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PubMed2000

Paratuberculosis in Iceland: epidemiology and control measures, past and present.

Paratuberculosis as well as the slow virus infections maedi/visna and jaagsiekte came to Iceland in 1933 when 20 sheep of the Karakul breed were imported from Halle, Germany. At least five of these sheep were subclinical carriers of paratuberculosis. Within 16 years paratuberculosis together with the other Karakul diseases (maedi/visna and jaagsiekte) almost ruined sheep farming, the main agricultural industry in Iceland. The first clinical case of paratuberculosis in sheep was confirmed in 1938, and in cattle in 1944. The first cattle cases of paratuberculosis appeared on farms where the disease had been prevalent in sheep for years. The virulence in cattle appeared to be considerably lower than in sheep. Extensive measures were used to control the spread of paratuberculosis in sheep. Hundreds of kilometres of fences were put up and used together with natural geographic borders to restrict the movement of sheep from infected areas. Serological and other immunological tests were also used to detect and dispose of infected individuals. These measures proved inadequate and the disease could not be eradicated. Culling and restocking of uninfected sheep in endemic areas eradicated maedi/visna and jaagsiekte but not paratuberculosis. Experiments showed that vaccination against paratuberculosis could reduce mortality in sheep by 94%. Vaccination of sheep in endemic areas has been compulsory in Iceland since 1966 and as a result losses have been reduced considerably. Today, serology is used to detect and control infection in cattle herds. Furthermore, serology is used to control vaccination of sheep and screen for infection in non-endemic areas. The complement fixation (CF) test for paratuberculosis has been used until now, but recently we have started comparing the CF test with the CSL absorbed ELISA test.

Animal Husbandry↗
PubMed2004

Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen.

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.

Animals↗
PubMed2005

Evaluation of five antibody detection tests for diagnosis of bovine paratuberculosis.

Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was > or =99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA "D" had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r(2) = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.

Animals↗
PubMed2006

Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp. paratuberculosis infection in the red deer (Cervus elaphus).

Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (10(9) CFU/animal) or medium (10(7) CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (10(3) CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines.

Animals↗
PubMed2002

Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.

Animals↗
PubMed2004

Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.

Animals↗
PubMed2004

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.

Animals↗
PubMed2006

Identification of diagnostic proteins in Mycobacterium avium subspecies paratuberculosis by a whole genome analysis approach.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne's disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these animals. As with many other animal diseases, efforts need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed by veterinarians or animal producers without expensive laboratory equipment. With the genome sequence of M. paratuberculosis now complete, we have taken a different strategy to identify novel proteins that are present uniquely in M. paratuberculosis and are antigenic in the context of infected cattle. Through a whole genome comparison of M. paratuberculosis with other sequenced mycobacterial genomes, we identified a collection of more than 90 genes that are present uniquely in M. paratuberculosis. This list has been further trimmed to 39 after amplification using polymerase chain reaction of unique genes using the genomic deoxyribonucleic acid template from several mycobacterial species and isolates. A selection of the remaining genes has been cloned and expressed in Escherichia coli and purified by affinity chromatography. Successfully purified proteins were analyzed using sera from rabbits immunized with M. paratuberculosis. Furthermore, to identify antigens in the context of disease, sera from cattle with Johne's disease as well as healthy control cattle are used in immunoassays. Using this methodology, we identified the first protein antigens specific to M. paratuberculosis.

Animals↗
PubMed1991

Treatment of Mycobacterium paratuberculosis infection in ruminants.

Paratuberculosis is a chronic, debilitating, fatal condition that usually is clinically undetectable until the onset of copious diarrhea. Paratuberculosis is caused by an acid-fast organism, M. paratuberculosis. Successful eradication of paratuberculosis depends on the early detection of infected animals, thereby allowing removal of carrier animals from the herd. Treatment for paratuberculosis is therefore rarely indicated or undertaken; however, treatment may be considered for animals of exceptional genetic value or companion animals. Antimicrobials reviewed in this article for the treatment of paratuberculosis include isoniazid, rifampin, streptomycin, amikacin, clofazimine, and dapsone. Treatment of paratuberculosis requires daily medication for extended periods and results in palliation of the disease rather than a definitive cure. The treatment for paratuberculosis recommended by the authors is isoniazid at 20 mg/kg administered orally every 24 hours for the rest of the animal's life. When the animal has acute onset of diarrhea, rifampin at 20 mg/kg every 24 hours is also administered orally. In severe, imminently life-threatening cases, an aminoglycoside should be administered concurrently for 3 to 8 weeks. This protocol (isoniazid, rifampin, and an aminoglycoside) will help ensure that Mycobacteria organisms are sensitive to at least two of the antibiotics. Rifampin treatment can be discontinued if clinical signs of paratuberculosis disappear and the cost of therapy is judged excessive. The combined therapeutic approach has been used in three animals, and the results are presented in this article. Because isoniazid, rifampin, and some aminoglycosides are not approved for use in food animals in the United States of America, the meat or milk from treated animals should not be used for human consumption.

Animals↗
PubMed1998

Thermal tolerance of Mycobacterium paratuberculosis.

D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) of Mycobacterium paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71 degrees C). Viable M. paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for the M. paratuberculosis strains tested were generally linear, with R2 of > or = 0.90, but a few curves (R2, 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similar D values in milk and in lactate solution. However, D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. D values for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains of M. paratuberculosis in milk at 71 degrees C (D71 degrees C) was 11.67 s. Pooled D62 degrees C, D65 degrees C, and D68 degrees C of clinical M. paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11 degrees C. The D values of clumped and single M. paratuberculosis cells were not significantly different. The D values of all M. paratuberculosis strains tested were considerably higher than those published for Listeria, Salmonella, and Coxiella spp. and estimated for Mycobacterium bovis, indicating that M. paratuberculosis is more thermally tolerant. This study supports the premise that M. paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 10(1) cells/ml.

Animals↗
PubMed2000

Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium subsp. paratuberculosis viability.

Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20 degrees C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log(10) concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10(3) cells of M. avium subsp. paratuberculosis per ml.

Cheese↗
PubMed2004

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis.

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.

Amino Acid Sequence↗
PubMed2006

Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n=2), human (n=3), sheep (n=2), and bison (n=1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.

Animals↗
PubMed1997

Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies.

Proteins secreted by Mycobacterium species have been suggested as major immune targets in the early phase of infection. In this study, we sought to identify specific antigens in culture filtrates and in soluble cell extracts of Mycobacterium paratuberculosis. The release of antigens into the culture medium during growth of the bacilli and the distribution of specific epitopes within the Mycobacterium species were investigated by immunoblot analysis with monoclonal antibodies (MAbs) raised against M. paratuberculosis antigens. MAb B6A interacted with a cellular antigen with an apparent molecular mass of 34.5 kDa in lysates of M. paratuberculosis. MAb B6A did not interact with lysates from any other mycobacterial species, suggesting recognition of an M. paratuberculosis species-specific epitope. MAb FL1-A1 reacted with an antigen of 44.3 kDa in M. paratuberculosis and a 9-kDa antigen in Mycobacterium kansasii. MAb PII-B1 reacted with concanavalin A (ConA)-binding cellular and filtrate molecules of M. paratuberculosis and with lysates of Mycobacterium kansasii and Mycobacterium avium 18. The affinity-purified glycosylated antigens migrated as a diffuse band of between 35 and 45.6 kDa and reacted strongly with ovine and bovine paratuberculosis serum and polyclonal serum against M. tuberculosis lipoarabinomannan antigens. These glycoconjugates were the earliest antigens detected in culture filtrates of M. paratuberculosis. Deglycosylation of the ConA-binding molecules with alpha-mannosidase enzyme abolished the reaction with MAb PII-B1 and with bovine but not ovine paratuberculosis serum, suggesting selective immunogenicity in the different animal species.

Animals↗
PubMed2001

Fibronectin attachment protein homologue mediates fibronectin binding by Mycobacterium avium subsp. paratuberculosis.

Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis.

Adhesins, Bacterial↗
PubMed2000

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls.

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

Adult↗
PubMed2004

Amplified fragment length polymorphism reveals genomic variability among Mycobacterium avium subsp. paratuberculosis isolates.

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.

Base Sequence↗
PubMed2003

Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates.

BACKGROUND: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. RESULTS: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. CONCLUSIONS: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Bacterial Proteins↗