Helicobacter pullorum outbreak in C57BL/6NTac and C3H/HeNTac barrier-maintained mice.

Helicobacter pullorum is a bacterial pathogen in humans. By using microaerobic culture techniques, H. pullorum was isolated from the feces of barrier-maintained mice and identified, on the basis of biochemical, restriction fragment length polymorphism, and 16S rRNA gene sequence analyses. This finding presents an opportunity to study H. pullorum pathogenesis in mice.

Rapid onset of ulcerative typhlocolitis in B6.129P2-IL10tm1Cgn (IL-10-/-) mice infected with Helicobacter trogontum is associated with decreased colonization by altered Schaedler's flora.

Infection with Helicobacter trogontum, a urease-positive helicobacter isolated from subclinically infected rats, was evaluated in B6.129P2-IL10(tm1Cgn) (interleukin-10(-/-) [IL-10(-/-)]) and C57BL/6 (B6) mice. In a first experiment, IL-10(-/-) mice naturally infected with Helicobacter rodentium had subclinical typhlocolitis but developed severe diarrhea and loss of body condition with erosive to ulcerative typhlocolitis within 1 to 3 weeks of experimental infection with H. trogontum. A second experiment demonstrated that helicobacter-free IL-10(-/-) mice dosed with H. trogontum also developed severe clinical signs and typhlocolitis within 2 to 4 weeks, whereas B6 mice colonized with H. trogontum were resistant to disease. In a third experiment, using helicobacter-free IL-10(-/-) mice, dosing with H. trogontum resulted in acute morbidity and typhlocolitis within 8 days. Acute typhlocolitis was accompanied by signs of sepsis supported by degenerative hemograms and recovery of Escherichia coli and Proteus spp. from the livers of infected mice. Quantitative PCR data revealed that H. rodentium and H. trogontum may compete for colonization of the lower bowel, as H. trogontum established higher colonization levels in the absence of H. rodentium (P < 0.003). H. trogontum-induced typhlocolitis was also associated with a significant decrease in the levels of colonization by five of eight anaerobes that comprise altered Schaedler's flora (P < 0.002). These results demonstrate for the first time that H. rodentium infection in IL-10(-/-) mice causes subclinical typhlocolitis and that infection with H. trogontum (with or without H. rodentium) induces a rapid-onset, erosive to ulcerative typhlocolitis which impacts the normal anaerobic flora of the colon and increases the risk of sepsis.

Progressive proliferative and dysplastic typhlocolitis in aging syrian hamsters naturally infected with Helicobacter spp.: a spontaneous model of inflammatory bowel disease.

Helicobacter spp. have been implicated in a variety of gastrointestinal tract diseases, including peptic ulcer disease, gastric cancer, and inflammatory bowel disease (IBD), in humans and animals. Although most models of IBD are experimentally induced, spontaneous or natural models of IBD are rare. Herein, we describe a long-term study of chronic, progressive lesions that develop in the distal portion of the large bowel of unmanipulated Syrian hamsters naturally infected with Helicobacter spp. Twenty-four Syrian hamsters of three age groups (group A, 1 month [n = 4], group B, 7-12 months [n = 12], group C, 18-24 months [n = 12]), underwent complete postmortem examination. Results of microbial isolation and polymerase chain reaction and restriction fragment length polymorphism analyses confirmed the presence of Helicobacter spp. infection in the distal portion of the large bowel of all animals. Additionally, confounding pathogens, such as Clostridium difficile, Lawsonia intracellularis, and Giardia spp. that can cause proliferative enteritis, were absent in the hamsters of this study. Histopathologic scores for inflammation (P < 0.01), hyperplasia (P < 0.01), and dysplasia (P < 0.05) were significantly higher in the ileocecocolic (ICC) junction of animals in group C, relative to group A. Dysplastic lesions of various grades were detected in 5 of 11 hamsters in group C. Interestingly, the segment of the bowel that is usually colonized by Helicobacter spp. in hamsters had the most severe lesions. One hamster of group C developed a malignant fibrous histiocytoma, whereas another hamster developed a round cell sarcoma originating from the ICC junction. Thus, lesions in the distal portion of the large bowel of aging hamsters naturally colonized with Helicobacter spp. warrants developing the hamster as an animal model of IBD and potentially IBD-related cancer.

Smoking prevalence and knowledge of associated risks in adult attenders at day centres for people with learning disabilities.

BACKGROUND: Increased longevity among people with learning disabilities is accompanied by an increase in morbidity. A possible explanation is that living in the community and a move to greater independence may bring higher health risks through obesity and smoking. The study aimed to see if rates of smoking have increased from earlier published rates and to ascertain the awareness of the risks of smoking among people with learning disabilities. METHODS: A total of 435 people attending four social services day centres in a large urban area were assisted to complete a questionnaire. RESULTS: Twenty-seven (6.2%) reported that they currently smoked. Those with mild disabilities were much more likely to smoke than those with more severe disabilities and they also reported smoking more heavily. For those with mild levels of learning disability, a higher than expected proportion living in hospital and staffed housing smoked, a lower proportion living with parents smoked but for those living independently the proportion who smoked was no higher than expected. Smokers were more knowledgeable about the risks than non-smokers even if the level of learning disability was controlled for. Only a third of smokers were concerned about the risks. CONCLUSIONS: The study provides no evidence that rates of smoking are increasing among people with learning disabilities nor that those living independently were more likely to smoke. Knowledge of health risks is poor across the group, but higher among the smokers who were unlikely to express concern about the risks. This may indicate that more support may be needed along with health education in this group.

Failure of surface ring mutant strains of Helicobacter mustelae to persistently infect the ferret stomach.

Helicobacter mustelae, the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genus Helicobacter, including H. pylori. The unique ring structures are composed of a protein named Hsr. To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free for H. mustelae were inoculated with an Hsr-deficient mutant strain or the wild-type H. mustelae strain. Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain. However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced. This decline contrasted the robust antral colonization by the wild-type strain. The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets. Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutant H. mustelae strain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain. Scant colonization by the Hsr-negative H. mustelae strain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response.

Helicobacter cetorum sp. nov., a urease-positive Helicobacter species isolated from dolphins and whales.

A novel helicobacter with the proposed name Helicobacter cetorum, sp. nov. (type strain MIT 99-5656; GenBank accession number AF 292378), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins (Lagenorhynchus acutus) and from the feces of three captive cetaceans (a Pacific white-sided dolphin [Lagenorhynchus obliquidens]; an Atlantic bottlenose dolphin [Tursiops truncatus]; and a beluga whale [Delphinapterus leucas]). The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy. Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals. In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria. Helicobacter cetorum is a fusiform gram-negative bacterium with a single bipolar flagellum. The isolates grow under microaerobic conditions at 37 and 42 degrees C but not at 25 degrees C. H. cetorum is urease, catalase, and oxidase positive, and it is sensitive to cephalothin. The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant. By 16S rRNA sequencing it was determined that H. cetorum represented a distinct taxon that clusters most closely with H. pylori. Further studies are necessary to determine the role of H. cetorum in the development of gastric ulcers and gastritis of cetaceans. This is the first description and formal naming of a novel Helicobacter species from a marine mammal.

Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age.

The bacterium Helicobacter pylori colonizes the gastric mucosa of half of the human population, resulting in chronic gastritis, ulcers, and cancer. We sequenced ten gene fragments from pairs of strains isolated sequentially at a mean interval of 1.8 years from 26 individuals. Several isolates had acquired small mosaic segments from other H. pylori or point mutations. The maximal mutation rate, the import size, and the frequency of recombination were calculated by using a Bayesian model. The calculations indicate that the last common ancestor of H. pylori existed at least 2,500-11,000 years ago. Imported mosaics have a median size of 417 bp, much smaller than for other bacteria, and recombination occurs frequently (60 imports spanning 25,000 bp per genome per year). Thus, the panmictic population structure of H. pylori results from very frequent recombination during mixed colonization by unrelated strains.

Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach.

Fumarate reductase (FRD) is the key enzyme in fumarate respiration induced by anaerobic growth of bacteria. In Helicobacter pylori, this enzyme appears to be constitutively expressed under microaerobic conditions and is not essential for its survival in vitro. In this study, the role of FRD in the colonization of H. pylori was investigated using a mouse model. The frdA gene coding for subunit A of FRD, and two control genes, copA and copP associated with the export of copper out of H. pylori, were inactivated by insertion of the chloramphenicol acetyltransferase cassette into these individual genes. The isogenic mutants of H. pylori strain AH244 were obtained by natural transformation. Seventy-five ICR mice (15 mice/group) were orogastrically dosed with either the wild type H. pylori strain AH244, its isogenic mutants, or Brucella broth (negative control). Five mice from each group were killed at 2, 4 and 8 weeks post-inoculation (WPI), respectively. H. pylori colonization was not detected in mouse gastric mucosa infected with the frdA mutant at any time point in the study by both quantitative culture and PCR. In contrast, the mice inoculated with either wild type AH244, copA or copPH. pylori mutants became readily infected. These data indicate that FRD plays a crucial role in H. pylori survival in the gastric mucosa of mice. Given that FRD, present in all H. pylori strains, is immunogenic in H. pylori -infected patients and H. pylori growth in vitro can be inhibited by three anthelmintics (morantel, oxantel and thiabendazole), this enzyme could potentially be used both as a novel drug target as well as in the development of vaccines for H. pylori prevention and eradication.

Cytolethal distending toxin in avian and human isolates of Helicobacter pullorum.

Helicobacter pullorum has been isolated from the feces and livers of poultry and is associated with human gastroenteritis. Discrimination of this organism from other enterohepatic Helicobacter species and Campylobacter species has proven difficult. H. pullorum from both avian and human clinical sources has DNA sequence homology and cytotoxic activity that represent a new member of the cytolethal distending toxin (CDT) family of bacterial toxins. CDT is a potential virulence factor in H. pullorum that may serve as a distinguishing phenotype and aid in identification of this organism in veterinary and human clinical samples.

Helicobacter pylori gastritis in cats with long-term natural infection as a model of human disease.

A natural infection with Helicobacter pylori (H. pylori) in domestic cats (Felis cattus) less than 2 years of age has been well described in a closed colony of animals. Six cats from this colony that were serially evaluated by culture, polymerase chain reaction, and light and electron microscopy for a period of 3 years demonstrated persistent gastric colonization with a single cag(-) vac(+) strain of H. pylori. In these cats, as well as five other 5- to 6-year-old cats that were examined, a long-term infection resulted in chronic diffuse lymphofollicular atrophic gastritis with areas of mucosal dysplasia in the antrum and predominantly midsuperficial gastritis in the body and cardia. Topographically, the distribution of lesions was similar in both young and older cats and closely resembled that found in humans, with the most severe changes occurring in the gastric antrum. Few granulocytes and no significant elevation in mast cells were seen in older H. pylori-infected cats compared with uninfected controls; however, marked increases in interepithelial globule leukocytes and numerous active mucosal lymphoid follicles were present in infected animals. Indices of gastritis were significantly greater in older infected cats when compared with uninfected controls and younger cats (P < 0.05). The antral cell proliferation index of infected older cats was significantly (P = 0.021) greater than that of uninfected controls. Apoptotic indices of the gastric antrum and body of infected cats were significantly (P = 0.01) increased versus controls. Chronic infection with H. pylori in cats shares many features of long-term H. pylori infection in humans, including the development of preneoplastic processes. This similarity provides useful, comparative insights into host-pathogen interactions.

Colonization and tissue tropism of Helicobacter pylori and a novel urease-negative Helicobacter species in ICR mice are independent of route of exposure.

BACKGROUND: In humans, Helicobacter pylori is known to colonize the stomach and to induce persistent gastritis; selected reports also suggest it causes extragastric disease, including hepatitis. H. pylori and a novel urease-negative Helicobacter sp. induce gastritis and typhlocolitis, respectively, when inoculated orally into mice. Experimental typhlocolitis and hepatitis have been caused by intraperitoneal (i.p.) injection of H. hepaticus, H. bilis, and the novel Helicobacter spp. However, the route by which i.p.-inoculated organisms localize to specific areas of the gastrointestinal system is unknown. MATERIALS AND METHODS: To determine whether Helicobacter spp. can be isolated from blood, can preferentially colonize specific tissues, and can cause pathological changes, we inoculated 6-week-old outbred mice orally or intraperitoneally with H. pylori or a novel Helicobacter sp. RESULTS: When these mice were inoculated by the i.p. route, H. pylori was cultured from lungs, spleen, liver, cecum, and stomach on day 1 after inoculation, from liver and stomach mucosa on day 3 after inoculation, and from the stomach on day 30 after inoculation, suggesting preferential colonization of the stomach. After inoculation by the i.p. route, the novel intestinal Helicobacter sp. was cultured from the blood, lungs, spleen, liver, kidneys, cecum, and feces but not from stomach mucosa on day 1 after inoculation. By day 30 after inoculation, the novel Helicobacter sp. was cultured from cecum and feces only, suggesting that it had preferentially colonized the lower bowel. By the i.p. route, the novel Helicobacter sp. induced hepatitis that persisted for 30 days after inoculation. Though mice inoculated intraperitoneally with H. pylori developed an acute hepatitis, the liver lesion began to resolve 30 days after inoculation. Mice inoculated orally with either H. pylori or the novel Helicobacter sp. did not have hepatitis on day 30 after inoculation but developed 100% colonization of stomach and cecum, respectively. CONCLUSION: The isolation of H. pylori and the novel Helicobacter sp. from multiple tissues infers that a transient helicobacter bacteremia occurs when Helicobacter spp. are injected intraperitoneally, but organisms are cleared rapidly from nontarget tissues and preferentially colonize specific regions of the gastrointestinal tract.

High-salt diet induces gastric epithelial hyperplasia and parietal cell loss, and enhances Helicobacter pylori colonization in C57BL/6 mice.

A high-salt diet in humans and experimental animals is known to cause gastritis, has been associated with a high risk of atrophic gastritis, and is considered a gastric tumor promoter. In laboratory rodents, salt is known to cause gastritis, and when coadministered, it promotes the carcinogenic effects of known gastric carcinogens. Because Helicobacter pylori has been associated with a progression from gastritis to gastric cancer, we designed a study to determine whether excessive dietary NaCl would have an effect on colonization and gastritis in the mouse model of H. pylori infection. Seventy-two, 8-week-old female C57BL/6 mice were infected with H. pylori strain Sydney, and 36 control mice were dosed with vehicle only. One-half of the infected and control mice were fed a high-salt diet (7.5% versus 0.25%) for 2 weeks prior to dosing and throughout the entire experiment. Twelve infected and 6 control animals from the high-salt and normal diet groups were euthanized at 4, 8, and 16 weeks. At 8 and 16 weeks postinfection (WPI), the colony-forming units per gram of tissue were significantly higher (P < 0.05) in the corpus and antrum of animals in the high-salt diet group compared with those on the normal diet. Quantitative urease was significantly higher (P < 0.05) at 4 and 8 WPI in the corpus and antrum of animals on the high-salt diet when compared with controls. At 16 WPI, mice in both the normal and the high-salt diet groups developed moderate to marked atrophic gastritis of the corpus in response to H. pylori infection. However, the gastric pits of the corpus mucosa in mice on the high-salt diet were elongated and colonized by H. pylori more frequently than those in mice on the normal diet. The high-salt diet was also associated with a significant increase in proliferation in the proximal corpus and antrum and a multifocal reduction in parietal cell numbers in the proximal corpus, resulting in the elongation of gastric pits. We conclude that excessive NaCl intake enhances H. pylori colonization in mice and in humans and that chronic salt intake may exacerbate gastritis by increasing H. pylori colonization. Furthermore, elevated salt intake may potentiate H. pylori-associated carcinogenesis by inducing proliferation, pit cell hyperplasia, and glandular atrophy.

Ranitidine bismuth citrate and clarithromycin, alone or in combination, for eradication of Helicobacter mustelae infection in ferrets.

OBJECTIVE: To determine whether ranitidine bismuth citrate, clarithromycin, or a combination of ranitidine bismuth citrate and clarithromycin would be efficacious in eradication of Helicobacter mustelae infection in ferrets. ANIMALS: 60 seven-month-old ferrets. PROCEDURE: To determine dosages of clarithromycin and ranitidine bismuth citrate that would suppress growth of, but not eradicate infection with, H mustelae, ferrets (n = 6/group) were treated p.o. with clarithromycin or ranitidine bismuth citrate at various dosages. Efficacy of treatment was then determined by treating ferrets with clarithromycin alone, ranitidine bismuth citrate alone, or clarithromycin and ranitidine bismuth citrate. Gastric biopsy specimens were obtained before, during, and at various times after treatment and submitted for quantitative bacterial culture and histologic evaluation. Minimum concentrations of clarithromycin that inhibited 90% of the growth of isolates obtained before and after treatment were determined. RESULTS: Dosages of clarithromycin and ranitidine bismuth citrate that suppressed growth of H mustelae were 12.5 and 24 mg/kg of body weight, p.o., every 8 hours, respectively. Infection was not eradicated in ferrets treated with ranitidine bismuth citrate alone but was eradicated in all 6 ferrets treated with clarithromycin and ranitidine bismuth citrate and in 4 of 6 treated with clarithromycin alone. A decrease in susceptibility to clarithromycin was detected for H mustelae isolates obtained after treatment. Mild or moderate antral gastritis was observed even in ferrets from which infection was eradicated. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of ranitidine bismuth citrate and clarithromycin was efficacious in eradicating H mustelae infection from ferrets.

Proliferative enterocolitis associated with dual infection with enteropathogenic Escherichia coli and Lawsonia intracellularis in rabbits.

Both enteropathogenic Escherichia coli (EPEC) and an obligate intracellular bacterium, previously referred to as an intracellular Campylobacter-like organism and now designated Lawsonia intracellularis, have been reported as causes of enterocolitis in rabbits. An outbreak of enterocolitis in a group of rabbits, characterized by an unusually high rate of mortality, was found to be associated with dual infection with EPEC and L. intracellularis. The EPEC strain was found to have eaeA gene homology but was negative for afrA homology. The absence of the afrA gene, which encodes the structural subunit for the AF/R1 pilus, indicates that this rabbit EPEC strain is distinct from the prototypic RDEC-1 strain. This finding suggests that rabbit EPEC strains widely reported in Western Europe, which lack AF/R1 pili, are also present in rabbits in the United States. Dual infection with these two pathogens in rabbits has not been previously reported and may have contributed to the unusually high mortality observed in this outbreak.

Chronic active hepatitis induced by Helicobacter hepaticus in the A/JCr mouse is associated with a Th1 cell-mediated immune response.

Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-gamma) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-gamma in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.

Inflammatory large bowel disease in immunodeficient rats naturally and experimentally infected with Helicobacter bilis.

Proliferative and ulcerative typhlitis, colitis, and proctitis were found incidentally in a breeding colony of male athymic nude (Cr:NIH-rnu) rats. Within the crypts of the large intestine, modified Steiner's silver stain revealed spiral organisms that were identified by culture, polymerase chain reaction, and sequencing to be Helicobacter bilis. The large bowel disease was reproduced in H. bilis-free male athymic nude rats that were injected intraperitoneally with a culture of H. bilis from the affected colony. The organism was isolated from the feces and cecum of the experimentally infected rats. H. bilis should be considered a potential pathogen in immunocompromised rats. The infection in immunocompromised rats may serve as an animal model for inflammatory large bowel disease.

Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis.

BACKGROUND & AIMS: Cancer of the gallbladder is the number one cause of cancer mortality in Chilean women. Incidence rates for this tumor vary widely on a worldwide basis, being approximately 30 times higher in high-risk than in low-risk populations, suggesting that environmental factors such as infectious microorganisms, carcinogens, and nutrition play a role in its pathogenesis. Because several Helicobacter sp. colonize the livers of animals and induce hepatitis, the aim of this study was to determine whether Helicobacter infection was associated with cholecystitis in humans. METHODS: Bile or resected gallbladder tissue from 46 Chileans with chronic cholecystitis undergoing cholecystectomy were cultured for Helicobacter sp. and subjected to polymerase chain reaction (PCR) analysis using Helicobacter-specific 16S ribosomal RNA primers. RESULTS: Recovery of Helicobacter sp. from frozen specimens was unsuccessful. However, by PCR analysis, 13 of 23 bile samples and 9 of 23 gallbladder tissues were positive for Helicobacter. Eight of the Helicobacter-specific PCR amplicons were sequenced and subjected to phylogenetic analysis. Five sequences represented strains of H. bilis, two strains of "Flexispira rappini" (ATCC 49317), and one strain of H. pullorum. CONCLUSIONS: These data support an association of bile-resistant Helicobacter sp. with gallbladder disease. Further studies are needed to ascertain whether similar Helicobacter sp. play a causative role in the development of gallbladder cancer.

Promotion of ulcerative duodenitis in young ferrets by oral immunization with Helicobacter mustelae and muramyl dipeptide.

BACKGROUND: The purpose of this study was to determine whether oral immunization of ferret kits with a whole-cell sonicate of Helicobacter mustelae lysate (Hml) and the adjuvant muramyl dipeptide (MDP) would reduce the incidence of natural colonization with H. mustelae and the extent of Helicobacter-associated gastritis by enhancing the host mucosal immune response. MATERIALS AND METHODS: Between the ages of 4 and 11 weeks, 44 ferret kits were gavaged with Hml and various doses of MDP. The extent of gastritis and duodenitis and the immune response to H. mustelae were evaluated. RESULTS: All kits became colonized naturally with H. mustelae and the majority developed mild to severe gastritis and duodenitis. Kits that received Hml with MDP developed significantly greater inflammation of the gastric antrum and duodenum, as compared to kits vaccinated with Hml alone. Vaccination with Hml and 50 micrograms of MDP was associated with severe lesions in the proximal duodenum characterized by accumulation of mononuclear inflammatory cells, mucosal erosion, and ulceration. Although serum antibody specific for H. mustelae in 4-week-old kits was approximately 50% of adult levels, a finding attributable to passively acquired maternal antibody, both systemic and mucosal antibody levels became depressed over time despite oral vaccination. The humoral immune response was sufficiently low to prevent detection of any significant dose effect of MDP on antibody levels among experimental groups. CONCLUSIONS: Oral vaccination of young ferrets with Hml and 50 micrograms MDP increased the risk of Helicobacter-associated mucosal ulceration in the proximal duodenum, which was associated with low humoral (but significant cell-mediated) immune responses to H. mustelae. In retrospect, the frequency of vaccination may have suppressed the systemic humoral immune response, thereby promoting mucosal damage by H. mustelae. The 50-microgram dose of MDP enhanced the cell-mediated immune response, which indirectly contributed to development of severe lesions. The increased frequency of mucosal damage associated with this vaccination regimen enhances the value of the ferret model for studying duodenal ulceration secondary to Helicobacter infection.

Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay.

The identification of a new murine pathogen, Helicobacter hepaticus, and its association with chronic active hepatitis and liver tumors prompted an evaluation of the prevalence of H. hepaticus in commercially available mice. Of the 28 different strains or stocks, totaling 160 mice from four major commercial vendors, cultured for H. hepaticus, 100% of mice from two outbred strains from one vendor were infected with H. hepaticus, whereas 9 of 13 inbred mouse strains from another vendor were infected. This high prevalence of H. hepaticus established a need for a rapid and reproducible, noninvasive assay for the screening of colony-maintained mice being used for biomedical research. The culturing of fecal material by using 0.45-microns-pore- size filtration for H. hepaticus consistently yielded reproducible results but required extended periods of time. (1 to 3 weeks) to obtain a definitive answer. Although it is rapid, the use of a direct PCR-based detection assay with fecal specimens is restricted by inhibitory agents. to circumvent these inhibitory agents and to augment our H. hepaticus culture technique, we have developed a novel PCR system in which the bacteria are isolated from fecal material in the presence of polyvinylpyropyrollidone and lysed by treatment with Chelex 100. The PCR is performed with Tth polymerase supplemented with a polymerase enhancer. By this PCR method, 24 H. hepaticus culture-positive and 30 H. hepaticus culture-negative fecal samples were correctly identified. Moreover, two samples which were PCR positive and culture negative initially were positive by both methods upon retesting of fresh material. Southern blot hybridizations and sequencing of PCR products showed them to be H. hepaticus specific. A comparison of results obtained under identical conditions indicated a 100-fold increase in sensitivity with Tth polymerase over Taq polymerase. This PCR method can be used as a noninvasive means of rapidly screening large numbers of colony mice for H. hepaticus.

Detection of Clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia.

Four specific-pathogen-free rabbits with anorexia died peracutely; decreased fecal output, nasal exudate, and labored breathing were the only other clinical abnormalities observed in two of the rabbits before death. The animals, three juveniles and one adult, were on a standard polyclonal antibody production regimen and had received immunizations approximately 2 weeks before presentation. External examination revealed distended abdomen and perineal fecal staining. At necropsy the small intestine was distended with fluid, and the cecum was distended with chyme. The small intestines and cecum had marked serosal hyperemia. Anaerobic bacterial culture techniques were used to isolate Clostridium difficile from the small intestine (3/4) and cecum (2/4). In all cases C. difficile toxin B was detected at high titers (10(2) to > 10(5)) in the small intestine by cytotoxicity assay with HeLa 229 cell culture. In two of the four rabbits C. difficile was isolated, and cytotoxin titers were detected at 10(1) and 10(4) in the cecum of affected rabbits. Toxin B was neutralized with C. sordellii antiserum but not C. spiroforme antiserum. In addition, toxin A was detected in each of the cytotoxin B-positive samples by a commercial toxin A enzyme immunosorbent assay. In vitro production of toxins A and B was detected from each culture isolate after incubation in chopped meat broth. These cases are noteworthy because spontaneous (nonantibiotic-associated) C. difficile enterotoxemia has not been previously reported in rabbits. Also the toxins of clostridial organisms are usually documented in the cecum, not the small intestine, of rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)