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Journal of Clinical Microbiology, October 2006, p. 3799-3803, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.01482-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Acquisition of Helicobacter pylori Infection in Rhesus Macaques Is Most Consistent with Oral-Oral Transmission

Jay V. Solnick,1,2,3* Julie Fong,3 Lori M. Hansen,3 Kikuko Chang,3 Don R. Canfield,4 and Julie Parsonnet5

Departments of Internal Medicine,1 Medical Microbiology & Immunology,2 Center for Comparative Medicine,3 California National Primate Research Center, University of California, Davis, Davis, California,4 Departments of Internal Medicine and Health Research and Policy, Stanford University Medical School, Stanford, California5

Received 18 July 2006/ Accepted 23 July 2006


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ABSTRACT
 
Socially housed rhesus monkeys rapidly acquired Helicobacter pylori infection, although the organism was rarely cultivated from saliva, feces, or the environment. Since the concentrations of H. pylori in vomit were compatible with what is known about the infectious dose, our results are most consistent with an oral-oral means of transmission.


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TEXT
 
Epidemiological and molecular fingerprinting data suggest that the transmission of Helicobacter pylori occurs from person to person during childhood, without an animal or environmental reservoir (2, 9, 10, 16-18). The relative importance of fecal-oral transmission versus that of oral-oral transmission is controversial (7, 13, 14). H. pylori DNA is often found in feces from infected individuals by PCR, but cultivation of H. pylori from feces (6, 8, 15) or contaminated water (12) is uncommon. The identification of H. pylori in vomitus, dental plaque, or saliva has also been achieved more often by PCR (1, 11) than by culture (15). Rhesus macaques are commonly infected with H. pylori, which is transmitted among socially housed animals at an early age (3, 4, 19, 20), a feature that mimics the transmission among humans living in developing countries. In this report we describe studies performed with the rhesus macaque to better characterize the shedding of H. pylori from naturally and experimentally infected animals and to study the variables that influence transmission.

Animals and procedures. Male and female rhesus macaques were located at the California National Primate Research Center (CNPRC), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. Specific pathogen-(H. pylori)-free (SPF) monkeys were hand reared in the nursery from the day of birth and challenged with H. pylori by previously described methods (19, 21). Animals naturally infected with H. pylori, which is endemic at CNPRC, were born and reared in outdoor field cages and ranged in age from 2.5 to 13 years. Vomiting was induced in monkeys sedated with ketamine (10 mg/kg of body weight administered intramuscularly) by intravenous administration of sterile 0.6 M lithium chloride (5 ml/kg). Quantitative cultures of gastric biopsy specimens were performed as described previously (21). Similar methods were used to culture H. pylori from vomit, saliva (diluted 1:1 with phosphate-buffered saline [PBS; pH 7.2]), and feces (diluted 1:4 with PBS and centrifuged at 82 x g for 1 min to remove large particles), except that selection was with 25 g/liter amphotericin B, 100 g/liter bacitracin, 1.65 g/liter polymyxin B, 5.35 g/liter nalidixic acid, and 50 g/liter vancomycin (ABPNV; all antibiotics were from Sigma) and the calculated limit of detection was 10 to 50 CFU/ml. The mean (standard error [SE]) recoveries from H. pylori-spiked feces and saliva performed in four to six independent trials were 104% (SE, 18%) and 120% (53%), respectively. Recovery from vomit was somewhat lower and more variable (mean, 60%; SE, 16%; n = 18). All procedures were approved by the CNPRC Research Advisory Committee and by the University of California, Davis, Chancellor's Animal Use and Care Administrative Advisory Committee. Student's two-tailed t test with the assumption of equal variance was used for all statistical comparisons unless otherwise noted.

Cultivation of H. pylori J166 from experimentally infected animals. On a total of 15 occasions, experimentally infected animals (n = 5) were induced to vomit from 1 to 20 weeks postchallenge and H. pylori from the stomach (antrum and corpus), vomit, and saliva was quantified. The mean bacterial density in the gastric antrum (log10 5.76 CFU/g) was significantly greater than that in the corpus (log10 3.95 CFU/g) (P < 0.01) (Fig. 1). In the antrum (but not the corpus) there was a trend toward a greater bacterial load at week 1 postinoculation (mean, log10 6.43 CFU/g) than at subsequent time points (mean, log10 4.17 CFU/g) (P = 0.08). Of the 15 vomit samples obtained from animals with documented gastric infection, H. pylori was cultured from 12 (80%). The bacterial load in vomit correlated significantly with that in the gastric antrum (Pearson correlation coefficient [R] = 0.51; P = 0.05) (Fig. 2A), in which the bacterial density was the greatest, but not with that in the corpus (R = 0.30; P > 0.05). H. pylori was never isolated from saliva before the animals vomited, but 10 of 14 samples (71%) obtained from animals after they vomited were positive. Fresh postprandial fecal samples (n = 62) for the isolation of H. pylori were obtained from 10 monkeys between 1 day and 2 months after challenge with H. pylori J166. All samples were negative.


Figure 1
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  		FIG. 1. Mean (SE) log10 H. pylori CFU/g of gastric tissue in the antrum and corpus of rhesus macaques infected experimentally with strain J166 (black bars) or naturally with H. pylori strains endemic at CNPRC (gray bars).


Figure 2
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  		FIG. 2. Scattergram of log10 H. pylori CFU/ml of vomit compared with CFU/g of tissue in the antrum of monkeys infected with H. pylori J166 (A) or compared with CFU/g of tissue in the corpus of animals naturally infected with H. pylori (B). Lines shows the least-squares best fit.

Cultivation of H. pylori from naturally infected animals. On a total of 16 occasions, naturally infected animals (n = 4) were induced to vomit and H. pylori in the stomach (antrum and corpus), vomit, and saliva was quantified. The mean bacterial density in the gastric antrum (log10 4.99 CFU/g) was significantly less than that in the corpus (log10 6.24 CFU/g) (P < = 0.05) (Fig. 1). Of the 16 vomit samples obtained from animals with documented gastric infection, H. pylori was cultured from 14 (88%). The bacterial load in vomit correlated significantly with that in the gastric corpus (R = 0.57; P < 0.05) (Fig. 2B), in which the bacterial density was the greatest, but not that in the antrum (R = 0.17; P was not significant). H. pylori was never isolated from saliva before the animals vomited, but 10 of 16 samples (63%) obtained from animals after they vomited were positive. Fresh postprandial fecal samples (n = 63) for isolation of H. pylori were obtained from 17 naturally infected macaques (n = 17). All samples were negative.

Transmission of H. pylori by exposure to fomites. In order to determine if H. pylori could be transmitted without direct animal-to-animal contact, we placed SPF monkeys (targets; n = 8) individually for 24 h in cages that had been occupied during the previous 24 h by a monkey infected with H. pylori J166 (n = 4) or a naturally infected monkey (n = 4). Each target monkey was placed in the cage occupied by each source monkey (a J166-infected or a naturally infected monkey) six times, for a total of 24 exposure days. Of the four target monkeys exposed to source monkeys infected with H. pylori J166, three remained uninfected and one was infected after 12 exposure days. Repetitive extragenic palindromic PCR (Rep-PCR) (21) performed with the DNA from the H. pylori strain isolated from this monkey showed that the fingerprint was identical to that from H. pylori J166 (data not shown). No target monkeys were infected after exposure to cages occupied by naturally infected source monkeys. Considering the eight target monkeys together, infection occurred at an incidence of 1 per 176 days of exposure (7 x 24 = 168 days for 7 uninfected monkeys, plus 1 x 6 days for the single infected monkey, if it is assumed that infection occurred halfway through the observation period).

SPF monkeys placed in cages formerly occupied by infected monkeys could potentially have had physical contact with an infected monkey in an adjacent cage. We therefore next performed a similar experiment with 4 SPF macaques and 14 naturally infected rhesus macaques, but all possibility of physical contact was eliminated. SPF and infected animals were housed on opposite sides of a "corncrib," an outdoor enclosure measuring 415 ft2 that has two areas separated by a 10-ft-long chute through which animals could be moved from one side to the other. Twice per week the SPF animals were removed to a holding cage, infected animals were transferred via the chute to the opposite side of the corncrib, and SPF animals were placed in the side previously occupied by the infected animals. All SPF animals were biopsied every 2 weeks to evaluate environmental transmission. Since H. pylori is unlikely to survive under ambient conditions for more than 24 h, exposure days were assumed to be only the first day after transfer. After 19 weeks of exposure (4 animals x 19 weeks x two exposures/week = 152 animal exposure days), no transmission was documented.

Cultivation of H. pylori from fomites. In order to determine if H. pylori could be cultivated from fomites, we used transport swabs (Fisher) to sample feces and multiple surfaces of four cages that housed a strain J166-infected source monkey during the previous 24 h. Swabs were streaked on brucella agar plates with ABPNV, and H. pylori was identified in the usual manner. Sampling was repeated on three occasions. H. pylori was isolated from a fecal swab on one occasion and was demonstrated by Rep-PCR to be strain J166. No other samples were positive. We also swabbed feces (n = 19) and numerous environmental surfaces (n = 22) in outdoor field cages, which measure 100 by 200 ft and which house approximately 60 to 120 monkeys, most of which are infected with H. pylori (19). All except one of the samples were negative for H. pylori; one fecal swab was positive.

Transmission of H. pylori during social housing. We previously showed that the incidence of H. pylori transmission to socially housed newborn macaques was 40% by 12 weeks of age and 90% at 1 year of age (20). Since H. pylori transmission may be different among newborn animals whose social exposure during the first year of life is primarily to their dam, we examined H. pylori transmission in 1- to 3-year-old SPF animals (n = 6) that were socially housed with naturally infected monkeys (n = 11) of similar ages. All 17 animals were housed together for 3 months in a "corncrib." Biopsy specimens were obtained from the SPF animals approximately every 2 weeks. By the end of the observation period, four of six SPF monkeys were infected. Kaplan-Meier survival analysis (Fig. 3) showed that the transmission of H. pylori among the 1- to 3-year-olds in the corncrib occurred at a rate comparable to that observed previously in newborn monkeys socially housed together with their dams (20). Since newborns and juveniles differ not only in age but also by the presence of breast-feeding (newborns are uniformly breast-fed but juveniles are not) and the nature of their social contact (dam versus peers), these data suggest that transmission of H. pylori in this setting is not affected by these variables.


Figure 3
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  		FIG. 3. Kaplan-Meier curves showing percentage of macaques uninfected with H. pylori over time during social housing of 1- to 3-year-old animals (•; n = 6) and newborn animals ({circ}; n = 20) studied during the first 180 days of life (20).

Fecal challenge. Although we and others have infrequently isolated H. pylori from feces, transmission might occur via an infectious but not cultivatable form of the organism. To address this possibility, three SPF monkeys were inoculated with a pooled 20% (wt/vol) suspension (in brucella broth) of feces collected from six monkeys naturally infected with H. pylori. The suspension was passed once through a sterile tea strainer to remove large particles, and then 20 ml of the freshly prepared suspension was administered by gavage to each animal every other day for 3 days. Culture of the suspension on ABPNV was negative for H. pylori. Antrum and corpus biopsy specimens collected 2 and 4 weeks after challenge were negative for H. pylori in all three challenged animals. Control cultures of feces spiked with laboratory-grown H. pylori showed that approximately 50% of the inoculum was recovered 1 h after mixing and filtering, which suggests that viable H. pylori was not affected by the preparation.

Perspective. Rhesus monkeys in a social environment with a high prevalence of H. pylori rapidly acquire infection (Fig. 3), as do humans, even though the organism cannot typically be cultivated from saliva, feces, or environmental surfaces. The rare cultivation of H. pylori from fomites or transmission without social contact and the inability to infect animals with direct inoculation of feces from infected animals suggest that direct animal-to-animal contact is generally required for transmission. Since the concentrations of H. pylori in vomitus are compatible with what is known about the infectious dose in macaques (21) and humans (5), our results are most consistent with an oral-oral means of transmission.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grant RR-14298 to J.V.S. from the National Center for Research Resources.


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Comparative Medicine, University of California, Davis, Davis, CA 95616. Phone: (530) 752-1333. Fax: (530) 752-7914. E-mail: jvsolnick{at}ucdavis.edu. Back


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Journal of Clinical Microbiology, October 2006, p. 3799-3803, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.01482-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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