Diagnosis of Helicobacter spp. infection in canine stomach

A total of 75 biopsied samples of cardia, fundus, body, and pyloric antrum from necropsied dogs that were submitted to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University from April 2003 to June 2004 were investigated. The objectives of this study were to determine the prevalence of Helicobacter spp. in canine stomach by polymerase chain reaction (PCR) in comparison to histochemistry versus immunohistochemistry (IHC), and to correlate these diagnostic methods with the clinical significance in infected dogs. Histopathological results revealed 60.0% (45/75) of samples to be positive, and consisted of mild gastritis in 64.44% (29/45), moderate gastritis in 11.11% (5/45), and severe gastritis in 24.44% (11/45). The proportion showing no histopathological lesions was 40.0% (30/75). Helicobacter spp. were localized to the luminal crypt in 18.67% (14/75), gastric pit in 22.67% (17/75), gastric gland in 21.33% (16/75), and gastric epithelium in 8% (6/75). The percentages of positive samples of Helicobacter spp. diagnosed by hematoxylin and eosin stain (H&E), Warthin Starry stain (WSS), IHC with rabbit polyclonal anti-H. pylori antibody, and PCR were 17.3% (13/75), 46.7% (35/75), 30.7% (23/75), and 10.7% (8/75), respectively. No significant differences weree observed in histopathological changes in portions of the stomach (p>0.05). The diagnosis of Helicobacter spp. by PCR in comparison to that by WSS and IHC was not significantly different (p>0.05). There were no relationships between pathological studies using H&E, WSS, and IHC, and especially between PCR and clinical signs of Helicobacter spp. infections in canine stomachs (p>0.05). The present study revealed significantly different levels of correlation for Helicobacter spp. detection between H&E and WSS (p<0.001). Results indicate that the method of choice for diagnosis of Helicobacter spp. infection in canine stomach is dependent on the purpose of study and appropriate specimen collection.     

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.     

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.     

Identification of Helicobacter DNA in feline pancreas,liver, stomach,and duodenum: Comparison between findings in fresh and formalin-fixed paraffin-embedded tissue samples

The clinical significance of Helicobacter spp. in feline digestive organs needs to be evaluated and formalin-fixed and paraffin-embedded (FFPE) tissue samples provide an invaluable source for molecular studies. In this study, we performed a PCR assay to investigate the presence of Helicobacter DNA in digestive organs from seven cats and compared this occurrence in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue samples from the same organs. The present study identified Helicobacter DNA in the pancreas, liver, stomach, and duodenum in fresh tissue samples but only in the stomach in FFPE samples. To our knowledge this is the first time that Helicobacter DNA have been identified in the feline pancreas. This study indicates that it is important to be aware of differences between results when analyzing FFPE samples compared to fresh tissue samples, especially regarding longer DNA fragments (>200 bp (base pairs)).     

Identification of Helicobacter spp. in oral secretions vs. gastric mucosa of stray cats

The definite mode of transmission of Helicobacter infection is largely unknown. This study was carried out primarily, to determine the existence of Helicobacter spp. in the oral secretions of stray cats as one of the possible routes of transmission and secondly, to evaluate the accordance between oral and gastric colonization of Helicobacter spp. in these cats. Forty-three adult stray cats were thus studied for the presence of Helicobacter species by quantitative rapid urease test (RUT), cytology and PCR. Helicobacter spp. were found in the oral secretions and gastric biopsies of 93% and 67.5% of the stray cats, respectively. There was not, however, any agreement observed between Helicobacter colonization at these two locations, at neither genus nor species level. These findings suggest that the oral cavity is routinely exposed to transient forms of bacteria and may temporarily harbor Helicobacter spp. Thus, oral cavity as a source of Helicobacter spp. may act as a reservoir for transmission and may not necessarily reflect the colonization status of the gastric mucosa.     

Identification of Helicobacter DNA in feline pancreas, liver, stomach, and duodenum: comparison between findings in fresh and formalin-fixed paraffin-embedded tissue samples

The clinical significance of Helicobacter spp. in feline digestive organs needs to be evaluated and formalin-fixed and paraffin-embedded (FFPE) tissue samples provide an invaluable source for molecular studies. In this study, we performed a PCR assay to investigate the presence of Helicobacter DNA in digestive organs from seven cats and compared this occurrence in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue samples from the same organs. The present study identified Helicobacter DNA in the pancreas, liver, stomach, and duodenum in fresh tissue samples but only in the stomach in FFPE samples. To our knowledge this is the first time that Helicobacter DNA have been identified in the feline pancreas. This study indicates that it is important to be aware of differences between results when analyzing FFPE samples compared to fresh tissue samples, especially regarding longer DNA fragments (>200 bp (base pairs)).     

Gastric carcinoma in canines and humans,a review

Gastric carcinoma (GC) is the most common neoplasm in the stomach of dogs. Although incidence in the general population is reported to be low, breed‐specific GC has a high incidence. Median age at presentation ranges from 8 to approximately 10 years. The disease is mostly located in the lesser curvature and antropyloric region of the stomach. Unfortunately, diagnosis is usually made when the disease is at an advanced stage and, therefore, prognosis is poor. Due to similarities in clinical presentation, diagnosis, histology and prognosis, canine GC may serve as a valuable model for human GC. Extensive pedigrees of canine gastric carcinoma cases could reveal insights for human gastric carcinoma. Putative species differences include the role of Helicobacter in pathogenesis, the wide array of genetic data and screening available for humans, and treatment protocols that are available for human GC.     

Laparoscopic esophagopexy,fundopexy, and hiatal herniorrhaphy for refractory regurgitation in a racing Alaskan husky sled dog

A 2-year-old intact female Alaskan husky sled dog was presented with a history of chronic exercise-induced regurgitation refractory to medical management. Previous diagnostics were unremarkable except for an endoscopic examination and histopathologic evaluation of the upper gastrointestinal tract that revealed the presence of Helicobacter spp. and mild non-specific inflammation of the proximal duodenum. A laparoscopic hiatal herniorrhaphy, esophagopexy, fundopexy, and ovariectomy were performed without complications in anesthesia or surgery and clinical improvement was observed with continued follow-up for 8 months after surgery.Key clinical message:Surgical treatment for hiatal hernia may be considered in racing Alaskan sled dogs with regurgitation refractory to gastric protectant therapy.     

Survey of Helicobacter infection in domestic and feral cats in Korea

Discovery of Helicobacter (H.) pylori has led to a fundamental change in our understanding of gastric diseases in humans. Previous studies have found various Helicobacter spp. in dogs and cats, and pets have been questioned as a zoonotic carrier. The present study surveyed the Helicobacter infections and investigated the presence of H. felis and H. pylori infections in domestic and feral cats in Korea. Sixty-four domestic cats and 101 feral cats were selected from an animal shelter. Saliva and feces were evaluated by Helicobacter genus-specific polymerase chain reaction (PCR). Genus-specific PCR positive samples were further evaluated for H. felis and H. pylori using specific primer pairs. Thirty-six of 64 (56.3%) samples from domestic cats and 92 of 101 (91.1%) samples from feral cats were PCR positive; the positive rate of feces samples was higher than that of saliva samples in both groups. H. felis and H. pylori species-specific PCR was uniformly negative. The prevalence of Helicobacter spp. in feral cats was approximately two-fold higher than that of domestic cats. The fecal-oral route may be more a common transmission route not only between cats but also in humans.     

Detection of Arcobacter species in gastric samples from swine

Swine stomachs were surveyed for evidence of Arcobacter spp. and Helicobacter spp. infections associated with gastric ulceration. A nested PCR test targeted to the 16S rRNA was developed to detect many Arcobacter spp. and Helicobacter spp. An internal oligonucleotide probe was used for differentiation and confirmation of the PCR product. Tissue samples were obtained from the nonglandular and glandular regions of 86 swine stomachs. Evidence of infection with these microbes was detected in 51%, with 77% of the positive samples being identified as A. butzleri using a highly specific probe. Nonglandular stomach samples (44%) were more likely to be positive by PCR than samples from the glandular (23%) region. Gross lesions of any stage of gastric ulceration, ranging from parakeratosis, erosions and ulceration, were observed in 24% of stomachs examined. Of 21 samples with lesions, 52% were positive by the broadly reactive PCR assay for Arcobacter spp. and Helicobacter spp. The majority of PCR-positive samples (75%) had no gross lesions. When a single step PCR assay that was more specific for Arcobacter spp. was used on the nonglandular stomach samples, 10.4% of the 86 samples were positive. Arcobacter spp. were culture from four of the sample stomachs. Partial sequencing of the 16S rRNA gene identified the isolates as A. butzleri (n = 2), A. cryaerophilus, (n = 1), and a mixed culture of A. butzleri and another Arcobacter spp. (n = 1). A single step PCR assay targeted to the urease gene and culturing methods were used to screen for H. pylori or other closely related urease positive bacteria, but none were found.     

A molecular epidemiological survey of Babesia, Hepatozoon,Ehrlichia and Anaplasma infections of dogs in Japan

Tick-borne diseases are often encountered in canine clinical practice. In the present study, a molecular epidemiological survey of dogs in Japan was conducted to understand the prevalence and geographical distribution of Babesia spp., Hepatozoon spp., Ehrlichia spp. and Anaplasma spp. Pathogen-derived DNA in blood samples obtained from 722 dogs with a history of exposure to ticks and/or fleas was examined by PCR. The prevalence of Babesia gibsoni, Babesia odocoilei-like species, Hepatozoon canis and Ehrlichia spp./Anaplasma spp. was 2.4% (16/722), 0.1% (1/722), 2.5% (18/722) and 1.5% (11/722), respectively. While B. gibsoni and Ehrlichia spp./Anaplasma spp. were detected in the western part of Japan, H. canis was detected in Tohoku area in addition to western and central parts of Japan.     

Prevalence and molecular characterization of Babesia in pet dogs in Shenzhen,China

Canine babesiosis is an important global tick-borne infectious disease of domestic dogs and wild canids. B. gibsoni and B. vogeli are the most widespread species mainly endemic in dog population in southern and eastern regions of China. In this study, 272 blood samples were collected from pet dogs in five districts of Shenzhen, China. Babesia DNA was detected in 30 samples with an overall prevalence of 11.0 % in pet dogs in Shenzhen. The difference in the positive rate between female and male pet dogs, among different breed, and among the five sampling sites was not significant, while the age and health status of pet dogs significantly influence the positive rate of Babesia spp. infection. Sequencing results showed the presence of only B. vogeli in all of the samples tested. Sequence comparison and phylogenetic analysis revealed that all 30 strains of Babesia identified in this study shared the highest identity with B. vogeli. In sum, all results of this study showed a considerable high infection rate of Babesia spp. in pet dogs, and B. vogeli was the only Babesia species infecting pet dogs in Shenzhen city. Such information is necessary to help to elucidate the epidemiology of canine babesiosis in Shenzhen, and it is necessary to actively monitor this disease in pet dogs.     

Endoscopically Visualized Lesions,Histologic Findings,and Bacterial Invasion in the Gastrointestinal Mucosa of Dogs with Acute Hemorrhagic Diarrhea Syndrome

Background

Etiology of hemorrhagic gastroenteritis (HGE) syndrome in dogs is unknown and histopathologic and microbial investigations have only been performed post mortem.

Objective

To identify characteristic intra vitam endoscopic and histologic mucosal lesions, as well as bacterial species, within the mucosa of dogs with HGE.

Animals

Ten dogs diagnosed with HGE were included. Eleven dogs with gastroduodenoscopy and different intestinal diseases were used as controls for microbial changes. Dogs pretreated with antibiotics or diagnosed with any disease known to cause bloody diarrhea were excluded from the study.

Methods

In this prospective study, gastrointestinal biopsies were collected from 10 dogs with HGE. Endoscopic and histologic changes were assessed according to WSAVA guidelines. Biopsies from the stomach, duodenum, ileum, and colon were investigated by histology and by immunohistochemistry for the presence of Clostridium spp. and parvovirus. The first duodenal biopsy taken with a sterile forceps was submitted for bacterial culture.

Results

Acute mucosal lesions were only found in the intestines, not in the stomach. Clostridium spp., identified as Clostridium perfringens in 6/9 cases, were detected on the small intestinal mucosa in all dogs with HGE, either by culture or immunohistopathology. In the control group, C. perfringens could only be cultured in one of 11 dogs.

Conclusions and Clinical Importance

The results of this study demonstrate an apparent association between C. perfringens and the occurrence of acute hemorrhagic diarrhea. The term “HGE,” which implies the involvement of the stomach, should be renamed as “acute hemorrhagic diarrhea syndrome.”     

A mixed population of Helicobacter pylori,Helicobacter bizzozeronii and “Helicobacter heilmannii” in the gastric mucosa of a domestic cat

Background

The presence of Helicobacter within the gastric mucosa is responsible for producing pathology in many animal species, including man. Since humans have been shown to harbour many of the same bacterial species as domestic carnivores, concern over their zoonotic potential has been growing. Helicobacter pylori, a class 1 carcinogen responsible for cases of gastritis and gastric cancer in humans, produces similar pathology in pet carnivores and is considered an example of anthroponosis. The case here presented refers to a 13 year-old mixed breed spayed female cat seen at necropsy.

Findings

Stomach samples were analysed for the presence of Helicobacter spp. by cytology, histopathology and PCR. Mild mucosal atrophy was observed in the fundus and antrum, while lymphoplasmocytic infiltrates where noted in the lamina propria of the antrum. Helicobacter-like organisms were observed in the corpus and antrum, occupying gastric glands and surface mucosa. It was possible to detect Helicobacter spp., H. pylori, H. heilmannii and H. bizzozeronii in the fundus, corpus and antrum by PCR, while in the antrum PCR samples were positive for H. pylori.

Conclusions

The spayed female under study could represent either a yet un-described population of domestic cats infected with H. pylori or a case of anthroponosis.     

Canine vector-borne disease in travelled dogs in Germany--a retrospective evaluation of laboratory data from the years 2004-2008

When importing dogs from various Mediterranean countries into Western Europe canine vector-borne infections are often considered as a major issue. Several diseases including babesiosis, leishmaniosis, hepatozoonosis, canine heartworm disease or ehrlichiosis can potentially be endemic in this region and pose a potential health risk for travelling dogs. Information on such infections in travelled dogs is scarce and therefore this study has been undertaken to examine the frequency of vector-borne infections in travelled dogs from the years 2004-2008. A total of 997 samples were screened by direct and/or indirect methods. Total seroprevalence was 7.5% with individual seroprevalence for the 3 species Leishmania spp., Ehrlichia canis and Babesia canis spp. ranging from 3.1 to 4.9%. Total detection rate for pathogens by direct methods was 3.5%. Ninteen Giemsa-stained blood smears were positive for large Babesia. None of the samples screened for microfilariae by Knott's test or for Dirofilaria immitis antigen by DiroChek^® were positive. Using PCR methods Leishmania-DNA was detected in 1/42 samples but none of 59 animals screened for E. canis-DNA was positive. The prevalence values as established by indirect and direct pathogen detection are considered as rather low.     

Detection of Atypical Cultivable Canine Gastric Helicobacter Strain and its Biochemical and Morphological Characters in Naturally Infected Dogs

Helicobacter‐like organisms are frequently found in canine stomachs, but the relationship between such organisms and gastric pathology has not been established. However, some such organisms have zoonotic importance. The aims of this study were to evaluate the morphological and biochemical characteristics of cultivable canine gastric Helicobacter‐like organisms (GHLOs) in pets and stray dogs and their prevalence in these two groups of dogs. Specimens were taken by gastroscopy from 30 clinically healthy stray dogs and 30 pet dogs. Cultures were positive from biopsies of 11/30 of stray and 6/30 of pet dogs. The isolated Helicobacters were observed by light microscopy and studied by biochemical, physiological and PCR analysis. Some of the isolated GHLO's displayed atypical shapes that were similar to Helicobacter pylori or Helicobacter acinonychis in stray dogs’ cytological examinations. They had 2–3 helices and were smaller than other canine GHLOs. One of these atypical Helicobacter strains was cultured. It was not possible to distinguish such strains by routine PCR and biochemical evaluations. Electron microscopy showed a smaller Helicobacter (2 μm in length) with 2 or 3 helixes. This study demonstrates that not all canine gastric Helicobacters are 5–15 μm in length, as has been previously proposed, and portrays the need for further investigation of canine GHLOs.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.     

Inductively coupled plasma mass‐spectrometric determination of platinum in excretion products of client‐owned pet dogs

Residues of antineoplastic drugs in canine excretion products may represent exposure risks to veterinary personnel, owners of pet dogs and other animal care‐takers. The aim of this study was to measure the extent and duration of platinum (Pt) excretion in pet dogs treated with carboplatin. Samples were collected before and up to 21 days after administration of carboplatin. We used validated, ultra‐sensitive, inductively coupled plasma‐mass spectrometry assays to measure Pt in canine urine, faeces, saliva, sebum and cerumen. Results showed that urine is the major route of elimination of Pt in dogs. In addition, excretion occurs via faeces and saliva, with the highest amounts eliminated during the first 5 days. The amount of excreted Pt decreased over time but was still quantifiable at 21 days after administration of carboplatin. In conclusion, increased Pt levels were found in all measured excretion products up to 21 days after administration of carboplatin to pet dogs, with urine as the main route of excretion. These findings may be used to further adapt current veterinary guidelines on safe handling of antineoplastic drugs and treated animals.     

Retrospective Biomolecular Investigation of Coxiella burnetii and Leptospira spp. DNA in Cases of Abortion,Stillbirth and Neonatal Mortality in Dogs and Cats

Abortion and neonatal mortality are events that can occur in breeding bitches and queens. It has been reported that up to 55% and 33% of these cases remain without a known cause, respectively, in canine and feline pregnancies. Unusual abortigenic and potentially zoonotic agents, including Coxiella burnetii and Leptospira spp., may be involved in these cases. C. burnetii is able to cause reproductive disorders in cattle, sheep and goats, and cases of abortion have been observed in dogs and cats. Moreover, several outbreaks of C. burnetii infection in humans have been caused by delivering bitches and queens, and some of these animals experienced abortion. Leptospira interrogans sensu lato is able to cause abortion or stillbirth in several animal species and its abortigenic role has occasionally been described in bitches and queens. The aim of this study was to search for C. burnetii and Leptospira spp. DNA in a retrospective series of 103 cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the identification of possible infectious agents. One hundred and fifty-one specimens were tested using PCR assays and found negative for C. burnetii and Leptospira DNA. However, in 49 samples (47.6%) other infectious causes of abortion, stillbirth, and neonatal mortality were identified. These results showed that C. burnetii and Leptospira spp. are probably not common abortigenic agents or causes of neonatal deaths in dogs. However, given the potential abortigentic and zoonotic role of these agents, surveillance of canine and feline abortion, stillbirth, and neonatal mortality could be advisable for a systematic investigation of these events.