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PubMed1994

Effect of combination therapy in Mycobacterium paratuberculosis infected rabbits.

Rabbits, infected with M paratuberculosis of caprine origin, were treated with streptomycin sulphate and rifampicin in combination with levamisole hydrochloride for a period of 2 months. Marked clinical response including significant rise in total serum protein, albumin and globulin were recorded in treated groups. Highly significant higher leucocyte migration inhibition indicating enhanced CMI reaction, occurred in rabbits treated for 4 weeks. Complete elimination of M. paratuberculosis from faeces, intestines and mesenteric lymph nodes was observed. They were also absent from spleen, kidneys, lungs and liver of rabbits treated with rifampicin and levamisole, whereas were present in lungs of the rabbits administered with streptomycin and levamisole. Absence of characteristic lesions of paratuberculosis and evidence of regenerative reaction were observed in visceral organs of rabbits treated with rifampicin and levamisole. Effectiveness of rifampicin is attributed to its effect on lymphocyte, the primary cell involved in cell mediated immunity. Rifampicin-levamisole combination appeared superior to streptomycin-levamisole in eliminating the infection of M. paratuberculosis from infected rabbits. Approximate cost of treatment was calculated to be Rs 8.50 per kg body weight. Cattle, sheep and goats, if treated in early stage of infection may recover from paratuberculosis.

Animals↗
PubMed1995

Mycobacterium paratuberculosis and inflammatory bowel disease: frequency distribution in serial colonoscopic biopsies using the polymerase chain reaction.

An association between Mycobacterium paratuberculosis and Crohn's disease is suspected but the evidence remains controversial. Using a one-step DNA extraction procedure with the thermophilic protease PRETAQ and amplification by the polymerase chain reaction, M. paratuberculosis DNA was detected in 22% of patients with Crohn's disease, and in 13% of patients with ulcerative colitis. M. paratuberculosis DNA was not found in any biopsy tissue from control non-inflammatory bowel disease patients. The biopsy tissues in which M. paratuberculosis was detected all came from regions which were inflamed when viewed microscopically. Overall, 7.7% of biopsies from such inflamed areas were positive. This low frequency of detection could be explained on the basis of extremely low abundance of the organism in relation to the area of mucosa sampled, or be consistent with a non-aetiological role for M. paratuberculosis in inflammatory bowel disease.

Animals↗
PubMed2006

Mitogen activated protein kinase p38 pathway is an important component of the anti-inflammatory response in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes.

We investigated the role of cell signaling through the mitogen-activated protein kinase-p38 (MAPK p38) pathway on the antimicrobial functions and cytokine expression by bovine monocytes after ingestion of Mycobacterium avium subsp. paratuberculosis. We evaluated the dynamic secretion of interleukin (IL)-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) as well as phagosome acidification and organism killing at several time points after in vitro infection of bovine monocytes with M. avium subsp. paratuberculosis. Monocytes treated with M. avium subsp. paratuberculosis had a significant increase in IL-10 expression at 2, 4, and 6h post-infection and an increase expression of TNF-alpha at 2, 4, 6, and 24h post-infection. In contrast, IL-12 expression did not increase at any time point post-infection. Moreover, MAPK p38 was rapidly phosphorylated at 10 and 60 min after M. avium subsp. paratuberculosis ingestion. Chemical inhibition of the MAPK p38 signaling pathway (SB203580) resulted in decreased expression of IL-10 and increased expression of IL-12 at 6h post-infection. Chemically blocking the MAPK p38 pathway also increased acidification of phagosomes as well as increasing the capacity of macrophages to kill organisms. Taken together, these results indicated that selective activation of MAPK p38 may be a major mechanism exploited by M. avium subsp. paratuberculosis to circumvent the antimycobacterial effects of mononuclear phagocytes.

Animals↗
PubMed2001

Etiology of Crohn's disease: the role of Mycobacterium avium paratuberculosis.

Crohn's disease is a chronic inflammatory bowel disease characterized by transmural inflammation and granuloma formation. Several theories regarding the etiology of Crohn's disease have been proposed, one of which is infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), which causes a similar disease in animals, and is present in the human food chain. Considerable evidence supports the presence of M. paratuberculosis in the intestinal tissues of many patients with Crohn's disease including culture, detection of homologous mycobacterial DNA, detection of the mycobacterial insertion sequence IS900 by both PCR and in situ hybridization in tissues, and a serologic immune response to recombinant M. paratuberculosis antigens. Despite this evidence, and our personal belief that M. paratuberculosis is a cause of Crohn's disease, widespread acceptance of this hypothesis will require evidence that specific anti-mycobacterial chemotherapy will cure the disease.

Cell Wall↗
PubMed1985

A glycolipid antigen specific to Mycobacterium paratuberculosis: structure and antigenicity.

Mycobacterium paratuberculosis (National Animal Disease Center strain 18 and American Type Culture Collection strain 19698), the causative agent of Johne Disease (bovine paratuberculosis), contains a major immunoreactive glycopeptidolipid (polar GPL-I) that has been isolated and characterized. (formula; see text) Thus, the glycolipid antigen belongs to the polar mycoside C glycopeptidolipid family present in other mycobacterial species. The distal 2,3-di-O-methyl-alpha-L-fucopyranosyl-(1----3)-alpha-L-rhamnopyranosi de unit, the obvious antigen determinant, appears to be characteristic of M. paratuberculosis. The glycolipid can be recognized readily in isolates of M. paratuberculosis by TLC and its presence may be used as a characteristic marker of the infectious agent. The polar glycopeptidolipid was highly reactive in ELISA against serum from an animal hyperimmunized with M. paratuberculosis strain 18, indicating its basic immunogenicity.

Amino Acid Sequence↗
PubMed2004

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Amino Acid Sequence↗
PubMed1988

Antigenic relationship between Mycobacterium paratuberculosis and Mycobacterium avium.

Four prototype strains of Mycobacterium paratuberculosis contained the type-specific glycopeptidolipid antigen of serovar 8 of the M avium complex. This glycolipid was distinguished by a 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl terminal unit. Of 59 low-passage, field isolates of M paratuberculosis, 2 contained this antigen, and these 2 isolates were indistinguishable from M avium serovar 8. However, most M paratuberculosis isolates had no characteristic surface glycopeptidolipid. Seemingly, M paratuberculosis, long regarded as a single species and the causative agent of bovine paratuberculosis, is not a homogeneous taxon. Most isolates obtained from infected ruminants may be antigenically defective, variants of M avium and, thereby, more successful pathogens.

Antigens, Bacterial↗
PubMed1994

In vivo administration of a monoclonal antibody against the type I IL-1 receptor inhibits the ability of mice to eliminate Mycobacterium paratuberculosis.

The purpose of this study was to determine the influence of endogenous interleukin-1 (IL-1) on resistance to paratuberculosis infection in experimentally infected gnotobiotic mice. Following a 6-month treatment with prednisolone to facilitate bacillary multiplication, control mice substantially reduced the numbers of M. paratuberculosis in the liver and ileum. In contrast, mice injected with a monoclonal antibody against the type I IL-1 receptor failed to reduce the numbers of M. paratuberculosis in the liver and ileum and exhibited more liver granulomas, which contained numerous acid-fast bacilli. These results indicate a significant role for endogenous IL-1 in host defense against experimental M. paratuberculosis infection in mice.

Animals↗
PubMed1996

Low calcium diet and 1,25-dihydroxyvitamin D(3) infusion modulate immune responses during Mycobacterium paratuberculosis infection in beige mice.

A 12-month study was conducted to evaluate the effects of feeding a low calcium (Ca) diet or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) infusion on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Male beige mice 6-8 weeks of age were assigned to one of the following treatments: (1) non-infected, (2) infected,(3) non-infected/1,25(OH)(2)D(3), (4) infected/1,25(OH)(2)D(3), and (5) infected/low Ca (0.15 percent) diet. Infected mice were inoculated intravenously with live M. paratuberculosis. At 1, 6 and 12 months postinfection, mice in Treatments 3 and 4 were implanted subcutaneously with mini-osmotic pumps to deliver 1,25(OH)(2)D(3). Infusion with 1,25(OH)(2)D(3) exacerbated M. paratuberculosis infection in most tissues at all time points. Mice infused with 1,25(OH)(2)D(3) had higher bacterial counts in spleen, liver, and ileum compared with control infected mice after 1 month of infection. In contrast, feeding a low Ca diet reduced the number of viable organisms cultured from the liver and ileum of infected mice. Plasma Ca and 1,25(OH)(2)D(3) were increased in mice infused with 1,25(OH)(2)D(3) at all time points but values for low Ca mice were not different than for non-infused mice. Splenocyte production of TNF, IL-1 and IL-6 was higher for mice fed the low Ca diet compared with control infected mice after 1 month of infection. Inducible IL-6 activity remained higher for this treatment at 6 months postinfection. These results suggest that feeding a low Ca diet to mice chronically infected with M. paratuberculosis appears to enhance their ability to clear the infection in a manner distinct from any effect of 1,25(OH)2D3.

Animals↗
PubMed2004

Certification of herds as free of Mycobacterium paratuberculosis infection: actual pooled faecal results versus certification model predictions.

Dutch dairy herds closed for at least 3 years with no history of paratuberculosis were recruited for a study on herd-certification. One hundred dairy herds were tested for Mycobacterium paratuberculosis at 6-month intervals by pooled faecal culture (five individual animal samples per pool) with solid media. Ninety of the herds completed 9 herd tests and 10 herds dropped out of the study for reasons other than a paratuberculosis diagnosis. Of the 90 herds completing the full study, 61% eventually were found to be M. paratuberculosis-infected. The number of infected herds detected decreased with each round of testing. Assuming that all infected herds had been detected by the ninth herd test, the observed percentage of herds that were truly noninfected (P-free) after each round of testing was calculated. The observed P-free was compared to the predicted P-free based on a previously reported herd-certification model. The P-free predicted by the model was significantly different from the observed P-free. When a single assumption in the model was changed and a diagnostic sensitivity of 40-50% was selected, the predicted P-free closely approximated the observed P-free for the 90 Dutch dairy herds studied. The critical assumption that was changed for Version 2.0 of the model was within-herd infection prevalence for infected but test-negative herds after each round of serial testing. Model Version 1.0 had assumed a 50% decrease in within-herd prevalence but Version 2.0 assumed a stable within-herd prevalence. Culture of pooled faecal samples provides a high-sensitive, high-specific, low-cost test for herd-certification programs.

Animals↗
PubMed2004

Longitudinal study of interferon-gamma, serum antibody and milk antibody responses in cattle infected with Mycobacterium avium subsp. paratuberculosis.

During a 2-year study period, 252 animals from dairy herds infected with Mycobacterium avium subsp. paratuberculosis, and 119 animals from non-infected herds were subjected to repeated blood and faecal sampling. Animals were retrospectively grouped by infection status as infected, exposed (culture negative animals from infected herds), or non-infected animals, and by age, 12-23 months (1+ year), 24-35 months (2+ years), or 36 months and older (3+ years). Samples were collected for culture of faeces, assessment of interferon-gamma (IFN-gamma) secreted by M. paratuberculosis antigen stimulated whole-blood lymphocytes (IFN-gamma test), and measurement of antibody responses against M. paratuberculosis in serum and milk by an in-house absorbed ELISA. The IFN-gamma test diagnosed higher proportions of infected and exposed animals than the antibody ELISAs. The highest sensitivity of IFN-gamma test was in infected cattle of 2+ years of age. Receiver-operating characteristic (ROC) analyses supported the assumption that the IFN-gamma test had a better performance than antibody tests of animals of 1+ and 2+ years of age. However, for animals of 3+ years all tests performed equally well. Application of single sampling compared with repeated samplings showed better performance of the IFN-gamma test by repeated samplings, and the milk antibody ELISA in animals of 3+ years of age performed significantly better with repeated sampling compared with single sampling. In conclusion, the IFN-gamma test may be applied for screening of cattle of 1 and 2 years of age for exposure to M. paratuberculosis and the antibody ELISAs from 3 years of age.

Age Factors↗
PubMed2005

Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis.

Paratuberculosis in goats occurs worldwide causing considerable economic losses mainly due to reduced milk production. Nowadays, there is still relatively little knowledge about the epidemiology of this disease in goats, and only a few epidemiological studies have been carried out in goats naturally infected with Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis). The objective of this study was to characterize forty four clinical caprine isolates of M. a. paratuberculosis by different molecular techniques (pulsed-field gel electrophoresis [PFGE], restriction fragment length polymorphism analysis coupled with hybridization to IS900, and IS1311 polymerase chain reaction-restriction enzyme analysis) to determine the most useful technique for molecular typing of caprine isolates, as well as to disclose the genetic variation amongst caprine isolates and the relationship with strains isolated from other animal species. PFGE was found to be the most discriminative technique identifying a total of 13 'multiplex' PFGE profiles, ten of which were novel profiles found only in caprine isolates to date. All isolates were genotyped as Type II strains, except two isolates that resembled the intermediate group referred as Type III.

Animals↗
PubMed2005

Detection of Mycobacterium avium subsp. paratuberculosis in intestinal and lymph node tissues of water buffaloes (Bubalus bubalis) by PCR and bacterial culture.

The efficacy of bacterial culture and IS900-specific polymerase chain reaction (PCR) was compared for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) from the intestinal and mesenteric lymph node tissues of water buffaloes (Bubalus bubalis) showing lesions of paratuberculosis (Johne's disease). Out of 20 (4.9%) animals showing histological lesions suggestive of paratuberculosis, 14 (70%) and 6 (30%) were positive in the PCR and bacterial culture, respectively. The results of this study suggested that PCR was more sensitive than bacterial culture in detection of subclinical paratuberculosis in water buffaloes. The bacterial concentration from large amount of tissues by differential and density gradient centrifugation method was found to facilitate the diagnosis by smear examination and PCR. The specificity of the PCR was confirmed by the product size and restriction digestion pattern of the amplicons. The sequence analysis of the amplified products (626bp of IS900 gene) from buffalo strain showed more than 97% homology with the published sequences.

Animals↗
PubMed1994

Histopathological and morphometrical comparison of granulomatous lesions in BALB/c and C3H/HeJ mice inoculated with Mycobacterium paratuberculosis.

Susceptibility to Mycobacterium paratuberculosis strain ATCC 19698 infection was compared histologically and morphometrically in BALB/c (Bcgs) and C3H/HeJ (Bcgr) mice. The granulomatous lesions, containing M. paratuberculosis, in the liver and spleen of BALB/c mice increased progressively until 32 weeks after intraperitoneal inoculation. In contrast, both the granulomatous lesions and number of bacteria decreased drastically in C3H/HeJ mice beginning at 6 weeks after inoculation. There were histological differences in granulomatous lesions between BALB/c and C3H/HeJ mice, indicating differing functional activity of cellular immunity. Intestinal lesions similar to those seen consistently in bovine paratuberculosis were observed first at 6 weeks after infection and later became disseminated widely, especially in BALB/c mice. Our results suggest that the innately susceptible BALB/c mice and the innately resistant C3H/HeJ mice may be useful and convenient models for further studies of immunoregulation and antimicrobial therapy of paratuberculosis.

Animals↗
PubMed2003

Analysis of repeated tests for interferon-gamma (IFN-gamma) response and faecal excretion for diagnosis of subclinical paratuberculosis in Danish cattle.

A total of 315 cattle were tested for infection with Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) at three consecutive samplings, using the interferon-gamma (IFN-gamma) test on whole blood and bacteriological culture of faecal samples. Of 205 cattle from 10 infected herds 99 (48%) were positive in the IFN-gamma test on at least one sampling using "IDEXX-criteria" for interpretation, and of 110 cattle from five non-infected herds three (3%) were positive. Forty-four animals from infected and one from non-infected herds tested positive at all three samplings. Although support for the specificity of the IFN-gamma test was provided by these results, they also indicate problems with false positives. Approximately half of the positive animals did not give the same result at all three samplings, indicating that repeated testing increases the chance of detecting reactors. Changing, or fluctuating, IFN-gamma test results occurred most frequently in animals younger than 1 year, indicating that the IFN-gamma test should be applied only to animals 1 year and older. M. paratuberculosis was isolated from 16 (4%) of 371 cattle, all from infected herds. Fifteen culture-positive cattle tested positive at least once in the IFN-gamma test. It was not possible to predict from the IFN-gamma test result the number of animals that would eventually develop disease. However, the test may be useful to detect animals that have been exposed to M. paratuberculosis earlier in their lives, and the testing of young cattle could be included in a control program to check for the effectiveness of preventing transmission of infection to calves and to identify animals at risk of developing disease later in their lives.

Animals↗
PubMed1998

Early immunopathological events in experimental ovine paratuberculosis.

An experimental oral infection of neonatal (< 2 weeks old) lambs with a cervine isolate of Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis), the causal agent of ruminant paratuberculosis (Johne's disease) was used to investigate bacteriological, histopathological and immunological changes during the early (up to 8 weeks) post-infection phase. In vitro culture for mycobacteria was positive in one faecal and three mesenteric lymph node (MLN) samples from the eight infected lambs. All mycobacterial isolates from MLN were identified as M.a. paratuberculosis by polymerase chain reaction (PCR). Small-to-medium sized focal granulomata were observed in jejunal (JPP) and ileal Peyer's patches (IPP) from four of the eight infected lambs. Compared with controls, JPP from all infected lambs had significantly (p < 0.05) higher proportions of CD8+ and CD2+ lymphocytes, and there were significantly (p < 0.05) fewer cells expressing B lymphocyte-associated markers in IPP and MLN. The T/B cell ratio was significantly (p < 0.05) increased in both JPP and MLN from infected lambs. The expression of a range of genes for cytokines was examined using specific reverse transcriptase PCR (RT-PCR) amplification of messenger RNA (mRNA) template isolated from MLN, JPP and IPP from both groups of animals. Densitometric analyses indicated that, in infected animals, MLN expressed significantly (p < 0.05) more mRNA for TNF-alpha: JPP had significantly increased (p < 0.05) mRNA for GM-CSF and significantly decreased (p < 0.05) mRNA for IL-4 and IFN-gamma. Infected lambs had significantly (p < 0.05) decreased titres of both circulating IgG and gut mycobacteria-associated IgG antibody. Infection was not associated with any consistent changes in lymphocyte reactivity to specific mycobacterial antigens, IFN-gamma release into supernatants from in vitro intestinal lymphocyte cultures or gut IgA antibody levels.

Animals↗
PubMed1998

Dietary calcium modulates Mycobacterium paratuberculosis infection in beige mice.

A 6-month study was conducted to evaluate the effects of feeding different levels of dietary calcium (Ca) on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Beige mice, averaging 8 weeks of age, were randomly assigned to one of the following dietary treatments: 1) 0.02% Ca, 2) 0.15% Ca, 3) 0.45% Ca, and 4) 1.0% Ca. Mice were infected intraperitoneally with 10(8) CFU viable M. paratuberculosis for 1, 3, and 6 month periods. Plasma Ca levels was unaffected by dietary Ca (x = 7.3 mg/dl). Plasma levels of 1,25(OH)2D3 was elevated significantly in 0.02% and 0.15% Ca groups compared to other treatments at the end of each period, with the highest levels observed for 0.02% Ca mice and intermediate values for 0.15% Ca mice. One month after infection, numbers of viable M. paratuberculosis cultured from the spleen were significantly reduced for 0.15% Ca mice, whereas the number of bacteria isolated from the liver and mesenteric lymph node (MLN) were higher for the 0.02% Ca group. There were no differences in bacterial numbers in the ileum although they tended to be higher for the 0.02% Ca group. Three months after infection, bacterial numbers in the spleen, ileum, and MLN did not differ across treatments, however, significantly lower numbers were found in the liver of 1.0% Ca mice. Reduced bacterial counts were also observed in the liver of 0.15%. 0.45%, and 1.0% Ca mice after a 6-month infection period compared to the 0.02% Ca group, with the lowest numbers isolated from the 1.0% Ca mice. Numbers of viable bacteria cultured from the ileum and MLN after 6 months of infection were also significantly reduced in 1.0% Ca mice. These results suggest that Ca metabolism is an important modulator of M. paratuberculosis infection.

Animals↗
PubMed1998

A study of immunological responses of sheep clinically-affected with paratuberculosis (Johne's disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology.

Nineteen adult sheep diagnosed as having clinical paratuberculosis (Johne's disease) and 16 unaffected controls were examined in this study. Animals were tested for the presence of circulating antibodies of Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and lymphocytes derived from the blood, mesenteric lymph nodes and intestines were examined for cell-mediated immune (CMI) responses to Johnin pure protein derivative (Johnin-PPD: J-PPD). Bacteriological examinations were carried out on faeces and tissues and any mycobacterial isolates identified as M. a. paratuberculosis (IS900+) or M. avium ssp. silvaticum (M. a. silvaticum) (IS901+) by polymerase chain reaction (PCR). Full necropsy and histopathological studies were performed and diseased animals were categorised on the basis of having a lepromatous or tuberculoid form of intestinal pathology. Unaffected control sheep were generally antibody-negative and demonstrated varying CMI responses to J-PPD. Clinically-affected animals were almost always antibody-positive with variable CMI responses. A correlation was observed between the histological lesion type in the intestine and the cellular immune response. Tuberculoid-type lesions were associated with strong CMI responses in lymphocytes derived from the peripheral blood, mesenteric lymph node and intestine, whereas lepromatous-type lesions were associated with weak CMI responses in all tissues examined.

Animals↗