Correlation between megaloileitis and antibodies to Bacillus piliformis in laboratory rat colonies.

Rat colonies in which antibodies to Bacillus piliformis were detected in animals examined at the age of 8 to 15 weeks were compared with rat colonies where no such antibodies were present. The seropositive colonies had a low incidence of megaloileitis in 5-week-old rats of Sprague-Dawley stock and some few inbred strains. In seronegative colonies, no megaloileitis was detected. In rats with megaloileitis, significantly high titers to B. piliformis were noted and the agents could be identified in the ileal mucosa by immunofluorescence technique.     

Demonstration of antibodies to Bacillus piliformis in SPF colonies and experimental transplacental infection by Bacillus piliformis in mice.

Antibodies to Bacillus piliformis were demonstrated by the immunofluorescence antibody technique in sera from mice and rabbits from SPF breeding colonies. Mice in various stages of pregnancy were experimentally infected with Bacillus piliformis and killed 2 to 3 days later. The organism was demonstrated in the uterus, foetal membranes and in the liver of the foetuses. Infection was not limited to any particular stage of pregnancy.     

Detection of serum antibodies to Bacillus piliformis in mice and rats using an enzyme-linked immunosorbent assay.

Rats and mice were infected with Bacillus piliformis organisms at a dosage which resulted in clinical signs of Tyzzer's disease in gerbils. Although rats and mice did not show clinical signs of disease, rising antibody titers to B. piliformis were detected by enzyme-linked immunosorbent assay (ELISA) 2 to 6 weeks post-inoculation and remained at positive levels 11 weeks post-inoculation. Western blot analyses of sera from experimentally infected animals revealed banding patterns nearly identical to those obtained using hyperimmune serum. Results indicated that elevated ELISA titers reflected production of specific antibodies directed against antigens of B. piliformis. ELISA and Western blot analyses of naturally infected animals yielded similar results. These findings suggest that immunoassays such as ELISA can be used to detect subclinically infected rats and mice in the absence of clinical signs or histopathologic evidence of Tyzzer's disease.     

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.     

Novel method for screening bacterial colonies for phosphatase activity.

Current methods for screening large numbers of bacterial colonies for phosphatase activity, rely heavily on the use of colorimetric assays. While such methods have been applied extensively in the laboratory, they are not without their drawbacks. We here describe a precipitating fluorescent probe that can be used to screen phosphatase activity in bacterial colonies. This probe can be incorporated directly into agar plates used to culture the organisms of interest. The approach offers several advantages over current methodologies including the ability to monitor the development of phosphatase activity with colony development, and the ability to distinguish between activity arising from cell-bound and cell-free enzyme. This enzyme probe was successfully used to detect and isolate phosphatase-producing bacteria from activated sludge.     

Green marker for colonies of Bacillus subtilis

A Bacillus subtilis plasmid encoding a green fluorescence protein gene (gfp) was constructed. The fluorescence of B. subtilis colonies having this plasmid on agar plates was so high that they could be readily discerned visually under UV light. The fluorescence could be effectively expressed in three ways (i) through use of a strong bsr promoter (blasticidin S resistance gene), (ii) by efficient translation with the bsr translation system, and (iii) through increase in the copy number per cell. The high stability of the GFP plasmid was demonstrated by using more complicated growth conditions without any antibiotic for selection.     

Studies on Tyzzer's disease: transplacental transmission of Bacillus piliformis in rats.

Clinically healthy rats with antibodies to Bacillus piliformis were given prednisolone in the last week of pregnancy. B. piliformis was demonstrated in the livers of their offspring. None of the dams or the young rats showed clinical signs of disease. Antibodies to B. piliformis were found in the young rats at birth, and presisted for several months. The importance of potential transplacental infections when attempting to establish colonies free from B. piliformis in discussed.     

Studies on Tyzzer's disease: application of immunofluorescence for detection of Bacillus piliformis and for demonstration and determination of antibodies to it in sera from mice and rabbits.

Bacillus piliformis antigens were demonstrated in smear preparations from infected mouse livers by direct immunofluorescence technique. Mouse serum antibodies against B. piliformis were demonstrated by indirect immunofluorescence technique. The test was employed quantitatively both on sera from experimentally infected mice and on sera from clinically healthy mice from colonies infected with B. piliformis, and could be used for the quantitative demonstration of antibodies in sera from a stock of rabbits with Tyzzer's disease. It was found very useful for the detection of subclinical infection.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

Patterns of gene expression in Bacillus subtilis colonies.

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.     

A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.     

Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.     

Serum antibody prevalence for Herpesvirus sylvilagus, Bacillus piliformis and California serogroup arboviruses in cottontail rabbits from Pennsylvania

A serologic survey of 60 eastern cottontail rabbits (Sylvilagus floridanus) from three counties in Pennsylvania was conducted in March 1983. Serum antibody prevalences for Herpesvirus sylvilagus and La Crosse virus (California serogroup) were less than 4%. There was no evidence of previous exposure to either Jamestown Canyon or snowshoe hare viruses (California serogroup). Antibody to trivittatus virus (California serogroup) was found in 60% of the 20 cottontails from York County. No cottontails had antibodies to Bacillus piliformis, the etiologic agent of Tyzzer's disease.     

The influence of Bacillus piliformis (Tyzzer) infections on the reliability of pharmacokinetic experiments in mice

The half-lives of warfarin and trimethoprim were significantly longer in mice acutely infected with Bacillus piloformis and in mice which ad clinically recovered from previous experimental infection with the organism. The volume of distribution of trimethoprim but not of warfarin was significantly greater in infected mice than in controls. Body clearances of warfarin was significantly reduced in both disease states. For trimethoprim this parameter was only reduced in the acute state of the disease. The importance of careful control of Tyzzer's disease in laboratory animals for use in pharmacological research is stressed.     

Efficient screening for catalytic antibodies using a short transition-state analog and detailed characterization of selected antibodies.

One of the major obstacles to acquiring catalytic antibodies is that it requires labor-intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition-state analog which contained only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three-step screening process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; and step 3, competitive-inhibition by the short TSA2. Only 22. 8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.     

Anti-nef antibodies and other predictors of disease progression in HIV-1 seropositive injecting drug users.

A cross-sectional and retrospective longitudinal study has been conducted in three Italian infectious disease centres to evaluate the role of anti-nef antibodies and other markers (HIV-1 p24 antigen, p24 Ag; Beta 2-microglobulin, B2-M; and number of CD4+ lymphocytes) as predictors of disease progression in HIV seropositive injecting drug users (IDUs). The selected patients were: 1) HIV-seropositive IDUs in different stages of HIV infection; 2) HIV-seropositive IDUs who had developed AIDS, from whom serial serum samples were available during the asymptomatic stage, and 3) HIV seropositive IDUs who remained asymptomatic through a follow-up period of the same duration as the patients who developed AIDS. Absence of anti-nef antibodies was associated with symptomatic HIV infection. A significant association between the absence of anti-nef antibodies, the presence of p24 Ag, high levels of B2-M, a number of CD4+ lymphocytes less than 500/ml at first visit and disease progression was found. Subjects who were persistently positive for antibody to nef were less likely to develop AIDS than those who were transiently or persistently negative. This difference was statistically significant (p = 0.03). The results of this study show that absence or disappearance of anti-nef antibodies may be used as predictor of disease evolution in HIV seropositive IDUs. This study also confirms the usefulness of other markers, such as p24 Ag, B2-M and number of CD4+ lymphocytes previously shown to be predictive of rapid disease progression for predicting the course of HIV seropositive IDUs.     

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

Cells of Bacillus subtilis recA1 are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system.     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Evaluation of antigen panels for ELISA monitoring of mouse colonies for antibodies to Pasteurellaceae

Pasteurellaceae infection in mice may be monitored by the detection of serum antibody using enzyme-linked immunosorbent assay (ELISA). We re-evaluated our standard antigen panel comprising Pasteurella pneumotropica and a V-factor requiring Haemophilus species (strain H21) by studying their serological relationship with Actinobacillus muris and 'Haemophilus influenzae-murium'. Serologically, A. muris and 'H. influenzae-murium' were found to be unrelated and to differ from P. pneumotropica and Haemophilus strain H21. These four antigens were used for monitoring breeding and experimental mouse colonies for a period of four years. The addition of 'H. influenzae-murium' antigen to the standard panel of antigens significantly increased the proportion of sera and serum panels showing anti-Pasteurellaceae antibody activity, but the addition of A. muris antigen did not.