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.     

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: 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.     

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.     

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.     

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.     

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.     

Pre-symptomatic detection of prions by cyclic amplification of protein misfolding

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals. At present, it is not possible to recognize individuals incubating the disease before the clinical symptoms appear. We investigated the effectiveness of the "Protein Misfolding Cyclic Amplification" (PMCA) technology to detect the protease-resistance disease-associated prion protein (PrP(res)) in pre-symptomatic stages. PMCA allowed detection of PrP(res) in the brain of pre-symptomatic hamsters, enabling a clear identification of infected animals as early as two weeks after inoculation. Furthermore, PMCA was able to amplify minute quantities of PrP(res) from a variety of experimental and natural TSEs. Finally, PMCA allowed the demonstration of PrP(res) in an experimentally infected cow 32 month post-inoculation, that did not show clinical signs and was negative by standard Western blot analysis. Our findings indicate that PMCA may be useful for the development of an ultra-sensitive diagnostic test to minimize the risk of further propagation of TSEs.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

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.     

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.     

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.     

Selective medium for Corynebacterium kutscheri and localization of the organism in mice and rats

A new selective medium for isolation of Corynebacterium kutscheri (CK) from animals suffering subclinical infection was made by adding furazolidone, nalidixic acid and corimycin to the heart infusion agar base, this being named FNC agar. The FNC agar inhibited the growth of gram-negative rods, Pseudomonas aeruginosa, Proteus sp. and gram-positive cocci but did not affect the growth of CK. When this medium was used to isolate CK from the oral cavity and cecal contents of mice and rats, two of 6 conventional mouse colonies and three of 8 conventional rat colonies were found to be infected, with isolation of the organism from 19 mice and 12 rats in total. The animals showed neither clinical signs nor lesions, but the antibody was positive in 11 mice and 10 rats. In mice and rats inoculated orally with 4 x 10(8) and 1 x 10(9) organisms, respectively, CK was isolated for 20 weeks post-inoculation by use FNC agar. The isolation rate of the organism was the highest in the oral cavities of both inoculated mice and rats, and also in the submaxillary lymph nodes of the inoculated rats. The organism was also recovered from the cecal contents of more than half of the inoculated mice and rats. Thus, it was considered that FNC agar was useful in isolating CK from the oral cavity and cecal contents of mice and rats with subclinical infection of the organism.     

Tyzzer's disease as a complication of canine distemper in a raccoon

Intercurrent canine distemper and Tyzzer's disease were diagnosed in a mature raccoon (Procyon lotor) submitted for necropsy. Clinical, gross and microscopic findings characteristic of canine distemper virus (CDV), included ataxia, dyspnea, suppurative conjunctivitis, interstitial pneumonitis and generalized lymphocytolysis. Inclusion bodies typical of CDV infection were present in many epithelial tissues. Acute multifocal hepatic necrosis and acute segmental necrotizing enteritis were attributed to the presence of Bacillus piliformis organisms in these lesions, confirmed by special stains and electron microscopy. This is apparently the first reported case of Tyzzer's disease in a raccoon.     

Clinical Picture and Antibody Response to Experimental Sarcoptes scabiei var. vulpes Infection in Red Foxes (Vulpes vulpes)

Three red foxes (Vulpes vulpes) were experimentally infected with Sarcoptes scabiei isolated from a naturally infected wild red fox. A fourth red fox served as a control. The first signs of sarcoptic mange became evident on the 31st day post infection (dpi). The signs gradually increased thereafter and between dpi 49 and 77 characteristic lesions of hyperkeratosis developed. Two of the infected foxes developed severe sarcoptic mange, and one of these animals died on dpi 121. The third fox developed a chronic hyperkeratotic lesion on its back, at the site where the mites had been applied. On dpi 127 the surviving foxes were treated systemically with ivermectin, and within 4 weeks the skin lesions had healed except on the pinnae of one animal. Antibodies to S. scabiei var. vulpes were demonstrated in the infected foxes by an ELISA with which seroconversion was seen around 4 weeks post infection (wpi). Western blot analysis of sequential sera of the infected animals demonstrated antibody activity consistently after the 2nd wpi. The fourth, non-infected, fox did not show any skin lesions throughout the experimental period nor any specific antibodies to S. scabiei var. vulpes. kw|Keywords|k]sarcoptic mange; k]red fox; k]serodiagnosis; k]ELISA     

Enterotoxaemia involving Clostridium perfringens iota toxin in a hysterectomy-derived rabbit colony

During an explosive outbreak of fatal enteropathic disease involving Clostridium perfringens iota (i) toxin, a total of 183 deaths occurred in 18 weeks. The clinical signs and post-mortem findings are reported. Examinations for virus, Bacillus piliformis and coccidia were negative. Clostridium perfringens i toxin was detected in 22 of 27 animals examined (81.5%), but clostridia were not isolated. Various treatments were attempted. It is concluded that i toxin and the syndrome described are closely related.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

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.