Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants. It is a gram-positive, acid-fast bacterium that survives in the environment and is resistant to heat and pasteurization. MAP has been detected in pasteurized milk and dairy products through contamination of raw milk from infected animals. This poses a potential risk to human health as MAP may play a role in Crohn's disease. Improved diagnostics, therapeutics, and management practices are needed to control MAP in animal populations and minimize risks to food safety.

1. Mycobacterium avium: A food borne pathogen of concern

2. HistoryParatuberculosis was first described in 1895 by Johneand FrothinghamIdentified in granulomatous lesions in the intestines of affected cattle that stained acid-fast indicating of Mycobacterial organism. The organism was cultured from cattle in 1910 and was classified as a Mycobacterium by Twort and Ingram (1910, 1912)

3. Mycobacterium aviumsubspecies paratuberculosis (MAP)Gram positive rod (0.5 x 1.5 micron)Acid fastFacultative intracellular Obligate parasitic pathogenRequires iron for growth

4. Virulence FactorsIntracellular pathogenGrow and multiply inside macrophagesChemically resistant Mycobacterial cell wall that is resistant to destruction or penetration Ability to neutralize antibacterial chemicals produced inside macrophagesToxic chemical components of the Mycobacterial cell wall

5. Complete Genome Sequence of MAP K-10Single circular chromosome of 4.8 Mb and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon.

6. In silico analysis identified >3,000 genes with homologs M .tuberculosis Availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology of MAP

7. Enables the development of new generations of diagnostic tests for Johne's disease. (Bannantineet al., 2005)

8. Natural ReservoirsNatural reservoir – wild animal population (Ruminants, Swine, Rabbit, Deer, Bison, Elk)MAP can survive – 250 days in water feces, cattle slurryManure from infected animal most common mode of contaminationVertical transmission during pregnancy

9. Disease Digestive tract route of entrance of MAPMultiplication of MAP in Intestinal mucosaPhagocytosis by macrophages Via lymph channelsInfiltration regional lymph node

10. CauseDecreased absorption & chronic diarrheaMuscle wasting and loss of weightSeverity leads to deathLeads to :Reduced milk yieldReduced meat yieldReduced reproductive performance

11. Potential Human Exposure to MAP

12. Presence of MAP in Milk & Milk ProductsMAP has been detected in the following :ColostrumRaw MilkPasteurized MilkPowdered MilkCheeseGoat and Sheep Milk

13. MAP in ColostrumColostrum good sample for MAP isolationEarly potential exposure of dairy calfMAP detected in udder tissue supramammary lymph nodes (Chiodiniet al, 1984)(Streeter et al, 1995)

14. MAP in Raw MilkSources of contamination: Direct shedding in milk

15. Fecal contamination

16. Mixing contaminated milkMAP isolated from – Supramammary lymph node; deep udder tissue(Sweeney et al ., 1992)

17. Incidence of MAP in Raw Milk

18. Pasteurized MilkMAP survival depends on initial load

19. 12/27 HTST pasteurized milk MAP positive(Grant et al, 2005) Standard pasteurization temperature fails to guarantee full inactivation of milk 6log10 - 85% reduction (Doherty et al, 2002)MAP isolated from milk treated at 82.5 °C(Slanaet al, 2008)Homogenization and pasteurization (Grant et al,2005)

20. Survival: Heat ResistanceIn most cases a 3 - 4 log kill Achieved with PasteurisationSurvival depends on initial contamination level

21. MAP in Powdered milkCoffee cream, whole milk powder, half-fat milk, skimmed milk,and baby food can also be contaminated

22. Children's at higher risk

23. Crohn’s disease in children's in Europe, 2004

24. Baby food contamination - 51 different samples, from 7 European countries were examined in which 25 (49.0%) samples were found positive. (Hruskaet al, 2005)MAP in cheeseMAP has been detected from market cheese(Clark et al,2006)Sub pasteurization temp treatment of milk for cheese production insufficient for MAP inactivation (Pearce et al,2001)

25. Occurrence of MAP in Cheese by PCR Greece 50 % CZ 12 % USA 5 % (Ayeleet al, 2004)

26. MAP in Sheep & Goat Raw Milk 104 sheep and goat milk sample analyzed in UK PCR - 1%(Grant et al, 2001)340 goat milk sample analyzed in Norway IMS-PCR- 7.1 %(Djanneet al, 2003)In India, MAP isolated from milk and feces of infected goat (Singh and Vihan 2004)

27. Effect of Food Processing Steps on MAPClarification, centrifugation, separation, standardization and homogenizationHomogenization – increases MAP count Centrifugation and microfiltration – removes MAP 95-99.9% (Grant et al, 2005) Homogenization and Pasteurization – more effective for MAP inactivation (Grant et al, 2005)

28. Processing of Dairy ProductsNaCl has little or no effect in cheeseLow pH significantly contribute MAP inactivationRipening of cheese significantly lower MAPTemp and low pH – most important factor in MAP inactivation during ripening Persistence of MAP in cheese High conc. of MAP in raw milk Short ripening period(Spahr and Schafroth, 2001)

29. MAP in Retail Dairy Products

30. MAP in Raw Meat ProductMAP isolated from GI tract and other organs of culled dairy animals. (Antognoliet al, 2008)Meat contaminated with MAP by Dissemination of pathogen in tissue

31. Fecal contamination

32. Fleece contamination

33. Wool and skin

34. Redistribution during washingDetection Mycobacterium aviumsubsp. ParatuberculosisCulture methodPCR based methodELISA

35. MAP Detection-Culture MethodMedia –Herrold’s Egg Yolk Medium (HEYM)Antibiotics- PANTA- Polymyxin B, Amphotericin B, Nalidix Acid, Trimethoprin, Azocillin VAN - Vancomycin, Amphoterin B, Nalidxic AcidAdditive- Mycobactin JDecontamination of sample: 1) NaOH 2) HPC

36. DisadvantageLong culture period – 4 - 12 weekFastidious growth requirementsContaminationAdvantagesGold standard

37. Simple and widely used methodPCR Based MethodsIS 900 – for M. paratuberculosisIS 901 – for M. aviumIS 1245 – for Mycobacterium avium complexhsp X gene – putative heat shock proteinF57 – diagnostic probe for MAPReal Time PCR- IS 900 sequence – (Khareet al, 2004) F57 – (Stephan et al, 2007)

38. DrawbacksPCR inhibitors - present in fecal, milk, milk product samplesCant differentiate between live and dead cellChances of cross amplificationSome protocols lack sensitivity

39. ELISAELISA tests based on:IFN–Υ - Expression of IFN-Υincreases during infectionProtoplasmic antigen (PPA-3) – first used antigen (Sweenayet al 1994) Lipoarabinomannan polysaccharide antigen (LAM)

40. AdvantagesDisadvantage Early detection is not possible Cross reactivityFalse positive result in case of immunizationCan performed similarly for all ruminantsSame test for milk and serum samplesRapid and Low priceSensitivity of ELISA Subclinical Infected Animal – 15-57%

41. Clinically Infected Animals – 89 -95 % Treatment No drug approved