Pathways and Genes Associated with Immune Dysfunction in Sheep Paratuberculosis.

Multibacillary and paucibacillary paratuberculosis are both caused by Mycobacterium avium subspecies paratuberculosis. Multibacillary lesions are composed largely of infected epithelioid macrophages and paucibacillary lesions contain T cells but few bacteria. Multibacillary disease is similar to human lepromatous leprosy, with variable/high levels of antibody and a dysfunctional immune response. Animals with paucibacillary disease have high cell-mediated immunity and variable levels of antibody. This study aims to characterize the immunological dysfunction using TruSeq analysis of the ileocaecal lymph node that drains disease lesions. Immune dysfunction is highlighted by repression of TCR/CD3 genes, T cell co-receptors/co-stimulators, T cell activation and signal-transduction genes. Inflammation was an acute phase response and chronic inflammation, with little evidence of acute inflammation. The high levels of immunoglobulin and plasma cell transcripts is consistent with the anti-MAP antibody responses in paratuberculosis sheep. Also notable was the overwhelming reduction in mast cell transcripts, potentially affecting DC activation of the immune response. This study also shows that there were no fundamental differences in the gene expression patterns in multibacillary and paucibacillary disease, no shift in T cell genes from Th1 to Th2 pattern but rather an incremental decline into immune dysfunction leading to multibacillary pathology.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn's disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.

M. paratuberculosis and Parkinson's disease--is this a trigger.

Genetic linkage studies and genome wide analysis have provided insights into complex medical diseases. Mycobacterium avium ss. paratuberculosis (MAP) causes Johne's disease, an important enteric inflammatory disease mostly studied in ruminant animals. MAP is also the putative cause of Crohn's disease. Moreover, MAP has been linked to other inflammatory diseases: sarcoidosis, Blau syndrome, autoimmune diabetes, autoimmune thyroiditis and multiple sclerosis. Genetic studies reveal an association between Parkinson's disease (PD), leprosy and Crohn's disease and since discovered, these findings have been considered "surprising". Autophagy and ubiquitin-proteosome systems are cellular systems that both fight intracellular pathogens (xenophagy) and maintain cellular protein quality control. PD is a common neurodegenerative disease that manifests clinically as a profound movement disorder. The recognized genetic defects of PD create disruption of cellular homeostasis that result in protein folding abnormalities of PD called Lewy bodies. Those same genetic defects are associated with susceptibility to intracellular pathogens, including mycobacteria. It is now understood that PD Lewy body pathology starts in the enteric nervous system and "spreads" to the brain in a retrograde fashion via the vagus nerve. This is the same process by which prions affect the brain. Lewy body pathology of the enteric nervous system predates the Lewy body pathology of the central nervous system (CNS) by years or even decades. This article proposes that genetic defects associated with PD also result in a permissive environment for MAP infection--ineffective xenophagy. It postulates that beginning as an enteric infection, MAP--via the vagus nerve--initiates a pathologic process that results in a targeted neuroinvasion of the CNS. The article proposes that MAP infection and resultant PD pathology are due, in the genetically at-risk and age dependant, to the consumptive exhaustion of the protein quality control systems.

Comparative immunological and microbiological aspects of paratuberculosis as a model mycobacterial infection.

Paratuberculosis or Johne's disease of livestock, which is caused by Mycobacterium avium subsp. paratuberculosis (MAP), has increased in prevalence and expanded in geographic and host ranges over about 100 years. The slow and progressive spread of MAP reflects its substantial adaptation to its hosts, the technical limitations of diagnosis, the lack of practical therapeutic approaches, the lack of a vaccine that prevents transmission and the complexity and difficulty of the on-farm control strategies needed to prevent infection. More recently evidence has accumulated for an association of MAP with Crohn's disease in humans, adding to the pressure on animal health authorities to take precautions by controlling paratuberculosis. Mycobacterial infections invoke complex immune responses but the essential determinants of virulence and pathogenesis are far from clear. In this review we compare the features of major diseases in humans and animals that are caused by the pathogenic mycobacteria M. ulcerans, M. avium subsp. avium, M. leprae, M. tuberculosis and MAP. We seek to answer key questions: are the common mycobacterial infections of humans and animals useful "models" for each other, or are the differences between them too great to enable meaningful extrapolation? To simplify this, the immunopathogenesis of mycobacterial infections will be defined at cellular, tissue, animal and population levels and the key events at each level will be discussed. Many pathogenic processes are similar between divergent mycobacterial diseases, and at variance between virulent and avirulent isolates of mycobacteria, suggesting that the research on the pathogenesis of one mycobacterial disease will be informative for the others.

Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease.

Although Crohn's disease is considered to be autoimmune in origin, there is increasing evidence that it may have an infectious cause. The most plausible candidate is Mycobacterium avium subspecies paratuberculosis (MAP). Intriguingly, Koch's postulates may have been fulfilled for MAP and Crohn's disease, even though they still have not been met for Mycobacterium leprae and leprosy. In animals MAP causes Johne's disease, a chronic wasting intestinal diarrhoeal disease evocative of Crohn's disease. Johne's disease occurs in wild and domesticated animals, including dairy herds. Viable MAP is found in human and cow milk, and is not reliably killed by standard pasteurisation. MAP is ubiquitous in the environment including in potable water. Since cell-wall-deficient MAP usually cannot be identified by Ziehl-Neelsen staining, identification of MAP in human beings requires culture or detection of MAP DNA or RNA. If infectious in origin, Crohn's disease should be curable with appropriate antibiotics. Many studies that argue against a causative role for MAP in Crohn's disease have used antibiotics that are inactive against MAP. However, trials that include macrolide antibiotics indicate that a cure for Crohn's disease is possible. The necessary length of therapy remains to be determined. Mycobacterial diseases have protean clinical manifestations, as does Crohn's disease. The necessity of stratifying Crohn's disease into two clinical manifestations (perforating and non-perforating) when interpreting the results of antibiotic therapy is discussed. Rational studies to evaluate appropriate therapies to cure Crohn's disease are proposed.

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

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

Paratuberculosis.

Paratuberculosis (Johne's disease) is a chronic, wasting, widespread mycobacteriosis of ruminants. It involves extensive mycobacterial shedding, which accounts for the high contagiousness, and ends with a fatal enteritis. Decreases in weight, milk production, and fertility produce severe economic loss. The DNA of the etiological agent (Mycobacterium paratuberculosis) has a base composition (66 to 67% G+C) within the range of that of mycobacteria (62 to 70% G+C), a size (4.4 x 10(6) to 4.7 x 10(6) bp) larger than that of most pathogenic mycobacteria (2.0 x 10(6) to 4.2 x 10(6) bp), and a high relatedness (> 90%) to Mycobacterium avium DNA. However, the DNAs of the two organisms can be distinguished by restriction fragment length polymorphism analysis. M. paratuberculosis genes coding for a transposase, a cell wall-associated protein (P34), and two heat shock proteins have been cloned and sequenced. Nucleic acid probes (two of which are species specific) are used, after PCR amplification, for M. paratuberculosis identification in stools and milk. As in leprosy, with disease progression, cellular immune reactions decrease and humoral immune reactions increase. Cutaneous testing with sensitins, lymphocyte proliferation assays, and cytokine tests are used to monitor cellular immune reactions in paratuberculosis, but these tests lack specificity. Complement fixation, immunodiffusion, and enzymometric tests based on antibodies to M. paratuberculosis extracts, to mycobacterial antigen complex A36, to glycolipids, and to proteins help identify affected cattle but are not species specific. The carboxyl-terminal portion of the 34-kDa cell wall-associated A36 protein (P34) carries species-specific B-cell epitopes and is the basis for an enzyme-linked immunosorbent assay. Diagnostic tests for paratuberculosis are also used in Crohn's disease, a chronic human ileitis mimicking Johne's disease, in which isolates identified as M. paratuberculosis have been found.

Genetic relatedness among Mycobacterium paratuberculosis and M. avium complex.

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis of total DNA was performed with 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

Insertion elements in mycobacteria.

We have isolated and characterised a repetitive element from the genome of Mycobacterium paratuberculosis. This repetitive element shows many features characteristic of a bacterial insertion element.

Pathology of spontaneous and experimental infection of North American wild ruminants with Mycobacterium paratuberculosis.

Spontaneous paratuberculosis was studied in free-ranging and captive bighorn sheep (Ovis canadensis), and Rocky Mountain goats (Oreamnos americanus). Lesions of paratuberculosis in these species resembled the disease in domestic sheep and goats. Mycobacterium paratuberculosis cultured from bighorn sheep was used to orally infect bighorn x mouflon (Ovis musimon) hybrid sheep, elk (Cervus elaphus nelsoni), mule deer (Odocoileus hemionus), and white-tailed deer (Odocoileus virginianus). Clinical paratuberculosis developed only in mule deer and was characterized by poor growth and diarrhea. Gross lesions were mild in all species. Enlargement of mesenteric lymph nodes was mild to moderate; the wall of the distal small intestine was affected minimally. Focal to diffuse infiltrates of epithelioid macrophages and giant cells occurred in the cortex of mesenteric lymph nodes, around mesenteric lymphatics, and in the intestinal mucosa. Extraintestinal lymph nodes, spleen, liver, and lung were involved in some animals; focal necrosis and mineralization was present in all species but was severe and widespread in the cervids.