Charles O. Thoen

Genomic Fingerprinting and Development of a Dendrogram for Brucella spp. Isolated from Seals, Porpoises, and Dolphins

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.

Immunohistochemical Detection of Mycobacterium Paratuberculosis in Formalin-Fixed, Paraffin-Embedded Bovine Tissue Sections

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Tuberculosis in Commercial Emus (Dromaius novaehollandiae)

Extensive granuloma formation typical of tuberculosis was observed in a mature female emu. The diagnosis was confirmed by demonstration of acid-fast bacilli in lesions and culture of a Mycobacterium with growth characteristics resembling M. avium from liver tissue. Individual emus on the affected farm and an epidemiologically related unit gave a positive skin reaction to intradermal M. avium tuberculin. The implication of tuberculosis in commercial emus is noted in relation to the growth of the industry in North America and to management and commercial practices that encourage dissemination of infection within the species and to other exotic and domestic animals.

Potential use of lymphocyte blastogenic responses in diagnosis of bovine tuberculosis

Abstract A comparison of in vitro lymphocyte responses and delayed type tuberculin skin test responses was made in an animal experimentally exposed to a Mycobacterium bovis -infected animal and in cattle naturally infected with M. bovis . Tuberculin skin tests did not suppress in vitro lymphocyte responses to M. bovis PPD and to M. avium PPD tuberculin. The whole blood test used in these studies provided for considerable savings in time as compared to use of purified lymphocytes for evaluating in vitro cellular responses. Variations in the responsiveness of lymphocytes to specific mycobacterial antigens was observed, therefore, it is recommended that profiles be established using three or more tests conducted at 14-day intervals.

An enzyme-labeled antibody test for detecting antibodies in chickens infected with Mycobacterium avium serotype 2.

An enzyme-labeled antibody test was used for detecting antibodies in serums from chickens infected experimentally with Mycobacterium avium serotype 2. Positive ELA reactions were observed in the serums of each of 8 chickens 2, 4, 6, and 8 weeks after infection; no reactions were observed in uninfected controls. Tuberculin skin tests did not induce positive ELA test reactions in uninfected chickens.

Japanese quail: susceptibility to avian tuberculosis.

Japanese quail (Coturnix coturnix japonica), also called coturnix, is used widely as an experimental bird in avian research. Being small and weighing 100 to 500 g, it requires much less cage space than does the chicken. The coturnix is relatively easy to raise; it appears to thrive on commercially prepared turkey feed (1-3). The coturnix is easy to handle also, and has a large accessible wing vein, resembling the cephalic vein of the chicken, which permits ease of intravenous injection as well as ease of obtaining blood specimens. In our studies of the virulence for fowl of certain mycobacteria isolated from various avian and mammalian species, including man, we had been using chickens which, when full grown, each required cage space at least 10 times that needed for a pair of coturnix. We now have had 3 years of experience with the coturnix in studies on the virulence of Mycobacterium avium. Thirty-six strains have been tested in at least two coturnix each. This report describes the routes of experimental inoculation, the lesions produced, and the results of the tuberculin test.

Characterization of Circulating Antibodies Against Chlamydia psittaci in Turkeys

Plasma and joint fluids from turkeys experimentally inoculated with Chlamydia psittaci strain TT3 were evaluated by immunoblotting to identify antibodies elicited by chlamydial antigens during the course of infection. Protein antigens from elementary bodies of TT3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose before being probed with plasma or synovial fluid from TT3-inoculated birds. The major outer-membrane protein (MOMP), the 60,000-molecular-weight proteins, and a 97,400-molecular-weight protein were the predominant antigens recognized by IgG in the plasma and joint fluids. Plasma IgG specific for the 97,400 protein band was first detectable at day 10 postinoculation (PI). Antibodies to the 60,000-molecular-weight protein and MOMP were first detected at days 14-17 PI and at days 7-10 PI, respectively, in some birds, and as late as days 36-42 PI and days 42-70 PI in others. The antibodies were still present at day 142 PI. Immunoblotting techniques indicated that the antigens to which these antibodies were reacting were protein. These observations may have implications for the development of serodiagnostic assays as well as the identification of potential proteins for subunit immunogens in birds.

Development of a whole blood lymphocyte stimulation assay for detecting hypersensitivity to PPD in cattle experimentally infected with Mycobacterium bovis

Abstract A whole blood lymphocyte stimulation assay utilizing the uptake of tritiated thymidine was developed for the detection of Mycobacterium bovis sensitivity in cattle. Results on eight M. bovis infected animals (six to ten weeks after infection) and eight control animals show that satisfactory lymphocyte stimulation can be obtained using heparinized whole blood diluted 10-fold in tissue culture medium and cultured with purified protein derivative (PPD) for three to seven days. Infected animals exhibited significantly greater stimulation when cultured with PPD than did control animals.

Chemical Destruction of Mycobacterium bovis in Milk

The tuberculocidal activity of phenol and 1-Stroke Environ was tested using five Mycobacterium bovis strains added separately to five samples of untreated cows milk. The tuberculocidal activity of each disinfectant was significantly improved (P = .005) by increasing exposure temperature from 4 to 23 C or by increasing exposure time from 1 to 6 h or by increasing disinfectant concentrations two-fold. Environ diluted 1:8 or phenol diluted 1:32 killed each of the five strains of M. bovis suspended in untreated milk (6 mg/ml) during a 6-h exposure at 23 C. Either disinfectant could be used to destroy M. bovis in unsalable milk from tuberculous cows scheduled for slaughter.

Immunoblots of outer-membrane proteins of chlamydiae with sera from turkeys experimentally exposed to avian and mammalian Chlamydia psittaci.

Outer-membrane protein (OMP)-enriched preparations from avian (turkey/TT3 and parrot/VS1) and mammalian (sheep abortion/B577) strains of Chlamydia psittaci were compared by immunoblotting using sera from turkeys exposed to these strains. Turkeys inoculated with avian chlamydiae became infected and developed strong serological responses, but turkeys inoculated with B577 failed to develop detectable serological responses. Sera from turkeys exposed to either of the two avian strains could be differentiated on the basis of immunoreaction patterns with OMPs of homologous and heterologous strains. Fewer bands and often weaker reactions were detected using sera from TT3- and VS1-inoculated birds with the heterologous avian strain. Sera from turkeys inoculated with either avian strain reacted with the 97,400-molecular weight (MW) protein. The sera reacted with the major outer-membrane protein (MOMP) of the homologous strains but not consistently with the MOMP of the heterologous strain. Results suggest that the 97,400-MW protein is highly immunogenic for turkeys and antigenically more complex than the MOMP.