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Mycobacterium paratuberculosis avium
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Mycobacterium paratuberculosis avium

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Mycobacterium paratuberculosis avium

food pathogen of concern

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  • 1. Mycobacterium avium: A food borne pathogen of concern
  • 2. History
    Paratuberculosis was first described in 1895 by Johneand Frothingham
    Identified 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 fast
    Facultative intracellular
    Obligate parasitic pathogen
    Requires iron for growth
  • 4. Virulence Factors
    Intracellular pathogen
    Grow and multiply inside macrophages
    Chemically resistant Mycobacterial cell wall that is resistant to destruction or penetration
    Ability to neutralize antibacterial chemicals produced inside macrophages
    Toxic chemical components of the Mycobacterial cell wall
  • 5. Complete Genome Sequence of MAP K-10
    • Single 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 Reservoirs
    Natural reservoir – wild animal population
    (Ruminants, Swine, Rabbit, Deer, Bison, Elk)
    MAP can survive – 250 days in water feces, cattle slurry
    Manure from infected animal most common mode of contamination
    Vertical transmission during pregnancy
  • 9. Disease
    Digestive tract route of entrance of MAP
    Multiplication of MAP in Intestinal mucosa
    Phagocytosis by macrophages
    Via lymph channels
    Infiltration regional lymph node
  • 10. Cause
    Decreased absorption & chronic diarrhea
    Muscle wasting and loss of weight
    Severity leads to death
    Leads to :
    Reduced milk yield
    Reduced meat yield
    Reduced reproductive performance
  • 11. Potential Human Exposure to MAP
  • 12. Presence of MAP in Milk & Milk Products
    MAP has been detected in the following :
    Colostrum
    Raw Milk
    Pasteurized Milk
    Powdered Milk
    Cheese
    Goat and Sheep Milk
  • 13. MAP in Colostrum
    Colostrum good sample for MAP isolation
    Early potential exposure of dairy calf
    MAP detected in udder tissue supramammary lymph nodes (Chiodiniet al, 1984)
    (Streeter et al, 1995)
  • 14. MAP in Raw Milk
    Sources of contamination:
    • Direct shedding in milk
    • 15. Fecal contamination
    • 16. Mixing contaminated milk
    MAP isolated from – Supramammary lymph node; deep udder tissue
    (Sweeney et al ., 1992)
  • 17. Incidence of MAP in Raw Milk
  • 18. Pasteurized Milk
    • MAP 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 Resistance
    In most cases a 3 - 4 log kill Achieved with Pasteurisation
    Survival depends on initial contamination level
  • 21. MAP in Powdered milk
    • Coffee 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 cheese
    • MAP 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 MAP
    Clarification, centrifugation, separation, standardization and homogenization
    Homogenization – 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 Products
    NaCl has little or no effect in cheese
    Low pH significantly contribute MAP inactivation
    Ripening of cheese significantly lower MAP
    Temp 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 Product
    MAP 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 washing
  • Detection Mycobacterium aviumsubsp. Paratuberculosis
    Culture method
    PCR based method
    ELISA
  • 35. MAP Detection-Culture Method
    Media –Herrold’s Egg Yolk Medium (HEYM)
    Antibiotics-
    PANTA- Polymyxin B, Amphotericin B,
    Nalidix Acid, Trimethoprin, Azocillin
    VAN - Vancomycin, Amphoterin B, Nalidxic Acid
    Additive- Mycobactin J
    Decontamination of sample:
    1) NaOH 2) HPC
  • 36. Disadvantage
    Long culture period – 4 - 12 week
    Fastidious growth requirements
    Contamination
    Advantages
    • Gold standard
    • 37. Simple and widely used method
  • PCR Based Methods
    IS 900 – for M. paratuberculosis
    IS 901 – for M. avium
    IS 1245 – for Mycobacterium avium complex
    hsp X gene – putative heat shock protein
    F57 – diagnostic probe for MAP
    Real Time PCR-
    IS 900 sequence – (Khareet al, 2004)
    F57 – (Stephan et al, 2007)
  • 38. Drawbacks
    PCR inhibitors - present in fecal, milk, milk product samples
    Cant differentiate between live and dead cell
    Chances of cross amplification
    Some protocols lack sensitivity
  • 39. ELISA
    ELISA tests based on:
    IFN–Υ - Expression of IFN-Υincreases during infection
    Protoplasmic antigen (PPA-3) – first used antigen
    (Sweenayet al 1994)
    Lipoarabinomannan polysaccharide antigen (LAM)
  • 40. Advantages
    Disadvantage
    Early detection is not possible
    Cross reactivity
    False positive result in case of immunization
    Can performed similarly for all ruminants
    Same test for milk and serum samples
    Rapid and Low price
    Sensitivity of ELISA
    • Subclinical Infected Animal – 15-57%
    • 41. Clinically Infected Animals – 89 -95 %
  • Treatment
    • No drug approved
    • 42. Expensive and unrewarding
    • 43. Antibiotic therapy – No complete cure
    • 44. Antibiotics used - Clofazimine or Isoniazid and either Rifabutin or Ethambutol
    Treatment of goat affected with MAP
    Streptomycin, Rifampicin, Levamisole
    (Das et al, 1992)
  • 45. Vaccination
    Heat killed or modified live preparation of M. paratuberculosis strain 18- reduces incidence
    Provides partial protection
    Decreases the No. of MAP shedding in feces
    (Kormendy, 1994)
    Disadvantage
    Positive antibody test, which may interfere
    with serological testing
  • 46. Management
    Over all cleanliness of farm
    Manure handling
    Care of new borne calf
    Breed selection – jersey and Cuernsey more
    susceptible
    Routine check up – ELISA, PCR
  • 47. MAP a human pathogen ?
    Chron’s disease in human, a sever inflammatory enteritis involving the terminal ileum
    Clinical symptoms of Crohn’s disease closely mimic those found in animals with Johne’s disease
    M. paratuberculosis has been isolated from biopsy tissues Crohn’s disease patients
    Epidemiological evidence correlating exposure to M. paratuberculosis with incidence of Crohn’s disease is not readily available (Stabel, 1997)
  • 48. Probiotics and MAP
    • Recent study shows presence of MAP in pasteurized milk and other dairy products such as cheese, yoghurt, baby foods
    • 49. Map growth was inhibited (delayed) when supplemented with supernatants from a number of Lb. paracasei isolates
    • 50. When co-inoculated with probiotic strains in sterile milk for 48 h (pH < 4.5) MAP could not be detected by culture method up to 50 days
    • 51. In vitro inhibitory effect of some lactobacilli on MAP, may be due to factors other than acid production. (Donaghyet al,2005)
  • Conclusions
    Economic losses of $1.5 billion/year
    Pasteurized milk, cheese, other dairy products may not be always free of MAP
    Contaminated baby food with MAP expose children and immuno-compromised people at high risk
    Effectiveness of pasteurization affected by initial concentration of MAP in raw milk
  • 52. Conclusions
    New technologies are required for the early detection of infected animals
    • Identification and characterization of antigen protein that are specific to MAPisnecessary for improved vaccine development
    • 53. In the current state of knowledge, magnitude and potential consequences of the presence of MAP in dairy products on retail sale must not be ignored.