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Improving Food Safety by Poultry Carcass Mapping

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Presentation on theme: "Improving Food Safety by Poultry Carcass Mapping"— Presentation transcript:

1 Improving Food Safety by Poultry Carcass Mapping
Thomas P. Oscar, Ph.D. USDA, ARS Princess Anne, MD

2

3 Definition: Poultry Carcass Map
Map of the distribution of pathogens on the carcass of poultry. Salmonella Campylobacter Listeria

4 Military Map: War on Pathogens
Location Number Types

5 Why Poultry Carcass Maps?
To better assess and manage food safety risks!

6 Microbial Ecology Pathogen is a minority member of the microbial community
Unattached surface water layer Attached biofilms Entrapped skin crevices feather follicles deep tissues The second concept of carcass mapping that I would like to highlight is the importance of microbial ecology on the process of producing an accurate map of the target on the carcass. This slide lists all of the possible ways in which a microbial target can be associated with the poultry carcass. Although there are many sampling methods, such as rinsing, swabbing and sponging, that could be used to map a microbial target on the poultry carcass, we prefer the whole part incubation method because we believe it has the best chance of detecting and enumerating all of the target microbes on the carcass. Use of a method that does not fully recover the target can result in an inaccurate carcass map, which in turn could result in an incorrect food safety decision. One of the methods that can be used to enumerate unattached, entrapped and biofilm microbes on the carcass part is to determine the detection time in the incubation fluid. Later in the presentation I will show the results of a study in which we developed a standard curve for enumerating Salmonella on whole chicken parts based on detection time by drop plating.

7 Carcass Sampling Methods
Rinse Swab Sponge Excision These methods will not produce an accurate carcass map

8 Whole Carcass Enrichment
Detects all pathogens present. Can be used to determine pathogen numbers.

9 Whole Carcass Enrichment Concepts and principles for enumeration
< 1 CFU/ml > 3 CFU/ml = pathogen = pre-enrichment broth = competitor = carcass part

10 Whole Sample Enrichment Standard Curve for Enumeration
Oscar, T. P. (2004) J. Food Prot. 67(6):

11 Poultry Carcass Mapping Concepts and Principles
Cornish Game Hen

12 Step 1 Divide Carcass into 12 Parts
A: Wing, left 41 g G: Back, rib 67 g B: Wing, right 41 g C: Breast, left front 62 g D: Breast, right front 60 g A B G H D F J L C E I K E: Breast, left back 35 g F: Breast, right back 37 g I: Thigh, left 48 g J: Thigh, right 50 g K: Drumstick, left 45 g H: Back, sacral 77 g L: Drumstick, right 48 g

13 Step 2 Spot Inoculate with 1 to 106 CFU of a MDR Salmonella
A: Wing, left 0.5-1 log G: Back, rib 3.5-4 log B: Wing, right 0.5-1 log D: Breast, right front 1.5-2 log C: Breast, left front 1.5-2 log A B G C D E: Breast, left back 2.5-3 log E F F: Breast, right back 2.5-3 log H I J I: Thigh, left 4.5-5 log J: Thigh, right 4.5-5 log K L K: Drumstick, left 5.5-6 log H: Back, sacral 3.5-4 log L: Drumstick, right 5.5-6 log

14 Distribution of Salmonella in a 5 μl drop of a 3 log/ml diluted culture
Oscar, T. P. (2009) J. Food Prot. 72:

15 Step 3 Incubate whole parts in 300 ml BPW at 40C and 100 rpm for 24 h

16 Step 4 Sample and drop plate (2 μl) onto XLH-CATS @ 1, 2, 3, 4, 5, 6, 7, 8, 24 h

17 Step 5 Incubate XLH-CATS plates @ 38C for 24 h

18 Step 6 Capture the Image

19 Step 7 Convert to Monochrome Image
A) 10-7 (3 log10 CFU/ml) B) 10-6 (4 log10 CFU/ml) C) 10-5 (5 log10 CFU/ml) D) 10-4 (6 log10 CFU/ml)

20 A) 1 h B) 2 h C) 3 h E) 5 h F) 6 h D) 4 h G) 7 h H) 8 h I) 24 h

21 Step 8 Count pixels The number of pixels in the monochrome image were then counted using Image J software.

22 Step 9 Determine Detection Time
To determine detection time, the pixels per drop were graphed as a function of sampling time and then fit to a sigmoid equation. Detection time was the time for the drop to reach 50% of confluent growth. Each line represents a different whole part and each pair of lines represents one of the six doses of Salmonella inoculated. As expected, the higher the dose, the shorter the detection time.

23 Step 10 Develop a Standard Curve
No = 7.78 ± 0.61 – (0.995*DT) To develop the standard curve, the dose of Salmonella inoculated on the whole part was graphed as a function of detection time. Detection time was not affected by the type of chicken part. However, variation of detection time within a dose and among chickens was observed and was most likely due to differences in the number and types of competing microorganisms present on the individual chickens and in the whole part incubations. Although this standard curve is specific for DT104 and Cornish Game Hen parts, it is possible to develop more global standard curves based on PCR detection time during whole part incubations and then use those standard curves to quantitatively map the distribution of Salmonella contamination on the chicken or turkey carcass.

24 Limitation: Only MDR strains Solution: Real-time PCR or Impedance

25 Qualitative Map Cornish Game Hen @ Retail

26 Qualitative Map Obtain chickens from retail
Divide carcass into 12 parts Incubate part in BPW (300 ml) Transfer 0.1 ml of BPW enrichment to RV broth A B Streak RV broth enrichment onto XLT4 G C D E F Grow suspect colony in BHI broth H I To map the distribution of Salmonella on the Cornish Game Hen carcass we obtained birds at retail. We then divided the carcass into 12 parts. The parts were then incubated in buffered peptone water. After 24h of pre-enrichment, the samples were selectively enriched in RV broth followed by selective plating on XLT4 agar. One suspect colony was selected per plate and preserved for subsequent serotyping and pulsotyping. J Store isolate at –70C K L Serotype, R-type and pulsotype

27 Antibiotic Resistance Profile Ax-Am-Ce-Cef-K-Su-Te
Salmonella serotype Antibiotic Resistance Profile Thompson Kentucky Typhimurium Glostrup Enteritidis n % Pansusceptable 1 3 5 2.8% Ax-Am-Ce-Su-Te 136 137 75.7% Ax-Am-Ce-K-Su-Te 24 13.3% Am-Cm-Su-Te 7 3.9% Ax-Am-Ce-Te 0.6% Ax-Am-Ce-Cef-Su-Te G-Su-Te 4 2.2% Ax-Am-Su-Te Ax-Am-Ce-Cef-K-Su-Te 2 171 181 1.1% 94.5% 1.7%

28 Since we only picked one isolate per positive part, this graph for isolates per bird as a function of time also reflects the number of positive parts per bird. The number of isolates per bird or positive parts per bird ranged from 0 to 12 with an average of 3.1 and median of 2. These results indicate that there is considerable variation in the number of positive parts per bird.

29 Parts = 181/840 (21.5%) Carcasses = 40/70 (57.1%) 37 different patterns

30 April 17, 2006 April 25, 2006 May 22, 2006 May 30, 2006 June 5, 2006
B A B A B Pos. G G G C D C D C D Neg. E F E F E F H H H I J I J I J K L K L K L May 30, 2006 June 5, 2006 June 12, 2006 A B A B A B G G G C D C D C D E F E F E F H H H I J I J I J K L K L K L

31 April 17, 2006 April 25, 2006 May 22, 2006 Pos. Neg. G: Back, rib
Thompson Pansusceptable L: Drumstick, right Kentucky AxAmCeSuTe C: Breast, left front Typhimurium AxAmCeSuTe Neg. A B A B A B G G G C D C D C D E F E F E F H H H I J I J I J K L K L K L

32 A B G H D F J L C E I K July 31, 2006 June 19, 2006 Sept. 18, 2006 July 17, 2006 Sept. 25, 2006 Oct. 16, 2006 Pos. Neg.

33 n =33 with > 1 contaminated part
October 16, 2006 A: Wing, left Typhimurium Pansusceptable G: Back, rib Typhimurium AxAmCeKSuTe Pos. C: Breast, left front Kentucky AxAmCeTe A B Neg. G C D E F H I J K L n =33 with > 1 contaminated part Serotype 12.1% (4/33) Ab resistance pattern 33.3% (11/33) PFGE pattern 100% (33/33)

34 Nov. 6, 2006 Nov. 13, 2006 Nov. 27, 2006 A B A B A B Pos. G G G C D C D C D Neg. E F E F E F H H H I J I J I J K L K L K L Dec. 4, 2006 Dec. 4, 2006 Jan. 8, 2006 A B A B A B G G G C D C D C D E F E F E F H H H I J I J I J K L K L K L

35 PFGE patterns = 100% similarity
Dec. 4, 2006 Pos. A: Wing, left Typhimurium AxAmCeSuTe G: Back, rib Typhimurium AxAmCeSuTe B: Wing, right Typhimurium AxAmCeSuTe Neg. A B G C D E F H I J K L H: Back, sacral Typhimurium AxAmCeSuTe L: Drumstick, right Typhimurium AxAmCeSuTe PFGE patterns = 100% similarity

36 Jan. 22, 2007 Feb. 5, 2007 Feb. 20, 2007 A B A B A B Pos. G G G C D C D C D E F E F E F Neg. H H H I J I J I J K L K L K L Feb. 26, 2007 March 5, 2007 March 12, 2007 A B A B A B G G G C D C D C D E F E F E F H H H I J I J I J K L K L K L

37 PFGE patterns = 100% similarity
March 5, 2007 Pos. A: Wing, left Typhimurium AxAmCeSuTe G: Back, rib Typhimurium AxAmCeSuTe B: Wing, right Typhimurium AxAmCeSuTe Neg. D: Breast, right front Typhimurium AxAmCeSuTe C: Breast, left front Typhimurium AxAmCeSuTe A B G C D E: Breast, left back Typhimurium AxAmCeSuTe E F F: Breast, right back Typhimurium AxAmCeSuTe H I J K L K: Drumstick, left Typhimurium AxAmCeSuTe H: Back, sacral Typhimurium AxAmCeSuTe L: Drumstick, right Typhimurium AxAmCeSuTe PFGE patterns = 100% similarity

38 March 26, 2007 April 2, 2007 April 9, 2007 April 16, 2007
B A B A B Pos. G G G C D C D C D Neg. E F E F E F H H H I J I J I J K L K L K L April 16, 2007 April 23, 2007 April 30, 2007 A B A B A B G G G C D C D C D E F E F E F H H H I J I J I J K L K L K L

39 April 9, 2007 Pos. Neg. G: Back, rib Glostrup GSuTe B: Wing, right
D: Breast, right front Glostrup GSuTe C: Breast, left front Glostrup GSuTe A B G C D E F H I J K L L: Drumstick, right Typhimurium AxAmCeSuTe

40 A B G H D F J L C E I K Jan. 28, 2008 Feb. 14, 2008 Feb. 28, 2008 March 5, 2008 April 2, 2008 April 9, 2008 Pos. Neg.

41 April 2, 2008 April 9, 2008 Pos. Neg. A: Wing, left Typhimurium
AxAmCeCefKSuTe A: Wing, left Typhimurium AxAmCeSuTe Neg. A B A B G G C D C D E F E F H H I J I J K L K L H: Back, sacral Typhimurium AxAmCeSuTe H: Back, sacral Typhimurium AxAmCeSuTe

42 April 20, 2008 June 3, 2008 July 14, 2008 Sept. 20, 2008 Pos. Neg. A B
H D F J L C E I K April 20, 2008 June 3, 2008 Pos. Neg. A B G H D F J L C E I K July 14, 2008 Sept. 20, 2008

43 April 20, 2008 Pos. Neg. D: Breast, right front Typhimurium AxAmCeSuTe
J I: Thigh, left Enteritidis Pansusceptable K L H: Back, sacral Typhimurium AxAmCeSuTe

44 Population Map (n = 70) Poultry Inspection WPE of all edible parts
A: Wing, left 41 g 27.1% G: Back, rib 67 g 38.6% B: Wing, right 41 g 24.3% D: Breast, right front 60 g 25.7% C: Breast, left front 62 g 28.6% A B G C D E: Breast, left back 35 g 7.1% E F F: Breast, right back 37 g 8.6% H I J I: Thigh, left 48 g 17.1% J: Thigh, right 50 g 12.9% K L K: Drumstick, left 45 g 10.0% H: Back, sacral 77 g 34.3% L: Drumstick, right 48 g 24.3%

45 Carcass hung upside down and washed from back to front
Wings versus Thighs Fisher's exact test P value 0.0371 P value summary * One- or two-sided Two-sided Significant? (P<0.05) Yes Data analyzed Positive Neg. Total A + B 36 104 140 I + J 21 119 57 223 280 Explanation Carcass hung upside down and washed from back to front

46 Left versus Right Drumstick
Fisher's exact test P value 0.0420 P value summary * One- or two-sided Two-sided Significant? (P<0.05) Yes Data analyzed Positive Neg. Total K 7 63 70 L 17 53 24 116 140 Explanation Viscera hung on right drumstick

47 Contamination from crop
Fine Map: Sub-parts Part code Part Weight, g Positive Total Incidence Gskin Back, rib 5.2 6 0.0% Ginside 62.9 5 83.3% The second dimension of carcass mapping is to increase the fineness of the map by separating the carcass part or region into sub-parts. This slide shows the results obtained when the rib back and sacral back were separated into outside and inside parts. The results for rib back, which was a carcass hot spot, indicated that the Salmonella contamination was more often associated with the inside of the carcass part rather than the skin. Thus, this is an example, where increasing the fineness of the map has the benefit of more precisely identifying the location of the Salmonella contamination. Explanation Contamination from crop

48 Future Mapping Research
Broilers and Turkeys Other Plants Process Steps Sub-part maps Other Pathogens Other Foods Quantitative In the future, we would like to work closely with FSIS to map the distributions of target organisms on the carcasses of broilers and turkeys. In the immediate future we would like to initiate a study with broilers to examine the impact of sampling methods and plant-to-plant variation on the carcass map for pathogen and indicator organism contamination as well as serotype and pulsotype patterns. This study would also involve the development of the relevant standard curves for quantitative mapping of target organisms on whole parts from broilers.

49 Take Home Message: Correct Approach = Accurate Map = Good Food Safety Decisions = Improved Food Safety Location Number Types

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