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A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect Bacillus piliformis isolate-specific antibodies in serum specimens from rats and gerbils experimentally infected with B. piliformis isolates R1, R2, or M. Detection was based on the ability of serum antibodies to block binding of B. piliformis isolate-specific monoclonal antibodies to purified B. piliformis flagella. Application of this assay to serum specimens collected from sham-infected or experimentally infected rats and gerbils demonstrated that the serum specimens were capable of specifically inhibiting the binding of B. piliformis isolate-specific monoclonal antibodies to homologous flagella preparations (70% inhibition) only when the serum specimens were from animals infected with the homologous B. piliformis isolate. Only one false-negative and false-positive result were obtained when 80 serum specimens were tested by this competitive inhibition ELISA. In addition, we demonstrated that little nonspecific inhibition of monoclonal antibody binding occurred (30% inhibition) in this immunoassay specific inhibition of monoclonal antibody binding by serum was due to serum antibody and a serum's ability to inhibit binding of monoclonal antibodies to purified B. piliformis flagella was correlated with antibody reactivity with B. piliformis flagella but not with serum antibody reactivity to whole B. piliformis organisms. These results suggest that this monoclonal antibody-based competitive inhibition assay could be successfully applied to the serologic identification of isolates involved in naturally occurring B. piliformis infections in laboratory animals.

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.

Two isolates of Bacillus piliformis originally obtained from rats from Japan and Indiana were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Protein and antigen profiles revealed heterogeneity between the two isolates, demonstrating that more than one isolate of B. piliformis is capable of infecting rats. Results of parallel infection and transmission studies with the two isolates were almost identical. Orally inoculated rats remained asymptomatic; however, enzyme-linked immunosorbent assay results revealed a significant increase in serum antibodies to B. piliformis. Formalin-killed B. piliformis elicited no serum antibody response among rats inoculated orally, indicating that viable organisms, capable of replicating within the host, are needed to induce a systemic humoral response. Naive rats and weanling gerbils were housed on soiled bedding from the experimentally infected, asymptomatic, seropositive rats. Although gerbils showed no clinical signs or histopathologic evidence of Tyzzer's disease, rats housed on bedding collected 1 or 2 weeks postinoculation seroconverted and remained seropositive but asymptomatic throughout the study. These results demonstrate that subclinically infected rats are capable of transmitting B. piliformis to naive rats and suggest that the histopathologic evaluation of sentinel gerbils may not be an effective method for detecting all strains of B. piliformis.