RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins.

Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and for microbial competition. These T6SS machines are loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control with RsmA repressing most known T6SS-related genes. Moreover, each of the H2- and H3-T6SS clusters encodes a sigma factor activator (SFA) protein called, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins necessary for the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of the H2-T6SS-linked VgrGs and their effector arsenal to enable very effective interbacterial killing. Sfa2 is specific as Sfa3 from the H3-T6SS cannot complement loss of Sfa2. Our study further delineates the regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely enabling P. aeruginosa to adapt to a range of environmental conditions.

Endoscopy Training in Small Animal Internal Medicine: A Survey of Residency Training Programs in North America.

Competency in multiple endoscopic techniques is a major goal of small animal internal medicine (SAIM) residency programs. Training relies predominantly on mentored supervision of procedures performed on patients. Supplementation of this apprenticeship model with classroom sessions and hands-on laboratories can be advantageous to trainees and patients. Few veterinary resources describe supplemental training options, and no single source exists for mentors to consult for program development. The purpose of this study was to describe the supplemental training opportunities currently available to SAIM residents at academic hospitals in the US and Canada and to compare their timing during the residency, resident and faculty time commitment, and perceived helpfulness. Data were collected by an electronic survey distributed to one faculty member per institution. The response rate was 80% (24/30). Most programs (22/24; 92%) offered some form of supplemental training, including classroom sessions (9/24) and hands-on laboratories using physical models (7/24), virtual reality simulators (2/24), and cadaver (2/24) and anesthetized (2/24) dogs. Fifteen programs provided residents with the opportunity to attend external endoscopy workshops. Only three programs required any training prior to residents performing procedures on patients. There was considerable variability in training between programs, precluding statistical comparisons. The survey identified topics for classroom sessions and several inexpensive physical models, rated very or extremely helpful, that would be suitable for programs with limited budgets. A human-based virtual reality simulator was also rated highly by two programs. Comprehensive, external workshops evoked numerous positive comments with perceived value ranging from somewhat to extremely helpful.

Evaluation of a Human Virtual-Reality Endoscopy Trainer for Teaching Early Endoscopy Skills to Veterinarians.

Competency in flexible endoscopy is a major goal of small animal internal medicine residency training programs. Hands-on laboratories to teach entry-level skills have traditionally used anesthetized laboratory dogs (live dog laboratory [LDL]). Virtual-reality endoscopy trainers (VRET) are used for this purpose in human medicine with the clear benefits of avoiding live animal use, decreasing trainee stress, and allowing repeated, independent training sessions. However, there are currently no commercially available veterinary endoscopy simulators. The purpose of the study was to determine whether a human VRET can be a reasonable alternative to a LDL for teaching early veterinary endoscopy skills. Twelve veterinarians with limited or no endoscopy experience underwent training with a VRET (n = 6) or a LDL (n = 6), performed two recorded esophagogastroduodenoscopies (EGD) on anesthetized dogs for evaluation purposes (outcomes laboratory), and then underwent training with the alternative method. Participants completed questionnaires before any training and following each training session. No significant differences were found between training methods based on: measured parameters from the outcomes laboratory, including duration of time to perform EGD; evaluators' assessment of skills; and, assessment of skills through blinded review of the esophageal portion of EGD recordings. The VRET was less stressful for participants than the LDL (p = .02). All participants found that the VRET was a useful and acceptable alternative to the LDL for training of early endoscopy skills. Based on this limited study, VRET can serve as a reasonable alternative to LDL for teaching endoscopy skills to veterinarians.

Methotrexate Administration to Patients With Presumed Ectopic Pregnancy Leads to Methotrexate Exposure of Intrauterine Pregnancies.

OBJECTIVE: To report clinical experience with methotrexate (MTX) treatment for suspected but not definite ectopic pregnancy (EP). METHODS: This was a retrospective cohort study. All patients treated with MTX for presumed EP between 2000 and 2016 were included. Demographic, clinical, sonographic, and outcome data were collected and analyzed. RESULTS: A total of 820 patients were treated with MTX, 692 (84.4%) of which were lacking definitive features of EP; 155 (22.4%) failed to follow up until complete resolution and were excluded. Retrospective sonographic categorization was applied to 537 patients; of those patients, 393 (73.2%) were categorized as probable EPs, 136 (25.3%) pregnancies of unknown location (PULs), and 8 (1.5%) probable intrauterine pregnancies (IUPs). Sixteen were eventually diagnosed with IUP: 6 from the probable EPs, 9 from the PULs, and 1 from the probable IUP group. Patients with final diagnosis of IUP had higher values of ß-human chorionic gonadotropin as well as lower prevalence of adnexal mass (38% versus 74%; P = .003), higher prevalence of intracavitary fluid (44% versus 9%; P = .0004) and thicker endometrium (17.1 ± 11.8 versus 9.7 ± 5.6; P = .04). None of the sonographic parameters were able to distinguish patients with IUP. One patient of the 16 with IUP was diagnosed with a viable pregnancy, and 7 additional patients had a possible viable pregnancy. None of them elected to continue the pregnancy. CONCLUSIONS: Most patients with suspected EP who are eligible for medical treatment lack definitive sonographic features of EP. Treatment with MTX in such cases should be delayed, as clinically reasonable, to improve the diagnosis and prevent inadvertent administration of MTX to patients with a viable IUP.

Hysteroscopic morcellation: review of the manufacturer and user facility device experience (MAUDE) database.

STUDY OBJECTIVE: To investigate the number and type of adverse events associated with hysteroscopic morcellation of intrauterine disease. DESIGN: Systematic review of Manufacturer and User Device Experience (MAUDE) database from 2005 to June 2014 (Canadian Task Force classification III). SETTING N/A PATIENTS: Women undergoing hysteroscopic surgery for removal of intrauterine polyps or myomas with use of a reciprocating morcellator. INTERVENTIONS: The MAUDE database was searched for the key words "Hysteroscope," "Hysteroscopic reciprocating morcellator," "Interlace," "MyoSure," "Smith & Nephew," and "TRUCLEAR," to identify reported incidences of device malfunction, injury, or death. A total of 119 adverse events were analyzed. Reports were reviewed individually and categorized by date of occurrence, type of morcellation device, type of complication, and a brief description. Each company was contacted to provide an estimate of the number of procedures performed or units sold to date. MEASUREMENTS AND MAIN RESULTS: From 2005 to June 2014, 119 adverse events were reported to the MAUDE database. On the basis of severity, adverse events were categorized as major or minor complications. Major events included intubation/admission to an intensive care unit (n = 14), bowel damage (n = 12), hysterectomy (n = 6), and death (n = 2). Minor events included uterine perforation requiring no other treatment (n = 29), device failure (n = 25), uncomplicated fluid overload (n = 19), postoperative bleeding controlled using noninvasive measures (n = 6), and pelvic infection (n = 4). These events were then categorized according to manufacturer. The number of adverse events reported to the MAUDE database was divided by the total units sold as a surrogate for the estimated number of procedures performed. Understanding the limitation of the numbers used as a numerator and denominator, we concluded that adverse events complicated hysteroscopic morcellation in <0.1% cases. CONCLUSIONS: The suction-based, mechanical energy, rotating tubular cutting system was developed to overcome adverse events that occur during traditional resectoscopy. On the basis of acknowledged limited information from the MAUDE database, it seems that life-threatening complications such as fluid overload, uterine perforation, and bleeding do occur with hysteroscopic morcellation but less frequently than with traditional electrocautery.

Low-intensity urbanisation alters community composition across multiple trophic levels on Lipsi Island, Greece.

Urbanisation has reduced the abundance and diversity of many taxonomic groups, and the effects may be more pronounced on islands, which have a smaller regional species pool to compensate. Green spaces within urban environments may help to safeguard wildlife assemblages, and the associated habitat heterogeneity can even increase species diversity. Here, total abundance and species diversity of butterflies, birds, and vegetation at nine rural and nine urban locations were quantified on Lipsi Island, Greece. Sites were assessed using Pollard walks for butterflies, point-count surveys for birds, and quadrats for vegetation. There was no significant difference in the abundance or species diversity of butterflies or vegetation among rural and urban locations, which could pertain to the low building density within urbanised areas and the minimal extent of urbanisation on the island. However, urban areas hosted a significantly greater abundance, richness, and diversity of birds compared to rural sites. The community composition of butterflies, birds, and vegetation also differed significantly between urban and rural locations, highlighting the impact of urbanisation on species across a broad range of trophic groups. This study contributes to ecological knowledge on the impacts of urbanisation across multiple trophic levels in island ecosystems, with comparisons across a gradient of island size and urbanisation intensity needed in future research.

A myristoylated alanine-rich C kinase substrate-related peptide suppresses cytokine mRNA and protein expression in LPS-activated canine neutrophils.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand-1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.

Effect of a combined aspiration and core biopsy technique on quality of core bone marrow specimens.

For cases requiring both a bone marrow aspirate and core biopsy, using the same needle and site (i.e., a combined technique) can decrease time, expense, and discomfort compared with the independent (direct) collection of each specimen. The benefits of the combined approach should not be achieved at the expense of specimen quality. In this study, core bone marrow samples obtained from the proximal humerus of 26 dogs by a combined technique immediately posteuthanasia were compared with core samples obtained directly from the opposite humerus. Both core samples from each dog were compared for relative overall quality. Biopsies were unsuccessful in four dogs using the combined technique and in one dog by the direct technique. Marrow length was shorter and hemorrhage artifact was more common using the combined technique. There were no differences in cellularity, megakaryocyte count, the myeloid/erythroid ratio (M/E ratio), iron stores, or diagnostic quality. Direct core biopsy remains the ideal technique; however, the degree of compromise was small in the samples obtained from the combined technique. For clinical patients, the advantages of the combined technique must continue to be weighed against potential loss of diagnostic sensitivity.

Cervical lung lobe herniation in dogs identified by fluoroscopy.

This study aimed to determine the frequency of cervical lung lobe herniation (CLLH) in dogs evaluated fluoroscopically and to identify associated characteristics. Reports of diagnostic procedures and patient summaries from 2008 to 2010 were reviewed retrospectively. Signalment, body weight, duration of cough, presence of heart murmur and airway collapse, and radiographic findings were compared between dogs with and without CLLH. Of the 121 dogs that were examined, CLLH occurred in 85 (70%). The extra-thoracic trachea kinked during herniation in 33 (39%) dogs with CLLH. Collapse of the intra-thoracic trachea (assessed fluoroscopically or bronchoscopically) and collapse of major bronchi (assessed fluoroscopically) were strongly associated with CLLH. Although redundant dorsal tracheal membrane on radiographs was associated with CLLH, extra-thoracic tracheal collapse, assessed fluoroscopically or bronchoscopically, was not. No other associations were found. Cervical lung lobe herniation was present in most dogs evaluated during cough and was associated with intra-thoracic large airway collapse, but not duration of cough.

Herniation du lobe pulmonaire cervical chez les chiens identifié par fluoroscopie. Cette étude a visé à déterminer la fréquence de l'herniation du lobe pulmonaire cervical (HLPC) chez les chiens évalués par fluoroscopie et à identifier les caractéristiques connexes. Des rapports des procédures diagnostiques et des sommaires des patients de 2008 à 2010 ont été examinés rétrospectivement. Le signalement, le poids corporel, la durée de la toux, la présence d'un souffle cardiaque et de l'affaissement des voies aériennes ainsi que les constatations radiographiques ont été comparés entre les chiens avec et sans HLPC. Parmi les 121 chiens qui ont été examinés, HLPC s'est produite dans 85 cas (70 %). La trachée extra-thoracique s'est tordue durant l'herniation chez 33 (39 %) des chiens atteints de HLPC. L'affaissement de la trachée intra-thoracique (évalué par fluoroscopie ou bronchoscopie) et l'affaissement des bronches majeures (évalué par fluoroscopie) étaient fortement associés à HLPC. Même si la membrane trachéale dorsale redondante sur les radiographies était associée à HLPC, l'affaissement trachéal extra-thoracique, évalué par fluoroscopie ou bronchoscopie, ne l'était pas. Aucune autre association n'a été trouvée. L'herniation du lobe pulmonaire cervical était présente chez la plupart des chiens évalués durant la toux et était associée à l'affaissement des grandes voies aériennes intra-thoraciques, mais non à la durée de la toux.(Traduit par Isabelle Vallières).

Dirofilaria immitis Identified in Galapagos Sea Lions (Zalophus wollebaeki): A Wildlife Health and Conservation Concern.

The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Epigenetic upregulation of Schlafen11 renders WNT- and SHH-activated medulloblastomas sensitive to cisplatin.

BACKGROUND: Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma. METHODS: SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo. RESULTS: High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells. CONCLUSIONS: High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Factors associated with clinical interpretation of tracheal wash fluid from dogs with respiratory disease: 281 cases (2012-2017).

BACKGROUND: Clinicians face several dilemmas regarding tracheal washes (TWs) for the diagnosis of respiratory disease, including method and prediction of bacterial growth from cytology results. OBJECTIVE: To compare cytology and culture of endotracheal and transtracheal washes and identify factors associated with discordancy and bacterial growth. ANIMALS: Two hundred forty-five dogs with respiratory disease. METHODS: Retrospective study. Tracheal wash submissions were included if cellularity was sufficient for cytologic interpretation and aerobic cultures were performed. Collection technique, cytology, bacterial growth, and antibiotic history were analyzed. RESULTS: Fewer transtracheal specimens (9/144, 6.3%) were excluded for hypocellularity than endotracheal (28/174, 16.1%); otherwise, results were similar and were combined. Of 281 specimens with cellularity sufficient for interpretation, 97 (34.5%) had bacteria on cytology and 191 (68.0%) had bacterial growth. Cytology positive/culture negative discordancy was uncommon (8/97, 8%). Cytology negative/culture positive discordancy was frequent (102/184, 55.4%), but occurred less often (28/184, 14.2%) when only 1+ growth or greater was considered positive. Oropharyngeal contamination was associated with bacterial growth, but not discordancy. No association was found between antibiotic administration and bacterial growth. CONCLUSIONS AND CLINICAL IMPORTANCE: Endotracheal wash fluid, in particular, should be screened for gross mucus or turbidity to maximize the likelihood of an adequate specimen. Otherwise, endotracheal and transtracheal specimens were similar. Presence of bacteria on cytology was a good predictor of any growth, while their absence was a good predictor of the absence of growth of 1+ or more. Recent antibiotic usage should not discourage TW culture if there is compelling reason to avoid delay.

Naturally occurring mutations in the canine CFTR gene.

Naturally occurring cystic fibrosis (CF)-causing mutations in the CFTR gene have not been identified in any nonhuman animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans (e.g., bronchiectasis and pancreatitis), we hypothesized that dogs with these disorders likely have a higher expression rate of CFTR mutations than the at-large population. Temporal temperature-gradient gel electrophoresis (TTGE) was used to screen canine CFTR in 400 animals: 203 dogs diagnosed with pancreatitis, 23 dogs diagnosed with bronchiectasis, and 174 dogs admitted to clinics for any illness (at-large dogs). Twenty-eight dogs were identified with one of four CFTR missense mutations. P1281T and P1464H mutations occur in relatively unconserved residues. R1456W is analogous to the human R1453W mutation, which has approximately 20% of normal CFTR function and is associated with pancreatitis and panbronchiolitis. R812W disrupts a highly conserved protein kinase A recognition site within the regulatory domain. We conclude that naturally occurring CFTR mutations are relatively common in domestic dogs and can be detected with TTGE. No substantive differences in mutation frequency were observed between the at-large, pancreatitis, and bronchiectasis dogs.

Expiratory dysfunction in young dogs with golden retriever muscular dystrophy.

Respiratory disease is a leading cause of morbidity in people with Duchenne muscular dystrophy and also occurs in the golden retriever muscular dystrophy (GRMD) model. We have previously shown that adult GRMD dogs have elevated expiratory flow as measured non-invasively during tidal breathing. This abnormality likely results from increased chest and diaphragmatic recoil associated with fibrosis and remodeling. Treatments must reverse pathologic effects on the diaphragm and other respiratory muscles to maximally reduce disease morbidity and mortality. Here, we extended our work in adults to younger GRMD dogs to define parameters that would be helpful in preclinical trials. Tidal breathing spirometry and respiratory inductance plethysmography were performed in GRMD dogs at approximately 3 and 6 months of age, corresponding to approximately 5-10 years in DMD, when clinical trials are often conducted. Expiratory flows were markedly elevated in GRMD versus normal dogs at 6 months. Values increased in GRMD dogs between 3 and 6 months, providing a 3-month window to assess treatment efficacy. These changes in breathing mechanics have not been previously identified at such an early age. Expiratory flow measured during tidal breathing of unsedated young GRMD dogs could be a valuable marker of respiratory mechanics during preclinical trials.

Failure to identify an association between serologic or molecular evidence of Bartonella infection and idiopathic rhinitis in dogs.

OBJECTIVE: To determine whether infection with or exposure to Bartonella spp was associated with idiopathic rhinitis in dogs. DESIGN: Case-control study. ANIMALS: 44 dogs with idiopathic nasal discharge and 63 age- and weight-matched control dogs without nasal discharge and no clinical signs of bartonellosis. Procedures-Serum was tested for antibodies against Bartonella henselae and Bartonella vinsonii subsp berkhoffii with indirect fluorescent antibody assays. Blood was tested for Bartonella DNA with a PCR assay. RESULTS: Results of the antibody and PCR assays were negative for all 44 dogs with idiopathic nasal discharge. One control dog had antibodies against B henselae; a second control dog had positive PCR assay results. We did not detect a significant association between assay results and group designation. CONCLUSIONS AND CLINICAL RELEVANCE: The present study failed to confirm an association between idiopathic rhinitis and exposure to or infection with Bartonella spp in dogs. Findings do not rule out the possibility that Bartonella infection may cause nasal discharge in some dogs, but the failure to find any evidence of exposure to or infection with Bartonella spp in dogs with idiopathic nasal discharge suggested that Bartonella infection was not a common cause of the disease.

Quantification of mucin gene expression in tracheobronchial epithelium of healthy dogs and dogs with chronic bronchitis.

OBJECTIVE: To develop a real-time PCR assay for the quantification of mucin gene expression in tracheobronchial brushing specimens from dogs and compare mucin gene expression in specimens from dogs with naturally occurring chronic bronchitis with that in specimens from healthy dogs. ANIMALS: 7 healthy dogs and 5 dogs with chronic bronchitis. PROCEDURES: Primers that were designed to span the predicted intron-exon boundaries of a canine MUC5AC-like gene were used to develop a real-time PCR assay for quantification of expression of that gene. Total mRNA was isolated from tracheobronchial brushing specimens obtained from dogs with and without bronchitis during anesthesia; MUC5AC-like gene expression in those samples was quantified by use of the real-time PCR assay. RESULTS: The PCR assay was sensitive and specific for the target sequence, the predicted amino acid sequence of which had greatest homology with human, porcine, and rat MUC5AC. The assay was able to quantify the target over a wide dynamic range. Dogs with chronic bronchitis had a 3.0-fold increase in the quantity of MUC5AC-like mRNA, compared with healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The ability to measure mucin gene expression from tracheobronchial brushing specimens collected from client-owned dogs during routine bronchoscopy should prove to be a useful tool for the study of bronchitis in dogs and expand the usefulness of airway inflammation in dogs as a model for bronchitis in humans.

Pulmonary lesions in cats with diabetes mellitus.

Diabetes mellitus (DM) is a common endocrinopathy of cats and humans. Although few studies have examined the effects of DM on the pulmonary system, changes in pulmonary function and immunology in humans with type I and II diabetes, and pulmonary lesions in a murine diabetic model have been documented. Our objective was to determine whether pulmonary lesions occurred in cats with DM. Medical records and necropsy evaluations of 42 cats with DM were compared with those of 45 age-matched, nondiabetic cats for the presence of clinical evidence of respiratory disease and pulmonary histopathological findings at the time of necropsy. No statistical difference was noted in the presence of clinical evidence of respiratory disease between cats with diabetes and control cats. Nevertheless, there was a significant association between the presence of abnormal pulmonary histopathology and DM (P = .018, odds ratio = 3 inclusive of all cats; P = .005, odds ratio = 5 when non-DM cats with overt clinical evidence of respiratory disease were excluded). Pulmonary abnormalities detected by histopathological examination in cats with diabetes included congestion and edema, histiocytosis, pneumonia, smooth muscle hypertrophy, fibrosis, mineralization, neoplasia, and type II pneumocyte hyperplasia. The observed association between DM and pulmonary lesions in cats, independent of clinical evidence of respiratory disease, emphasizes the need for careful assessment of the respiratory tract in sick cats with diabetes.

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.

OBJECTIVE: To determine whether bronchial brushings from dogs with chronic cough have increased numbers of goblet cells and WBCs, compared with numbers for healthy dogs, or have differing WBC populations, compared with populations in bronchoalveolar lavage (BAL) fluid obtained from dogs with chronic cough. ANIMALS: 9 healthy dogs and 10 dogs with chronic cough. PROCEDURE: Specimens were collected by use of bronchoscopy. Cellular composition was determined for brushings, and results from dogs with chronic cough were compared with those from healthy dogs. Cellular composition of brushings was compared with composition of BAL obtained from dogs with chronic cough. RESULTS: Brushings from healthy dogs contained a median of 2.9 x 10(6) epithelial cells, comprising 100% epithelial cells (96% ciliated, 3% goblet, and 1% other) and no WBCs. Brushings from dogs with chronic cough had 4.5 x 10(6) epithelial cells, comprising 93% epithelial cells (86% ciliated, 2% goblet, and 12% other). Dogs with chronic cough had significantly greater percentages of WBCs (7%) and neutrophils (6%), compared with values for healthy dogs. Five dogs with chronic cough had no neutrophilic inflammation evident in BAL, but 4 of these had evidence of neutrophilic inflammation in brushings. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils, but not goblet cells, were increased in brushings from dogs with chronic cough. Analysis of bronchial brushings provides information about airway inflammation that differs from that found by examination of BAL in some dogs with chronic cough and is a more sensitive indicator of airway inflammation than cytologic examination of BAL in these dogs.