Interclinician agreement on the recognition of selected respiratory clinical signs in dogs and cats with abnormal breathing patterns.

In humans, classification of abnormal breathing patterns (ABP) and recognition of ancillary respiratory signs are difficult, as reflected by poor-to-moderate interclinician agreement. The aims of this study were to assess interclinician agreement for respiratory sign recognition in dogs and cats and evaluate the influence of clinical experience on agreement. Dogs and cats with ABP were recruited from three hospitals. Included animals were evaluated by three clinicians at each hospital before therapeutic intervention. Consensual definitions for each respiratory clinical sign were provided to all clinicians. Interclinician agreement was measured via Fleiss' kappa and intraclass correlation coefficient statistics. Influence of clinical experience on interobserver agreement was studied via mixed-effects logistic regression. One-hundred and fifteen dogs and 49 cats with ABP were recruited. Out of 12 clinical signs evaluated, only stertor (kappa, 0.80), stridor (kappa, 0.64), attenuation of heart/lung sounds (kappa, 0.60), and goose honking (kappa, 0.84) in dogs, and stertor (kappa, 0.65) and open-mouth breathing (kappa, 0.75) in cats, were considered sufficiently reliable among clinicians. Agreement on respiratory rate estimation was good in both species (intraclass correlation coefficient, 0.75). The greater the difference in clinical experience between two clinicians, the lower the odds of agreement between the two clinicians' respiratory physical examination findings. Interclinician agreement was demonstrated to be poor for recognition of most respiratory clinical signs in dogs and cats. Teaching and clinical experience acquisition should be encouraged to improve respiratory clinical sign recognition.

Association between respiratory clinical signs and respiratory localization in dogs and cats with abnormal breathing patterns.

The diagnostic values of respiratory signs have been under-investigated in pets. The study aim was to explore commonly assumed associations between respiratory signs and disease localization in pets with abnormal breathing patterns (ABP). Dogs and cats with ABP presenting to three hospitals were included if investigations permitted disease localization. Hypothesized associations between respiratory signs and disease location were evaluated via mixed-effects logistic regression. Sensitivity, specificity, and positive diagnostic likelihood ratio were calculated. One-hundred and fifteen dogs and 49 cats with ABP were recruited. Confirmed associations included: inspiratory effort with extra-thoracic airway disease (odds ratio [OR], 9.1; 95% confidence interval [95% CI] 3.0-27.2); expiratory effort with intra-thoracic airway disease (OR, 6.5; 95% CI, 2.3-18.1); paradoxical breathing and attenuation of heart/lung sounds with pleural space disease (paradoxical breathing: OR, 4.5; 95% CI 1.7-12.1; sound attenuation: OR, 11.5; 95% CI 4.0-33.3); decreased nasal airflow and stertor with nasal/pharyngeal disease (nasal airflow: OR, 26.2; 95% CI 8.1-84.8; stertor: OR, 155.2; 95% CI 24.9-968.8); stridor with laryngeal or tracheal disease (laryngeal disease: OR, 39.9; 95% CI 7.6-209.0; tracheal disease: OR, 32.4; 95% CI 4.2-248.0); tracheal sensitivity with bronchial disease (OR, 3.8; 95% CI 1.5-9.6); crackles with pulmonary or bronchial disease (pulmonary disease: OR, 5.4; 95% CI 2.1-13.8; bronchial disease: OR, 3.9; 95% CI 1.6-9.8); and goose honking with tracheal disease (all dogs with goose honking had tracheal involvement). Select respiratory signs provide guidance to localize and prioritize causes of the underlying respiratory disease in pets, allowing targeted interventions in animals with ABP.

Lung ultrasound nodule sign for detection of pulmonary nodule lesions in dogs: Comparison to thoracic radiography using computed tomography as the criterion standard.

Thoracic radiography (TR), the most common screening test for pulmonary metastases in dogs, can fail to detect small lesions <3 mm. Lung ultrasonography (LUS) is a widely available imaging modality capable of detecting peripheral nodules but is underutilized for this purpose. Thoracic computed tomography (CT) is the criterion standard for diagnosis of lung metastases and nodular disease but is less practical for a variety of reasons. We hypothesized that LUS would be more sensitive but less specific at detecting nodules consistent with metastatic pulmonary disease in dogs compared to TR, using CT as the criterion standard. This was a masked, single-center prospective study of 62 client-owned dogs evaluated for respiratory signs or pulmonary metastatic neoplasia screening using TR, LUS and CT. Dogs were included if metastatic pulmonary disease was a differential. All imaging modalities were scored as having nodules (yes/no) and other types of pathologic lesions were recorded. Sensitivity (Se), specificity (Sp) and positive (LR+) and negative likelihood ratios (LR-) were determined for TR and LUS. For TR, Se and Sp were 64% and 73%, and LR+ and LR- were 2.37 and 0.49, respectively. For LUS, Se and Sp were 60% and 65% and LR+ and LR- were 1.71 and 0.62, respectively. The results of the study indicate that LUS had a similar Se to TR, with both modalities missing nodules when used for screening. The low Sp and LR- suggests caution should be used when assuming TR and LUS rule out the presence of nodules.

Blood cultures and blood microbiota analysis as surrogates for bronchoalveolar lavage fluid analysis in dogs with bacterial pneumonia.

BACKGROUND: Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. RESULTS: Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). CONCLUSIONS: Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

Discrimination between respiratory and non-respiratory sound waveforms in dogs using acoustic wave recordings: An objective metric of cough.

Cough is an important respiratory protective mechanism, which when persistent also contributes to disease pathology. It is therefore both a marker for and a target of therapeutic intervention. In dogs, assessment of cough is subjective, generally based on owner's perceptions of clinical signs. In humans, acoustic cough monitoring provides objective data on cough frequency by examining acoustic waveforms. We hypothesized that healthy mesocephalic dogs would demonstrate characteristic cough waveforms which could be distinguished from other acoustic behaviors (AB); whine, bark, growl, lick, drink, chew and throat-clear. Data were obtained from 10 healthy employee-owned dogs. Acoustic behaviors were recorded using a CTA-laryngeal-microphone and analyzed using RavenPro bioacoustics software for nine objective acoustic parameters (AP). Similarity between AB were assessed using a one-way analysis of similarity (ANOSIM) with a P<0.001 significance level. Inter- and intra-group statistical analysis was performed using a one-way ANOVA on ranks with P<0.05 significance level. With the exception of throat-clear, cough was dissimilar to every other evaluated AB (P<0.0001), with significant differences in one or more of the analyzed waveform parameters (P<0.001 for each). No between-subject differences were identified between cough and throat-clear groups for any parameter. All other behaviors showed statistically significant within-group variation (P<0.001). Cough and throat-clear (a clinically similar mechanism to protect the airways) have repeatable acoustic features that are distinguishable from other common AB and are consistent between dogs. Acoustic monitoring may provide an objective means for evaluating cough frequency and intensity in dogs with respiratory disease and assessing response to therapeutic intervention.

Pulmonary hypertension secondary to respiratory disease and/or hypoxia in dogs: Clinical features, diagnostic testing and survival.

Pulmonary hypertension (PH) is associated with substantial morbidity and if untreated, mortality. The human classification of PH is based on pathological, hemodynamic characteristics, and therapeutic approaches. Despite being a leading cause of PH, little is known about dogs with respiratory disease and/or hypoxia (RD/H)-associated PH. Therefore, our objectives were to retrospectively describe clinical features, diagnostic evaluations, final diagnoses and identify prognostic variables in dogs with RD/H and PH. In 47 dogs identified with RD/H and PH, chronic airway obstructive disorders, bronchiectasis, bronchiolar disease, emphysema, pulmonary fibrosis, neoplasia and other parenchymal disorders were identified using thoracic radiography, computed tomography, fluoroscopy, tracheobronchoscopy, bronchoalveolar lavage, and histopathology. PH was diagnosed using transthoracic echocardiography. Overall median survival was 276.0 days (SE, 95% CI; 216, 0-699 days). Dogs with an estimated systolic pulmonary arterial pressure (sPAP) ≥47mmHg (n=21; 9 days; 95% CI, 0-85 days) had significantly shorter survival times than those <47mmHg (n=16; P=0.001). Estimated sPAP at a cutoff of ≥47mmHg was a fair predictor of non-survival with sensitivity of 0.78 (95% CI, 0.52-0.94) and specificity of 0.63 (95% CI, 0.38-0.84). Phosphodiesterase-5 (PDE5) inhibitor administration was the sole independent predictor of survival in a multivariable analysis (hazard ratio: 4.0, P=0.02). Canine PH is present in a diverse spectrum of respiratory diseases, most commonly obstructive disorders. Similar to people, severity of PH is prognostic in dogs with RD/H and PDE5 inhibition could be a viable therapy to improve outcome.

Aerodigestive disorders in dogs evaluated for cough using respiratory fluoroscopy and videofluoroscopic swallow studies.

Aerodigestive diseases, hybrid disorders representing a pathologic link between respiratory and alimentary tracts, may manifest with respiratory signs without gastrointestinal signs. These are underdiagnosed in dogs due to poor clinical recognition and diagnostic limitations. We hypothesize that a subset of dogs presenting for cough without gastrointestinal signs would have occult aerodigestive disorders identified using videofluoroscopic swallow study (VFSS). Data were retrospectively obtained from 31 client-owned dogs presenting for cough, with thoracic radiographs, and a VFSS between April 2015 and December 2017. Exclusion criteria were cough of cardiac origin or gastrointestinal signs within 6 months. Swallow study parameters included pharyngeal/esophageal motility, laryngeal obstruction/defects, penetration-aspiration, reflux, excessive aerophagia, megaesophagus (ME), lower-esophageal sphincter achalasia-like syndrome (LES-AS), and sliding hiatal hernia (HH). The median (interquartile range) duration of cough was 4 (2-8) months. Thoracic radiographs were unremarkable in 11 dogs, with aspiration pneumonia suspected in seven. In 25/31 dogs (81%), VFSS abnormalities were detected and some dogs had more than one defect: pharyngeal (n=10) or esophageal hypomotility (n=10), reflux (n=9), penetration-aspiration (n=8), excessive aerophagia (n=6), laryngeal obstruction (n=3), ME (n=3), HH (n=2), and LES-AS (n=1). A respiratory disorder causing cough was identified in 17 dogs with VFSS abnormalities (laryngeal obstruction/defect and airway disease including chronic or eosinophilic bronchitis, tracheal/mainstem bronchial collapse, bronchiectasis, and bronchomalacia). An alimentary disorder identified on VFSS in absence of a discrete respiratory disorder causing cough was diagnosed in eight dogs. In conclusion, canine aerodigestive disorders can manifest as cough without alimentary signs. VFSS is a useful diagnostic to determine the contribution of esophageal/gastrointestinal pathology in dogs with cough.

Clinical efficacy of tadalafil compared to sildenafil in treatment of moderate to severe canine pulmonary hypertension: a pilot study.

INTRODUCTION: Canine pulmonary hypertension (PH) is associated with high morbidity and mortality. Tadalafil, a phosphodiesterase-5 inhibitor used commonly in humans with PH, has not been evaluated in a clinical trial in dogs with naturally occurring PH. Our objectives were to compare the efficacy of tadalafil and sildenafil on PH assessed by peak tricuspid regurgitant flow velocity, estimated systolic pulmonary arterial pressure gradient, voluntary activity, quality of life, and safety profiles in dogs with moderate to severe PH. ANIMALS: Twenty-three dogs with echocardiographic evidence of moderate to severe PH were enrolled. METHODS: A prospective short-term, randomized, double-blinded pilot study was carried out. Dogs with PH were randomly allocated to receive sildenafil or tadalafil for 2 weeks and assessed via echocardiography, activity monitors, and owner-reported outcomes. RESULTS: Collectively, phosphodiesterase-5 inhibition significantly decreased (improved) quality of life scores (p = 0.003) and visual analog score (p = 0.024) without significant between-treatment difference of these variables. Phosphodiesterase-5 inhibition did not significantly affect peak tricuspid regurgitant flow velocity (p = 0.056) or voluntary activity (p = 0.27). A total of 33% (7/21) of dogs experienced at least one adverse event during the study (tadalafil, n = 5; sildenafil, n = 2) with no significant difference between treatment type and incidence of adverse events (p = 0.36). DISCUSSION: In this pilot study, phosphodiesterase-5 inhibition led to apparent improvement in quality of life scores without documenting superiority of tadalafil over sildenafil. CONCLUSION: Tadalafil at a dose of 2 mg/kg once daily appears to be a viable alternative to sildenafil in dogs with moderate to severe PH.

The utility of point-of-care ultrasound right-sided cardiac markers as a screening test for moderate to severe pulmonary hypertension in dogs.

Dogs with respiratory disease can develop pulmonary hypertension (PH), a comorbid condition that can impact therapy and prognosis. Without confirmation using the criterion standard of echocardiography, this complication may be missed. Point-of-care ultrasound (POCUS) is a simple, non-invasive screening test that may suggest PH. It was hypothesized that in dogs POCUS right-sided cardiac markers (R-SCM) at the subxiphoid view would predict moderate to severe PH confirmed by echocardiography. Forty-three client-owned dogs that underwent respiratory evaluation with POCUS and echocardiography were included. POCUS R-SCM evaluated in the subxiphoid view included subjective caudal vena cava distention (CVCsx), CVCsx >1cm, gallbladder wall edema and ascites. PH was defined by tricuspid regurgitation pressure gradient (TRPG) as mild (30-49.9mmHg), moderate (50-74.9mmHg) or severe (>75mmHg). POCUS subxiphoid views were blindly evaluated post hoc and compared to echocardiography. Chi square test and one-way ANOVA were used to evaluate correlations between POCUS R-SCM and echocardiographic diagnosis of moderate to severe PH. Twenty-six dogs with PH, and 17 dogs without PH, were enrolled. There was no significant difference in the presence or absence of any R-SCM between dogs with and without PH. When dogs with no PH and mild PH were grouped and compared to dogs with moderate to severe PH (i.e., dogs for which treatment for PH would be recommended), no significant differences in R-SCM were noted. POCUS R-SCM using the CVCsx view was not a sensitive screening test to identify dogs with PH in this study population.

Mechanical dilation, botulinum toxin A injection, and surgical myotomy with fundoplication for treatment of lower esophageal sphincter achalasia-like syndrome in dogs.

BACKGROUND: Megaesophagus (ME) carries a poor long-term prognosis in dogs. In people, lower esophageal sphincter (LES) achalasia is a rare cause of ME that may respond to targeted intervention. Dogs with lower esophageal sphincter achalasia-like syndrome (LES-AS) have been described recently, warranting investigation of analogous targeted treatment. HYPOTHESIS/OBJECTIVES: Evaluate response of dogs with LES-AS to LES mechanical dilation and botulinum toxin A (BTA) injections, with or without surgical myotomy and fundoplication. We hypothesized that clinical and videofluoroscopic swallow study (VFSS) features of LES-AS would improve after treatment targeting functional LES obstruction. ANIMALS: Fourteen client-owned dogs with LES-AS diagnosed by VFSS. METHODS: Retrospective study. Dogs diagnosed with LES-AS underwent treatment between April 2015 and December 2017. Outcome measures included client perception of clinical severity, body weight (BW), body condition score (BCS), regurgitation frequency, and VFSS parameters (ME, esophageal motility, gastric filling). Dogs with positive responses were considered candidates for LES myotomy with fundoplication. RESULTS: By a median IQR of 21 (IQR, 14-25) days after mechanical dilation and BTA, clients reported clinical improvement in 100% of dogs, BW increased 20.4% (IQR, 12.7%-25%), pre- and post-treatment BCS was 3 (IQR, 3-4) and 5 (IQR, 4-5), respectively, and frequency of regurgitation decreased by 80% (IQR, 50%-85%). Duration of effect was 40 (IQR, 17-53) days. Despite clinical improvement, ME and abnormal esophageal motility persisted in 14 dogs. Six dogs subsequently underwent myotomy and fundoplication and maintained improvement observed after mechanical dilation and BTA. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with LES-AS experienced significant, temporary, clinical improvement after mechanical dilation and BTA. Preliminary results suggest myotomy with fundoplication provide lasting clinical benefit despite persistence of ME.

Pharmacokinetics and dynamics of mycophenolate mofetil after single-dose oral administration in juvenile dachshunds.

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young-aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose-bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole-blood mitogen-stimulated T-cell proliferation was determined using a flow cytometric assay. Plasma MPA Cmax (mean ± SD, 9.33 ± 7.04 µg/ml) occurred at <1 hr. The AUC0-∞ (mean ± SD, 12.84±6.62 hr*µg/ml), MRTinf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.

Standardization of a Videofluoroscopic Swallow Study Protocol to Investigate Dysphagia in Dogs.

BACKGROUND: Videofluoroscopic swallow study (VFSS) is the gold standard for diagnosis of dysphagia in veterinary medicine but lacks standardized protocols that emulate physiologic feeding practices. Age impacts swallow function in humans but has not been evaluated by VFSS in dogs. HYPOTHESIS/OBJECTIVES: To develop a protocol with custom kennels designed to allow free-feeding of 3 optimized formulations of contrast media and diets that address limitations of current VFSS protocols. We hypothesized that dogs evaluated by a free-feeding VFSS protocol would show differences in objective swallow metrics based on age. ANIMALS: Healthy juvenile, adult, and geriatric dogs (n = 24). METHODS: Prospective, experimental study. Custom kennels were developed to maintain natural feeding behaviors during VFSS. Three food consistencies (thin liquid, pureed food, and dry kibble) were formulated with either iohexol or barium to maximize palatability and voluntary prehension. Dogs were evaluated by 16 swallow metrics and compared across age groups. RESULTS: Development of a standardized VFSS protocol resulted in successful collection of swallow data in healthy dogs. No significant differences in swallow metrics were observed among age groups. Substantial variability was observed in healthy dogs when evaluated under these physiologic conditions. Features typically attributed to pathologic states, such as gastric reflux, were seen in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Development of a VFSS protocol that reflects natural feeding practices may allow emulation of physiology resulting in clinical signs of dysphagia. Age did not result in significant changes in swallow metrics, but additional studies are needed, particularly in light of substantial normal variation.

Precision Medicine in Cats: Novel Niemann-Pick Type C1 Diagnosed by Whole-Genome Sequencing.

State-of-the-art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state-of-the-art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole-genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 25× genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann-Pick type C1 on cat chromosome D3.47456793 caused by an adenine-to-cytosine transversion, c.1322A>C. The cat was homozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population.

Long-term evaluation of mesenchymal stem cell therapy in a feline model of chronic allergic asthma.

BACKGROUND: Mesenchymal stem cells (MSCs) decrease airway eosinophilia, airway hyperresponsiveness (AHR), and remodelling in murine models of acutely induced asthma. We hypothesized that MSCs would diminish these hallmark features in a chronic feline asthma model. OBJECTIVE: To document effects of allogeneic, adipose-derived MSCs on airway inflammation, AHR, and remodelling over time and investigate mechanisms by which MSCs alter local and systemic immunologic responses in chronic experimental feline allergic asthma. METHODS: Cats with chronic, experimentally induced asthma received six intravenous infusions of MSCs (0.36-2.5 × 10E7 MSCs/infusion) or placebo bimonthly at the time of study enrollment. Cats were evaluated at baseline and longitudinally for 1 year. Outcome measures included: bronchoalveolar lavage fluid cytology to assess airway eosinophilia, pulmonary mechanics and clinical scoring to assess AHR, and thoracic computed tomographic (CT) scans to assess structural changes (airway remodelling). CT scans were evaluated using a scoring system for lung attenuation (LA) and bronchial wall thickening (BWT). To assess mechanisms of MSC action, immunologic assays including allergen-specific IgE, cellular IL-10 production, and allergen-specific lymphocyte proliferation were performed. RESULTS: There were no differences between treatment groups or over time with respect to airway eosinophilia or AHR. However, significantly lower LA and BWT scores were noted in CT images of MSC-treated animals compared to placebo-treated cats at month 8 of the study (LA P = 0.0311; BWT P = 0.0489). No differences were noted between groups in the immunologic assays. CONCLUSIONS AND CLINICAL RELEVANCE: When administered after development of chronic allergic feline asthma, MSCs failed to reduce airway inflammation and AHR. However, repeated administration of MSCs at the start of study did reduce computed tomographic measures of airway remodelling by month 8, although the effect was not sustained at month 12. Further study of MSC therapy including repeated MSC administration is warranted to assess impact on remodelling in chronic asthma.

Neonatal aerosol exposure to Bermuda grass allergen prevents subsequent induction of experimental allergic feline asthma: evidence for establishing early immunologic tolerance.

Allergic asthma is increasing in industrialized countries, especially in children. Rodent and human studies suggest an opportunity to "prevent" asthma in the perinatal period. The aims of this study were to create a more "natural" model of feline asthma by exposing offspring of asthmatic queens to Bermuda grass allergen (BGA) by inhalation only, and to investigate maternal-fetal-infant interactions in the development of asthma. Kittens from asthmatic queens were divided into four groups: maternal exposure to aerosolized BGA during the third trimester, neonatal exposure to aerosolized BGA in the first three months of life, both maternal and neonatal exposure, or saline control. Kittens failing to achieve an asthmatic phenotype based on bronchoalveolar lavage fluid (BALF) analysis by 6 months underwent traditional sensitization: adjuvanted allergen injection, intranasal allergen, and aerosol challenges. BALF was collected at 3, 4 and 6 months, and after sensitization at 8 months, and analyzed for eosinophil counts and BGA-specific IgG and IgA. Intradermal testing (IDT) was performed at 6 and 7 months. At six months none of the kittens had airway eosinophilia, BGA-specific IgG or IgA, and were non-responsive to IDT. After sensitization, kittens receiving neonatal aerosolization failed to develop airway eosinophilia as seen in the controls. Kittens exposed to BGA aerosols, either in-utero or neonatally, continued to lack IDT response. Chronic exposure to BGA aerosols failed to induce asthma in kittens, and instead tolerized the kittens to BGA. This is the first evidence that neonatal intervention could potentially "prevent" allergic asthma in cats.

Comparison of direct and indirect bronchoprovocation testing using ventilator-acquired pulmonary mechanics in healthy cats and cats with experimental allergic asthma.

Airway hyperresponsiveness (AHR) is a key feature of asthma and can be measured using bronchoprovocation. Direct (methacholine, MCh) or indirect (adenosine-5-monophosphate, AMP; or mannitol) bronchoprovocants are used in human patients, the latter inducing AHR only with pre-existing airway inflammation. The present study compared the responses to direct (MCh) and indirect (mannitol, AMP) bronchoprovocation in healthy and asthmatic cats (n=6/group). The order of bronchoprovocant was randomized using a published table of random numbers and there was a 1-month washout before crossover to the next treatment. Pulmonary mechanics were measured in anesthetized and mechanically ventilated cats using a critical care ventilator. Saline at baseline and increasing doses of each bronchoprovocant were aerosolized for 30 s, followed by 4 min of data collection between doses. The endpoint for each bronchoprovocant was reached when airway resistance exceeded 200% of baseline values (EC200Raw). There was a significant difference (P<0.001) in the airway response of asthmatic vs. healthy cats over the range of MCh concentrations, despite there being no significant difference in the EC200Raw between the groups. Response to MCh was significantly greater (P<0.05) in asthmatic than in healthy cats at MCh concentrations as low as 0.0625 mg/mL. For AMP, a small subset of asthmatics (n=2/6) responded at low concentrations; four asthmatic cats and all healthy cats failed to respond even to the highest concentrations of AMP. One asthmatic cat but no healthy cats responded to mannitol. In conclusion, MCh discriminated asthmatic from healthy cats but neither AMP nor mannitol was an effective bronchoprovocant in this model.

Endothelin-1 concentrations in bronchoalveolar lavage fluid of cats with experimentally induced asthma.

BACKGROUND: There is a need for biomarkers for diagnosis, therapeutic monitoring, and prognosis for asthma in cats. Endothelin-1 (ET-1) is implicated in the pathogenesis of inflammatory airway diseases in other species but not the cat. OBJECTIVE: To conduct a prospective experimental study to show that experimentally asthmatic cats, but not control cats without airway inflammation, would have increased concentrations of ET in BALF. ANIMALS: Eleven healthy, adult research cats. METHODS: Prospective experimental study. Six healthy cats without airway inflammation were used as controls. Asthma was induced using Bermuda grass allergen (BGA) in 5 cats. Collection of BALF for total nucleated cell and differential counts was performed. The concentration of ET-1 in cell-free BALF samples was determined. Data were analyzed using a Mann-Whitney U-test with P < .05 considered significant. RESULTS: The median [range] BALF total cell numbers, eosinophil numbers, and eosinophil percentages were significantly higher in the cats following experimental induction of asthma (1,870 cells/µL [1,450-3,440], 711 cells/µL [356-1,686] and 38% [20-49]) compared to baseline control parameters (462 cells/µL [239-780], 18 cells/µL [18-62] and 3.5% [0-8]) (P < .01). The median [range] BALF ET concentration was also significantly higher after induction of asthma (1.393 fmol/mL[0.977-2.247]) compared to healthy control cats (0.83250 fmol/mL [0.625-1.038]) (P = .012). CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests that BAL ET-1 concentration can be used to differentiate normal cats from those with experimentally induced asthma. If the same holds true for cats with naturally developing asthma, BAL ET-1 may prove a useful diagnostic biomarker for asthma.

Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (<2 and 2-6 years) (p<0.001) and fewer %CD4+CD25+FoxP3+ Tregs in the tumor draining lymph nodes of OSA patients compared to the unrelated lymph node (p=0.049). However, there was no significant difference in % Tregs in the peripheral blood or lymph nodes between the control dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA.

Endocrine and immunologic effects of inhaled fluticasone propionate in healthy dogs.

BACKGROUND: Inhaled glucocorticoids reduce airway inflammation while minimizing systemic effects in several species. HYPOTHESIS: Inhaled fluticasone suppresses the hypothalamic-pituitary-adrenal axis (HPAA), modifies immune function, and induces clinical signs to a lesser extent than PO-administered prednisone in dogs. ANIMALS: Seven healthy adult pet dogs. METHODS: Dogs were randomized to 1 of 3 treatment groups in a crossover design: fluticasone propionate (220 mug actuation of a metered dose inhaler delivered via a spacer and mask, q12h), placebo (spacer and mask alone, q12h), or prednisone (1 mg/kg PO q24h). Each treatment was administered for 3 weeks followed by a 4-week washout. Appetite, attitude, and water consumption were recorded during the last week of each treatment period. Urine cortisol : creatinine ratios, ACTH stimulation tests, white blood cell counts, lymphocyte phenotype, and serum IgM and IgA concentrations were recorded at each baseline and after the last day of each treatment. Clinical observations were expressed descriptively. Friedman's test was applied to all data comparisons. Pairwise comparisons were made with a mixed model analysis when data were normally distributed, whereas signed rank tests were used otherwise (significance P-value <.01). RESULTS: Appetite and water consumption increased during prednisone treatment. Peak serum cortisol concentrations post-ACTH were significantly decreased in prednisone- and fluticasone-treated dogs compared with placebo (prednisone > fluticasone). Serum IgM concentrations were significantly decreased in dogs treated with prednisone. CONCLUSIONS AND CLINICAL IMPORTANCE: As used, fluticasone suppresses the HPAA to a lesser extent than prednisone and may avert systemic signs associated with PO-administered glucocorticoids in dogs.