The issue of Norovirus in leafy greens and raspberries Ann De Keuckelaere, Ambroos Stals, Liesbeth Jacxsens, Andreja Rajkovic, Mieke Uyttendaele Ghent University, Belgium 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University Background / introduction Food borne viruses = viruses infectious to humans that can be transmitted through food Noroviruses (NoV) & Hepatitis A virus (HAV) – Need specific host cells to replicate è cannot multiply in the environment or on foods. – Viral particles are excreted in stool or vomit è high concentrations (1011 virus particles/g). – Very low infectious dose (<100 virus particles). – Do not cause spoilage or visible contamination of food products. – High environmental resistance (heat, high or low pH, drying, light and UV exposure) 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 2 Background / introduction Norovirus (genus) Ø 5 genogroups: mainly GI & GII infectious to humans Ø Infectious dose: ~18 NoV particles Ø Incubation period: 12-‐48 h Ø Symptoms: nausea, vomiting, diarrhea, Fever, “stomach flu” mild disease AAA VPg AA A 27-32 nm ssRNA Ø Transmission: Faecal-‐oral spread Fresh produce infected food-‐handlers (e.g. pickers…) contaminated water and surfaces 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 3 Background / introduction Leafy Greens Soft Red Fruits LETTUCE Risk factors RASPBERRIE Contaminated pesticide water S P-‐H• Contaminated irrigation P-‐H• water (large surface area) H • Contaminated food handlers H • Contaminated fruit pickers P • Risk of cross-‐contamination P • (cross-‐contamination due to due to washing washing) + for both lettuce and raspberries multiple outbreaks and reported RASFF available 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 4 Background / introduction NoV Outbreaks In the EU (2010) N = 84 Fruit and berries and products thereof Vegetables and products thereof September/October 2012 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 5 Background / introduction The challenges of working on NoVs Molecular detection methods Infectious OR non-‐infectious? Cultivation is NOT possible Use of surrogates for survival & inactivation No enrichment of samples is possible Ø Murine norovirus (MNV) Ø MS2 Ø and others Importance of elution/extraction protocol to achieve a low detection limit Precious fecal samples 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 6 Objectives / Research question Microbiological risk assessment Task. Elaboration of virus detection methods • Selection viral extraction method for fresh produce • Optimization of viral concentration method for water Task Development of Sampling and testing scheme Task Development of training program in virus detection in fresh produce and water Task Collection of Norovirus data in fresh produce • Lettuce and irrigation water sampling • Raspberry batch testing Task Risk assessment for Norovirus in raspberries 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 7 RISK ASSESSMENT DATA GATHERING METHODS Experimental set-‐up / Approach Detection method soft red-‐fruits & vegetables Stals et al. (2011) Evaluation and selection of detection method in (irrigation) water 1 Selection & optimalization of swabbing method 2 Batch 5 Batch testing testing IQF mixed/minced raspberries raspberries (puree) HAS lettuce open field Construction flowchart farm to fork for ≠ raspberries products Data gathering to model cross-‐3 contamination @ washing stage Quantitative exposure model for ü IQF raspberries 6 ü raspberry puree Review on RA that use water in production process of fresh 4 produce Lettuce/water/hands Quantitative exposure model for fresh cut lettuce 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 8 METHODS Results Detection method soft red-‐fruits & vegetables Stals et al. (2011) Evaluation and selection of detection method in (irrigation) water Selection & optimalization of swabbing method 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 9 Results: Evaluation and selection of detection method in (irrigation) water • Evaluation use of surrogate viruses MNV-‐1 and MS2 phages • Validation selected method for detection Norovirus, but also Hep. A virus & rotavirus 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 10 Results RISK ASSESSMENT DATA GATHERING LETTUCE HAS lettuce open field Lettuce/water/hands Data gathering to model cross-‐ contamination @ washing stage Review on RA that use water in production process of fresh produce Quantitative exposure model for fresh cut lettuce 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 11 Results: HAS for lettuce in open field Ø All water and lettuce samples negative for MS2 Presence of NoV Ø Water (2 – 5 L) Hands Le1uce (2 x 10 g) (Ct-‐value) 1 -‐ -‐ -‐ 2 -‐ -‐ -‐ 3 -‐ -‐ -‐ 1 -‐ -‐ 1 (38.12) 2 -‐ -‐ 1 (37.81) 3 -‐ -‐ -‐ 1 -‐ -‐ 1 (35.94) 2 -‐ -‐ 1 (38.70) 3 -‐ -‐ -‐ 0/27 0/21 4/27 Visit Farmer 1 Farmer 2 Farmer 3 FRNA phages GI & GII NoV All 4 lettuce samples positive for GI NoV 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 12 Results: Wash experiment Goal: To examine the potential of washing water as a vehicle of transmission of norovirus (Quantitatively) Useful data/insights for future RA in the fresh produce supply chain WB1 inoculated with: Experimental set-‐up: • MS2 (3 ≠ conc.) • MNV-‐1 (1 conc.) WB1 WB2 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 13 Results: Wash experiment WB1: approx. 4.5 log MS2/ml Transmission between WBs • Contamination level of WB1 ≈ Cte • Cont. level of WB2 ↗ Gll ca. 0.7 log ∆ WB1 MS2 concentra1on (PFU/ 10 g) * Transmission to lettuce • Overall transfer ratio of MS2 from WB1 to lettuce = 0.5% ± 0.2% • Significant influence WB2 but 1.00E+05 1.00E+04 1.00E+03 1.00E+02 ** 1.00E+01 1.00E+00 0 * : below detection limit 1 2 x 200 g washed 3 4 A whole batch of lettuce can get contaminated due to initial contamination of WB1 with pathogens 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 14 Results: Review RA that use water in production process of fresh produce Enteric viruses 41 RA studies were selected (1992-‐2013) Characterization of the selected literature studies W-‐perspective (‘water’ or ‘environmental’) Bacteria Protozoa Helminths F-‐ perspective (‘food’ or ‘agriculture’) F&W-‐perspective In-‐depth analysis of selected literature studies Ø Ø Ø Ø Ø Prevalence and concentration of m.o. in environment Transmission routes Growth/removal/survival/inactivation of m.o. Consumer behavior Dose-‐response relation & risk characterization ü Used strategies ü Assumptions ü Proxy data ü Data gaps Used acceptable risk level – Risk mitigation strategies under study 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 15 Future perspectives LETTUCE Review on RA that use water in production process of fresh produce Quantitative exposure model for fresh cut lettuce First draft stage of article Expected submission: Summer 2014 Start: April 2014 (post Veg-‐i-‐Trade via personal Ph.D sholarship from Flemish Government) 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 16 RISK ASSESSMENT DATA GATHERING Results RASPBERRIE S Batch testing IQF raspberries Batch testing mixed/minced raspberries (puree) Construction flowchart farm to fork for ≠ raspberries products Quantitative exposure model for ü IQF raspberries ü raspberry puree 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 17 Results: Batch testing of processed raspberries Ø 1 batch => 5 samples Ø EU origin (2011-‐2012) ü Enumeration of E. coli and coliforms ü Detection GI and GII NoV (in 2 x 10 g) with RT-‐qPCR 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 18 Results: Batch testing of processed raspberries Molecular detection Extraction and concentration step Detection protocol NoV in raspberries Process Control => Efficiency extraction process Control for inhibition during RT-‐qPCR => 1/10 dilutions Control qPCR ⇒ Sequencing amplicon 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 19 Results: Batch testing of processed raspberries Criteria to determine if 20 g raspberry sample is NoV positive: 1. Ct < Ct LOD (Ct > LOD è background) Ø LOD = 500 GC/20 g • • If Ct < LOQ è quantification Ø LOQ = 5000 GC/20 g If LOQ < Ct < LOD è presence/absence 2. Confirmation of the authenticity of amplicon by sequencing (exclude false-‐positive results via plasmid contamination) 3. At least 1/2 10g subsamples shows positive qPCR signal (GI, GII or both) 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 20 Results: Batch testing of processed raspberries Results Raspberry puree (14 batches) Ø Company A 2/7 batches positive for NoV • Batch 1: 4/5 samples positive & quantifiable • Batch 2: 2/5 samples positive & quantifiable Ø Company B 0/7 batches positive for NoV Results IQF Raspberries (12 batches) ü 2/14 positive (GI & GII) ü High levels of NoV ü Multiple samples per batch positive Batches of concern Ø Company C 1/7 batches positive for GI NoV • Batch 5: 1/5 samples positive but not-‐quantifiable • 2 samples of 2 other batches positive RT-‐qPCR signals (GII NoV) but inconclusive results by sequencing Ø Company D 0/5 batches positive for NoV ü 1/12 positive ü Low levels ü Max 1 sample/ batch 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et Batch NOT of concern al. , Ghent University 21 Results: Batch testing of processed raspberries Batch testing relevant YES or NO ? Ø The limitation of testing: If n = 5 units are sampled of one batch, and all negative (c= 0) ⇒ is corresponding to a 95% probability of rejection of a batch that contains 45% defective units (= 20 g portions being contaminated with NoV). Thus still 5% probability to accept the highly contaminated batch. And e.g. if 30% defective units, even higher chance of accepting the batch Sampling cannot guarantee absence of NoV positive units in a batch !!! Ø The limitations of present detection methods 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 22 Results: Batch testing of processed raspberries Batch testing relevant YES or NO ? Ø The limitation of testing Ø The limitations of present detection methods: ⇒ Have relatively low recovery efficiency (generally < < 20%) ⇒ Have high limit of detection (2-‐steps RT-‐qPCR: LOD = 500 GC/20 g) ⇒ Present methods are very expensive and laborious + big variability between different labs ⇒ What is relevance of finding genomic copies? Detection and ü Interpretation of results is crusial interpretation is work of specialists ü Inclusion of necessary controls is a prerequisite 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 23 Results: Quantitative NoV exposure model By dr. Ambroos Stals & prof. Liesbeth Jacxsens Objective: development of an exposure model to describe the possible contamination of raspberries by human noroviruses Effect of intervention measures? Output model: # NoV particles/kg raspberry puree # NoV particles/250g IQF raspberry package 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 24 Results: Quantitative NoV exposure model Raspberry farm #2 Raspberry farm #3 … Raspberry farm #10 NoV contamination Collection centers (POL/BEL) IQF raspberries Raspberry puree Mixing + homogenization Heat treatment: • None • 75°C – 30s • 90°C – 30s NoV homogenization NoV inactivation Module 3 NoV 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent Uconsumption niversity 25 Consumption by customer (e.g. via raspberry cake) Module 2 Division into small packages (250 g) Module 1 Raspberry farm #1 • sdf Results: Quantitative NoV exposure model ContaminaGon • sdf sources NoV contamination site IntervenGon measures • Water used for pesGcide spraying Module 1 (pre-harvest stage) • NoV inactivating pesticide (Vertimec) • NoV shedding food pickers Module 1 (harvest stage) • Handwashing after “restroom” visit Module 2 Processing • Heat treatment (only for raspberry puree) • 10 farms: 5 small – 3 medium – 2 large • 245 food pickers in total • 10-hour working day 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 26 Results: Quantitative NoV exposure model Scenarios Basic scenario • 5/245 NoV shedding food pickers on 10 farms • Constant NoV prevalence in water used for pesticide spraying • No intervention measures Effect of # NoV shedding food pickers • Scenario: 0/245 NoV shedding food pickers • Scenario: 20/245 NoV shedding food pickers Effect intervention measures • Scenario: hand washing aZer restroom visit • Scenario: use of NoV inacGvaGng VerGmec pesGcide Effect pesticide abuse • Scenario: 4 fold overusage of pesGcides Deliverable D6.10: Microbiological risk assessment output for viruses and protozoa on fresh produce. By dr. Ambroos Stals & prof. Liesbeth Jacxsens 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 27 Results: Quantitative NoV exposure model Results for Raspberry puree Treatment No heat treatment Mild heat treatment (75°C – 30s) Harsh heat treatment (90°C – 30s) Effect of heat treatment on basic scenario Mean NoV level (NoV par1cles/ kg) 14,1 ± 145,0 0,2 ± 2,1 0,0007 ± 0,007 = 5% ,2 olog 10 f 1 kNg oV / samples: 11 ton >200 NoV raspberries parGcles/kg 90 % of 1 kg samples: <3 NoV parGcles/kg Mild heat treatment is sufficient in most cases NoV particles / kg raspberry puree 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 28 Results: Quantitative NoV exposure model Results for IQF raspberries How many NoV in a 250g (=115 rasp) raspberry package? No packages with > 1 NoV particle Basic + Hand + Hand Scenario 3b scenario washing disinfecGon (20 shedding FP) Mean # NoV in 250g raspberry package via NoV shedding FP Mean # NoV in 250g raspberry package via healthy FP (thus via pesGcide water) Total # NoV par1cles in 250g package of IQF raspberries 1 % of packages: > 18 NoV particles 3.0 0.0 0.0 7.3 0.1 0.1 0.1 0.1 3.1 0.1 0.2 7.4 Hand washing proved to be a very effective intervention measure at harvest stage 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 29 Lessons learnt ü Genuine NoV possitive lettuce and raspberry samples were found Those who search will find BUT inclusion of controls and correct interpretation is essential ü Batch testing for NoV in raspberries on a regular basis is NOT a guarantee for food safety – testing is always too little too late Need of preventive approach ! ü Hand washing = effective intervention measure @ harvest for ü Cross-‐contamination during processing can have MAJOR influence on NoV contamination level of fresh produce è contamination of whole batch è maintain good water quality Disinfectants 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 30 Future perspectives • Further research on positive raspberry samples on infectivity => long range PCR & cell-‐binding PCR (dr. Dan Li) • Finishing review on RA that use water in the production process of fresh produce (Ann & Liesbeth) • Start of quantitative exposure assessment for viruses on lettuce using model of Irene on E. coli (Ann & Liesbeth) 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 31 References / Publications • • • • • De Keuckelaere, A., et al. (2013). Evaluation of viral concentration methods from irrigation and processing water. Journal of Virological Methods 187(2), 294-‐303. De Keuckelaere, et al. (2013b). Performance of two real-‐time RT-‐PCR assays for the quantification of GI and GII noroviruses and Hepatitis A virus in environmental water samples. Food Analytical Methods 6(4), 1016-‐1023. Holvoet, K., et al. (2014). Quantitative study of cross-‐contamination with Escherichia coli, E. coli O157, MS2 phage and murine norovirus in a simulated fresh-‐ cut lettuce wash process. Food Control 37, 218-‐227. De Keuckelaere, A. et al. (submitted 2014). Semi-‐direct lysis of swabs and their efficiency to recover human noroviruses GI and GII from surfaces. Food and Environmental Virology. De Keuckelaere, A. et al. (submission after S-‐A). Batch testing for noroviruses in frozen rasberries. International Journal of Food Microbiology. • Ph.D’s expected: Ann De Keuckelaere -‐> submission October 2014 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University 32 This is a work of many The VIRUS team: With a lot of help of: dr. Ambroos Stals Coordinator & Promotor: dr. Dan Li ir. Ann De Keuckelaere Ø lab technicians: Elien & Marcus Ø thesis students Ø cooperation with several companies Ø many others … Ø Link with raspberry production : Coordinator risk University of assessment Belgrade dr. Andreja Rajkovic & colleaques 6th Veg-‐i-‐Trade CM mee1ng, March 2014, Pretoria, SA De Keuckelaere et al. , Ghent University
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