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PubMed1992

[Experimental paratuberculosis in sheep after intravenous or oral inoculation: pathogenicity and biologic diagnosis].

An experimental paratuberculosis study was performed in sheep. One group of 6 lambs was inoculated intravenously with the equivalent of 50 mg (wet weight) of live bacilli, another group of 6 lambs was inoculated orally by placing 500 mg (wet weight) of live organisms in milk feed and a group of 3 lambs was used as controls. The degree of cellular immunity was followed by examining delayed hypersensitivity using 3 allergens (bovine tuberculin PPD, avian tuberculin PPD and johnine PPD) and that of humoral immunity using complement fixation test, agar gel immunodiffusion test and ELISA. The elimination of bacilli in the faeces was examined simultaneously. After 2 years no macroscopic or microscopic lesion was observed in intravenously inoculated lambs and in those exposed orally to M paratuberculosis; cultures were negative. It appears that domestic sheep were able to control the infection. Nevertheless, most of them developed cellular and humoral immunity against paratuberculosis antigen. The best results were obtained in intravenously inoculated lambs.

Animals↗
PubMed1991

Serologic enzyme-linked immunosorbent assay responses of calves vaccinated with a killed Mycobacterium paratuberculosis vaccine.

The purpose of this study was to document the effect of calfhood vaccination for Mycobacterium paratuberculosis on a serologic ELISA. Fifteen calves vaccinated with a killed paratuberculosis vaccine and 5 unvaccinated control calves were tested from the first through the fifteenth month of life. Age of vaccination ranged from 5 to 40 days. Blood samples were collected prior to vaccination and periodically thereafter. Serum antibody was analyzed by use of the ELISA. All calves were ELISA-negative prior to vaccination. Thirteen of 15 vaccinated calves became ELISA-positive between 2 and 6 months after vaccination. The unvaccinated cohort remained ELISA-negative. Wide-spread use of vaccine may interfere with diagnosis of paratuberculosis and with control programs that are based on serologic tests that measure humoral antibody.

Animals↗
PubMed1986

Immunohistochemical distribution of immunoglobulin and secretory component in the ileum of normal and paratuberculosis-infected cattle.

The immunohistochemical distribution of IgA, IgG, IgM and secretory component in the ileum of 10 normal and 21 paratuberculosis-infected cattle was investigated. Semi-quantitative analysis of the number of each class of Ig-containing cells in the lamina propria mucosa of infected ileums showed that IgG and IgM-containing cells and total Ig-containing cells were significantly more numerous than those in the normal ileums. There was no significant difference in the numbers of IgA-containing cells between the two groups of cattle. The distribution of IgA, IgM and SC was basically similar in the two groups. However, IgG-containing cells characteristically accumulated around the granulomas. It was considered that excessive local production of Ig in the intestinal mucosa, along with subsequent formation of immune complex or release of histamine from mast cells, could account for the occurrence of diarrhoea and participate in the pathogenesis of bovine paratuberculosis. A comparison of the local immunological state in paratuberculosis and Crohn's disease was made.

Animals↗
PubMed1988

The distribution of ferritin, lactoferrin and transferrin in granulomatous lymphadenitis of bovine paratuberculosis.

Immunohistochemical examination of iron-binding proteins was carried out in the formalin-fixed mesenteric lymph nodes of normal cattle and of cattle with paratuberculosis. Ferritin (FT) and lactoferrin (LF) were found in the granulomas in ileal lymph nodes from six infected cattle. A weak reaction for transferrin (TF) was found in granulomas of a lymph node from one of the infected cattle. FT was found in the macrophages in the medullary sinuses of normal and infected nodes; however, the reaction in infected nodes was generally stronger than that in normal ones. LF in the macrophages was found in only two infected nodes. Neutrophils in both normal and infected cattle always reacted strongly for LF. The TF was always found in the blood vessels and intracellular space. These results suggest that: (1) FT and LF may be important in vivo sources of iron for Mycobacterium paratuberculosis, since their own iron-binding compounds are considered to acquire iron from FT and LF in vitro; (2) the increase in FT and LF in the granulomas may be related to inflammatory hyposideraemia associated with paratuberculosis and (3) epithelioid and giant cells may have a different iron metabolism, from normal macrophages.

Animals↗
PubMed1989

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.

Animals↗
PubMed1990

Growth of Mycobacterium paratuberculosis in radiometric, Middlebrook and egg-based media.

The ability of BACTEC radiometric 7H12 broth, Middlebrook 7H10 Tween broth, Middlebrook 7H10 agar, and Herrold's egg-yolk medium to provide early detection of Mycobacterium paratuberculosis was evaluated. The minimum detection times in days for the various media were: 7H12, 9; 7H10 agar, 23 (plate), 28 (slant); 7H10 Tween broth; 27; and Herrold's egg-yolk medium, 43 (plate), 49 (slant). The radiometric broths provided the earliest detection of M. paratuberculosis, and 3625 organisms ml-1 were required to produce a positive, radiometric growth-index reading. Of the non-radiometric plate and slant media evaluated, microscope examination of the translucent 7H10 agar plate resulted in the earliest detection and highest mean colony counts (387) as compared with Herrold's egg-yolk agar plate (208). Similar results were noted for 7H10 and Herrold's egg-yolk agar slants; however, accurate colony counts could not be determined because of confluent growth. All media were supplemented with 2 micrograms ml-1 of mycobactin J and excess amounts of this supplement inhibited the growth of M. paratuberculosis in radiometric 7H12 media.

Animals↗
PubMed1988

Effect of glucocorticoids on cows suspected of subclinical infection with M. paratuberculosis.

Glucocorticoids were administered to 10 heifers suspected of subclinical infection with Mycobacterium paratuberculosis. Three animals remained untreated. M. paratuberculosis was isolated from the internal organs of 2 animals after this treatment but not from any of the control group. Delayed type hypersensitivity and lymphocyte reactivity towards Johnin and purified protein derivates of M. avium and M. bovis were depressed. A sharp increase in total leucocyte count, due to an increase in neutrophil numbers, occurred. In the three untreated animals these parameters did not change during the experiment. A decrease of specific immunological reactivity towards M. paratuberculosis occurred, but not to such an extent that clinical disease developed.

Animals↗
PubMed1992

Induction of cellular immune reactions by A36, an antigen complex of Mycobacterium paratuberculosis: comparison of A36 and johnin components.

Paratuberculosis (Johne's disease) is a chronic enteritis syndrome of ruminants, which is due to infection by Mycobacterium paratuberculosis. Cutaneous testing with proteins extracted from a mycobacterial culture fluid (johnin-PPD) is currently used to evaluate the cellular immune status. We have compared the components of johnin-PPD with those of the A36 complex, a thermostable macromolecular antigen (TMA) present in the cytoplasm and associated with the cell wall of M. paratuberculosis. The presence in the johnin-PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal antibodies, which bind respectively to the 65-kDa M. leprae heat shock protein, the 28-kDa M. leprae superoxide dismutase, and M. tuberculosis lipoarabinomannan, recognized components of the johnin-PPD. The ability of A36 to trigger delayed hypersensitivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36-sensitized mice, matched that of johnin-PPD. The homology levels of T epitopes between A36 and the TMA complexes of M. phlei, M. bovis, M. tuberculosis and M. avium were estimated, in a lymphoproliferation assay, to be 51, 52, 59 and 94% respectively. A strong cross-reactivity of A36 with an M. leprae sonicate was also observed by cutaneous testing. The A36 components within the 45.2-26.8-kDa and the 21.6-19.8-kDa ranges were proved to induce the proliferation of T lymphocytes from sensitized mice. This work supports the possible use of the A36 complex, and of some of its components, for cutaneous tests and lymphocyte proliferation assays, in order to monitor cellular immunity in Johne's disease.

Animals↗
PubMed1986

A reappraisal of the complement fixation test using soluble Mycobacterium avium antigen for the detection of M. paratuberculosis infection in cattle.

Serums from 263 cattle suspected of having paratuberculosis on the basis of clinical signs, were tested for antibodies to Mycobacterium paratuberculosis with a complement fixation test (CFT) employing a heat extracted, soluble M. avium antigen. Microscopic examination confirmed that 172 (65.4%) clinically affected animals had paratuberculosis, the remainder being disease-free. The specificity and sensitivity of the CFT was 92.3% and 74.4% respectively. Phenol treatment of serums before testing was compared with no treatment and was found to have no significant effect on the CFT titres. Results obtained are discussed in relation to the cause of false negative and false positive reactions.

Animals↗
PubMed1985

Isolation of Mycobacterium paratuberculosis from intestinal mucosa and mesenteric lymph nodes of goats by use of selective Dubos medium.

To isolate Mycobacterium paratuberculosis from contaminated material, a selective medium, selective Dubos medium (SDubos), was developed by supplementing conventional Dubos medium (CDubos) with carbenicillin, polymyxin, trimethoprim, and amphotericin B. The intestine and mesenteric lymph nodes of 1,501 goats were cultured in parallel on SDubos and CDubos after decontamination with oxalic acid. The contamination rate was reduced more than 150 times by the use of SDubos. The number of positive specimens (18, or 1.2%), i.e., those which revealed growth of M. paratuberculosis, was too small to evaluate the number of specimens which could have been missed due to contamination on CDubos. However, the number of specimens positive on SDubos (16, or 89%) showed that the antibiotics used were not harmful to M. paratuberculosis.

Animals↗
PubMed1987

Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species.

DNA extracted from an unclassified Crohn's disease-isolated Mycobacterium strain was cloned. The recombinant clones were radiolabeled and hybridized to restriction digests of mycobacterial DNA transferred to nylon membranes. Restriction fragment length polymorphisms (RFLPs) were identified that distinguished between mycobacterial DNA samples. Quantitative estimates of frequencies of DNA base substitution were also obtained. No RFLPs were detected between the DNA of three unclassified Crohn's disease-isolated mycobacteria and Mycobacterium paratuberculosis, although several RFLPs were detected that distinguished between M. paratuberculosis and both M. avium complex serovars 2 and 5. The frequency of DNA base substitution between M. paratuberculosis and M. avium complex serovar 2 was measured as 0.87 (+/- 1.2)%.

Cloning, Molecular↗
PubMed1991

Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis.

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.

Animals↗
PubMed1977

Effect of freezing on the viability of Mycobacterium paratuberculosis in bovine feces.

Three bovine fecal specimens were cultured for Mycobacterium paratuberculosis before freezing and after frozen storage at -70 degrees C for 3 and 15 weeks. The losses in viability from 0 to 3 weeks of storage were significant (P = 0.01) for all three samples. The losses in viability between 3 and 15 weeks of storage were not significant (P greater than 0.05) for two specimens in which the M. paratuberculosis occurred naturally, but the loss was significant (P = 0.01) in a simulated specimen (M. paratuberculosis culture added to feces from a healthy cow).

Animals↗
PubMed1987

Economic losses due to paratuberculosis in dairy cattle.

The results of a study of the economic losses caused by paratuberculosis in dairy cattle are reported. The losses in production and the determination of lost future income due to premature disposal are emphasised. A decrease in milk production of 19.5 per cent compared with the lactation two years before culling was recorded in animals showing clinical signs of paratuberculosis. The decrease in production in the last lactation but one compared with the previous lactation was 5 per cent. In animals with non-clinical forms of paratuberculosis these decreases in production were 16 per cent and 6 per cent, respectively.

Age Factors↗
PubMed1978

Pathological evaluation of paratuberculosis in naturally infected cattle.

Thirty-two of 51 cattle infected with Mycobacterium paratuberculosis had chronic enteritis, chronic lymphangitis or mesenteric lymphadenopathy, or all three, at slaughter. Granulomatous inflammatory lesions were mild to advanced and predominantly involved the distal small intestine. Rectal involvement was seen only in five cattle. Fourteen had microgranulomas in the liver. There were three cytological forms of macrophages: histiocytic, polygonal and epithelioid. The latter two types had engulfed moderate numbers of acid-fast bacilli. The histiocytic macrophages usually were packed with acid-fast bacilli. Except in the liver and occasionally its nodes, remote lesions of paratuberculosis were not found in other organs. One animal had endocardial and aortic calcifications. Most cattle with signs of diarrhea had globule leukocytes in or around myenteric ganglion cells. The thymus of 3- to 8-year-old cattle with clinical signs frequently had mild to advanced involution. The thymus of similarly aged infected animals without clinical signs, and of paratuberculosis-negative animals, had not involuted.

Animals↗
PubMed1992

Effects of dialyzable lymph node extracts on lymphoblast proliferative capacity of blood mononuclear cells in cattle with chronic paratuberculosis.

Dialyzable lymph node extracts (DLE) containing transfer factor prepared from calves sensitized to Mycobacterium paratuberculosis and keyhole-limpet hemocyanin (KLH) were administered to 4 adult cows with chronic paratuberculosis. Cutaneous delayed hypersensitivity, lymphocyte blastogenesis, monocyte migration-inhibition, and lymphoblast proliferative capacity as a reflection of interleukin-2 (IL-2) activity were measured in response to M bovis purified protein derivative, johnin, and KLH before and after treatment with DLE. Change in cutaneous delayed hypersensitivity was not evident after DLE treatment. Alterations in histologic features of pre- and posttreatment sections of ileum and mesenteric lymph nodes were not detected. Lymph node extract treatment significantly (P less than 0.05) increased IL-2 activity and migration-inhibition in response to johnin and KLH in vitro. Treatment had no effect on lymphocyte blastogenesis. The data indicate that cattle with chronic paratuberculosis may benefit from DLE treatment, by virtue of increased IL-2 activity, and that effects of DLE are at least partially mediated by an increase in IL-2 activity.

Animals↗
PubMed1992

Measurement of lymphoblast proliferative capacity of stimulated blood mononuclear cells from cattle with chronic paratuberculosis.

Concanavalin A (conA) blast proliferation as a quantitative measure of lymphoblast proliferative capacity by blood mononuclear cell supernatants was measured in cattle naturally infected with Mycobacterium paratuberculosis and in healthy control cattle. Blast cell proliferation was significantly reduced in infected animals, compared with control cattle when blood mononuclear cells were stimulated with conA. Proliferation was significantly greater than media control when M bovis purified protein derivative and johnin were used to stimulate cells from the infected group. After sensitizing control and affected cattle with M paratuberculosis bacterin (live M bovis and keyhole limpet hemocyanin in Freund's incomplete adjuvant), infected animals had no difference in blast cell proliferative capacity with the mycobacterial antigens and conA stimulation, whereas healthy animals had significantly increased blast proliferation in response to all the sensitizing antigens. The blast cell proliferative capacity in infected animals with keyhole limpet hemocyanin stimulation was increased significantly after sensitization; however, it remained significantly less than that in the sensitized control group. These data indicate that cattle naturally infected with M paratuberculosis probably produce suboptimal interleukin-2 (IL-2) activity in response to a potent IL-2 inducer (conA) and fail to optimize IL-2 activity when sensitized with a potent immunogen (keyhole limpet hemocyanin).

Animals↗
PubMed1991

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats.

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.

Animals↗