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PubMed1997

Relation between serologic response and pathologic findings in sheep with naturally acquired paratuberculosis.

OBJECTIVE: To investigate the relation between lesions and serologic response in sheep with naturally acquired paratuberculosis and to evaluate the efficacy of agar gel immunodiffusion (AGID) and ELISA as diagnostic tests. ANIMALS: 134 adult sheep, culled by the owner from flocks with history of severe chronic weight loss. PROCEDURE: Before euthanasia, blood samples were collected and serum was obtained. AGID and ELISA were performed, using a protoplasmic antigen of Mycobacterium paratuberculosis. Sensitivity and specificity of these techniques were evaluated, using histopathologic findings as a reference. Histologic studies were carried out focusing on specimens containing intestinal organized lymphoid tissue. RESULTS: Of the sheep examined, 46.3% had lesions that were classified in 5 categories, according to their location, extension, cellular types, and presence of acid-fast bacilli. Regarding serologic test results, 37.1% of sheep with lesions had a positive AGID result, and all sheep without lesions had negative results. However, ELISA positivity was slightly greater--48.4% of sheep with lesions; however, by use of ELISA, 11.1% of sheep without lesions had positive results. CONCLUSION: A close relation between pathologic findings and serologic response was determined, especially between presence of acid-fast bacilli (potentially heavily shedding sheep) in the intestinal lesions and positivity to serologic tests, especially AGID. CLINICAL RELEVANCE: On the basis of these results, together with its high specificity, reliability, and the difficulty of fecal specimen culture in sheep, AGID is proposed as a useful tool for seeking evidence of paratuberculosis in live sheep, and therefore, is suggested as the basis for control programs in areas in which the disease is a serious threat to sheep production.

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↗
PubMed1996

Differences in the kinetics of T cell accumulations in C3H/HeN (Bcg-resistant) and C57BL/6 (Bcg-susceptible) mice infected with Mycobacterium paratuberculosis.

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and- resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, gamma delta TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL, and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or gamma delta T cells. Significant increases in accumulation of CD8+ and gamma delta T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or gamma delta T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and gamma delta T cells may play a role in the increased suspectibility of C57BL/6 mice to infection with M. paratuberculosis.

Animals↗
PubMed1996

Increased intestinal TNF-alpha, IL-1 beta and IL-6 expression in ovine paratuberculosis.

Mycobacterium avium subspecies paratuberculosis is an intracellular parasite of intestinal macrophages and causes a chronic granulomatous enteritis in sheep and other ruminants (paratuberculosis or Johne's disease). Macrophages can be produced a variety of immunoregulatory cytokines that may influence mycobacterial killing and produce disordered inflammation within the gut. In this study, messenger RNA (mRNA) was extracted from intestinal tissue from control and multibacillary diseased sheep and profiles for the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, transforming growth factor-beta1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF) were semi-quantified using reverse transcriptase polymerase chain reactions (RT-PCR). Infected intestinal tissues had significantly increased mRNA for TNF-alpha, IL-1beta and IL-6 but TGF-beta1 and GM-CSF mRNA levels were significantly different from controls. Supernatants from in vitro intestinal cultures were assayed for TNF-alpha activity using the PK(15)-1512 cytotoxicity bioassay and levels were significantly raised in diseased samples. TNF-alpha was not detected in any serum samples. Further analysis on intestinal tissues from sheep with the different, paucibacillary, form of the disease showed significant elevation of TNF-alpha mRNA but not other cytokines tested. Increased pro-inflammatory cytokine expression in the intestine coincident with a failed or misdirected immune response may contribute to the pathogenesis of paratuberculosis and the persistence of a chronic inflammatory state.

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↗
PubMed2005

Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn's disease at 5q31.

Campylobacter jejuni and Mycobacterium paratuberculosis have been implicated in the pathogenesis of Crohn's disease. The presence of bacterial metabolites in the colonic lumen causing a specific breakdown of fatty acid oxidation in colonic epithelial cells has been suggested as an initiating event in inflammatory bowel disease (IBD). l-Carnitine is a small highly polar zwitterion that plays an essential role in fatty acid oxidation and ATP generation in intestinal bioenergetic metabolism. The organic cation/carnitine transporters, OCTN1 and OCTN2, function primarily in the transport of l-carnitine and elimination of cationic drugs in the intestine. High-resolution linkage disequilibrium mapping has identified a region of about 250kb in size at 5q31 (IBD5) encompassing the OCTN1 and -2 genes, to confer susceptibility to Crohn's disease. Recently, two variants in the OCTN1 and OCTN2 genes have been shown to form a haplotype which is associated with susceptibility to Crohn's. We show that OCTN1 and OCTN2 are strongly expressed in target areas for IBD such as ileum and colon. Further, we have now identified a nine amino acid epitope shared by this functional variant of OCTN1 (Leu503Phe) (which decreases the efficiency of carnitine transport), and by C. jejuni (9 aa) and M. paratuberculosis (6 aa). The prevalence of this variant of OCTN1 (Phe503:Leu503) is 3-fold lower in unaffected individuals of Jewish origin (1:3.44) compared to unaffected individuals of non-Jewish origin (1:1). We hypothesize that a specific antibody raised to this epitope during C. jejuni or M. paratuberculosis enterocolitis would cross-react with the intestinal epithelial cell functional variant of OCTN1, an already less efficient carnitine transporter, leading to an impairment of mitochondrial beta-oxidation which may then serve as an initiating event in IBD. This impairment of l-carnitine transport by OCTN1 may respond to high-dose l-carnitine therapy.

Amino Acid Sequence↗
PubMed2006

Protein extraction from Mycobacterium avium subsp. paratuberculosis: Comparison of methods for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis, native PAGE and surface enhanced laser desorption/ionization time of flight mass spectrometry.

Mycobacterium paratuberculosis causes Johne's disease, a chronic bowel disease in ruminants worldwide and is currently incurable. This study was conducted to compare methods for examining the proteome of M. paratuberculosis. SDS-PAGE, native PAGE and SELDI-TOF-MS were compared and the efficacy of various lysis buffers was assessed. Chaotropic agents (Urea CHAPS and potassium thiocyanate) and non-ionic detergent (Tween20 and Triton X-100) extracts were compared on three different ProteinChip surfaces along with two energy absorbing molecules (EAM): EAM-1 proprietary formulation and sinapinic acid (Ciphergen). Urea CHAPS was efficient for extraction of proteins and their detection on all the ProteinChip surfaces. However, potassium thiocyanate was the most effective buffer, leading to detection of the greatest number of protein peaks on the immobilized metal affinity chromatography (IMAC) surface. Sinapinic acid was more efficient than the EAM-1 proprietary formulation and resulted in additional peaks with higher intensity for both the low and the medium molecular weight range proteins. Intra-chip and inter-chip coefficient of variation for mass/charge varied from 0.01% to 0.07% and 0.00% to 0.08%, respectively. SELDI-TOF-MS was an efficient tool for the protein profiling of M. paratuberculosis and will be useful for investigation of novel proteins, although SDS-PAGE/2D gel electrophoresis is recommended for study of high molecular weight species. All buffers were suitable for protein extraction for SDS-PAGE, while Tween20 was best for native PAGE.

Bacterial Proteins↗
PubMed2006

Decision analysis model for paratuberculosis control in commercial dairy herds.

A previous economic test-and-cull decision analysis model has been strengthened and updated with current epidemiologic information. Created using Excel and PrecisionTree software, the model incorporates costs and benefits of herd management changes, diagnostic testing, and different management actions based on test results to control paratuberculosis in commercial dairy herds. This novel "JD-Tree" model includes a herd management decision node (four options), a test/no test decision node (two options), a diagnostic test choice decision node (five options), test result chance nodes (four levels of possible results), and test action decision nodes (three options; cull, manage, no action). The model culminates in a chance node for true infection status. Outcomes are measured as a net cost-benefit value to the producer. The model demonstrates that improving herd management practices to control infection spread (hygiene) is often more cost-effective than testing; not all herds should test as part of a paratuberculosis control program. For many herds, low-cost tests are more useful than more sensitive, higher cost tests. The model also indicates that test-positive cows in early stages of infection may be retained in the herd to generate farm income, provided they are managed properly to limit infection transmission. JD-Tree is a useful instructional tool, helping veterinarians understand the complex interactions affecting the economics of paratuberculosis control and to define the accuracy and cost specifications of better diagnostic tests.

Animals↗
PubMed2005

A survey for Mycobacterium avium subspecies paratuberculosis in the Royal Zoological Society of Antwerp.

Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.

Animals↗
PubMed2000

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation.

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.

Animals↗
PubMed2000

Bulk-tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds.

Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk-collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown.

Animal Husbandry↗
PubMed2000

Soil type as a putative risk factor of ovine and caprine paratuberculosis seropositivity in Spain.

Relationships between soil type and ovine and caprine paratuberculosis in the Avila region (central Spain) were evaluated using data from a cross-sectional study of the most-important diseases of small ruminants in this Spanish region between 1996 and 1997. Questionnaire data from 61 herds (38 ovine and 23 caprine) and 1451 serum samples (1041 ovine and 410 caprine) were used. Herd paratuberculosis (herds were scored as positive to paratuberculosis if any of the serum samples was positive in an agar-gel immunodifussion) was the outcome of interest, whereas soil type in the municipality where farms were located was the predictor variable. Other variables related to soil and soil usage, and herd size, replacement, main food production and animal species were also introduced into the multivariable logistic regression. The final model contained only two independent variables: the predictor variable soil type (coded as two dummy variables ST-1 and ST-2) and herd size (dichotomized at the highest quartile). The estimated Odds Ratios were 25.9 (95% CI: 1.6, 411) for ST-1 (entisols as soil type) and 3.5 (95% CI: 0.3, 45) for ST-2 (inceptisols as soil type).

Animals↗
PubMed1999

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.

Animals↗
PubMed1997

The contribution of molecular biology to Mycobacterium avium subspecies Paratuberculosis research.

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacterium avium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.

Animals↗
PubMed1999

Serum antibodies to Mycobacterium paratuberculosis in patients with Crohn's disease.

Mycobacterium paratuberculosis has been suggested as a causative organism of Crohn's disease. Despite a long-term debate to prove this possibility, the role of this bacteria in the pathogenesis of Crohn's disease is still a subject of controversy. In the present study, serum antibodies (IgG, IgA, and IgM) to the protoplasmic antigen of M. paratuberculosis were quantified in patients with Crohn's disease and in control subjects by using an enzyme-linked immunosorbent assay whose specificity was increased by preabsorbing the sera with cell extracts of Mycobacterium phlei. As compared to normal controls (1/20; 0.062+/-0.022), a significant difference was seen in the antibody-positive prevalence rate and mean values of the serum IgG titer in patients with Crohn's disease (5/13; 0.102+/-0.039) (P < 0.05), but not in patients with ulcerative colitis (2/20; 0.065+/-0.035) and tuberculosis (0/4; 0.053+/-0.008). No significant differences were seen in the antibody-positive prevalence rate and mean values of the serum IgA and IgM titers among the four study groups. These results indicate the unique immune response to M. paratuberculosis in patients with Crohn's disease, suggesting that this organism may play some role in the pathogenesis of Crohn's disease.

Adolescent↗
PubMed2000

The pathology of goat paratuberculosis: gross and histopathological lesions in the intestines and mesenteric lymph nodes.

From a pathological examination of the intestinal tracts of 1590 goats killed at slaughterhouses in the Fars Province of Iran, 59 cases (3.71%) were suspected, on gross examination, of having paratuberculosis. The diagnosis was confirmed by histopathological study and Ziehl-Neelsen staining of direct smears of rectal faeces. On the basis of severity of involvement of the terminal ileum and mesenteric lymph nodes, the microscopic lesions were classified to mild, moderate and severe forms. Caseous necrosis and calcification were observed only in the mesenteric lymph nodes. High numbers of acid-fast organisms were present in the epithelioid macrophages of the intestine but were inapparent or sparse in the mesenteric lymph nodes. On microscopic examination, 13.5% of the suspected animals were found to have paratuberculosis, in comparison with 3.38% by direct faecal smears. In addition, 30.5% and 15.3% of the animals were diagnosed as having eosinophilic enteritis and linguatulosis, respectively. These findings stress the importance of a careful histopathological examination of the intestines and mesenteric lymph nodes for the diagnosis of paratuberculosis in goats.

Animals↗