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PubMed1995

Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.

Animals↗
PubMed1998

Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures.

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

Animals↗
PubMed1993

Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease.

A species-specific recombinant clone (F57) was obtained from a genomic library of Mycobacterium paratuberculosis in the transcription vector pGem 3Z. This clone proved to be specific for all mycobacteria tested, including M. avium, and was able to recognize all of the tested M. paratuberculosis strains isolated from animals and humans (patients with Crohn's disease). The F57 insert was sequenced and a segment of 620 bp with a G + C content of 58.9% was identified. Comparison of the sequence with sequences in the EMBL and UGEN data banks revealed the uniqueness of the F57 sequence, which had no resemblance to other known genes.

Animals↗
PubMed2000

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.

Animals↗
PubMed1992

Effects of mycobactin J and lactoferrin supplementation of drinking water on the in vivo multiplication of Mycobacterium paratuberculosis in gnotobiotic mice.

In this study the effect of supplementation of drinking water with mycobactin J or lactoferrin on the multiplication of Mycobacterium paratuberculosis in gnotobiotic mice was investigated. The results indicated that neither mycobactin J nor lactoferrin, at the doses used, appeared to have any effect on fecal shedding or tissue burdens of M. paratuberculosis.

Administration, Oral↗
PubMed1991

Associations between dairy production indices and lipoarabinomannan enzyme-immunoassay results for paratuberculosis.

Data from an epidemiological study in Ontario, involving 304 dairy herds, were used to identify associations between selected production indices and lipoarabinomannan antigen serological test results for paratuberculosis (LAM-ELISA). Analyses were conducted at both the herd and individual cow levels of organization. After analytically controlling for management and cow factors in the respective regression models, positive serological paratuberculosis status (as defined by the LAM-ELISA test), was associated with higher milk somatic cell counts at both the herd average (p less than 0.01), and individual cow levels of organization (p less than 0.0001). In contrast, LAM-ELISA test results were consistently not associated with calving intervals in either the herd average or individual cow level analyses. Associations between LAM-ELISA results and milk production were inconsistent. No associations were found at the herd level of organization, and LAM-ELISA results were not associated with a change in breed class average (BCA) for milk, between the previous and the most recent lactations of individual cattle. However, at the individual cow level, LAM-ELISA results were positively associated with higher milk production as measured by the current BCA (p less than 0.05), and individual cow average kg of milk produced per year of life since two years of age (p less than 0.0001).

Animals↗
PubMed1994

Mycobacterium paratuberculosis infection in two llamas.

Mycobacterium paratuberculosis was diagnosed in 2 llamas by bacteriologic culturing and DNA probe testing of fecal and intestinal specimens. Diarrhea and weight loss were observed in both animals. Lesions in the gastrointestinal tracts of the 2 llamas were similar to pathologic changes reported in cattle infected with M paratuberculosis.

Animals↗
PubMed1994

Herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in Wisconsin.

A random sample of Wisconsin dairy herds, stratified by herd size, were tested for paratuberculosis by use of an absorbed ELISA procedure. The ELISA was optimized for overall accuracy by means of receiver operating characteristic curve analysis, and had a sensitivity and specificity of 50.9 and 94.9%, respectively. Herd prevalence was analyzed for correlation with responses to a management practices questionnaire completed by the herd owners. One hundred and fifty-eight herds and 4,990 cattle were tested. Of these, 50% of herds and 7.29% of cattle had positive test results. Calculation of true prevalence from the apparent prevalence indicated that 4.79% of cattle and 34% of the Wisconsin dairy herds tested had serologic evidence of paratuberculosis. Among the 54 herds classified as positive on the basis of true prevalence estimation, the mean number of test positive cattle was 20.3%. The geographic distribution of herds with positive results was not uniform. More infected herds were found in the southern and western districts of Wisconsin than in the eastern district. The west-central district had a larger number of infected herds than did other districts. By use of chi 2 analysis, the only management factor found to be significantly associated with herd prevalence was housing of calves after weaning (P = 0.03). Specifically, in herds with higher prevalence, calves were separated after weaning into calf barns and hutches rather than into pens in the cow barn more often than in herds with lower prevalence. This factor was also considered significant by use of logistic regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Husbandry↗
PubMed1996

Comparison of milk with serum ELISA for the detection of paratuberculosis in dairy cows.

OBJECTIVE: To compare milk with serum ELISA for detection of antibodies to Mycobacterium paratuberculosis. DESIGN: Epidemiologic survey. ANIMALS: 821 Missouri dairy cattle of 12 herds that were brucellosis certified and Dairy Herd Improvement Association members. PROCEDURE: Milk and serum samples obtained concurrently from Missouri dairy herds were tested by use of a standard ELISA procedure. Concurrent collection of milk and serum samples controlled for interactions such as colostral antibodies and the effect of time. On the basis of milk and serum ELISA values, disease prevalence and correlation between milk and serum test results were determined. RESULTS: Correlation measures on individual animals indicated low correlation of milk and serum ELISA values. McNemar's chi 2 of 7.6 was significant (P = 0.05). Analysis correlation was low (kappa = 0.08), as was regression analysis (R2 = 0.02). CLINICAL IMPLICATIONS: Milk ELISA for the detection of exposure to M paratuberculosis lacked correlation with serum ELISA. Further evaluation to determine sensitivity and specificity of milk ELISA will augment the usefulness of milk ELISA as a herd screening test.

Animals↗
PubMed1995

Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows.

Mycobacterial culture was performed on colostrum, milk, and feces from 126 clinically normal cows of a single herd with high prevalence of Mycobacterium paratuberculosis infection. Thirty-six (28.6%) cows were determined to be shedding the organism in the feces. Of the 36 fecal Culture positive cows, M paratuberculosis was isolated from the colostrum of 8 (22.2%) and from the milk of 3 (8.3%). Cows that were heavy fecal shedders were more likely to shed the organism in the colostrum than were light fecal shedders.

Animals↗
PubMedDate not supplied

Use of an experimental chicks model for paratuberculosis enteritis (Johne's disease).

Developments in the diagnosis and treatment of paratuberculosis is constrained by the lack of an experimental animal model. To investigate this problem conventional chicks immunodepressed by a Cyclophosphamide injection and concurrent inoculation of Infectious Bursal Disease Virus (IBDV) were infected with Mycobacterium paratuberculosis and kept for 4 months. The immunodepressed chicks eliminated mycobacteria with their faces from the first month until the third month and developed typical intestinal lesions of mycobacterial infection characterized by aggregation of macrophages with monocytes and lymphocytes. Diarrhoea was absent. The number of lymphocytes decreased by about 80%. The serological tests carried out with Complement Fixation test were negative. For the positive bacteriology and typical granulomatous lesions, the conventionally reared chicks proved to be a useful laboratory model for reproduction of Johne's disease.

Animals↗
PubMed1999

Relation between pathologic findings and cellular immune responses in sheep with naturally acquired paratuberculosis.

OBJECTIVE: To investigate the relation between lesions and cellular immune response-based tests in sheep with naturally acquired paratuberculosis and to evaluate efficacy of an interferon-gamma (IFN-gamma) assay and the intradermal skin test. ANIMALS: 110 and 88 adult sheep were used to evaluate the IFN-gamma assay and skin test, respectively. PROCEDURE: Before euthanasia, heparinized blood was collected and incubated with avian purified protein derivative (PPD). Interferon-y production was assessed, using a commercial enzyme immunoassay. Skin testing in the caudal fold also was performed, using avian PPD. Sensitivity and specificity of these techniques were evaluated, using histopathologic findings as reference. RESULTS: Of the sheep examined, 61 (55.4%) had lesions that were classified in 5 categories, according to location, extension, cellular types, and presence of acid-fast bacilli. Regarding cellular immunity test results, 55.4% of sheep with and 6.1 % without pathologic changes had positive results of the IFN-gamma assay. Skin test sensitivity (55.6%) and specificity (100%) were slightly greater. CONCLUSION: Close association between pathologic findings and cellular immune response was apparent. Most sheep with focal (tuberculoid) and some with diffuse lesions (borderline tuberculoid), all with scant or no acid-fast bacilli in the intestine, had positive results of the IFN-gamma assay and skin test. Most sheep with diffuse lesions and high numbers of bacilli (borderline lepromatous) were test negative. CLINICAL RELEVANCE: Cellular immune response-based and serologic tests are useful tools for seeking evidence of paratuberculosis. Each technique detects different types of immune-reacting sheep, and their simultaneous use may detect most infected animals.

Animals↗
PubMed1995

Ex vivo induction of TNF-alpha and IL-6 mRNA in bovine whole blood by Mycobacterium paratuberculosis and mycobacterial cell wall components.

Johne's disease is a chronic enteritis of cattle and other ruminant species that is of worldwide economic importance. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using reverse transcriptase polymerase chain reaction (RT-PCR) and specific bovine oligonucleotide cytokine primers and probes for bovine TNF-alpha and IL-6, we examined the ex vivo expression of mRNA for these inflammatory cytokines in whole blood from healthy cattle. Cytokine mRNA levels increased after a brief incubation of bovine whole blood with Mycobacterium paratuberculosis or its lipoarabinomannan (LAM). Muramyl dipeptide (MDP) and Escherichia coli LPS also stimulated TNF-alpha and IL-6 mRNA expression. Several strains of M. paratuberculosis were tested and found to have similar abilities to stimulate TNF-alpha and IL-6 mRNA expression. Several strains of the closely related Mycobacterium avium, and the unrelated saprophyte, Mycobacterium phlei, had somewhat less ability to stimulate TNF-alpha and IL-6 mRNA expression.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Antibodies to Mycobacterium paratuberculosis in patients with Crohn's disease.

IgG antibodies against Mycobacterium paratuberculosis protoplasmic antigen were looked for by an enzyme immunosorbent assay in patients with Crohn's disease, Ulcerative colitis, active pulmonary tuberculosis, past pulmonary tuberculosis, and in healthy controls. Serum reactivity for these antibodies was not correlated to PPD skin test positivity without history of mycobacterial disease. A cutoff based on the mean absorbance value of a pool of healthy blood donors was chosen. Positive values were found in 12/24 (50%) patients with active pulmonary tuberculosis and 2/10 (20%) skin-test-positive subjects with past pulmonary tuberculosis as expected, because antigenic sharing is common among different mycobacteria. The control group of blood donors showed borderline positivities in 3/149 cases (2.01%). Positive values were found in 4/108 (3.70%) patients with Crohn's disease and 2/40 (5%) with ulcerative colitis. We conclude that our data do not support a causal relationship between the M. paratuberculosis and Crohn's disease, but occasional patients with inflammatory bowel diseases show unexpected positivities for these mycobacterial antibodies.

Adolescent↗
PubMed1999

Characterization of Mycobacterium paratuberculosis p36 antigen and its seroreactivities in Crohn's disease.

Recent data using improved cultural, molecular, and serological techniques have strengthened the association of Mycobacterium paratuberculosis with Crohn's disease, an inflammatory bowel disease (IBD) with unknown etiology. To provide more evidence of an etiological association, antibody reactivities of Crohn's disease patients were tested by immunoblotting against M. paratuberculosis-recombinant antigens. A clone containing a 1,402-bp insert and expressing a 36K-antigen (p36) was analyzed. No homology was found between the deduced amino acid sequence of p36 and any protein sequences compiled in the GenBank indicating that p36 is a novel mycobacterial protein. The reactivity of 199 serum samples was tested against the p36 by immunoblotting technique. Sera from 77 of 89 (86.5%) Crohn's disease patients and 16 of 18 (89%) sera from patients with tuberculosis and leprosy reacted with p36 compared to 5 of 42 (12%) ulcerative colitis and non-IBD control sera (p < 0.0001). In addition, p36 reacted to all sera from 10 normal controls that were Bacillus Calmette-Guerin (BCG)-immunized and only to 10% of 40 normal controls that were not BCG-immunized. The fact that sera from Crohn's disease patients reacted to p36 with the same high frequency as the sera from patients that were exposed to mycobacterial antigens further supports the hypothesis of the mycobacterial etiology in Crohn's disease.

Antibodies, Bacterial↗
PubMed1991

Lysis and fractionation of Mycobacterium paratuberculosis and Escherichia coli by matrix solid-phase dispersion.

A novel method for the lysis and subsequent fractionation of bacterial constituents from Mycobacterium paratuberculosis strain 19698 (M. paratuberculosis) and Escherichia coli strain DH5 alpha utilizing the technique of matrix solid-phase dispersion (MSPD) is described. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain cellular lysis. The blended material was used to prepare a column which was sequentially eluted with solvents of increasing polarity. Fractionation of cellular components was confirmed by analysis of the solvent extracts. The possible applicability of the MSPD technique as a general method for the lysis and fractionation of bacterial components is proposed.

Bacteriological Techniques↗
PubMed1988

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis.

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.

Animals↗