Helicobacter – An Emerging New Zoonotic Pathogen

The genus Helicobacter contains 35 named species and numerous provisionally named species. It is likely that several novel Helicobacter species await discovery. Members of this genus are microaerobic, have a fusiform or curved to spiral rod morphology and are motile by flagella that vary in number and location among different species (Vandamme et al., 1990). All known Helicobacters live in human and animal hosts, where colonization occurs primarily in the gastrointestinal tract. The type species, Helicobacter pylori (H. pylori), was isolated from the stomach of humans and has been associated with a variety of gastric anomalies including gastritis, peptic ulcer disease, gastric carcinoma, and gastric mucosaassociated lymphoma (Parsonnet, 1998). Like H. pylori, other species of Helicobacter have also been shown to colonize the stomach and cause disease in animals. Gastric colonizers include H. felis, H. mustelae, H. acinonychis, H. bizzozeronii, H. heilmannii, H. salomonis, and a recently isolated novel Helicobacter sp. of dolphins (Hodzic et al., 2001). Several species of Helicobacter have been identified in rodents, including the species H. hepaticus, H. bilis, H. muridarum, H. aurati, H. cinaedi, H. cholecystus, H. trogontum, H. rodentium, and a bacterium morphologically resembling H. Flexispira taxon 8 (formerly Flexispira rappini) (Hodzic et al., 2001). Evidence is accumulating that especially pigs, dogs, and cats constitute reservoir hosts for gastric Helicobacter species with zoonotic potential.


Introduction
The genus Helicobacter contains 35 named species and numerous provisionally named species. It is likely that several novel Helicobacter species await discovery. Members of this genus are microaerobic, have a fusiform or curved to spiral rod morphology and are motile by flagella that vary in number and location among different species (Vandamme et al., 1990). All known Helicobacters live in human and animal hosts, where colonization occurs primarily in the gastrointestinal tract. The type species, Helicobacter pylori (H. pylori), was isolated from the stomach of humans and has been associated with a variety of gastric anomalies including gastritis, peptic ulcer disease, gastric carcinoma, and gastric mucosaassociated lymphoma (Parsonnet, 1998). Like H. pylori, other species of Helicobacter have also been shown to colonize the stomach and cause disease in animals. Gastric (Hodzic et al., 2001). Evidence is accumulating that especially pigs, dogs, and cats constitute reservoir hosts for gastric Helicobacter species with zoonotic potential.

History of Helicobacter research
The first well-known report of gastric Helicobacters was by Bizzozero in Turin in 1893 (Bizzozero, 1893). Bizzozero was a well-known anatomist, famous already for his proof that all dividing cells required cell nuclei (Castiglioni, 1947). In his anatomical observations of the gastric mucosa of dogs, Bizzozero reported "spirochetes" inhabiting the gastric glands (Figura & Orderda, 1996) and even the canaliculi of the parietal cells. In hand-drawn color illustrations, Bizzozero showed gram-negative organisms with approximately 10 wavelengths within the parietal cells and gastric glands. We now know these organisms variously identified as H. canis, H. felis , and/or H. heilmannii (Heilmann & Borchard, 1991). Bizzozero's work was extended by Salomon, who was able to propagate these spiral organisms in mouse stomachs after feeding ground-up gastric mucosa from cats and dogs to his mouse colony (Salomon, 1896 (Chen et al., 1995). Warren had observed patients with spiral organisms on their gastric mucosa since 1979 and had documented the inflammation associated with the bacteria by the time he and Marshall began a concerted attempt to study the organisms in patients with various upper gastrointestinal symptoms. After August 1981, the team studied patients attending for endoscopy and was able to demonstrate the gramnegative bacteria on Gram stains but could not culture them at that time. They tentatively treated one patient with tetracycline and were able to observe a decrease in the number of neutrophils in the gastric mucosa as well as apparent disappearance of the bacteria. They recognized, however, that anecdotal evidence of the bacteria's role in gastric inflammation was of little value and therefore commenced a study in 100 consecutive endoscopy patients to try to culture the bacteria, as well as determine their association with gastritis and/or other clinical syndromes. Initially, they did not focus specifically on the etiology of peptic ulcer disease, although they were aware that gastritis was strongly associated with duodenal and gastric ulcers, as well as with gastric cancer (Warren & Marshall, 1983).

Classification of Helicobacter species
The genus Helicobacter contains 35 named species and numerous provisionally named species. It is likely that several novel Helicobacter species await discovery. Members of this genus are microaerobic, have a fusiform or curved to spiral rod morphology and are motile by flagella that vary in number and location among different species (Vandamme et al., 1990). All known Helicobacters live in human and animal hosts, where colonization occurs primarily in the gastrointestinal tract. The type species, H. pylori, was isolated from the stomach of humans and has been associated with a variety of gastric anomalies including gastritis, peptic ulcer disease, gastric carcinoma, and gastric mucosa-associated lymphoma (Parsonnet, 1998 (Hodzic et al., 2001). A number of Helicobacter species may confound experimental data because of their association with disease progressing in various kinds of animals (Chin et al., 2000, Ward et al., 1994, Eaton et al., 1996. H. hepaticus and H. bilis were initially reported as pathogens associated with hepatitis and inflammatory bowel diseases (Shomer et al., 1997, Ward et al., 1996, and H. typhlonicus caused proliferative typhlocolitis in SCID mice ( Franklin 1999). H. suncus was isolated from house musk shrews as a pathogenic agent (Goto et al., 2000).
Most routine laboratories apply the same basic biochemical tests for the identification and differentiation of all Campylobacter-like organisms and would fail to identify many Helicobacter species. Although the number of Helicobacter species encountered in human clinical samples is fairly small, the lack of application of highly standardized procedures and the well-known biochemical inertness of Campylobacter-like organisms render biochemical identification of all of these bacteria very difficult. Whereas Arcobacter strains can be differentiated from Campylobacter and Helicobacter strains by their ability to grow in air and at low temperature (Vandamme et al., 1991), there are no clear biochemical characteristics to separate the genus Helicobacter from the genus Campylobacter. Theoretically, one has to differentiate over 35 validly named species and subspecies, as well as various unnamed taxa. An overview of biochemical and other methods to differentiate Campylobacter and Arcobacter species was described earlier (Vandamme, 2000). A summary of the characteristics of cultivated Helicobacter species shows that discrimination between some species may rely on only one differential feature. Moreover, some species, notably H. pylori and H. acinonychis, and H. felis and H. bizzozeronii, cannot be differentiated with conventional phenotypic tests.

Clinical sequels of Helicobacter infection
Helicobacter pylori (H. pylori) is a Gram-negative, spiral-shaped, microaerophilic bacterium that infects the human gastric mucosa (Warren and Marshall, 1983). Chronic infection is thought to be associated with chronic active gastritis, peptic ulcer and gastric malignancies, such as mucosa-associated B cell lymphoma and adenocarcinoma (NIH, 1994). In particular, this organism has been categorized as a class I carcinogen by the World Health Organization (International Agency for Research on Cancer, 1994) and previous studies have confirmed that long-term infection with H. pylori induces adenocarcinoma in Mongolian gerbils (Honda et al., 1998;Watanabe et al., 1998). The association between H. pylori and gastric cancer has been explained by two possible mechanisms.
Gastric mucosal infection with H. pylori is accompanied by infiltration of neutrophils, and activated inflammatory cells are known to produce oxygen radicals (Evans et al., 1995;Ramarao et al., 2000). Oxygen radicals are known as inducers and initiators because they cause direct DNA damage (Clemens, 1991), but the relationship of these radicals with the onset of gastric cancer has not been sufficiently explored. Ammonia/ammonium concentrations increase in the gastric mucosa due to infection with H. pylori, and Tsujii et al. have found that ammonia acts as a promoter in a rat model of gastric cancer induced by Nmethyl-N-nitro-N-nitrosoguanidine (MNNG) (Tsujii et al., 1992). To consider the association between H. pylori infection and the onset of diffuse type of gastric cancer, unlike intestinal type gastric cancer, the process from infection with H. pylori through gastric mucosal atrophy, intestinal metaplasia, and development of cancer must be excluded (Correa et al., 1994;Fay et al., 1994). Direct evidence must therefore be found to indicate progression from infection with H. pylori through persistent inflammatory cell infiltration resulting in DNA damage by oxygen radicals, point mutations of genes, and finally carcinogenesis.

Host ranges of Helicobacters
Since H. muridarum was first reported in the intestinal mucosal of mice and rats (Lee 1992), additional Helicobacter species have been isolated from laboratory animals. Several Helicobacter species such as H. hepaticus (Fox 1994), H. muridarum, H. bilis , H. rodentium (Shen et al., 1997), Flexispira rappini (Schauer et al., 1993), H. typhlonicus (Franklin et al., 1999) have been identified in rodents. The genus Helicobacter contains 24 named species and numerous provisionally named species. It is likely that several novel Helicobacter species await discovery. Members of this genus are microaerobic, have a fusiform or curved to spiral rod morphology and are motile by flagella that vary in number and location among different species (Vandamme et al., 1990). All known Helicobacters live in human and animal hosts, where colonization occurs primarily in the gastrointestinal tract. The type species, H. pylori, was isolated from the stomach of humans and has been associated with a variety of gastric anomalies including gastritis, peptic ulcer disease, gastric carcinoma, and gastric mucosa-associated lymphoma (Parsonnet, 1998

Transmission of Helicobacters
In-depth knowledge of the transmission patterns may constitute important information for future intervention strategies. In the absence of consistent and verified environmental reservoirs, a predominantly person-to-person transmission has been postulated. H. pylori infection is associated with poor living conditions, and possible transmission routes are fecal-oral, oral-oral, or gastro-oral, but firm evidence is lacking (Torres et al., 2000). Young children are particularly vulnerable to infection by transmission of H. pylori from their infected parents, especially infected mothers (Rothenbacher et al., 1999), and it is generally believed that such transmission is influenced by socio-economic status. However, little is known about how and when maternal transmission occurs during perinatal period, especially whether this occurs before or after parturition. In the present study, we examined these issues in an experimental murine model, Mongolian gerbil model that have been reported as a most optimal laboratory animal model to study H. pylori in vivo (Hirayama et al., 1996).
In the previous study, Lee & Kim (2006) examined these issues in an experimental murine model, Mongolian gerbil model that have been reported as a most optimal laboratory animal model to study H. pylori. Pregnant Mongolian gerbils, infected experimentally with H. pylori, were divided as four groups. Following the experimental design, the stomachs of the mother and litters were isolated and assessed for transmission of H. pylori at prenatal period, parturition day, 1-week old age and 3-week old age respectively. Bacterial culture and polymerase chain reaction (PCR) was used to examine the presence of transmitted H. pylori. All litters showed no transmission of H. pylori during pregnancy and at parturition day. However, they reveled 33.3% and 69.6 % at 1-week old age and 3-week old age respectively by PCR. These results suggested that vertical infection during prenatal period or delivery procedure is unlikely as a route of mother-to-child H. pylori infection. It might be acquired H. pylori through breast-feeding, contaminating saliva and fecal-oral during cohabitat (Lee & Kim, 2006).
Half of the world's population is estimated to be infected with H. pylori and the infection is mainly acquired in early childhood but the exact routes of transmission remain elusive. Infected mothers are generally considered to be the main source of the pathogen ( Graham et al., 1991). There is an obvious public health impact of H. pylori infection and thus, to design targeted and costeffective prevention strategies, elucidation of the mode of transmission for this bacteria is crucial (Fendrick et al., 1999). It is known that H. pylori infection is typically acquired in early childhood and usually persists throughout life unless specific treatment is applied (Crone & Gold 2004). Definitive modes of transmission have not yet been characterized and the principal reservoir appears to be humans. Person-to-person transmission via fecal-oral, oraloral and gastro-oral routes have been proposed (Mladenova et al., 2006). Numerous studies also indicate low socioeconomic status, including domestic overcrowding in childhood, as major risk factors for higher infection prevalence rates (Frenck & Clemens 2003). Little is known about when and how often maternal transmission of H. pylori occurs during perinatal stage. In the previous study, Lee & Kim (2006) examined these issues in an experimental murine model.
The results of the vertical-transmission experiment indicated that vertical transmission of H. pylori was not occurred at pregnant and delivery staged. However, they reveled 33.3% and 69.6 % at lactating and weaning stage respectively. Recent epidemiological studies in humans suggest that the acquisition of H. pylori occur during childhood. For example, Rothenbacher et al (2000) reported that H. pylori acquisition seems to occur mainly between the first and second year of life: that is, after the age of weaning. Our results are in agreement with this report. Also, Rothenbacher et al (2000) reported that infected parents, especially infected mothers, play a key role in the transmission of H. pylori within families. Maternal contact behaviour during the breastfeeding period may be responsible for the high frequency of maternal transmission (Kurosawa et al., 2000). Our results also showed that the maternal-transmission of H. pylori was not observed during pregnancy and delivery stage, but detected at lactating and weaning stage. On the basis of these findings, vertical infection during pregnancy or at delivery is unlikely as a route of mother-to-child H. pylori infection. Lee & Kim (2006) suggested that H. pylori infection of transplacental route during pregnancy might not be occurred and that H. pylori transmission by discharges of uterine or vagina, obstetric delivery tract, during parturition might not be occurred. It might be acquired H. pylori through breast-feeding, contaminating saliva and fecal-oral during co-habitat.

Diagnostic methods of Helicobacters
To detect Helicobacter species, serologic tests (Livingston et al., 1997), the culture method (Russel et al., 1995), and the PCR (Engstrand et al., 1992) have been used. Serologic test may be not available for animal screening because of absence of available species-specific antibodies against Helicobacter species. Also, culture assay is labor-intensive. It has been reported that PCR assays is easy and useful method and can be performed even on feces as a noninvasive means of rapidly screening large numbers of animals for Helicobacter species (Beckwith et al., 1997). However, those kinds of PCR assays requires multiple assays because of a lot of Helicobacter species (Grehan et al., 2002). There is no doubt that a bacteriological culture is the best method for diagnosing a bacterial infection. However, it is not easy to cultivate Helicobacters because the specimens are usually obtained from several different locations by biopsy or necropsy. In addition, the sensitivity of the culture-isolation method is low (Hammar et al., 1992). Therefore, a culture is not considered to be the most practical diagnostic method. As a result, the CLO test and staining methods are preferred in many clinical laboratories. Nonetheless, they also have problems such as accuracy of species-specific identification (Megraud, 1997). PCR which is a specific and sensitive molecular method for detecting Helicobacter DNA, can supplement the above methods. However, PCR methods using species-specific primers require multiple assays because of a lot of Helicobacter species (Grehan et al., 2002). In this study, the RNA polymerase ß-subunitcoding gene (rpoB) was used for the detection of novel Helicobacter species by a simple PCR analysis. rpoB is an important transcription apparatus in all microorganisms. Because this region is highly conserved, this rpoB DNA PCR could be used as a consensus PCR analysis method to detect Helicobacter species. Therefore, it is clear that PCR methods targeting a stable gene such as rpoB would give more reliable results. Mutiple PCR assays using Helicobacter species-specific primers may be considered an expensive, laborious, and thus impractical procedure for many samples in clinical laboratory settings. On the other hand, this consensus PCR can be used alone without multiple assays. Therfore, the cost, which is higher than those of other methods, including culture, will be reduced. Helicobacter species may be identified in this single PCR and the presence of a novel species may be detected. Fecal samples may be stored at room temperature for up to a week without affecting the outcome of PCR for Helicobacter species (Beckwith et al., 1997). Therefore, monitoring of Helicobacter infection could be conducted very easily by this consensus PCR with feces. In the previous study, the consensus PCR using rpoB primers was able to detect successfully Helicobacter species (Kim & Kim, 2004). A set of primers (HF, 5'-ACTTTAAACGCA TGAAGATAT-3'; and HR, 5'-ATATTTTGACCTTCTGGGGT-3') was used to amplify rpoB DNA (458 bp) encompassing the Rif r region. Amplification of rpoB DNAs (458 bp) from Helicobacter species PCR products was electrophoresed on a 1.2% agarose gel.

Preventive and therapeutic methods of Helicobacters
Various pharmacological regimens have been studied in the treatment of H. pylori infection. Antibiotics (Fera et al., 2001), proton-pump inhibitors (Park et al., 1996), H 2 -blockers (Sorba et al., 2001), and bismuth salts (Midolo et al., 1997) are suggested standard treatment modalities, which are typically combined in dual, triple and quadruple therapy regimens in order to eradicate H. pylori infection (Worrel et al., 1998). Some problems may arise upon administration of these eradication regimens, i.e. the cost (Worrel et al., 1998), the efficacy of antibiotics regarding the pH (for instance, amoxicillin is most active at a neutral pH and tetracycline has greater activity at a low pH) (Worrel et al., 1998) and resistance to the antibiotics (Ferrero et al., 2000). However, above 15% of the patients undergoing such drug regimens experienced therapeutic failure (Worrel et al., 1998). screened for anti-H. pylori activity, exhibiting Rheum palmatum, Rhus javanica, Coptis japonica and Eugenia caryophyllata strong anti-H. pylori activity (Bae et al., 1998). Cranberry juice possesses modest anti-H. pylori activity (Burger et al., 2000). The anti-H. pylori activities of Aristolochia paucinervis, black myrobalan and cinnamon were also examined (Gadhi et al., 2001). Anti-H. pylori compounds from the Brazilian medicinal plant Myroxylon peruiferum have successfully isolated (Ohsaki 1999). Extracts and fractions from seven Turkish plants were also demonstrated to elicit anti-H. pylori activity (Yesilada et al., 1999). The leaves, roots and stems of Korean and Japanese wasabi exhibited bactericidal activities against H. pylori, having the leaves the highest bactericidal activity (Shin et al., 2004). In addition, some flavonoids and isoflavonoids isolated from licorice such as licochalcone A, licoisoflavone B, and gancaonols have been reported to exhibit inhibitory activities against H. pylori (Fukai et al., 2002

Helicobacters as an emerging new zoonotic pathogen
The genus Helicobacter contains at least 24 named species and an additional 35 or more novel Helicobacters wait formal naming (Fox, 2002). Members of this genus are microaerobic, have a fusiform or curved to spiral rod morphology and are motile by flagella that vary in number and location among different species (Vandamme et al., 1990). All known Helicobacters live in human and animal hosts, where colonization occurs primarily in the gastrointestinal tract. The type species, H. pylori, was isolated from the stomach of humans and has been associated with a variety of gastric anomalies including gastritis, peptic ulcer disease, gastric carcinoma, and gastric mucosa-associated lymphoma (Parsonnet, 1998 (Hodzic et al., 2001). The initial interest in animal Helicobacters arose from the need for a suitable animal model for studying H. pylori infection, and subsequently from an ecological perspective . However, there have been recent concerns regarding the potential of animals, notably domestic pets, to be a source of zoonotic Helicobacter i n f e c t i o n . D o g s a n d c a t s u s e d f o r b i o m e d i c a l r e s e a r c h h a v e b e e n occasionally found to harbor H. pylori strains (Handt et al., 1995), while H. felis has been implicated as a potential human pathogen in a few cases (Wegmann et al., 1991). H. pylori has also been found in pet animals, and it can promote gastritis when introduced into specific-pathogen-free cats. The significance of this infection as a cause of gastritis in pet dogs and cats is nevertheless unclear. The main gastric Helicobacter species in dogs and cats are primarily H. heilmannii (formerly "Gastrospirillum hominis") and H. felis. These two species are collectively referred to as gastric Helicobacter-like organisms (GHLO) because they cannot be distinguished by light microscopy. So far, H. heilmannii has not been reliably cultured in vitro.

Conclusions
Clinical symptoms associated with non-H. pylori Helicobacters in humans can be characterized by atypical complaints such as acute or chronic epigastric pain and nausea. Other aspecific symptoms include hematemesis, recurrent dyspepsia, irregular defecation frequency and consistency, vomiting, heartburn, and dysphagia, often accompanied by a decreased appetite. Evidence is accumulating that especially pigs, dogs, and cats constitute reservoir hosts for gastric Helicobacter species with zoonotic potential. The recent successes with in vitro isolation of these fastidious microorganisms from domestic animals open new perspectives for developing typing techniques that can be directly applied on gastric biopsies from humans. These techniques should make it possible to determine whether animal and human strains belonging to the same Helicobacter species are clonally related.