Helicobacter mustelae Isolation from Feces of Ferrets: Evidence To Support Fecal-Oral Transmission of a Gastric Helicobacter

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1 INFECTION AND IMMUNITY, Feb. 1992, p /92/ $02.00/0 Copyright 1992, American Society for Microbiology Vol. 60, No. 2 Helicobacter mustelae Isolation from Feces of Ferrets: Evidence To Support Fecal-Oral Transmission of a Gastric Helicobacter J. G. FOX,'* B. J. PASTER,2 F. E. DEWHIRST,2 N. S. TAYLOR,' L.-L. YAN,1 P. J. MACUCH,2 AND L. M. CHMURA' Division of Comparative Medicine, Massachusetts Institute of Technology, , 37 Vassar Street, Cambridge, Massachusetts ,' and Forsyth Dental Center, Boston, Massachusetts Received 10 July 1991/Accepted 13 November 1991 Helicobacter mustelae has been isolated from stomachs of ferrets with chronic gastritis and ulcers. When H. mustelae is inoculated orally into H. mustelae-negative ferrets, the animals become colonized and develop gastritis, a significant immune response, and a transient hypochlorhydria. All of these features mimic Helicobacterpylori-induced gastric disease in humans. Because the epidemiology of H. pylori infection is poorly understood and its route of transmission is unknown, the feces of weanling and adult ferrets were cultured for the presence of H. mustelae. H. mustelae was isolated from the feces of 11 of 36 ferrets by using standard helicobacter isolation techniques. H. mustelae was identified by biochemical tests, ultrastructural morphology, reactivity with specific DNA probes, and 16S rrna sequencing. H. mustelae was not recovered from 20-week-old ferrets which had been H. mustelae positive as weanlings, nor was H. mustelae recovered from 1-year-old ferrets. Isolation of H. mustelae from feces may correspond to periods of transient hypochlorhydria, or H. mustelae may be shed in feces intermittently. The H. mustelae-colonized ferret provides an ideal model for studying the pathogenesis and transmission of H. pylori-induced gastric disease. Several gastric Helicobacter spp. have a narrow host specificity; Helicobacter pylori infects humans, gnotobiotic pigs, gnotobiotic dogs, and, to a lesser extent, nonhuman primates, whereas H. mustelae infects ferrets (1, 8, 11, 12, 14, 16, 18, 30-32, 39). H. felis has a wider host range and colonizes the gastric mucosa of dogs, cats, ferrets, and rodents (14, 26-29). In studies of H. mustelae infection in ferrets, it was ascertained that ferret kits are initially colonized with the organism soon after weaning (at 6 weeks of age) and subsequently become persistently infected (7, 14). Oral inoculation of H. mustelae into specific-pathogen-free ferrets results in the development of gastritis similar to that observed in cases of natural H. mustelae infection (9, 15). Interestingly, these ferrets developed a transient hypochlorhydria approximately 3 to 4 weeks after being dosed with the bacteria. Hypochlorhydria is also associated with H. pylori infection in some human cases (20, 34, 43). The purpose of this report is to document isolation of H. mustelae from the feces of both weanling and young adult ferrets. MATERIALS AND METHODS Animals. Two groups of ferrets were examined in this study. In the first group, 26 ferrets were monitored at the ages of 9 and 20 weeks for the presence of H. mustelae or Campylobacter spp. in their feces. In group 2, an additional 10 ferrets (4 who were 8 months of age and 6 who were 12 months of age) were also screened for the same organisms. The ferrets in group 2 were not available for further study. Ferrets were maintained in animal resources accredited by the American Association for Accreditation of Laboratory Animal Care. Ferrets were housed singly in stainless steel caging measuring 25 by 31 by 19 in. (1 in. = 2.54 cm). Ferrets were given water ad libitum initially and fed Purina Cat Chow; at 12 weeks of age, half of the animals in group 1 were placed on a high-fat (22%) pelleted diet; the remaining half * Corresponding author. 606 were fed the high-fat formula plus a retinoid given orally via gavage five times per week. The feeding protocol was relevant to another study involving these ferrets. Microbiology. Freshly collected fecal swabs were streaked on CVA medium containing cefoperazone, vancomycin, and amphotericin B (Remel). Duplicate plates were then incubated at 37 and 42 C for 3 to 5 days in vented jars containing N2, H2, and CO2 (80:10:10). Bacteria were identified as Campylobacter-like organisms (CLOs) by Gram stain, morphology under phase microscopy, and whether they were oxidase positive, catalase positive or negative, or urease positive or negative. All CLOs had further biochemical tests performed on them as previously described (8, 14) (Table 1). Urease activity was assessed 5 min after inoculation of a culture into urease broth by the method of Hazell et al. (22). All other tests were read after 3 days of incubation. Electron microscopy. Negatively stained specimens for electron microscopy were prepared as follows. Cells from agar media were harvested after 1 to 2 days of growth and suspended in 1 ml of 10 mm Tris buffer, ph 7, at a concentration of 108 cells per ml. A drop of this suspension was placed on a Formvar-coated, carbon-reinforced copper grid (300 mesh), and the excess fluid was drawn off with filter paper. After 90 s, cells on the grid were stained with 1% (wt/vol) phosphotungstic acid, ph 6.5, for 10 to 15 s. Samples were examined with a JEOL JEM electron microscope operating at 100 kv. RNA isolation. RNA was isolated and partially purified by a modification of the procedure of Pace et al. as previously described (36, 37). 16S rrna sequencing. rrna sequences were determined by using a modification of the Sanger dideoxy chain termination technique in which primers complementary to conserved regions of the 16S rrna were elongated by using avian myeloblastosis virus reverse transcriptase (25). The details of our protocol have been described previously (4, 37).

2 VOL TABLE 1. Biochemical characteristics of fecal Helicobacter spp. from ferrets Presence or absence in: Characteristica H. Other mustelae Helicobacter spp. Oxidase production + + Catalase production + Urease production + H2S production Lead acetate disk-bap + + TSI agar - - Motility + + Hippurate hydrolysis Yellow pigment Growth at: 250C C C + + Growth in: 1.5% NaCl 2.0% NaCl 3.0% NaCl 1.0% glycine + + Triphenyltetrazolium chloride ( ,Lg/ml) Trimethylamine N-oxide + + (anaerobically) Susceptibility to: Nalidixic acid + + Cephalothin - + Metronidazole + + a Abbreviations: BAP, blood agar plate; TSI, triple sugar iron. Data analysis. A program set for data entry, editing, sequence alignment, secondary-structure comparison, similarity matrix generation, and dendrogram construction for 16S rrna data was written in Microsoft QuickBASIC for use on IBM PC-AT and IBM PC-AT-compatible computers. RNA sequences were entered and aligned as previously described (37). Our sequence data base contains approximately 300 sequences, including sequences determined in our laboratory, previously published sequences, and unpublished sequences provided to us by other scientists. Probe design and labeling. Aligned 16S rrna sequences were examined to locate regions of 24 to 30 bases which were unique for target species or, in the case of genus probes, were identical for all members of a genus but different from sequences in all other genera in our data base. Probes complementary to these targets were synthesized commercially (Midland Certified Reagent Co., Midland, Tex.). The sequences and target locations of the probes are shown in Table 2. Probes were 5' end labeled with [a-32p]atp by using T4 polynucleotide kinase (42). Probes were purified by chromatography on a Sep-Pak C18 column (42). Probes were validated for specificity by using the following reference strains: H. mustelae R PT H. MUSTELAE IN FERRET FECES 607 (=ATCC 43772T), 88-7, 1649, and 104; H. pylori ATCC 43504T, , , C014, Win 1, Gr 1, and 1030-L H; H. felis CS1T (=ATCC 49179T), CS2, CS3, CS4, DS1, DS2, and DS3; H. fennelliae CCUG 18820T; H. muridarum STiT; Wolinella curva ATCC 35244; Wolinella recta FDC 371; Campylobacter upsaliensis CCUG 14913T; Campylobacter jejuni ; Campylobacter coli 85-1; Campylobacter concisus FDC 484T (=ATCC 33237T); Escherichia coli K-12; Fusobacterium nucleatum subsp. nucleatum ATCC 25586T; Prevotella intermedia (Bacteroides intermedius) ATCC 25611T; Actinobacillus actinomycetemcomitans ATCC 29522; Eikenella corrodens 23834T; and Capnocytophaga gingivalis ATCC 33624T. Whole-cell preparations. Through methods developed in our laboratory, approximately 108 bacterial cells harvested from agar plates for hybridization to the probes were spotted onto Whatman 541 filter paper. The filters were placed, with cells facing upwards, on 3MM chromatography paper soaked in 1 M NaI. After 30 min of incubation at 37 C, the filters were neutralized on 1.5 M NaCl-0.1 M Tris for 5 min at room temperature. The filters were air dried and then baked at 80 C under vacuum for 2 h. Hybridization conditions. Baked filters were prehybridized at 50 C in a solution containing final concentrations of the following: 25% formamide (Fisher Scientific Co., Fair Lawn, N.J.), 4x SSC (20x SSC is 3 M NaCl and 0.3 M sodium citrate, ph 7.0), 25 mm Na2PO4, 1 mm disodium EDTA, salmon testes DNA (type III; Sigma Chemical Co., St. Louis, Mo.), lx Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), and 0.1% sodium dodecyl sulfate. Hybridizations were carried out for 1 to 2 h. After two rinses in lx SSC at room temperature, the filters were washed in 1 x SSC at 50 C for 15 min. Radioactivity was determined with a Betascope 603 (Betagen, Waltham, Mass.), which allows the rapid imaging of radioactive patterns, usually in less than 15 min. Additional probes were tested on the same filters by stripping the filters with 0.1 x SSC at 75 C for 1 h and repeating the hybridization procedure. ELISA for H. mustelae antibody. Antigen was prepared from whole-cell extracts by previously published methods for the H. mustelae enzyme-linked immunosorbent assay (ELISA) used in our laboratory (9, 10). Three isolates of H. mustelae (including ATCC 43772) from gastric biopsy samples of ferrets were used. The ELISA was carried out by the methods of Fox et al. (9, 10). The titer of serum samples was expressed as the dilution of serum giving a reading equal to the mean plus 2 standard deviations of the negative control values. Sera. Sera for ELISA evaluation from the 26 H. mustelaeinfected ferrets in group 1 were collected at ages 9, 19, and 27 weeks. Nucleotide sequence accession numbers. The sequences for the rrnas of the two organisms examined in this study are available from GenBank under the accession numbers TABLE 2. DNA probes used Target taxon Location Sequence All Helicobacter spp '-TCTCAGGCCGGATACCCGTCATAGCCT-3' H. mustelae '-ACAGGATTTCACATCTGACTTATTACTC-3' H. mustelae '-GAATAACAGTTTCAAATGCAGTTCTGTAG-3'

3 608 FOX ET AL. INFECT. IMMUN. w FIG. 1. H. mustelae with lateral and polar sheathed flagella. Note doughnut-shaped flagellum insertion sites (arrowheads). Magnification, x20,400. M35048 for H. mustelae and M12345 for Helicobacter sp. strain RESULTS Culture of H. mustelae from feces. Twenty-four of 26 9-week-old ferrets in group 1 and three of the 8-month-old ferrets in group 2 had CLOs isolated from their feces. Eleven of these strains (eight from group 1 and three from group 2) were motile, nonhemolytic, gram-negative, slightly curved rods and were catalase, oxidase, and strongly urease positive. In addition, the organisms grew at 37 and 42 C in 0.4% triphenyltetrazolium chloride and 0.1% trimethylamine N-oxide anaerobically, were hippurate negative, produced H2S on lead acetate disks but not in triple sugar iron agar, and did not grow on 1.5, 2, and 3% NaCl or at 25 C. The 11 strains were sensitive to nalidixic acid and metronidazole but resistant to cephalothin. All of these phenotypic and biochemical criteria were compatible with a classification of H. mustelae (Table 1). The 16 other CLOs from group 1 were catalase and urease negative but oxidase positive. All other biochemical reactions were similar to those of H. mustelae. However, the organisms were sensitive to cephalothin, nalidixic acid, and metronidazole. All 26 ferrets from group 1 were negative for Helicobacter spp. when their feces were cultured at 20 weeks. Electron microscopy. The ultrastructural analysis of strain 292-ElA from group 1 depicted short, slightly curved rods 2 by 0.5,um with four or more sheathed flagella; the sheathed flagella were usually arranged in a bipolar and lateral format. Insertion points, when visible, were slightly convex doughnut-shaped structures and measured 80 by 100 nm. All of these features have been noted previously by us and are typical of characteristics ascribed to H. mustelae (8, 11, 35) (Fig. 1). ELISA. At 9 to 10 weeks of age, all but 1 of the 26 ferrets from group 1 had antibody titers of <1:64; 1 ferret had a titer of 1:274. When bled 9 weeks later, eight ferrets had a two- to threefold increase in titer of immunoglobulin G to H. mustelae. The titer of antibody to H. mustelae in the 26 ferrets at 27 weeks continued to rise; the majority had titers of 1:128, with four having a greater-than-fourfold increase in titer (1:294 to 1:2,358), indicating active infection. DNA probe analysis. Twenty-three of the fecal isolates (20 from group 1 and 3 from group 2) and the reference organisms were examined with all-helicobacter species- and H. mustelae-specific DNA probes. The all-helicobacter probe recognized all strains of Helicobacter spp. but did not hybridize with any other bacterial strains examined. The H. mustelae probe was specific only for the four reference strains of H. mustelae examined. Twenty-two of the 23 clinical isolates were recognized by the all-helicobacter probe. Six of the urease-positive isolates were recognized by the H. mustelae probes; the remaining five urease-positive Helicobacter spp. are being further characterized. RNA sequence analysis. 16S rrna sequencing provided definitive molecular evidence that one of the fecal isolates was H. mustelae. Comparing 1,385 bases of the 16S rrna

4 VOL. 60, 1992 sequence for urease-positive H. mustelae 292-ElA with those of the type strain of H. mustelae (GenBank accession number M35048) demonstrated 100% sequence identity (38). Because of the high specificity of the species-specific probe for H. mustelae, it is likely that the remaining five ureasepositive isolates that were recognized by this species-specific probe are indeed additional strains of H. mustelae. The sequence of 16S rrna from the catalase-negative, ureasenegative isolate was only 97.2% similar (1,387 bases) to the sequence for that of H. mustelae. This level of similarity (see GenBank) indicates that strain represents a new species of Helicobacter. Further definition and naming of this new Helicobacter species will be addressed in a subsequent publication. DISCUSSION Recently, animal models have been used to study both the pathogenesis and epidemiology of gastric Helicobacter infections. Use of H. pylori has been restricted in animal models because of its host specificity; H. pylori is infectious only for humans, germ-free pigs, germ-free dogs, and nonhuman primates (1, 24, 34, 39). Fortunately, models being studied by using other gastric Helicobacter spp. are providing insight into these processes. In a series of experiments, H. felis caused persistent gastritis when given orally to germ-free mice, germ-free rats, and specific-pathogen-free mice (13, 26, 27). However, H. felis could not be transmitted from infected animals to uninoculated controls despite prolonged contact of the rodents in the same cage. Even given the coprophagous nature of rodents, these experiments indicated that fecal-oral spread of H. felis did not occur. In addition, H. felis could not be isolated from the intestines of infected or contact mice or rats (14, 26, 27). In limited gnotobiotic-dog experiments, both H. pylori and H. felis were transmitted to control littermates (29, 39). The explanation provided was that the dogs spread the helicobacters via oral-oral contact, whereas rodents did not because of their inability to vomit. Another animal model studied is the ferret infected either naturally or experimentally with H. mustelae (9, 15, 17). Natural H. mustelae infection in the ferret occurs shortly after weaning (7, 14). Every ferret with chronic gastritis we have thus far examined also has H. mustelae isolated from the gastric mucosa, while specificpathogen-free ferrets not infected with H. mustelae do not have gastritis (7, 9, 15). H. mustelae, like H. pylori, clearly adheres to the gastric mucosa and has urease subunits similar to those reported for H. pylori; both urease production and adhesion are cited as potentially important virulence factors in the pathogenesis of H. pylori gastroduodenal disease (6, 9, 35). We recently fulfilled Koch's postulates by demonstrating that H. mustelae isolated from a ferret with gastritis and inoculated into previously H. mustelae-negative ferrets without gastritis readily colonized the gastric mucosa, could be reisolated, and upon reisolation produced an immune response and caused chronic gastritis; all of these features were noted in ferrets naturally colonized with H. mustelae (9, 14, 15). In the same study, the experimentally infected ferrets developed a transient hypochlorhydria approximately 4 weeks after being dosed with H. mustelae (15). This period coincided with heavy H. mustelae colonization of the fundus (15). We hypothesize that the ferrets sampled in this study were naturally colonized at 5 to 6 weeks of age; when sampled at 9 to 10 weeks of age, they were hypochlorhydric. The hypochlorhydric state may have allowed larger numbers of H. MUSTELAE IN FERRET FECES 609 H. mustelae to exit the stomach and facilitated subsequent recovery from the feces. Cultures negative for H. mustelae from feces of the same ferrets at 20 weeks of age support this theory. The ferrets from group 1 were in a nutritional study which precluded gastric biopsy and measurement of gastric ph. Active infection of ferrets at 9 to 27 weeks of age was based on elevated H. mustelae immunoglobulin G antibody, which is an accurate indication of persistent infection in the ferret and is consistent with our previous finding that virtually 100% of the ferrets from this source are colonized with the organism (7, 9, 11, 14). However, isolation of H. mustelae from the feces of 8-month-old ferrets in the second group suggests that (i) ferrets with acidic gastric ph can shed the organism in feces, (ii) H. mustelae-associated hypochlorhydria can exist for longer periods in selected ferrets, or (iii) H. mustelae is shed in the feces intermittently. The mode of transmission of H. pylori is poorly defined. Person-to-person transmission is strongly suspected because of the clustering of H. pylori infections, with a higher prevalence of H. pylori antibody in families of infected children and in institutionalized patients than in the healthy population (2, 5, 23, 24, 32). Gastroenterologists performing endoscopies are at a higher risk of H. pylori infection than age-matched physicians in other specialties (33). latrogenic transmission has also been recorded, suggesting either fomite or direct transmission (20, 21, 43). However, despite the arguments that the epidemiologic characteristics support fecal-oral (as well as oral-oral) spread, H. pylori to date has not been isolated from feces (19). Reports also have associated H. pylori infection with hypochlorhydria. One volunteer who ingested H. pylori had confirmed elevated gastric ph (39). In recent iatrogenic infections with H. pylori, similar occurrences of transient gastric achlorhydria were noted in two patients 2 weeks after infection with H. pylori; in each case, the hypochlorhydria lasted for approximately 2 months (20, 41). Interestingly, the high prevalence of H. pylori infection noted in populations residing in New Orleans, Colombia, and Peru corresponds with high rates of chronic atrophic gastritis (3, 10, 40). As this type of gastritis increases in severity, there is a corresponding loss of parietal cell mass and an attendant hypochlorhydria. In theory, these infected people could shed H. pylori in the feces for extended periods, contaminate the environment (e.g., water), and serve as the primary sources of fecal-oral H. pylori transmission. In summary, the identification of H. mustelae in the feces of ferrets was based on several criteria used for microbial identification; all of the data, including phenotypical, biochemical, DNA hybridization, and RNA sequence, confirmed that several of the fecal CLOs were H. mustelae (8, 11, 14, 35, 38). We have therefore conclusively demonstrated for the first time recovery of a gastric Helicobacter species from the feces of an infected host. This finding supports the argument that H. pylori, like H. mustelae, can be spread via the fecal-oral route. The ferret should provide an ideal model for further exploration of the epidemiology and pathogenesis of Helicobacter-induced gastric disease. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants RR01046 and RR07036 from the Division of Research Resources, National Institutes of Health; grant P01-CA26731 from the National Cancer Institute; and grants DE and DE from the National Institute of Dental Research.

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