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ANSES Nancy, French National Reference

Laboratory

WHO collaborating centre for research on zoonosis

OIE reference laboratory for rabies

CRL for rabies

NRL for rabies and echinococcosis for agriculture ministry

MINISTERE DE L’AGRICULTURE

ET DE LA PÊCHE

Flotation/sieving method to detect Echinococcus multilocularis eggs by real-time PCR in soil

samples and application to the study of their transfer from faeces to soil

Umhang G, Bastien M, Renault C, Faisse M, Boucher JM, Caillot C,

Hormaz V, Poulle ML, Boué F.

University of Reims Champagne Ardenne, SFR Cap Santé, EA 3800

PROTAL, 51092 Reims cedex, France

National Reference Laboratory for Echinococcus spp.,

Wildlife Surveillance and Eco-Epidemiology Unit,

More than 1.5 billion people (24% of the world’s population) are infected with soil-transmitted helminths worldwide.

Introduction

In Europe echinococcosis, is one the main parasitic disease of concern according to the European Food Safety Authorities.

Amongst Echinococcus species, Echinococcus multilocularis, causing alveolar echinococcosis, is currently a real threat to public health in Europe, with a larger endemic area than previously thought.

E. multilocularis Life Cycle

Definitive Host

Protoscolex

Protoscolex

Human AE larval stage

Stade larvaire

Ver adulte

Œufs

Dissemination of egg in the environment

by faeces

Intermediate Host

Adult phase in the intestine of fox

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In humans, the long asymptomatic period (from five to 15 years) makes the identification of the source of infection difficult or impossible. Some recurrent potential risk factors for developing alveolar echinococcosis in Europe have been identified as “dog or cat ownership,” “living in a rural area,” “having a kitchen garden,” “farming” and “handling foxes”.

Introduction Risk in Kitchen garden

Risk in Kitchen garden

Environmental contamination ? Faeces in garden? Egg on/in the soil ?

In this context, the aim of our study was to develop a flotation/sieving method for the detection of E. multilocularis eggs in soil samples by real-time PCR

To illustrate the utilization of this method, soil samples collected in kitchen garden were tested in order to improve our understanding of the transfer of eggs from hosts to the environment.

Objectives

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Origins of soil samples:

From our experimental station that is surrounded by a double fence since 20 years. No wild foxes in this area

Soil samples free of Em eggs

Mat-Met : validation

Origins of E. multilocularis eggs

Faecal samples removed from the colon of a naturally infected fox diagnosed by the SCT method exhibiting no worms of the taenia genus. Eggs were isolated using home made micropipette Exclusive presence of Em eggs confirm by individual testing of 20 eggs randomly selected

Mat-Met: validation

10 g soil spiked with Em egg

Adapted from the flotation methods of Mathis et al., 1996

Step 1: separation of eggs from soil particules

Centrifugation 1000g 15 min

Discard supernatant

Suspension with 10 ml 0,2% Tween20

Mat-Met: validation

Step 2: Flotation with Zinc chloride

Suspension with 15 ml Zinc chloride

(density 1,42)

Centrifugation 1000g 20 min

supernatant

Filtration 20µm nylon

mesh

Mat-Met: validation

Step 3: Washing and concentration of eggs

Rinsed with 50 ml 0,2% Tween20

Discard supernatant

Centrifugation 1000g 15 min

1 ml with eggs For DNA extraction

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DNA extraction and qPCR assays

NucleoSpin Tissue kit (Macherey-Nagel)

The qPCR reactions were performed in duplicate, on a RotorGene thermocycler (Qiagen)

Echinococcus multilocularis primers and hydrolysis probes for qPCR, mitochondrial rrnL gene (Knapp et al, 2016)

rrn-Em forward primer - CTGTGATCTTGGTGTAGTAGTTGAGATTT

rrn-Em reverse primer - GGCTTACGCCGGTCTTAACTC

rrn-Em probe FAM - TGGTCTGTTCGACCTTTTTAGCCTCCAT-TAMRA

rrn-Em reverse primer (SEQ-PCR) GGGGTCAATCACAACAACCC

Internal control: plasmid DNA construction with it’s specific Probe

All qPCR Em-positive samples were confirmed by sequencing the long PCR products of a second real-time PCR, performed on the same gene.

Mat-Met: DNA extraction and qPCR assays

We have tested 10 g of naïve soil samples spiked with Em eggs

Method sensitivity for Em eggs detection in soil

Number of eggs spiked

0

1

3

5

10

10 eggs 05 eggs 03 eggs 01 egg No egg

qPCR amplification

We have tested 10 g of naïve soil samples spiked with Em eggs

Method sensitivity for Em eggs detection in soil

Number of eggs spiked

replicate

Positive results sensitivities

0

15

0 --

1 5 33,3 %

3 10 66,7 %

5 12 80,0 %

10 25 25 100,0 %

The flotation/sieving method coupled to qPCR is enabled the systematic detection of

10 eggs of E. multilocularis in 10 g of soil sample.

Study area

kitchen garden experimentation

Ardennes Departement

Endemic area for E. multilocularis

Foxes Prevalence ≈ 40 %

(Combes et al. 2012)

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Study area

kitchen garden experimentation

9 Rural Villages

50 familial Kitchens gardens

enclosed

Continuous fence

around the

garden/property

open

≥ 1.20m

Fence

Permeable Fence

Open door

Characterization of kitchen garden

half - open

Study area

kitchen garden experimentation

9 Rural Villages

50 familial Kitchens gardens

10 open

31 half- open

9 enclose

Feces and 5 soil samples were collected during winter period

★★★ Num

ber

of

faec

es p

er 1

00

m2 p

rosp

ecte

d

(mea

n ±

sd)

N=100

Collect of faeces in Kitchen garden

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Analyze of faeces in Kitchen garden

★★★ Num

ber

of

faec

es p

er 1

00

m2 p

rosp

ecte

d

(mea

n ±

sd)

N=100

6/86 7%

(2.6–14.6)

24/69 34,8%

(23,5-47,6)

2/18 11,1%

(1,4-34,7)

E. multilocularis faeces qPCR occurence

Collect of soil in Kitchen garden (preliminary results)

250 soil samples collected

Using flotation/sieving method 26/250 (10,4%) soil samples were positives for E. multilocularis.

21/50 (42%) of Kitchen garden are positive for E. multilocularis.

With restriction because we don’t know if the eggs detected are infectious or not

At garden level: Situation is different: in 21 kitchen gardens we found a least one positive soil samples.

Collect of soil in Kitchen garden (preliminary results)

Repartition of positive kitchen gardens is variable from one village to another

Positive garden

Negative garden

Conclusion:

- The Flotation/sieving method to detect Echinococcus multilocularis eggs using real-time PCR in soil is a reliable technique to detect 10 Em eggs in 10 g of soil

- Preliminary results in kitchen garden show that contamination with egg(/DNA) of Echinococcus multilocularis in garden is probably under evaluated if we consider only feces

Evaluation of E. multilocularis in environmental samples such as soil, vegetables, fruits and water can improve

our understanding of sources of human cases of alveolar echinococcosis.

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Thank you for your

attention