Upload
trinhtu
View
218
Download
4
Embed Size (px)
Citation preview
31/05/2017
1
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
31/05/2017
2
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
31/05/2017
3
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
31/05/2017
4
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)
31/05/2017
5
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
31/05/2017
6
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.