Transmission of canine gastric Helicobacter salomonis infection from dam to offspring and between puppies.

H. bizzozeronii CCUG 35045, a new canine gastric Helicobacter spp. was used for experimental infection of four weaned puppies at 7 weeks of age. Controls were four nonchallenged puppies. The puppies originated from two dams which had Helicobacter salomonis infection in biopsy samples taken 3 weeks before the delivery but which had urease, brush cytology and culture-negative biopsy samples taken 7 weeks after antimicrobial treatment (metronidazole, amoxicillin, bismuth subcitrate). Both dams were detected urease- and Helicobacter-positive again three and a half months after therapy. Dam B was shown to be colonised with the similar genotype of H. salomonis for more than 2 years. Unexpectedly, H. salomonis was also cultured from gastric biopsy samples of the nonchallenged puppies three times during 7 months. When H. salomonis isolates of dams and puppies were studied by ribotyping (HaeIII, ClaI or PstI) they were shown to be identical although the HaeIII and PstI REA patterns of dam A differed from the patterns of dam B and nonchallenged group by one fragment. PFGE pattern analysis of NotI digests, however, revealed that the isolates of the puppies were identical with the isolates of dam B, and differed from the isolates of dam A. The isolates of the dams and puppies in the nonchallenged group were metronidazole-resistant. The antimicrobial therapy had merely suppressed, but not eradicated, the infection from dams. These studies suggested that puppies may acquire gastric Helicobacter infection from dams during the lactation period and puppies can infect each other during their early life. PFGE pattern analysis was shown to be a more distinguishing method than ribotyping to study the similarity of the isolates.

Comparison of diagnostic methods for detecting gastric Helicobacter-like organisms in dogs and cats.

Diagnostic methods for detecting gastric Helicobacter-like organisms (GHLOs) in dogs and cats were compared. Samples for brush cytology, the urease test and histological examination were collected post mortem from the fundus, corpus and antrum of 10 dogs (17 sample sites from each animal) and 10 cats (14 sample sites each). Samples of tissue from the fundus or corpus were taken for transmission electron microscopy and culture from three and eight dogs, respectively, and from six cats that gave a positive urease test with samples from these regions. In all dogs and in six of the 10 cats, GHLOs were detected by at least one of three methods (brush cytology, urease test or histological examination) in all regions. By brush cytology, GHLOs were demonstrated in all samples from the dogs and the positive cats. In cats, the urease test (60 min) gave a positive result in every sample site; in dogs it gave a positive result in 100% of the corpus samples, in 95% of the fundus samples and in 62% of the antral samples. Histological examination revealed GHLOs in all samples from the fundus and corpus of the dogs and of the positive cats; and in 74% and 91.7% of the antral samples of the dogs and cats, respectively. GHLOs were seen in all dogs and cats studied by transmission electron microscopy, and culture of gastric tissue was successful in 3/8 dogs and 1/6 cats. In this study, brush cytology was thus the most sensitive method for demonstrating GHLOs.

Lung injury leading to respiratory distress syndrome in young Dalmatian dogs.

A progressive pulmonary disease resulting in severe respiratory failure and death in an average of 3 weeks was diagnosed in 11 young Dalmatian dogs. The dogs were from 4 litters, all genetically related by a common ancestor. The initial clinical signs were tachypnea and noisy respiration. Respiratory distress developed shortly before death and was characterized by strenuous and rapid respirations, along with cyanosis and vomiting. On blood gas analysis, there were severe arterial hypoxemia, hypercapnia, and marked alveolar-arterial oxygen difference. Radiographically, a diffuse pattern of alveolar, interstitial, and peribronchial densities was observed in the lungs. Most dogs developed pneumomediastinum and gastroesophageal intussusception in the terminal phase of the disease. There was no response to treatment with antibiotics, corticosteroids, diuretics, or oxygen. At necropsy, the lungs were wet, heavy, and relatively airless. Absence of 1 kidney in 2 dogs and severe internal hydrocephalus in 2 dogs were additional necropsy findings. Pulmonary histopathology included metaplasia and atypia of the alveolar and bronchiolar epithelium, a nonpurulent inflammatory reaction characterized mainly by mononuclear cells and macrophages, eosinophilic hyaline membrane formation, and focal pulmonary fibrosis. The histological manifestations were typical of acute lung injury. Clinically, the findings were consistent with adult respiratory distress syndrome (ARDS), except for the relatively long course. No known risk factors for ARDS, such as trauma, toxin exposure, infection, or endotoxemia could be identified. The relationship of the other abnormalities (ie, renal aplasia, hydrocephalus) to the pulmonary disease also remains obscure. An inherited defect is suspected, because segregation analysis of the 4 litters suggests autosomal recessive inheritance.

Detection and effects of helicobacters in healthy dogs and dogs with signs of gastritis.

OBJECTIVES: To determine prevalence, colonization density, and distribution of helicobacters and gastric histologic findings in healthy dogs and dogs with signs of gastritis; to evaluate association of colonization density and gastric inflammation; and to compare the number of Helicobacter spp with degree of inflammation. DESIGN: Cross-sectional prevalence survey. ANIMALS: 25 healthy dogs and 21 dogs with signs of gastritis. PROCEDURE: During endoscopy, gastric mucosal biopsy specimens were obtained from healthy and affected client-owned dogs. Histologic and cytologic evaluation and results of a urease test were used for detecting helicobacters, which were identified definitively by use of transmission electron microscopy and bacterial culture. RESULTS: Helicobacters were detected in all 25 healthy and 20 of 21 affected dogs. Cytologic examination was a more sensitive method than histologic examination or the urease test. Helicobacters were found least frequently and in fewest number in the antrum in both groups of dogs. Gastric inflammation was evident in both groups of dogs and did not differ significantly between groups. A significant association was not detected between colonization density or the number of Helicobacter spp and degree of gastric inflammation. In both groups, H bizzozeronii, H felis, and H salomonis were cultured. CLINICAL IMPLICATIONS: Histologically verified chronic gastritis is common in dogs with signs of gastritis as well as in healthy dogs. Colonization density of helicobacters was not associated with degree of gastric inflammation in the dogs of our study. It remains to be determined whether certain strains of Helicobacter spp can induce gastritis in dogs.

Effect of triple therapy on eradication of canine gastric helicobacters and gastric disease.

Nine helicobacter-positive pet dogs with upper gastrointestinal signs were studied to evaluate the effect of a triple therapy, normally applied to humans for the eradication of gastric helicobacters, on clinical signs and gastric histology, as well as the recurrence of helicobacters after eradication in an extended follow-up in four dogs. Endoscopy was performed at entry to the study and repeated after eradication therapies and additional treatments. If the triple therapy (amoxycillin, metronidazole and bismuth subcitrate) failed, tetracycline and omeprazole were prescribed. Additional therapies were instituted if clinical signs persisted after eradication therapies. Helicobacter status was verified from gastric biopsy specimens by the urease test and histological examination, and in a few dogs also by brush cytology. Triple therapy eradicated gastric helicobacters in 7/9 dogs; gastric helicobacters were also eradicated in one dog treated with tetracycline and omeprazole. Eradication of helicobacters resulted in significant improvement, but not total resolution, of clinical signs. Subsequent additional therapies resulted in further alleviation of clinical signs. Neither triple therapy nor additional therapies had a significant effect on gastric histological changes. Gastric helicobacters recurred in 4/4 dogs within three years of the eradication treatment. Because canine gastric helicobacters alone were not definitively shown to induce clinical signs, routine eradication therapy seems not to be warranted at present.

Prolonged-release hydroxypropyl methylcellulose matrix tablets of furosemide for administration to dogs.

Furosemide is a problematic drug in a prolonged-release product because its absorption is site specific, taking place mainly in the upper parts of the alimentary tract. The aim of the study reported here was to develop prolonged-release furosemide formulations for dogs. The type of preparation selected was a hydroxypropyl methylcellulose (HPMC) matrix tablet. Evaluation was based on dissolution studies, on in vivo disintegration studies in the canine stomach and on bioavailability studies in Beagle dogs. The variables tested were the viscosity grade of the polymer, the amount of polymer and presence or absence of an alkaline compound (potassium carbonate) in the formulation. When potassium carbonate was included, furosemide was absorbed so slowly that drug administration once daily would give plateau drug plasma concentrations, even though the elimination half-life of furosemide is only about one hour. In vitro dissolution tests gave a wrong indication of the in vivo behaviour of the products. Thus, in vivo studies are important from the very beginning in the development of new drug products for dogs.

Culture and characteristics of Helicobacter bizzozeronii, a new canine gastric Helicobacter sp.

Organisms whose cells were large, tight spirals were isolated from gastric biopsies of dogs. Touch cytology samples from all of the dogs contained large spiral organisms. Characteristics of 10 strains are described. These organisms were 5 to 10 microns long by 0.3 microns wide, and each cell had 10 to 20 sheathed flagella at both ends of the cell. The cells did not have periplasmic fibrils. These organisms were microaerophilic and grew at 37 and 42 degrees C but not at 25 degrees C on brain heart infusion agar containing blood. They did not grow on brucella blood agar. They were catalase and oxidase positive, hydrolyzed urea but not hippurate, reduced nitrate, and were resistant to nalidixic acid but susceptible to cephalothin and metronidazole. In contrast to Helicobacter felis, they hydrolyzed indoxyl acetate. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of all of the strains were similar, and the protein patterns of these organisms differed from those of other Helicobacter spp. Dot blot DNA-DNA hybridization experiments revealed that the new strains were closely related to each other but clearly different from H. felis, Helicobacter pylori, Helicobacter mustelae, and Campylobacter jejuni. The name Helicobacter bizzozeronii sp. nov. is proposed for these organisms. Our results suggest that other "uncultured" gastric helicobacters may be cultured if optimal culture conditions are found.

Helicobacter salomonis sp. nov., a canine gastric Helicobacter sp. related to Helicobacter felis and Helicobacter bizzozeronii.

During a study of the prevalence and distribution of gastric helicobacters in domestic pets, a novel group of Helicobacter-like organisms were identified. These "Helicobacter group 2" strains were initially distinguished from the species Helicobacter felis and Helicobacter bizzozeronii by their cellular morphology and the type of motility exhibited. Bacterial cells were only slightly spiral, 5 to 7 microns long, and 0.8 to 1.2 microns wide and showed an unusual slow wavelike motion. Each cell had tufts of sheathed flagella at one or both ends. Phylogenetic analysis by 16S ribosomal DNA sequence comparison revealed that H. felis, H. bizzozeronii, "Gastrospirillum hominis" 2, and the new group of helicobacters formed a distinct cluster with intraspecies similarity values of more than 98%. These taxa were clearly separated from all other known Helicobacter species. Dot blot DNA-DNA hybridization studies indicated that the Helicobacter group 2 strains are genetically homogeneous and distinct from other canine and feline gastric helicobacters. Quantitative DNA-DNA hybridization experiments showed that Helicobacter group 2 strains exhibit > 90% DNA homology to each other, but < 39% homology to the phylogenetically related taxa H. felis and H. bizzozeronii. We propose the name Helicobacter salomonis for the novel Helicobacter group 2 strains. The type strain is H. salomonis Inkinen (= CCUG 37845).

Prolonged-release hard gelatin capsules of furosemide for the treatment of dogs.

The object of this study was to examine whether prolonged-release hard gelatin capsule formulations could be developed for dogs. Different viscosity grades of hydroxypropyl methylcellulose (HPMC) and sodium carboxymethylcellulose (NaCMC) were used to control drug release. Furosemide was chosen because of its wide use in the management of heart failure in dogs. In vitro, selecting different viscosity grades allowed good control of drug release, whereas in vivo the difference between formulations was clearly smaller. Although all formulations gave prolonged release, both inter- and intra-individual variation in the plasma concentration-time curves was high. It is difficult to develop prolonged-release formulations for drugs such as furosemide with highly variable pharmacokinetic properties. However, hard gelatin capsules containing hydrophilic polymers could still be a suitable choice for some drugs.

Occurrence and topographical mapping of gastric Helicobacter-like organisms and their association with histological changes in apparently healthy dogs and cats.

The occurrence and topographical mapping of the gastric Helicobacter-like organisms (GHLOs) and their association with histological changes were studied in apparently healthy dogs and cats. Multiple samples were collected for histological examination from the fundus, corpus and antrum of the stomach of 10 dogs and 10 cats. Fundus and corpus were also sampled for transmission electron microscopy (three dogs, six cats), and for culture (eight dogs, six cats). In all dogs, GHLOs were detected in the fundus and corpus, and in the antrum of nine dogs, and significantly more often in the fundus and corpus (in all sample sites examined) than the antrum (P < 0.01). In cats, GHLOs were demonstrated in 6/10 individuals, and in all regions and sample sites. In dogs GHLOs were detected in all sample sites of the fundus and corpus. Lymphocytes, plasma cells and lymphocyte aggregates were found in all dogs in all regions; there were significantly more plasma cells in the antrum than in the corpus (P < 0.05). Neutrophils were found in six dogs, and eosinophils in seven dogs. In cats, lymphocyte aggregates were found only in GHLO-positive cats, which also had more lymphocytes in the fundus and corpus than GHLO-negative ones (P < 0.5). In dogs, no statistically significant association was found between the number of GHLOs and inflammatory parameters. Four dogs showed histological changes comparable to mild chronic gastritis and another six dogs to mild active chronic gastritis. Mild chronic gastritis was found in the antrum of all cats, and it occurred significantly more often in the antrum than in other regions (P < 0.01). In cats, there was a statistically significant association between GHLOs and chronic gastritis in the fundus and corpus (P < 0.05). GHLOs resembling human 'Helicobacter heilmannii' were identified in all the dogs and cats studied by electron microscopy, and Helicobacter felis in one dog in addition. Culture was successful in three dogs and one cat; 'H, heilmannii' was identified in two of the dogs, and H. felis in the third dog and the cat. GHLOs were found to be common in apparently health dogs and cats. Based on the results of this study, one sample from the fundus and corpus is enough to demonstrate GHLOs. In cats, GHLOs may cause histological changes comparable to chronic gastritis, but in dogs this association remain unclear. It is also questionable if the histological criteria for human gastritis, used in the present study, are suitable for dogs and cats.

Isolation and identification of Helicobacter spp. from canine and feline gastric mucosa.

It is known that virtually all healthy adult dogs and cats harbor spiral helicobacters in their gastric mucosa. Three species, Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis have been isolated in vitro from the gastric mucosa of these animals. The aims of this study were to evaluate the efficacy of an isolation method for canine and feline gastric helicobacters that has been developed at the University of Helsinki; to estimate the prevalence and distribution of these taxa in the samples examined; and to assess the efficacy and validity of an extensive set of standardized conventional phenotypic tests, whole-cell protein profiling, and ultrastructural analysis in identifying the different species isolated from canine and feline gastric mucosa. We cultured 95 and 22 gastric mucosal biopsies from dogs and cats, respectively. Twenty-one H. bizzozeronii strains, 8 H. felis strains, 8 H. salomonis strains, 3 mixed cultures, 2 "Flexispira rappini"-like organisms, and 3 as yet uncharacterized strains were isolated from the dogs, and 3 H. felis strains were isolated from the cats. The methods used here yielded Helicobacter isolation rates of 51% from dogs and 13.6% from cats, which exceed those reported previously. The main difficulties were primary isolation, mixed cultures, and identification to the species level. In the species identification, a detailed morphological examination was found to yield important phenotypic characteristics. A large panel of biochemical and tolerance tests did not clearly differentiate the closely related species H. bizzozeronii, H. felis, and H. salomonis. Highly standardized whole-cell protein profiling was shown to be an excellent method for species identification. Improvements in culture conditions for these bacteria are still needed, especially for cats. A genetic identification method not requiring culture is needed for future studies of these very fastidious helicobacters, as the clinical significance and ecology of these species within the gastric mucosa of the domestic carnivores remain largely unknown.