Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a newly recognized bacterium associated with chronic active hepatitis, hepatic carcinoma, and inflammatory bowel disease in mice. Currently, fecal or tissue PCR, fecal culture, or histologic examination of silver-stained liver sections is used to diagnose H. hepaticus infection. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of H. hepaticus infection in mice with a membrane digest preparation of H. hepaticus as the antigen. Sera from mice positive for H. hepaticus by PCR or histologic examination (n = 88), positive for Helicobacter bilis by PCR (n = 13), positive for other helicobacters (not identifiable to species level) by PCR (n = 25), or negative for all Helicobacter species by PCR (n = 162) were used to evaluate the ELISA. Results indicated that ELISA provided 93.2% sensitivity, 94% specificity, 87.2% positive predictive value, and 96.9% negative predictive value. Cross-reactive antibodies were detected in some mice infected with helicobacters not identifiable to species level. To further define ELISA sensitivity and specificity, groups of 10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter muridarum, or H. bilis. Sera were collected and examined by the ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and maintained ELISA reactivity throughout the 18-week study, while mice infected with H. muridarum and H. bilis were negative by ELISA. These results indicate that this reported ELISA is highly sensitive and specific for the serodiagnosis of H. hepaticus infection in mice.

Behavioral and physiologic effects of inapparent wound infection in rats.

There is a common notion that rats are resistant to postoperative wound infection because many recover from surgery performed under nonsterile conditions. As a result, nonaseptic surgical techniques are used commonly in rat surgery. Our aim was to determine if these techniques cause wound infection and, if so, whether or not the infection, inapparent to casual observation, creates measurable changes in rat physiology and behavior. Rats subjected to craniotomies or laparotomies and inoculated with 10(8) Staphylococcus aureus or Pseudomonas aeruginosa or sterile saline were tested for open-field activity, freezing behavior, home-cage behavior score, and wheel-running activity. Physiologic indices included lactate dehydrogenase, blood glucose, plasma fibrinogen, complete blood counts, wound bacterial counts and histology scores, body temperature, and body weight. Although no clinical signs were detected by postoperative observation, rats inoculated with bacteria were significantly less active in the open field and the duration of freezing behavior was shorter. Plasma fibrinogen, serum glucose, total white blood cell counts, and wound histology scores were significantly altered in the bacteria-inoculated rats. These findings underscore the need for sterile techniques in rat surgery to avoid confounding experimental data.

Evaluation of subcutaneous chambers as an alternative to conventional methods of antibody production in chickens.

We compared antibody levels among serum, egg yolk extract, and granuloma fluid in chickens immunized with bovine serum albumin (BSA). One group of hens was immunized by intramuscular and subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, followed by two subsequent booster injections in incomplete Freund's adjuvant. Two other groups were surgically implanted with plastic, perforated wiffle balls (subcutaneous chambers). After a 30-day recovery period, one of the groups with subcutaneous chambers was immunized with BSA in sterile water with two subsequent boosts. The other group was injected with only sterile water. Serum samples, eggs, and granuloma fluid were collected biweekly and analyzed to determine specific IgG, total IgG, and total protein. The subcutaneous chambers were well tolerated. Quantitative ELISAs of serum, egg yolk extract, and granuloma fluid specimens disclosed that specific antibody levels were present in all specimens by 2 weeks after primary immunization. During the course of the experiment, specific antibody levels of serum and egg yolk specimens were significantly higher than those of granuloma fluid (P less than 0.05). However, an additional injection of antigen into the subcutaneous chambers resulted in specific antibody levels in granuloma fluid specimens that were comparable to those of serum and egg yolk extract. Use of subcutaneous chambers in chickens may be a viable alternative to routine antibody production methods.

Evaluation of a subcutaneously implanted chamber for antibody production in rabbits.

Polyclonal antibody production in subcutaneous chambers was compared to traditional antibody production methods in rabbits. The chamber, a sterilized plastic wiffle golf ball that had been surgically implanted in the subcutis of the thoracic region, was immunized via a percutaneous injection of antigen into the core of the ball through one of the perforations in the chamber wall. Rabbits bearing chambers were immunized on the same schedule and with the same concentrations of antigens as were provided the adjuvant injected rabbits. Fluid volumes of 12 to 22 ml could be removed from each chamber at weekly intervals. Chamber antibody to specific microbial antigens was equal to or better than serum antibody produced to the same antigens with Freund's or acrylamide adjuvants. The comfort of the rabbit, the ease in chamber immunization, and the recovery of high titer antibody in large volumes make the subcutaneous chamber an attractive method for polyclonal antibody production.

Treatment of Trichophyton mentagrophytes infection in rabbits.

Copper sulfate and a metastabilized chlorous acid/chlorine dioxide (MECA) compound were evaluated for efficacy in treating Trichophyton mentagrophytes infection in rabbits. Forty rabbits naturally infected with T mentagrophytes were allotted to 5 treatment groups: nontreated control, sham dip, 1% copper sulfate dip, MECA dip, and MECA spray. Results of lesion culture, regression of gross lesions, and histopathologic findings were documented. Copper sulfate and MECA were effective in treating clinical dermatophytosis and in decreasing T mentagrophytes carrier status in rabbits.

Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node.

Bacterial translocation is defined as the passage of indigenous bacteria from the gastrointestinal (GI) tract through the lamina propria to the mesenteric lymph nodes (MLN) and other organs. We compared the relative abilities of various aerobic, facultatively anaerobic, and obligately anaerobic bacteria to translocate from the GI tract to the MLN in gnotobiotic mice colonized with single strains of bacteria. Indigenous gram-negative enteric bacilli translocated in large numbers to the MLN, whereas gram-positive bacteria translocated at intermediate levels and obligately anaerobic bacteria at only very low levels. Our results suggest that enteric bacilli such as Escherichia coli, Proteus, and Enterobacter are associated with a higher incidence of bacteremia in debilitated patients, because these bacteria translocate more efficiently from the GI tract than do other bacteria, especially obligate anaerobes.

Efficacy of commercial vaccines for protecting guinea pigs against Bordetella bronchiseptica pneumonia.

Autogenous bacterins are recommended to protect guinea pigs (Cavia porcellus) against pneumonia due to Bordetella bronchiseptica. Bordetella vaccines are available commercially for several other animal species. The substantial antigenic cross-reactivity among Bordetella isolates from various animal species suggests that immunity resulting from use of these vaccines might protect guinea pigs. Groups of ten individually housed Hartley guinea pigs from a colony free of Bordetella were vaccinated with one of two commercial porcine B. bronchiseptica vaccines, a human DPT vaccine (which includes a Bordetella pertussis component), or an autogenous B. bronchiseptica bacterin. Twenty-one days following vaccination, the animals were challenged with an intranasal dose of 10(6) virulent B. bronchiseptica cells. The animals were euthanized and necropsied 15 days after challenge. The nares, nasopharynx, distal trachea and lungs were cultured. All nonvaccinated control animals developed acute signs of pneumonia, while none of the vaccinated animals developed clinical signs of disease or gross lesions. The frequency of B. bronchiseptica isolation from the lungs of animals in each vaccine group was reduced. However, approximately 70% of all animals in each vaccine group harbored B. bronchiseptica in the trachea, and almost all harbored B bronchiseptica in the nares and nasopharynx. The porcine vaccines appeared to afford protection against acute pulmonary disease in the guinea pig.

Salmonella enteriditis serotype Amsterdam in a commercial rat colony.

Salmonella enteriditis serotype Amsterdam was isolated from the feces of 10 of 25 barrier maintained Fischer 344 rats in a commercial colony of over 35,000 animals. During the subsequent 3-month period, a small percentage of asymptomatic rats remained culture-positive for Salmonella despite an extensive program of killing animals with positive fecal cultures. The failure of this procedure to eradicate Salmonella from the colony led to the eventual destruction of the entire colony. Salmonella enteriditis serotype Amsterdam was cultured from wild rodent feces on a cracked well head casing which covered the shaft to the well that provided water for the barrier facility; an identical Salmonella serotype was also isolated from an animal care technician working with animals in this barriered colony. The technician was asymptomatic, and the organism was cultured on only one occasion. This outbreak was retrospectively attributed to water-borne Salmonella which apparently survived treatment with a faulty chlorinator.

Relationship between cecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes.

Translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes (MLN) and other organs. The extent of translocation of certain indigenous, oxygen-tolerant bacteria from the cecum to the MLN, spleen, liver, kidney, and peritoneal cavity were determined in diassociated or triassociated gnotobiotic mice. Minimal bacterial translocation occurred to the spleen, liver, kidney, or peritoneal cavity. However, most bacterial strains readily translocated to the MLN. The percentage of the total population of each bacterial strain in the ceca was compared with the percentage of the total population of that strain in the MLN. There was a direct relationship between the numbers of a particular bacterial strain populating the ceca of diassociated or triassociated mice and the numbers of viable bacteria of this strain present in the MLN. Thus, the cecal population level of a particular bacterial strain determined the numbers of viable bacteria of this strain translocating to the MLN. The translocation of these bacterial strains from the gastrointestinal tract is an important first step in the pathogenesis of infection caused by members of the normal intestinal microflora.

Kinetic analysis of bacterial clearance in mice using the ESTRIPc and KINET microcomputer programs.

Two BASIC microcomputer programs, ESTRIPc and KINET, were used to analyze the kinetics of bacterial clearance from the blood and mesenteric lymph nodes of mice. Because of the similarities between the clearance of bacteria and the clearance of drugs from tissue, blood pharmacokinetic techniques were applied to the analysis of bacterial clearance data. The ESTRIPc program, developed for pharmacokinetic analysis and modified for the study of bacterial clearance, was employed to fit the experimental data of bacterial survival versus time to a polyexponential equation with 1, 2, or 3 terms. The KINET program, written specifically for kinetic analysis of bacterial clearance, uses the biexponential equation constants derived with ESTRIPc to calculate half-life values, rate constants, and other useful kinetic parameters. The combined use of these programs permits precise comparisons of the clearance rates of different bacterial species from the blood or tissues of experimental animals.

Hydrolytic enzymes of anaerobic bacteria isolated from human infections.

Thirty-three strains of anaerobic bacteria isolated from human clinical specimens were examined for the presence of heparinase, hyaluronidase, chondroitin sulfatase, gelatinase, collagenase, fibrinolysin, lecithinase, and lipase activities. Pronounced heparinase activity was limited to species of the genus Bacteroides. A number of species of the genera Bacteroides and Clostridium produced hyaluronidase and chondroitin sulfatase. Gelatinase, collagenase, and fibrinolysin activities were encountered in isolates of the genera Bacteroides, Clostridium, and Peptostreptococcus. All strains capable of degrading collagen also hydrolyzed other protein substrates. Lipolytic activity was minimal among these anaerobic bacteria. No specific hydrolytic activity was consistently associated with the isolates.