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The use of polymerase chain reaction (PCR) to confirm presence of selected pathogenic bacteria along milk value chain in Tanga region

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Presentation by F Shija, G Misinzo, H Nonga, LR Kurwijila, K Roesel and D Grace at the 14th international conference of the Association of Institutions for Tropical Veterinary Medicine (AITVM), Johannesburg, South Africa, 25-29 August 2013.

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The use of polymerase chain reaction (PCR) to confirm presence of selected pathogenic bacteria along milk value chain in Tanga region

  1. 1. F Shija, G Misinzo, H Nonga, LR Kurwijila, K Roesel and D Grace Presented at the 14th International Conference of the Association of Institutes for Tropical Veterinary Medicine (AITVM) Johannesburg, South Africa 25-29 August 2013
  2. 2.  Food-borne diseases are a threat and are responsible for 33-90% cases of mortality to children  Bacterial milk contamination causes: ◦ Milk spoilage ◦ Milk-born zoonotic diseases  Up to 90% diary related diseases are due to pathogenic bacteria from milk
  3. 3.  Dairy industry in most African countries is dominated by unpasteurized milk and informal markets  PCR is a technique which utilizes primer sets to detect presence of pathogens in a sample
  4. 4.  Risks of milk safety hazards in informal market are high and unknown in Tanzania  Previous studies have been on the specific risks associated with pathogenic microbes along the milk chain (e.g. Swai and Schoonman 2011; Kaiza et al. 2011)
  5. 5.  PCR detection of milk bacterial contaminants is powerful, gives reliable information on pathogens in milk  Results of the study will be used to improve food safety throughout smallholder and informal milk value chain in Tanzania
  6. 6. Main Objective: To assess milk handling practices, bacterial contamination and determine selected milk borne zoonotic pathogens along the dairy value chain in Lushoto and Handeni districts of Tanga region Specific objectives: 1. To assess the possible sources of microbial contamination of milk from farm to consumer 2. To establish total plate count of bacteria and coliforms in milk from Lushoto and Handeni districts 3. To establish the prevalence of Escherichia coli O157:H7 and Brucella abortus in milk using polymerase chain reaction
  7. 7. Study area: Tanga region of the North-eastern part of Tanzania Why Tanga?: Well coordinated dairy value chain ◦ Long history of smallholder dairy farming organized under TDCU ◦ Well developed TFL, one of the biggest supplier of processed milk in Tanzania Study design o A cross–sectional study
  8. 8. Study area
  9. 9.  A total of 93 (65 farmers, 28 retailers) interviewed  A total of 166 milk samples from farmers, vendors, restaurants/kiosks, collection centres and consumers
  10. 10. Chains identified-Lushoto district 1. 2. 3. 4. 5. 6. 7. 8. Farmer Consumer Farmer Sub Col centre (no chilling ) CC(chilling) Factory Consumer Farmer CC(chilling) Factory Consumer Farmer CC Consumer Farmer CC Restaurant/kiosk Consumer Farmer Selling point Consumer Farmer Restaurant Consumer Farmer/Processor Consumer
  11. 11. Chains identified – Handeni district 1. Farmer Consumer 2. Farmer Vendor Consumer 3. Farmer Vendor Collection Centre Factory consumer 4. Farmer Collection centre consumer 5. Farmer Vendor Restaurant Consumer
  12. 12. Escherichia coli O157:H7 (O157-3 and O157-4) Brucella abortus (BRU P5 and BRU P8 )
  13. 13. Microbiological isolation, PCR and statistical data analysis (stata IC 11)
  14. 14. General practices during milking storage and delivery Variable Sources of water Milking practices Containers storage used for Containers used for delivery/transportation Means of delivery Category Tap Wells Dams and/or streams Cleaning animal shed before milking Wash hands before milking Wash cow's teats before milking Wash hands after milking wide necked aluminium vessel milk Wide necked plastic vessel Used water and oil bottles Cooking pan "sufuria" wide necked aluminum vessel Wide necked plastick vessel Used water and oil bottles Cooking pan "sufuria" Others e.g traditional pots On foot By bicycle By motorcycle No. (%) farmers respondents 26 (40.0) 21 (32.3) 19 (29.3) 28 (43.1) 46 (70.7) 41 (63.1) 47 (72.3) 2 (03.1) 56 (86.1) 6 (09.2) 1 (01.5) 0 (0.0) 38 (58.5) 8 (12.3) 3 (4.6) 16 (24.6) 37 (56.9) 9 (13.8) 3 (4.6)
  15. 15. Milk marketing channels
  16. 16. Total plate counts and coliform plate counts for milk actors in the chains Variable Observation s Mean (log10 cfu/ml) Std. Dev (log10) Min Max Total Plate Count Farmers 21 5.3 5.4 3.3 5.8 Vendors 5 5.8 5.7 4.6 6.1 Restaurants 7 0 5.3 Farmers 22 4.8 4.9 2.5 5.5 Vendors 4 4.8 5.1 3.3 5.4 Restaurants 7 3.6 3.9 0 4.3 4.9 4.9 Coliform plate count
  17. 17. Detection of B. abortus by PCR using BRU P5 and BRU P8 primer pairs targeting 16S-23S gene at between 500 to 600 bp. Note that lane M is a molecular weight marker while lanes A, C, D, E, F, G, H, J, K, M, O, P and Q are positive amplicons whereas lane B, I, L and N are negative amplicons. R is a positive control. 42% positive 600 bp 500 bp
  18. 18. Positive samples for B. abortus in Lushoto and Handeni districts Source of milk samples Lushoto (%) Handeni (%) Consumers (2.2) (9.5) Restaurant (4.4) (4.8) Farmers (31.1) (26.2) Vendors - (7.1) (37.8) (47.6) Total Both districts (%) (5.7) (4.6) (28.7) (4.6) (42.5)
  19. 19. Detection of E. coli using O157-3 and O157-4 primer pairs targeting hyla A between at 356 bp. Note that lane M is a molecular weight marker, lane A to K are negative amplicons while lane L is a positive control M 600 bp 500 bp A B C D E F G H I J K L
  20. 20.  Poor hygienic practices at milking and selling places contributes to increase in microorganisms  Lack of knowledge on zoonotic diseases and their causes in farmers contributed to poor unhygienic practices in milky activities  The prevalence of B. abortus suggests high contamination rate- relates to findings by Schoonman and Swai (2005)
  21. 21.     Veterinary/extension services should be provided to livestock farmers on proper animal husbandry and control of diseases. Responsible authorities must ensure that existing regulations are instituted and where possible there should be a mandatory screening of milk before sales to the public. Consumer practices such as milk boiling should be further encouraged. Further study to relate the findings with human brucellosis in that area should be carried out.
  22. 22.  The Federal Ministry for Economic Cooperation and Development, Germany through the Safe Food, Fair Food project  Sokoine University of Agriculture (SUA)  International Livestock Research Institute (ILRI)  AITVM

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