Gastric Helicobacter

The discovery of 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. Investigation of the relationship of gastric disease to Helicobacter spp. in other animals has resulted in the discovery of H. mustelae in ferrets with gastritis and peptic ulcers, H. acinonychis in cheetahs with severe gastritis, and H. Heilmannii in pigs with gastric ulcers. The presence of gastric Helicobacter-like organisms (HLO) in the stomachs of dogs and cats has been known for many years but the relationship of those organisms to gastric disease remains controversial.

Helicobacter spp. Infecting the Stomachs of Dogs and Cats

Helicobacter are spiral-shaped or curved, or sometimes coccoid gram-negative bacteria that inhabit the glands, parietal cells and mucus of the stomach. The large gastric HLO are morphologically indistinguishable by light microscopy, in which they are seen as large, 5-12µ long spirals. They have been classified into several Helicobacter spp. on the basis of 16s rRNA sequencing, DNA hybridization, and electron microscopic appearance. H. felis, "H. heilmannii", H. bizzozeronii, and H. pametensis have been detected in the gastric mucosa of pet cats. H. pylori has been isolated from the stomachs of a group of colony housed cats, but not pet cats. H. bizzozeronii, H. heilmannii, H. felii, H. salomonis, F. rappini and H. bilis have been identified in dogs.

How common is Infection with Gastric Helicobacter spp.?

There is a high prevalence of gastric Helicobacter infection: HLO have been observed in gastric biopsies from 41-100% of clinically healthy and 57-100% of vomiting cats. The prevalence of individual Helicobacter spp. has not been thoroughly investigated, as it requires specialized techniques. H.felis has been cultured from 3/21 Helicobacter infected cats in Finland, whereas "H. heilmannii" was identified by PCR in 38/49 Swiss cats. PCR studies in Helicobacter infected cats in the USA have identified 18/36 with "H. heilmannii", 6/36 with H. felis (4/36 co-infected with H. felis and "H. heilmannii"), 2/36 H. bizzozeronii and 10 cats with unclassified Helicobacter spp. Broadly similar results have been observed in 50 German cats, though H. bizzozeronii infected up to 30% of cats. Electron microscopic examination of gastric biopsies from infected cats has demonstrated co-infection with spiral organisms of differing morphology-H. felis, H. Heilmannii, H. bizzozeronii, and other large gastric spiral organisms.

H. pylori infection has been reported in a group of laboratory cats in the USA, but has not been reported in pet cats in the USA or Europe to date. It has been proposed that H. pylori is an anthroponosis--an animal infection with a human pathogen.

Several studies have shown that gastric Helicobacter are common in dogs, with a prevalence ranging from 67-100% in healthy pet dogs, 74-90% in dogs presented with vomiting and 100% in laboratory beagles. In dogs H. bizzozeronii is the most prevalent species followed by "H. heilmannii" and H.salomonis, with H. felis less common. Coinfection with one or more spp, usually H. bizzozeronii and H. heilmannii, is apparent in about 15-20% of dogs. H.pylori has not been found in pet dogs.

Are Cats and Dogs a Zoonotic Risk?

"H. heilmannii", the predominant species in pet cats, and 20-40% of pet dogs, is also found in the mucosa of 0.4-4% of people. H. heilmannii type 1 is the principal subtype in people and is thought to be acquired by zoonotic transmission from dogs, cats or pigs which are commonly infected with H. heilmannii-like organisms (HHLO).

To provide a more informed estimate of the zoonotic potential of cats, gastric DNA from cats (45 American and German) infected with

H. heilmannii, was amplified with primers against H. heilmannii ureB and 16s rDNA genes and sequenced. Fluorescence in situ hybridization (FISH) with eubacterial and H. heilmannii specific probes was employed to directly visualise H. heilmannii subtypes and their intragastric distribution (Priestnall et al). ureB sequences of H. heilmannii amplicons clustered with human and feline isolates of H. heilmannii and were distinct from the Helicobacter heilmannii-like organisms, H. felis, H. salomonis and H. bizzozeronii. 16S rDNA sequences in cats and dogs were not consistent with H. heilmanii Type 1 and clustered predominantly with types 2 and 4. No obvious differences in sequences were observed between cats from different countries. FISH failed to definitively characterize H. heilmannii subtypes present in 14 of 15 cats. As H. heilmannii type 1 is the dominant species in infected people, the zoonotic risk posed by cats and dogs is likely small.

In another study (Van den Bulck et al) gastric biopsy samples from humans with histological evidence of non-Helicobacter pylori spiral bacteria (n = 123) and samples from the gastric antrum, corpus, and cardia from dogs (n = 110) and cats (n = 43) were subjected to a multiplex PCR, enabling the identification of Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, and "Candidatus Helicobacter suis." Single infections with "Candidatus Helicobacter suis," H. felis, H. bizzozeronii, H. salomonis, a hitherto unknown genotype of a non-H. pylori spiral organism (Helicobacter-like organism 135 [HLO135]), and H. pylori were identified in 30.9%, 8.9%, 2.4%, 11.4%, 7.3%, and 8.9% of the human biopsy samples, respectively. Mixed infections (16.3%) with two or even three of these were also found. Although the majority of human non-H. pylori organisms are Helicobacter species naturally occurring in the stomachs of pigs, cats, and dogs, the frequent identification of H. salomonis in human gastric biopsy samples is in contrast to its rare identification in pet carnivore samples, suggests other sources of infection.

Do Helicobacter spp Cause Gastric Disease?

The cause of gastritis in cats and dogs is seldom determined and is usually been attributed to dietary allergy or intolerance, parasites, or a reaction to bacterial antigens. The association of Helicobacter infection with gastric disease in humans, ferrets, cheetahs, pigs and experimentally infected laboratory animals suggests that spiral organisms may have a role in the pathogenesis of gastritis in cats. The results of studies of cats and dogs with naturally acquired gastric Helicobacter Infection can be summarized as follows:

The Relationship of Infection to Clinical Signs

The high prevalence of gastric colonization with HLO in healthy and sick cats indicates that there is no simple "infection = disease" relationship. An uncontrolled treatment trial of dogs and cats with gastritis and Helicobacter infection showed that clinical signs in 90% of 63 dogs and cats responded to treatment with a combination of metronidazole, amoxicillin and famotidine, and that 74% of 19 animals re-endoscoped had no evidence of Helicobacter in gastric biopsies. Controlled clinical trials are required to confirm these observations (see treatment).

The Relationship of Infection to Gastric Histopathology

The majority of studies in cats and dogs with naturally acquired Helicobacter infections demonstrate that the fundus and cardia are more densely colonized with bacteria than the pylorus. Large HLO colonize the superficial mucus and gastric glands, and may also be observed intracellularly. Degeneration of gastric glands, with vacuolation, pyknosis and necrosis of parietal cells is more common in infected than uninfected dogs and cats.

The gastric mucosal inflammation present in Helicobacter infected dogs and cats is generally mononuclear, and ranges from mild to moderate in severity. A correlation between the presence of HLO and the extent of histopathological changes in the gastric corpus has been demonstrated in cats. The paucity of uninfected animals has hampered most investigations.

We have analyzed cytokines in gastric mucosa from 8 uninfected and 20 infected cats. Infected cats have upregulation of IL-8 (P=0.001) and IL-1ß (P=0.01), but not IFN-γ or IL-10.The evaluation of cytokines complements histopathology and should be useful when evaluating grading systems for inflammation. Gastric lymphoid hyperplasia is more common and more extensive in Helicobacter infected, than uninfected cats. Studies in cats that have examined full thickness gastric biopsies have demonstrated a strong association between infection and lymphoid follicle hyperplasia. In addition to this local immune response, a systemic immune response characterized by circulating anti-Helicobacter IgG has been detected in sera from naturally infected cats. To date there has been no association made between Helicobacter infection and gastrointestinal ulcers or gastric neoplasia in cats. However the relatively low prevalence of these diseases coupled with the small number of animals evaluated to date means that such a relationship cannot be discounted.

Studies of the immune and inflammatory changes in 30 dogs with naturally acquired gastritis have shown that mucosal pathology is related to cytokine mRNA expression (neutrophils to IL-8 and IFN-γ, macrophages and lymphocytes to IFN-γ, and fibrosis to IL-1ß) (Wiinberg et al). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histological severity correlated with atrophy, infiltration with lymphocytes and macrophages, and the expression of IL-10 and IFN-γ. Helicobacter spp infection was associated with increased expression of TGF-ß, and fibrosis. Macrophages, T-cells, and epithelial metaplasia were more frequent in uninfected than infected dogs. Circulating anti-Helicobacter IgG was higher in uninfected than infected dogs. Studies in Korea have also failed to show a relationship of Helicobacter infection to pro-inflammatory cytokine upregulation (Hwang et al).

Further Defining the Pathogenicity of Individual Helicobacter Species

Studying the effects of Helicobacter on the gastric mucosa of cats presenting with spontaneous disease is limited by the variability in the host makeup and the infecting Helicobacter species (and possible strain variation too). In contrast to humans, in whom H. pylori infection predominates, the investigation of pathogenicity in cats is complicated by the fact that they can be colonized by a variety of Helicobacter spp., and simultaneous colonization with multiple species has been frequently observed. To overcome some of these difficulties and improve our understanding of cat-Helicobacter interactions we have employed experimental infections with H. felis and H. pylori.

Studies of the pathogenicity of H. felis in laboratory cats have demonstrated gastritis, lymphoid follicular hyperplasia and seroconversion.

H. pylori infection in cats is associated with a moderately severe to severe gastritis yet clinical signs associated with gastritis, such as inappetance and vomiting, are generally absent. It serves as a model for investigating feline gastric mucosal responses and the mechanisms of Helicobacter pylori colonization, persistence and disease.

Analyses of gastric juice and biopsies from kittens in an H. pylori-infected cat colony, using rapid urease tests, ureB PCR and histopathology demonstrated H. pylori in nine of 17 kittens by eight weeks and in 16 of of these same 17 kittens by 14 weeks of age. UreB RLP patterns and sequences of PCR products from gastric mucosa were identical in mothers and kittens. Bacterial densities were similar in the pylorus, fundus and cardia. Infection was associated with circulating anti-Helicobacter IgG antibodies and significant (P<0.05) gastric inflammation and lymphoid follicles; the pathology resembled that often seen in infected human infants.

In more chronically infected cats we sought to measure the development of host inflammatory and immune responses, and their relationship to the putative bacterial virulence factors cag pathogenicity island (cagPAI), vacA allele and oipA in combination with bacterial colonization density in a feline model of the early stages of H. pylori infection (Straubinger et al). Infecting H. pylori strains were positive for vacAs1 but lacked the cagPAI and an active oipA gene. Colonization density was uniform throughout the stomach. Up-regulation of IFN-γ, IL-1α, IL-1ß, IL-8, and increased severity of inflammatory infiltrates and fibrosis were observed in infected cats. The median number and total area of lymphoid aggregates were five and ten times greater, respectively, in the stomachs of infected than uninfected cats. Secondary lymphoid follicles in uninfected cats were rare and positive for BLA.36 and B220 but negative for CD3 and CD79α, whereas in infected cats they were frequent and positive for BLA.36, CD79α, and CD3 but negative for B220. Cats infected with H. pylori can also develop antigastric antibodies that cross-react with Helicobacter antigens. Changes in gastric acid secretion and serum gastrin which are known to occur in humans with H. pylori infection have recently been demonstrated in cats with experimental H. pylori infection.

These findings have implications for the development of gastric inflammation and possibly GI lymphoma. The methods developed to study these research cats are now being employed to evaluate cast with spontaneous gastritis. Further studies are ongoing to try to determine the genetic attributes of H. pylori that impact the host response.

Eradicating Helicobacter spp in Cats and Dogs

The general lack of knowledge of the pathogenicity of gastric Helicobacter spp. has meant that veterinarians are faced with the dilemma of either treating, or ignoring, spiral bacteria observed in biopsies from patients with chronic vomiting and gastritis. Eradication of H. pylori infection in symptomatic humans has been associated with resolution of symptoms and gastric abnormalities. In light of their pathogenicity in man and other animals it would seem reasonable that eradication of gastric Helicobacter spp. is considered prior to initiating treatment with immunosuppressive agents to control gastritis. An uncontrolled treatment trial of dogs and cats with gastritis and Helicobacter infection lends support to this approach. Clinical signs in 90% of 63 dogs and cats responded to treatment with a combination of metronidazole, amoxicillin and famotidine, and 74% of 19 animals re-endoscoped had no evidence of Helicobacter in gastric biopsies. However, controlled therapeutic studies in asymptomatic cats suggest that it is difficult to eradicate gastric Helicobacter spp. in cats with azithromycin, tinidazole, bismuth and ranitidine, or clarithromycin, metronidazole, bismuth and ranitidine for four or seven days. After three weeks of amoxicillin, metronidazole and omeprazole, cats with H. pylori infection were culture negative, but five out of six cats were positive in a species specific PCR in dental plaque, saliva and/or gastric fluid samples. Amoxicillin and metronidazole does not appear to be an effective treatment in dogs either, with a high failure rate 1 month after antibiotics.

It is unclear if in most studies antibiotic failure was due to reinfection or recrudescence, although the persistence of Helicobacter by PCR suggests recrudescence is likely. These findings contrast markedly with studies in H. pylori infected people where 80% cure rates with 1%/yr reinfection are observed.

We have recently performed a study in H. pylori infected cats to determine if differences in H.pylori genotype, and antibiotic sensitivity explain the difficulty in eradicating Helicobacter in cats: DNA sequences of a variety of genes from H. pylori isolated from cat strains matched those of H. pylori strains of human origin. Isolates from different cats differed in growth rate in culture, intrinsic susceptibility to metronidazole and their ability to colonize stomachs of H. pylori-free cats. The pattern of metronidazole sensitivity and the role of genes metabolizing metronidazole were similar in strains from people and cats. Further tests showed that chronically infected cats could be cured of resident H. pylori with amoxicillin (20mg/kg PO BID 14days), metronidazole (10-15mg/kg PO BID) and clarithromycin (7.5mg/kg PO BID). This yielded 100% eradication in 12/12 cats 1 month post treatment.

Non- gastric Helicobacter spp.

Cholangiohepatitis / cholangitis complex in cats is an ill-defined inflammatory disorder of the hepatobiliarytree, that is one of the most common hepatic disorders in cats .It has been sub-categorized as suppurative and non-suppurative to reflect the relative proportions of neutrophils to lymphocytes and plasma cells, and the degree of bile duct hyperplasia and fibrosis. Suppurative cholangiohepatitis is often associated with a short duration of illness, moderate elevations of liver enzymes, jaundice, fever, neutrophilia and being male. Non-suppurative cholangiohepatitis is similarly characterized by jaundice and elevated hepatic enzymes, but a longer duration of clinical signs, hepatomegaly and a protein rich abdominal effusion, hyperglobulinemia and lymphocytosis are observed more frequently than in cats with suppurative cholangiohepatitis.

Bacterial infections, most frequently enteric species, have been implicated in acute cholangiohepatitis, and a clinical response to antimicrobial therapy has been observed in a cat with Enterobacter associated cholangiohepatitis. Concurrent pancreatic or intestinal inflammation and cholestasis (intra- or extrahepatic) are also frequently diagnosed in cats with both suppurative and non-suppurative cholangiohepatitis and may facilitate bacterial colonization, possibly by ascending infection of the biliary tree. However, a cause and effect relationship of these potential etiologies to cholangiohepatitis has yet to be demonstrated.

In people, rodents and dogs there is evidence that inflammation and/or neoplasia of the liver and biliary tract are associated with infection with Helicobacter spp. Helicobacter DNA or organisms, have been identified in the liver, bile or gallbladder of people with chronic cholecystitis, cholestatic liver disease and hepatobiliary carcinoma and cirrhosis. In mice H. hepaticus have been associated with hepatitis, hepatocellular carcinoma, and inflammatory bowel disease and Helicobacter bilis with hepatitis and typhlitis. H. cholecystus has been cultured from the gallbladders and pancreas of hamsters with cholangiohepatitis and pancreatitis. H. canis has been cultured from the liver of a young dog with hepatitis. To date there are no reports of Helicobacter spp associated liver disease in cats. However, as cats, like humans and other species, harbor Helicobacter in their stomachs, i.e., H. felis, H. heilmanii and H. bizzozzeronii, and H. canis, bilis, cinaedi and flexispira have been cultured from feces it is possible that Helicobacter species have a role in hepatic disease in cats.

It is against this background that we examined the role of Helicobacter spp. in cholangiohepatitis in cats, by evaluating archived hepatic tissue samples from cats with and without cholangiohepatitis for the presence or absence of Helicobacter spp using PCR, immunocytochemistry and silver staining.

Tissue blocks from 32 cats with cholangiohepatitis were identified in the pathology database for the period 1992-2001 (Greiter-Wilke et al). Tissue blocks from a group of 13 cats with non-inflammatory liver disease, and 4 with normal hepatic histology served as a control group. Positive PCR results of liver samples were obtained from 2 cats with cholangiohepatitis, and 1 cat with PSVA. Sequence analysis indicated homology with H. nemestrinae / pylori in one Ch cat, and H. bilis in the PSVA cat. Two amplicons of different sizes persistently detected in the third cat, were consistent with H nemestrinae / pylori and H. fenelliae/cinaedii. PCR of gastric tissue samples yielded sequences with close homology to gastric Helicobacter spp, e.g., H. heilmannii. No Helicobacter-like organisms were identified in hepatic tissue by Steiner stain or immunocytochemistry. FISH using a eubacterial probe identified a semicurved bacterium in the intrahepatic bile duct of one cat with cholangiohepatitis.

Recent studies of cats with lymphocytic cholangitis (Boomkens et al) detected H. pylori like sequences in the bile of 4/15cats with lymphocytic cholangits, 8/51 cats with non-LC cholangitis and 7/12 healthy cats.

The Helicobacter sequences identified in thee studies are consistent with those associated with liver disease in other species. Further, prospective studies are warranted to elucidate the role of Helicobacter and other infectious agents in liver disease in cats.

Acknowledgements

Dr. Simpson is supported by a grant from the US public health service (DK 002938). I gratefully acknowledge the generous support provided by numerous graduate students, post-docs, technicians and external collaborators--some of whom are mentioned in the references below!

References

1.  Boomkens SY, Kusters JG, Hoffmann G, Pot RG, Spee B, Penning LC, Egberink HF, van den Ingh TS, Rothuizen J. Detection of Helicobacter pylori in bile of cats. FEMS Immunol Med Microbiol. 2004 Nov 1;42(3):307-11

2.  Greiter-Wilke,A, E. Scanziani, S. Soldati, P.L. McDonough, S.A. Center, M. Rishniw K.W. Simpson. Evaluation of the association of Helicobacter with feline cholangiohepatitis JVIM 2006, In press.

3.  Hwang CY, Han HR, Youn HY. Prevalence and clinical characterization of gastric Helicobacter species infection of dogs and cats in Korea. J Vet Sci. 2002 Jun;3(2):123-33.

4.  Priestnall SL, Wiinberg B, Spohr A, Neuhaus B, Kuffer M, Wiedmann M, Simpson KW. Evaluation of "Helicobacter heilmannii" subtypes in the gastric mucosas of cats and dogs. J Clin Microbiol. 2004 May;42(5):2144-51.

5.  Straubinger RK, Greiter A, McDonough SP, Gerold A, Scanziani E, Soldati S, Dailidiene D, Dailide G, Berg DE, Simpson KW. Quantitative evaluation of inflammatory and immune responses in the early stages of chronic Helicobacter pylori infection. Infect Immun. 2003 May;71(5):2693-703.

6.  Van den Bulck K, Decostere A, Baele M, Driessen A, Debongnie JC, Burette A, Stolte M, Ducatelle R, Haesebrouck F. Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats. J Clin Microbiol. 2005 May;43(5):2256-60.

7.  Wiinberg B, Spohr A, Dietz HH, Egelund T, Greiter-Wilke A, McDonough SP, Olsen J, Priestnall S, Chang YF, Simpson KW. Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection. J Vet Intern Med. 2005 Jan-Feb;19(1):4-14.