Histopathologic observations in weanling B6C3F1 mice and F344/N rats and their adult parental strains.

Weanling Fischer 344/N (F344) rats and the first filial hybrid of C57BL/6 x C3H (B6C3F1) mice and retired breeders from the parental stocks of these strains were monitored over a 5-yr-period by examining the histopathology of selected organs and comparing those results to viral and mycoplasmal serology and the intestinal tract bacterial flora of each animal on an individual basis. Serology gave no evidence of viral infection, but Mycoplasma arthriditis antibodies were detected. Reactivity of serum of adult C57BL/6 female mice with control cells or media (tissue culture, TC) was seen in a significant number of mice. TC reactivity correlated positively with lymphoid perivascular infiltrates, predominantly of the lungs, suggesting an allergic response in development of the lesions. Other lesions of note consisted of Harderian gland inflammation of rats, focal necrotizing lesions of the liver of both species, and thickening of the pleura and adjacent pulmonary interstitium of weanling rats. Embolization of bacteria from the gastrointestinal tract to the liver was considered a possible cause of the liver necrosis in both species. Although lesions of the lung and Harderian gland of the rats are similar to those caused by known viral agents, the cause of the latter could not be determined as these animals were negative for viral antibodies and the former was considered to be related to incomplete pulmonary development in the young rat. Features differentiating the lesions observed in animals of this survey from those caused by viral infection are discussed.

Cultivation of Bacillus piliformis (Tyzzer) in mouse fibroblasts (3T3 cells).

Bacillus piliformis was successfully propagated in a continuous mouse-embryo fibroblast cell line (3T3). 3T3 monolayers were successfully inoculated with a variety of infected materials including yolk sac and liver suspensions, minced yolk sac and liver, and primary chicken-embryo liver-cell cultures. An initial decrease in B. piliformis number was noted after inoculation of the cell layer with 2.6 X 10(5) organisms followed by a peak bacterial count at 48 h. Bacillus piliformis remained infectious for mice after 22 passages in 3T3 cell monolayers. Limited growth of B. piliformis was obtained in cell lines of mouse connective-tissue origin (L-929) and mouse liver origin (NCTC 1469).

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.

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.

Isolation, propagation, and characterization of a newly recognized pathogen, cilia-associated respiratory bacillus of rats, an etiological agent of chronic respiratory disease.

A Gram-negative, filamentous, rod-shaped bacillus which failed to grow in cell-free media was isolated in apparently pure culture from the bronchial scraping and washing of a laboratory rat suffering from chronic respiratory disease by inoculating embryonated chicken eggs via the allantoic route. None of the embryos died during 20 serial passages at weekly intervals. The bacillus was reisolated in embryonated eggs from cesarean-derived barrier-maintained N:SD(SD) rats 8 and 12 weeks after intranasal inoculation with 10th-passage allantoic fluid. The inoculated rats were housed in Horsfall-type units and remained free from other known respiratory pathogens, including mycoplasmas and murine viruses, throughout the study. The bacillus colonized the ciliated epithelial cells of the respiratory tract and caused a marked peribronchial infiltration and hyperplasia of mononuclear cells which progressed with time. The bacillus, ca. 0.2 micron wide by 4 to 6 micron long, stained very poorly with basic aniline dyes but was readily demonstrated with the Warthin-Starry silver technique. It was heat labile (56 degrees C for 30 min); spore forms were not observed. It withstood freeze-thawing and was successfully stored at -70 degrees C. Although no visible means of locomotion was observed with the electron microscope, a slow gliding motility, sometimes with bending and flexing of bacilli apparently adherent to the glass surface, was observed with phase microscopy. As an etiological agent of chronic respiratory disease of rats, this cilia-associated respiratory bacillus (tentatively designated the CAR bacillus) may be the first recognized gliding bacterium known to cause disease in a warm-blooded vertebrate.

Experimentally induced Tyzzer's disease in Mongolian gerbils (Meriones unguiculatus).

Tyzzer's disease was induced in outbred, 3- to 4-week-old female Mongolian gerbils (Meriones unguiculatus) by oral inoculation of either a yolk sac preparation containing spores or a suspension of infected mouse liver. Clinical signs and lesions were similar regardless of the inoculum. Nests of Bacillus piliformis were seen in follicle associated epithelium of ileal Peyer's patches by 24 hours post-inoculation. Disease severity peaked at the fifty through seventh days. Many animals died during this time, and they had lesions in the intestinal tract, liver, and myocardium. Lesions began to resolve in animals surviving past the seventh day of infection. Because of the unique susceptibility of gerbils to Bacillus piliformis infection, they may be useful as an aid to study the natural course of Tyzzer's disease and as sentinel animals. They also may serve as a valuable diagnostic tool as the recipient of suspect material.

Pathology and diagnosis of mousepox.

The pathologic changes of mousepox were studied during an outbreak at the National Institutes of Health in 1979. The most consistent lesions were necrosis of lymphatic tissues, especially the spleen, lymph nodes, and Peyer's patches. Hepatic necrosis and jejunal hemorrhage also were found. In two transmission studies, the disease was experimentally induced in BALB/cAnN and C3H/HeN-nu mice. Athymic mice were found to be highly susceptible, and they developed fulminant disease. The diagnosis was confirmed by demonstration of pox virions in infected tissues by electron microscopy, staining of viral antigen by immunoperoxidase methods, and by isolation of the virus in chorioallantoic membranes of hen's eggs and in cultures of chick embryonic cells.

A study of mouse strains susceptibility to Bacillus piliformis (Tyzzer's disease): the association of B-cell function and resistance.

Tests were conducted on 11 inbred strains of mice and an NIH outbred stock. It was found that only the CBA/N and C3. CBA/N mice (strains deficient in IgM production) were highly susceptible to Bacillus piliformis infection. Susceptibility to infection was determined by induction of typical surface liver lesions and the ability to maintain serial passage without concurrent administration of cortisone. Mice deficient in T-cell function (Nu/Nu/++) were as resistant to Bacillus piliformis infection as intact immunologically competent mice. The data suggested that resistance to Tyzzer's disease was, at least in part, a B-cell function.

Effect of heat and selected chemical disinfectants upon infectivity of spores of Bacillus piliformis (Tyzzer's disease).

Spores of Bacillus piliformis of rabbit origin were harvested from the yolk sac of previously inoculated hen's eggs and subjected to various heat or chemical disinfectant treatments. Subsequently, spores were tested for infectivity in embryonated eggs inoculated via the yolk sac route, and others were treated with trypsin and then tested in embryonated eggs. The spores were not affected by the heat treatment of 60 degrees C but were rendered noninfective with treatments of 70 degrees C and 80 degrees C. Infectivity was not restored by treatment with trypsin. Infectivity was not lost when spores were treated with a phenolic germicidal detergent, ethanol or either of two quaternary ammonium compounds containing 9% or 17% benzalkonium chloride. A graded effect was observed with formaldehyde solution and an iodophor. Spores were rendered noninfective after treatment with peracetic acid (1.0%) and a wetting agent, sodium alkylarylsulfonate or sodium hypochlorite solution (0.3%) for 5 minutes, and infectivity was not restored by trypsin treatment. The probable means of transmission of Bacillus piliformis was discussed and sodium hypochlorite solution (0.3%) was recommended as a surface disinfectant in animal facilities as an aid to the prevention and control of Tyzzer's disease.

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.

Tyzzer's Disease of Rabbits: Isolation and Propagation of Bacillus piliformis (Tyzzer) in Embryonated Eggs.

Bacillus piliformis (Tyzzer) was isolated from the liver of rabbits with Tyzzer's disease and serially passaged in embryonated hens' eggs. Weanling rabbits given the 32nd egg passage developed lesions typical of Tyzzer's disease and died. B. piliformis was reisolated from the liver of these rabbits in embryonated eggs. Outside the host cell, the motile vegetative phase appeared to be unstable, and no means was found to preserve its viability; the results of titrations were believed to be dependent upon the resistant stage or spore. The spore withstood repeated freeze and thaw and was resistant to heat treatment of 56 C for 1 hr but not 80 C for 0.5 hr. None of several antibacterial substances tested in embryonated eggs was completely inhibitory; B. piliformis was resistant to sulfamethazine and chloramphenicol. The taxonomic position of this pleomorphic, gram-negative, sporeforming, pathogenic bacterium which appears to grow only in certain cells of several species remains unresolved.