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Mycobacterium paratuberculosis avium
- 1. Mycobacterium avium: Afood borne pathogen of concern
- 2. HistoryParatuberculosis was firstdescribed 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 pathogenGrowand 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 Sequenceof MAP K-10Single circular chromosome of 4.8 Mb and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon.
- 6. In silico analysisidentified >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 developmentof 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 tractroute 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 Exposureto MAP
- 12. Presence of MAPin Milk & Milk ProductsMAP has been detected in the following :ColostrumRaw MilkPasteurized MilkPowdered MilkCheeseGoat and Sheep Milk
- 13. MAP in ColostrumColostrumgood 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 RawMilkSources of contamination: Direct shedding in milk
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- 17. Incidence of MAPin Raw Milk
- 18. Pasteurized MilkMAP survivaldepends on initial load
- 19. 12/27 HTST pasteurizedmilk 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 ResistanceInmost cases a 3 - 4 log kill Achieved with PasteurisationSurvival depends on initial contamination level
- 21. MAP in PowderedmilkCoffee cream, whole milk powder, half-fat milk, skimmed milk,and baby food can also be contaminated
- 22. Children's athigher risk
- 23. Crohn’s disease inchildren'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 MAPin 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 FoodProcessing 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 DairyProductsNaCl 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 RetailDairy Products
- 30. MAP in RawMeat 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
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- 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 andwidely 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 basedon:IFN–Υ - Expression of IFN-Υincreases during infectionProtoplasmic antigen (PPA-3) – first used antigen (Sweenayet al 1994) Lipoarabinomannan polysaccharide antigen (LAM)
- 40. AdvantagesDisadvantage Early detectionis 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%
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- 43. Antibiotic therapy –No complete cure
- 44. Antibiotics used -Clofazimine or Isoniazid and either Rifabutin or EthambutolTreatment of goat affected with MAPStreptomycin, Rifampicin, Levamisole(Das et al, 1992)
- 45. VaccinationHeat killed ormodified live preparation of M. paratuberculosis strain 18- reduces incidenceProvides partial protectionDecreases the No. of MAP shedding in feces(Kormendy, 1994)Disadvantage Positive antibody test, which may interfere with serological testing
- 46. ManagementOver all cleanlinessof farmManure handlingCare of new borne calfBreed selection – jersey and Cuernsey more susceptibleRoutine check up – ELISA, PCR
- 47. MAP a humanpathogen ?Chron’s disease in human, a sever inflammatory enteritis involving the terminal ileumClinical symptoms of Crohn’s disease closely mimic those found in animals with Johne’s diseaseM. paratuberculosis has been isolated from biopsy tissues Crohn’s disease patientsEpidemiological evidence correlating exposure to M. paratuberculosis with incidence of Crohn’s disease is not readily available (Stabel, 1997)
- 48. Probiotics and MAPRecentstudy shows presence of MAP in pasteurized milk and other dairy products such as cheese, yoghurt, baby foods
- 49. Map growth wasinhibited (delayed) when supplemented with supernatants from a number of Lb. paracasei isolates
- 50. When co-inoculated withprobiotic strains in sterile milk for 48 h (pH
- 51. In vitro inhibitoryeffect of some lactobacilli on MAP, may be due to factors other than acid production. (Donaghyet al,2005)ConclusionsEconomic losses of $1.5 billion/yearPasteurized milk, cheese, other dairy products may not be always free of MAPContaminated baby food with MAP expose children and immuno-compromised people at high riskEffectiveness of pasteurization affected by initial concentration of MAP in raw milk
- 52. ConclusionsNew technologies arerequired for the early detection of infected animalsIdentification and characterization of antigen protein that are specific to MAPisnecessary for improved vaccine development
- 53. In the currentstate of knowledge, magnitude and potential consequences of the presence of MAP in dairy products on retail sale must not be ignored.