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[Mycobacteria identified in a centre for veterinary research between 1973 and 1979 (author's transl)].

During the years 1973 to 1979 the Central Laboratory of Veterinary Research in Alfort identified 425 strains of mycobacteria: 93 were pure cultures received from other laboratories, and the remaining 332 were isolated in this laboratory from 590 pathological specimens of different origin and from 12 samples from the environment. Sixteen species were identified, using biochemical criteria and drug sensitivity. "Tuberculous" mycobacteria (Mycobacterium bovis, M. tuberculosis and M. africanum) where the most frequent (58.5%), and "non-tuberculous" mycobacteria (41,5%) were identified as M. avium-intracellulare (136 strains), M. peregrinum (10 strains), M. paratuberculosis (5 strains), M. terrae (5 strains), M. chelonei (4 strains), M. fortuitum (2 strains), and M. aurum, M. vaccae, M. marinum and M. gordonae (1 strain of each). From the specimens received for isolation the more frequent organisms isolated were M. bovis (55%) and M. avium-intracellular (32%).

Animals↗

Fatty acid and polar lipid analysis as tools in the identification of Mycobacterium leprae and some related slow-growing mycobacterial species.

Some species of slow-growing Mycobacteria, including Mycobacterium leprae (1 strain), M. lepraemurium (2 strains), M. paratuberculosis (12 strains) and a group of 12 M. avium-like strains (isolates from wild animals) were examined by gas chromatography (GC) for cellular fatty acids and by thin-layer chromatography (TLC) for polar lipids. All the GC patterns, including that of M. leprae, contained high levels of tuberculostearic-, stearic-, octadecenoic- and palmitic acid. Tetradecanoic-, pentadecanoic-, hexadecenoic- and heptadecanoic acid were also generally present but in lower concentrations. In addition to these acids shared by all strains, each bacterial species or group was found to exhibit compounds which were not detected (or detected in considerably lower quantities) in the other taxa examined. Thus each bacterial species or group could be distinguished by their GC profiles. The corresponding TLC patterns were also rather complex. A total of 39 different spots were distinguished. A few of these were shared by all strains, some were characteristic of certain species or groups, whereas others were strain-specific. Both M. leprae and M. lepraemurium shared several features with the other strains but could be distinguished from each other and from the others by their patterns of slow-moving (polar) spots. The 12 M. avium-like strains were divided into two main groups, one with only a few slow-moving spots (rough stains), and one with several slow-moving spots (smooth strains) which included the M. avium reference strains.

Chromatography, Gas↗

Paramphistomum microbothrioides in American bison and domestic beef cattle.

Intestinal paramphistomiosis was diagnosed in a bull and cow with severe diarrhea and weight loss, and subsequent investigation suggested that the infection was transmitted from American bison (Bison bison). The rumen fluke, Paramphistomum microbothrioides, was recovered from the rumen of 3 bison on the farm or origin. Single operculate eggs of P microbothrioides measuring 130 x 69 micrometers were recovered from the feces of the bull, from 9 of 10 bison, and from 3 of 9 other beef cattle. Snails of the genera Physa and Helisoma were collected at the farm of origin. Ostertagiosis and paratuberculosis were considered in the differential diagnosis but were ruled out on the basis of fecal, plasma, and mucosal tests. In view of the lack of approved drugs for treatment of paramphistomes in the United States, treatment was attempted with an 8 times normal dose of thiabendazole (528 mg/kg). Advice was given on grazing management strategies designed to reduce exposure of susceptible cattle to metacercariae, and the owner was advised to drain a large snail habitat.

Animals↗

Evaluation of lymphocyte stimulation tests for diagnosis of bovine tuberculosis in elk (Cervus elaphus).

Lymphocyte stimulation tests (LST), performed using 6 antigen preparations, were compared individually and in pairs. The tests were performed on 433 blood samples collected from elk in Mycobacterium bovis-infected herds. These elk were killed as part of Agriculture and Agri-Food Canada's bovine tuberculosis eradication policy, and mycobacterial culture results were obtained from tissues of each animal. The LST, which had the highest total sum of sensitivity and specificity, was a comparative test that used M bovis purified protein derivative (PPD) and M paratuberculosis (johnin) PPD. This test had a sensitivity of 76%, with confidence limits (CL) of 63 to 85% for this estimate, and specificity of 77% (CL, 72 to 81%). The LST, using only M bovis PPD antigen, had a sensitivity of 70% (CL, 57 to 80%) and specificity of 74% (CL, 69 to 79%); when it was compared with culture results, using the kappa statistic, agreement was only 32%. This indicated that the LST identified different elk than did M bovis isolation tests.

Animals↗

Molecular definition of unique species status of Mycobacterium w; a candidate leprosy vaccine strain.

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cultivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers such as M. fortuitum, M. smegmatis, M. chelonae and M. vaccae. However, M. w was not fully identical to any one of these. In the present study, a molecular biology approach was used to define the species identity of M. w in a manner that allows reliable comparison to be made with over 30 known mycobacterial species. A 383-bp region, present at the amino terminus of the conserved mycobacterial 65-kDa gene, has been polymerase chain reaction (PCR) amplified in M. w and DNA sequence was determined. A comparison of the M. w DNA sequence with those of M. tuberculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a species-specific polymorphism, i.e., the presence of nucleotide substitutions unique to M. w. In an alternate approach, a 441-bp region, also a part of the 65-kDa gene, has been PCR amplified in M. w and a Hae III restriction pattern was generated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results.

Despite the widespread use of polymerase chain reaction (PCR) for diagnosis of infectious diseases, the technology has not been generally introduced into routine diagnostic laboratories. One of the most serious problems which has influenced the acceptance of this technology is the occurrence of false positive PCR results. This study describes the experience, in a hospital laboratory setting, of using PCR for the diagnosis of heat-labile enterotoxin-producing E. coli, M. tuberculosis, M. paratuberculosis and human papillomavirus. Results indicate that a build-up of amplicons, generated during the amplification process in the laboratory, is the main source of PCR-contamination. Protocols are described that include both physical and chemical procedures to prevent contamination. The use of photo-induced psoralen is recommended for those laboratories already involved in PCR work where amplicons are likely to be present. An enzymatic system (uracil-N-glycosylase) was evaluated and is recommended for workers intending to start diagnostic PCR. Attention was given to simple control measures which are easily implemented in a routine diagnostic laboratory. Protocols such as these are likely to have a major impact on the introduction of PCR-based methods into routine laboratories.

Bacteria↗

Viral association with Crohn's disease.

Crohn's disease is an inflammatory disorder of the gastrointestinal tract, the cause of which remains unknown. Since the first description by Dalziel in 1913 (1), similarities between Crohn's disease and intestinal mycobacterial infection, particularly Johne's disease in ruminants, have been widely recognized (2, 3). After Mitchell and Rees demonstrated the transmission of granulomata from Crohn's disease by injecting intestinal homogenates into the footpads of mice (4), there followed many studies attempting to identify infective agents within the bowel of patients with Crohn's disease. Although Mycobacterium paratuberculosis has been identified in intestinal tissue from a proportion of patients with Crohn's disease, a convincing role for this agent in the aetiology of Crohn's disease has not been established (5). Likewise, extensive studies into bacterial (6-9) and viral (10) agents potentially associated with Crohn's disease have been inconclusive, although recent ultrastructural observations of viral particles within submucosal granulomata have added a new impetus to the search (11, 12). This review examines the evidence for an association between Crohn's disease and viral infection from epidemiological studies, transmission and cell culture, specific immunological responses, ultrastructure and from molecular biological techniques.

Antibodies, Viral↗

[Seroepidemiologic investigations in the Alpine ibex (Capra i. ibex) of Piz Albris in the canton of Grigioni (Switzerland)].

Sero-epidemiological investigations in wild animals may allow to assess distribution of selected pathogens that sometimes seem to be involved in sanitary interrelationships between wild and domestic ungulates sharing the same areas. Serological studies were carried out to investigate the prevalence of antibody against 8 pathogens in Alpine ibex of Albris colony (Grisons, Switzerland). Investigated sera came from 89 animals shot by gamekeepers in 1990-1991. Antibody against smooth Brucella, Coxiella burnetti, Leptospira interrogans, Borrelia Burgdorferi, Mycobacterium paratuberculosis, BHV-1 and ovine-caprine lentiviruses were not detected in the tested sera. However, 31% of sera analysed were found to be positive for Chlamydia psittaci. Three sera showed high antibody titres ( > or = 1/128) suggestive of active infection in the animals. Any influence of Chlamydia psittaci in reproductive performance of free-ranging alpine ibex should be investigated through isolation of the agent. Results are discussed with reference to methods used and with epidemiological picture in Switzerland and were compared with results of serological investigations carried out in ibex in Italy and France.

Animals↗

Cross-reactive and species specific Mycobacterium tuberculosis antigens in the immunoprofile of Schaumann bodies: a major clue to the etiology of sarcoidosis.

Sarcoidosis, once thought to be a variant of tuberculosis, is currently listed as a disease of unknown etiology. The present study was initiated by unpublished observations that Schaumann bodies-the laminated inclusions often encountered in sarcoid granulomas-cross-reacted with commercial polyclonal antibodies to Mycobacterium bovis, Mycobacterium duvalii and Mycobacterium paratuberculosis. Given the broad cross-reactivity of many mycobacterial antigens, those findings lacked specificity but warranted in depth probing of the immunoprofile of the bodies, particularly for specific mycobacterial antigens. Formalin-fixed tissue from eight patients with an established diagnosis of sarcoidosis was studied with panels of antibodies against both common cytoplasmic proteins and various mycobacterial antigens, using a labeled streptavidin-biotin-alkaline phosphatase technique. Our findings indicate that Schaumann bodies are indeed residual bodies of heterophagic mycobacterial derivation. They immunostained intensely for the lysosomal proteins muramidase and CD68, variably for some cytoskeletal proteins (tubulin, desmin, vimentin) and not at all for cytokeratin, muscle actin, alpha-1-antichymotrypsin and ferritin. Both cross-reactive and species specific antigenic determinants of M. tuberculosis complex were shown to be present. Affinity absorption with killed intact bacilli H37 Rv resulted in virtually equal loss of binding by all polyclonal antimycobacterial antibodies to cross-reactive ligands in Schaumann bodies. In addition, the bodies were clearly labeled with the monoclonal antibodies TB68 and TB71, known to recognize species specific epitopes of Mycobacterium tuberculosis complex. Although obtained on a small number of cases, our findings uphold Schaumann's original postulate that the laminated calcific inclusions represent remnants of "transformed tubercle bacilli".

Antibodies, Monoclonal↗

A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus).

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph node (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84.97%) and 88% [CL 77.94%], respectively, and the specificity of both was 89% [CL 85.92%]). The sensitivity and specificity of histopathology II were 89% (CL 79.95%) and 77% (CL 72.81%), respectively. The highest sensitivity estimates (93-95% [CL 84.98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%] were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results showed that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.

Abattoirs↗

[Frequency and cost of health problems in Swiss dairy cows and their calves (1993-1994)].

Between July 1993 and July 1994 morbidity and management information related to dairy cows and their calves up to the age of 8 weeks were recorded in 113 randomly selected dairy herds. Also recorded were any costs incurred through disease and prevention. Blood and faeces were analysed with respect to selected pathogens. The health problems most frequently diagnosed in cows were reproductive and udder diseases. Calves suffered most often from diarrhea, omphalitis and pneumonia. The directly disease-related costs per cow-year on average amounted to CHF 139.44 and CHF 4.18 per calf. For prevention, farmers spent on average CHF 10.18 per cow-year. Results from the laboratory analyses indicate that in 68.1% of the farms antibodies against Leptospira hardjo and in 61.9% against Coxiella burnetii were detected. In 8.0% of the farms antibodies against Mycobacterium paratuberculosis were found. Antibodies against BVD virus was present in 99.4% of the farms. Cows from 63.7% farms were infected with gastrointestinal strongylids. Veterinary assistance was required on average 1.96 times per cow-year. In almost all reproductive and puerperal disease cases a veterinarian was consulted while lameness in the majority of cases was treated by the owner. The veterinary profession was hardly ever involved in disease prevention.

Animals↗

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Animals↗

Health evaluation of free-ranging guanaco (Lama guanicoe).

Twenty free-ranging guanaco (Lama guanicoe) in Chubut Province, Argentina, were immobilized for health evaluations. All but two animals appeared to be in good condition. Hematology, serum chemistry, and vitamin and mineral levels were measured, and feces were evaluated for parasites. Serology tests included bluetongue, brucellosis, bovine respiratory syncitial virus, bovine viral diarrhea/mucosal disease, equine herpesvirus 1, infectious bovine rhinotracheitis, Johne's disease (Mycobacterium paratuberculosis), foot and mouth disease, leptospirosis (17 serovars), parainfluenza-3, and vesicular stomatitis. Blood samples from 20 domestic sheep (Ovis aries) maintained in the same reserve with the guanaco were also collected at the same time for serology tests. No guanaco had positive serologic tests. Sheep were found to have antibody titers to bovine respiratory syncytial virus, Johne's disease, leptospirosis, and parainfluenza-3. There was no apparent difference in external appearance or condition, or statistical difference in blood test values, between the animals that were positive or negative for parasite ova.

Analgesics, Opioid↗

[Mycobacterial intestinal disease in woodpigeons (Columbia palumbus) (author's transl)].

During and shortly after the second world war, an infection bearing a resemblance to avian tuberculosis was observed in woodpigeons in Denmark and Great Britain. These birds had been found dead or been shot. The patogenic agent, however, could not be isolated by the usual methods. In the Netherlands, the disease was also detected in woodpigeons and occasionally in psittacine birds. The histological changes bore a resemblance to those observed in Johne's disease. Detailed bacteriological and experimental studies showed that there were two different infections. One agent was a mycobacterium of the species, which could not be grown on the usual culture media for M. tuberculosis, whereas it could on the media used in the culture of M. paratuberculosis, particularly Smith's medium. The bacterium also soon becomes rough on this culture medium. As a result, differentiation of serological types by Schaefer's method failed. The other type of mycobacterium (which indeed causes a similar form of intestinal disease) could be readily cultured and was identified as M. avium type 2. The former mycobacterium is still nameless in point of fact but is sometimes wrongly referred to as Mycobacterium columbae. This rod was not found to be pathogenic for the domesticated pigeon (Columba livia), not even when intestinal mucosa containing large numbers of bacteria and obtained from a diseases woodpigeon which had died recently, was inoculated orally in recently hatched specimens of the domesticated pigeon. To account for the appearance of tuberculosis in native woodpigeons, it is suggested that low plasma transferrin levels could result in marked susceptibility to infections such as tuberculosis and trichomoniasis.

Animals↗

Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease.

BACKGROUND/AIMS: In order to assess the potential impact of bacterial eradication on recurrence rates, the prevalence of various enteropathogenic bacteria and toxins in chronic inflammatory bowel diseases (CIBD) was prospectively examined. METHODOLOGY: Stool, sera and gut tissue samples from a total of 59 patients (33 males, 26 females; mean age: 42 years +/- 14; 21 Crohn's disease, 14 ulcerative colitis, 24 controls) were examined for the presence of enteropathogenic bacteria by culture, immunoblotting and PCR. RESULTS: Conventional cultures failed to detect obligate pathogenic bacteria. By PCR, mycobacteria were found in 85% of all groups, with mycobacterium paratuberculosis not detected. Yersinia species were observed in 63% of patients with Crohn's disease, in 46% of patients with ulcerative colitis, and in 36% of the control patients. Pathogenic E. coli were identified in stool samples of three patients with ulcerative colitis (21%) by amplifying the EAE-gene, one of whom exhibited shiga-like-toxin as well. CONCLUSIONS: We concluded that mycobacteria do not play a causative role in CIBD. Yersinia species seem to persist in intestinal tissue in CIBD patients without adequate immune response and might, thus, contribute to tissue destruction. E. coli infections contribute to the disease process in a small group of patients with ulcerative colitis and their eradication might eliminate the need for immediate surgical intervention.

Adult↗