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Immunodiffusion analysis shows that Mycobacterium paratuberculosis and other mycobactin-dependent mycobacteria are variants of Mycobacterium avium.

Antigenic analysis by immunodiffusion has been applied to 74 strains of mycobactin-dependent mycobacteria. Thirty-eight strains were of Mycobacterium paratuberculosis from cases of Johne's disease of cattle or goats. The remaining cultures were obtained from a variety of animals and included the wood pigeon bacillus. Rabbit antisera were raised to some of the strains and these, together with antisera to M. avium and M. intracellulare, were used to examine sonicate preparations of all the cultures. All were found to be antigenically identical with M. avium and none were found to belong to M. intracellulare. A predominance of the cultures from Johne's disease belonged to the potential brunense subspecies of M. avium, and the remainder together with the majority of the other mycobactin-dependent strains belonged to the type subspecies. In view of these findings the separate species status of M. paratuberculosis is refuted and some difficulty remains in the nomenclature of strains giving rise to Johne's disease.

Animals

Some diagnostic features of the pathogenesis of bovine paratuberculosis (Johne's disease) and serum biochemical changes after oral reinfection.

Ten bull-calves were infected with 10(8) viable cells of Mycobacterium paratuberculosis per os. During the 400-day period of observation faecal and blood samples were taken from animals at 30-day intervals. Faecal samples were examined microscopically, blood samples by the CFT, AGID and LST tests. Intradermal allergic tests were carried out at PI (post infection) days 92, 217, 336, using mammalian, avian and johnin PPD. In the period of study, these efficiency indices showed fluctuations characteristic of the given tests. In the period between PI day 160 and 400 fifteen biochemical parameters were measured monthly, TRP, ALP, TRIG and CHOL were reduced by day 400, pointing to disorders of digestion and absorption. Increased activities of CK, ALD, LDH, alpha-HBDH and ALT indicated skeletal muscle and/or liver damage in the first place. Serum CK, ALD activities and TRIG and TRP concentrations may serve as useful complementary values to the specific diagnosis of paratuberculosis, particularly in the advanced stage of the disease.

Animals

Mycobacterial antigen detection by immunohistochemistry in goat paratuberculosis.

An immunohistochemical method (PAP) for a microscopic diagnosis of paratuberculosis in goats is described as an alternative method to the Ziehl-Neelsen technique. Mycobacterial antigens are only found in enteric and mesenteric lymph node lesions. This method is the most sensible, particularly in cases in which there are no paratuberculosis lesions and the Ziehl-Neelsen technique shows no acid-fast bacilli (AFB).

Animals

Immunohistochemical distribution of ferritin, lactoferrin, and transferrin in granulomas of bovine paratuberculosis.

Granulomatous lesions of bovine paratuberculosis contained ferritin, lactoferrin, and a small amount of transferrin, as demonstrated by the immunohistochemical method. Macrophages in the normal bovine ileum did not contain lactoferrin and transferrin; however, ferritin was found in individual macrophages of Peyer's patches. These results may help elucidate the relationship between intracellular growth of Mycobacterium paratuberculosis and the presence of iron-binding proteins in the granulomas.

Animals

Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves.

Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.

Animals

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.

Animals

Prophylactic effect of monensin sodium against experimentally induced paratuberculosis in mice.

Monensin sodium (0, 15, or 30 mg/kg of complete feed) was fed ad libitum for 1 week to female mice (strain C57BL6/J) that were genetically susceptible to infection with Mycobacterium paratuberculosis. Ten mice in each of the 3 groups were inoculated intraperitoneally with M paratuberculosis (10(9) organisms). Sterile saline solution was injected intraperitoneally into 10 other mice in each group. Rations were continued for 50 days, then mice were euthanatized, and body weight, splenic weight, and hepatic weight for each mouse were recorded. Ratios of body weight to splenic weight and of body weight to hepatic weight were calculated for each mouse. Hepatic granulomas in 50 light microscopic fields were counted, and presence of acid-fast organisms in those granulomas was recorded. Infected mice given monensin had higher body weight and fewer hepatic granulomas than did mice not given monensin. Although hepatic granulomas were fewer in these mice, they contained acid-fast organisms. Effects of 15 mg of monensin and those of 30 mg of monensin/kg of complete feed were not different.

Animals

[Biologic characteristics of mycobacterium strains isolated from cattle from herds with clinical paratuberculosis].

In the period from 1983 to 1986, bacteriological examination for paratuberculosis was performed in 263 samples of lymph nodes, intestinal mucous membrane and excrements of cattle, kept on a farm where clinical paratuberculosis occurred. Seventy-nine strains of mycobacteria were isolated during the culturing. On selective agar medium with mycobactin as the growth stimulator, 71 strains were isolated which had failed to grow on the conventional mycobacterium-culturing media. In the subculture, the dependence of mycobacteria on the mycobactin declined and the number of mycobacterium strains growing in the subculture on conventional mycobacting-free media doubled. Two thirds of the mycobacteria which did not depend on mycobactin during growth exhibited the same antigenic properties as Mycobacterium avium 1, 2, 3, 8 during serotypification. Ability to induce sensibility to PPD avian tuberculin or paratuberculin was demonstrated during the bioassays of mycobactin. Almost a half of the strains inducing animals' sensitivity to the above-mentioned allergens were found to be virulent to pullets that had tuberculosis in their parenchymatous organs. Of the laboratory animals, the highest virulence of the mycobactin-dependent mycobacterium strains was demonstrated in mice subjected to intravenous infection, accompanied by hyperplasia of the spleen, with reisolation of the mycobacterium culture within six eight weeks after infection.

Animals

Use of an enzyme-linked immunosorbent assay to estimate prevalence of paratuberculosis in cattle of Florida.

A survey was conducted from 1986 through 1987, for which an ELISA was used to obtain information on the prevalence of Mycobacterium paratuberculosis infection in cattle of Florida. Results revealed prevalence of 8.6% in beef cattle and 17.1% in dairy cattle. In beef and dairy cattle, prevalence increased with increasing herd size. It was concluded that ELISA-detectable circulating antibodies to M paratuberculosis are widespread in cattle of Florida.

Animals

Genetic relatedness among Mycobacterium paratuberculosis and M. avium complex.

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis of total DNA was performed with 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

DNA Probes

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay.

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.

Animals

Comparison of two methods for isolation of Mycobacterium paratuberculosis from bovine fecal samples.

Fecal samples from 131 cattle clinically suspect for paratuberculosis were cultured bacteriologically, using the traditional sedimentation processing method and a processing method that included a centrifugation step. Of 16 samples that were contaminated, 6 were culture-positive on at least 1 medium and by 1 processing method. Ten of 131 (7.6%) fecal samples processed by both methods were lost because of contamination. The number of culture-positive samples (using both processing methods) were 65 of 121 (53.7%) on media without miconazole and 60 of 121 (49.6%) on media with miconazole. Seven of the 121 (5.8%) samples were culture-positive, using centrifugation, after 16 weeks' incubation at 37 C. Thirteen of 60 (21.7%) isolates were obtained only with centrifugation, and 10 of these had low colony counts, suggesting that a centrifugation step may have concentrated microorganisms that would have gone undetected without centrifugation. Six of 60 (10%) isolates positive for M paratuberculosis on the sedimentation method were negative on the centrifugation method. Contamination rates were significantly (P less than 0.001) increased when centrifugation was used. The miconazole significantly (P less than 0.001) decreased contamination rates when centrifugation was used.

Animals

Experimental paratuberculosis (Johne's disease)--studies on biochemical parameters in cattle.

Ten male Holstein-Friesian calves naturally infected by Mycobacterium paratuberculosis were experimentally re-infected orally at an average of 17 days. Monthly measurements were conduced of the following activities, in the period between post infection days 160 and 400: total protein (TPR), albumin (ALB), cholesterol (CHOL), triglycerides (TRIG), Zn and Cu concentrations as well as sorbitol dehydrogenase, lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), gamma-glutamyltransferase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase and fructose-1,6-diphosphate aldolase (ALD). TPR, ALB, TRIG, and CHOL were reduced by day 400, in conjunction with disorders of digestion and absorption. Increased activities of CK, ALD, LDH, alpha-HBDH, AST and ALT primarily indicated damage to skeletal muscle and/or liver. Serum CK and ALD activities as well as TRIG and TPR concentrations may serve as aids to specific diagnosis of paratuberculosis, particularly in the advanced stage of the disease.

Animals

Characterization of total deoxyribonucleic acid of Mycobacterium paratuberculosis (ATCC 19698) and of M. avium complex (ATCC 25291) using restriction enzymes.

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis was conducted of Total DNA using 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

Animals

Survival of Mycobacterium paratuberculosis in slurry.

Cattle and swine slurry and a mixture of equal parts of both, was mixed with a culture of M. paratuberculosis, 0.1 mg per ml (1 mg = 33 X 10(6) viable units) and stored under anaerobic conditions at 5 degrees and 15 degrees C. At 5 degrees C the survival time for M. paratuberculosis was 252 days in all 3 kinds of slurry, and at 15 degrees C it was 98 days in cattle slurry, 182 days in swine slurry, and 168 days in mixed slurry.

Animal Husbandry

Skin testing, fecal culture, and lymphocyte immunostimulation in cattle inoculated with Mycobacterium paratuberculosis.

Fourteen calves at 21 days of age were experimentally inoculated with 100 mg (wet weight) of Mycobacterium paratuberculosis. Three calves were inoculated orally, 4 intravenously, and 7 subcutaneously. Lymphocyte immunostimulation, fecal culture, and intradermal tuberculin skin testing were done between 112 to 150 days following exposure. Lymphocyte immunostimulation test results, conducted at 112 days after inoculation, showed all animals positive to Mycobacterium avium purified protein derivative. Fecal culture results, taken at 120 days after inoculation, showed that 2 of 3 animals inoculated intravenously were positive, whereas only 2 of 7 inoculated subcutaneously were positive (8 of 14 total were positive). Intradermal skin testing results at 150 days with M avium purified protein derivative showed 13 of the 14 calves were positive. Calves were examined at necropsy 153 days after inoculation, and M paratuberculosis was isolated from tissues of each of the 14 calves.

Animals

Prevalence of Mycobacterium paratuberculosis in ileocecal lymph nodes of cattle culled in the United States.

During 1983 and 1984, ileocecal lymph node specimens were obtained from clinically normal cattle at 76 US Department of Agriculture-inspected cull cattle slaughterhouses in 32 states and Puerto Rico. Mycobacterium paratuberculosis was isolated from the lymph nodes of 119 of 7,540 cattle. The prevalence of bovine paratuberculosis was 1.6% overall, with 2.9% in dairy culls and 0.8% in beef culls. The prevalence for females and males did not appear to differ significantly.

Animals