Isolation of bacteria other than Helicobacter pylori from stomachs of squirrel monkeys (Saimiri spp.) with gastritis.

Gastric biopsy specimens obtained from 12 squirrel monkeys (Saimiri spp.) were investigated by culture for the presence of bacteria. The stomachs of two monkeys with gastritis were colonized with gram-negative, urease-positive bacteria, identified as Ochrobactrum anthropi by the Vitek and API NFT methods (BioMérieux). A third monkey with gastritis was positive for Aeromonas salmonicida and Pseudomonas vesicularis (both urease-negative). No Helicobacter pylori was isolated from squirrel monkeys. Light microscopic and transmission electron microscopic examination revealed that the O. anthropi isolates were covered by extracellular material, indicating a capsule. Characterization of the O. anthropi urease revealed Michaelis-Menten constants (Km values) of 6.2 and 4.0 mM urea for the ureases of O. anthropi isolates S664 and S1835, respectively, and 3.7 for type strain 49188. Western blot analysis using H. pylori- and H. felis-specific antibodies detected shared antigenic epitopes between the ureases of H. pylori, H. felis, and O. anthropi. The apparent molecular mass of the urease enzymes of the O. anthropi isolates was determined on 6% nondenaturing gels to be approximately 82 kDa. Antimicrobial susceptibility tests, using the MicroScan method (Dade International), revealed multidrug resistance for the O. anthropi isolates with susceptibilities for the antibiotics amikacin, ciprofloxacin, gentamicin, cefoperazone, tobramycin, imipenem, and trimethoprim/sulfamethoxazole.

Molecular epidemiology, pathogenesis and prevention of gastric cancer.

Cancer of the stomach is one of the most commonly diagnosed malignancies and remains an important cause of mortality world wide. This type of cancer is not uniformly distributed among populations but shows a marked variation in both incidence and mortality. Although gastric cancer is declining in many parts of the world, the reasons for this decline are not well understood and its etiology remains unclear. Several factors are suspected to play a role in gastric carcinogenesis, including the effects of diet, exogenous chemicals, intragastric synthesis of carcinogens, genetic factors, infectious agents and pathological conditions in the stomach (such as gastritis). A new look at the results of epidemiological and experimental studies is important for the establishment of strategies for control. Since cancer of the stomach has a very poor prognosis in its more advanced stages, such a control program must have its main focus on primary prevention. This review describes our knowledge about cancer of the stomach regarding epidemiology, pathogenesis and prevention.

Helicobacter pylori in liquid culture: evaluation of growth rates and ultrastructure.

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (.01% wt.vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140-200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 10 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs, clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at -80 degrees C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order).

Gastric ulcer associated with a Helicobacter-like organism in a cougar (Felis concolor).

A 12-year-old captive female cougar (Felis concolor) died following perforation of a gastric ulcer. Histologically, erosions and ulcers were present in the antral area of the stomach. Fibroplasia and infiltrates of neutrophils bordered the ulcers. Modified Steiner silver stain revealed numerous tightly coiled helical bacteria. The bacteria stained positively with a rabbit polyclonal anti-Helicobacter pylori antibody. Morphology, location, and positive immunohistochemical staining suggests that the organism is a Helicobacter.

Immunogenicity and safety of recombinant Helicobacter pylori urease in a nonhuman primate.

Groups of squirrel monkeys (Saimiri spp.), predetermined to be free of Helicobacter infections in the gastric mucosa, were immunized orally with 0.5-4.5 mg of Helicobacter pylori recombinant urease (rUrease) and 25-500 micrograms of Escherichia coli heat-labile enterotoxin (LT) adjuvant. Oral immunization with rUrease resulted in a markedly elevated serum immunoglobulin G (IgG) antibody response with peak levels at 45 days after immunization. No significant gastric inflammation or cytotoxicity was evident in rUrease immunized monkeys as determined by light and electron microscopy. Twenty-five micrograms of LT was a sufficient and safe adjuvant dosage, whereas higher dosages resulted in diarrhea and lethargy. Animals developed a serum IgG antibody response to LT that did not impede the production of anti-rUrease antibody levels. The results of this investigation indicate that rUrease is immunogenic in a nonhuman primate.

Adaptation of the [13C]urea breath test as a noninvasive method for detection of Helicobacter pylori infection in squirrel monkeys (Saimiri spp.).

The [13C]urea breath test was adapted for use in squirrel monkeys (Saimiri spp.) for identification of experimentally induced infection with Helicobacter pylori, the bacterium causing gastric ulcer in humans. A canine anesthesia inhalation mask was modified with a volume-reducing insert allowing sufficient breath collection from these small primates within 30 sec. Fourteen milligrams of [13C urea per kilogram of body weight was adequate for clear distinction between experimentally infected and noninfected animals. Initial infection of five squirrel monkeys resulted in increased 13CO2 in breath within 3 days after inoculation with H. pylori. Additional inoculation with H. pylori superimposed on an existing gastric population caused a transient increase in breath 13CO2 values, which gradually declined over the following 15 days. Breath test results indicating H. pylori infection were confirmed by high [13C] concentration in blood, by urease-positive culture, modified Steiner stain reaction, and Western blot analysis. This modified [13C]urea breath test provides a rapid, reproducible, noninvasive method for screening small primates used as nonhuman models for the study of gastric infection with H. pylori.

Attachment of Mycoplasma mobile 163 K to piscine gill arches and rakers--light, scanning and transmission electron microscopic findings.

Explant cultures of gill arches and rakers were established to evaluate the attachment and colonization characteristics as well as the cytotoxic effects of the piscine bacterium, Mycoplasma mobile 163 K on piscine gill epithelium. Light, scanning and transmission electron microscopy were applied in this study and revealed heavy colonization of mycoplasmas on gill rakers, resulting in severe tissue damage of the gill epithelium. The complications for the function of the gills during breathing, the mechanisms of cytotoxicity, and the validity of this newly-established in vitro model are discussed in detail. In addition, anatomical specialities designated as spikes were identified on the inner surface of the gill rakers from trout; these could be used in the differentiation of fish.

Characterization of cytopathogenicity of aquarium seal mycoplasmas and seal finger mycoplasmas by light and scanning electron microscopy.

Two recent mycoplasma isolates (one from an aquarium seal and one from a patient with the clinical entity known as seal finger) have been evaluated for cytopathogenicity in tracheal explant cultures. Examinations were performed in direct comparison to Mycoplasma phocidae, an isolate from an epidemic of seal pneumonia along the New England coast (USA) in 1979-1980. The study revealed similar inhibitory effects on ciliated tracheal epithelial cells and clumping of cilia by attached mycoplasmas; no exfoliation of respiratory epithelial cells was observed. In addition, cytopathic effects caused by the mycoplasmas were distinguished from effects of aging in non-infected explants under long-term in-vitro cultivation conditions. The general meaning of mycoplasmas in seals is discussed in detail in this paper.

Cytopathogenicity of Mycoplasma fermentans (including strain incognitus).

Mycoplasma fermentans strain incognitus, an organism recently identified in tissues of patients with AIDS and in tissues of otherwise healthy adults with an acute fatal respiratory disease, was evaluated for cytopathogenicity for tracheal tissue in vivo and in vitro. In this study, the organism produced a chronic infection of the lower respiratory tract in LEW rats following intranasal inoculation and induced both ciliostasis and cytopathology in experimentally infected tracheal explants from rats. The time of onset of ciliostasis, type of cytopathogenicity, and localization of organism in strain incognitus were different from those in other strains of M. fermentans as well as other species of mycoplasmas isolated from humans. The results strongly support, but do not prove, that M. fermentans strain incognitus is an unusually invasive mycoplasma, as it was the only strain found within respiratory epithelial cells both in vivo and in vitro. Detection of the organism within the lamina propria also supported the organism's invasive potential. Further study of both the in vivo and in vitro models should provide insights into this potentially unique mycoplasma-host relationship.

Protein and antigen variability among strains of Mycoplasma arthritidis.

Six strains of Mycoplasma arthritidis isolated from different host tissues were examined for differences in their proteins and antigens by using one- and two-dimensional electrophoretic techniques as well as immunoblotting. One-dimensional electrophoresis revealed differences in concentrations of individual bands, but not differences in the overall banding pattern. By two-dimensional electrophoretic analysis, 25 proteins were identified as strain-variable whereas the majority of protein spots was strain-constant (about 195 after IEF-2D-PAGE and 145 after NEPHGE-2D-PAGE). Immunoblot analysis using an antiserum against the type-strain of Mycoplasma arthritidis (PG 6) revealed size-heterogeneity of antigens of all six strains. An epitopic relationship between these size-variant antigens could be demonstrated by using monospecific antibodies produced against some of these antigens of Mycoplasma arthritidis. Furthermore, we describe a highly variable antigen of Mycoplasma arthritidis similar to that shown previously in Mycoplasma pulmonis.

Cytopathic effects of Mycoplasma pulmonis in vivo and in vitro.

This study was performed to evaluate the cytopathic features resulting from Mycoplasma pulmonis infection of tracheal organ cultures compared to with those seen in in vivo infection and to use this system to determine possible differences in cytopathic effects in two M. pulmonis variants found to cause different diseases in vivo. The attachment of M. pulmonis to respiratory epithelium was similar in vivo and in vitro. Cytopathic effects seen in both systems were also similar in loss of tight junctions between cells and exfoliation of respiratory cells, resulting in exposure of the subepithelial layer. These similarities indicate that the observed tissue damage is initiated by the mycoplasmas rather than by immunologic host responses but does not exclude the possibility that host responses may subsequently contribute to the cytopathological events. Comparison of the effects of the two variants (one known to cause death in vivo) did not reveal differences in vitro. This suggests that host factors (not present in vitro) may account for differences in virulence. Detailed in vitro studies allowed the identification of the time frame corresponding to the in vivo infection and also revealed the limitations of the in vitro system.

Protein and antigen heterogeneity among strains of Mycoplasma fermentans.

The proteins and antigens of three strains of Mycoplasma fermentans were compared with those of a mycoplasma, designated "Mycoplasma incognitus," recently identified in tissues of AIDS patients. Previous studies have shown that "M. incognitus" is most likely not a new species but rather a strain of M. fermentans. In the present study, one- and two-dimensional electrophoretic analysis demonstrated the expected similarity between these mycoplasmas, but it also demonstrated several distinct protein differences. Nine proteins were identified as strain variable by two-dimensional gel electrophoresis. Also, immunoblot analysis using rabbit antiserum against the type strain of M. fermentans (strain PG 18) documented the occurrence of size heterogeneity in at least one and possibly two other antigens.