Régulation des populations de Nématodes gastro-intestinaux ...

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Résultats sequence were then quantified by spectrometry to establish the number of target copy/µl. 10- fold dilution series were prepared from the stock plasmid solution corresponding to 10 7 to 10 1 copies of the target sequence. Serial dilution of the plasmid was then used as an external standard, allowing the determination of the number of copies of the target cDNA in each assay. Each sample for each investigated cytokine was performed in duplicate. Ovine Beta- actin was used as a housekeeping gene to normalize expression between different samples. The results obtained for each cytokine are expressed as a ratio of the number of cytokine mRNA copies / Beta-actin mRNA copies. A non-template control reaction (NTC) was included in each run. Finally, specificity of amplification reaction was assessed by acquisition of a dissociation curve. Data was obtained by slowly increasing the temperature of PCR reaction from 60 to 95°C; denaturation of non-specific PCR products is followed by a faster decrease of fluorescence than for specific products. The melting profile of distinct PCR product was obtained by plotting the first derivative (-dF/dT) of fluorescence (F) versus temperature (T). 2.6. Histology At necropsy, two tissue samples from the fundic area of the abomasal wall were taken; one was fixed in 10% formalin for conventional histology and immunohistochemistry (IHC), while the second was stored at –70°C for frozen IHC analysis. 2.6.1. Conventional histology and IHC with paraffin-embedded samples Abomasal tissue was paraffin-embedded and 3 µm sections were mounted on glass slides. Eosinophils and globule leukocytes were counted on haematoxylin-eosin stained slides whereas mast cells were counted after Giemsa staining. Immunohistochemistry was performed using antibodies already described for cell phenotyping on paraffin-embedded tissues in sheep (Lacroux et al., 2006). This antibody set allows specific labeling of 176
Résultats macrophages/monocytes (mouse anti-human CD68 antibody, clone Ki-M6, Serotec, 1:150 dilution), B lymphocytes (mouse anti-human CD20-like antibody, clone BLA-36, Novocastra, 1:50 dilution), or T lymphocytes (rabbit anti-human CD3 antibody, A 0542, DAKO, 1:100 dilution). 2.6.2. IHC on frozen samples Five µm sections of abomasal tissue were obtained using a Leika cryomicrotome. Slides were fixed for 20 min in an –20°C acetone bath before drying and directly processed for IHC. Monoclonal antibodies directed against CD4 (mouse anti-ovine CD4, SEROTEC, reference MCA2213, 1:2000 dilution), or CD8 (mouse anti-bovine CD8, SEROTEC, reference MCA837G, 1:800 dilution) antigens were used. For each type, cells were counted on five randomly selected fields at high magnification (x 400). The results were expressed as the sum of five fields. 2.7. Statistical analysis Comparisons between the three groups (previously-infected, primary-infected and uninfected control), between the two necropsy dates (D49 and D75) within a group, and between the two breeds for a given group were performed using Kruskal-Wallis non parametric tests (SYSTAT software). P < 0.05 was considered significant. Kinetics of egg excretions and blood eosinophil counts were compared between groups using analysis of variance with repeated values (SYSTAT software). 177
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N° d’ordre : 2346 THESE présent
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A mon très cher et respecté « co
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4.3.3. Mise en place et rôle des e
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ANNEXE A L’ARTICLE « HAEMONCHUS
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Un problème économique … et sci
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1. GENERALITES SUR LES STRONGLES GA
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*La vaccination contre Trichostrong
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3.2.3. La sélection de la résista
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LIEU DE L’ETUDE RACES ETUDIEES /
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*Réduction de la taille des vers a
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ISOTYPE Type d’antigène Concentr
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6.1.4. Immunohistochimie sur coupes
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Procédures expérimentales éviden
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Résultats CHAPITRE V : RESULTATS 1
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Résultats - à la polarisation Th1
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Vet. Res. 37 (2006) 1-16 © INRA, E
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ut no clinical signs of coccidiosis
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gene sequence was not known (Eotaxi
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-20 °C acetone bath before drying.
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Th 2 response in H. contortus infec
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significant systemic CEL 3 specific
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3.5.4. Reduced fecal egg output At
Page 125 and 126: populations in sheep exposed to con
Page 127 and 128: Résultats Annexe à l’article «
Page 129 and 130: Résultats Les corrélations entre
Page 131 and 132: Article II : Immune responses in la
Page 133 and 134: 1. INTRODUCTION Résultats Due to t
Page 135 and 136: 2. MATERIAL AND METHODS 2.1. Experi
Page 137 and 138: Résultats RNA were digested with 1
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Page 143 and 144: 3. RESULTS 3.1. Dynamics of the wor
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Page 147 and 148: 4. DISCUSSION Résultats In this st
Page 149 and 150: Résultats nematode infections elic
Page 151 and 152: REFERENCES Résultats Balic, A., Bo
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Page 155 and 156: Table 1 : Schematic description of
Page 157 and 158: Table 3 : Reagents and procedures u
Page 159 and 160: Table 5 : Correlations between cell
Page 165 and 166: Résultats PARTIE II : ETUDE COMPAR
Page 167 and 168: Résultats Article III : Dynamics o
Page 169 and 170: 1 - INTRODUCTION Résultats The con
Page 171 and 172: 2 - MATERIALS AND METHODS 2.1. Expe
Page 173 and 174: Résultats of water. Larvae were co
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Page 181 and 182: Résultats in INRA 401 lambs and bl
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Page 185 and 186: Résultats terms of female size, eg
Page 187 and 188: Résultats Gauly, M., Kraus, M., Ve
Page 189 and 190: Table 1 : Schematic description of
Page 191 and 192: Table 3 : Total worm burden, length
Page 193 and 194: Epg Epg 30000 25000 20000 15000 100
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Page 197 and 198: Discussion générale CHAPITRE VI :
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Page 209 and 210: Discussion générale connaissance
Page 211 and 212: Références bibliographiques Adams
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Références bibliographiques Yazwi
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