Lack of association between the occurrence of Crohn's disease and occupational exposure to dairy and beef cattle herds infected with Mycobacterium avium subspecies paratuberculosis.

A cross-sectional survey was conducted to identify associations between Crohn's disease (CD) and Mycobacterium avium ssp. paratuberculosis (Map) exposure. A questionnaire was used to collect information on exposure to cattle infected with Map, and personal and family history of CD in dairy and beef cattle producers with and without Map-infected herds, and in veterinarians who did or did not have contact with Map-infected herds. Cases of CD were selected from respondents and matched 1:4 with controls on occupation, age, and sex. Multivariable conditional logistic regression was used to assess associations between Map exposure and CD. There were 3 cases of CD in 702 producers and 4 cases in 774 veterinarians, yielding a prevalence of 0.47%. No association was found between exposure to JD and CD in any phase of the analysis. However, the number of cases of CD is not large and limits the power to detect important differences.

Flow microfluorometric quantitation of the blastogenic response of lymphocytes.

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.

Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli.

DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1. Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme. A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.

Selective media for isolation of Brucella abortus strain RB51.

Brucella abortus strain RB51 (SRB51) is the standard vaccine used to protect cattle against brucellosis and is currently being used to vaccinate bison in the United States (US). Currently available media for culture of Brucella have not been evaluated for their ability to support growth of SRB51. In this study, five selective media for isolating brucellae, four commercially available media for gram-negative bacteria, and tryptose agar with 5% bovine serum (TSA) were compared to two SRB51 selective media developed in this study (rifampin brucellae medium (RBM), and malachite green brucellae medium (MGB)), for their ability to support growth and enhance recovery of SRB51. Four of the five media currently used for isolation of brucellae and two of the four media used for other Gram-negative bacteria did not support growth of SRB51. Modified Kuzdas and Morse (MKM), Brilliant Green, Skirrow's, RBM, and MGB supported growth of SRB51 in a manner similar to TSA. Recovery of SRB51 from tissues of SRB51-vaccinated bison was attempted on TSA, MKM, RBM, and MGB. From a total of 436 samples, SRB51 was isolated from 9.6, 4.3, 5.5, and 9.0% on TSA, MKM, RBM, or MGB media, respectively. Strain RB51 was recovered on only one medium (nine on TSA; three on RBM; and 9 on MGB) from 21 samples. Overgrowth of contaminating bacteria prevented potential detection of SRB51 from 9. 4, 5.5, 0.07, and 5.9% of samples on TSA, MKM, RBM, or MGB, respectively. These data suggest that the use of RBM and MGB, in combination with TSA, enhances the ability to recover SRB51 from tissue samples.

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.

Evaluation of a potassium chloride extract of Brucella abortus in an ELISA for detecting Brucella antibodies in bulk tank milk samples from cows.

An enzyme-linked immunosorbent assay (ELISA) was developed using as antigen a potassium chloride extract of Brucella abortus strain 1119-3 for detecting Brucella antibodies in bulk tank samples of cow's milk. Three-hundred-thirty-four Milk Ring Test (MRT) suspicions milk samples originating from cattle herds in 13 states and 106 BRT negative milk samples were analyzed. Fifty-four of 334 MRT suspicious milk samples were positive on ELISA; bacteriologic examinations revealed B. abortus field strain was isolated from cows in 15 herds, B. abortus strain 19 was isolated from cows in 16 herds and serologic suspects were reported in 6 of the other 23 herds. Two-hundred-fifty-eight (85.6%) of the 301 MRT suspicious samples were negative on ELISA; field investigations and/or serologic tests on cattle failed to disclose Brucella infection in these herds. Suspicious ELISA reactions were detected in 22 MRT suspicious bulk tank milk samples; serologic suspects were reported in 8 of the 22 herds. No false positive ELISA reactions were detected in the 106 MRT negative bulk tank milk samples collected from dairy herds in 7 states.

Paratuberculosis in cattle: a comparison of three serologic tests with results of fecal culture.

Feces and blood were collected from cattle in 13 herds known to be infected with Mycobacterium paratuberculosis to evaluate a complement-fixation (CF) test, an agar gel immunodiffusion (AGID) test and an enzyme-linked immunosorbent assay (ELISA) for the serologic diagnosis of paratuberculosis. M. paratuberculosis was isolated from the feces of 36 of 192 cattle examined. Twenty-three culture-positive animals had CF test titers regarded as suspect or positive, 10 were positive by the AGID test and 34 were suspect or positive by the ELISA. Of the 156 culture-negative animals, the CF test agreed on 136, the ELISA on 129 and the AGID on 151.

Experimental exposure of llamas (Lama glama) to Brucella abortus: humoral antibody response.

Positive antibody reactions to brucella were observed in the sera of four llamas receiving Brucella abortus Strain 19 subcutaneously at 2-3 weeks post-exposure (PE) using five of eight conventional brucella serologic tests and an ISU-ELISA. Positive brucella antibody reactions were detected in sera of four llamas exposed by intraocular instillation (IOI) of 1.02x10(8) (high dose) B. abortus Strain 2308 at 16-35 days PE using seven of eight serologic tests or an ISU-ELISA. Brucella antibody was also detected in sera of four llamas exposed by IOI of 9x10(5) (low dose) B. abortus using each of four agglutination tests, Complement Fixation test, PCFIA, the rivanol test and the ISU-ELISA at 16-35 days PE. Positive reactions were observed using the Card test, BAPA, SPT, STT, the rivanol test, the PCFIA, and the ISU-ELISA on sera collected on days 42-70 PE, except on one llama, given the low dose; that llama was negative on the PCFIA on day 42. Positive or suspicious reactions were not detected in sera of controls, receiving saline subcutaneously, using the routine tests, with the exception of the CFT. The B. abortus Strain 2308 was isolated from tissues of seven of eight llamas exposed to virulent B. abortus Strain 2308.

Mycobacterium bovis infection in baboons (Papio papio).

Two tuberculin-positive baboons in a primate colony were found to have grossly visible tuberculous lesions in the liver, spleen, lung, and mediastinal lymph nodes on necropsy. Results of histopathologic examination of the tissues showed granulomas with Langhans giant cells. An acid-fast organism was isolated from tissues of each baboon; the isolates were identified as Mycobacterium bovis by being negative for niacin production and nitrate reduction and by their susceptibility to thiophen-2-carbosylic acid hydrazide and to 5% glycerol.

Some immune responses in cattle exposed to Mycobacterium paratuberculosis after injection with modified-live bovine viral diarrhea virus vaccine.

Delayed-type hypersensitivity responses to Mycobacterium paratuberculosis purified protein derivative (PPD) were decreased in cows experimentally exposed to M. paratuberculosis 7 days after exposure to a modified-live bovine viral diarrhea virus (ML-BVDV) vaccine. In vitro lymphocyte blastogenic responses to phytohemagglutinin were decreased in each of 3 cows 7 days after exposure to ML-BVDV vaccine. Also, decreased lymphocyte blastogenic responses to M. paratuberculosis PPD were observed in cultures of 2 of 3 cows 7 days after exposure to ML-BVDV vaccine. No significant differences in enzyme-linked immunosorbent assay reactions were detected in sera of M. paratuberculosis-infected cattle collected before and at 4 and 12 weeks after exposure to ML-BVDV vaccine.

Control of Johne's disease in four commercial dairy herds in Iowa.

A 6-year study was conducted in 4 dairy herds in Iowa in which Johne's disease was diagnosed previously. Fecal specimens were collected at 6-month intervals from animals 2 years of age and over for mycobacteriologic examination. Serum samples were obtained at 3-month intervals and tested by enzyme-linked immunosorbent assay (ELISA). The antigen used in the ELISA was a potassium chloride extract of a field strain of Mycobacterium paratuberculosis. The ELISA reactions were observed in 87% of the cows from which M. paratuberculosis was isolated. Dairy producers that participated in the Johne's control program reported reduced economic losses. Increased income was attributed to improved milk production, increased value of vaccinated animals sold as replacements to other dairy herds in which Johne's disease had been diagnosed, and the increased market value of slaughter animals removed from the herd.

Evaluation of an enzyme immunoassay for diagnosis of bovine leptospirosis caused by Leptospira interrogans serovar hardjo type hardjo-bovis.

Sensitivity and specificity of 4 different antigen preparations from Leptospira interrogans serovar hardjo were compared in an enzyme immunoassay for detection of antibodies against serovar hardjo type hardjo-bovis in serum. Two antigens prepared using detergents showed serogroup cross-reactivity. A mechanically extracted membrane and a lipopolysaccharide antigen showed a high degree of leptospiral serogroup specificity. The lipopolysaccharide antigen was the most suitable antigen for detection of anti-hardjo antibodies. Enzyme immunoassay was more sensitive than the microscopic agglutination test for detecting antibodies in serum from experimentally and naturally infected cattle. It was not possible to differentiate vaccinated from infected animals or to detect a secondary immune response in vaccinated animals that were subsequently infected.

Serum biochemical and hematologic values of normal and Mycobacterium bovis-infected American bison.

Hematologic and serum biochemical tests were used to monitor the health of 3 groups of bison in an experimental study of tuberculosis. Bison were randomly assigned to Mycobacterium bovis-infected, M. bovis-sensitized, and uninfected control groups. Hematologic measurements included total and differential leukocyte counts, hemoglobin (Hb), packed cell volume (PCV), fibrinogen, and plasma proteins. Biochemical tests included serum urea nitrogen, creatinine, aspartate amino transferase, sorbitol dehydrogenase, serum calcium, and serum phosphorus. There were no significant differences (P = 0.05) in any test values between groups of bison. The bison data were combined and compared to similar data of cattle. The mean values for PCV and Hb were higher than values (PCV 24-46%, Hb 8-15 g/dl) for cattle. Mycobacterium bovis-infected bison had a slight increase in the number of blood monocytes and lymphocytes when compared to the uninfected bison but were within the normal ranges for bison and cattle. Other hematologic parameters were within normal ranges reported for cattle. Creatinine levels in all bison were above the normal range (1.0-1.5 mg/dl) for cattle. Phosphorus levels for M. bovis-infected and M. bovis-sensitized bison exceeded the normal range (5.6-8.0 mg/dl) reported for cattle. The level for uninfected bison was near the upper limit of normal for cattle. Mean values for other serum biochemical tests were within the normal ranges reported for cattle.

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.

Mycobacterium bovis infection in North American elk (Cervus elaphus).

A naturally occurring outbreak of Mycobacterium bovis infection in captive wild elk (wapiti) in Montana was confirmed by mycobacteriologic examination. Twenty-eight of 143 elk responded to M. bovis purified protein derivative (PPD) tuberculin injected intradermally in the cervical region (SCT). The results of comparative cervical tuberculin skin tests conducted within 9 days of SCT revealed greater responses to M. bovis PPD tuberculin than to M. avium PPD tuberculin in 23 of 28 elk responding. At necropsy, several grossly visible tuberculous lesions were observed in the parenchyma of the lung, thoracic lymph nodes, and submandibular lymph nodes. Microscopic examination of appropriately stained tissue sections revealed the presence of granulomatous lesions containing acid-fast bacilli. An enzyme-linked immunosorbent assay (ELISA) was developed using a sarkosyl extract of M. bovis (antigen) and peroxidase-labeled protein G (conjugate); reactions were detected in the sera of 8 of 9 elk responding to M. bovis PPD tuberculin. Lymphocyte blastogenic assay responses were detected using M. bovis antigens in 7 of 9 elk positive on skin tests using M. bovis PPD.

Isolation of Mycobacterium avium serotype 3 from a white-headed tree duck (Dendrocygna viduata).

Tuberculous lesions were observed at necropsy in the liver of 1 of 10 White-headed Tree ducks (Dendrocygna viduata) imported from Nigeria. Microscopic examination revealed granulomas with acid-fast bacilli; Mycobacterium avium serotype 3 was isolated. Two chickens inoculated intraperitoneally with the isolant had gross and microscopic granulomas in the liver at necropsy 62 days after inoculation. Isolants from chickens were serologically similar to the strain isolated from the duck.

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.

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.

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.

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.