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Multilocus enzyme electrophoresis analysis of the Mycobacterium avium complex and other mycobacteria.

Multilocus enzyme electrophoresis analysis was used to evaluate the Mycobacterium avium complex (MAC), M. paratuberculosis, and nine other mycobacterial species. The average number of alleles per locus was 2.8 for the 35 MAC and 2 M. paratuberculosis strains which represented 24 electrophoretic types (ETs) and two distinct groups. The M. avium group was resolved into 17 ETs and contained the M. paratuberculosis ET. The M. intracellulare group consisted of six ETs. There was complete agreement between Gen-Probe identification and group placement by multilocus enzyme electrophoresis. The mean genetic diversity per locus for the 24 MAC ETs was 0.38. This procedure subdivided some serovars and, if implemented, should prove to be a powerful epidemiologic tool for the MAC. Eleven additional ETs were formed after the data for the other mycobacterial species were pooled with those for the MAC.

Alleles↗

Identification of Mycobacterium avium complex in sarcoidosis.

Cell wall-defective bacteria which later reverted to acid-fast bacilli have been isolated from sarcoid tissue. These have not been conclusively shown to be mycobacteria. Specific PCR assays were applied to identify mycobacterial nucleic acids in these cultured isolates and in fresh specimens obtained from patients with sarcoidosis. Positive amplification and hybridization were observed with Mycobacterium avium complex- and/or Mycobacterium paratuberculosis-specific probes in five of the six cultured isolates and two fresh skin biopsy samples and one cerebrospinal fluid specimen. There was no amplification or hybridization with Mycobacterium tuberculosis or M. avium subsp. silvaticum probes, respectively. Patients' sera were also tested for antibody reactivities by immunoblotting with M. paratuberculosis recombinant clones expressing the 36,000-molecular-weight antigen (36K antigen) (p36) and the 65K heat shock protein (PTB65K). All seven sarcoidosis, four of six tuberculosis, and all six leprosy patient serum specimens showed strong reactivity with p36 antigen. In contrast, 13 of 38 controls showed only weak reactivity with p36 (P = 0.002 for controls versus sarcoidosis samples). Similarly, PTB65K reacted with high intensity with sera from 5 of 5 sarcoidosis, 5 of 6 tuberculosis, and 5 of 6 leprosy patients, compared with its low-intensity reaction with 5 of 22 controls (P = 0.001 for controls versus sarcoidosis samples). This study demonstrates the isolation and/or identification of M. paratuberculosis or a closely related M. avium complex strain from sarcoid skin lesions and cerebrospinal fluid. Furthermore, the reactivity of antibodies in sarcoid patient sera against p36 and PTB65K antigens was comparable to the reactivity of sera obtained from patients with known mycobacterial disease. Collectively, these data provide further support for the theory of the mycobacterial etiology of sarcoidosis.

DNA, Bacterial↗

Rapid identification of mycobacteria from AIDS patients by capillary electrophoretic profiling of amplified SOD gene.

Aim-Rapid differentiation of mycobacterial species at the genomic level.Methods-The manganese superoxide dismutase (SOD) gene (464 bp) and 16SrRNA (353 bp) from 104 isolates (18 species) of mycobacteria were amplified using polymerase chain reaction (PCR). Products were sequenced and a phenogram of SOD sequences derived. PCR products of SOD gene were digested with HaeIII, and restriction fragment profiles visualised using capillary electrophoresis.Results-Novel SOD sequences were found for M szulgai, M marinum, M phlei, M smegmatis, M chelonei, M paratuberculosis, M malmoense, M intracellulare serotype 7, M intracellulare serotype 18, and M celatum types 1, 2, and 3. Phylogenetic analysis indicated that 18 of 19 species studied had 8-29% interspecies and <6% intraspecies sequence diversity in the SOD gene. No consistent differences were detected between AIDS and non-AIDS isolates. M paratuberculosis showed a unique SOD sequence with a 1.1% (SD 0.5%) diversity from M avium. Capillary electrophoresis profiles were able to differentiate 16 of 18 species within 24 hours.Conclusions-A phenogram of SOD sequences clearly delineated all mycobacterial species and showed two distinct clusters, fast growing species, and the M avium complex (MAC). Within the MAC, M avium (five types), M intracellulare (five types), M scrofulaceum (two types), and M paratuberculosis (one type) could be demonstrated. Phylogenetic diversity of M celatum from MAC, previously suggested by 16SrRNA data, was confirmed. This simple and rapid method for DNA extraction, in conjunction with capillary electrophoresis of SOD restriction fragments, allows rapid identification of mycobacterial isolates.

Journal Article↗

Selection for immune response in goats: the effect of immunization procedure on antibody response to diphtheria toxoid and human serum albumin.

The serum antibody titers to diphtheria toxoid and human serum albumin were determined in 103 goat kids from lines selected for 12 yr for high or low antibody response to diphtheria toxoid. In the 12th yr, six groups of kids were immunized with different preparations of the antigens. In all groups but one, the antigens were emulsified in Freund's incomplete adjuvant with added sonicated Mycobacterium paratuberculosis. The groups received the following treatments: Group 1 was immunized with both antigens mixed in the same syringe, Group 2 got both antigens injected separately, Group 3 got both antigens injected separately, but with a lower concentration of M. paratuberculosis, Group 4 was immunized with diphtheria toxoid only, Group 5 was immunized with human serum albumin only, and Group 6 was immunized with both antigens mixed, but without any M. paratuberculosis. The animals were immunized at 4 wk of age, and the antibody titers were determined 3 wk later by ELISA and passive hemagglutination. The mean antibody titers to both antigens were different between the selected lines (P less than .03). There was no effect of separate vs combined injections of antigens. However, there were indications of antigen suppression or competition between the antigens. Animals receiving only one antigen seemed to mount a higher antibody response to that antigen than did animals immunized with two antigens.

Animals↗

Johne's disease in New Zealand: the past, present and a glimpse into the future.

Johne's disease is endemic in cattle and sheep in New Zealand and is spreading in farmed deer. - Procedures for the control of Johne's disease in New Zealand have been sporadic and often ineffective. Characterisation of strains of Mycobacterium paratuberculosis using molecular biological techniques have revealed two distinct strain types. One type has been found in cattle, goats and deer, and the other in sheep, goats and deer. Vaccination has been identified as an important method for the control of Johne's disease in New Zealand but the side effects of the current oil-based vaccines severely limit their use. There is increasing international concern that M. paratuberculosis may cause Crohn's disease in humans. Demands for food free of M. paratuberculosis would have very serious consequences for the meat and dairy industries in New Zealand and overseas. The cattle, sheep, goat and deer industries need to jointly develop a strategy for the control of Johne's disease in New Zealand.

Journal Article↗

Immunosuppression in goats by dexamethasone and cyclophosphamide.

The influence of dexamethasone and cyclophosphamide on the goat immune system was investigated. Seven goats, with a previous contact with caprine herpesvirus type 1 (CHV-1), were used. All had been vaccinated with live Mycobacterium paratuberculosis vaccine. Six goats were injected intravenously (i.v.) with dexamethasone daily for 5 days (2.5-4 mg/kg BW per day). Three also received 25 mg/kg BW of cyclophosphamide on day 0. The seventh goat was not treated. Dexamethasone alone caused depression, slight lymphopenia and fall in tuberculin reaction. Dexamethasone plus cyclophosphamide caused a severe clinical reaction, marked leukopenia (lymphopenia and polymorphopenia), fall in tuberculin reaction and significant increase in CHV-1 neutralizing antibody titres. M. paratuberculosis antibody reaction was variable and thus difficult to be assessed. CHV-1 was not isolated.

Animals↗

First evidence of Johne's disease in farmed red deer (Cervus elaphus) in Belgium.

In a deer farm, chronic diarrhoea was seen in a 4-year-old hind. This animal died in poor condition on the farm and Johne's disease was suspected. Ziehl-Neelsen staining of the faeces of this hind were positive for the presence of clumps of small acid-fast bacilli, but faecal cultures remained negative. Direct and indirect tests were performed on 24 hinds and stags (yearlings, 2- and 4-year-old animals). The indirect tests performed were serology (Mycobacterium paratuberculosis antibody ELISA, HerdChek, Idexx), comparative cervical skin test (CCT) and lymphoproliferation test (LT) using Mycobacterium bovis purified protein derivative (PPD) and Mycobacterium avium PPD as antigens. Three positive serological results, three positive CCT and eight positive LT were observed in hinds and stags older than 2 years. No positive serological results were observed in the yearling group, whereas some sensitisation was observed in the CCT as well as in the LT for the same group of animals. The degree of concordance between these indirect tests was poor. The three seropositive animals were slaughtered and subjected to post-mortem examination. Histopathology was performed on mesenteric lymph nodes and on the terminal ileum. Visual changes in some mesenteric lymph nodes were observed, no gross lesion was seen in the intestine. Although Ziehl-Neelsen staining yielded no positive results, a catarrhal focal necrotic enteritis associated with a granulomatous lymphadenitis compatible with Johne's disease was evidenced. The mycobacterial cultures on organ samples from slaughtered animals were positive after 2 months for M. avium subspecies paratuberculosis and negative for M. bovis and M. avium. This is the first description of Johne's disease in a deer farm in Belgium.

Animal Husbandry↗

Comparative sensitivity of various faecal culture methods and ELISA in dairy cattle herds with endemic Johne's disease.

In three New South Wales dairy cattle herds with endemic Johne's disease, prevalence rates by faecal culture were determined to be 12, 18 and 22%, respectively. Whole herd faecal culture was shown to detect markedly more infected cattle than whole herd testing by the EMAI absorbed ELISA, particularly in the two herds with greatest prevalence. In the three study herds, five methods for whole herd faecal culture were compared in each. These included two methods based on primary culture on Herrold's egg yolk medium with mycobactin J (HEYM): (1) conventional decontamination with sedimentation and primary culture on HEYM; (2) Whitlock decontamination and culture on HEYM. The remaining three methods were based on radiometric (BACTEC) culture: (3) decontamination and filtration to BACTEC medium; (4) modified Whitlock decontamination to BACTEC medium and (5) Whitlock decontamination to BACTEC medium. For BACTEC cultures, two methods were compared as confirmatory tests for Mycobacterium paratuberculosis: mycobactin dependence on conventional subculture to HEYM and IS900 PCR analysis of radiometric media. Among 179 cattle tested simultaneously by all five culture methods, 38 cattle were confirmed to be shedding M. paratuberculosis. In identifying shedder cattle, method 5 was the most sensitive, followed by methods 2, 4, 1, and 3 was the least sensitive. The number of BACTEC cultures confirmed by mycobactin dependence or PCR was similar.

Animals↗

The effect of Johne's vaccination on tuberculin testing in farmed red deer (Cervus elaphus).

AIM: To assess the degree of interference with bovine tuberculin testing in farmed red deer that vaccination of young deer with an oil-adjuvanted vs aqueous formulation of live attenuated Mycobacterium paratuberculosis Strain 316F vaccines would be likely to cause, and to compare immunological responses between vaccine formulations. METHODS: Five-month-old red deer (n = 45) were randomly allocated to three treatment groups of 15 animals, which received either no vaccine, a single 2-ml dose of an oil-adjuvanted formulation or two 2-ml doses, 6 weeks apart, of an aqueous formulation of live attenuated M. paratuberculosis Strain 316F vaccine injected subcutaneously (S/C) in the neck (Control, Oil-adjuvant Ptb, and Aqueous Ptb groups, respectively). Injection- site reactions were described and measured on Weeks 3, 6 and 9. Animals were weighed and lymphocyte transformation tests (LTT) and antibody enzyme-linked immunosorbent assays (ELISA) using avian, bovine and Johnin tuberculin were conducted on blood samples collected at Weeks 0, 6, 12, 15, 24, 27, 36 and 39. A bovine mid-cervical skin test (MCT) was applied at Week 12, and comparative cervical skin tests (CCTs) at Weeks 24 and 36. At Week 42, the animals were slaughtered at a commercial deer slaughter premises and subjected to rigorous meat inspection. RESULTS: Two animals were eliminated at the start of the trial due to a positive cross-reaction with bovine tuberculin in the initial LTT. Almost all animals reacted to the MCT at Week 12, with mean skin thicknesses of 3.9, 2.9 and 1.0 mm for the Oil-adjuvant Ptb, Aqueous Ptb and Control groups, respectively. When the CCT was conducted at Week 24, 2/15 Oil-adjuvant Ptb, 2/14 Aqueous Ptb and 1/14 Control animals were classified as CCT-positive to bovine tuberculin. By Week 36, all animals were CCT-negative. The Oil-adjuvant Ptb vaccination resulted in high persistent levels of antibody that reacted with bovine tuberculin, compared with negligible levels in the Aqueous Ptb group. Overall, a single dose of the Oil-adjuvant Ptb vaccine in deer stimulated a vigorous, cross-reactive immune response, evidenced by high LTT, skin-test and antibody reactions to bovine tuberculin, with both cell-mediated and humoral characteristics. By comparison, two doses of the Aqueous Ptb vaccine produced less cross-reactivity and a bias towards a cell-mediated response. The Oil-adjuvant Ptb vaccine resulted in moderate injection site lesions that were quite persistent, whereas the Aqueous Ptb vaccine resulted in smaller nodules that regressed more quickly. CONCLUSIONS: Vaccination of farmed deer with an oil-adjuvanted Johne's vaccine has the potential to cause significant interference with routine tuberculin skin testing. The cross-reactivity should decline with time and the CCT should be able to clear MCT-positives, but there is a risk of false-positives to the blood test for tuberculosis (BTB), due to high persistent levels of antibody. The CCT could be used as a primary skin test in vaccinated deer on some farms. The Aqueous Ptb caused fewer problems with skin testing and produced significantly less bovine antibody than the Oil-adjuvant Ptb, but stimulated persistent cell-mediated immune responses that may provide some protection against Johne's disease.

Animals↗

USE OF ARTHROBACTER TERREGENS FOR BIOASSAY OF MYCOBACTIN.

Reich, Claude V. (Johns Hopkins-Leonard Wood Memorial Leprosy Research Laboratory, Johns Hopkins University, Baltimore, Md.), and John H. Hanks. Use of Arthrobacter terregens for bioassay of mycobactin. J. Bacteriol. 87:1317-1320. 1964.-Arthrobacter terregens was used to assay mycobactin, a growth factor for Mycobacterium paratuberculosis. Within 7 days, A. terregens gave a linear photometric growth response to mycobactin in the range of 0.05 to 0.2 mug/ml. Preparations found to be active (or inactive) by this assay produced corresponding effects on the growth of M. paratuberculosis after 6 weeks to 4 months. Mycobactin was produced routinely from pellicles of M. phlei on a peptone-glycerol-beef heart infusion medium, and was extracted from both cells and medium by organic solvents. The mycobactin content per cell rose rapidly after the third day and attained a maximum at 4 to 6 days. The decline to less than one-half this value by the tenth day was associated with excretion into the medium. Production on synthetic media occurred after increasing the usual levels of asparagine. The demonstrated effects of crude mycobactin on the donor strain were (i) to catalyze the onset of growth and (ii) to reverse the effect of conditions which cause the formation of abnormal cells.

Animals↗

Relationship between negative skin test with Johnin after vaccination and post mortem findings.

One hundred and seventy-six cattle, vaccinated against paratuberculosis in the first month of life with a whole-cell inactivated vaccine, containing Mycobacterium paratuberculosis suspended in a water/oil emulsion, were tested with intradermal Johnin between the ages of five and 14 months. Negative results in the skin test occurred more frequently in animals found positive in post mortem examinations (P = 0.008) using histological, microscopical and cultural techniques.

Animals↗

Johne's disease in Canada Part I: clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds.

Recent international developments in the area of infectious disease control and nontariff trade barriers, along with possible zoonotic concerns, have provoked a revival of interest in Johne's disease in Canada and elsewhere. The bacterium causing Johne's disease, Mycobacterium avium subspecies paratuberculosis, is distributed worldwide and causes chronic granulomatous enteritis, also known as paratuberculosis, in domestic and exotic ruminants, including cattle. The subclinical form of this disease results in progressive weight loss, reduced milk production, lower slaughter value, and premature culling, with possible impacts on fertility and udder health. Eventually, infection can lead to the clinical form that manifests as chronic diarrhea, emaciation, debilitation, and eventual death. Currently, available tests to detect infected animals produce many false-negative results and some false-positives, particularly in subclinically infected animals, thus making their interpretation and utilization challenging in control programs. The objective of this 2-part review is to critically review the literature about Johne's disease in dairy cattle for bovine practitioners in Canada. Part I covers the clinical stages, pathophysiology, diagnosis, and prevalence of infection in Canada, while Part II discusses impacts, risk factors, and control programs relevant to Canadian dairy farms. By reviewing the scientific literature about Johne's disease, control of the disease could be pursued through informed implementation of rational biosecurity efforts and the strategic use of testing and culling.

Animals↗

Evaluation of defined media suitable for isolation of auxotrophic mutants of mycobacteria.

In order to investigate the production of auxotrophs of slow growing mycobacterial pathogens, an evaluation of defined liquid and solid media suitable for Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium paratuberculosis was undertaken. Five defined liquid media were evaluated. A number of solid support matrices was added to the liquid media to produce solid media for evaluation. Tween 80 was added to all media to produce diffuse homogeneous growth. Although satisfactory growth in defined liquid media was achieved with all strains tested, poor growth was generally observed in the equivalent defined solid medium for strains belonging to the tuberculosis complex. Only M. paratuberculosis produced satisfactory growth on defined solid media, especially on Middlebrook 7H9. The inhibitory effect of Tween 80 and the presence of inhibitory factor(s) in agar were confirmed. Partial removal or inactivation of these factors by the addition of absorbents to the media was achieved.

Agar↗

Mycobacterial proteome extraction: comparison of disruption methods.

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.

Animals↗

Unexpected detection of DNA by nucleic acid sequence-based amplification technique.

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.

DNA, Bacterial↗

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium.

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.

Animals↗

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera.

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.

Animals↗

Johne's disease eradication and control: regulatory implications.

The time has come for the livestock industry and the veterinary profession to take Johne's disease seriously in the United States. The continued spread of the disease with accompanying economic hardship to infected herds and the possibility that M. paratuberculosis may be a food-borne pathogen makes inaction a costly proposition. Efforts have started at the national level with NJWG to have guidelines written for a national control program for Johne's disease. A national control program is desirable because it would provide uniformity to control efforts. Veterinarians and livestock owners should be aware of the effort and let their views be known. In addition, national program guidelines already have been written (see the appendix) to certify test-negative herds for paratuberculosis. States that do not offer the certification program should consider it. A list of test-negative herds for Johne's disease that livestock owners could use to buy low risk animals would be a significant step forward in our efforts to control Johne's disease. Federal regulations concerning Johne's disease are outdated and should be changed to facilitate participation by the livestock industry in a Johne's disease control and eradication program.

Animals↗