Mycobacterium paratuberculosis avium


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Mycobacterium paratuberculosis avium

food pathogen of concern

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Mycobacterium paratuberculosis avium

  1. 1. Mycobacterium avium: A food borne pathogen of concern<br />
  2. 2. History<br />Paratuberculosis was first described in 1895 by Johneand Frothingham<br />Identified in granulomatous lesions in the intestines of affected cattle that stained acid-fast indicating of Mycobacterial organism<br />. <br /> The organism was cultured from cattle in 1910 and was classified as a Mycobacterium by Twort and Ingram (1910, 1912)<br />
  3. 3. Mycobacterium aviumsubspecies paratuberculosis (MAP)<br />Gram positive rod (0.5 x 1.5 micron)<br />Acid fast<br />Facultative intracellular<br /> Obligate parasitic pathogen<br />Requires iron for growth<br />
  4. 4. Virulence Factors<br />Intracellular pathogen<br />Grow and multiply inside macrophages<br />Chemically resistant Mycobacterial cell wall that is resistant to destruction or penetration<br /> Ability to neutralize antibacterial chemicals produced inside macrophages<br />Toxic chemical components of the Mycobacterial cell wall<br />
  5. 5. Complete Genome Sequence of MAP K-10<br /><ul><li>Single circular chromosome of 4.8 Mb and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon.
  6. 6. In silico analysis identified >3,000 genes with homologs </li></ul> M .tuberculosis<br /><ul><li> Availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology of MAP
  7. 7. Enables the development of new generations of diagnostic tests for Johne's disease.</li></ul> (Bannantineet al., 2005) <br />
  8. 8. Natural Reservoirs<br />Natural reservoir – wild animal population <br /> (Ruminants, Swine, Rabbit, Deer, Bison, Elk)<br />MAP can survive – 250 days in water feces, cattle slurry<br />Manure from infected animal most common mode of contamination<br />Vertical transmission during pregnancy<br />
  9. 9. Disease <br />Digestive tract route of entrance of MAP<br />Multiplication of MAP in Intestinal mucosa<br />Phagocytosis by macrophages <br />Via lymph channels<br />Infiltration regional lymph node<br />
  10. 10. Cause<br />Decreased absorption & chronic diarrhea<br />Muscle wasting and loss of weight<br />Severity leads to death<br />Leads to :<br />Reduced milk yield<br />Reduced meat yield<br />Reduced reproductive performance<br />
  11. 11. Potential Human Exposure to MAP<br />
  12. 12. Presence of MAP in Milk & Milk Products<br />MAP has been detected in the following :<br />Colostrum<br />Raw Milk<br />Pasteurized Milk<br />Powdered Milk<br />Cheese<br />Goat and Sheep Milk<br />
  13. 13. MAP in Colostrum<br />Colostrum good sample for MAP isolation<br />Early potential exposure of dairy calf<br />MAP detected in udder tissue supramammary lymph nodes (Chiodiniet al, 1984)<br />(Streeter et al, 1995)<br />
  14. 14. MAP in Raw Milk<br />Sources of contamination:<br /><ul><li> Direct shedding in milk
  15. 15. Fecal contamination
  16. 16. Mixing contaminated milk</li></ul>MAP isolated from – Supramammary lymph node; deep udder tissue<br />(Sweeney et al ., 1992)<br />
  17. 17. Incidence of MAP in Raw Milk<br />
  18. 18. Pasteurized Milk<br /><ul><li>MAP survival depends on initial load
  19. 19. 12/27 HTST pasteurized milk MAP positive</li></ul>(Grant et al, 2005) <br /><ul><li>Standard pasteurization temperature fails to guarantee full inactivation of milk</li></ul> 6log10 - 85% reduction (Doherty et al, 2002)<br /><ul><li>MAP isolated from milk treated at 82.5 °C</li></ul>(Slanaet al, 2008)<br />Homogenization and pasteurization <br />(Grant et al,2005)<br />
  20. 20. Survival: Heat Resistance<br />In most cases a 3 - 4 log kill Achieved with Pasteurisation<br />Survival depends on initial contamination level<br />
  21. 21. MAP in Powdered milk<br /><ul><li>Coffee cream, whole milk powder, half-fat milk, skimmed milk,and baby food can also be contaminated
  22. 22. Children's at higher risk
  23. 23. Crohn’s disease in children's in Europe, 2004
  24. 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)</li></li></ul><li>MAP in cheese<br /><ul><li>MAP has been detected from market cheese</li></ul>(Clark et al,2006)<br /><ul><li>Sub pasteurization temp treatment of milk for cheese production insufficient for MAP inactivation (Pearce et al,2001)
  25. 25. Occurrence of MAP in Cheese by PCR </li></ul> Greece 50 %<br /> CZ 12 %<br /> USA 5 % (Ayeleet al, 2004)<br />
  26. 26. MAP in Sheep & Goat Raw Milk <br />104 sheep and goat milk sample analyzed in UK<br /> PCR - 1%(Grant et al, 2001)<br />340 goat milk sample analyzed in Norway<br /> IMS-PCR- 7.1 %(Djanneet al, 2003)<br />In India, MAP isolated from milk and feces of infected goat (Singh and Vihan 2004)<br />
  27. 27. Effect of Food Processing Steps on MAP<br />Clarification, centrifugation, separation, standardization and homogenization<br />Homogenization – increases MAP count <br />Centrifugation and microfiltration – removes MAP 95-99.9% (Grant et al, 2005) <br />Homogenization and Pasteurization – more effective for MAP inactivation (Grant et al, 2005) <br />
  28. 28. Processing of Dairy Products<br />NaCl has little or no effect in cheese<br />Low pH significantly contribute MAP inactivation<br />Ripening of cheese significantly lower MAP<br />Temp and low pH – most important factor in MAP inactivation during ripening <br />Persistence of MAP in cheese<br /> High conc. of MAP in raw milk<br /> Short ripening period<br />(Spahr and Schafroth, 2001)<br />
  29. 29. MAP in Retail Dairy Products<br />
  30. 30. MAP in Raw Meat Product<br />MAP isolated from GI tract and other organs of culled dairy animals. <br />(Antognoliet al, 2008)<br />Meat contaminated with MAP by<br /><ul><li> Dissemination of pathogen in tissue
  31. 31. Fecal contamination
  32. 32. Fleece contamination
  33. 33. Wool and skin
  34. 34. Redistribution during washing</li></li></ul><li>Detection Mycobacterium aviumsubsp. Paratuberculosis<br />Culture method<br />PCR based method<br />ELISA<br />
  35. 35. MAP Detection-Culture Method<br />Media –Herrold’s Egg Yolk Medium (HEYM)<br />Antibiotics- <br /> PANTA- Polymyxin B, Amphotericin B, <br /> Nalidix Acid, Trimethoprin, Azocillin<br /> VAN - Vancomycin, Amphoterin B, Nalidxic Acid<br />Additive- Mycobactin J<br />Decontamination of sample:<br /> 1) NaOH 2) HPC<br />
  36. 36. Disadvantage<br />Long culture period – 4 - 12 week<br />Fastidious growth requirements<br />Contamination<br />Advantages<br /><ul><li>Gold standard
  37. 37. Simple and widely used method</li></li></ul><li>PCR Based Methods<br />IS 900 – for M. paratuberculosis<br />IS 901 – for M. avium<br />IS 1245 – for Mycobacterium avium complex<br />hsp X gene – putative heat shock protein<br />F57 – diagnostic probe for MAP<br />Real Time PCR- <br /> IS 900 sequence – (Khareet al, 2004)<br /> F57 – (Stephan et al, 2007)<br />
  38. 38. Drawbacks<br />PCR inhibitors - present in fecal, milk, milk product samples<br />Cant differentiate between live and dead cell<br />Chances of cross amplification<br />Some protocols lack sensitivity<br />
  39. 39. ELISA<br />ELISA tests based on:<br />IFN–Υ - Expression of IFN-Υincreases during infection<br />Protoplasmic antigen (PPA-3) – first used antigen<br /> (Sweenayet al 1994) <br />Lipoarabinomannan polysaccharide antigen (LAM)<br />
  40. 40. Advantages<br />Disadvantage <br />Early detection is not possible <br /> Cross reactivity<br />False positive result in case of immunization<br />Can performed similarly for all ruminants<br />Same test for milk and serum samples<br />Rapid and Low price<br />Sensitivity of ELISA<br /><ul><li> Subclinical Infected Animal – 15-57%
  41. 41. Clinically Infected Animals – 89 -95 % </li></li></ul><li>Treatment <br /><ul><li>No drug approved
  42. 42. Expensive and unrewarding
  43. 43. Antibiotic therapy – No complete cure
  44. 44. Antibiotics used - Clofazimine or Isoniazid and either Rifabutin or Ethambutol</li></ul>Treatment of goat affected with MAP<br />Streptomycin, Rifampicin, Levamisole<br />(Das et al, 1992)<br />
  45. 45. Vaccination<br />Heat killed or modified live preparation of M. paratuberculosis strain 18- reduces incidence<br />Provides partial protection<br />Decreases the No. of MAP shedding in feces<br />(Kormendy, 1994)<br />Disadvantage<br /> Positive antibody test, which may interfere <br /> with serological testing<br />
  46. 46. Management<br />Over all cleanliness of farm<br />Manure handling<br />Care of new borne calf<br />Breed selection – jersey and Cuernsey more <br /> susceptible<br />Routine check up – ELISA, PCR <br />
  47. 47. MAP a human pathogen ?<br />Chron’s disease in human, a sever inflammatory enteritis involving the terminal ileum<br />Clinical symptoms of Crohn’s disease closely mimic those found in animals with Johne’s disease<br />M. paratuberculosis has been isolated from biopsy tissues Crohn’s disease patients<br />Epidemiological evidence correlating exposure to M. paratuberculosis with incidence of Crohn’s disease is not readily available (Stabel, 1997)<br />
  48. 48. Probiotics and MAP<br /><ul><li>Recent study shows presence of MAP in pasteurized milk and other dairy products such as cheese, yoghurt, baby foods
  49. 49. Map growth was inhibited (delayed) when supplemented with supernatants from a number of Lb. paracasei isolates
  50. 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. 51. In vitro inhibitory effect of some lactobacilli on MAP, may be due to factors other than acid production. (Donaghyet al,2005)</li></li></ul><li>Conclusions<br />Economic losses of $1.5 billion/year<br />Pasteurized milk, cheese, other dairy products may not be always free of MAP<br />Contaminated baby food with MAP expose children and immuno-compromised people at high risk<br />Effectiveness of pasteurization affected by initial concentration of MAP in raw milk<br />
  52. 52. Conclusions<br />New technologies are required for the early detection of infected animals<br /><ul><li>Identification and characterization of antigen protein that are specific to MAPisnecessary for improved vaccine development
  53. 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.</li></li></ul><li>