Developing a Biosensor Method for On-farm Detection of Campylobacter in Broiler Chickens

Transcription

1 Developing a Biosensor Method for On-farm Detection of Campylobacter in Broiler Chickens Dr Lynn McIntyre Dept. Food Science and Agri-Food Supply Chain Management Sensors in Food and Agriculture 1-2 December 2015; Cambridge

2 Harper Adams University A dedicated agri-food university 134 academic staff 3,000 students BSc, MSc, PhD Research activities in food safety, crop production, animal health and welfare, entomology, aquaculture, precision agriculture

3 On 650 ha (1,605 acre) farm in Shropshire county, UK

4 Campylobacter jejuni First described in 1886 Commensal organism in birds, cattle, pigs and sheep Commonly associated with raw poultry meat, raw milk, raw meat & untreated water Unusual physiology Unable to grow below 30 C (optimum ~42 C) Microaerophilic (5% O 2 ) Sensitive to various stresses

5 Why is it important? It is the most significant cause of foodborne illness in the developed world! Estimated 9 million cases every year in the EU Acknowledged high prevalence of pathogen in raw chicken (65% %) Low infective dose (<500 cells) Infection can lead to long-term sequelae Paralysis, reactive arthritis, death Significant overall burden and cost ( )

6 Campylobacteriosis in New Zealand (Baker et al., 2006) 18,000 16,000 14,000 Notifications Hospitalisations 1,200 1,000 Notifications 12,000 10,000 8,000 6,000 4,000 2, Hospitalisations Year 0

7 New Zealand chicken production (Baker et al., 2006)

8 The impact of regulation on human health

9 Number of lab-confirmed cases of campylobacteriosis in the UK FSA Annual Science Report, 2013/14

10 Burden of disease (FSA, 2014)

11 The Campylobacter Joint Government- Industry Strategy (2010) Research priorities Understanding current practice and potential intervention strategies (from Farm to Fork) The biology of the host and pathogen Development of novel (on-farm) detection and diagnostic tools Flock monitoring Intervention strategy Reduced contamination at retail

12 How does chicken become contaminated? Colonisation of birds onfarm (biosecurity /depopulation) Cross-contamination of birds during transportation Cross-contamination of carcasses/offal during processing

13 Baseline data and targets FSA Campylobacter Risk Management Programme u < 10 2 CFU/g CFU/g > 10 3 CFU/g Baseline % 31% 27% Target 2013 improvement improvement 19% Target 2015 improvement improvement 10%

14 So why did the poultry industry want a novel detection method? Existing Standard Methods Time consuming (several hours up to 5 days) Laborious, requires skilled trained personnel Needs to be performed in laboratory environment Desirable Method for Flocks Monitoring Rapid result Simple (easy to perform and interpret) Low cost On site testing

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16 But keep in mind.

17 and

18 Alternative Campylobacter detection methods PCR Optigene Lateral flow Electronic nose Speedy Breedy

19 How would a rapid test improve the situation? Immediate changes to farm practices Rescheduling of flock slaughter Longer-term monitoring of farm practices Significant changes to production No thin Shorter period between thin and depletion

20 Vantix biosensor: potentiometric screen printed sensor

21 Voltage (mv) Biosensor-based immunoassay for Campylobacter detection Detection antibody Analyte (cell) HRP HRP substrate Potentiometric measurement Potential difference Product Sensor reader device Time (s) Measurement output Capture antibody Sensor surface Reference electrode Working electrode Disposable screenprinted sensor strip 30 min total assay time Signal reads as Relative Voltage = Sample mv baseline mv Q: Sensitivity? Specificity? Performance in real life samples?

22 Where we thought we would go with this Lab-based On farm

23 Methodology Preliminary Study Antibody selection; collation of targets; determination of assay format & initial assay performance; culture-based and PCR-based method adaptation as control methods Performance Optimisation Study Assay tests against different target cell concentrations; optimisation of assay components & test conditions; determination of detection limit Specificity Study Assay tests against various bacterial target strains & species and against various non target bacterial isolates relevant to poultry Evaluation using Farm Samples (Biosensor vs Culture and PCR Testing) Tests on different sample types collected from flocks of different age at different commercial farms; selection of suitable sampling method and assay validation

24 Relative Voltage (mv) Method Sensitivity Initial assay conditions Optimised assay conditions Vantix blocking agent replacing BSA Detection limit Detection limit Detection limit E E E E E E+08 Campylobacter cell concentration (CFU/ml)

25 Relative Voltage (mv) Method Specificity E+05 CFU/ml 1.00E+06 CFU/ml 1.00E+08 CFU/ml Campylobacter spp. (32 isolates) S. aureus S. Typhimurium E. coli

26 Relative Voltages (mv) Which Farm Samples to test? A. Boot cover B. Litter C. Faecal droppings D. Cloacal swab unspiked negative samples A B C D High background noise on boot cover, litter and faecal droppings

27 Optimising Cloacal Swab Detection Cloacal swab (with BSA) unspiked spiked at 1.00E+05 CFU/ml Cloacal swab (with Vantix block) unspiked spiked at 1.00E+05 CFU/ml A B C D 0-20 A B C D N = 12; A negative (4) B positive at 1.00E+04 CFU/ml (1) C positive at 1.00E+05 CFU/ml (6) D positive at 1.00E+06 CFU/ml (1)

28 What we've realised Lab-based On farm

29 10 BBSRC/Innovate UK Campylobacter E-nose project Air Sampling 2 Different Chicked Sheds and 2 Controls Divergence Observations (axes F1 and F2: %) 5 F2 (0.00 %) 0-5 Pimlico air Wing air kitchen control wing control F1 ( %)

30 Acknowledgements Fittonia Elgina Faccenda Foods Ltd Harper Adams University Vantix Diagnostics