Adherence protects the binding sites of Helicobacter pylori urease from acid-induced damage.

Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.

Enhanced reduction of Helicobacter pylori load in precolonized mice treated with combined famotidine and urease-binding polysaccharides.

The present study investigated the effect of a model urease-binding polysaccharide in combination with a histamine H(2) receptor antagonist on Helicobacter pylori colonization in vivo. Euthymic hairless mice were treated daily with dextran sulfate via drinking water and/or famotidine via intragastric gavage starting at 1 week postchallenge with a CagA(+) VacA(+) (type 1) strain of H. pylori. Treatment of precolonized mice for 2 weeks with dextran sulfate combined with famotidine yielded a group mean bacterial load (per 100 mg of gastric tissue) of log(10) 1.04 CFU, which was significantly lower than those of the famotidine (log(10) 3.35 CFU, P < 0.01) and dextran sulfate (log(10) 2.45 CFU, P < 0.05) monotherapy groups and the infected nontreated group (log(10) 3.64 CFU, P < 0.01). Eradication was achieved after 2 weeks of treatment in 50% or more of the test mice using drug combinations (1 or 2 weeks of famotidine plus 2 weeks of dextran sulfate) versus none in the monotherapy and positive control groups. The enhanced activity of the drug combination may be related to the daily pattern of transient acid suppression by famotidine inducing periodic bacterial convergence to superficial mucus sites penetrated by dextran sulfate from the lumen. Increased urease-dextran sulfate avidity was observed in vitro in the presence of famotidine and may partly account for the enhanced activity. With potential utility in abbreviating treatment time and eradication of antibiotic-resistant strains, the use of urease-targeted polysaccharides concurrently with a gastric acid inhibitor warrants consideration as an additional component of the standard multidrug chemotherapy of H. pylori infection.

Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides.

BACKGROUND & AIMS: The significance of acid-primed recognition of ligands by Helicobacter pylori urease is unknown. This study aimed to further characterize the specificity of urease adherence in vitro and verify whether specific inhibition will translate into in vivo suppression of colonization. METHODS: A highly sensitive competitive enzyme-linked ligand capture assay was used to quantify the capacity of each test inhibitor to compete with labeled mucin for binding sites on immobilized native urease. A model polymer that strongly bound urease was used in an in vivo trial using euthymic hairless mice as an infection model. RESULTS: The blockage of urease-gastric mucin interaction by certain inhibitors revealed an acid-functional lectin-like activity by urease, specifically recognizing bacterial lipopolysaccharides and certain species of polysaccharides, nonbacterial glycolipids, and glycoproteins. Dextran sulfate significantly (P < 0.01) suppressed colonization of mice by H. pylori when given before and/or after challenge. CONCLUSIONS: The acid-driven high-affinity adherence of H. pylori urease to mucin and lipopolysaccharides contributes to gastric mucosal colonization by the bacterium based on in vivo targeting experiments using specific polysaccharides in a mouse model with acute infection. Acid-functional urease-homing polysaccharides that can interfere with urease-mucin or H. pylori whole cell-mucin interaction in vitro can significantly interfere with colonization by the bacterium in vivo.

Affinity purification of Helicobacter pylori urease. Relevance to gastric mucin adherence by urease protein.

A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.

A euthymic hairless mouse model of Helicobacter pylori colonization and adherence to gastric epithelial cells in vivo.

The hairless mouse strain NS:Hr/ICR was examined as a potential small animal model of Helicobacter pylori colonization, adherence to gastric epithelial cells in vivo, and gastritis. Among several small animals tested, NS:Hr/ICR mice proved to be the most highly susceptible to H. pylori infection. Challenge with clinical isolates of H. pylori consisting of either phenotype I or II (VacA and CagA positive and negative, respectively) resulted in colonization by mucus-resident and epithelial cell-adherent bacterial populations. Cell-adherent bacteria resisted 80 cycles of top-speed Vortex washing and were recovered only by homogenization of serially washed glandular stomach tissue, indicating intimate association with the mucosal surface. Immunoperoxidase staining of paraffin sections of gastric tissue from infected mice revealed H. pylori antigens localized in the glandular region of the mucosa, with some colonized areas seen in the vicinity of submucosal mononuclear cell infiltration. The latter inflammatory reaction was observed as a function of the H. pylori phenotype (only type I induced inflammation) and the challenge dose (only those mice challenged with 10(8) CFU or higher showed the reaction). The NS:Hr/ICR strain of mice is a suitable miniature model of H. pylori infection and may prove useful in the quest for an efficacious mode of treatment for this common infection in humans.

Oral passive immunization against experimental salmonellosis in mice using chicken egg yolk antibodies specific for Salmonella enteritidis and S. typhimurium.

The efficacy of chicken egg yolk homotypic antibodies specific for outer membrane proteins (OMP), lipopolysaccharide (LPS) or flagella (Fla) in controlling experimental salmonellosis in mice was investigated. Mice challenged orally with 2 x 10(9) c.f.u. of Salmonella enteritidis or 2 x 10(7) c.f.u. of S. typhimurium were orally treated with 0.2 ml anti-OMP, -LPS or -Fla yolk antibody three times a day for three consecutive days. In mice challenged with S. enteritidis, antibody treatment resulted in a survival rate of 80%, 47% and 60% using OMP, LPS or Fla specific antibodies respectively, in contrast to only 20% in control mice. In the S. typhimurium trial, survival rate was 40%, 30% and 20% using OMP, LPS or Fla specific antibodies respectively in contrast to 0% in control mice. In vitro adhesion of S. enteritidis and S. typhimurium to HeLa cells was significantly reduced by anti-OMP, -LPS, and -Fla homotypic antibodies. Results suggest that egg yolk antibodies specific for Salmonella OMP, LPS, and Fla may protect mice from experimental salmonellosis when passively administered orally. Of these antibodies, anti-OMP exhibited the highest level of protection in vivo and in vitro.

Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies.

OBJECTIVE: To protect neonatal calves against fatal salmonellosis within the first 2 weeks after birth, using chicken egg yolk antibodies specific against Salmonella typhimurium or S dublin. ANIMALS: 38 neonatal Holstein calves from Salmonella-free farms. PROCEDURE: After removal of the lipid components with hydroxypropylmethylcellulose phthalate, egg yolk antibodies were spray dried. At 4 days of age, calves were challenge exposed by oral inoculation with 10(11) virulent S typhimurium (experiment 1) or S dublin (experiment 2). Starting from the challenge-exposure day, egg yolk antibody preparations were administered orally 3 times a day for 7 to 10 days. RESULTS: In passive immunization trials, the orally administered antibodies conferred dose-dependent protection against infection with each of the homologous strains of Salmonella. Within 7 to 10 days after challenge exposure, all control calves died, whereas low-titer antibody-treated calves had 60 to 100% mortality. Only fever and diarrhea, but no deaths (P < 0.01), were observed in calves given the highest titer of antibody. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with that in control calves, survival was significantly higher among calves given antibodies with titers of 500 (P < 0.05) and 1,000 (P < 0.01) homotypic for S typhimurium and with titer of 5,000 (P < 0.01) for S dublin. Egg yolk antibodies specific for whole cell S typhimurium or S dublin are protective against fatal salmonellosis when given in sufficiently high concentration, and may be clinically useful during a salmonellosis outbreak.

Effect of oral egg antibody in experimental F18+ Escherichia coli infection in weaned pigs.

The protection conferred by egg antibody specific for F18-fimbriae against infection with F18+ Escherichia coli was studied in controlled passive immunization trials involving weaned pigs. Parameters of protection consisted of body weight gain, frequency and severity of diarrhea and recovery of the challenge strain of F18+ E. coli. Weaned pigs at four weeks of age were challenge exposed once daily for three days by oral inoculation with 10(11) cfu of virulent F18+ E. coli followed by daily administration of antibody supplemented feed for 9 days starting from the first challenge day 0. Results showed a dose-dependent response to antibody treatment. The group of pigs given 1:50 titer of antibody in feed had less frequency of diarrhea (P < 0.01-0.05), higher rate of gain (P < 0.01) and lower isolation rate of challenge strain in rectal and intestinal swabs (P < 0.01) compared to non-treated control. In the same manner, the anti-F18 antibody significantly reduced adherence of F18+ E. coli to pig intestinal epithelial cells in vitro (P < 0.01). Results suggest that egg antibodies specific for the F18 fimbriae is a suitable immunotherapeutic agent for pigs infected with F18+ E. coli and that pigs can be protected from overt clinical disease and the subsequent reduced performance arising from infection with this pathogen.

Field evaluation of chicken egg yolk immunoglobulins specific for bovine rotavirus in neonatal calves.

The oral efficacy of chicken egg yolk immunoglobulins (yIg) specific for bovine rotavirus (BRV) serotypes G6 and G10 in protecting neonatal calves was examined in a herd of cattle under field conditions. In one of the three trials, yIg-treated calves tested under high relative humidity (RH) showed a significantly increased mean body weight (P < 0.05) and a decrease in number of calves shedding high titer of BRV (G6) in stool compared to control calves (P < 0.01), suggesting that our yIg product was effective in a field condition with an epidemic outbreak of BRV diarrhea.

Passive protection of neonatal calves against bovine coronavirus-induced diarrhea by administration of egg yolk or colostrum antibody powder.

The protective effect of egg yolk and colostrum powders prepared from hens and cows vaccinated with inactivated bovine coronavirus (BCV) antigen was evaluated in a challenge model with a virulent BCV strain. Twenty three calves from BCV-free herds were randomly divided into control and several treatment groups. All calves were orally challenged with 1 x 10(9) TCID50 of the virulent Kakegawa strain of BCV at 24 to 36 h after birth. Calves in treatment groups received either egg yolk powder or cow colostrum containing BCV specific antibodies. Daily treatment with these antibody preparations started 6 h until 7 days post-challenge. Control calves which received no antibody had severe diarrhea and all died within 6 days after infection. In contrast, calves fed milk containing egg yolk or colostrum with neutralization titers of 1:2560 or 1:10,240 respectively all survived and had positive weight gain unlike the other treatment groups. These results indicate that the orally administered egg yolk and colostrum powders protected against BCV-induced diarrhea in neonatal calves and that the egg yolk used provided a higher degree of protection compared to colostrum powder on a titer basis. Treatment with whole egg yolk from immunized hens therefore provides a more efficacious alternative to the existing methods of specific passive protection against BCV.

Passive protection against bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg yolk.

Chicken egg yolk immunoglobulins (yIg) specific against bovine rotavirus (BRV) serotypes 6 (strain Shimane) and 10 (strain KK-3) were used for oral passive immunization of suckling mice against experimental BRV challenge. The protective capacity of the antibody preparation was tested using different concentrations of yIg against a challenge dose of 10(7.5) TCID50 for Shimane and 10(7.0) TCID50 for KK-3 strain. There was a significant homotypic (P < 0.05) and heterotypic (P < 0.01) protection using 160 anti-Shimane or 160 anti-KK-3 neutralizing antibody titer (NAT) compared to control mice given yIg derived from eggs of mock-immunized (control) hens. The titer of infectious BRV recovered from intestinal tissue or luminal chyme decreased with increasing homotypic yIg NAT. A decrease in degree and duration of BRV antigen localization in the villus epithelial lining was observed in mice treated with homotypic yIg at optimum dose for prevention of diarrhea. The NAT in sera of challenged mice increased with decreasing NAT in the yIg given before challenge suggesting that protection was dose-dependent. The present findings indicate that a passive protection could be achieved by the use of yIg against BRV-induced diarrhea in this murine model.

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.

Relationship of prevalence and intensity of infection to morbidity in schistosomiasis japonica: a study of three communities in Leyte, Philippines.

To determine whether prevalence and intensity of infection are factors in morbidity in schistosomiasis japonica, a cross-sectional study was undertaken in three villages in Leyte, Philippines, namely, Santol (A), Santa Rosa (B), and Macanip (C). Kato thick-smear fecal examination and egg counts were made on 289 of 341 residents in Village A (85%), 824 of 1,008 in Village B (82%), and 1,113 of 1,241 in Village C (90%). Prevalences of 26%, 39%, and 44%, respectively, were found in the three villages, the majority of their populations (56-74%) remaining uninfected. Most of the infected persons (17-30% of the total population) had light infections (10-100 eggs/g feces). Moderately infected persons (101-400 eggs/g) comprised a smaller segment (7-14%), while a very small proportion (2-7%) had heavy infections (greater than or equal to 401 eggs/g). Age prevalence and egg excretion peaked earlier in the areas with higher prevalence (B and C) than in the area with the lowest prevalence (A). There was no relationship between area prevalence and mean egg count. Symptoms of inability to work, weakness, abdominal pain, and diarrhea correlated with the presence of infection in the area with the highest prevalence (C), but not in the area with the lowest prevalence (A). Except for diarrhea, there was no relationship between symptoms and intensity of infection. Very few persons presented with hepatomegaly and/or splenomegaly (1-5%). The frequency of liver enlargement on the midsternal (measuring 3-6 cm and 6 cm or more) and midclavicular line (2-4 cm), as well as spleen enlargement (Hackett 2 or greater), correlated with the presence but not with the intensity of infection. Hepatomegaly was sex- and age-related, being most common among males and among adolescents aged 10-14 years.