) [120-355]
2625(1301) [683-7108]
Differential Macrophages
119 (35.5) [47.6-234]
5560(83.2) [451-4478]
Lymphocytes 31.5(14.2) [2.40-78.1]
161 (7.60) [142-178]
Neutrophils 47.9 (9.34) [28.4-74.2]
808 (381) [54.6-2061]
Eosinophils 3.27(1.53)
[0-7.10] 92.3(3.72) [0 – 355]
Mast Cells 2.07(1.02) [0 – 7.10]
21.18 (3.72) [0 – 71.1]
Plasma Cells 0 0
106
4.7.3 Infectious disease
There were nine samples from six dogs that cultured bacteria from bronchoalveolar
lavage fluid (Appendices 1, 2, 5). These dogs were all diagnosed with either a
bacterial or aspiration pneumonia based upon history, clinical examination and
diagnostic investigation (Appendix 1). Cell counts are outlined in Table 31.
Table 31 : Cell counts from nine BAL samples from six dogs categorized as
infectious disease. Cell counts are presented as mean (standard deviation)
and [range].
Cells Unadjusted BAL fluid
cell count (cells/µL)
Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 3144(523)
[160-15250]
11207(1248)
[638 – 55953]
Differential
Macrophages
540(78.6)
[4.80 – 686]
1167(294)
[9.91- 2219]
Lymphocytes 118(55.1)
[9.60 – 458]
301(24.9)
[19.8-1679]
Neutrophils
824 (393)
[18.7 – 3403]
9586(1197)
[70.1 – 51757]
Eosinophils 1.23(0.20)
[0 – 8.20]
10.6(0.87)
[0 – 78.6]
Mast Cells 0.80(0.32)
[0 – 3.40]
2.50 (0.97)
[0 – 8.90]
Plasma Cells 0
0
107
4.7.4 Non-infectious disease
Fourteen bronchoalveolar lavage samples from 8 dogs with non-infectious
respiratory disease included those diagnosed with pulmonary fibrosis (n=6), non-
cardiogenic oedema (n=5), two with bronchointerstitial pneumonia and one dog with
phaeochromocytoma (Appendices 1, 2, 5). Cell counts are listed in Table 32.
Table 32: Cell counts from 14 BAL samples from eight dogs with categorsied
as non-infectious disease. Cell counts are presented as mean (standard
deviation) and [range] (*outlier removed)
Cells Unadjusted BAL fluid
cell count (cells/µL)
Urea- adjusted fluid cell
count (cells/µL)
Total Nucleated 265 (100)
[90.0 – 1110]
2346(579)
[256 – 7000]
Differential
Macrophages
136(49.1)
[10.8 – 289]
647(284)
[80.5 – 2304]
Lymphocytes 27.5(10.1)
[0 – 98.6]
213(106)
[0 – 1345]
Neutrophils 139(59.7)
[10.0 – 888]
1155(463)
[25.6 – 5740]
Eosinophils 22.3(10.6)
[0 – 167]
184(90.3)
[0 – 1120]
Mast Cells 0.31(0.15)
[0 – 2.50]
5.58(2.05)
[0 – 37.8]
Plasma Cells 0*
[0 – 20.3]
0*
[0 – 277]
108
4.7.5 Inflammatory disease category
Forty one samples from 28 dogs were included in the inflammatory respiratory
disease category. Four dogs with SLE were included in this category given the
resulting inflammation caused by the immune response. One dog diagnosed by
biopsy and later necropsy with sterile pyogranulomatous disease was included. The
remaining samples were from dogs diagnosed with chronic bronchitis. Cell counts of
inflammatory disease are listed in Table 33.
Table 33: Cell counts from 41 BAL samples from 28 dogs with disease
categorized as Inflammatory Disease. Cell count presented as mean (standard
deviation) and [range]
Cells Unadjusted BAL fluid cell count (cells/µL)
Urea- adjusted fluid cell count (cells/µL)
Total Nucleated 937(464)
[8.00 – 11000]
2873(837)
[28.6-20240]
Differential
Macrophages
203(32.1)
[0.56-1079]
1061(146)
[1.99 – 5032]
Lymphocytes 52.0(11.5)
[0 – 440]
193(30.1)
[0 – 810]
Neutrophils 414(54.6)*
[0 – 10010]
1106(171)*
[0-18418]
Eosinophils 12.0(4.18)
[0 – 126]
59.4(15.2)
[0 – 386]
Mast Cells 1.60(0.66)
[0 – 25.8]
9.55(3.62)
[0 – 128]
Plasma Cells 1.90(0.83)
[0 – 34.4]
8.30(3.44)
[0 – 171]
109
4.8 Analysis Summary
4.8.1 Analysis between specific respiratory diseases
The statistical association between diseases is listed in Appendix 7. When cell
counts were adjusted for dilution, data distribution was normal in the larger disease
groups. In some disease groups including sterile pyogranulomatous disease,
phaeochromocytoma and pulmonary carcinoma, the sample size was too small to
detect the distribution (Tables 19-21).
There was a significant difference between urea adjusted and unadjusted total cell
counts and differential cell counts for all cell types except plasma cells when results
from all disease states were pooled (p<0.05) as shown in Table 34.
Table 34: Statistical difference between unadjusted and urea adjusted cell
counts in pooled BALF from all respiratory diseases.
Parameter P Value P value After removal of outliers
Total Cell Count 0.00378 3.084 x 10-9
Macrophages 2.27 x 10-6 7.9 x 10-6
Lymphocytes 3.319 x 10-5 1.59 x 10-5
Neutrophils 0.07556 0.0004547
Eosinophils 0.002608 0.002934
Mast cells 0.00606 0.00597
Plasma Cells 0.2049 0.2049
110
After correction for dilution, there was no significant difference between total cell
counts between individual disease states (p=0.43). There was also no significant
difference in differential cell counts between disease states with two exceptions. A
significant difference was detected in dogs with pulmonary fibrosis in the
percentage of adjusted eosinophils (p=0.001) and adjusted plasma cells (p=0.006)
compared to dogs with other respiratory diseases. Adjusted mast cells were
significantly different (p=0.022) in dogs with laryngeal paralysis (Figure 16,17)
compared to dogs with other respiratory disease states.
Figure 16: Total cell counts of respiratory diseases diagnosed adjusted for
dilution by urea (ooutlier)
111
Figure 17: Comparison of differential cell counts between diseases
Key:
A: Immune mediated disease
B: Phaeochromocytoma
C: Pyogranulomatous Disease
D: Laryngeal Collapse
E: Laryngeal Paralysis
F: Neoplasia
G: Chronic Bronchitis
H: Pulmonary Fibrosis
I: Aspiration Penumonia
J: Non cardiogenic oedema
K: Bronchointerstitial Pneumonia
L: Bacterial Pneumonia
112
Key:
A: Immune mediated disease
B: Phaeochromocytoma
C: Pyogranulomatous Disease
D: Laryngeal Collapse
E: Laryngeal Paralysis
F: Neoplasia
G: Chronic Bronchitis
H: Pulmonary Fibrosis
I: Aspiration Penumonia
J: Non cardiogenic oedema
K: Bronchointerstitial Pneumonia
L: Bacterial Pneumonia
113
Key:
A: Immune mediated disease
B: Phaeochromocytoma
C: Pyogranulomatous Disease
D: Laryngeal Collapse
E: Laryngeal Paralysis
F: Neoplasia
G: Chronic Bronchitis
H: Pulmonary Fibrosis
I: Aspiration Penumonia
J: Non cardiogenic oedema
K: Bronchointerstitial Pneumonia
L: Bacterial Pneumonia
114
4.8.2 Assessment of broad respiratory groups
The difference in cell count was assessed between diseases grouped as infectious,
non-infectious, inflammatory, neoplastic and those with upper respiratory tract disease.
No significant difference (p=0.49) was found between different respiratory groups after
correction of total and differential cell counts for dilution using urea (Appendix 8).
Corrected cell counts for broad disease processes is demonstrated in Figure 18 and
shown in Table 35. Unadjusted cell counts are demonstrated in Figure19and listed in
Table 35. These could also not be used individually to differentiate between specific
respiratory disease groups. When uncorrected values were compared, a significant
difference was dected in the mean total cell count of dogs with infectious disease when
compared to other categories (p=0.008) (Appendix 8)
115
Table 35: Broad respiratory disease group cell counts. Cell counts are reported
as mean and standard deviation (Adjusted= urea corrected for dilution); URTD:
Upper Respiratory Tract Disease
Inflammatory
Non-infectious
Infectious URTD Neoplasia
Unadjusted cell count
677 8.00- 11000
342 90.0- 1340
2813 160- 15250
206 120- 355
162 1.00-300
Adjusted cell count
2460 28.6- 20240
2530 256- 7000
10091 638-55953
2625 683- 7108
1322 104-2654
Unadjusted Macrophage
179 0.56- 1079
167 10.8- 1059
483 4.80- 2745
119 47.6 234
71.5 27.0-123
Adjusted Macrophage
965 1.99- 5032
900 80.5- 3891
1061 9.91- 2904
1539 451- 4478
647 31.3-1088
Unadjusted lymphocytes
41.8 0- 440
32.7 0- 201
106 4.92- 458
31.5 2.40- 78.1
9.21 6.30-12.4
Adjusted lymphocytes
179 0- 810
26.0 0- 1345
275 19.8- 1678
161 142 178
81.3 7.29-157
Unadjusted neutrophils
413 0- 10010
137 10.0- 888
735 18.7- 3403
49.6 28.4- 74.2
78.0 15.2-162
Adjusted neutrophils
1119 0- 18418
1135 25.6- 5740
8613 70.1- 51756
808 54.6- 2061
564 65.6-1433
Unadjusted eosinophils
12.0 0- 126
22.2 0- 167
1.98 0- 8.20
3.28 0- 7.10
2.95 0-6.00
Adjusted eosinophils
59.4 0- 386
182 0- 1120
15.9 0-78.6
92.3 0- 355
29.8 0
53.1
Unadjusted mast cells
1.60 0- 25.8
1.27 0- 13.4
0.56 0- 3.40
2.07 0-7.10
0
Adjusted mast cells
9.54 0- 128
7.25 0- 49.3
1.69 0- 8.90
21.2 0- 71.1
0
Unadjusted plasma cells
1.62 0- 34.4
2.40 0- 20.3
0 0 0
Adjusted plasma cells
6.75 0- 171
23.3 0- 277
0 0 0
116
Figure 18: Urea adjusted total cell count for dogs grouped on disease process
(ooutlier)
117
Figure 19: Total cell counts for broad categories of respiratory disease not
adjusted for dilution (bar represents mean and lines range ooutlier)
118
Chapter 5 DISCUSSION
The aim of the study was to determine if BAL cell counts corrected for dilution with urea
to represent PELF could distinguish between different respiratory diseases. Both
specific respiratory diseases and broad disease categories including inflammatory,
neoplastic, non-infectious, upper respiratory tract disease and infectious groups were
evaluated. Since urea-adjusted nucleated and differential cell count of BAL fluid
collected from dogs with diagnosed respiratory disease has not previously been
reported, the first hypothesis was that the use of this technique would allow accurate
quantification of the cellular composition of PELF. The second hypothesis was that
BALF corrected for dilution by urea would identify cell counts that could distinguish
between different disease processes.
The first hypothesis was proven; all samples had cell numbers calculated in PELF
based on the relative concentration of urea in plasma and BALF, thus allowing
accurate quantification of PELF. However, this study failed to prove the second
hypothesis, in that the results did not demonstrate any difference between diseases
and disease groups when unadjusted and adjusted total and differential cells counts
were compared. Thus the second hypothesis of the study was rejected.
5.1 Diagnosis of respiratory disease processes
The cytological responses demonstrated in BALF with respiratory disease has been
used previously to describe broad categories of disease including; acute neutrophilic,
chronic-active, chronic and eosinophilic inflammation, haemorrhage and neoplasia,
defined according to relative differential cell counts.
In this study we used an alternate approach, starting with known respiratory diseases
in clinical patients, and retrospectively applying BALF cell counts modified by the
endogenous marker urea present in PELF, to correct for dilution. We were interested
to determine if infectious, neoplastic, inflammatory or non-inflammatory responses
could be distinguished from one another by corrected cell counts.
119
Each group was assessed to determine if absolute or differential cell counts could be
used with confidence to discriminate between the different disease processes.
Unfortunately, no significant difference was identified between groups when cell counts
were adjusted for dilution (p=0.49) suggesting this technique is not of clinical value
during the diagnostic process for dogs with respiratory disease. There was no
identifiable threshold that separated the patients with infectious disease from those
with inflammatory or non inflammatory disease. In this study the total and differential
counts of BALF cells were not useful for identifying patients with infectious respiratory
disease.
The unadjusted total cell count, before correcting for dilution was increased in
infectious disease but when adjusted cell counts were assessed there was still no
significant difference between disease categories. This contrasts with a previous study
(Peeters et al, 2008) that found a statistically significant difference between lower
respiratory tract infection and chronic bronchitis (the infectious and inflammatory
categories in this study) when nucleated cell count and the percentage of neutrophils
were examined. While the relative percentage of neutrophils can be compared
between these two studies, they vary in that total cell counts cannot be compared, and
Peeters et al (2008) used BALF without correction for dilution.
In contrast, the neutrophilic inflammation found in cytology associated with neoplasia,
chronic bronchitis or inflammatory disease and non-infectious disease in our study is
consistent with the cytologic groups reported by Hawkins et al (1990). Furthermore,
neutrophil counts cannot statistically distinguish disease categories. The raw and
absolute cell counts between this study and that of Peeters et al (2008) were also
markedly different, and meaningful comparison is hampered by a lack of
standardisation between studies (refer to Table 36).
120
Table 36: Total nucleated cell count (TCC) and neutrophils (Neuts) in BAL from
dogs. A comparison between Peeters et al, 2008 unadjusted cell counts and
unadjusted (unadj) and adjusted (Adj) cell count in this study based on final
diagnosis. Cell counts reported as mean and range of cell counts reported. LRTI
= lower respiratory tract infection; CB = chronic bronchitis; Infect. Dis=
Infectious disease group; Inflamm. Dis= Inflammatory disease group.
Peeters et al This study
LRTI CB
Infect.
Dis.
Unadj.
Infect.
Dis.
Adj.
C.B
Unadj.
C.B
Adj.
Inflam.
Dis.
Unadj.
Inflam.
Dis.
Adj.
TCC
mean
range
7990
735
2040
991
250
1807
275
1887
200-41700 70-13000 160-15250 637-55953 10-11000 219-20240 8-11000 28-20240
Neuts.
mean
range
7270
242
679
8970
30
260
71
550
1917-7830 0-728 18-3403 70-51756 0-10010 0-18418 0-10010 0-18418
The results of this study also showed that BAL cytology was insensitive for detecting
pulmonary neoplasia. The cell counts did not differentiate neoplastic respiratory
disease from the other disease processes and none of the respiratory neoplastic
diseases were diagnosed by BAL cell cytology.
Of interest was a dog diagnosed with phaeochromocytoma (Table 20). A BAL was
performed in this dog as it presented with clinical signs suggestive of respiratory
disease including tachypnoea, exercise intolerance and intermittent respiratory noise.
On retrospective analysis this dog did not have concurrent respiratory disease despite
the clinical signs, which resolved after resection of the phaeochromocytoma. The
121
inclusion of this dog in the study was considered valid, and demonstrates that systemic
disease such as a phaeochromocytoma, presenting with respiratory clinical signs, can
produce abnormal cell counts in BALF and PELF, consistent with significant respiratory
diseases and processes. This suggests that BAL assessment cannot be used a ‘gold
standard’ for diagnosis of respiratory disease, and supports the proposal that BAL
cytology should be used only as an ancillary test in respiratory disease diagnosis.
There was no statistically significant difference between this dog’s total unadjusted cell
count in BALF (p=0.78) or (adjusted cell count) in PELF (p=0.44). Differential cell
counts were also unable to differentiate this condition from other respiratory disease.
However, this may represent a type II error given there was only one dog that
underwent BAL diagnosed with phaeochromocytoma. As discussed in section 5.2 and
5.3, type I and II errors may have occurred in other analyses thus masking a significant
difference between respiratory disease groups. A larger study with greater numbers of
patients within each group would be required to identify any difference.
5.2 Pulmonary Epithelial Lining Fluid Recovery
One possible explanation for a lack of apparent difference between adjusted cell
counts could be due to inaccuracy in the technique itself. Various reports of urea used
as an endogenous marker of PELF dilution in BALF have identified sources of potential
error. The technique assumes that the concentration of urea between PELF and
plasma is the same. However prolonged BAL dwell times permit urea to move into the
bronchopulmonary segment and hence may overestimate PELF recovery and reduce
relative corrected cell counts (Rennard et al, 1986; Marcy et al, 1987; McGorum et al,
1993a).
Although attempts were made to standardise dwell time in this study, the data suggests
that this was not the case. This study planned to limit this phenomenon by using a
standardised, rapid BAL technique with BAL recovery occurring 30 seconds after
instillation of saline so that corrected cell counts were minimally affected by variable
recovery. However, for at least one sample, dilution was calculated to be greater than
100% and after review of the video recording of the BALF collection, the dwell time was
122
determined to have exceeded 30 seconds. By minimising the impact of urea diffusion,
correction of cell counts by the endogenous marker urea is a sensitive indicator of
PELF dilution. It is unlikely that the disease process affected the recovery of BALF
given this has not previously been recorded in any of the diseases diagnosed and urea
has equilibrated in the alveolus prior to the procedure (Rennard et al, 1986). It is also
considered unlikely that the disease process in this particular dog altered the diffusion
of urea into the alveolar space, as this was the only sample from dogs with bacterial
pneumonia where BALF recovery was assessed to be greater than one hundred
percent. However a greater sample size would help clarify the likelihood of increased
or altered urea permeability in the various disease states.
Another source of potential error could be due to performance of procedure by different
clinicians with varying expertise and clinical techniques. This appeared to be the most
important factor to account for variability in BALF recovery. Another possible reason for
lack of apparent difference between adjusted cell counts is poor recovery of PELF.
There were 3 samples in which PELF recovery was 0.053, 0.8 and 2.5 percent and 18
samples with PELF recovery <10 percent and 22 samples in which recovery was only
10-20% of PELF (Appendix 2). This was not consistent with one disease process and
occurred in dogs with chronic bronchitis which represented the most frequent disease
identified. Poor recovery of PELF was likely operator dependant. Other reasons such
as sub-optimal equipment including suction traps and the suction machine used may
have caused this potential increased PELF recovery but owing to the retrospective
assessment of data, the exact reason could not be identified. Diagnostic testing was
subject to multiple clinicians’ techniques and skills despite the standardisation of
collection times and sample processing.
Another possibly more useful interpretation of the variable PELF recovery may be in
interpretation of results of cell counts. Poor PELF recovery was identified in three
samples with 0.053, 0.8 and 2.5% calculated. These samples are less likely to have
representative cell count and components of PELF. Thus, urea adjusted BALF may be
used in interpreting whether a representative sample was attained of PELF or whether
just infused fluid was re-aspirated. This may be a more significant finding, particularly if
123
deciding a diagnostic sample was obtained to represent a disease process. This is
consistent with previous studies that have looked to reduce the variability in
interpretation of BALF (Mills and Litster, 2005; Rennard et al, 1986; Vail et al 1995).
5.3 Total Cell Counts
One of the hypotheses of the study was to determine if cell counts in BALF when
corrected for dilution could distinguish between different respiratory diseases and
disease processes. The inability to detect a statistical difference in total and differential
cell counts between the individual respiratory diseases diagnosed in this study may
represent a type II error due to the large variability in cell counts and small sample
sizes. A power analysis may have better identified a minimal sample size required to
increase the ability to detect a difference between these respiratory diseases and
respiratory disease groups.
The power of this study could be increased with a greater number of dogs included and
a multi-centre approach to increase case numbers and potentially varied respiratory
disease exposure. There were a number of diseases types that included only one dog
and no dogs with eosinophilic bronchopneumopathy were diagnosed during the study
period, so that this disease process could not be assessed and compared with others.
Increased numbers of dogs may also decrease any variability of the effect of increased
permeability of the alveolocapillary barrier to urea rather than truly decreased
concentrations of inflammatory cells in PELF.
A large number of parameters influence data obtained from analysis of BALF,
including the BAL procedure itself, the period between collection and processing of the
fluid, and techniques of processing and examining the slide (Dehard et al, 2004). Of
these parameters, all were standardised except for the operator in the BAL procedure
itself which accurately reflects the normal clinical environment.
Cell counts corrected for dilution in the broader respiratory groups could not
differentiate between these respiratory groups implying the total cell numbers (p=0.49;
Appendix 8) and differential cell numbers (p=0.36 - 0.63; Appendix 8) of PELF in
different disease states are not statistically different.
124
Results of the current study revealed a total cell count (TCC) after correction for
dilution of 5778 cells/µL in the six dogs diagnosed with pulmonary fibrosis. A previous
study identified BALF TCC was <805 cells/µL in nine dogs (Krafft et al, 2011), however
this study did not account for dilution of PELF, thus highlighting the differences when
assessing different studies and the benefit of cell count standardisation.
125
5.4 Differential Cell Counts
The only significant difference detected in this study was for differential counts in dogs
with pulmonary fibrosis.Traditionally the differentiation of pulmonary fibrosis from other
chronic respiratory diseases, especially chronic bronchitis, is difficult. Auscultation,
thoracic radiography, bronchoscopy, and BALF analysis have poor specificity. While
the biomarker endothelin-1 and high resolution computed tomography can potentially
increase the accuracy of diagnosis (Krafft et al, 2011), further diagnostic tests may
increase the specificity of diagnosis. Results of the current study revealed significantly
higher differential eosinophil cell count (p=0.001) and plasma cell count (p=0.006) in
the six dogs diagnosed with pulmonary fibrosis compared to other respiratory disease.
In the current study study neutrophils were increased to 4.5% in the dogs with
pulmonary fibrosis. In comparison, a previous study of dogs with pulmonary fibrosis
(Krafft et al, 2011) reported neutrophils were increased to seventy five percent.
According to the classification scheme of Hawkins (Hawkins et al 1990; Hawkins et al,
1995), the study of Krafft et al (2011) would classify the BALF as neutrophilic and
consistent with non- bacterial infection (protozoa, fungal, rickettsial) or chronic
bronchitis. This was not the case in this study; when assessing the total differential cell
count of PELF, neutrophils were not significant in differentiating between any
respiratory disease. This result or lack of difference between differential cell counts
may indicate a wide variability in cell populations in BALF within a respiratory disease
or that cytological categories should consider broader disease entities and group
diseases more loosely.
The increased eosinophil cell count in dogs with pulmonary fibrosis in this study may
also represent a type I error as a result of the small sample size or reflect an unknown
aetiology of disease where fibrosis is an end- stage of an inflammatory response.
Larger numbers of dogs are required to further assess this difference between
respiratory diseases.
Mast cells in PELF were significantly increased (p=0.022) in dogs with laryngeal
paralysis when compared to the other respiratory diseases. Other abnormalities that
often occur with laryngeal paralysis such as megaoesophagus, bronchopneumonia,
126
narrowing or dilation of the extra thoracic trachea, hiatal hernia and gastro-
oesophageal reflux (Burbridge, 1995), were not reported in these dogs and thus could
not account for this finding. It is more likely this represents a type II error as only two
dogs were diagnosed with laryngeal paralysis.
In a previous study by Peeters et al (2000), the percentage of polymorphonucleocytes
in unadjusted BALF was significantly different between dogs with lower respiratory tract
infection and dogs with chronic bronchitis, pulmonary carcinoma or pulmonary
blastomycosis. This was not evident in either unadjusted or urea adjusted BALF in the
current study (Appendix 2; table 9, 13, 20; figures 6, 10). Given there was no statistical
difference prior to adjustment for PELF dilution, mathematical error and dilution are not
the cause of this difference between studies. While bacterial pneumonia would be
considered likely to cause a significant increase in neutrophils, it may not have
occurred in this current study as bronchoscopic findings and cytological changes are
inconsistently present as has been previously documented (Hawkins, 1999; Peeters et
al, 2000).
Pulmonary infiltrates and significant inflammation may not be present early in the
course of disease and the discrepancy between this study and the study by Peeters et
al (2000) may be a result of the timing of sample collection in the disease. Alternatively
it may be a result of the small numbers of dogs (n= 4) diagnosed with bacterial
pneumonia in this study and thus a type II error. False positive cultures that can occur
in the healthy tracheobronchial tree in dogs due to airway colonisation rather than true
infection was also ruled out in this study as all dogs were retrospectively analysed and
the definitive diagnosis was based upon gross bronchoscopic, radiological,
haematological, cytological, culture, historical and physical findings and response to
treatment. Thus it is unlikely the difference in results between studies was due to
incorrect diagnosis.
127
5.5 The effect of Mycoplasma spp on total and differential cell counts
Of fifty six samples in this study tested for Mycoplasma spp., twenty seven BAL
samples in five respiratory disease groups were found to be positive for Mycoplasma
spp. There was no statistically significant association with any one respiratory disease.
A retrospective assessment of treatment of these cases demonstrated doxycycline was
the antibiotic chosen in all cases to treat potential Mycoplasma spp. infection. In all
cases, none of the dogs improved with treatment for Mycoplasma spp. alone, which
may support the hypothesis that this is a commensal organism of the respiratory tract.
Alternatively, Mycoplasma spp. may be an opportunisitic organism that needs
consideration in treatment of all respiratory diseases in effecting response to any
treatment regime.
The role of Mycoplasma spp. as a primary cause of respiratory disease in dogs and
cats has been debated. A number of studies have shown several Mycoplasma spp. to
be inhabitants of the eye, joints, alimentary, respiratory and genital tracts, suggesting it
may be a causal factor of inflammation in these locations (Rosendal, 1990; Rosendal,
1982). Another study investigated Mycoplasma spp. as a primary pathogen and found
that its presence was indicative of disease (Chandler and Lappin, 2002).
In the current study, there was a greater percentage of lymphocytes in BALF in dogs
with chronic bronchitis when Mycoplasma spp. was present (p=0.01).It has been
postulated that Mycoplasma spp. interferes with antigenic processing by macrophages
which then interferes with cytokine signaling for T-lymphocyte stimulation (Roitt et al,
1969) and thus inhibits lymphoid blast transformation. This mechanism is likely to be
complex and a proposed mechanism of altered lymphocyte activity and number may
be a result of mycoplasma interfering with antibody production (Kaklamanis et al,
1969). An alternate explanation may involve the expansion of the macrophage
population after early depression of phagocytic activity that might stimulate
autoreactive clones of T- and B- lymphocytes as is seen in mice infected with
Mycoplasma arthritidis (Kaklamani et al, 1993). It was out of the scope of this study to
investigate the causality of increased differential lymphocyte counts in dogs testing
positive for Mycoplasma spp.
128
There was no other statistical difference in total or differential cell counts in any other
respiratory disease state in dogs that tested positive for Mycoplasma spp. This may be
a result of the small sample size of dogs with respiratory disease other than chronic
bronchitis.
5.6 Effect of dynamic airway collapse on cell counts
There was no significant effect of tracheobronchomalacia or dynamic airway disease
on recovered BALF cell counts in dogs with chronic bronchitis (Table 33).
Bronchomalacia was not a frequent finding in dogs with tracheal collapse (Appendix 4).
Tracheobronchomalacia is diagnosed by documentation of a reduction in airway
diameter of the trachea or bronchi during bronchoscopy. It has previously been
hypothesised that bronchomalacia is common in dogs with tracheal collapse and is
associated with inflammatory airway disease (Jokinen et al, 1977; Johnson and
Pollard, 1977). However in this study, there was no significant evidence of increased
inflammation in dogs with chronic bronchitis that had airway collapse, thus a role for
chronic inflammation in malacic airway changes could not be confirmed in this study.
This is also consistent with a previous study (Johnson and Pollard, 2010) that could not
confirm a role for airway inflammation in bronchomalacia.
This study also supports results of previous studies (Johnson and Pollard, 2010;
Johnson and Fales, 2001) that failed to establish a role for bacterial infection in
tracheal collapse, although this may be a type II error due to the small numbers of dogs
identified with tracheobronchomalacia.
129
5.7 Conclusions
Unfortunately this study did not assist in further discrimination of respiratory disease
based on standardisation of BALF cell counts using urea standardisation thus
discarding our second hypothesis. The value of BAL cytology as an ancillary test
remains high in distinguishing respiratory disease, however comparison on retrieved
cell counts and percentages can be highly variable between studies given the volumes
of lavage fluid and varied PELF dilutions retrieved.
While this study did not identify a significant difference between cell counts
representative of PELF in different respiratory diseases, greater numbers of cases with
a larger number of respiratory diseases are likely to be required to differentiate
diseases with acceptable sensitivity and specificity. If broad respiratory disease
processes are considered, a significant difference in cell counts representing PELF
could not be identified in this study.
Currently, no ‘gold standard’ can be used to discriminate different respiratory diseases
and different respiratory disease processes. Disease identification continues to rely on
a combination of history, physical, haematological, radiological, gross bronchoscopic,
cytological examination and culture of BALF.
130
References
a. Randox Urea Rx Daytona Laboratory reference UR3825. Randox Laboratories Ltd,
Ardmore, Diamond Rd, Crumlin, Co.Antrium, United Kingdom
b. R statistics package available at http://www.r-project.org. R foundation; Vienna
Austria Accessed July, 2012
Adams FH, Moss AJ, Fagan L (1963) The tracheal fluid in the fetal lamb Biol Neonat 5
pp151-158
Alberts WM (2004) Eosinophilic interstitial lung disease Curr Opin Pulm Med 10(5) pp
419-424
Amis TC, McKiernan BC (1986) Systematic identification of endobronchial anatomy
during bronchoscopy in the dog. Am J Vet Res 47(12) pp 9-57.
Armbrust LJ, Biller, DS, Bamford A, et al (2012) Comparison of three-view thoracic
radiography and computed tomography for detection of pulmonary nodules in dogs
with neoplasia J Am Vet Med Assoc 240 pp 1088–1094
Au JJ, Weisman DL, Stefanacci JD, et al (2006) Use of computed tomography for
evaluation of lung lesions associated with spontaneous pneumothorax in dogs: 12
cases (1999–2002). J Am Vet Med Assoc 228(5) pp733–7.
Avni T, Levy I, Sprecher H et al (2012) Diagnostic accuracy of PCR alone compared to
galactomannan in bronchoalveolar lavage fluid for diagnosis of invasive pulmonary
aspergillosis: a systematic review J of Clin Microbiol 50(11) pp 3652-3658
Bailie WE, Stowe EC , Schmitt AM (1978) Aerobic Bacterial Flora of Oral and Nasal
Fluids of Canines with Reference to Bacteria Associated with Bites J of Clin Microbiol
7(2) pp 223-231
Barcante JM , Barcante P, Ribero TA, et al. (2008) Cytological and parasitological
analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum
infection in dogs. Veterinary Parasitology 158, pp 93-102.
131
Basset G, Bouchonnet F, Crone C et al (1988) Potasium transport across rat alveolar
epithelium: evidence for an apical Na+-K+ pump J Physiol 400 pp 529-543
Bayat S, Menaouar A, Anglade D et al (1998) In vivo quantitation of epithelial lining
fluid in dog lung Am J Respir Crit Care Med 158 pp 1715-1723
Bayat S, Louchahi K, VerdièreB,et al (2004) Comparison of 99mTc-DTPA and urea for
measuring cefipime concentrations in epiethlial lining fluid Eur respir J 24 pp150-156
Baughman RP, Bosken CH, Loudon RG et al (1983) Quantitation of bronchoalveolar
lavage with methylene blue. Am Rev Respir Dis 128 pp 266-270
Bell DY, Haseman JA, Spock A et al (1981) Plasma proteins of the bronchoalveolar
surface of the lungs of smokers and nonsmokers. Am Rev Respir Dis 124 pp 72-79
Benumof JL (2013) Respiratory Physiology and Respiratory Function During
Anesthesia (Chap 5) in Benumof and Hagberg's Airway Management pp 118- 158
Hagberg CA (ed) Saunders, Elsevier Philadelphia PA
Bolt G, Monrad J, Koch J et al (1994) Canine angiostronglylosis: a review Vet
Rec135(19) pp 447-452Boswood A, Dukes-McEwan J, Loureiro J et al (2008) The
diagnostic accuracy of different natriuretic peptides in the investigation of canine
cardiac disease J. of Sm Anim Pract 49 pp 26–32
Bourque AC., Conboy G, Miller IM et al. (2008) Pathological findings in dogs naturally
infected with Angiostrongylus vasorum in Newfoundland and Labrador, Canada.
Journal of Veterinary Diagnostic Investigation 20 pp 11-20
Brandendistel LJ, Vogler GA, Franck PA (1992) Bronchoalveolar eosinophilia in
random-source versus purpose-bred dogs. Lab Anim Sci 42 pp 491-496
Brown NO, Noone KE, Kurzman ID (1983) Alveolar lavage in dogs. Am J Vet Res 44
pp 335-337.
Brownback KR and Simpson SQ (2013) Association of bronchoalveolar lavage yield
with chest computed tomography findings and symptoms in immunocompromised
patients Ann Thorac Med 8(3) pp 153-159
132
Brownlie (2004) Mycoplasmas associated with canine infectious respiratory disease.
Microbiology 150 pp 3491-3497
Brownlie SE (1990) A retrospective study of diagnosis of 109 cases of canine lower
respiratory disease J Small Anim Pract 31 pp 371-376
Burbridge HM (1995) A review of laryngeal paralysis in dogs Br Vet J 151 pp 71-82
Burbridge H, Goulden BE, Jones BR (1991) Neurogenic laryngeal paralysis in the dog
New Zealand Vet J 39 pp 83-87
Byers DE andHoltzman MJ (2011) Alternatively Activated Macrophages and Airway
Disease Chest 140(3) pp 768-774
Cannon MS, Wisner ER, Johnson LR et al (2009) Computed tomography bronchial
lumen to pulmonary artery diameter ratio in dogs without clinical pulmonary disease
Veterinary Radiology & Ultrasound 50(6) pp 622–624.
Carlisle CH, Atwell RB (2008) A survey of heartworm in dogs in Australia. Aust Vet J
61(11) pp 356-360
Chalker VJ (2005) Canine Mycoplasmas Res Vet Sci 79(1) pp 1-8
Chalker VJ, Owen WMA, Paterson C et al (2004) Mycoplasmas associated with canine
infectious respiratory disease. Microbiology 150 pp 3491-3497
Chalker VJ, Owen WM, Paterson C et al (2009) Tools used in the diagnosis and
staging of lung cancer: what’s old and what’s new? Q J Med 102:443–448
Chandler JC, Lappin MR (2002) Mycoplasma respiratory infections in small animals: 17
cases (1988-1999) J.Am Anim Hosp Assoc 38 pp 111-119
Cheng PW, Boat TF, Shaikh S et al (1995) Differential effects of ozone on lung
epithelial lining fluid volume and protein content. Exp Lung Res 21 pp3 51–365
Chandler JC, Lappin MR (2002) Mycoplasmal respiratory infections in small animals;
17 cases (1988-1999) J Am Anim Hosp Assoc 38 pp 111-119
133
Chen S, Sorrell T, Nimmo G et al (2000) Epidemiology and hostand variety-dependent
characteristics of infection due to Cryptococcus neoformans in Australia and New
Zealand. Australasian Cryptococcal Study Group. Clin Infect Dis 31 pp 499–508.
Chotirmall SH, Al-Alawi M, Mirkovic B et al (2013) Aspergillus-Associated Airway
Disease, Inflammation, and the Innate Immune Response Biomed Res Int
2013:723129. doi: 10.1155/2013/723129
Clerxc C, Peeters D, Snaps F et al (2000) Eosinophilic bronchopneumopathy in dogs J
Vet Intern Med 14 (3) pp 282-291
Clercx C, Peeters D, German AJ et al (2002) An immunologic investigation of canine
eosinophilic bronchopneumopathy J Vet Intern Med 16 (3) pp229-237
Clercx C ,Peeters D (2007) Canine Eosinophilic Bronchopneumopathy Vet Clin Small
Anim Pract 34 pp 917-935
Cobben NAM, Jacobs JA, van Dieijen-Visser et al (1999) Diagnostic value of BAL fluid
cellular profile and enzymes in infectious pulmonary disorders Eur Respir J 14 pp 496-
502
Cohn L (2010) Pulmonary parenchymal disease in Textbook of Veterinary Internal
Medicine 7th ed. Ettinger SJ and EC Feldman eds pp 1096-1119 Saunders Elsevier,
Miss USA
Cohn LA (2007) Respiratory defenses in Health and Disease Vet Clin Small Anim Pract
37 pp 845-860
Corcoran BM, Thoday KL, Henfrey JI (1991) Pulmonary infiltration with eosinophils in
14 dogs. J Small Anim Pract 32 (10) pp494-502
Corcoran BM, Cobb M, Martin MWS et al (1999) Chronic Pulmonary disease in West
Highland white terriers Vet Rec 144 pp 611-616
Cowell R, Tyler R and C Baldwin (1987) Trantracheal and bronchial washes (in) Cowell
R, Tyler R (eds) Diagnostic cytology of the dog and cat pp167-177 American
Veterinary Publications, Goleta, Calif, USA
134
Crager CS (1992) Canine primary ciliary dyskinesia Compend Contin Ed Pract Vet: Sm
Anim Pract 14 pp 1440-1445
Crawford PC, Dubovi EJ, Castleman WL et al (2005) Transmission of equine influenza
to dogs. Science 310 pp 482–485.
Cray C, Rodriguez M, Zaias J et al (2009) Effects of Storage Temperature and Time on
Clinical Biochemical Parameters from Rat Serum J Am Assoc Lab Anim Sci 48(2) pp
202–204.
Creevy KE (2009) Airway Evaluation and Flexible Endoscopic Procedures in Dogs and
Cats: Laryngoscopy, Transtracheal Wash, Tracheobronchoscopy, and Bronchoalveolar
Lavage Vet Clin Small Anim 39 pp 869–880
Cross C, Rieben PA, Salisbury PF (1960) Urea permeability of alveolar membrane;
hemodynamic effects of liquid in the alveolar spaces. Am J Physiol 198 (5) pp 1029-
1031
Currie DC, Cooke JC, Morgan AD (1987) Interpretations of bronchograms and chest
radiographs in patients with chronic sputum production Thorax 42 pp 278-284
Dallman MJ, Martin RA, Roth L (1988) Pneumothorax as the primary problem in two
cases of bronchioloalveolar carcinoma in the dog J Am Anim Hosp Assoc 24 pp 710-
714
Davis GS, Giancola MS, Costanza MC et al (1982) Analyses of sequential samples of
human healthy volunteers Am Rev Respir Dis 126 pp 611-616
Dehard S, Bernaerts F, Peeters D et al (2008) Comparison of Bronchoalveolar Lavage
Cytospins and Smears in Dogs and Cats J Am Anim Hosp Assoc 44 pp 285-294.
De Brauwer EI, Jacobs JA, Nieman F et al (2000) Cytocentrifugation conditions
affecting the differential cell count in bronchoalveolar lavage fluid. Anal Quant Cytol
Histol 22 pp 416-422
De Francesco TC, Rush JE, Rozanski EA et al (2007) Prospective Clinical Evaluation
of an ELISA B-Type Natriuretic Peptide Assay in the Diagnosis of Congestive Heart
Failure in Dogs Presenting with Cough or Dyspnea J Vet Intern Med 21 pp 243–250
135
De Lorenzi D, Bertoncello D, Drigo M (2009) Bronchial abnormalities found in a
consecutive series of 40 brachycephalic dogs J Am Vet Med Assoc 235 (1) pp 835-840
De Brauwer EI, Drent M, Mulder PG et al (2000) Differential cell analysis of
cytocentrifuged bronchoalveolar fluid samples affected by the area counted Anal Quant
Cytol Histol 22 pp143-149.
Dehard S, Bernaerts F, Peeters D et al (2008) Comparison of Bronchoalveolar Lavage
Cytospins and Smears in Dogs and Cats J Am Anim Hosp Assoc 44 pp 285-294.
Drent M, Cobben NAM, Henderson RF et al (1996) Usefulness of lactate
dehydrogenase and its isoenzymes as indicator of lung damage or inflammation. Eur
Respir J 9 pp 1736-1742.
Drobatz DC, Silverstein KJ (2010) Clinical Evaluation of the Respiratory Tract in
Textbook of Veterinary Internal Medicine 7th Ed Ettinger SJ and EC Feldman Saunders
Elsevier St Louis, Miss pp 1055-1066
Dubovi EJ, BL Njaa BL(2008) Canine Influenza Vet Clin Small Anim 38 pp 827-835
Duncan C, Stephen C, Campbell J (2006) Clinical characteristics and predictors of
mortality for Cryptococcus gattii infection in dogs and cats of southwestern British
Columbia Can Vet J. 47(10) pp 993–998.
Duncan JR, Prasse KW, Mahaffey EA (1994) Veterinary Laboratory Medicine. Clinical
Pathology 3rd Ed. Iowa State University Press, Ames Iowa pp 162-184
Dyce KM, Sack WO,Wensing CJG (1987) Textbook of Veterinary Anatomy WB
Saunders Co Philadelphia PA pp 143-162
Edwards DF, Patton CS, Kennedy JR (1992) Primary ciliary dyskinesia in the dog
Problems in Veterinary Medicine 4 pp 291-319
Effros RM, Feng NH, Mason G et al (1990) Solute concentrations of the pulmonary
epithelial lining fluid of anaesthetized rats J Appl Physiol 68 (1) pp 275-281
Effros RM, Murphy C, Ozker K et al (1992) Kinetics of urea exchange in air-filled and
fluid- filled rat lungs. Am J Physiol 263 pp L619-L626
136
Effros RM, Jacobs E, Hacker A et al (1993) Reversible inhibition of urea exchange in
rat hepatocytes J Clin Invest 91 pp 2821-2828
Effros RM, Murphy C, Hacker A et al (1994) Reduction and uptake of methylene blue
from rat air-spaces. J Appl Physiol 77 pp 1460-1465
Eid AA, Keddisii JI,Kinasewitz GT (1999) Hypoalbuminemia as a cause of Pleural
Effusions Chest 115 pp 1066-1069
Epstein SE, Mellema MS,Hopper K (2010) Airway microbial culture and susceptibility
patterns in dogs and cats with respiratory disease of varying severity J of Vet Emerg
and Crit Care 20(6) pp 587-594
Eriksson M, von Euler H, Ekman E et al (2009) Surfactant Protein C in Canine
Pulmonary Fibrosis J Vet Intern Med 23 pp 1170–1174
Erasmus JJ, Truong MT, Munden RF (2005) CT, MR, and PET imaging in staging of
non-small-cell lung cancer. Semin Roentgenol 40 pp 126-142.
Erles K,Brownlie J (2008) Canine respiratory coronavirus: an emerging pathogen in the
canine infectius respiratory disease complex Vet Clin Small Anim 38 pp 815-825
Ettinger S (2010) Disease of the Trachea and Upper Airways in Textbook of Veterinary
Internal Medicine 7th ed. Ettinger SJ and EC Feldman eds pp 1066-1088 Saunders
Elsevier, Miss USA
Evans HE, De Lahundra A. eds (2013) Miller’s anatomy of the dog. Fourth ed.
Philadelphia: Elsevier Saunders, 2013 p359.
Fenlon HM, Doran M, Sant SM et al (1996) High-resolution chest CT in systemic lupus
erythematosus. Am J Roentogenol 166 pp 301–7.
Fine DM, DeClue AE, Reinero CR (2008) Evaluation of circulating amino terminal-pro-
B-type natriuretic peptide concentration in dogs with respiratory distress attributable to
congestive heart failure or primary pulmonary disease J.Am Vet Med Assoc 232 (11)
pp 1674-1679
137
Fleury-Feith J, Escudier E, Pocholle MJ, et al (1987) The effects of cytocentrifugation
on differential cell counts in samples obtained by bronchoalveolar lavage. Acta Cytol
31 pp 606-610.
Fonfara S, de la Heras Alegret L, German AJ et al (2011) Underlying diseases in dogs
referred to a veterinary teaching hospital because of dyspnea: 229 cases (2003-2007).
J Am Vet Med Assoc. 239(9) pp 1219-24.
Foster SF, Martin P, Braddock JA et al (2004) A restrospective analysis of feline
bronchoalveolar lavage cytology and microbiology (1995-2000) J Feline Med Surg 6 pp
189-198
Fraczek MG, Kirwan MB, Moore CB et al (2014) Volume dependency for culture of
fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative
PCR Mycoses 57(2):69-78. doi: 10.1111/myc.12103.
Frericks BB, Meyer BC, Martus P et al (2008) MRI of the Thorax During Whole-Body
MRI: Evaluation of Different MR Sequences and Comparison to Thoracic Multidetector
Computed Tomography (MDCT) J. of Mag Res Imag 27(3) pp538–545
Gibson PG, Allen CJ, Yang JP (1993) Intra-epithelial mast cells in allergic and non-
allergic asthma Am Rev Respir Dis 148: 80-86
Glick JH (1969) Serum lactate dehydrogenase isoenzyme and total lactate
dehydrogenase values in health and disease, and clinical evaluation of these tests by
means of discriminant analysis. Am J Clin Pathol 52 pp 320-328.
Goggs R, Benigni L, Fuentes VL et al (2009) Pulmonary thromboembolism J. of Vet
Emerg and Crit Care 19(1) 2009, pp 30–52
Gülmez I, Oğuzkaya F, Bilgin M et al (1999) Posterior mediastinal goiter. Monaldi Arch
Chest Dis 54:402–403
Graber SE , Krantz SB (1978) Erythropoietin and the control of red cell production Ann
Rev Med 29 pp 51-66
Guleria R, Pangtey G (2007) Lung in SLE Indian Journal of Rheumatology 2(4) pp.
131–132
138
Harvey CE (1982) Stenotic nares surgery in brachycephalic dogs. J Am Anim Hosp
Assoc 18 pp 535-537
Hawkins EC, DeNicola DB , Kuehn NF (1990) Bronchoalveolar lavage in the evaluation
of pulmonary disease in the dog and cate. State of the Art. J. Vet Int Med 4(5) pp 267-
264
Hawkins EC, Morrison WB, DiNicola DB et al (1993) Cytologic analysis of
bronchoalveolar lavage fluid from 47 dogs with multicentric malignant lymphoma J Am
Vet Assoc 203 pp 1418-1425
Hawkins EC, De Nicola DB, Plier ML (1995) Cytological analysis of bronchoalveolar
lavage fluid in the diagnosis of spontaneous respiratory tract disease in dogs: a
retrospective study. J Vet Intern Med 9 pp 386-392.
Hawkins EC (1999) Pulmonary parenchymal diseases. In Ettinger SJ, Feldman EC,
eds. Textbook of Veterinary Internal Medicine, 5th Ed. Philadelphia, PA: WB Saunders
pp1061-1091
Hawkins EC, Basseches J, Berry CR, Stebbins ME and KK Ferris (2003)
Demographic, clinical and radiographic features of bronchiectasis in dogs: 316 cases
(1988-2000). J Am Vet Med Assoc 223 pp 1628-1635
Hawkins EC (2004) Bronchoalveolar lavage. In Textbook of Respiratory Disease in
Dogs and Cats. King LG, ed. Missouri: Saunders, pp 118-128.
Hawkins EC, Rogala AR, Large EF et al (2006) Cellular composition of bronchial
brushings obtained from healthy dogs and dogs with chronic cough and cytologic
composition of bronchoalveolar lavage fluid obtained from dogs with chronic cough.
Am J Vet Res 67(1) pp160-167
Hawkins EC, DeNicola DB (1990) Cytologic analysis of tracheal wash specimens and
bronchoalveolar lavage fluid in the diagnosis of mycotic infections in dogs. J Am Vet
Med Assoc 197 pp 79–83.
139
Hawkins EC, Clay LD, Bradley JM et al (2010) demographic and Historical Findings,
Including Exposure to Environmental Tobacco Smoke in Dogs with Chronic Cough J
Vet Intern Med 24 pp 825-831
Hediger Ma, Smith CP, You G et al (1996) Structure, regulation and physiological roles
of urea transporters Kidney Int 49 pp 1615-1623
Heikkilä HP, Lappalainen AK, Day MJ et al (2011) Clinical, bronchoscopic,
histopathologic, diagnostic imaging, and arterial oxygenation findings in West Highland
White terriers with idiopathic pulmonary fibrosis J Vet Intern Med 25 pp 433-439
Hirt RA, Wiederstein I, Denner EB et al (2010) Influence of the collection and
oxygenation method on quantitative bacterial composition in bronchoalveolar lavage
fluid samples from healthy dogs. Vet J. 184(1) pp 77-82
Hunninghake GW, Kawanami O, Ferrans VJ et al (1981) Characterization of the
inflammatory and immune effector ceels in the lung parenchyma of patients with
interstitial lung disease Am J of Resp Res 123 pp407-412
Huston SM, Mody CH (2009) Cryptococcosis: an emerging respiratory mycosis Clin
Chest Med 30(2) pp 253-264
Johnson LR (2009) Respiratory Diagnostics. Abstracts European Veterinary
Conference Voorjaarsdagen 2009
Johnson LR, Fales WH (2001) Clinical and microbiologic findings in dogs with
bronchoscopically diagnosed tracheal collapse: 37 cases (1990–1995). J Am Vet Med
Assoc 219 pp 1247–1250
Johnson EG, Wisner ER, Marks SL et al (2006) Contrast enhanced CT thoracic duct
lymphography. Paper presented at: the annual conference of the American college of
veterinary radiology. Chicago: 2006.
Johnson EG, Wisner EK (2007) Advances in respiratory Imaging Vet Clin Small Anim
37 pp 879–900
Johnson LR,Pollard RE (2010) Tracheal Collapse and Bronchomalacia in Dogs: 58
Cases (7 /2001 –1 /2008) J Vet Intern Med 24 pp 298–305
140
Johnson LR, Lappin MR, Baker DC (1999) Pulmonary Thromboembolism in 29 Dogs:
1985–1995 J Vet Intern Med 13 pp338–345
Johnson LR, Queen EV, Vernau W et al (2013) Microbiologica and Cytologic Analysis
of Bronchoalveolar Lavage Fluid from Dogs with Lower Respiratory Tract Infection: 105
Cases (2001-2011) J Vet Intern Med 27 pp 259-267
Jokinen K, Palva T, Sutinen S et al (1977) Acquired tracheobronchomalacia. Annals
Clin Res 9 pp 52–57.
Jones KP, Edwards JH, Reynolds (1990) A comparison of albumin and urea as
reference markers in bronchoalveolar lavage fluid from patients with interstitial lung
disease. Eur Resp J 3 pp 152-156
Jordan HE, Mullins ST, Stebbins ME (1993) Endoparasitism in dogs: 21,583 cases
(1981-1990) J Am Vet Med Assoc 203 pp 547-549
Kaklamani E, Karalis D, Koumandaki Y et al (1993) The effect of Mycoplasma
arthritidis infection on the kinetics of colloidal carbon clearance in mice. Immunol &
Medical Microbiol 6 (4) pp 299-305
Kaklamanis E,Pavlatos M (1969) Inhibition of antibody formation and lymphocyte blast
transformation by mycoplasma. Communication at the meeting of British Society for
Immunology, 23rd October
Kelly CA, Kotre CJ, Ward C et al (1987) Anatomical distribution of bronchoalveolar
lavage fluid as assessed by digitial subtraction radiography. Thorax 42 pp 624-628
Kerl ME (2010) Acid-Base, Oximetry, and Blood Gas in Textbook of Veterinary Internal
Medicine 7th ed. Ettinger SJ and EC Feldman eds pp467-471 Saunders Elsevier, Miss
USA
Kerl ME (2003). Update on canine and feline fungal diseases. Vet Clin North Am Small
Anim Pract. 33 pp721–747
Kim JS, Lee KS, Koh EM et al (2000) Thoracic involvement of systemic lupus
erythematosus: clinical, pathologic, and radiologic findings. J Comput Assist Tomogr
24 pp 9–18.
141
King RR, Zeng QY, Brown DJ et al (1988) Bronchoalveolar lavage cell populations in
dogs and cats with eosinophilic pneumonitis (abstr) in Proceedings of the Seventh
Veterinary Respiratory Symposium. Chicago: The Comparative Respiratory Society
Klech H, Pohl W (1989) Technical recommendations and guidelines for
bronchoalveolar lavage (BAL). Report of the European society of pneumology task
group on BAL. Eur Respir J 2 pp 561-585.
Kogan DA, Johnson LR, Sturges BK et al (2008) Etiology and clinical outcome in dogs
with aspiration pneumonia: 88 cases (2004-2006) J Am Vet Med Assoc 233(11) pp
1748-175
Kotoulas C, Panagiotou I, Tsipas P et al (2014) Experimental studies in the bronchial
circulation. Which is the ideal animal model? J Thorac Dis 6(10) pp1506-1512
Krafft E, Heikkilä HP, Jespers P et al (2011) Serum and Bronchoalveolar Lavage Fluid
Endothelin-1 Concentrations as Diagnostic Biomarkers of Canine Idiopathic Pulmonary
Fibrosis J Vet Intern Med 25:990–996
Kvito- White H, Balog K, Scott-Moncrieff JC et al (2012) Acquired Bilateral Laryngeal
Paralysis Associated with Systemic Lupus Erythematosus in a Dog J Am Anim Hosp
Assoc 48 (1) pp 60-65
Leonard HC (1960) Collapse of the larynx and adjacent structures in the dog J Am Vet
Med Assoc 137 pp 360-363
Liam CK, Pang YK, Poosparajah S. (2007) Diagnostic yield of flexible bronchoscopic
procedures in lung cancer patients according to tumour location. Singapore Med J.
48(7) pp 625-31.
Lobetti R (2000) Common variable immunodeficiency in miniature dachshunds affected
with Pneumonocystis carinii pneumonia. J Vet Diagn Invest 12 pp 39–45.
Lott JA, Nemensanszky E (1986) Lactate dehydrogenase In: Clinical enzymology, a
case oriented approach. Lott JA, Wolf PL, eds. Chicago, PA: Year Book Medical
Publishers, pp 213-244.
142
Ludwig LL, Simpson AM, Han E (2010) Pleural and Extrapleural Diseases in Textbook
of Veterinary Internal Medicine 7th ed. Ettinger SJ and EC Feldman eds pp1125-1137
Saunders Elsevier, Miss USA
Maack T (2006) The broad homeostatic role of natriuretic peptides Arq Bras Endocrinol
Metabol 50 pp 198-207
MacDonald KA, Kittleson MD, Munro C et al (2003) Brain Natriuretic Peptide
Concentration in Dogs with Heart Disease and Congestive Heart Failure J Vet Intern
Med 17 pp 172–177
Macey RI (1984) Transport of water and urea in red blood cells Am J Physiol 246 pp
C195-C203
Macready DM, Johnson LR, Pollard RE (2007) Fluoroscopic and radiographic
evaluation of tracheal collapse in 62 dogs. J Am Vet Med Assoc 230 pp 1870–6.
Maggiore AD (2014) Tracheal and Airway Collapse in Dogs Vet Clin Small Anim 44 pp
117–127
Malik, R, Dill-Macky E, Martin P et al (1995) Cryptococcosis in dogs: a retrospective
study of 20 cases J Med Vet Mycol 33(5) pp 291-297
Marcy TW, Merrill WW, Rankin JA et al (1987) Limitations of using Urea to Quantify
Epithelial Lining Fluid Recovered by Bronchoalveolar Lavage Am Rev Respir Dis 135
pp 1276-1280
McCullough S and J Brinson (1999) Collection and Interpretation of Respiratory
Cytology. Clin Tech Sm Anim Pract 14(4) pp 220-226
McGorum BC, Dixon PM, Halliwell REW et al (1993) Comparison of cellular and
molecular components of bronchoalveolar lavage fluid harvested from different
segments of the equine lung. Res Vet Sci 55 pp 57-59
McGorum BC, Dixon PM, Halliwell REW et al (1993a) Evaluation of urea and albumin
as endogenous markers of dilution of equine bronchoalveolar lavage fluid Res in Vet
Sci 55 pp 52-56
143
McKieranan BC, Smith AR, Kissil M (1984) Bacterial isolates from the lower trachea of
clinically healthy dogs J Am Anim Hosp Assoc 20 pp 139-142
McKiernan BC (2000) Diagnosis and treatment of canine chronic bronchitis. Twenty
years of experience. Vet Clin North Am Small Anim Pract 30 pp 1267-1278
Melamies MA; Järvinen A-K, Seppälä KM (2011) Comparison of results for weight-
adjusted and fixed-amount bronchoalveolar lavage techniques in healthy Beagles Am J
Vet Res 72 pp 694–698
Miller CJ (2007) Approach to the Respiratory Patient Vet Clin Small Anim 37 pp 861–
878
Mills PC,Litster A (2005) Using urea dilution to standardize components of pleural and
bronchoalveolar lavage fluids in the dog New Zeal Vet J 53 (6) pp 423-427
Mills PC,Litster A (2006) Using urea dilution to standardize cellular and non-cellular
components of pleural and bronchoalveolar lavage (BAL) fluids in the cat J Feline Med
& Surg 8 pp 105-110
Mordelet-Dambrine M, Arnoux A, Stanislas-Leguern G et al (1984) Processing of lung
lavage fluid causes variability in bronchoalveolar cell count. Am Rev Respir Dis 130 pp
305-306.
Morgan E,d S Shaw S (2010) Angiostrongylus vasorum infection in dogs: continuing
spread and developments in diagnosis and treatment Journal of Small Animal Practice
51 pp 616–621
Mullins MD, Becker DM, Hagspiel KD et al (2000) The role of spiral volumetric
computed tomography in the diagnosis of pulmonary embolism. Arch Intern Med
160(3) pp 293–298.
Myer CW (1980) Radiography Review: The Vascular and bronchial patterns of
pulmonary disease Vet Radiol and Ultrasound 21(4) pp 156–160
Naidich DP, Marshall CH, Gribbin C et al (1990) Low-dose CT of the lungs: preliminary
observations. Radiology 175 pp 729-731.
144
Neofytos D, Horn D, Anaissie E et al (2009) Epidemiology and outcome of invasive
fungal infection in adult hematopoietic stem cell transplant recipients: analysis of
multicenter prospective antifungal therapy (PATH) alliance registry Clinical Infectious
Diseases 4(3) pp 265-273
Nielson DW (1986) Electrolyte composition of pulmonary alveolar subphase in
anaesthetized rabbits J Appl Physiol 60 pp 972-979
Nolan TJ,Smith G (1995) Time series analysis of the prevalence of endoparasitic
infections in cats and dogs presented to a veterinary teaching hospital Vet Parasitol 59
pp 87-96
Norman BC (2010) Transtracheal wash and Bronchoscopy in Textbook of Veterinary
Internal Medicine 7th ed. Ettinger SJ and EC Feldman eds pp 406-408 Saunders
Elsevier, Miss USA
Normand IC, Olver RE, Reynolds EOR et al (1971) Permeability of lung capillaries and
alveoli to non-electrolytes in the foetal lamb J Physiol 219 pp 303-330
Norris CR, Griffey SM, Samii VF et al (2001) Comparison of results of thoracic
radiography, cytologic evaluation of bronchoalveolar lavage fluid, and histologic
evaluation of lung specimens in dogs with respiratory tract disease: 16 cases (1996-
2000). J Am Vet Med Assoc 218 pp 1456-1461.
Norris CR, Griffey SM, Samii VF et al (2002a) Thoracic radiography, bronchoalveolar
lavage cytopathology, and pulmonary parenchymal histopathology: a comparison of
diagnostic results in 11 cats. J Am Anim Hosp Assoc 38 pp 337-345.
Norris C, Griffey S, Walsh P (2002) Use of keyhole lung biopsy for diagnosis of
interstitial lung diseases in dogs and cats: 13 cases (1998-2001) J Am Vet Med Assoc
221 pp 1453-1459
Ohno Y, Koyama H, Nogami M et al (2007) STIR turbo SE MR imaging vs.
coregistered FDG-PET/CT: quantitative and qualitative assessment of N-stage in non-
small-cell lung cancer patients. J Magn Reson Imaging 26 pp 1071-1080.
Ortega R, Hansen CJ, Elterman K et al (2011) Pulse Oximetry N Engl J Med 364 p 33.
145
Oyama MA, Rush JE, Rozanski EA et al (2009) Assessment of serum N-terminal pro-
B-type natriuretic peptide concentration for differentiation of congestive heart failure
from primary respiratory tract disease as the cause of respiratory signs in dogs J Am
Vet Med Assoc 235 pp 1319–1325
Padrid P (2000) Pulmonary Diagnostics. Vet Clin Nth Am: Sm Anim Pract 30(6) pp
1187-1205
Padrid PA, Hornoff WJ, Kurpurshoek CJ et al (1990) Canine chronic bronchitis J Vet
Intern Med 4 pp 172-180
Padrid PA, Feldman BF, Funk K et al (1991) Cytologic, microbiologic, and biochemical
analysisof bronchoalveolar lavage fluid obtained from 24 healthy cats AmJ of Vet Res
52 pp1300-1307
Park SW, Lee YM, Jang AS et al (2004) Development of chronic airway obstruction in
patients with eosinophilic bronchitis: a prospective follow-up study. Chest 125 pp
1998–2004.
Peeters DE, McKiernan BC, Weisiger RM et al (2000) Quantitative bacterial cultures
and cytological examination of bronchoalveolar lavage specimens in dogs. J Vet Intern
Med.14(5) pp 534-541
Peterson BT, Griffith DE, Tate RW and et al (1993) Single-cycle bronchoalveolar
lavage to determine solute concentrations in epithelial lining fluid. Am Rev Respir Dis
147 pp 1216-1222
Pink JJ, Doyle RS, Hughes JML et al (2006) Laryngeal collapse in seven
brachycephalic puppies J Sm Anim Pract 47 pp131-135
Polverino M, Polverino F, Fasolino M et al (2012) Anatomy and neuro-pathophysiology
of the cough reflex arc Multidisciplinary Respiratory Medicine 7:5 doi:10.1186/2049-
6958-7-5
Pusch R, Kleen M, Habler O et al (1997) Biochemical and cellular composition of
alveolar epithelial lining fluid in anaesthetized healthy lambs. Eur J of Med Res 2 pp
499-505
146
Radhakrishan A, Drobatz KJ, Culp WT et al (2007) Community-acquired infectious
pneumonia in puppies: 65 cases (1993-2002) J Am Vet Med Assoc 230(10) pp 1493-
1497
Raffan E, Loureiro J, Dukes-McEwan J et al (2009) The cardiac biomarker NT-proBNP
is increased in dogs with azotemia J. Vet Intern Med 23(6) pp.1184 – 1189
Rajamäki MM, Järvinen AK, Sorsa T et al (2002) Clinical findings, bronchoalveolar
lavage fluid cytology and matrix metalloproteinase-2 and -9 in canine pulmonary
eosinophilia Vet J 163 (2) pp168-181
Rajamäki MM, Järvinen AK, Saari SA et al(2001) Effect of repetitive bronchoalveolar
lavage on cytologic findings in healthy dogs. Am J Vet Res. 62(1) pp13-6.
Rajamaki MM, Jarvinen AK, Sorsa T et al (2006) Elevated levels of fragmented
laminin-5 gamma-2 chain in bronchoalveolar lavage fluid from dogs with pulmonary
eosinophilia Vet J 171(3) pp 562-565
Randolph JF, Moise NS, Scarlett JM et al (1993) Prevalence of mycoplasmal and
ureaplasmal recovery from tracheobronchial lavages and prevalence of mycoplasmal
recovery from pharyngeal swab specimens in dogs with or without pulmonary disease .
Am J Vet Res 54 pp 387-391
Rathbun SW, Raskob GE, Whitsett TL (2000) Sensitivity and specificity of helical
computed tomography in the diagnosis of pulmonary embolism: a systematic review.
Ann Intern Med 132(3) pp 227– 232.
Rebar AH, DeNicola DB, Muggenburg BA (1980) Bronchopulmonary lavage cytology in
the dog: Normal Findings Vet Pathol 17 pp 294-304
Reinero CR, Cohn LA (2007) Interstitial lung diseases. Vet Clin Small Anim Pract 37 pp
937-947
Rennard SI, Basset G, Lecossier D et al (1986) Estimation of volume of epithelial lining
fluid recovered by lavage using urea as a marker of dilution J Appl Physiol 60 pp 532-
538
147
Restrick L, Sampson AP, Piper PJ et al (1995) Inulin as a marker of dilution of
bronchoalveolar lavage in asthmatic and normal subjects. Am J Respir Crit Care Med
151 pp 1211-1217
Richardson MD, Page ID (2017) Aspergillus serology: Have we arrived yet? Medical
Mycology 55, pp 48–55 doi: 10.1093/mmy/myw116
Riise GC, Andersson B, Ahlstedt S (1996) Bronchial brush biopsies for studies of
epithelial inflammation in stable asthma and non obstructive chronic bronchitis. Eur
Resp J 9 pp 1665-1671
Riise GC, Larsson S, Andersson BA (1992) A bronchoscopic brush biopsy study of
large airway mucosal pathology in smokers with chronic bronchitis and in healthy
nonsmokers Eur Respir J 5 pp 382-386
Roitt JM, Greaves MF, Torrigiani G et al (1969) The cellular basis of immunologic
responses. A synthesis of some current views Lancet 2 (7616) pp367-71.Rosendal S
(1990) Mycoplasmal infections in Greene CE (ed) Infectious diseases of the dog and
cat pp 446-449 WB Saunders, Philadelphia, USA
Rosendal S (1982) Canine mycoplasmas; their ecologic niche and role in disease J
Am Vet Med Assoc 180 pp 1212-1214
Roudebush P (1990) Tracheobronchoscopy. Vet Clin North Am Small Anim Pract.
20(5) pp 1297-314.
Rycroft AN, Tsounakou E, V Chalker V (2007) Serological evidence of Mycoplasma
cynos infection in canine infectious respiratory disease. Vet Microbiol 120 pp 358-362
Saltini C, Hance AJ, Ferrans VJ et al (1984) Accurate quantification of cells recovered
by bronchoalveolar lavage. Am Rev Respir Dis130 pp 650-658.
Sant SM, Doran M, Fenlon HM et al (1997) Pleuropulmonary abnormalities in patients
with systemic lupus erythematosus: assessment with high resolution computed
tomography, chest radiography and pulmonary function tests. Clin Exp Rheumatol 15
pp 507–513.
148
Saunders HM, Keith D (2004) Thoracic imaging. in Respiratory disease in dogs and
cats. King LG ed Philadelphia: WB Saunders pp. 72–92.
Schmiedt C, Kellum H, Legendre AM et al (2006) Cardiovascular Involvement in 8
Dogs with Blastomyces dermatitidis Infection J Vet Intern Med 20 pp 1351–1354
Schonhöfer B, Wenzel M, Geibel M, et al (1998) Blood transfusion and lung function in
chronically anemic patients with severe chronic obstructive pulmonary disease. Crit
Care Med 26 pp 1824–1828
Schreiber G, McCrory DC. (2003) Performance characteristics of different modalities
for diagnosis of suspected lung cancer: summary of published evidence. Chest.123 (1
Suppl) pp115S-128S.
Schuller S, Fredericksen, M, Schroder, H et al (2005) Differentiation of canine
intrathoracic neoplasia and inflammation by computed tomography Berliner und
Munchener Tierarztliche Wochenschrift 118 (1-2) pp 76 - 84
Scott M, Dennis J, Watson G et al (1993) Bronchoalveolar lavage of histologically
normal and diseased canine lung lobes. Vet Pathol 30 (5) p 433
Schulze HM,Rahilly LJ (2012) Aspiration pneumonia in Dogs: Pathophysiology,
Prevention, and Diagnosis Compendium:Cont Ed Vet ppE1-E6
Sigrist, NE, Adamik KN, Doherr MG et al (2011) Evaluation of respiratory parameters
at presentation as clinical indicators of the respiratory localization in dogs and cats with
respiratory distress J of Vet Emerg and Crit Care 21(1) pp 13–23
Silverstein D,Drobatz KJ (2006) Clinical Evaluation of the Respiratory Tract in
Textbook of Veterinary Internal Medicine 6th ed. Chap 212 pp1206 -1217 Ettinger SJ
and Feldman EC (eds) Elsevier Saunders Miss. USA
Silverstein D,Drobatz KJ (2010) Clinical Evaluation of the Respiratory Tract in
Textbook of Veterinary Internal Medicine 7th ed. Chap 227 pp1055 -1066 Ettinger SJ
and Feldman EC (eds) Elsevier Saunders Miss. USA
Similowski T, Agustí A, MacNee W et al (2006) The potential impact of anaemia of
chronic disease in COPD Eur Resp J 27(2) pp 390-396
149
Simón F,Siles- Lucas, M, Morchón R et al (2012). Human and Animal Dirofilariasis: the
Emergence of a Zoonotic Mosaic Clin Microbiol Rev. 25(3) pp 507–544.
Simón F, Genchi C, Prieto G et al.( 2001). Immunity in the vertebrate hosts, p 218 In
Simón F, Genchi C (ed), Heartworm infection in humans and animals. Ediciones
Universidad de Salamanca, Salamanca, Spain.
Slade R, Crissman K, Norwood J et al (1993) Comparison of antioxidant substances in
bronchoalveolar lavage cells and fluid from humans, guinea pigs and rats. Exp Lung
Res 19 pp 469-484
Sone S (1986) Role of alveolar macrophages in pulmonary neoplasias Biochimica et
Biophysica Acta 823 pp 227-245
Smith MM (2000) Diagnosing Laryngeal Paralysis J Am Anim Hosp Assoc 36 (5) pp
383-384
Syrä P, Heikkilä HP, Lilja-Maula L et al (2013) The Histopathology of Idiopathic
Pulmonary Fibrosis in West Highland White Terriers shares Features of Both Non-
specific Interstitial Pneumonia and Usual Interstitial Penurmonia in Man J Comp Pathol
149 pp 303-313
Sparkes A, Wotton P, Brown P (1997) Tracheobronchial washing in the dog and cat In
Practice May 19 (5) pp 257-259
Stanley BJ, Hauptman JG, Fritz MC et al (2010) Esophageal dysfunction in dogs with
idiopathic laryngeal paralysis: A controlled cohort study Vet Surg 39 pp 139-149
Starr TW, MuIIey RC (1988) Dirofilaria immitis in the Dingo (Canis familiaris dingo) in
aTropical Region of the Northern Territory, Australia.Journal of Wildlife Diseases 24(1)
pp. 164-165
Suter PF, Carrig CB, O’Brien TR et al (1974) Radiographic recognition of primary and
metastatic pulmonary neoplasms of dogs and cats J Am Vet Radiol Soc 15 pp 3-25
Sweeney CR, Rossier Y, Ziemer EL et al.(1992) Effects of lung site and fluid volume
on results of bronchoalveolar lavage fluid analysis in horses. Am J Vet Res 53 pp
1376-1379.
150
Syrjä P, Heikkilä HP Lilja-Maula L et al (2013) The Histopathology of Idiopathic
Pulmonary Fibrosis in West Highland White Terriers shares features of both non-
specific interstitial pneumonia and unusual interstitial pneumonia in Man J Comp Path
149 pp 303-313
Taboada J (1991) Pulmonary diseases of potential allergic origin Semin Vet Med Surg
(Small Anim) 6(4) pp 278-285
Tangner CH, Hobson HP (1982) A retrospective of 20 surgically managed cases of
collapsed trachea. Vet Surg 11 pp 146–149.
Taubert A, Pantchev N, Vrhovec MG et al (2009) Lungworm infections
(Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus) in dogs and
cats in Germany and Denmark in 2003-2007.Vet Parasitol. 159(2) pp 175-80.
Taylor AE, Guyton AC,Bishop VS (1965) Permeability of the alveolar membrane to
solutes. Circ Res 16 pp 353-362
Tebb AI, Johnson VS,Irwin PJ (2007) Angiostrongylus vasorum (French heartworm) in
a dog imported into Australia Aust Vet J 85 1&2 pp 23-28
Thrall DE (1998) ‘The Canine Lung’ in Thrall DE, ed. Textbook of Veterinary Diagnostic
Radiology, WB Saunders Co, Phil 3rd ed Chap 32 pp366-383
Vail DM, Mahler PA, Soergel SA et al (1995) Differential cell analysis and phenotypic
subtyping of lymphocytes in bronchoalveolarlavage fluid from clinically normal dogs.
Am J of Vet Res 56 pp 282-285
Venker-van Haagan AJ, Vroom MW, Heijn A et al (1985) Bronchoscopy in small animal
clinics: An analysis of the results of 228 bronchoscopies J Am Anim Hosp Assoc 21 pp
521-526
Ward C, Duddridge M, Fenwick J et al (1992) The Origin of Water and Urea Sampled
at Bronchoalveolar Lavage in Asthmatic and Control Subjects. Am Rev Respir Dis 146
pp 444-447
151
Ward C, Fenwick J, Booth H et al (1997) Albumin is not suitable as a marker of
bronchoalveolar lavage dilution in interstitial lung disease Eur Resp J 10 (9) pp 2029-
2033
Watson PJ, Wotton P, Eastwood J et al (2006) Immunoglobulin Deficiency in Cavalier
King Charles Spaniels with Pneumocystis Pneumonia J Vet Intern Med 20 pp 523–527
Watson PJ, Herrtage ME, Peacock MA (1999) Primary ciliary dyskinesia in
Newfoundland dogs Vet Rec 144 pp 718-725
Ward C, Effros RM,Walters EH (1999) Assessment of epithelial lining fluid dilution
during braonchoalveolar lavage Eur Respir Rev 9 (66) pp32-37
West JB (2008) Respiratory Physiology. The Essentials Eighth Ed Lippincott, Williams
and Wilkins Philadelphia PA
Willcox M, Kervitsky A, Watters LC et al.(1988) Quantification of cells recovered by
bronchoalveolar lavage. Comparison of cytocentrifuge preparations with the filter
method. Am Rev Respir Dis138 pp 74-80.
Wood EF, O’Brien RT, Young KM (1998) Ultrasound-guided fine-needle aspiration of
focal parenchymal lesions of the lungs in dogs and cats. J Vet Intern Med 12 pp 338–
342.
Woodard PK, Sostman HD, MacFall JR et al. (1995) Detection of pulmonary embolism:
comparison of contrast-enhanced spiral CT and time-of-flight MR techniques. J Thorac
Imaging 10(1) pp 59–72.
Woods KS, Defarges AMN, Abrams-Ogg ACG et al (2014) Comparison of Manual and
Suction Pump Aspiration Techniques for Performing Bronchoalveolar Lavage in 18
Dogs with Respiratory Tract Disease J Vet Intern Med 28 pp 1398–1404
Yohn SE, Hawkins EC, Morrison WB et al (1994) Confirmation of a pulmonary
component of multicentric lymphosarcoma with bronchoalveolar lavage in two dogs J
Am Vet Med Assoc 204 pp 97-101
152
Zoia A, Slater LA, Heller J et al (2009) A new approach to pleural effusion in cats:
markers for distinguishing transudates from exudates J.of Feline Medicine and Surgery
11 pp 847-855
153
APPENDICES
154
Appendix 1: Tests used for Respiratory Diagnosis
. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
1 Chronic
Bronchitis √ √ √ √
3 view x √ (-) √ √ x x x x
2 Chronic
Bronchitis √ √ √ √
3 view x √ (-) √ √ x x x x
3 Chronic
Bronchitis √ √ √ √ 3 view x
√ P.
aeruginosa
(broth)
√ √ x √ √ (-) x
4 Chronic
Bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (-) x
upper
airway
exam
5 Chronic
Bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x
upper
airway
exam
6
Pneumonia-
Infectious-
Bacterial
√ √ √ √ 3 view x
√
B.bronchise
ptica
√ √ x √ √(-) x
155
. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
7 Laryngeal
Paralysis √ √ √ √ 3 view x X √ √ x x x x
upper
airway
exam
8 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x √ x x
9 Chronic
bronchitis √ √ √ √ 3 view x
√ Mixed
bacterial
flora
(broth)
√ √ x x √ (+) x
10 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x
11 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ √ x √ (+) x
12 Pulmonary
carcinoma √ √ √ √ 3 view √ X √ √ √ x x x
surgery,
coags
13 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ x x √ x
upper
airway
exam
156
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
14 Chronic
bronchitis √ √ √ √ 3 view x
√ scanty
mixed
bacterial
√ √ x x √ (+) x ultrasound
15 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (-) √
echocardio
gram
16
Sterile
pyogranulomat
ous disease
√ √ √ √ 3 view √ √ (-) √ √ √ √ √ (+) x mycobacte
rium PCR
17 Chronic
Bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (+) √
echocardio
gram
18
Neoplasia-
met-
Haemangiosar
coma
√ √ √ √ 3 view x √ (-) √ √ √ x x x abdomince
ntesis
19
Neoplasia-
met-
Haemangiosar
coma
√ √ √ √ 3 view x √ (-) √ √ √ √ x x abdominoc
entesis
20 Aspiration
Pneumonia √ √ √ √ 3 view √
√
Enterobacter √ √ x x √(-) x ultrasound
157
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
21 Aspiration
Pneumonia √ √ √ √ 3 view √
√
Enterobacter √ √ x x √(-) x ultrasound
22 SLE- systemic
immune √ √ √ √ 3 view x √ (-) √ √ x √ √(-) x
ana, csf,
arthrocente
sis
23 SLE- systemic
immune √ √ √ √ 3 view x √ (-) √ √ x √ √(-) x
ana, csf,
arthrocente
sis
24 SLE- systemic
immune √ √ √ √ 3 view x √ (-) √ √ x √ √(-) x
ana, csf,
arthrocente
sis
25 Pulmonary
fibrosis √ √ √ √ 3 view √ √ (-) √ √ √ x √ (+) x
26 Pulmonary
fibrosis √ √ √ √ 3 view √ √ (-) √ √ √ x √ (+) x
27 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ √ x √ (+) x
PM report
28 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ √ x √ (+) x
158
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
Cardio
gram
other
29
Pneumonia-
infectious-
bacterial
√ √ √ √ 3 view x √ Klebsiella
pneumoniae √ √ x x x x
30
Pneumonia-
infectious-
bacterial
√ √ √ √ 3 view x
√
Pasteurella
sp
√ √ x x √ (+) x
31 Aspiration
pneumonia √ √ √ √ 3 view x
√
Enterobacter
cloacae
√ √ x √ x x arthrocente
sis, CSF
32 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) √
33
Bronchointers
titial
pneumonia-
non infectious
√ √ √ √ 3 view x
√ Mucoid
coliform
(broth)
√ √ √ √ √(-) x
34
Bronchointers
titial
pneumonia-
non infectious
√ √ √ √ 3 view x
√ Mucoid
coliform
(broth)
√ √ √ √ √(-) x
35 Pulmonary
fibrosis √ √ √ √ 3 view √ X √ √ √ x x √
159
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
36 Chronic
Bronchitis √ √ √ √ 3 view x √ (-) √ √ √ x √ (+) x abdo us
37
Pneumonia-
infectious-
bacterial
√ √ √ √ 3 view X √ coliform √ √ x x √ (+) √
38 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ √ x √(-) x
39 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ √ x √(-) x
40 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x
41 Aspiration
pneumonia √ √ √ √ 3 view x √ E.coli √ √ x √ x x
42 Pulmonary
fibrosis √ √ √ √ 3view √ √ (-) √ √ x √ √ (+) √
43 Pulmonary
fibrosis √ √ √ (-) √ √ x x √ (+) √
44 Systemic
immune-med √ √ √ √ 3 view √ √ (-) √ √ √ √ x √
ANA
160
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
45 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ √ √ x √
46 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x
fluoro
airway
47 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x
48 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x
49 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x fluroscopy
50 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-)
51 Laryngeal
collapse √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x
upper
airway
exam
52 Laryngeal
collapse √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x
upper
airway
exam
161
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
Cardio
gram
other
53
Non
cardiogenic
oedema (Upper
airway
obstruction)
√ √ √ √ 3 view √
√
Pseudomon
as sp
√ √ x x √ (+) x
upper
airway
exam
54
Non
cardiogenic
oedema (Upper
airway
obstruction)
√ √ √ √ 3 view √
√
Pseudomon
as sp
√ √ x x √ (+) x
upper
airway
exam
55
Non cardiogenic
oedema (Upper
airway
obstruction)
√ √ √ √ 3 view √
√
Pseudomon
as sp
√ √ x x √ (+) x
upper
airway
exam
56 Chronic
Bronchitis √ √ √ √ 3 view √ √ (-) √ √ √ x √(-) x
57 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ x √(-) x
58 Chronic
Bronchitis √ √ √ √ 3 view x
√
Pasteurella
sp
√ √ x x √ (+) x
upper
airway
exam,
cortisols
162
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
59 Chronic
bronchitis √ √ √ √ 3 view x
√
Pasteurella
sp
√ √ x √ (+) x
60
Non cardiogenic
oedema (near
drowning)
√ √ √ √ 3 view √ √ (-) √ √ x x √(-) x
61
Non cardiogenic
oedema (near
drowning)
√ √ √ √ 3 view √ √ (-) √ √ x x √(-) x
62 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ x √ √ (+) x
63 Chronic
bronchitis √ √ √ √ 3 view √ √ (-) √ √ x √ √ (+) x
64 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √(-) x
65 Chronic
bronchitis √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x fluoroscopy
66 Pulmonary
fibrosis √ √ √ √ 3 view x √ (-) √ √ x x √ (+) x fluoroscopy
163
No. Diagnosis history physical cbc biochem
Thoracic
rad
thorax ct culture bal
Response
to tx
Lung
biopsy/ PM
Faecal
float
mycoplasma
PCR test
Echo
cardio
gram
other
67
Pneumonia-
infectious-
bacterial
√ √ √ √ 3 view x
√ B.
bronchisepti
ca
√ √ x √ √ (+) x fluoroscopy
68
Pneumonia-
infectious-
bacterial
√ √ √ √ 3 view x
√ B.
bronchisepti
ca
√ √ x √ √ (+) x fluoroscopy
69 Systemic immune
mediated disease √ √ √ √ 3 view x √ (-) √ √ √ x x x
ANA,
arthrocente
sis, UPC
70 Systemic immune
mediated disease √ √ √ √ 3 view x √ (-) √ √ √ x x x
ANA,
arthrocente
sis, UPC
71 Laryngeal
paralysis √ √ √ √ 3 view x
√
Pasteurella
multocida
√ √ x x √(-) x
upper
airway
exam
72 Phaeochromocyto
ma √ √ √ √ 3 view x √ (-) √ √ √ (adrenal) x x x
ECG,
gastrin,
serial BP,
u/sound
164
165
Appendix 2: Raw and processed BALF data
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Tracheal
collapse +
Bronchitis
1 8.4 0.91 9.230769 355 3276.923 10.83
43% non
react,
152.65,
1409.08
33% act,
117.15,
1081.38
6%, 21.3,
196.62
11%,
39.05,
360.46
6%, 21.3,
196.62
1%, 3.55,
32.77 0
Tracheal
collapse +
Bronchitis
2 8.4 1.51 5.562913 200 1112.583 17.98
20% non
react, 40,
222.52
51% act,
102, 567.42
20%, 40,
222.52
5%, 10,
55.63 2%, 4, 22.25
1%, 2,
11.13 1%, 2, 11.13
Chronic
bronchitis 3 4.7 0.73 6.438356 140 901.370 15.53
9% 12.6,
81.12
67%act 93.8,
603.92
5% 7,
45.07
20% 28,
180.27 0 0 0
Dynamic
airway
collapse +
bronchitis
4 3.2 0.64 5 260 1300 20 30% 78,390 6% 15.6,
78
64%,
166.4,
832
0 0 0
166
and
hypolastic
trachea
Dynamic
airway
collapse +
bronchitis
and
hypoplastic
trachea
5 3.2 0.39 8.205129 340 2789.74 11.25 30%, 102,
836.92
6% 20.4,
167.38
64%,
217.6,
1785.43
0 0 0
Bordatella
bronchisepti
ca
pneumonia
6 4.6 0.48 9.583334 164 1571.67 10.43 12%, 19.68,
188.60
3%, 4.92,
47.15
80% ,
131.2,
1257.34
(50%
degen, )
5%, 8.2,
78.58 0 0
Laryngeal
Paralysis +
mild large
bronchi
collapse
7 3.5 1.82 1.923077 355 682.69 37.71 66%, 234,
450.57
22%, 78.1,
150.2
8%,
28.4,
54.62
2%, 7.1,
13.65
2%, 7.1,
13.65
0
167
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Chronic
bronchitis 8 5.6 0.9 6.222223 150 933.34 16.07
62% act, 93,
578.67
17% non
react, 25.5,
158.67
11%, 16.5,
102.67
10%, 15
93.34 0 0 0
Grade II
tracheal
collapse +
chronic
bronchitis +
mycoplasm
9 6.6 0.21 31.42857
1 22 691.43 3.18
9%, 1.98,
62.23
2%, 0.44,
13.83
72%,
15.84,
497.83
17%, 3.74,
117.54 0 0
Chronic
bronchitis +
dynamic
airway Dx
10 7.6 0.58 13.10344
8 384 5031.72 7.63
100%, 384,
5031.72 0 0 0 0 0
Chronic
bronchitis +
dynamic
airway Dx
11 7.6 1.45 5.241379 200 1048.27 19.08 88%, 176
922.48 0
12%,
24,
125.79
0 0 0
pulmonary
carcinoma +
diffuse
intralesional
12 4.6 0.52 8.846154 300 2653.85 11.30 41%, 123,
1088.08
3%, 9,
79.62
54%,
162,
1433.08
2%, 6, 53.08 0 0
168
pyogran
inflamm
Bronchiecta
sia (chronic
bronchitis)
13 6.9 3.92 1.760204 420 739.29 56.81 50%, 210,
369.64
3%, 12.6,
22.18
43%,
180.6,
317.89
4%, 16.8,
29.57 0 0
Chronic
bronchitis-
collapse
mainstem
bronchi +
mycoplasm
a +
14 6.1 1.46 4.178082 1030 4303.42 23.93
81%, 834.3,
3485.77
6% act,
61.8, 258.2
11%, 103,
473.37
2%,
20.6,
86.07
0 0 0
Dynamic
airway
collapse +
Chronic
bronchitis +
bronchiecta
sis
15 7.8 1.57 4.968153 860 4272.61 20.13 79%, 679.4,
3375.36
2%, 17.2,
85.45
11%,
94.6,
469.99
1%, 8.6,
42.72
3%, 25.8,
128.18
4%, 34.4,
170.90
Pyogran.
pneumonia
+
Mycoplasma
16 5.8 0.47 12.34043
6 58 715.75 8.10
44%,25.52,
314.93
14%, 8.12,
100.21
42%,
24.36,
300.62
0 0 0
Bronchiect
Dynamic
airway
collapse +
17 8.9 0.71 12.53521
1 145 1817.61 7.98
25.5%,
36.96,
463.49
43.5%,
63.08,
790.66
21.5%,
31.18,
390.79
3%, 4.35,
54.53 0
1.5%, 2.175,
27.26
169
Mycoplasma
+
severe
bronchointe
rstitial.
Central Dx
18 9.4 8.12 1.157635 90 104.19 86.38
30%,27,
31.26
(52% act)
7% 6.3,
7.29
63%,
56.7,
65.64
0 0 0
severe
bronchointe
rstitial
Central Dx
19 9.4 0.74 12.70270
3 95 1206.76 7.87
68%, 64.6,
820.59
13%,
12.35,
156.88
16%,
15.2,
193.08
3%, 2.85,
36.20 0 0
Megaoesop
hagus +
severe
pneumonia
20 2.5 0.27 9.259259 1960 18148.15 10.8 16%, 313.6,
2903.70
6%, 117.6,
1088.89
78%,
1528.8,
14155.5
0 0 0
Megaoesop
hagus +
severe
pneumonia
21 2.5 1.04 2.403846 3740 8990.38 41.6 6%, 224.4,
539.42
3%, 112.2,
269.71
91%,
3403.4,
8181.25
0 0 0
SLE 22 5.8 0.73 7.945205 670 5323.29 12.59 26%, 174.2,
1384.05
7%, 46.9,
372.63
67%,
448.9,
3566.6
0 0 0
SLE 23 5.8 1.62 3.580247 790 2828.4 27.93 12%,94.8,
339.41
2%, 15.8,
56.57
86%,
679.4,
2432.42
0 0 0
170
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
SLE 24 5.8 4.87 1.190965 1310 1560.16 83.97 26%,340.6,
405.64
7%, 91.7,
109.21
67%,
877.7,
1045.31
0 0 0
Mycoplasma
+ idiopathic
pulmonary
fibrosis
25 3.5 0.45 7.777778 120 933.34 12.86 9%, 10.8, 84 11%, 13.2,
102.67
75%,
90,
700.01
6%, 7.2, 56 0 0
Mycoplasma
+ idiopathic
pulmonary
fibrosis
26 3.5 0.37 9.459459 120 1135.14 10.57 9%, 10.8,
102.16
11%, 13.2,
124.87
75%,
90,
851.36
6%, 7.2,
68.11 0 0
Dynamic
collapse
bronchi Mild
bronchitis.
Mycoplasma
+
27 5.6 0.28 20 60 1200 5.0 82% , 49.2,
984
10%, 6,
120
4%, 2.4,
48 4%, 2.4, 48 0
o
Dynamic
collapse
28 5.6 0.03 186.67 10 1866.67 .053 94%, 9.4, 4%, 0.4, 2%, 0.2, 0 0 0
171
bronchi Mild
bronchitis.
Mycoplasma
+
1754.67 74.67 37.33
Chronic
infection-
K.pneumoni
ae
29 4.5 1.2 3.75 170 637.5 26.67 53%, 90.1,
337.86
34%, 57.8,
216.75
11%,
18.7,
70.13
1%, 1.7,
6.38
1%, 1.7,
6.38 0
Chronic
infection-
Pasteurella
sp +
Mycoplasma
30 5.1 1.39 3.669065 15250 55953.24 27.25
4.5%,
686.25,
2517.9
3%, 457.5,
1678.6
92.5%,
14106.3
,
51756.7
5
0 0 0
Aspiration
pneumonia-
Enterobacte
r cloacae
31 7.6 3.68 2.065217 480 991.304 48.42 1%, 4.8, 9.91 2%, 9.6,
19.82
97%,
465.6,
961.56
0 0 0
Chro. inflam
bronchial +
pulm. Paren
Dx
32 4.7 4.46 1.053811 1240 1306.72 94.89
87%,
1078.8,
1136.86
6%, 74.4,
78.4
5%, 62,
653.34 0 0 2%, 24.8, 26.13
mild pulm
intersitial
inflam
33 7 1.36 5.147059 310 1595.59 19.43 82%, 254.2,
1308.38
6%, 18.6,
95.74
5%,
15.5,
79.78
7%, 21.7,
111.7 0 0
mild pulm
intersitial
34 7 2.52 2.777778 160 444.45 36 69%, 110.4, 13%, 20.8, 10%,
16, 8%, 12.8, 0 0
172
inflam 306.67 57.78 44.45 35.56
Chronic
pulmonary
inflam
35 3 0.22 13.63636 290 3954.54 7.34 25%, 72.5,
988.64
34%, 98.6,
1344.5
33%,
95.7,
1305
1%, 2.9,
39.55
GOBLET 7%,
20.3, 276.82
Dynamic
airway Dx +
mycoplasm
a +
36 8.4 2.84 2.957746 560 1656.34 33.81 79%, 442.4,
1308.51
6%, 33.6,
99.39
12%,
67.2,
198.76
2%, 11.2,
33.13
1%, 5.6,
16.56 0
Chronic
bronchitis
and
dynamic
airway
collapse
Mycoplasma
+
37 7.1 2.69 2.63940 340 897.4 37.89 69%, 234.6,
619.21
10%, 34
89.7
20%,
68,
179.48
0 1%, 3.4,
8.9
Chronic
Bronchitis 38 6.9 1.42 4.85915 250 1214.79 20.58
50%, 125,
607.4
24%, 60,
291.55
25%,
62.5,
303.7
1%, 2.5,
12.15
Common
Goblet and
ciliated
columnar
epithelial
Cult-, myco
-, Chronic
bronchitis
39 6.9 1.32 5.227272 160 836.36352 19.13 68%, 108.8,
568.72
23%, 36.8,
192.36
9%,
14.4,
75.27
0 0
Occ cilated
columnar
epiethlial
173
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Myco +,
Chronic
bronchitis,
mainstem
bronchial
collapse
40 10.8 1.98 5.454545 400 2181.81 18.33 7%, 28,
152.73
5%, 20,
109.1
86%,
344,
1876.36
2%, 8, 43.64 0
Clusters
columnar
epithelial cells
Bacterial
pneumonia 41 1.9 2.35
0.808510
6 3050 2465.96 123.68
90%, 2745,
2219.36
5%, 152.5,
123.3
5%,
152.5,
123.3
0 0 0
Pulm Fib,
Bact- 42 3.8 0.19 20 350 7000 5 2%, 7, 350 0
82%,
287,
5740
16%, 56,
1120
Myco +,
Bact -,
chronic
inflammator
y airway and
pulmonary
fibrotic
[parenchym
al Dx
43 3.8 0.73 5.205479 1110 5778.08 19.21 5%, 55.5,
288.9 0
80%,
888,
4622.46
15%, 166.5,
866.71 0 0
174
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Multi
systemic
immune
mediated
disease
44 3 0.84 3.571428 8 28.571 28 7%, 0.56,
1.99
1%, 0.08,
0.29
92%,
7.36,
26.28
0 0 0
Myco -, bact
-, chronic
bronchitis,
mild
dynamic
airway
collapse
45 7.5 1.65 4.545454 460 2090.91 22 63%, 289.8,
1317.27
9%, 41.4,
188.18
27%,
124.2,
564.54
<1%, 4.6,
20.9
<1%, 4.6,
20.9 0
Myco-, bact
-, mild
chronic
bronchitis
46 8.5 0.73 11.64383
56 120 1397.26 8.59
88%, 105.6,
1229.59
3%, 3.6,
41.92
6%, 7.2,
83.84
4%, 4.8,
55.89 0 0
Bronchiecta
sis and
severe
dynamic
airway dx
47 8.5 0.47 18.08510
6 160 2893.62 5.53
75%, 120,
2170.2
4%, 6.4,
115.74
9%,
14.4,
260.43
10%, 16,
289.36
2%, 3.2,
57.87 0
Chronic
bronchitis,
bronchiecta
48 8.5 0.47 18.09 160 2894.4 5.5 75%, 120,
2170.8
4%, 6.4,
115.78
9%,
14.4,
10%, 16,
289.4
2%, 3.2,
57.89 0
175
sis, myco- 260.49
Chronic
bronchitis,
mycopl –
bact -
49 3.9 3.02 1.29 170 219.30 77.44 41%, 69.7,
89.91
1%, 1.7,
2.19
45%,
76.5,
98.68
13%, 22.1,
28.51 0 0
Chronic
bronchitis,
mycopl –
bact -
50 3.9 0.89 4.38 250 1095 22.82 80%, 200,
876
6%, 15,
65.7
12%,
30,
131.4
1%, 2.5,
10.9
1%, 2.5,
10.9 0
Dynamic
airway
collapse +
laryngeal
collapse,
myco +
51 9 0.92 9.78 140 1369.20 10.2 34%, 47.6,
465.53
13%, 18.2,
178
53%,
74.2,
725.67
0 0 0
Dynamic
airway
collapse +
laryngeal
collapse,
myco +
52 9 1.41 6.38 210 1339.80 15.6 57%, 119.7,
763.68
13%, 27.3,
174.17
29%,
60.9,
388.54
0 0 0
Brachyceph
alic
syndrome +
heat stress
Myco+
53 6.8 0.51 13.33 90 1199.7 7.5 56%, 50.4,
671.83
9%, 8.1,
107.97
32%,
28.8,
383.9
2%, 1.8, 24 1%, 0.9,
12 0
176
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Brachyceph
alic
syndrome +
heat stress
Myco+
54 6.8 0.45 15.11 250 3777.5 6.62 61%, 152.5,
2304.28
12%, 30,
453.3
23%,
57.5,
868.83
3%, 7.5,
113.33
1%, 2.5,
37.8
0
Brachyceph
alic
syndrome +
heat stress
Myco+
55 6.8 0.17 40 90 3600 2.5 49%, 44.1,
1764
7%, 0.63,
252
44%,
39.6,
1584
0 0 0
Chronic
bronchitis,
myco -
56 8.1 1.29 6.27 300 1881 15.93 41%, 123,
771.21
20%, 60,
376.2
34%,
102,
639.54
5%, 15,
94.05 0 0
Chronic
bronchitis,
myco -
57 8.1 2.65 3.06 2520 7711.20 32.72 13%, 327.6,
1002.46
8%, 201.6,
616.9
74%,
1864.8,
5706.29
5%, 126,
385.56 0 0
Myco +,
pasteurella
58 4.9 2.66 1.84 11000 20240 54.29 4%, 440, 4%, 440, 91%,
10010, 1%, 110, 0 0
177
+, dynamic
airway
disease and
chronic
bronchial d.
809.6 809.6 18418.4 202.4
Myco +,
pasteurella
+, dynamic
airway
disease and
chronic
bronchial d.
59 4.9 1.9 2.57 880 2261.6 38.78 37%, 325.6,
836.79
8%, 70.4,
180.93
47%,
413.6,
1062.95
8%, 70.4,
180.93 0 0
Myco -,
bact-,
fungal-
neurogenic
oedema
(near
drowning)
60 4.8 1.48 3.24 130 421.20 30.84 55%, 71.55,
231.77
21%, 27.3,
88.45
24%,
31.2,
101.1
0 0 0
bact-,
fungal-
neurogenic
oedema
(near
drowning)
61 4.8 3.93 1.22 330 402.6 81.88 20%, 66,
80.52
8%, 26.4,
32.21
72%,
237.6,
289.87
0 0 0
Myco +,
cult-,
Normal
62 3.3 0.4 8.25 63 519.75 12.12 37%, 23.3,
192.3
25%,
15.75,
129.94
35%,
22.05,
181.92
1%, 0.63,
5.19
2%, 1.26,
10.4 0
178
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Bronchial
collapse,
myco +, cult
-
63 3.3 0.63 5.23 345 1807.14 19.09 76%, 262.2,
1373.43
20%, 69,
361.43
3%,
10.35,
54.21
0 1%, 3.45,
18.07 0
chronic
bronchitis ,
Myco neg,
contam
mixed flora,
64 5.8 0.05 116 20 2320 0.8 84%, 16.8,
1948.8
10%, 2,
232
5%, 1,
116
1%, 0.2,
23.2 0 0
Severe
bronchitis
and
degenerativ
e airway
diseas,
mycoplas +,
bact-
65 8.6 0.66 13.03 160 2084.85 7.6 39%, 62.4,
813.09
4%, 6.4,
83.39
42%,
67.2,
875.64
12%, 19.2,
250.18
1%, 1.6,
20.84
2% plasma,
3.2, 41.67
Severe
bronchitis +
degen air
dis, myco +,
bact -
66 8.6 2.34 3.67 1340 4924.79 27.2 79%, 1058.6,
3890.58
15%, 201,
738.72
2%,
26.8,
98.49
2%, 26.8,
98.49
1%, 13.4,
49.25
1% plasma,
13.4, 49.25
179
Diagnosis Dog Serum
Urea
BALF
Urea Urea ratio
Cell count
(cells/ul)
Urea adjusted
cell count
%
Recovery
ELF
Macrophage
(unadj, adj)
Lymphocy
tes
Neutro
phils eosinop
Mast
cells Plasma Cells
Mycoplas +,
brachyceph
alic airway
dx, no lung
pathol
67 4.3 0.99 4.34 2300 9989.9 23.02 80%, 1840,
7991.92
15%, 345,
1498.48
4%, 92,
399.59 1%, 23, 99.9 0 0
Mycoplas +,
bacterial
pneumonia
Bordatella
bronchisepti
ca
68 4.3 0.59 7.29 160 1166.4 13.72 18%, 28.8,
209.95
6%, 9.6,
69.98
71%,
113.6,
828.14
5%, 8, 58.32 0 0
Bact neg,
immune
mediated
polyarthritis
69 3.3 0.33 10 60 600 10 5%, 3, 300 17%, 10.2,
102
77%46.
2, 462 0
1%, 0.6,
6 0
Bact neg,
immune
mediated
polyarthritis
70 3.3 1.86 1.77 1070 1893.9 56.36 30%, 321,
568.17
5%, 53.5,
94.69
64%,
684.8,
1212.1
0 0
0
Pasteurella
multocida,
myco neg,
laryngeal
71 7.7 0.13 59.23 120 7107.6 1.68 63%, 75.6,
4477.8
2%, 2.4,
142.15
29%,
34.8,
2061.2
5%, 6,
355.38
1%, 1.2,
71.1 0
180
parálisis (
with low
grade
inflammatio
n)
Phaeochro
mocytoma, 72 6.6 2.58 2.558 100 255.8 39.1
88%, 88,
225.1 0
10%,
10,
25.58
1%, 1, 2.5 1%, 1,
2.5 0
181
Appendix 3: Signalment of dogs included
Dog Breed Age Sex Entire Weight
1 Jack Russell 15y Male Yes 9.6kg
2 Jack Russell 15y Male yes 9.6kg
3 Tenterfield Terrier 9y Male No 6.8kg
4 Staffordshire
Terrier 2y 3m Male Yes 19.1kg
5 Staffordshire
Terrier 2y 3m Male yes 19.1kg
6 Golden Retriever 21w Female yes 9.8kg
7 Pit Bull 16y 2m Male no 32.4kg
8 Labradoodle 10y 1m Male No 21.6kg
9 Jack Russell 11y 1m Male Yes 9.7kg
10 Pomeranian 12yo Male No 6.4kg
11 Pomeranian 12yo Male No 6.4kg
12 Whippet Cross 13y 2m Female No 15.9kg
13 German Shepherd 9y 5m Female No 23.4kg
14 Staffordshire terrier 14y 10m Female No 17.4kg
15 Shih Tzu 14y 5m Female No 10kg
16 Staffordshire terrier 7y 6m Male No 16.3
182
Dog Breed Age Sex Entire Weight
17 Maltese Cross 13y 7m Male No 5kg
18 Kelpie Cross 9y Male No 13.8kg
19 Kelpie Cross 9y Male No 13.8kg
20 Hungarian Visla 8y 9m Male No 32.2kg
21 Hungarian Visla 8y 9m Male No 32.2kg
22 Border Collie 5y 5m Female yes 23.9
23 Border Collie 5y 5m female Yes 23.9kg
24 Border Collie 5y 5m female Yes 23.9kg
25 West Highland
White Terrier 3y 7m Male Yes 8.8kg
26 West Highland
White Terrier 3y 7m Male Yes 8.8kg
27 Shih Tzu 10y 6m Male No 11.68kg
28 Shih Tzu 10y 6m Male no 11.68kg
29 Jack Russell
Terrier 10y 2m female No 8.1kg
30 Labrador 8m Female Yes 17.8kg
31 Bull Mastiff 3y 1m Female No 57.9
32 Golden Retriever 10y 8m Female No 33.7kg
183
Dog Breed Age Sex Entire Weight
33 Maltese Cross 7y 10m Male No 8.5kg
34 Maltese Cross 7y10m Male No 8.5kg
35 English Springer
Spaniel 11y 2m Female No 13.9kg
36 Miniature Poodle 12yo Female No 7.7kg
37 Poodle X 12y 7m Male no 6.2
38 Staffordshire
Terrier 12y 6m female no 20kg
39 Staffordshire
Terrier 12y 6m female no 20kg
40 Maltese 14y 8m female no 3.4
41 Labrador retriever 6m male yes 31kg
42 Rhodesian Ridge
Cross 9y 8m Male no 28.2
43 Rhodesian Ridge
Cross 9y 8m male no 28.2kg
44 Pugalier 2yo male no 18.3kg
45 West Highland
White 11y 6m male no 10.4kg
46 Lhasa Apso 5y 6m male no 7.8kg
184
Dog Breed Age Sex Entire Weight
47 Shih Tzu 13y 6m male no 6.8kg
48 Shih Tzu 13y 9m male no 6.8kg
49 Labrador retriever 11y 3m male yes 44.9kg
50 Labrador retriever 11y 3m male yes 44.9kg
51 Cavalier King
Charles Spaniel 9y male no 13.6kg
52 Cavalier King
Charles Spaniel 9y male no 13.6kg
53 Maltese Cross 7y 2m female no 8.1kg
54 Maltese Cross 7y 2m female no 8.1kg
55 Maltese Cross 7y 2m female no 8.1kg
56 Border Collie 13y2m female no 26.8kg
57 Border Collie 13y2m female no 26.8kg
58 West Highland
White 8y1m male yes 10.8kg
59 West Highland
White 8y1m male yes 10.8kg
60 Staffordshire terrier 1y male yes 18.2kg
61 Staffordshire terrier 1y male yes 18.2kg
185
Dog Breed Age Sex Entire Weight
62 Labrador Retriever 11y 6m female No 33.8kg
63 Labrador Retriever 11y 6m female no 33.8kg
64 Pug 10y2m male no 14.7kg
65 Shih Tzu 11y6m male no 11.5kg
66 Shih Tzu 11y6m male no 11.5kg
67 Boston Terrier 4m Male no 7.9kg
68 Boston Terrier 4m male no 7.9kg
69 Rhodesian
Ridgeback 9y6m male no 33.9kg
70 Rhodesian
Ridgeback 9y6m male no 33.9kg
71 Poodle cross 15y 11m male no 13.6kg
72 Staffordshire terrier
Cross 15yo female no 20kg
186
Appendix 4: Mycoplasma spp. diagnosis in dogs included
Case # Mycoplasma
specifically
tested?
PCR Test
performed
Bacterial
culture
performed
Other bacteria
if cultured?
detected
1 no no yes No growth no
2 no no yes No growth no
3 yes yes Yes
+specific
mycoplasma
culture
Pseudomonas
aeruginosa
no
4 yes yes yes no no
5 yes yes yes no no
6 yes yes yes B.bronchiseptica no
7 no no no n/a no
8 no no yes no no
9 yes yes yes Mixed bacterial
flora
yes
10 yes yes yes No growth yes
11 yes yes yes No growth yes
12 no No no no no
13 yes yes yes no no
187
Case # Mycoplasma
specifically
tested?
PCR Test
performed
Bacterial
culture
performed
Other bacteria
if cultured?
detected
14 yes yes yes Scanty mixed
bacterial
yes
15 yes yes yes no no
16 yes Yes (+) yes no yes
17 yes Yes (+) yes no yes
18 no no yes No no
19 no no yes no no
20 yes Yes (-) yes Enterobacter no
21 yes Yes (-) yes Enterobacter no
22 yes Yes (-) yes no no
23 yes Yes (-) yes no no
24 yes Yes (-) yes no no
25 yes Yes (+) yes no yes
26 yes Yes (+) yes no yes
27 yes yes yes no yes
28 yes yes yes no yes
29 no no yes Klebsiella no
188
pneumoniae
30 yes Yes (+) yes Pasteurella sp yes
31 no no yes Enterobacter
cloacae
no
32 yes Yes (-) yes no no
33 yes Yes (-) yes Mucoid coliform no
34 yes Yes (-) yes Mucoid coliform no
35 no no no no no
36 yes Yes (+) yes no yes
37 yes Yes (+) no coliform yes
38 yes Yes (-) yes no no
39 yes Yes (-) yes no no
40 yes Yes (+) yes no yes
41 no no yes E.coli no
42 yes Yes (+) yes no yes
43 yes Yes (+) yes no yes
44 no no yes no no
45 yes no yes no no
46 yes Yes (-) yes no no
189
Case # Mycoplasma
specifically
tested?
PCR Test
performed
Bacterial
culture
performed
Other bacteria
if cultured?
detected
47 yes Yes (-) yes no no
48 yes Yes (-) yes no no
49 yes Yes (-) yes no no
50 yes Yes (-) yes no no
51 yes Yes (+) yes no no
52 yes Yes (+) yes no no
53 yes Yes (+) yes Pseudomonas
sp
yes
54 yes Yes (+) yes Pseudomonas
sp
yes
55 yes Yes (+) yes Pseudomonas
sp
yes
56 yes Yes (-) yes no no
57 yes Yes (-) yes no no
58 Yes Yes (+) yes Pasteurella Sp yes
59 yes Yes (+) yes Pasteurella Sp yes
60 yes Yes (-) yes no no
61 yes Yes (-) yes no no
190
Case # Mycoplasma
specifically
tested?
PCR Test
performed
Bacterial
culture
performed
Other bacteria
if cultured?
detected
62 yes Yes (+) yes no yes
63 yes Yes (+) yes no yes
64 yes Yes (-) yes no no
65 yes Yes (+) yes no yes
66 yes Yes (+) yes no yes
67 yes Yes (+) yes Bordatella
bronchiseptica
yes
68 yes Yes (+) yes Bordatella
bronchiseptica
yes
69 no no yes no no
70 no no yes no no
71 yes yes (-) yes Pasteurella
multocida
no
72 no no yes no no
191
Appendix 5: Classification of diseases diagnosed and presence of Mycoplasma spp.
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
1 Tracheal collapse +
Bronchitis
Chronic Bronchitis Tracheal collapse no
2 Tracheal collapse +
Bronchitis
Chronic Bronchitis Tracheal Collapse no
3 Chronic bronchitis Chronic Bronchitis n/a no
4
Dynamic airway collapse +
bronchitis and hypolastic
trachea
Chronic Bronchitis 1. Hypoplastic trachea
2. Dynamic airway
collapse (bronchial)
no
5
Dynamic airway collapse +
bronchitis and hypoplastic
trachea
Chronic Bronchitis 1. Hypoplastic trachea
2. Dynamic airway
collapse (bronchial)
no
6 Bordatella bronchiseptica
pneumonia
Pneumonia-
Infectious- Bacterial
no
7
Laryngeal Paralysis + mild
large bronchi collapse
Laryngeal Paralysis Bronchial collapse no
8 Chronic bronchitis Chronic bronchitis no
9
Grade II tracheal collapse +
chronic bronchitis +
mycopla Dx
Chronic bronchitis Tracheal collapse yes
10 Chronic bronchitis +
dynamic airway Dx
Chronic bronchitis 1. Tracheal collapse
2. Bronchial collapse
yes
11 Chronic bronchitis +
dynamic airway Dx
Chronic bronchitis 1. Tracheal collapse yes
192
2. Bronchial collapse
12
pulmonary carcinoma +
diffuse intralesional pyogran
inflamm
Pulmonary carcinoma no
13 Bronchiectasia (chronic
bronchitis)
Chronic bronchitis Bronchiectasis no
14
Chronic bronchitis- collapse
mainstem bronchi +
mycoplasma +
Chronic bronchitis Bronchial collapse yes
15
Dynamic airway collapse +
Chronic bronchitis +
bronchiectasis
Chronic bronchitis Bronchiectasis
Bronchial collapse
Tracheal collapse
no
16 Pyogran. pneumonia +
Mycoplasma
Sterile
pyogranulomatous
disease
yes
17 Bronchiect Dynamic airway
collapse + Mycoplasma +
Chronic Bronchitis Bronchiectasis
Bronchial collapse
yes
18
severe bronchointerstitial??
Central Dx -met
Neoplasia- met-
Haemangiosarcoma
no
19
severe bronchointerstitial??
Central Dx-met
Neoplasia- met-
Haemangiosarcoma
no
20 Megao + severe pneumonia Aspiration Pneumonia Megaoesophagus no
21
MegaO + severe
pneumonia
Aspiration Pneumonia Megaoesophagus no
22 SLE SLE-systemic immun. no
23 SLE SLE-systemic immun. no
193
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
24 SLE SLE- systemic immun no
25 Mycoplasma + idiopathic
pulmonary fibrosis
Pulmonary fibrosis yes
26 Mycoplasma + idiopathic
pulmonary fibrosis
Pulmonary fibrosis yes
27
Dynamic collapse bronchi
Mild bronchitis.
Mycoplasma +
Chronic bronchitis Bronchial collapse yes
28
Dynamic collapse bronchi
Mild bronchitis.
Mycoplasma +
Chronic bronchitis Bronchial collapse yes
29 Chronic infection-
K.pneumoniae
Pneumonia-
infectious- bacterial
no
30
Chronic infection-
Pasteurella sp +
Mycoplasma
Pneumonia-
infectious- bacterial
yes
31 Aspiration pneumonia-
Enterobacter cloacae
Aspiration pneumonia Immune polyarthritis no
32 Chro. inflam bronchial +
pulm. Paren Dx
Chronic bronchitis no
33 mild pulm intersitial inflam
Bronchointerstitial
pneumonia- non
infectious
no
34 mild pulm intersitial inflam
Bronchointerstitial
pneumonia- non
infectious
no
35 Chronic pulmonary inflam Pulmonary fibrosis no
194
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
36 Dynamic airway Dx +
mycoplasma +
Chronic Bronchitis Bronchial collapse yes
37
Chronic bronchitis and
dynamic airway collapse
Mycoplasma +
Pneumonia-
infectious- bacterial
Bronchial collapse yes
38 Chronic Bronchitis Chronic bronchitis no
39 Cult-, myco -, Chronic
bronchitis
Chronic bronchitis no
40
Myco +, Chronic bronchitis,
mainstem bronchial
collapse
Chronic bronchitis Tracheal collapse yes
41 Bacterial pneumonia Aspiration pneumonia no
42 Bact- Pulmonary fibrosis Chronic bronchitis yes
43
Myco +, Bact -, chronic
inflammatory airway and
pulmonary fibrotic
[parenchymal Dx
Pulmonary fibrosis Chronic bronchitis yes
44 Multi systemic immune
mediated disease
Systemic immune
mediated disease
no
45
Myco -, bact -, chronic
bronchitis, mild dynamic
airway collapse
Chronic bronchitis no
46 Myco-, bact -, mild chronic
bronchitis
Chronic bronchitis Bronchial collapse no
47 Bronchiectasis and severe
dynamic airway dx
Chronic bronchitis Bronchiectasis
Bronchial collapse
no
195
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
48 Chronic bronchitis,
bronchiectasis, myco-
Chronic bronchitis Bronchiectasis
Bronchial collapse
no
49 Chronic bronchitis, mycopl
–bact -
Chronic bronchitis no
50 Chronic bronchitis, mycopl
–bact -
Chronic bronchitis no
51 Dynamic airway collapse +
laryngeal collapse, myco +
Laryngeal collapse Bronchial collapse no
52 Dynamic airway collapse +
laryngeal collapse, myco +
Laryngeal collapse Bronchial collapse no
53 Brachycephalic syndrome +
heat stress Myco+
Non cardiogenic
oedema (Upper
airway obstruction)
yes
54 Brachycephalic syndrome +
heat stress Myco+
Non cardiogenic
oedema (Upper
airway obstruction)
yes
55 Brachycephalic syndrome +
heat stress Myco+
Non cardiogenic
oedema (Upper
airway obstruction)
yes
56 Chronic bronchitis, myco - Chronic Bronchitis no
57 Chronic bronchitis, myco - Chronic bronchitis no
58
Myco +, pasteurella +,
dynamic airway disease
and chronic bronchial d.
Chronic Bronchitis Infectious bacteria
Tracheal collapse
yes
59
Myco +, pasteurella +,
dynamic airway disease
and chronic bronchial d.
Chronic bronchitis Infectious bacteria
Tracheal collapse
yes
196
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
60
Myco -, bact-, fungal-
neurogenic oedema (near
drowning)
Non cardiogenic
oedema (near
drowning)
no
61 bact-, fungal- neurogenic
oedema (near drowning)
Non cardiogenic
oedema (near
drowning)
no
62 Myco +, cult-, Normal Chronic bronchitis Laryngeal paralysis
(subclin)
yes
63 Bronchial collapse, myco +,
cult -
Chronic bronchitis Laryngeal paralysis
(subclin)
yes
64 chronic bronchitis , Myco
neg, contam mixed flora,
Chronic bronchitis no
65
Severe bronchitis and
degenerative airway diseas,
mycoplas +, bact-
Chronic bronchitis Bronchial collapse yes
66
Severe bronchitis and
degenerative airway diseas,
mycoplas +, bact-
Pulmonary fibrosis Bronchiectasis yes
67 Mycoplas +, brachycephalic
airway dx, no lung pathol
Pneumonia-
infectious- bacterial
yes
68
Mycoplas +, bacterial
pneumonia
Bordatella bronchiseptica
Pneumonia-
infectious- bacterial
yes
69 Bact neg, immune mediated
polyarthritis
Systemic immune
mediated disease
no
70 Bact neg, immune mediated
polyarthritis
Systemic immune
mediated disease
no
197
Dog Clinician
Diagnosis
Disease Subclassification Mycoplasma
71
Pasteurella multocida,
myco neg, laryngeal
parálisis ( with low grade
inflammation)
Laryngeal paralysis no
72 Phaeochromocytoma, Phaeochromocytoma no
198
Appendix 6: Disease Group cell counts
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
inflamm 1 355 3276.923 117.15 1081.38 21.3 196.62 39.05 360.46 21.3 196.62 3.55 32.77 0 0
Inflamm 2 200 1112.583 102 567.42 40 222.52 10 55.63 4 22.25 2 11.13 2 11.13
Inflamm 3 140 901.370 93.8 603.92 7 45.07 28 180.27 0 0 0 0 0 0
Inflamm 4 260 1300 78 390 15.6 78 166.4 832 0 0 0 0 0 0
inflamm 5 340 2789.74 102 836.92 20.4 167.38 217.6 1785.43 0 0 0 0 0 0
inflamm 8 150 933.34 25.5 158.67 16.5 102.67 15 93.34 0 0 0 0 0 0
Inflamm 9 22 691.43 1.98 62.23 0.44 13.83 15.84 497.83 3.74 117.54 0 0 0 0
Inflamm 10 384 5031.72 384 5031.72 0 0 0 0 0 0 0 0 0 0
Inflamm 11 200 1048.27 176 922.48 0 0 24 125.79 0 0 0 0 0 0
inflamm 13 420 739.29 210 369.64 12.6 22.18 180.6 317.89 16.8 29.57 0 0 0 0
Inflamm 14 1030 4303.42 61.8 258.2 103 473.37 20.6 86.07 0 0 0 0 0 0
Inflamm 15 860 4272.61 679.4 3375.36 17.2 85.45 94.6 469.99 8.6 42.72 25.8 128.18 34.4 170.90
Inflamm 16 58 715.75 25.52 314.93 8.12 100.21 24.36 300.62 0 0 0 0 0 0
Inflamm 17 145 1817.61 36.96 463.49 63.08 790.66 31.18 390.79 4.35 54.53 0 0 2.175 27.26
199
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
Inflamm 22 670 5323.29 174.2 1384.05 46.9 372.63 448.9 3566.6 0 0 0 0 0 0
Inflamm 23 790 2828.4 94.8 339.41 15.8 56.57 679.4 2432.42 0 0 0 0 0 0
Inflamm 24 1310 1560.16 340.6 405.64 91.7 109.21 877.7 1045.31 0 0 0 0 0 0
Inflamm 27 60 1200 49.2 984 6 120 2.4 48 2.4 48 0 0 0 0
Inflamm 28 10 1866.67 9.4 1754.67 0.4 74.67 0.2 37.33 0 0 0 0 0 0
Inflamm 32 1240 1306.72 1078.8 1136.86 74.4 78.4 62 653.34 0 0 0 0 24.8 26.13
Inflamm 36 560 1656.34 442.4 1308.51 33.6 99.39 67.2 198.76 11.2 33.13 5.6 16.56 0 0
Inflamm 38 250 1214.79 125 607.4 60 291.55 62.5 303.7 2.5 12.15 0 0 0 0
Inflamm 39 160 836.36352 108.8 568.72 36.8 192.36 14.4 75.27 0 0 0 0 0 0
Inflamm 40 400 2181.81 28 152.73 20 109.1 344 1876.36 8 43.64 0 0 0 0
Inflamm 44 8 28.571 0.56 1.99 0.08 0.29 7.36 26.28 0 0 0 0 0 0
Inflamm 45 460 2090.91 289.8 1317.27 41.4 188.18 124.2 564.54 4.6 20.9 4.6 20.9 0 0
Inflamm 46 120 1397.26 105.6 1229.59 3.6 41.92 7.2 83.84 4.8 55.89 0 0 0 0
Inflamm 47 160 2893.62 120 2170.2 6.4 115.74 14.4 260.43 16 289.36 3.2 57.87 0 0
200
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
Inflamm 48 160 2894.4 120 2170.8 6.4 115.78
14.4
260.49 16 289.4 3.2 57.89 0 0
Inflamm 49 170 219.30 69.7 89.91 1.7 2.19 76.5 98.68 22.1 28.51 0 0 0 0
Inflamm 50 250 1095 200 876 15 65.7 30 131.4 2.5 10.9 10.9 10.9 0 0
Inflamm 56 300 1881 123 771.21 60 376.2 102 639.54 15 94.05 0 0 0 0
Inflamm 57 2520 7711.20 327.6 1002.46 201.6 616.9 1864.8 5706.29 126 385.56 0 0 0 0
Inflamm 58 11000 20240 440 809.6 440 809.6 10010 18418.4 110 202.4 0 0 0 0
Inflamm 59 880 2261.6 325.6 836.79 70.4 180.93 413.6 1062.95 70.4 180.93 0 0 0 0
inflamm 62 63 519.75 23.3 192.3 15.75 129.94 22.05 181.92 0.63 5.19 1.26 10.4 0 0
Inflamm 63 345 1807.14 262.2 1373.43 69 361.43 10.35 54.21 0 0 3.45 18.07 0 0
Inflamm 64 20 2320 16.8 1948.8 2 232 1 116 0.2 23.2 0 0 0 0
Inflamm 65 160 2084.85 62.4 813.09 6.4 83.39 67.2 875.64 19.2 250.18 1.6 20.84 3.2 41.67
Inflamm 69 60 600 3 300 10.2 102 46.2 462 0 0 0.6 6 0 0
Inflamm 70 1070 1893.9 321 568.17 53.5 94.69 684.8 1212.1 0 0 0 0 0 0
201
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
Non
infectious 66 1340 4924.79 1058.6 3890.58 201 738.72 26.8 98.49 26.8 98.49 13.4 49.25 13.4 49.25
Non
Infectious 60 130 421.20 71.55 231.77 27.3 88.45 31.2 101.1 0 0 0 0 0 0
Non
Infectious 61 330 402.6 66 80.52 26.4 32.21 237.6 289.87 0 0 0 0 0 0
Non
infectious 33 310 1595.59 254.2 1308.38 18.6 95.74 15.5 79.78 21.7 111.7 0 0 0 0
Non
infectious 34 160 444.45 110.4 306.67 20.8 , 57.78 16 44.45 12.8 35.56 0 0 0 0
Non
infectious 35 290 3954.54 72.5 988.64 98.6 1344.5 95.7 1305 2.9 39.55 0 0 20.3 276.82
Non infect 72 100 255.8 88 225.1 0 0 10 25.58 1 2.5 1 2.5 0 0
Non
Infectious 25 120 933.34 10.8 84 13.2 102.67 90 700.01 7.2 56 0 0 0 0
Non
Infectious 26 120 1135.14 10.8 102.16 13.2 124.87 90 851.36 7.2 7.2 0 0 0 0
Non
Infectious 42 350 7000 288.97 350 0 0 287 5740 56 1120 0 0 0 0
202
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
Non
Infectious 43 1110 5778.08 55.5 288.9 0 0 888 4622.46 166.5 866.71 0 0 0 0
Non
Infectious 53 90 1199.7 50.4 671.83 8.1 107.97 28.8 383.9 1.8 24 0.9 12 0 0
Non
Infectious 54 250 3777.5 152.5 2304.28 30 453.3 57.5 57.5 7.5
,
113.33 2.5 37.8 0 0
Non
Infectious 55 90 3600 44.1 1764 0.63 252 39.6 1584 0 0 0 0 0 0
infectious 68 160 1166.4 28.8 209.95 9.6 69.98 113.6 828.14 8 58.32 0 0 0 0
Infectious 41 3050 2465.96 2745 2219.36 152.5 123.3 152.5 123.3 0 0 0 0 0 0
infectious 6 164 1571.67 19.68 188.60 4.92 47.15 131.2 1257.34 8.2 78.58 0 0 0 0
Infectious 20 1960 18148.15 313.6 2903.7 117.6 117.6
1528.8
14155.5 0 0 0 0 0 0
Infectious 21 3740 8990.38 224.4 539.42 112.2 112.2 3403.4 8181.25 0 0 0 0 0 0
Infectious 37 340 897.4 234.6 619.21 34 89.7 68 179.48 0 0 3.4 8.9 0 0
Infectious 29 170 637.5 90.1 337.86 57.8 216.75 18.7 70.13 1.7 6.38 1.7 6.38 0 0
203
Category Dog
Cell count
(cells/ul)
adjusted
cell count
Mac.
unadj Mac. adj
Lymph
unadj
Lymph
adj
Neut
unadj
Neut
adj
Eos.
unadj
Eos.
adj
Mast
unadj
Mast
adj
Plasma
unadj
Plasma
adj
Infectious 30 15250 55953.24 686.25 2517.9 457.5 1678.6 14106.3, 51756.7
5 0 0 0 0 0 0
Infectious 31 480 991.304 4.8 9.91 9.6 19.82 465.6 961.56 0 0 0 0 0 0
URTD 71 120 7107.6 75.6 4477.8 2.4 142.15 34.8 2061.2 6 355.38 1.2 71.1 0 0
URTD 51 140 1369.20 47.6 465.53 18.2 178 74.2 725.67 0 0 0 0 0 0
URTD 52 210 1339.80 119.7 763.68 27.3 174.17 60.9 388.54 0 0 0 0 0 0
URTD 7 355 682.69 234 450.57 78.1 150.2 28.4 54.62 7.1 13.65 7.1 13.65 0 0
neoplasia 12 300 2653.85 123 1088.08 9 79.62 162 1433.08 6 53.08 0 0 0 0
Neoplasia 18 90 104.19
27
31.26 6.3 7.29 56.7 65.64 0 0 0 0 0 0
Neoplasia 19 95 1206.76 64.6 820.59 12.35 156.88 15.2 193.08 2.85 36.20 0 0 0 0
Legend: Eos.= Eosinophils, Mac.= macrophages, Neut.= neutrophils, adj.= adjusted, unadj= unadjusted
204
Appendix 7: Statistical P values of respiratory disease when comparing all respiratory diseases
Legend:
Adj.= adjusted
Non-cardio= non cardiogenic
Phaeochromo.= Phaeochromocytoma
Variable IM
MD
Ph
aeo
ch
rom
o.
Gra
n. D
isea
se
Lary
ng
eal
co
llap
se
Lary
ng
eal
para
lysis
Neo
pla
sia
Ch
ron
ic
bro
nch
itis
Pu
lmo
nary
fib
rosis
Asp
irati
on
pn
eu
mo
nia
No
n c
ard
io.
oed
em
a
Bro
nch
oin
t.
pn
eu
mio
nia
Bacte
rial
pn
eu
mo
nia
Overa
ll P
valu
e
Total Cell count .00007 8.4 x
10-4
Adj. cell count 0.435
Macrophage 0.0057 0.238
Adj. macrophage .083 0.736
Lymphocytes 0.542
Adj. lymphocytes 0.744
Neutrophils 0.0063 0.021
Adj. neutrophils 0.137
Eosinophils 0.0065 0.366
AdjEosinophils 0.0016 0.148
Mast cells 0.972
Adj Mast cells .021 0.66
Plasma cells 0.0241 0.626
Adj. plasma cells 0.0069 0.284
205
Appendix 8: Statistical analysis of broad disease category groups
Legend:
** Very few plasma cells in groups with very few data points
Adj.= adjusted
Variable
Infl
am
mato
ry
Dis
ea
se
No
n-
Infe
cti
ou
s
Dis
ea
se
Infe
cti
ou
s
Dis
ea
se
Up
per
Resp
irato
ry
Tra
ct
Dis
ea
se
Resp
irato
ry
Neo
pla
sia
Overa
ll P
valu
e
Total Cell count 0.007596 0.7833
Adj. cell count 0.4903
Macrophage 0.5273
Adj. macrophage 0.4682
Lymphocytes 07972.0
Adj. lymphocytes 0.6301
Neutrophils 0.2622
Adj. neutrophils 0.5967
Eosinophils 0.5292
Adj. Eosinophils 0.4106
Mast cells 0.934
Adj Mast cells 0.5855
Plasma cells 0.6423**
Adj. plasma cells 0.3594**