Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools.
Identifieur interne : 000035 ( France/Analysis ); précédent : 000034; suivant : 000036Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools.
Auteurs : Jenny Knapp [France] ; Laurence Millon [France] ; Lorane Mouzon [France] ; Gérald Umhang [France] ; Francis Raoul [France] ; Zeinaba Said Ali [France] ; Benoît Combes [France] ; Sébastien Comte [France] ; Houssein Gbaguidi-Haore [France] ; Frédéric Grenouillet [France] ; Patrick Giraudoux [France]Source :
- Veterinary parasitology [ 1873-2550 ] ; 2014.
English descriptors
- KwdEn :
- MESH :
- diagnosis : Echinococcosis.
- genetics : Echinococcus multilocularis.
- methods : Environmental Monitoring.
- parasitology : Feces, Foxes.
- Animals, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity.
Abstract
The oncosphere stage of Echinococcus multilocularis in red fox stools can lead, after ingestion, to the development of alveolar echinococcosis in the intermediate hosts, commonly small mammals and occasionally humans. Monitoring animal infection and environmental contamination is a key issue in public health surveillance. We developed a quantitative real-time PCR technique (qPCR) to detect and quantify E. multilocularis DNA released in fox faeces. A qPCR technique using a hydrolysis probe targeting part of the mitochondrial gene rrnL was assessed on (i) a reference collection of stools from 57 necropsied foxes simultaneously investigated using the segmental sedimentation and counting technique (SSCT) (29 positive for E. multilocularis worms and 28 negative animals for the parasite); (ii) a collection of 114 fox stools sampled in the field: two sets of 50 samples from contrasted endemic regions in France and 14 from an E. multilocularis-free area (Greenland). Of the negative SSCT controls, 26/28 were qPCR-negative and two were weakly positive. Of the positive SSCT foxes, 25/29 samples were found to be positive by qPCR. Of the field samples, qPCR was positive in 21/50 (42%) and 5/48 (10.4%) stools (2 samples inhibited), originating respectively from high and low endemic areas. In faeces, averages of 0.1 pg/μl of DNA in the Jura area and 0.7 pg/μl in the Saône-et-Loire area were detected. All qPCR-positive samples were confirmed by sequencing. The qPCR technique developed here allowed us to quantify environmental E. multilocularis contamination by fox faeces by studying the infectious agent directly. No previous study had performed this test in a one-step reaction.
DOI: 10.1016/j.vetpar.2013.12.023
PubMed: 24484767
Affiliations:
- France
- Bourgogne-Franche-Comté, Franche-Comté, Grand Est, Lorraine (région), Île-de-France
- Besançon, Malzéville, Paris
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pubmed:24484767Le document en format XML
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INRA, University of Franche-Comté, 25030 Besançon, France; Department of Parasitology-Mycology, University Hospital of Besançon, Besançon, France. Electronic address: jenny.knapp@univ-fcomte.fr.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France; Department of Parasitology-Mycology, University Hospital of Besançon, Besançon</wicri:regionArea><placeName><region type="region">Bourgogne-Franche-Comté</region><region type="old region">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Millon, Laurence" sort="Millon, Laurence" uniqKey="Millon L" first="Laurence" last="Millon">Laurence Millon</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France; Department of Parasitology-Mycology, University Hospital of Besançon, Besançon, France; Clinical Investigation Center (Inserm CIT 808), University Hospital of Besançon, Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France; Department of Parasitology-Mycology, University Hospital of Besançon, Besançon, France; Clinical Investigation Center (Inserm CIT 808), University Hospital of Besançon, Besançon</wicri:regionArea><placeName><region type="region">Bourgogne-Franche-Comté</region><region type="old region">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Mouzon, Lorane" sort="Mouzon, Lorane" uniqKey="Mouzon L" first="Lorane" last="Mouzon">Lorane Mouzon</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon</wicri:regionArea><placeName><region type="region" nuts="2">Bourgogne-Franche-Comté</region><region type="old region" nuts="2">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Umhang, Gerald" sort="Umhang, Gerald" uniqKey="Umhang G" first="Gérald" last="Umhang">Gérald Umhang</name><affiliation wicri:level="3"><nlm:affiliation>Nancy Rabies and Wildlife Laboratory, Technopole Agricole et Vétérinaire, BP 40009, 54220 Malzéville, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Nancy Rabies and Wildlife Laboratory, Technopole Agricole et Vétérinaire, BP 40009, 54220 Malzéville</wicri:regionArea><placeName><region type="region" nuts="2">Grand Est</region><region type="old region" nuts="2">Lorraine (région)</region><settlement type="city">Malzéville</settlement></placeName></affiliation></author><author><name sortKey="Raoul, Francis" sort="Raoul, Francis" uniqKey="Raoul F" first="Francis" last="Raoul">Francis Raoul</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon</wicri:regionArea><placeName><region type="region" nuts="2">Bourgogne-Franche-Comté</region><region type="old region" nuts="2">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Ali, Zeinaba Said" sort="Ali, Zeinaba Said" uniqKey="Ali Z" first="Zeinaba Said" last="Ali">Zeinaba Said Ali</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon</wicri:regionArea><placeName><region type="region" nuts="2">Bourgogne-Franche-Comté</region><region type="old region" nuts="2">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Combes, Benoit" sort="Combes, Benoit" uniqKey="Combes B" first="Benoît" last="Combes">Benoît Combes</name><affiliation wicri:level="3"><nlm:affiliation>French establishment for fighting zoonoses (ELIZ), Domaine de Pixerécourt, 54220 Malzéville, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>French establishment for fighting zoonoses (ELIZ), Domaine de Pixerécourt, 54220 Malzéville</wicri:regionArea><placeName><region type="region" nuts="2">Grand Est</region><region type="old region" nuts="2">Lorraine (région)</region><settlement type="city">Malzéville</settlement></placeName></affiliation></author><author><name sortKey="Comte, Sebastien" sort="Comte, Sebastien" uniqKey="Comte S" first="Sébastien" last="Comte">Sébastien Comte</name><affiliation wicri:level="3"><nlm:affiliation>French establishment for fighting zoonoses (ELIZ), Domaine de Pixerécourt, 54220 Malzéville, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>French establishment for fighting zoonoses (ELIZ), Domaine de Pixerécourt, 54220 Malzéville</wicri:regionArea><placeName><region type="region" nuts="2">Grand Est</region><region type="old region" nuts="2">Lorraine (région)</region><settlement type="city">Malzéville</settlement></placeName></affiliation></author><author><name sortKey="Gbaguidi Haore, Houssein" sort="Gbaguidi Haore, Houssein" uniqKey="Gbaguidi Haore H" first="Houssein" last="Gbaguidi-Haore">Houssein Gbaguidi-Haore</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon</wicri:regionArea><placeName><region type="region" nuts="2">Bourgogne-Franche-Comté</region><region type="old region" nuts="2">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Grenouillet, Frederic" sort="Grenouillet, Frederic" uniqKey="Grenouillet F" first="Frédéric" last="Grenouillet">Frédéric Grenouillet</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon</wicri:regionArea><placeName><region type="region" nuts="2">Bourgogne-Franche-Comté</region><region type="old region" nuts="2">Franche-Comté</region><settlement type="city">Besançon</settlement></placeName></affiliation></author><author><name sortKey="Giraudoux, Patrick" sort="Giraudoux, Patrick" uniqKey="Giraudoux P" first="Patrick" last="Giraudoux">Patrick Giraudoux</name><affiliation wicri:level="3"><nlm:affiliation>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France; Institut Universitaire de France, 75015 Paris, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Chrono-environnement, UMR UFC/CNRS 6249 aff. INRA, University of Franche-Comté, 25030 Besançon, France; Institut Universitaire de France, 75015 Paris</wicri:regionArea><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Paris</settlement></placeName></affiliation></author></analytic><series><title level="j">Veterinary parasitology</title><idno type="eISSN">1873-2550</idno><imprint><date when="2014" type="published">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Echinococcosis (diagnosis)</term><term>Echinococcus multilocularis (genetics)</term><term>Environmental Monitoring (methods)</term><term>Feces (parasitology)</term><term>Foxes (parasitology)</term><term>Real-Time Polymerase Chain Reaction</term><term>Sensitivity and Specificity</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Echinococcosis</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Echinococcus multilocularis</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Environmental Monitoring</term></keywords><keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Feces</term><term>Foxes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Real-Time Polymerase Chain Reaction</term><term>Sensitivity and Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The oncosphere stage of Echinococcus multilocularis in red fox stools can lead, after ingestion, to the development of alveolar echinococcosis in the intermediate hosts, commonly small mammals and occasionally humans. Monitoring animal infection and environmental contamination is a key issue in public health surveillance. We developed a quantitative real-time PCR technique (qPCR) to detect and quantify E. multilocularis DNA released in fox faeces. A qPCR technique using a hydrolysis probe targeting part of the mitochondrial gene rrnL was assessed on (i) a reference collection of stools from 57 necropsied foxes simultaneously investigated using the segmental sedimentation and counting technique (SSCT) (29 positive for E. multilocularis worms and 28 negative animals for the parasite); (ii) a collection of 114 fox stools sampled in the field: two sets of 50 samples from contrasted endemic regions in France and 14 from an E. multilocularis-free area (Greenland). Of the negative SSCT controls, 26/28 were qPCR-negative and two were weakly positive. Of the positive SSCT foxes, 25/29 samples were found to be positive by qPCR. Of the field samples, qPCR was positive in 21/50 (42%) and 5/48 (10.4%) stools (2 samples inhibited), originating respectively from high and low endemic areas. In faeces, averages of 0.1 pg/μl of DNA in the Jura area and 0.7 pg/μl in the Saône-et-Loire area were detected. All qPCR-positive samples were confirmed by sequencing. The qPCR technique developed here allowed us to quantify environmental E. multilocularis contamination by fox faeces by studying the infectious agent directly. No previous study had performed this test in a one-step reaction.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Bourgogne-Franche-Comté</li><li>Franche-Comté</li><li>Grand Est</li><li>Lorraine (région)</li><li>Île-de-France</li></region><settlement><li>Besançon</li><li>Malzéville</li><li>Paris</li></settlement></list><tree><country name="France"><region name="Bourgogne-Franche-Comté"><name sortKey="Knapp, Jenny" sort="Knapp, Jenny" uniqKey="Knapp J" first="Jenny" last="Knapp">Jenny Knapp</name></region><name sortKey="Ali, Zeinaba Said" sort="Ali, Zeinaba Said" uniqKey="Ali Z" first="Zeinaba Said" last="Ali">Zeinaba Said Ali</name><name sortKey="Combes, Benoit" sort="Combes, Benoit" uniqKey="Combes B" first="Benoît" last="Combes">Benoît Combes</name><name sortKey="Comte, Sebastien" sort="Comte, Sebastien" uniqKey="Comte S" first="Sébastien" last="Comte">Sébastien Comte</name><name sortKey="Gbaguidi Haore, Houssein" sort="Gbaguidi Haore, Houssein" uniqKey="Gbaguidi Haore H" first="Houssein" last="Gbaguidi-Haore">Houssein Gbaguidi-Haore</name><name sortKey="Giraudoux, Patrick" sort="Giraudoux, Patrick" uniqKey="Giraudoux P" first="Patrick" last="Giraudoux">Patrick Giraudoux</name><name sortKey="Grenouillet, Frederic" sort="Grenouillet, Frederic" uniqKey="Grenouillet F" first="Frédéric" last="Grenouillet">Frédéric Grenouillet</name><name sortKey="Millon, Laurence" sort="Millon, Laurence" uniqKey="Millon L" first="Laurence" last="Millon">Laurence Millon</name><name sortKey="Mouzon, Lorane" sort="Mouzon, Lorane" uniqKey="Mouzon L" first="Lorane" last="Mouzon">Lorane Mouzon</name><name sortKey="Raoul, Francis" sort="Raoul, Francis" uniqKey="Raoul F" first="Francis" last="Raoul">Francis Raoul</name><name sortKey="Umhang, Gerald" sort="Umhang, Gerald" uniqKey="Umhang G" first="Gérald" last="Umhang">Gérald Umhang</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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