Surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP): Implications for Johne's disease (JD) diagnosis and pathogenesis Ashutosh Wadhwa, Ph. D. Candidate Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, The University of Tennessee, Knoxville awadhwa @utk.edu February, 23 rd 2010, 12.20 PM, Room 160 PBB
Introduction Johne s disease (JD) - pronounced as YO-nees, is also called paratuberculosis, caused by Mycobacterium avium paratuberculosis (MAP). It is a chronic infection, causing reduction in milk production, malnutrition, weight loss and eventually death. The natural hosts for MAP are wild and domesticated ruminants, including dairy and beef cattle, sheep, goats, red deer, cervids, and camelids.
Introduction In the United States, JD has been found in 68 % of the dairy herd and causes an estimated annual loss of $220 million to the US dairy industry. No treatment available. Early screening and culling is the best practice to control. Crohn s disease (CD), a chronic inflammatory disease, also called as inflammatory bowel disease. Recent researches have shown that not all but at least some cases of CD may be caused by MAP.
MAP The Bug Obligate intracellular bacteria Small (0.5 x 1.5 micron) Rod shaped bacteria MAP has a single circular chromosome. It is - slow-growing, gram-positive, acid-fast positive and aerobic. The cell wall is made up of lipids and polysaccharides. MAP Unique since it does not produce mycobactin and depend on the host cell to provide iron. MAP share 95% of its genes and exhibit homologies of more than 99% between these genes with MAA.
Pathogenesis of JD
Progression of JD/Clinical Signs JD can be classified in 4 stages: 1. Silent infection: Young stock up to 2 years of age. No clinical or measurable clinical signs. 2. Subclinical infection: No clinical signs; but could be detected. 3. Clinical infection: I/P of 2 10 years - gradual weight loss, persistent or intermittent diarrhea and decreased milk production. 4. Advanced clinical infection: It arise if animals with clinical signs are not culled. More lethargic, weak and emaciated characterizing pipestream diarrhea, hypoproteinemia and intermandibular edema.
Diagnosis Fecal /Tissue culturing for MAP takes 6-8 weeks. Intradermal test- low sensitivity and specificity. Polymerase chain reaction (PCR) - IS900 insertion sequence, F57 Costly, lengthy procedure and specialized equipments. Complement Fixation test and agar gel immunodiffusion test can also be done but it suffers poor sensitivity.
Sero-Diagnosis and various antigens Recent reports suggest that Enzyme linked immunosorbant assays (ELISA) should be used for controlling the disease. ELISA s detect an optical density in milk or serum that correlates to antibody response to MAP. Various antigens internal proteins (MAP p35 K), alkyl hydroperoxide reductases C and D (AhpC and AhpD), recominant polypeptides. All suffer low sensitivity. Ethanol vortex ELISA (EVELISA)- Uses a major cell wall lipopeptide as an antigen- Sensitivity 95%. EVELISA on bovine serum samples
The antigen 200 ml of MAP culture Treatment with 70% ethanol and vortexing Ethanol Extract antigen
Recent research from our group
Research Objective I Optimization and evaluation of serological tests for JD. Need for a rapid and relatively cheap screening test with highest diagnostic sensitivity. Need of a Lab-on-a-chip technology for onsite detection of JD.
Proposed Method i. Testing field serum samples provided by collaborators. ii. Testing milk samples provided by collaborators. Proposed Lab-on-a-chip diagnostic device Primary antibody Solution dispensing channels iii. Development of a microfluidic diagnostic device for Johne s diagnosis. Wash Solutions Secondary antibody Reaction and detection channel Waste Layout of the microfluidic chip
Research Objective II Isolation and characterization of molecules in the ethanol extract of MAP. Need to identify sub-species specific molecules to explain why MAP occupies a specific biological niche. Host immune response to the surface associated lipopeptides of MAP.
Proposed Method i. Analysis of lipids in MAP ethanol extract using TLC ii. Purification of MAPspecific lipids using Cyclograph iii. Structural analysis of MAP-specific lipids using NMR and mass spectrometry Initial mass spectrometry results
Research Objective III In vivo studies on possible roles of MAPspecific lipids in pathogenesis of JD. Which response dominates first Cell mediated or humoral immune response? Effect of MAP specific surface molecules on cytokine production and gene expression.
Proposed Methods-III i. Effects of MAP-specific lipids on cell (macrophage, dendritic cell, epithelial cell) functions ii. Cytokine productions (BioPlex at Genomics hub) iii. Gene expression (Microarray)
Antibody Binding (Absorbance at 415 nm) Statistical Analysis All experiments will be conducted in duplicates or triplicates and repeated twice. The statistical difference of antibody binding among various sets of conditions and between negative and positive samples will be evaluated by using a Mann-Whitney U test. Statistical analysis and depiction of box plots will be conducted using a statistical software, R. Milk Dilution N P N P N P N P 1:2.5 1:5 1:10 1:20 Preliminary result from EV MILK ELISA
References 1. Nielsen, S.S. and N. Toft, Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-gamma assay and faecal culture techniques. Vet Microbiol, 2008. 129(3-4): p. 217-35. 2. Kennedy, D.J. and G. Benedictus, Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species. Rev Sci Tech, 2001. 20(1): p. 151-79. 3. Tiwari, A., et al., Johne's disease in Canada Part I: clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds. Can Vet J, 2006. 47(9): p. 874-82. 4. Ott, S.L., S.J. Wells, and B.A. Wagner, Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med, 1999. 40(3-4): p. 179-92. 5. Harris, N.B. and R.G. Barletta, Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin Microbiol Rev, 2001. 14(3): p. 489-512. 6. Momotani, E., et al., Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves. Vet Pathol, 1988. 25(2): p. 131-7. 7. Lugton, I., Mucosa-associated lymphoid tissues as sites for uptake, carriage and excretion of tubercle bacilli and other pathogenic mycobacteria. Immunol Cell Biol, 1999. 77(4): p. 364-72. 8. Chacon, O., L.E. Bermudez, and R.G. Barletta, Johne's disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Annu Rev Microbiol, 2004. 58: p. 329-63.
References..cont 9. Whitlock, R.H. and C. Buergelt, Preclinical and clinical manifestations of paratuberculosis (including pathology). Vet Clin North Am Food Anim Pract, 1996. 12(2): p. 345-56. 10. Collins, M.T., et al., Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens. J Clin Microbiol, 1990. 28(11): p. 2514-9. 11. Ellingson, J.L., C.A. Bolin, and J.R. Stabel, Identification of a gene unique to Mycobacterium avium subspecies paratuberculosis and application to diagnosis of paratuberculosis. Mol Cell Probes, 1998. 12(3): p. 133-42. 12. Cocito, C., et al., Paratuberculosis. Clin Microbiol Rev, 1994. 7(3): p. 328-45. 13. Sherman, D.M., et al., Comparison of the complement-fixation and agar gel immunodiffusion tests for diagnosis of subclinical bovine paratuberculosis. Am J Vet Res, 1990. 51(3): p. 461-5. 14. Collins, M.T., et al., Consensus recommendations on diagnostic testing for the detection of paratuberculosis in cattle in the United States. J Am Vet Med Assoc, 2006. 229(12): p. 1912-9. 15. Speer, C.A., et al., A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle. Clin Vaccine Immunol, 2006. 13(5): p. 535-40. 16. Eda, S., et al., New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry. Foodborne Pathog Dis, 2005. 2(3): p. 250-62.
Acknowledgment Dr. Shigetoshi Eda, Major Advisor Dr. Graham Hickling, Member Doctoral Committee Dr. Lisa Muller, Member Doctoral Committee Dr. Gina M. Pighetti, Member Doctoral Committee Ms. Cathy Scott, Research Associate, Center for Wildlife Health. Funding Sources:
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