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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↗
PubMed2001

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium.

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for the presence of the bacterium in modified BACTEC 12B medium, both before and after exposure to gamma irradiation. The water used to wash the larvae off the faecal cultures was also tested for the presence of the bacterium. Positive growth was confirmed as M. avium subspecies paratuberculosis by IS900 polymerase chain reaction and restriction endonuclease analysis of the product. M. avium subspecies paratuberculosis was detected in the unirradiated larval suspensions and wash waters of all three nematode species, and in the irradiated H. contortus larval suspension.

Animals↗
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↗
PubMed1993

Use of a dot enzyme-linked immunosorbent assay on absorbed sera for the diagnosis of bovine paratuberculosis.

This study describes the response of cattle to a dot enzyme-linked immunosorbent assay (ELISA) using sera absorbed with Mycobacterium phlei. Results obtained by visual observation are compared with those obtained using a densitometer. Infection status of cattle was determined by faecal culture. Cattle of different levels of exposure and disease manifestation were examined. A significantly higher dot ELISA response was observed (using both absorbed and non-absorbed sera) in animals with heavy shedding of M. paratuberculosis than in animals which tested negative by faecal culture or shed M. paratuberculosis at lower levels (P < 0.05). Paratuberculosis was diagnosed by visual determination of dot ELISA results using non-absorbed sera in 29 of 44 (65.9%) clinically-suspect animals giving positive results by faecal culture, and 85 of 93 (91.4%) cattle testing negative by faecal culture. With absorbed sera, the sensitivity of visual determination decreased to 15 of 44 (34.1%), while specificity increased to 91 of 93 (97.8%). Approximately 75% of cattle yielding positive results by dot ELISA were heavy bacterial shedders (> 1,500 colonies/g of faeces) at the time of serological testing. Comparison of the dot ELISA results determined visually with results obtained by objective densitometric measurement showed compatible specificity. Sensitivity of the dot ELISA was 65.9% for non-absorbed sera using visual evaluation and 87.5% using densitometric evaluation at a cut-off optical density value of 0.2. For absorbed sera, the values were 34.1% and 82.5%, respectively.

Absorption↗
PubMed2002

Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP.

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Blotting, Southern↗
PubMed1983

Experimental infection of some North American wild ruminants and domestic sheep with Mycobacterium paratuberculosis: clinical and bacteriological findings.

Mycobacterium paratuberculosis originally isolated from bighorn sheep (Ovis canadensis) with spontaneous paratuberculosis was used to orally inoculate Rocky Mountain elk (Cervus elaphus nelsoni) calves, mule deer (Odocoileus hemionus) fawns, white-tailed deer (Odocoileus virginianus) fawns, bighorn X mouflon (Ovis musimon) hybrid lambs, and domestic lambs. All experimentally exposed animals became infected. During the first year of infection, hybrid and domestic sheep were able to control the infection but infection was progressive in elk and deer. Clinical paratuberculosis occurred only in mule deer.

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↗
PubMed1991

An evaluation of selected screening tests for bovine paratuberculosis.

The objective of this study was to evaluate the performance of the lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA), carbohydrate antigen complement fixation (CH-CFT), and protein D antigen agar gel immunodiffusion (D-AGID) tests for bovine paratuberculosis, relative to histopathology, and to culture and isolation of Mycobacterium paratuberculosis from tissues and feces. Samples for test evaluation were collected from four sources including blood and tissues from 400 cull cows at three abattoirs in Ontario, blood and feces from a paratuberculosis survey of cattle from 120 dairy farms in Ontario, a serum bank containing samples from cattle from Ontario and Québec, and a bank of sera from cattle from Pennsylvania and the northeastern United States. The data were analyzed using receiver operator characteristic curves, estimates of relative sensitivity and specificity, and kappa statistics of agreement between tests. The LAM-ELISA performed significantly better than both the CH-CFT and the D-AGID tests. The LAM-ELISA was better at predicting fecal shedding status than tissue infection. However, the LAM-ELISA also had limitations. When interpreted as positive or negative (+/-), at a critical optical density of 0.675, its sensitivity and specificity relative to bacteriology were 49% and 87% respectively. Although the serological tests examined in this study provided some information, they did not predict well the infection status of individual animals.

Animals↗
PubMed1990

Gas chromatographic characterization of the relationship between some Mycobacterium avium and Mycobacterium paratuberculosis strains.

Mycobacterium avium strain P-55 and M. avium strain DENT differ from M. avium strain 16909-338 on the basis of their fatty acid spectra (C14:0, C18:0 and tuberculostearic [TBS] acids) studied by multivariate statistical analyses. Strains P-55 and DENT are closer to M. paratuberculosis strain 5889 than to M. avium strain 16909-338, a finding which is in harmony with earlier immunological observations. The recently isolated M. paratuberculosis strain 385 has proved different from M. paratuberculosis strain 5889.

Animals↗
PubMed1990

[Allergic and serologic diagnosis of paratuberculosis in cattle].

The use of allergenodiagnosis in cattle herds where paratuberculosis is to be eradicated depends on the allergenogenic properties of the causative agent of this disease. An evaluation of the reliability of an intradermal allergic test performed to diagnose paratuberculosis in cattle in the given area revealed that this method is important especially if the result of this test is explicitly negative. A cultivation proof of the causative agent of paratuberculosis is of greatest importance among bacteriological methods for infection diagnosis. The agreement of the bacteriological and serological examination which reached 80% in young cattle pointed out the high specificity of an indirect haemagglutination test. In cows, unlike calves and heifers, the specificity of diagnostic tests was considerably lower. It is recommended to use an elimination method mainly in young cattle: regular examinations of calves from their age of three months will help to form a group of heifers free of infection, observing the basic infection controlling measures and providing conventient diet.

Animals↗
PubMed1990

Preliminary observations on ovine paratuberculosis (Johne's disease) in Zambia.

In an imported flock of sheep from South Africa, an ewe became partially anorexic and gradually losing weight and conditions. The paratuberculosis (Johne's disease) was confirmed on clinical, faecal and histopathological examination. Cultural examination remained doubtful. Serological investigation of other sheep in the flock and at other farm reacted to antibodies of Mycobacterium paratuberculosis on complement fixation test. The study suggests that the disease is actively spreading. This is the first report of ovine paratuberculosis in the Republic of Zambia.

Animals↗
PubMed1988

Accidental self-inoculation with Mycobacterium paratuberculosis bacterin (Johne's bacterin) by veterinarians in Wisconsin.

We surveyed Wisconsin veterinarians to assess the frequency and severity of accidental self- and other human exposure to Mycobacterium paratuberculosis bacterin (Johne's bacterin). Of 199 veterinarians administering the bacterin to cattle, 22 reported one or more exposures, including 19 needle-stick exposures, 8 skin surface exposures, and 2 oral mucosa exposures. The mean incidence of needle sticks was 5.5/100 veterinarians/year of bacterin use or 1/1,000 doses administered. The mean total doses given in the needle-stick exposure group was 276 +/- 318 vs 80 +/- 268 in the group without needle-stick exposure, and the mean number of months administering the bacterin was 21.7 and 16.1, respectively; 63% of needle-stick exposures took place during the injection process. Five adverse reactions were reported, and each resulted from needle-stick exposure. The only systemic reaction followed an exposure to the original bacterin formulation of sonically ruptured M paratuberculosis in Freund incomplete adjuvant. The remaining reactions were to the current formulation of whole killed M paratuberculosis in mineral oil and ranged from a small nodule persisting for 4 to 6 months to painful inflammation of a finger persisting for 24 months. We anticipate an increase in incidence of these minimally debilitating injuries as the use and distribution of the bacterin expands. For hand wounds, we recommend conservative management. Surgical intervention should be considered if a granuloma persists and causes the patient functional difficulty.

Accidents, Occupational↗
PubMed1986

In vitro transformation of lymphocytes from blood and milk of cows with subclinical paratuberculosis.

Lymphocytes from blood or milk of 12 cows were evaluated in vitro for the lymphocyte's capability to proliferate in response to mitogens (phytohemagglutinin-A, concanavalin A, and pokeweed mitogen) and to an antigen prepared from Mycobacterium paratuberculosis (purified protein derivative, PPD-J). Responses of 4 control cows were compared with those of 4 cows subclinically infected with M paratuberculosis and with 4 apparently noninfected herdmates. Blood lymphocytes or milk lymphocytes from control cows had no detectable responses to PPD-J. Blood lymphocytes from infected cows had significant (P less than 0.05) responses to PPD-J, but milk lymphocytes from these cows did not. Conversely, milk lymphocytes from apparently noninfected herdmate cows had significant (P less than 0.05) responses to PPD-J, but blood lymphocytes from these cows did not. There were no significant differences in the responses of blood lymphocytes from control, noninfected, or infected cows to the mitogens. However, milk lymphocytes from infected cows had significantly (P less than 0.05) lower responses than did lymphocytes from the milk of control or noninfected cows to all mitogens. The decreased responsiveness of milk lymphocytes from cows subclinically infected with M paratuberculosis may indicate that immunocompetency of the mammary gland was altered.

Animals↗