Determination of the Prevalence of Helicobacter heilmannii-Like Organisms Type 2 (HHLO-2) Infection in Humans and Dogs Using Non-Invasive Genus/Species-Specific PCR in Korea

ABSTRACT

Helicobacter spp. may have multiple routes of transmission. It is unclear, however, whether the agent is zoonotic and therefore transmitted from an animal reservoir, including dogs. The aim of this population-based study was to assess the relationship between pet ownership or frequent exposure to dogs and Helicobacter spp. infection, especially focusing on HHLO-2 (Helicobacter heilmannii-like organisms type 2) in saliva and feces samples in Korea, using non-invasive genus/species-specific PCR. One hundred twenty-four eligible human subjects and 39 dogs participated in this study. Relativity of contact with dogs and Helicobacter spp. infection diagnosed by genus-specific PCR showed a statistically significant result (P<0.01), but in the relativity analyses between contact with dogs and H. pylori, H. felis and H. bizzozeronii infections diagnosed using species-specific PCR, only Helicobacter felis showed a statistically significant result. Although H. pylori infection showed a statistically significant relativity, no statistically significant association was found between veterinarian subjects and Helicobacter. spp., H. felis and H. bizzozeronii infections. On performing risk factor analyses of HHLO-2 infection by transmission, using matching species, between HHLO-2-positive dog owners and HHLO-2-positive dogs, Helicobacter felis infection showed an extremely significant relativity (P<0.0001), and Helicobacter bizzozeronii may also be a possible significant risk factor (P<0.01). These results suggest that HHLO-2 infection might be a zoonotic infection, because continuous contact with dogs was proved to be correlated with human H. felis and H. bizzozeronii infections in this study.

Helicobacter spp. are gram-negative and spiral-shaped bacteria. Till now, almost 24 Helicobacter spp. have been reported, and most are suspected or proven gastric or hepatic pathogens [1]. Spiral organisms, such as Helicobacter spp., have been observed in domestic feline and canine gastric tissues since the last 100 years. These gastricbacteria have received special attention, and it seems that the stomachs of many types of domestic or companion animal species harbor their own Helicobacter spp. with long-term evolution. The Helicobacter spp. infection rate is 40 to 80% throughout the world in humans, and the most common Helicobacter spp. infection in humans in half of the world’s population is H. pylori [2]. Recently, H.pylori was identified in domestic cats obtained from a commercial breeder, which suggests that pets may be a potentialsource of H. pylori infection [3].

General diagnostic methods for Helicobacter infection are divided into invasive and non-invasive methods [4]. ELISA has been developed and used for making the diagnosis of Helicobacter species, and direct observation of Helicobacter spp. organisms in biopsied specimens usually requires the use of special stains, e.g. Giemsa, WSS, Genta and alcian yellow-toluidine blue stains. Typically, H.pylori is not visualized by H&E staining [5]. Culture with biopsy of the historically affected region is the gold standard in invasive measures; however, this method is less sensitive in most Helicobacter species. In non-invasive methods, the PCR assay has a higher sensitivity and specificity than the other non-invasive methods, such as serological tests, and nested PCR analysis for Helicobacter spp. increases both the sensitivity and specificity than a single PCR analysis [6].

However, epidemiologic data of other Helicobacter spp., such as H. felis and H. bizzozeronii, are lacking to support the animal and public health implications. Helicobacter spp. have been detected continuously in humans, mainly H. pylori, and Helicobacter spp., such as H.felis and H. bizzozeronii, have been detected in pets in Korea [7]. Despite the advances in understanding the pathophysiology and virulence determinants of H. pylori infection, knowledge of H. felis or H. bizzozeronii is lacking till now [8]. Animal and public health implications of Helicobacter species have been continuously debated since their introduction to the seroepidemiologic studies of H. pylori with transmission occurring from cats to humans [9] or from sheep milk to humans [10].

Recently, circumstantial evidence has suggested that these bacteria, also referred to as “Helicobacter heilmannii-like organisms” (HHLO), may be transmitted through animals [11]. At least two genotypically different types of HHLO have been identified based on the analysis of 16S rRNA genes, and type II was found to be highly related to H. felis and H. bizzozeronii. Although HHLO-2 (HHLO type II) was found in a small percentage of 0.2 to 0.6% in humans, approximately 50% of non-pylori Helicobacter species inducing gastritis, gastric ulcer and MALT lymphoma in humans are associated with HHLO-2 [12]. The infection with HHLO-2 is common in domestic animals with or without gastric diseases, and various studies have reported the prevalence rate as 41 to 100% in dogs and 57 to 100% in cats [13, 14]. The identification of HHLO-2 in humans that have mostly been identified in pets arouses interest in the zoonotic potential of HHLO-2; however, this finding is not clear as yet.

The aims of this study were to evaluate the prevalence and morphologic types of Helicobacter spp. using Helicobacter genus-specific PCR and Helicobacter species-specific PCR and to assess the relation of exposure to pets with HHLO-2 prevalence in a population-based study of pet owners in Korea.

MATERIALS AND METHODS

Study population and sample preparation: One hundred and 24 human subjects were included in the “dog-contact risk factor group”. In detail, the dog-contact risk factor group was composed of 41 dog owners and 43 veterinarians who regularly contact dogs. In this group, 12 veterinarians who were themselves dog owners. Each of the dog owners including veterinarians was matched with their own 39 dogs by labeling for the statistical analyses. Forty human subjects with no history of pet ownership were included for the negative dog-contact factor (Supplemental Fig. 1 ). Saliva and feces samples were taken from all subject populations. The feces and saliva samples were taken by using sterilized cotton swabs and subsequently submerged in 500 µl of autoclaved phosphate buffered saline. DNA was extracted from 20 to 30 µl of each sample by using DNeasy Tissue Kit (Qiagen, Santa Clarita, CA, U.S.A.). The DNA samples were eluted in 200 µl volume and stored in a −20°C freezer until PCR was conducted.

Genus-specific PCR: Each DNA sample was amplified on Helicobacter 16S rRNA gene using C70 and B37 outer primers30, and subsequently, the PCR products were conducted nested PCR using C97 and C98 inner primer pair (Supplemental Table 1) [15]. The PCR mixture, total volume of 20 µl contained a final primer concentration of 0.5 µM, 1 µl DNA samples (0.3 µl for nested PCR) and 18 µl diethyl pyrocarbonate treated water, was added to Maxime PCR PreMix Kit (iNtRON Biotechnology Inc., Seoul, Korea). The PCR samples were heated at 95°C for 5 min followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 53°C for 45 sec and extension at 72°C for 3 min and finally extended at 72°C for 15 min using outer primers by using programmed temperature control system (PC808, Astec, Fukuoka, Japan). For nested PCR, the PCR products were heated at 94°C for 2.5 min followed by 35 cycles of denaturation at 94°C, annealing at 50.5°C, extension at 72°C for 1 min each and a final extension at 72°C for 15 min. The PCR products were electrophoresed by 1.5% agarose gels containing ethidium bromide in 0.5x TBE buffer and visualized on ultraviolet light illuminator.

Species- specific PCR: PCR amplifications of H. felis and H. bizzozeronii were performed using primer (Supplemental Table 1) which amplify the urease B gene of them [16]. PCR assay specific for H. pylori is also performed. The total volume of PCR mixture 20 µl including a final each primer concentration of 0.5 µM, 1 µl DNA samples (0.3 µl for nested PCR) and 18 µl diethyl pyrocarbonate treated water was added to Maxime PCR PreMix Kit (iNtRON Biotechnology Inc.). DNA extracts from pure cultures of H. pylori(HpKTCC H. pylori strain 114), H. felis(ATCC 49179) and H. bizzozeronii(ATCC 70030) served as positive controls.

For PCR amplification of H. pylori, the samples were heated at 95°C for 5 min and followed by 35 cycles at 94°C for 45 sec, at 59°C for 45 sec and at 72°C for 45 sec and a final extension at 72°C for 10 min using outer primes. Second round of PCR was performed at 95°C for 5 min, 30 cycles followed at 94°C for 45 sec, at 54°C for 45 sec and at 72°C for 30 sec and final extension at 72°C for 10 min. For H. felis-specific PCR, samples were heated at 94°C for 2.5 min once, followed by 40 cycles of denaturation at 94°C, annealing at 45°C, extension at 72°C for 1 min each with a final extension at 72°C for 15 min using outer primes. Second round of PCR was performed at 94°C for 2.5 min, 30 cycles followed at 94°C for 45 sec, at 50°C for 45 sec and at 72°C for 45 sec and final extension at 72°C for 15 min. The H. bizzozeronii-specific PCR was carried out following conditions, heated at 94°C for 2.5 min once and 33 cycles of at 94°C for 1 min, at 57°C for 1 min and at 72°C for 1 min. Final extension was performed at 72°C for 16 min using outer primes. Second round of PCR was performed at 94°C for 2.5 min, 30 cycles followed at 94°C for 45 sec, at 55.5°C for 45 sec and at 72°C for 45 sec and final extension at 72°C for 15 min. The PCR products were electrophoresed on ethidium-bromide stained 1.5% w/v agarose gels in 0.5x TBE buffer and visualized on ultraviolet light illuminator.

Nucleotide sequence analysis: In order to confirm the identity of H. pylori, H. felis and H. bizzozeronii specific PCR assay products with their target genes, after the PCR products of the specific size were extracted by commercial gel extraction kit (MEGAquick-spin, INTRON, Seoul, Korea), direct sequencing of the PCR products with specific primer was conducted by ABI Prism 3730 XL DNA Analyzer (PE Applied Biosystems, Foster City, CA, U.S.A.). The result of sequencing was compared to those present in databases using BLAST software.

Statistical analysis: Pearson’s chi-square test and Fisher’s exact test were used to find the dependence between 2 categories. To check for the independence of 2 categories, including positive/negative PCR detection, the results in human subjects/animal subjects were tested by chi-square analysis using contingency tables. After counting the positive results for Helicobacter spp., H. pylori, H. felis and H. bizzozeronii as the variables, the possibility of risk of transmission between animal and human subjects was assessed using Pearson’s chi-square test with Yates’ continuity correction or 2-tailed Fisher’s exact test for count data to rule out a variety of distribution assumptions. For this reason, Pearson’s chi-square test is vague when cell counts are less than 5. From the contingency tables, odd ratios for each Helicobacter spp. can be calculated for interpreting the possible risk factor analysis. Statistical analyses were conducted using the conventional statistical software R (version 2.15.2), and the significance was set at P<0.05.

RESULTS

Risk factor analyses by Genus/species-specific PCR: Genus/species-specific single PCR was performed using inner primer for 16S rRNA gene in Helicobacter spp. H. pylori, H. felis and H. bizzozeronii were detected by nested PCR assay (Supplemental Fig. 2). Direct sequencing was performed on 2 of each Helicobacter species-specific PCR product in both human and animal, and these PCR products were randomly selected. When the result of sequencing was compared to those present in databases using BLAST software, above 99% was represented homology to their specific Helicobacter species.

For the risk factor analyses, 124 eligible human subjects and 39 dogs participated in this study. Among the human subjects, 41 dog owners and 43 veterinarians including 12 veterinarians who were themselves dog owners were included in the statistical analysis. Relativity of contact with dogs and Helicobacter spp. infection diagnosed by genus-specific PCR showed a statistically significant result (P<0.01, Table 1), but in the relativity analyses between groups of humans with frequent contact with dogs and H. pylori, H. felis and H. bizzozeronii infections diagnosed using species-specific PCR, only H. felis showed a statistically significant result (Table 1). Although H. pylori infection showed a statistically significant relativity, no statistically significant association was found between the veterinarian subjects and Helicobacter. spp., H. felis and H. bizzozeronii infections (Table 2). On performing risk factor analyses of HHLO-2 infection by transmission, using matching species, between HHLO-2-positive dog owners and HHLO-2-positive dogs, H. felis infection showed an extremely significant relativity (P<0.0001, Table 3), and H. bizzozeronii may also be a possible significant risk factor (P<0.01, Table 3).

Table 1. Transmitted infection possibility of Helicobacter spp., Helicobacter pylori, Helicobacter felis and Helicobacter bizzozeronii in dog-contact risk factor.

		Helicobacter spp. a)		Helicobacter pylori b)		Helicobacter felis c)		Helicobacter bizzozeronii d)		Species Subtotal	Total
		Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive
Dog-Contact	Negative	19	21	34	6	39	1	24	16	40	160
	Positive	18	66	31	53	66	18	51	33	84	336

a) Pearson’s χ² test with Yates’ continuity correction: χ²=7.59, P <0.01, Fisher’s exact test for count data: odds ratio=3.28, P <0.01. b) Pearson’s χ² test with Yates’ continuity correction: χ²=23.23, P=not significant, Fisher’s exact test for count data: odds ratio=9.50, P=not significant. c) Pearson’s χ² test with Yates’ continuity correction: χ²=6.09, P <0.05, Fisher’s exact test for count data: odds ratio=10.50, P <0.01. d) Pearson’s χ² test with Yates’ continuity correction: χ²=0, P=not significant, Fisher’s exact test for count data: odds ratio=0.97, P=not significant.

Table 2. Transmitted infection possibility of Helicobacter spp., Helicobacter pylori, Helicobacter felis and Helicobacter bizzozeronii in veterinarian risk factor.

		Helicobacter spp. a)		Helicobacter pylori b)		Helicobacter felis c)		Helicobacter bizzozeronii d)		Species Subtotal	Total
		Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive
Veterinarian	Negative	10	31	20	21	31	10	28	13	41	164
	Positive	8	35	11	32	35	8	23	20	43	172

a) Pearson’s χ² test with Yates’ continuity correction: χ²=0.14, P=not significant, Fisher’s exact test for count data: odds ratio=1.40, P=not significant.

b) Pearson’s χ² test with Yates’ continuity correction: χ²=3.59, P<0.05, Fisher’s exact test for count data: odds ratio=2.73, P<0.05. c) Pearson’s χ² test with Yates’ continuity correction: χ²=0.14, P=not significant, Fisher’s exact test for count data: odds ratio=0.71, P=not significant. d) Pearson’s χ² test with Yates’ continuity correction: χ²=1.35, P=not significant, Fisher’s exact test for count data: odds ratio=1.85, P=not significant.

Table 3. Risk factor analyses of Helicobacter felis and Helicobacter bizzozeronii by matching species between dog-owner and dog.

Helicobacter felis a)		Dogs		Helicobacter bizzozeroniib)		Dogs
		Negative	Positive			Negative	Positive
Dog-owners	Negative	23	0	Dog-owners	Negative	18	1
	Positive	7	9		Positive	7	13

a) Pearson’s χ² test with Yates’ continuity correction: χ²=13.79, P<0.0001, Fisher’s exact test for count data: odds ratio=Inf*. *Infinite value describes the faulty cell data by excel program (0/0). b) Pearson’s χ² test with Yates’ continuity correction: χ²=12.62, P<0.001, Fisher’s exact test for count data: odds ratio=30.04, P <0.001.

DISCUSSION

The present study investigated the possible role of frequent contact with dogs or dog ownership in the transmission of Helicobacter spp. infection in a representative population sample comprising of dogs, dog owners, non-dog owners and veterinarians in Korea. We specifically tested the hypothesis that HHLO-2 may be a very important agent causing cross infection, because it has attracted attention as a zoonotic agent in recent studies [11, 17]. Dog owners and veterinarians were included as the subjects, because of their frequent contact with dogs. Overall, we found strong evidence for an increased risk of HHLO-2 infections, such as H. felis and H. bizzozeronii infections, associated with the presence of dogs in the household (dog owners group), and co-infection rates were relatively high in dog-contact group (42.85%) than no dog-contact group (7.50%). In addition, H. felis and H. bizzozeronii co-infection rate was most frequent in both human and animal subject (42.85% in human subject and 55.12% in animal subject). In the previous study about H. pylori, co-infection rate of H. pylori L-form and vegetable-form in human subject was reported as 78.38% [19], but other form of Helicobacter spp. co-infection rate report is extremely rare. Results indicated that in the group that was in frequent contact with dogs inside or outside the house (group in frequent contact with dogs), genus Helicobacter spp. (P<0.01) and H. felis(P<0.05) infection could be considered as a zoonotic infection. However, frequent contact with dogs did not have relativity as a risk factor in H. pylori and H. bizzozeronii infections, and this result supported the result of the previous studies [8]. Interestingly, the veterinary group in Korea showed a high prevalence of H. pylori infection with statistical significance as shown in Table 2.

Acquisition of HHLO-2, including H. felis or H. bizzozeronii, may occur through animal contact, contamination of the household environment and also provably through direct or indirect contact inside the house. Among these possible transmission routes, possibility of transmission via animal contact through the oral-oral or the fecal-oral route may be highest in dog owners as compared with non-dog owners. However, the other possible transmission routes are not apparent. A statistically strong positive relation between H. felis infection and contact with dogs was identified in this study. Therefore, the potential for zoonosis might be considered in H. felis infection, both from dogs to humans and from humans to dogs, indicating that dogs might be a source for this infection; and this result is in contrast with the result of previous studies that have considered a low possibility of zoonosis in H. pylori infection in Germany [8]. Further research should integrate the approaches to the environmental or cultural factors, including diet and other life-style variables with clear roles of individual factors.

Several reports described the isolation of H. pylori from cats, and a possible non-primate reservoir for H. pylori was also identified [13, 17, 18]. H. pylori has been discovered in the stomach of dogs, and H. pylori was found in 3 dogs with no clinical signs according to our PCR results (data not shown). This result may suggest that dogs could be the natural host for H. pylori, or it may be transmitted from other sources especially humans. Furthermore, on comparing the type of identified HHLO-2 in owners and their dogs, the dog owners group showed the presence of organisms identical to those in their dogs. It might be speculated that H. felis and H. bizzozeronii could be transmitted both from dogs to humans and from humans to dogs in a strong statistically significant manner. Although the other sources of infection, such as contaminated water or food, were considered, the possibility of these sources of infection is comparatively low in H. pylori infection. These data support that dog ownership was linked to a high socioeconomic status in the risk of transmitted HHLO-2 infection.

Treatment of Helicobacter infection consists of combination therapy with antimicrobial and anti-secretory drugs. It is already well-known that Helicobacter infection in dogs and cats can be eradicated with treatment, and it is also known which drugs are the best for use in the veterinary medical field. The exact details of transmission are still unclear. A strong incidence of HHLO-2 infection has been reported in dog owners, suggesting that transmission from animals to humans or from humans to animals is strongly possible. In this report, we have statistically significant supporting results in dog owners and veterinarians of HHLO-2 infection, which should be included as the subjects, because of their frequent contact with dogs. Overall, we found strong evidence for an increased risk of HHLO-2 infections, such as H. felis and H. bizzozeronii infections from this research, although infection-positive subjects showed no clinical signs (subclinical infection) mostly. Only few human subjects reported extremely sporadic vomit symptoms in the pre-investigation for sampling procedures.

In conclusion, we might suggest that HHLO-2 (Helicobacter heilmannii-like organisms type 2) infection might be zoonotic, because continuous contact with dogs was proved to be correlated with human H. felis and H. bizzozeronii infections in this study. However, this report never intended to criticize pet ownership, but to carry out more intensive prevention, treatment and socio-epidemologic research of Helicobacter felis and H. bizzozeronii infections, which should be considered in both the medical and veterinary fields.