217 lines
22 KiB
XML
217 lines
22 KiB
XML
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<mods:affiliation id="95C6236FBC9988D89A0EB97C682869F2">UMR BIPAR, Ecole Nationale Vétérinaire d’Alfort, ANSES, Université Paris-Est, INRA, National Reference Laboratory for Foodborne Parasites, Animal Health Laboratory, 14 rue Pierre et Marie Curie, 94700 Maisons-Alfort, France & UMR BIPAR, ANSES, Ecole Nationale Vétérinaire d’Alfort, INRA, Université Paris-Est, National Reference Laboratory for Foodborne Parasites, Animal Health Laboratory, 14 rue Pierre et Marie Curie, 94700 Maisons-Alfort, France</mods:affiliation>
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<mods:affiliation id="86162532721A562DB95787BF7F2A9CEB">Epidemiology Unit, Paris-Est University, Laboratory for Animal Health, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 94700 Maisons-Alfort, France</mods:affiliation>
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<mods:affiliation id="834B72E655538AA15D08F847B2D6BD94">INSERM, Université de Limoges, CHU Limoges, UMR 1094, Institut d’Epidémiologie et de Neurologie Tropicale, GEIST, & National Reference Center on Toxoplasmosis, Toxoplasma Biological Resources Center, CHU Limoges, 87042 Limoges, France</mods:affiliation>
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Bioassay for
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in mice
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The
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experiments were approved by the local Animal Research Ethics Committee (ComEth Anses, EnvA, UPE) of Maisons-Alfort. In order to limit suffering and distress, mice were acclimated for 7 days after their arrival. Cages were filled with paper strips. Animal health and behaviour were monitored daily. Mice were observed based on the following criteria:
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<paragraph id="525836D0E259FF988B31FCE1541EFCC1" blockId="3.[113,767,802,946]" pageId="3" pageNumber="4">– external physical appearance (disheveled or spiked hairs, watering eyes, bent back, tremors),</paragraph>
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If any of these criteria were critically altered, mice were subsequently euthanised and examined post-mortem to investigate the parasitic load. Euthanasia consisted in CO
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asphyxiation, followed by cervical dislocation.
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After approval from the local Animal Research Ethics Committee, between 5 and 11 heart samples, randomly chosen from among those with the highest titers of agglutinating antibodies, were bioassayed weekly in three outbred female Swiss Webster mice (Charles River Laboratory,
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). Additionally, nine seronegative hearts, randomly selected from the total number of samples, were bioassayed in mice. Briefly, each whole heart was mixed and incubated at 37 °C for 1.5 h with trypsin (final concentration 0.25%). The suspension was then filtered, pelleted by centrifugation, washed in saline, and resuspended in a saline solution containing penicillin G, streptomycin and amoxicillin to limit bacterial proliferation. This homogenate was inoculated intraperitoneally into three mice [
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<bibRefCitation id="36764B21E259FF988B3AFA375690F992" author="Afonso E & Poulle ML & Lemoine M & Villena I & Aubert D & Gilot-Fromont E." box="[122,135,1524,1547]" pageId="3" pageNumber="4" pagination="313 - 314" refId="ref10463" refString="1. Afonso E, Poulle ML, Lemoine M, Villena I, Aubert D, Gilot-Fromont E. 2007. Prevalence of Toxoplasma gondii in small mammals from the Ardennes region, France. Folia Parasitologica, 54, 313 - 314." type="journal article" year="2007">1</bibRefCitation>
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,
|
||
<bibRefCitation id="36764B21E259FF988BD6FA3756A7F992" author="Villena I & Aubert D & Gomis P & Ferte H & Inglard JC & Bisiaux H & Dondon JM & Pisano E & Ortis N & Pinon JM" box="[150,176,1524,1547]" pageId="3" pageNumber="4" pagination="4035 - 4039" refId="ref13215" refString="59. Villena I, Aubert D, Gomis P, Ferte H, Inglard JC, Denis- Bisiaux H, Dondon JM, Pisano E, Ortis N, Pinon JM. 2004. Evaluation of a strategy for Toxoplasma gondii oocyst detection in water. Applied Environmental Microbiology, 70, 4035 - 4039." type="journal article" year="2004">59</bibRefCitation>
|
||
]. Mice were monitored twice daily with food and water supplied
|
||
<emphasis id="6093EAC2E259FF988B92F9D05729F9B3" box="[210,318,1555,1578]" italics="true" pageId="3" pageNumber="4">ad libitum</emphasis>
|
||
. In case of acute toxoplasmosis, the mice were culled by CO
|
||
<subScript id="CE633495E259FF988A74F9F85729F9D2" attach="left" box="[308,318,1595,1611]" fontSize="7" pageId="3" pageNumber="4">2</subScript>
|
||
asphyxiation, followed by cervical dislocation, and samples of brain were taken for analysis. Mice were bled 4 weeks post-inoculation and their serum was tested at 1:25 dilution for
|
||
<taxonomicName id="95E74D53E259FF988A72F94E5782F93D" box="[306,405,1677,1700]" family="Sarcocystidae" genus="Toxoplasma" kingdom="Chromista" order="Eucoccidiida" pageId="3" pageNumber="4" phylum="Miozoa" rank="species" species="gondii">
|
||
<emphasis id="6093EAC2E259FF988A72F94E5782F93D" box="[306,405,1677,1700]" italics="true" pageId="3" pageNumber="4">T. gondii</emphasis>
|
||
</taxonomicName>
|
||
antibodies with the MAT. Later on, mice were culled 60 days post-inoculation by cervical dislocation, and their brains were examined for tissue cysts.
|
||
</paragraph>
|
||
<paragraph id="525836D0E259FF988B31F8DB5419F8A8" blockId="3.[113,526,1816,1841]" box="[113,526,1816,1841]" pageId="3" pageNumber="4">
|
||
<emphasis id="6093EAC2E259FF988B31F8DB5419F8A8" bold="true" box="[113,526,1816,1841]" pageId="3" pageNumber="4">
|
||
Genotyping of
|
||
<taxonomicName id="95E74D53E259FF988A71F8DB57B6F8A8" box="[305,417,1816,1841]" family="Sarcocystidae" genus="Toxoplasma" kingdom="Chromista" order="Eucoccidiida" pageId="3" pageNumber="4" phylum="Miozoa" rank="species" species="gondii">
|
||
<emphasis id="6093EAC2E259FF988A71F8DB57B6F8A8" bold="true" box="[305,417,1816,1841]" italics="true" pageId="3" pageNumber="4">T. gondii</emphasis>
|
||
</taxonomicName>
|
||
isolates
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="525836D0E259FF988BD9F89255DDFEBD" blockId="3.[113,768,1873,2049]" lastBlockId="3.[813,1467,239,292]" pageId="3" pageNumber="4">
|
||
Brain cysts from seropositive mice were isolated by percoll gradient centrifugation [
|
||
<bibRefCitation id="36764B21E259FF988A3EF8B3578FF81E" author="Cornelissen AW & Overdulve JP & Hoenderboom JM" box="[382,408,1904,1927]" pageId="3" pageNumber="4" pagination="103 - 108" refId="ref11109" refString="15. Cornelissen AW, Overdulve JP, Hoenderboom JM. 1981. Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation. Parasitology, 83, 103 - 108." type="journal article" year="1981">15</bibRefCitation>
|
||
]. DNA was extracted using a QIAamp DNA MiniKit (Qiagen, Courtaboeuf,
|
||
<collectingCountry id="2AF07640E259FF988938F84D54D6F83C" box="[632,705,1934,1957]" name="France" pageId="3" pageNumber="4">France</collectingCountry>
|
||
), and genotyping analysis of
|
||
<taxonomicName id="95E74D53E259FF988A32F86E540DF85D" authority="DNA" authorityName="DNA" box="[370,538,1965,1988]" family="Sarcocystidae" genus="Toxoplasma" kingdom="Chromista" order="Eucoccidiida" pageId="3" pageNumber="4" phylum="Miozoa" rank="species" species="gondii">
|
||
<emphasis id="6093EAC2E259FF988A32F86E57C2F85D" box="[370,469,1965,1988]" italics="true" pageId="3" pageNumber="4">T. gondii</emphasis>
|
||
DNA
|
||
</taxonomicName>
|
||
was performed with 15 microsatellite markers in a single multiplex PCR assay, as described elsewhere [
|
||
<bibRefCitation id="36764B21E259FF988A0FF829574BF798" author="Ajzenberg D & Collinet F & Mercier A & Vignoles P & Darde ML" box="[335,348,2026,2049]" pageId="3" pageNumber="4" pagination="4641 - 4645" refId="ref10544" refString="3. Ajzenberg D, Collinet F, Mercier A, Vignoles P, Darde ML. 2010. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. Journal of Clinical Microbiology, 48 (12), 4641 - 4645." type="journal article" year="2010">3</bibRefCitation>
|
||
]. All strains isolated were cell cultivated and banked in the
|
||
<taxonomicName id="95E74D53E259FF988F46FF2C5293FE9F" authorityName="Nicolle & Manceaux" authorityYear="1909" box="[1030,1156,239,262]" family="Sarcocystidae" genus="Toxoplasma" kingdom="Chromista" order="Eucoccidiida" pageId="3" pageNumber="4" phylum="Miozoa" rank="genus">
|
||
<emphasis id="6093EAC2E259FF988F46FF2C5293FE9F" box="[1030,1156,239,262]" italics="true" pageId="3" pageNumber="4">Toxoplasma</emphasis>
|
||
</taxonomicName>
|
||
Biological Resource
|
||
<collectingRegion id="9023F832E259FF988E36FF2C53ACFE9F" box="[1398,1467,239,262]" country="France" name="Centre" pageId="3" pageNumber="4">Center</collectingRegion>
|
||
(
|
||
<collectionCode id="34F6AE15E259FF988874FECE557CFEBD" box="[820,875,269,292]" country="United Kingdom" httpUri="http://biocol.org/urn:lsid:biocol.org:col:12685" lsid="urn:lsid:biocol.org:col:12685" name="Botanical Record Club" pageId="3" pageNumber="4" type="Herbarium">BRC</collectionCode>
|
||
, Reims).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |