Nov 13, 1991 - Support Fecal-Oral Transmission of a Gastric Helicobacter. J. G. FOX,'* B. J. PASTER,2 .... suspended in1 ml of 10 mM Trisbuffer, pH 7, at a concentration of 108 cells ... (wt/vol) phosphotungstic acid, pH 6.5, for 10 to 15 s. Samples were ... filters were neutralized on 1.5 M NaCl-0.1 M Tris for 5 min at room ...
Vol. 60, No. 2
INFECTION AND IMMUNITY, Feb. 1992, p. 606-611
0019-9567/92/020606-06$02.00/0 Copyright © 1992, American Society for Microbiology
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, 45-104, 37 Vassar Street, Cambridge, Massachusetts 021394307,' and Forsyth Dental Center, Boston, Massachusetts 022152 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. 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-12000 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,
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 *
37).
Corresponding author. 606
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TABLE 1. Biochemical characteristics of fecal Helicobacter spp. from ferrets Characteristica
Oxidase production Catalase production Urease production H2S production Lead acetate disk-BAP TSI agar Motility Hippurate hydrolysis Yellow pigment Growth at: 250C 370C
420C Growth in:
Presence or absence in: Other H. mustelae Helicobacter spp.
+ + +
+
+
+
+ +
-
-
+ +
+ +
+ +
+ +
+
+
+
+ + +
1.5% NaCl 2.0% NaCl
3.0% NaCl 1.0% glycine Triphenyltetrazolium chloride (400 ,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, 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 R85-13-6PT sequence
607
(=ATCC 43772T), 88-7, 1649, and 104; H. pylori ATCC 43504T, 3001-1, 1006-1, 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 84-161; 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, 0.0025 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
All Helicobacter spp.
274-300 588-615 623-651
H. mustelae H. mustelae
Sequence
5'-TCTCAGGCCGGATACCCGTCATAGCCT-3' 5'-ACAGGATTTCACATCTGACTTATTACTC-3'
5'-GAATAACAGTTTCAAATGCAGTTCTGTAG-3'
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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 266-11. 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
