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PubMed2001

Use of Hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. paratuberculosis did not significantly alter the number of apoptotic cells.

Animals↗

PubMed2006

Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (or paratuberculosis). Paratuberculosis is a chronic gastroenteritis mainly affecting cattle, sheep and other ruminants. MAP is also of concern due to the heretofore unresolved issue of its possible role in Crohn's disease in humans. We present here a review of MAP (i) mobile genetic elements; (ii) repetitive elements; (iii) single nucleotide polymorphisms; and (iv) whole-genome comparisons to study the molecular epidemiology of MAP. A summary of the findings to date is presented, and the discriminatory power, advantage and disadvantages of each of the methods are compared and discussed.

Animals↗

PubMed2004

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

Animals↗

PubMed1999

Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.

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↗

PubMed2001

Identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA.

A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA (RAPD). A primer OPE-20 (5'-AAC-GGT-GAC-C-3') was identified to be the most suitable primer when tested with four ATCC reference strains of M. paratuberculosis and eight well characterized field strains each of M. paratuberculosis and M. avium. Primer OPE-20 was further tested for its ability to identify and subtype 200 field isolates of M. paratuberculosis. The fingerprint patterns of M. paratuberculosis (n=212) consisted of five unique common fragments (620, 450, 310, 230, 180bp) and nine variable fragments resulting in six distinct genotypes. The DNA fingerprints of M. avium (n=8) consisted of a single common fragment of 620bp, and 15 variable fragments resulting in six different genotypes. The cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a different RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible and reliable technique for identification and sub-typing of M. paratuberculosis.

Animals↗

PubMed2001

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium.

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.

Animals↗

PubMed2002

Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences.

The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.

Animals↗

PubMed2006

Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity.

We analyzed the multilocus short sequence repeats (SSRs) of 211 and 56 isolates of Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium, respectively. The M. avium subsp. paratuberculosis isolates could be differentiated into 61 genotypes. The M. avium subsp. avium isolates showed limited diversity. These SSRs are stable and suitable for studying the molecular epidemiology of M. avium subsp. paratuberculosis.

Animals↗

PubMed1999

PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311.

Point mutations in the IS 1311 sequences from sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and M. avium subsp. avium (M. avium) were targeted to develop a polymerase chain reaction (PCR) that would be useful in the diagnosis and control of Johne's disease. Candidate PCR tests were evaluated for sensitivity, specificity and ease of interpretation of the restriction endonuclease analysis (REA) products. One IS 1311 PCR, amplifying a 608 base pair product, was shown to be suitable when the amplified product was digested with Hinf I and Mse I. The PCR detected 50 fg of template DNA from M. paratuberculosis strain 316 V, the equivalent of 10 organisms. The test was evaluated further using purified DNA from M. paratuberculosis and M. avium isolates and diagnostic samples including primary radiometric cultures. All 89 M. paratuberculosis samples were correctly identified and typed according to host species or IS 900 restriction fragment length polymorphism (RFLP) type. All 28 isolates of M. avium were also correctly identified. A second PCR/REA strategy based on a shorter fragment of IS 1311 was developed for formalin-fixed paraffin-embedded tissue samples. It correctly differentiated sheep and cattle strains of M. paratuberculosis in 27 tissue samples in which acid fast bacilli had been observed in Ziehl Neelsen stains and in which sufficient amplified product was present for REA with Hinf I. Both tests were specific for M. paratuberculosis when tested against 24 other mycobacterial species. These simple and rapid tests can be used on a range of diagnostic samples for the confirmation of Johne's disease and will be of benefit in control and eradication programmes for this disease.

Animals↗

PubMed1999

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.

Base Sequence↗

PubMed2005

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates.

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.

Conserved Sequence↗

PubMed2005

Genomic polymorphisms for Mycobacterium avium subsp. paratuberculosis diagnostics.

Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP(A)s) and those only present in the latter (LSP(P)s). We examined the distribution of 3 LSP(A)s and 17 LSP(P)s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP(A)8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP(P)s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.

Genome, Bacterial↗

PubMed2006

Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.

A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.

Animals↗

PubMed1990

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov.

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycobacterium avium↗

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↗

PubMed2004

Efficacy of pasteurization conditions for the inactivation of Mycobacterium avium subsp. paratuberculosis in milk.

Mycobacterium avium subsp. paratuberculosis, the causative agent of a chronic enteritis in ruminants (Johne's disease), has been linked to Crohn's disease in humans. This microorganism is shed by infected animals primarily in the feces but is also shed in the milk at much lower levels. Therefore, dairy products from infected animals may be one mode of transmission of this animal pathogen. This study was designed to evaluate the effectiveness of the holder and high-temperature short-time pasteurization standards on the destruction of M. paratuberculosis. One hundred eighty experiments were conducted in this study using a slug-flow pasteurizer unit and a laboratory scale pasteurizer unit. Ultrahigh-temperature milk was inoculated at two concentrations, 10(8) and 10(5) CFU/ml, with three different field strains of M. paratuberculosis. Five different time-temperature combinations were evaluated: 62.7 degrees C for 30 min, 65.5 degrees C for 16 s, 71.7 degrees C for 15 s, 71.7 degrees C for 20 s, and 74.4 degrees C for 15 s. Three replicates of each experiment were run for the pasteurizer unit, time-temperature combination, and strain of M. paratuberculosis. Treatment of milk regardless of bacterial strain or pasteurizer unit resulted in an average 5.0- and 7.7-log kill for the low and high concentrations of inoculum, respectively. Milk treated for cheese production (65.5 degrees C for 16 s) resulted in a much lower and more variable kill. Results from this study indicate that the current U.S. minimum standards for batch and high-temperature short-time pasteurization of grade A milk significantly reduced the survivability of M. paratuberculosis, but some bacteria survived subpasteurization heat treatment of milk used for cheese manufacture.

Animals↗

PubMed2003

Enhanced expression of interleukin-1alpha and tumor necrosis factor receptor-associated protein 1 in ileal tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis.

Infection with Mycobacterium avium subsp. paratuberculosis is associated with high levels of morbidity, decreased production, and early culling in dairy cattle. Clinical symptoms of Johne's disease include persistent diarrhea, inappetence, and resultant weight loss due to chronic inflammation of the small intestine. Although the presence or absence of intestinal lesions cannot be used as a definitive indicator of M. avium subsp. paratuberculosis infection, most infected cattle exhibit significant changes to intestinal mucosa, with the focus of pathology surrounding the ileal cecal junction. Typical pathology of M. avium subsp. paratuberculosis infection includes inflammation, thickening of the lumenal wall, and hyperplasia in draining lymph nodes. To further understand the pathology of Johne's disease, we compared the gene expression profiles of ileal tissues from Johne's disease-positive (n = 6), and Johne's disease-negative (n = 5) Holstein cattle. Gene expression profiles were compared with a bovine total leukocyte (BOTL-3) cDNA microarray. Genes that were expressed at significantly higher levels (>1.5-fold; P < 0.05) in tissues from Johne's disease-infected animals relative to noninfected animals included those encoding tumor necrosis factor receptor-associated protein 1 (TRAF1), interleukin-1alpha (IL-1alpha), MCP-2, N-cadherin, and beta1 integrin (CD29). Dramatic upregulation of IL-1alpha (21.5-fold) and TRAF1 (27.5-fold) gene expression in tissues of Johne's disease-positive cows relative to tissues from control cows was confirmed by quantitative real-time PCR. Western blot analysis confirmed that IL-1alpha and TRAF1 mRNA levels resulted in increased protein expression in tissues of Johne's disease-positive cattle relative to tissues from control cattle. High levels of IL-1alpha can produce symptoms similar to those found in clinical Johne's disease. Taken together, the data presented in this report suggest that many outward symptoms of Johne's disease may be due to IL-1alpha toxicity. In addition, enhanced levels of TRAF1 could result in cells within the lesions of Johne's disease-positive cattle that are highly resistant to TNF-alpha-induced signaling.

Animals↗