Reinhard Hirt

PCR-based genetic evidence for occurrence of Helicobacter pylori and novel Helicobacter species in the canine gastric mucosa

The canine gastric mucosa is known to be a habitat for various Helicobacter species. So far, five Helicobacter species have been described from the canine gastric mucosa, but histological studies have demonstrated a greater variety. In order to gain more information on diversity of canine gastric mucosa colonising helicobacters, biopsy samples of four pet dogs were examined by DNA-based techniques. PCR with a primer pair binding specifically to the 16S rDNA of the species of the genus Helicobacter and generating a fragment of approximately 400 bp indicated the presence of Helicobacter strains in the stomachs of the four dogs. PCR products were cloned into Escherichia coli DH10B and PCR-re-amplified 16S rDNA fragments were subjected to amplified ribosomal DNA restriction analysis (ARDRA) employing restriction enzyme HhaI. Restriction profiles indicated the presence of at least two different Helicobacter species in two dogs. Partial sequences of 16S rDNA of six clones were compared with sequences available in the EMBL data bank. Two sequences obtained from different dogs were identical with the corresponding sequences of Helicobacter pylori strains. Three sequences showed highest but moderate similarity values to H. pylori (96.6-98.0%) and one sequence to Helicobacter salomonis (97.3%). In contrast to previous reports our data implicate that the gastric mucosa of dogs may be colonised by strains of H. pylori or a very closely related species but they also confirm indications for the presence of so far uncultivated species of Helicobacter.

Classification of a Brevundimonas strain detectable after PCR with a Helicobacter-specific primer pair.

In a study for the isolation of new Helicobacter strains, biopsy samples were taken from the gastric mucosa of dogs and subjected to PCR amplification using a Helicobacter-specific primer pair (H276f and H676r, directed to the 16S rDNA) to identify members of this genus in the specimens. From one Helicobacter positive sample, a bacterial strain was isolated which displayed a characteristic band after PCR amplification with the Helicobacter-specific primer pair. The isolate designated H2/98-FUNDUS was motile, oxidase-, catalase- and aminopeptidase-positive and grew only under microaerophilic conditions at 37 degrees C. The bacterium was classified by a polyphasic approach, including analysis of the isoprenoid quinones, fatty acids, polar lipids and partial 16S rDNA sequence. Analysis of the 16S rDNA sequence (1003 bases) indicated that the strain H2/98-FUNDUS is a member of the genus Brevundimonas and most closely related to Brevundimonas aurantiaca DSM 4731T (99.5% sequence similarity). Isolate H2/98-FUNDUS contained a predominant ubiquinone Q-10 and a fatty acid profile with the major compounds C18:1 and C16:0. In the polar lipid extract, phosphatidylglycerol, six unknown phospholipids, one unknown phosphoglycolipid, two unknown glycolipids and two unknown aminolipids were detected. All these results indicate that H2/98-FUNDUS represents a new member of the genus Brevundimonas which gives a positive signal upon PCR employing the Helicobacter-specific primer pair.

Characteristics of canine nasal discharge related to intranasal diseases: a retrospective study of 105 cases

OBJECTIVES To compare characteristics of nasal discharge caused by different intranasal aetiologies in dogs. METHODS Medical records of 105 dogs with nasal discharge due to intranasal disease were retrospectively reviewed with special focus on composition, severity, duration and localisation of discharge. On the basis of diagnostic findings, cases were classified into different disease groups and characteristics of discharge were compared between groups. RESULTS Cases were classified as having non-specific rhinitis (n=42), nasal neoplasia (n=23), foreign bodies (n=21), nasal mycosis (n=7) and miscellaneous disorders (n=13). Dogs with foreign bodies or nasal mycosis were significantly younger. Mucous components of discharge occurred more often in non-specific rhinitis and nasal neoplasia, although haemorrhagic components predominated in nasal neoplasia when discharge lasted ê14 days. Pure or mixed haemorrhagic discharge was significantly more common with nasal neoplasia, foreign bodies and nasal mycosis. Purulent components were associated with longer duration of discharge and predominantly seen in non-specific rhinitis and foreign bodies. Dogs with foreign bodies were presented earlier and sneezing was more frequent. Nasal stridor was significantly more often observed in dogs with nasal neoplasia. CLINICAL SIGNIFICANCE Characteristics of nasal discharge and associated clinical signs might aid in planning the diagnostic approach, but a combination of diagnostic techniques is still required to confirm a diagnosis.

Evaluation of variables to describe the shape of volumetric capnography curves during bronchoconstriction in dogs

The aim of the study was to investigate changes in volumetric capnography (V(C)) variables during bronchoconstriction in dogs and compare it with total respiratory resistance (R(L)) measured with a Fleisch pneumotachograph. Six dogs were challenged with increasing concentrations of carbachol until obvious signs of bronchoconstriction were seen. All V(C) parameters were obtained before, directly after, 10 and 20 min after maximal bronchoconstriction. The slope of phase III (SIII) and airway and alveolar dead space parameters were significantly different from baseline directly after the challenge. The V(C) curve obtained a typical shape at the time of maximal bronchoconstriction and a trend to return to baseline shape was seen over time. A significant correlation was found for all aforementioned parameters with R(L). We conclude that the shape of the V(C) curve in combination with dead space calculation can be used to verify bronchoconstriction on a breath-to-breath basis.

Influence of the collection and oxygenation method on quantitative bacterial composition in bronchoalveolar lavage fluid samples from healthy dogs

The purpose of the study was to evaluate the effects on quantitative and qualitative microbial content of endoscopic bronchoalveolar lavage fluid (BALF) in healthy dogs using a laryngeal mask airway (LMA). It was hypothesised that oropharyngeal protection might prevent contamination of BALF with oropharyngeal microflora. Ten healthy Beagle dogs were randomly assigned to two groups and underwent endoscopic BAL on two occasions, either via an open unprotected oropharynx with oxygen supply provided via a nasal catheter (NT) or through a sterile LMA. For the second sampling, groups were switched. BALF analysis included quantitative microbial culture, nucleated cell counts and cytology. The mean (+/-SD) number of colony forming units (CFU)/mL found in the BALF using the LMA was 25,610+/-22,943 in the right lung (RL) and 22,510+/-18,779 in the left (LL). With the NT technique, the figures were 21,068+/-19,375 for the RL and 16,060+/-15,523 for the LL, respectively. Nucleated cell counts/microL were 691.0+/-181.6 (RL) and 734.0+/-171.6 (LL) for LMA, and 772.0+/-251.0 (RL) and 748+/-163.2 (LL) for NT. No significant differences were detected either in the number of CFU/mL or in the diversity of bacterial species with the two methods. A significant increase in BALF bacterial counts (with reduced species diversity) was observed on the second compared to the first sampling regardless of the method used. Protection of the oral cavity and oropharynx using an LMA had no significant influence on BALF bacterial counts. The findings suggest that with careful endoscope insertion, the risk of contamination of BALF by resident and transient oropharyngeal microflora can be negligible.

Inhaled budesonide therapy in cats with naturally occurring chronic bronchial disease (feline asthma and chronic bronchitis)

OBJECTIVES To describe the long term use of inhaled budesonide in cats with naturally occurring asthma and chronic bronchitis and to measure its effects. METHODS Owners of 43 cats diagnosed with asthma or chronic bronchitis, which had been prescribed 400 µg of inhaled budesonide twice daily, were contacted and information was retrieved by a questionnaire. Nineteen cats still receiving inhaled budesonide after more than 2 months were re-evaluated clinically and underwent barometric whole body plethysmography and adrenocorticotropic hormone-stimulation testing. RESULTS In 20 of the cats, therapy had been withdrawn by the owners. Cats (n=23) still receiving inhaled budesonide improved clinically and 19 cats that were reevaluated had significantly lower basal PENH (P=0·048) and higher PCPenh300 (P=0·049) values than before treatment. Corticosteroid-induced side effects were not observed in any cats but hypothalamic-pituitary-adrenal axis suppression was detected in 3 of 15 cases. CLINICAL SIGNIFICANCE Treatment with inhaled budesonide was well tolerated, resulting in improvement of clinical signs and barometric whole body plethysmography parameters. Although inhaled budesonide therapy was found to cause suppression of the hypothalamic-pituitary-adrenal axis in some cats, no cats showed clinical signs attributable to corticosteroid side effects.

Architecture and Inflammatory Cell Composition of the Feline Lung with Special Consideration of Eosinophil Counts

An increase in the number of eosinophils in bronchoalveolar lavage fluid (BALF) is a hallmark of feline asthma; however, a wide range in the percentage of eosinophils in BALF has been documented in healthy cats. In this study, BALF and lung tissue were collected from 15 cats without respiratory disease, BALF was taken from 15 cats with asthma and lung tissue was collected from six different asthmatic cats. Total nucleated cell count (TNCC) and inflammatory cell percentages were measured in BALF and lung tissue was evaluated microscopically. Asthmatic cats had a significantly higher eosinophil count in lung tissue, but BALF TNCC did not differ significantly between groups. Cats without respiratory signs had significantly more numerous macrophages and lymphocytes in BALF than asthmatics, but significantly lower percentages of eosinophils (4.2 ± 7.8% versus 49.4 ± 20.6%, P <0.001). In healthy feline airways a BALF eosinophil percentage of <5% can be expected. Dominant microscopical findings in feline asthma include high eosinophil counts, airway remodelling and inflammation. There is good correlation between the findings in BALF and tissue in feline asthma.

Volumetric capnography curves as lung function test to confirm bronchoconstriction after carbachol challenge in sedated dogs

This study investigated volumetric capnography (VC) in detecting airway responsiveness following airway challenge using carbachol in seven sedated dogs via face mask. Nebulised saline was administered, followed by increasing concentrations of nebulised carbachol until airflow limitation occurred (EP). Dead space (DS) variables and shape indices of the VC curve were calculated automatically after entering arterial carbon dioxide tension. Airway DS, airway DS to tidal volume (VT) ratio and the intercept of slope 2 of the VC curve decreased significantly at EP by 10%, 13% and 16%, respectively, minute ventilation, VT and alveolar DS increased significantly at EP by 49%, 22% and 200%, respectively. We conclude that VC and derived indices may be used to verify a reaction to airway challenge caused by carbachol in sedated dogs.

Out of Breath: Asthma in Humans and Their Animals

Asthma is a common chronic inflammatory disease of the airways characterized by reversible bronchoconstriction and airflow obstruction due to mucus hypersecretion, inflammatory infiltrates, and edema. Common symptoms are coughing, wheezing, and respiratory distress. The disease seems to be caused by a combination of genetic and environmental interactions. This short characterization of bronchial asthma does not only apply to humans but similarly to domestic animals, even though the pathophysiology as well as nomenclature may differ. Feline asthma (FA) is one of the most important syndromes out of a spectrum of chronic inflammatory airway diseases in cats. In dogs a syndrome termed eosinophilic bronchopneumopathy (EBP) exists, which shares some similarities with pulmonary eosinophilic syndromes in humans. Recurrent airway obstruction (RAO) or “heaves” is described as a naturally occurring asthma-like disease in horses. Therapeutic efforts in both human and animal patients target the underlying inflammatory response of the disease by administration of anti-inflammatory glucocorticoids. Bronchoconstriction can be prevented or eased by administration of anti-obstructive s2-sympathomimetics. The only curative treatment in allergic or IgE-mediated asthma is allergen-specific immunotherapy, which is readily applied in human asthmatic patients. In domestic animals, this therapeutic option is used only in experimental settings because successful identification of causative allergens is often difficult.

Immunophenotype of Peripheral Blood Lymphocytes in Dogs with Inflammatory Bowel Disease

Background Inflammatory bowel disease (IBD) is common in dogs. Despite the known importance of intestinal lymphocytes in its pathogenesis, little is known about the role of peripheral blood lymphocytes (PBLs) in IBD. Objectives The aims of this study were (1) comparison of PBLs analyzed by flow cytometry (FCM) in IBD dogs and healthy controls and (2) comparison of PBLs in IBD dogs at the time of diagnosis and in dogs in clinical remission. Animals Whole blood samples of 19 IBD dogs at the time of diagnosis and blood samples of 6 dogs in clinical remission were collected. Ten healthy dogs served as controls. Methods In this prospective observational study, PBLs were analyzed with multicolor FCM by staining with a panel of anticanine and cross‐reactive monoclonal antibodies against T‐ and B‐cell differentiation antigens, including CD45, CD3, CD4, CD8α, CD8β, TCRαβ, TCRγδ, CD79αcy, and CD21. Results The IBD patients’ PBLs had significantly decreased percentages of TCRγδ+ T lymphocytes (median: healthy dogs, 3.32; IBD dogs, 0.97; P = 0.03) and CD21+ B cells (median: healthy dogs, 27.61; IBD dogs, 17.26; P = 0.04). There were no significant differences in PBLs between pretreatment and follow‐up samples. Conclusions and Clinical Importance The differences between PBLs in healthy and IBD dogs analyzed by FCM indicate an imbalance of lymphocytes with different immunologic functions and emphasize the potential value of this technique in a larger cohort of dogs. The PBLs did not differ between IBD dogs before treatment and clinically well‐controlled dogs after treatment.