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.     

A serological survey on Bacillus piliformis infection in laboratory rabbits in Japan

A total of 544 rabbit sera obtained from 6 commercial breeding facilities and 9 research institutions during 1985-1990 were tested for Bacillus piliformis antibody by an enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody test (IFAT). The antibody was detected in 53 (14.2%) rabbits from 3 breeding facilities and 30 (17.4%) rabbits from 6 research institutions, indicating the prevalence of B. piliformis infection among laboratory rabbits in Japan. The overall agreement with ELISA for immune status was 96.9% (527/544) with IFAT. In tests of the ability of ELISA and IFAT to quantitate antibody, a correlation coefficient (r) of 0.86 (P less than 0.01) was obtained by plotting the measured average log of the ELISA titer against the corresponding log of the IFAT titer.     

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.     

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.     

Naturally occurring Bacillus piliformis infection (Tyzzer's disease) in guinea pigs

Two juvenile male Hartley guinea pigs (Cavia porcellus) were found dead 36 hours after receipt from a commercial source. Both guinea pigs had dependent, subcutaneous edema and excess serous fluid in their thoracic and abdominal cavities. Their livers were mottled and the cecal walls were reddened focally. Histopathologic exam revealed nests of Bacillus piliformis within the absorptive epithelial cells of the ileum, cecum and colon. Vegetative organisms and spore-like structures were observed in the cytoplasm of intestinal epithelial cells by electron microscopy. A diagnosis of Tyzzer's disease was made.     

An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of rabbit serum antibody directed against the causative agent of Tyzzer's disease, Bacillus piliformis. Ninety-four percent agreement was found between the ELISA and an indirect fluorescent antibody test. The sensitivity of the ELISA was 95% and its specificity was 92% as compared to the indirect fluorescent antibody test (IFAT). The rabbit origin B. piliformis isolate used in this ELISA was found to be cross-reactive by ELISA and IFAT to B. piliformis isolates of rat, gerbil and horse origin. This suggests that a single B. piliformis isolate may be used as antigen for an ELISA utilizable for multiple species.     

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.     

Rederivation of rat colonies seropositive for Bacillus piliformis and the subsequent screening for antibodies.

Latent infection of rats in a breeding colony with Bacillus piliformis detectable by antibodies to the agent in an immunofluorescence assay was eliminated by a combination of traditional rederivation techniques, using animal units not previously used for rat breeding, and the use of specific disinfection procedures. The success rate was apparently correlated with the use of peracetic acid instead of aldehyde products to decontaminate the animal unit.     

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.     

Studies on Tyzzer's disease in rats.

An outbreak of an epidemic disease occurred in a specified-pathogen-free (SPF) breeding colony of rats. The clinical signs and the post-mortem findings were characteristic for Tyzzer's disease. The causative agent, Bacillus piliformis, was demonstrated microscopically in ileum, liver and myocardium, and transmitted to mice where its pathogenicity appeared to be similar to that of another strain isolated from mice. B. piliformis from spontaneously-infected rats was demonstrated by indirect immunofluorescence technique. By means of the same technique it was found that the fluorescence antibody titre obtained of the individual sera from spontaneously-infected mice, rats and rabbits was the same, whether the antigen employed was organisms isolated from rats or mice. By testing sera from healthy rats in 3 different colonies by use of immunofluorescence technique, antibodies were found in several sera.     

Studies on Tyzzer's disease: acquired immunity against infection and activation of infection by immunosuppressive treatment.

The correlation between serum antibody titre and resistance to challenge infection with Bacillus piliformis was studied in naturally infected mice, in experimentally infected but recovered mice, and in mice treated with antigen prepared from infected livers. Irrespective of the way in which the antibodies were acquired resistance to infection was found to be related to the immunofluorescence antibody titre found. Experimentally infected but recovered mice, as well as rats with persistent antibodies to Bacillus piliformis, were given prednisolone in order to activate a possible persistent infection. Bacillus piliformis was detected in the rats, but not in the mice.     

Naturally occurring Tyzzer's disease and intestinal spirochetosis in guinea pigs

Bacillus piliformis infection (Tyzzer's disease) occurred in two young guinea pigs, causing unthriftiness and diarrhea which resulted in death. There was necrosis and inflammation of the ileum, cecum, and colon. Intestinal epithelial cells contained organisms resembling Bacillus piliformis. Spirochetes were found in the cecum and colon, mainly in crypts. Acute diarrhea occurred in another guinea pig which became cachetic and was killed. Histologically, large numbers of spirochetes were present in the wall of both the cecum and colon, and they were associated with severe necrosis and inflammation. Bacillus pilformis was not found in this animal.     

Subclinical infection and transmission of Tyzzer's disease in rats.

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.     

Experimentally induced Tyzzer's disease in the African white-tailed rat (Mystromys albicaudatus)

Tyzzer's disease was induced experimentally in nonimmunosuppressed, weanling Mystromys albicaudatus by oral inoculation with Bacillus piliformis spores. Focal areas of necrosis bordered by cells containing B. piliformis were observed first in the tunica muscularis of the intestine and in the periportal region of the liver 3 days post-inoculation, in the ventricular myocardium 7 days post-inoculation and in the brainstem 9 days post-inoculation. Healing in the tunica muscularis, liver and myocardium was accompanied by granuloma formation. The findings indicate that Mystromys are susceptible to B. piliformis infection. This is, to our knowledge, the first time brain lesions have been reported in any species following oral inoculation with B. piliformis. Tyzzer's disease should be considered as a possible diagnosis in Mystromys with hepatoenteritis, myocarditis, or indications of central nervous system disorders.     

Lesions of experimentally induced Tyzzer's disease in Syrian hamsters, guineapigs, mice and rats

The relative susceptibilities of C57BL/6NCR and BALB/cANNCR mice, F344/NCR rats, 2/NCR guineapigs and CR:RGH Syrian hamsters to Bacillus piliformis infection were determined by orally inoculating 20 weanling females from each species with suspensions of B. piliformis spores. Animals from each group were sequentially necropsied over 2 week periods to document the lesions produced. No significant gross or microscopic lesions were observed in the BALB mice or the Fischer rats. Gross and microscopic lesions were observed in the livers and intestines of many guineapigs and hamsters killed 3-14 days after inoculation. A large lesion was observed in the left cardiac ventricle of one C57BL mouse 10 days after inoculation. Warthin-Starry silver-stained tissue sections revealed clusters of B. piliformis within the cytoplasm of intestinal epithelial cells, smooth muscle cells, hepatocytes and myocytes bordering foci of necrosis in the intestines, liver and heart.     

Tyzzer's disease in free-living cottontail rabbits (Sylvilagus floridanus) in Maryland

Complement-fixing (CF) antibody to Bacillus piliformis antigen was found in 9 of 14 (64%) serum samples obtained from cottontail rabbits (Sylvilagus floridanus) killed in the wild. CF antibody was not present in the serum of 8 cottontail rabbits trapped as juveniles in the same geographic areas and held in captivity for 4 years. Sero-negative cottontail rabbits died acutely with lesions typical of Tyzzer's disease following the intragastric administration of 10(3.8) ELD50 of B. piliformis spores. The possible influence of Tyzzer's disease upon the cyclic population pattern of cottontail rabbits in the wild is discussed. A hypothesis is presented that B. piliformis spores passed in the feces of diseased wild animals could contaminate pastures, hay and grain, and thereby serve as sources of infection to other animals.     

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.     

Studies on Tyzzer's disease: a long-term study of the humoral antibody response in mice, rats and rabbits.

Mice treated with an antigen prepared from livers infected with Bacillus piliformis developed antibodies to the microorganism which reached a peak on the 7th day and disappeared within 40 days: antibody titres in experimentally-infected mice remained at a high level throughout life. The antibody titres in naturally-infected mice, rats and rabbits ramined positive throughout life and followed the same pattern as that of the experimentally-infected mice.