Helicobacter spp. infection in cats: evaluation of the humoral immune response and prevalence of gastric Helicobacter spp

The principal aims of this study were to evaluate the humoral immune response (IgG) of cats with gastric Helicobacter spp. infection, and to determine the prevalence of different types of Helicobacter spp. in the stomachs of cats. The Helicobacter infection status of 45 cats (12 healthy spay/neuter cats, 9 sick cats, 24 colony cats) was determined by evaluating endoscopic gastric biopsies for urease activity, presence of Helicobacter-like organisms (HLO) on histopathology, and genus and species-specific PCR. Serum samples were evaluated with a kinetic enzyme linked immunosorbent assay (ELISA) utilizing the high molecular cell-associated protein (HM-CAP) fraction of H. felis ATCC 49179.Seventeen of 45 cats were infected with Helicobacter spp.: "H. heilmannii" 9/17, H. felis 4/17, mixed "H. heilmannii" and H. felis 3/17, unclassified-Helicobacter spp. 7/17. H. pylori was not detected in any cat. Kinetic ELISA results were significantly higher for infected cats, than for uninfected cats. Cats infected with different Helicobacter spp. showed similar distribution of OD/min values. There were no effects of age or clinical signs on the results of kinetic ELISA. No correlation between colonization density and seroconversion was observed. There were statistically significant, but weak correlations between the degree of seroconversion and the degree of inflammation, and the number of lymphoid follicles. Infected cats had more severe inflammation in the pylorus and fundus than uninfected cats. Infected sick cats had a higher degree of pyloric, but not fundic inflammation, than healthy infected cats and uninfected sick cats.The results indicate that naturally acquired infection with gastric Helicobacter spp. is associated with seroconversion (IgG) in cats. The similar ELISA values in cats infected with a variety of Helicobacter spp. suggests substantial antigenic homology between different Helicobacter spp. The higher degree of inflammation in infected than uninfected cats, supports a role for Helicobacter as a cause of gastritis in cats.     

Histological and immunohistochemical detection of different Helicobacter species in the gastric mucosa of cats.

Detailed histopathological evaluation of the gastric mucosa of Helicobacter-infected cats is complicated by the difficulty of recognizing Helicobacter organisms on hematoxylin and eosin (HE)-stained sections and the ability of multiple Helicobacter species to infect cats. In this study, the presence and localization of different species of Helicobacter in the stomachs of cats was investigated using silver staining and immunohistochemistry. Five groups containing 5 cats each were established (group 1: urease negative and Helicobacter free; groups 2, 3, 4, and 5: urease positive and infected with Helicobacter heilmannii, unclassified Helicobacter spp., Helicobacter felis, and Helicobacter pylori, respectively). Gastric samples were evaluated by HE and silver staining and by immunohistochemistry with 3 different anti-Helicobacter primary antibodies. Helicobacter were detected by Steiner stain in all infected cats at the mucosal surface, in the lumen of gastric glands, and in the cytoplasm of parietal cells. In silver-stained sections, H. pylori was easily differentiated from H. felis, H. heilmannii, and unclassified Helicobacter spp., which were larger and more tightly coiled. No organisms were seen in uninfected cats. Helicobacter antigen paralleled the distribution of organisms observed in Steiner-stained sections for 2 of the 3 primary antibodies tested. The antisera were not able to discriminate between the different Helicobacter species examined. A small amount of Helicobacter antigen was present in the lamina propria of 3 H. pylori-, 3 H. felis-, and 1 H. heilmannii-infected cat. Minimal mononuclear inflammation was present in uninfected cats and in those infected with unclassified Helicobacter spp. and H. heilmannii cats. In H. felis-infected cats, lymphoid follicular hyperplasia with mild pangastric mononuclear inflammation and eosinophilic infiltrates were present. The H. pylori-infected cats had severe lymphoid follicular hyperplasia and mild to moderate mononuclear inflammation accompanied by the presence of neutrophils and eosinophils. These findings indicate that Steiner staining and immunohistochemistry are useful for detecting Helicobacter infections, particularly when different Helicobacter species can be present. Monoclonal antibodies specific for the different Helicobacter species could be important diagnostic aids. There appear to be differences in the severity of gastritis in cats infected with different Helicobacter species.     

Detection of Helicobacter spp. DNA in the oral cavity of dogs

The mode of acquisition of gastric Helicobacter spp. infection in dogs has not been determined. It is suspected that oral-oral and faecal-oral transmission may be involved. The present study sought to determine if Helicobacter spp. DNA is present in the oral cavity of healthy and vomiting dogs. Thirty-eight pet dogs (27 vomiting and 11 clinically healthy) were studied. The presence of Helicobacter spp. was determined by single and nested PCR evaluation of DNA extracted from saliva, dental plaque and gastric biopsy samples. Helicobacter spp. DNA was detected by nested PCR in 36 (94.7%) gastric biopsies, 17 (44.7%) dental plaque and 19 (50%) saliva samples out of the 38 dogs examined. Overall 27 (71.1%) dogs screened by nested PCR were found to harbour Helicobacter spp. DNA in the oral cavity (dental plaque and/or saliva). There was no significant difference in the prevalence of Helicobacter spp. DNA in the oral cavity of vomiting and healthy dogs, and the time from vomiting to oral sampling did not have significant impact. This study confirms the high prevalence of gastric Helicobacter spp. infection in dogs, and reveals that Helicobacter spp. DNA is detectable in the oral cavity of over 70% of dogs. These findings support the possibility of oral-oral transmission between dogs and that the canine oral cavity may act as source of non-pylori Helicobacter spp. infection for humans.     

Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii-like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.     

Occurrence and species level diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in healthy and diarrheic dogs and cats

In order to study the occurrence and co-infection of different species of Campylobacter, enteric Helicobacter and Anaerobiospirillum in dogs and cats and define a possible association between these microrganisms and gastrointestinal disorders, 190 dogs and 84 cats, either healthy or with diarrhea, were sampled between 2002 and 2003. Thirty-three C. upsaliensis, 17 C. jejuni, 2 C. helveticus, 1 C. lari isolates from dogs and 14 C. helveticus, 7 C. jejuni, 6 C. upsaliensis isolates from cats were identified using species-specific PCR and phenotypic tests. Whole cell protein profile analysis, phenotypic tests, PCR-RFLP of gyrB and a phylogenetic study of partial groEL and 16S rRNA sequences were used to identify 37 H. bilis, 22 H. canis and 14 H. cinaedi in dogs and 12 H. canis, 5 H. bilis and 2 H. cinaedi in cats. Whole cell protein profile analysis, phenotypic tests and species-specific PCR of 16S rRNA were used to identify 14 A. succiniciproducens, 12 A. thomasii isolates and one unidentified Anaerobiospirillum sp. isolate in dogs and 3 A. thomasii isolates in cats. Fifty-two animals (19%) were positive for the isolation of more than one genus. No significant statistical correlation was found between any isolates of Campylobacter, Helicobacter or Anaerobiospirillum spp. or the various co-infection rates, and the presence of diarrhea in either dogs or cats. Campylobacter isolates were also tested for antibiotic resistance using the agar dilution method.     

Gastric function in dogs with naturally acquired gastric Helicobacter spp. infection

The association of Helicobacter pylori with gastritis, peptic ulcers, and gastric neoplasia has led to fundamental changes in the understanding of gastric disease in humans. The relationship of Helicobacter spp. infection to gastric disease in dogs is unclear. The objective of this study was to determine if Helicobacter infection affects the gastric secretory axis of dogs. Eight Beagle dogs with naturally acquired Helicobacter spp. infection were studied before and after (4 and 29 days) the attempted eradication of Helicobacter spp. with a combination of amoxicillin, metronidazole, and famotidine (AMF). Six specific-pathogen-free, Helicobacter-free Beagle dogs served as controls. The electron microscopic appearance of spiral organisms in infected dogs indicated coinfection with Helicobacter felis- and H bizzozeronii-like organisms. Unstimulated gastric pH and fasting, postprandial, and bombesin-stimulated plasma gastrin were similar in both infected and uninfected dogs, although a trend (P = .09) toward higher meal-stimulated gastrin was observed in infected dogs at 60 minutes. Pentagastrin-stimulated maximal acid output (mmol HCI/kg0.75/hour) and titratable acidity (mmol HCl/mL) were similar in both infected and uninfected dogs, but gastric pH during maximal acid output was lower (P < .01) in uninfected dogs. Mild gastric inflammation was present in both infected and uninfected dogs. Gastric spiral organisms were undetectable in 6/8 infected dogs 4 days after AMF but had recurred in 8/8 dogs 29 days after AMF. Analysis of gastric DNA with Helicobacter-specific primers indicated persistence of Helicobacter DNA at 4 and 29 days after antibiotic therapy. Acid secretion, plasma gastrin, and mucosal inflammation were not affected by the transient suppression of Helicobacter spp. by AMF. These findings suggest that gastric secretory function in dogs is not markedly perturbed by naturally acquired Helicobacter spp. infection and that treatment with amoxicillin, metronidazole, and famotidine causes suppression rather than eradication of gastric Helicobacter spp. in dogs.     

Detection of Helicobacter spp. in gastric, fecal and saliva samples from swine affected by gastric ulceration

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.     

Malassezia spp. overgrowth in allergic cats

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.     

Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study

ABSTRACT: In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs.     

Giardia duodenalis in colony stray cats from Italy

Giardia duodenalis is the most common intestinal protozoan in humans and animals worldwide, including eight morphologically identical assemblages, infecting pets, livestock, wildlife and human beings. Assemblages A and B are those with the higher zoonotic potential, and they have been detected in several mammals other than humans; the others (C to H) show a higher host specificity. Cats can harbour both the specific Assemblage F and the zoonotic ones A and B. Several studies have been carried out on G. duodenalis genotypes in cats; however, the role of this species in the epidemiology of giardiasis is still poorly understood. In this scenario, the present study carried out the detection and genetic characterization at sub-assemblage level of G. duodenalis from colony stray cats in central Italy. In the period 2018–2019, 133 cat faecal samples were analysed for the presence of G. duodenalis cysts by a direct immunofluorescence assay. Positive samples were subsequently subjected to molecular analyses for assemblage/sub-assemblage identification. Forty-seven samples (35.3%) were positive for G. duodenalis cysts by immunofluorescence. G. duodenalis DNA was amplified at SSU-rDNA locus from 39 isolates: 37 were positive for zoonotic Assemblage A and 2 showed a mixed infection (A + B). Positive results for the β-giardin gene were achieved for 25 isolates. Sequence analysis revealed 16 isolates belonging to Sub-assemblage AII and 8 to Sub-assemblage AIII. One isolate resulted as ambiguous AI/AIII. Large sequence variability at the sub-assemblage level was detected, with several double peaks and mutations, making complex a proper isolate allocation. When compared with previous studies, the 35.3% prevalence of G. duodenalis in cats reported in the present article was surprisingly high. Moreover, all positive cats resulted to be infected with zoonotic assemblages/sub-assemblages, thus indicating stray cats as a possible source of human giardiasis and highlighting the sanitary relevance of cat colonies in the study area.     

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.     

Radiographic measurement of vertebral heart size in healthy stray cats

The aims of this study were to determine vertebral heart size (VHS) in stray cats and to compare different radiographic views. This study was performed on 50 adult stray cats. All cats were short-haired and non-obese and were considered to be healthy based on physical examination and electrocardiography. Left and right lateral, dorsoventral and ventrodorsal radiographs were taken. The long and short axes of the heart were measured in millimetres. The thoracic vertebral length spanned by each dimension was measured caudally from the fourth thoracic vertebra. Mean+/-SD and the correlation coefficient between the measurements were calculated with standard statistical software. The sum of the long and short axes of the heart expressed as VHS was 7.3+/-0.49 vertebrae in right lateral, 7.3+/-0.55 vertebrae in left lateral, 7.5+/-0.68 vertebrae in dorsoventral and 7.5+/-0.53 vertebrae in ventrodorsal. The differences between right and left lateral as well as dorsoventral and ventrodorsal views were not significant (P>0.05). Absolute measurements and vertebral heart scale values were slightly smaller than those reported in the literature for mixed population of cats. It is, therefore, important to take the breed in to account.     

In situ detection of urease-positive Helicobacter pylori-like organisms on swine gastric mucosa

The objective of this study was to improve the visual localization of urease activity of Helicobacter pylori-like organisms (HPLO) on swine gastric mucosa by in vitro optimization of the urea concentration and pH indicator of a urease test reagent. Five 21-day-old conventional pigs were infected orally with HPLO (3 pigs) or Brucella broth alone (2 pigs). At 17 d after infection the pigs were euthanized and their stomachs excised and tested for HPLO by a modified urease test formulation sprayed onto the gastric mucosa, as well as confirmatory culture and isolation of HPLO from urease-positive sites. This study showed improved detection of HPLO in porcine gastric mucosa with the use of a modified urease test formulation containing 5% urea and the pH indicator bromocresol purple compared with the use of a conventional formulation of 2% urea and phenol red. This test can readily be applied to achieve a presumptive diagnosis of HPLO in cases of gastritis or gastric esophageal ulceration in pigs.     

Helicobacter spp. from gastric biopsies of stranded South American fur seals (Arctocephalus australis)

Gastrointestinal lesions with uncertain etiology have been widely described among pinnipeds. The aim of our study was to investigate the presence of Helicobacter spp. in the gastric mucosa of South American fur seals (Arctocephalusaustralis). Gastric biopsies from thirteen seals, stranded on the shores of the Southwestern Atlantic Ocean in Argentina, were evaluated for the presence of Helicobacter spp. by PCR and DNA sequence analysis. Six gastric biopsies were positive for Helicobacter spp. Pairwise sequence comparisons showed less than 95% identity to novel Helicobacter spp. described from pinnipeds from North America and Australia. However, phylogenetic analysis revealed that the South American fur seal sequences clustered with 99-100% homology with H. cetorum, a species isolated from dolphins and whales. The presence of H. cetorum in pinnipeds, if confirmed by its isolation from the gastric mucosa of these mammals, demonstrates the wide host range of this bacterium in the marine environment.     

Antibodies to Neospora caninum in stray cats from north Italy

Antibodies to Neospora caninum were determined in serum samples from 282 stray cats coming from four colonies near Turin (north-west Italy). Sera were tested using a Neospora Agglutination Test (NAT). Seroprevalence was 24.8% at 1:80, 12.8% at 1:160 and 5.3% at 1:320 dilution. Seroprevalence in females and males, in different colonies and in different age classes, did not differ significantly. Our results confirm that domestic cats are exposed to N. caninum and the observed seroprevalences suggest that risk of exposure is high and N. caninum should be considered in differential diagnosis in cats with neurological signs.