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Global risk-ranking of foodborne parasites (2012) 80,00

Parasites transmitted by fresh produce: challenges in removal and inactivation

70,00 60,00 50,00 40,00 30,00 20,00 10,00 0,00

Lucy Robertson, NMBU, Oslo, Norway http://www.fao.org/3/a-i3649e.pdf Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Global risk-ranking of foodborne parasites (2012)

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Effects of treatments / processes • Removal

80,00

– Washing procedures – Washing may result in introduction or spread of localised contamination

70,00 60,00 50,00 40,00 30,00

• Inactivation

20,00

– Physical treatments – Chemical treatments

10,00 0,00

http://www.fao.org/3/a-i3649e.pdf Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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1

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Parasites in focus – removal by washing

How to determine removal? • Spiking experiments

Parasite

– Add parasite, conduct procedure, determine removal efficiency

Taenia solium (cestode)

• Experimental challenges – Uncertainty in the amount (concentration) of pathogen added – Assess removal by looking in the wash solution (amount removed) or remaining on the product (amount that is not removed) - or both? – How effective and efficient are methods for determining the amount of pathogen in the wash solution or on the product; what is the uncertainty and how can this be incorporated into the results? – Replicate experiments? – Contact time? Temperature? Spike size? Other factors? – Use of surrogates? Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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• Viability assay necessary or indicator of viability or infectivity

Removal from fresh produce by washing procedures Not investigated – eggs are considered to be sticky

Echinococcus spp. (cestode)

2+3

Toxoplasma gondii (protozoan)

4

Not investigated – but adherence considered to also be affected by produce (raspberries vs blueberrries)

Cryptosporidium spp. (protozoan)

5

Found to adhere strongly – apple peel, spinach leaves and zucchini investigated. Under 40 % (highest removal). Entry into stomata as well as adherence.

Not investigated – eggs are considered to be sticky (adhere to dog hair)

Ascaris lumbricoides (nematode)

9

Not investigated – but eggs are considered highly sticky due to adherent external layer with some hydrophobic properties

Fasciola spp. (trematode)

12

A few studies – 10 min running water may remove 50 %. Active attachment (secretion of adhesins or filaments)

Cyclospora cayetanensis (protozoan)

13

No documented studies, but considered very sticky (more so than Crypto), due to specific adhesins (have not been investigated)

Tenth Workshop of NRL for parasites, Rome; May 2015

Parasite Taenia solium (cestode)

– – – – – –

assessment of morphology, assessment of progress in embryonation/sporulation, inclusion or exclusion of specific “vital” dyes, fluorescent in situ hybridisation (FISH), infection of cell cultures, use of genes as viability markers, sometimes in combination with cell culture (CC-PCR), – infection of animal models (bioassay)

• Not all assays suitable for all parasites • Does viability correlate with infectivity? Norwegian University of Life Sciences

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Norwegian University of Life Sciences

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Parasites in focus – how to assess survival or inactivation -1

How to determine survival or inactivation?

Tenth Workshop of NRL for parasites, Rome; May 2015

Rank

Rank

Methods that have been used for assessing viability

1

• •

In vitro hatching and dye staining of oncospheres Bioassay

Echinococcus spp. (cestode)

2+3

• •

In vitro hatching and dye staining of oncospheres Bioassay

Toxoplasma gondii (protozoan)

4

• • • •

Observation of sporulation Cell culture Molecular assays (mRNA-based PCR viability assay) Bioassay

Cryptosporidium spp. (protozoan)

5

• • • •

Vital dye inclusion / exclusion Cell culture In vitro excystation Molecular assays (quantitative reverse transcription-PCR (qRT-PCR) to detect and quantify changes in gene expression) Marker molecule assays (e.g. amylopectin) Electrorotation Bioassay

• • • 7

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Parasites in focus – how to assess survival or inactivation - 2 Parasite

Rank

Physical treatments used in fresh produce industry • • • •

Chilling and freezing Heat treatment (blanching, pasteurisation) Drying Irradiation – Ionising (e.g., gamma irradiation) – Non-ionising (e.g., germicidal uv) • High pressure • Ultrasound (data on parasites in fresh produce lacking) • Novel methods (data on parasites in fresh produce lacking) – e.g. high-intensity electric field pulses and radio frequency

Methods that have been used for assessing viability

Ascaris lumbricoides (nematode)

9

• • • • •

Observation of embryonation Vital dye inclusion / exclusion Molecular assays (rRNA quantification) Electrorotation Bioassay

Fasciola spp. (trematode)

12

• • •

Morphology In vitro excystation Bioassay

Cyclospora cayetanensis (protozoan)

13

• • •

Observation of sporulation Vital dye exclusion Electrorotation

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Inactivation under chilling or freezing 1 Parasite Taenia solium (cestode)

Rank

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Inactivation under chilling or freezing 2

Reported survival

Parasite

Rank

Reported survival

1

Chilling: Formal expts. lacking but another species survived ca. 1 year at 4 C; known to survive well in cool conditions Freezing: Formal expts lacking, but expected to be similar to Echinococcus

Ascaris lumbricoides (nematode)

9

Chilling: Embyronation may be retarded under chilling, but able to survive and embryonate later. Freezing: Viability and infectivity demonstrated after weeks at 25C

Echinococcus spp. (cestode)

2+3

Chilling: Formal expts. lacking but expected to survive well in cool conditions (months or years) Freezing: Extremely freeze-tolerant; survive months at -18C and over a day at less than -50C

Fasciola spp. (trematode)

12

Chilling: Cooler temperatures favour survival. Can survive refrigeration temperatures for months. Freezing: Somewhat susceptible; may survive freezing to around -10C, but lose infectivity below -18C.

Toxoplasma gondii (protozoan)

4

Chilling: Sporulation may be retarded under chilling, but able to survive months and sporulate later, and sporulated oocysts survive chilling for months. Freezing: Somewhat susceptible, but some oocysts may survive temps to below -20C for periods of weeks

Cyclospora cayetanensis (protozoan)

13

Chilling: Sporulation retarded (takes around 10 days at around +25C), but some able to survive and sporulate later. Freezing: Somewhat susceptible, but some oocysts may survive temps to below -20C for brief periods (outbreak associated with frozen cake filling)

Cryptosporidium spp. (protozoan)

5

Chilling: Survive best in cool, damp conditions. Can survive refrigeration temperatures for months. Freezing: Somewhat susceptible, but some oocysts may survive temps to below -20C and blast freezing

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Inactivation under irradiation

Inactivation under heating or drying Parasite

Rank

Taenia solium (cestode)

1

Parasite

Reported survival Heating: Formal expts. lacking, but considered to be effective Drying: Data lacking, but likely to be effective

Echinococcus spp. (cestode)

2+3

Toxoplasma gondii (protozoan)

4

Heating: Loss of infectivity at 60C within 1 min Drying: Data lacking, but likely to be effective

Cryptosporidium spp. (protozoan)

5

Heating: Effective inactivation Drying: Effective inactivation

Ascaris lumbricoides (nematode)

9

Heating: Effective inactivation Drying: Effective inactivation

Fasciola spp. (trematode)

12

Heating: Data lacking, but effective inactivation expected Drying: Data lacking, but likely to be effective – anecdotal reports of surviving some drying on plants

Cyclospora cayetanensis (protozoan)

13

Heating: Loss of viability at 40-50C within hours Drying: Loss of infectivity within days

Heating: Effective inactivation Drying: Data lacking, but likely to be effective

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Rank

Taenia solium (cestode)

Reported survival

1

Hymenolepis diminuta eggs used as surrogate: egg hatching abrogated by exposure to 300-600 MPa for 1 min. Treatment Lou F, Neetoo H, Chen H, Li J Echinococcus spp. 2 + 3 at 400 MPa and above caused rupturing of the oncosphere. Inactivation of a human norovirus (cestode)

surrogate by high-pressure

Toxoplasma gondii Infectivity abrogated at pressures of 340 MPa for 1 min. processing:4 effectiveness, (protozoan)

mechanism, and potential

Cryptosporidium Infectivity in fruit juice completely abrogated after 60 sec. 550 application5in the fresh produce spp. (protozoan) MPa

industry. Appl Environ Microbiol.

Ascaris lumbricoides 9 Embryonation in eggs exposed to 241 -276 MPa for 60 sec or 2011 Mar;77(5):1862-71. (nematode) 1 min; delayed embryonation after lower exposures. Fasciola spp. (trematode)

12

No data

Cyclospora cayetanensis (protozoan)

13

Eimeria acervulina used as surrogate; infectivity aabrogated after HPP at 550 MPa.

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

Rank 1

Reported survival (ionising and non-ionising* irradiation) Ionising irradiation: Data lacking – see Echinococcus Non-ionising irradiation: Susceptible at 2,430-2,880 mJ/cm2

Echinococcus spp. (cestode)

2+3

Toxoplasma gondii (protozoan)

4

Ionising irradiation: Susceptible at 0.4 to 0.8 kGy Non-ionising irradiation: susceptible at 10 to 20 mJ/cm2 – but some studies show survivalat doses above 500 mJ/cm 2.

Cryptosporidium spp. (protozoan)

5

Ionising irradiation: 50 kGy to eliminate infectivity in mice. Non-ionising irradiation: susceptible at 15-25 mJ/cm2 pulsed UV

Ascaris lumbricoides (nematode)

9

Ionising irradiation: Susceptible at 2 -10 kGy Non-ionising irradiation: susceptible at 400mJ/cm2

Fasciola spp. (trematode)

12

Ionising irradiation: Susceptible at 0.7 kGy Non-ionising irradiation: Data lacking

Cyclospora cayetanensis (protozoan)

13

Ionising irradiation: Data lacking Non-ionising irradiation: Data lacking

Ionising irradiation: Susceptible at 0.4 kGy Non-ionising irradiation: Data lacking – see Taenia

Tenth Workshop of NRL for parasites, Rome; May 2015

*Note shadowing effect on uneven surfaces etc. Norwegian University of Life Sciences

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Chemical sanitizer treatments used in fresh produce industry

Inactivation under high pressure processing Parasite

Taenia solium (cestode)

15

• Main method of reducing microbial load - produce washing with disinfectants • Also to allow recirculation of wash water (high organic load) – Chlorine – commonest, cheapest – BUT carcinogenic potential (byproducts), banned in some countries – Gaseous chlorine dioxide – Sodium dichloroisocyanurate (NaDCC) – Hydrogen peroxide – Ozone – Organic acids – Potassium permanganate – Quaternary ammonium compounds (QACs) – not for organic produce Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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4

26.05.2015

Inactivation under gaseous chlorine dioxide or Na-dichloroisocyanurate

Inactivation under chlorination wash Parasite Taenia solium (cestode)

Rank 1

Reported survival Parasite

Only at very high doses (other Taenia egg species)

Taenia solium (cestode)

Echinococcus spp. (cestode)

2+3

Only effective at very high Ct

Toxoplasma gondii (protozoan)

4

Cryptosporidium spp. (protozoan)

5

Some effect with high Ct but negligible effect at Ct used in fresh produce industry.

Ascaris lumbricoides (nematode)

9

Ineffective

Fasciola spp. (trematode)

12

Studies lacking

Cyclospora cayetanensis (protozoan)

13

Contrasting results - may be effective at high Ct

Studies lacking – likely to be ineffective at Ct used in fresh produce industry

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Inactivation under hydrogen peroxide, ozone, organic acids Parasite Taenia solium (cestode)

Hydrogen peroxide

Ozone

Organic Acids

1

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Echinococcus spp. (cestode)

2+3

Toxoplasma gondii (protozoan)

4

Studies lacking

No effect detected

Studies lacking

Cryptosporidium spp. (protozoan)

5

Effective for juice treatment

Apple cider outbreak..?

Limited or negligible effects

Ascaris lumbricoides (nematode)

9

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Fasciola spp. (trematode)

12

Studies lacking

Studies lacking

No effect detected

Cyclospora cayetanensis (protozoan)

13

No effect detected

Studies lacking

Studies lacking

Norwegian University of Life Sciences

1

Chlorine dioxide

NaDCC

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Echinococcus spp. (cestode)

2+3

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Toxoplasma gondii (protozoan)

4

Studies lacking

Studies lacking

Cryptosporidium spp. (protozoan)

5

Some effect with high Ct.

Some effect reported

Ascaris lumbricoides (nematode)

9

Studies lacking – likely to be ineffective

Studies lacking – likely to be ineffective

Fasciola spp. (trematode)

12

Studies lacking

Studies lacking

Cyclospora cayetanensis (protozoan)

13

Limited effect with high Ct.

Some effect reported

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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Data gaps: removal

Rank

Tenth Workshop of NRL for parasites, Rome; May 2015

Rank

19

• Enumeration technique • Replicate experiments • Washing technique that is applicable to fresh produce industry • Impact of variables • Investigation of spread of parasites throughout lot during washing • Investigation of adherence and adherence properties • Acceptance that complete removal cannot be guaranteed – what is an acceptable limit?

Nestle, Lausanne, November 2014

Norwegian University of Life Sciences

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5

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Control in the food-chain

Data gaps: inactivation

• Prevention of contamination – – – –

Probably most important contol HACCP procedures, from farm to sale/consumption Be aware of likely sources of contamination (lifecycle information) Knowledge of extent of infection in animals / humans in contact with food-chain – Parasite transmission stages cannot proliferate as contaminants, but survival prolonged.

• Techniques for assessing viability and infectivity • Most research pointed at water industry (or lab decontamination) • Fresh produce industry very different • Replicate studies – how many replicates and which variables should be considered

• Disinfection one of most critical processing steps in freshcut vegetable production - disinfection or sanitization procedures will continue • Proper investigations necessary to give a basis for which of those available are most appropriate and effective

• Assume current chemical sanitizer treatments have minimal effect

Tenth Workshop of NRL for parasites, Rome; May 2015

Norwegian University of Life Sciences

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COST Action – EURO-FBP WG1: Regionspecific ranking & current surveillance systems

Norwegian University of Life Sciences

Tenth Workshop of NRL for parasites, Rome; May 2015

Food industry

Food analysis labs

Veterinary labs

• Link together dispersed groups

WG3: Interventions

Food supplychain operators

• Share competence, knowledge and expertise • Establish & promote collaboration and research

EUROFBP

22

Regulatory bodies Risk analysis groups Medical diagnostic labs

• Fill knowledge gaps WG2: Analytical and diagnostic methods for FBP

EURO-FBP, COST ACTION

• Focus on ESRs • Outcomes

WG4: Global trends, risk assessment and research agenda

Norwegian University of Life Sciences

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EURO-FBP, COST ACTION

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Who??

Horizontal and vertical integration

26 COST countries (11 inclusiveness countries) WG1

WG2

WG3

WG4 • Austria • Belgium • Bosnia + Herzegovina • Croatia • Czech Republic • Denmark • Estonia • France • Germany • Greece • Hungary • Iceland

INTESTINAL PROTOZOA TISSUE PROTOZOA NEMATODES CESTODES TREMATODES

EURO-FBP, COST ACTION

Norwegian University of Life Sciences

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• • • • • • • • • • • • • •

Ireland Italy Latvia Netherlands Norway Poland Portugal Romania Serbia Slovenia Spain Sweden Switzerland UK

EURO-FBP, COST ACTION

Norwegian University of Life Sciences

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Norwegian University of Life Sciences

28

WORK PLAN AND BUDGET – YEAR 1 Activity

Place

Proposed Dates

Nr. Partners Budget (€)

1st MC meeting

COST, Brussels

30th March

34

COST

1st exec. comm. meeting

Zagreb, Croatia

Sept / Oct?

45 ?

40,950

Workshop WG1 2nd WG meeting 3rd MC meeting

RIVM, Netherlands?

Early 2016

50?

57,500

STSM (WG 2 + 3)

tbd

tbd

8

10,000

Dissemination

website

Start Aug 2015

2nd MC meeting 1st WG meeting

4,500

TOTAL SCIENTIFIC COSTS

120,950

TOTAL INCLUDING FSAC (15 %)

139,092

EURO-FBP, COST ACTION PROPOSAL

Norwegian University of Life Sciences

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Tenth Workshop of NRL for parasites, Rome; May 2015

7