Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Gießen
DVG
2006
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| Ausgabe: | 1. Aufl. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VI, 93 S. Ill., graph. Darst. 21 cm |
| ISBN: | 3938026987 |
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| 245 | 1 | 0 | |a Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |c von Alessandro Broglia |
| 250 | |a 1. Aufl. | ||
| 264 | 1 | |a Gießen |b DVG |c 2006 | |
| 300 | |a VI, 93 S. |b Ill., graph. Darst. |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |a Berlin, Freie Univ., Diss., 2006 | ||
| 650 | 7 | |a ELISA |2 cabt | |
| 650 | 7 | |a Sheep Disease |2 cabt | |
| 650 | 7 | |a Parasitosis |2 cabt | |
| 650 | 7 | |a Diagnostic Techniques |2 cabt | |
| 650 | 7 | |a Immunodiagnosis |2 cabt | |
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Datensatz im Suchindex
| _version_ | 1804136402636505088 |
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| adam_text | TABLE OF CONTENTS
FIGURE LIST................................................................................. iv
TABLE LIST.................................................................................. vi
1. INTRODUCTION...........................................................................1
2. LITERATURE REVIEW..................................................................3
2.1. Taxonomy of Dicrocoelium spp..........................................................3
2.2. Species.........................................................................................3
2.3. Morphology........................................................................ 4
2.4. Life cycle............................................................................4
2.5. Epidemiology.......................................................................5
2.5.1. Environmental factors......................................................5
2.5.2. Intermediate hosts...........................................................6
2.5.3. Definitive host...............................................................7
2.6. Pathogenesis, clinical findings and lesions....................................9
2.7. Diagnosis.............................................................................11
2.7.1. Direct methods...............................................................11
2.7.2. Indirect methods.............................................................12
2.7.2.1. The indirect ELISA (Enzyme-Linked
Immunosorbent Assay)................................................ 13
2.7.2.2. Enzyme-Assisted Immunoelectroblotting...........................14
2.8. Treatment and control of dicrocoeliosis._________________________17
3. MATERIALS AND METHODS........................................_________20
3.1. Study area.............„.........................................„............................._..........20
3.2. Experimental infection_______________________________________22
33. Enzyme -Linked Immunosorbent Assay (ELISA)_________________23
3.3.1. Antigen preparation.........................................................23
3.3.2. Setting ELISA parameter..................................................24
3.3.2.1. Titration of antigen......................................................24
3.3.2.2. Titration of serum........................................................24
3.3.2.3. Conjugate and substrate...............................................24
3.3.2.4. Implementation of ELISA test.........................................25
3.3.2.5. Cut-off definition.........................................................26
3.3.3. Cross-reaction with other parasitic infections...........................26
3.3.3.1. Cross-reaction with Fasciola hepática and
Paramphistomum spp...................................................26
3.3.3.2. Cross-reaction with nematodeparasite infection...............27
3.4. Enzyme Immune Transfer Blotting (EITB)..................................28
3.4.1. Sample treatment............................................................28
3.4.2. SDSPAGE...................................................................29
3.4.3. Blotting.......................................................................30
3.4.4. Immunoassay................................................................31
3.4.5. Determination of protein molecular weight..............................32
3.5. Field serum samples of sheep flocks from Trentino (Italy)............. 33
3.5.1. Serum samples...............................................................35
4. RESULTS.....................................................................................37
4.1. Experimental infection....._......................................................................37
4.1.1. Ants collection...............................................................38
4.1.2. Establishment rate...........................................................39
4.2. ELISA test._______________________________________....................41
4.2.1. Determination of protein concentration of antigen solution...........41
4.2.2. Antigen and serum dilution................................................41
4.2.3. Cut off definition............................................................41
4.2.4. Temporal dynamics of IGg response
of experimentally infected lambs..........................................43
4.2.5. Cross reaction with other helminths......................................46
4.2.5.1. Cross -reaction with F. hepática and
Paramphistomum spp...................................................46
4.2.5.2. Cross-reaction with nematode parasite infection................48
43. EITB test.__________________________________________________50
4.3.1. Proteic fractions D. dendriticum antigens of by SDS-PAGE.........50
4.3.2. Antibody response dynamics in experimentally infected lambs......51
4.3.3. Comparative immunoblot with sera of lambs infected with F.
hepática.........................................................................53
4.3.4. Comparative immunoblot with sera of lambs infected with others
nematodes.......................................................................54
4.4. Experimental screening of dicrocoeliosis in sheep flocks in
North East Italy (Trentino Province)..........................................55
4.4.1. Detection of D. chinensis in red deer (Cervus elaphus) a province of
Trento...........................................................................57
5. DISCUSSION........................................................._________.....59
6. CONCLUSIONS.................................................._________._____67
7. SUMMARY...................................................................................69
8. ZUSAMMENFASSUNG...................................................................70
9. REFERENCES................................................................................71
10. APPENDIX........................................................................................................85
10.1. Reagents..........................................................................................85
10.1.1. Reagents for antigen preparation..........................................85
10.1.2. Reagent for ELISA test....................................................85
10.1.3. Reagents for SDS-PAGE...................................................86
10.1.4. Reagents for Immunoblot..................................................87
10.2. Tables___________________________..................................89
11. ACKNOWLEDGEMENTS____________________________________.....91
12. CURRICULUM VITAE_________________________________________92
13. SELBSTANDIGKEITSERKLÄRUNG__________________________.....93
FIGURE LIST
• Figure 1. Localization of Brandenburg in Germany (left) and eastern Brandenburg
districts (right) (1: Uckermark; 2: Barnim; 3: Märkisch-Oderland; 4: Oder-Spree)..........20
• Figure 2. Typical D. dendriticum biotope in eastern Brandenburg (Photo; R.Schuster).... 21
• Fig. 3. Scheme of the SemiDry blotting sandwich..............................................................30
• Fig.4. Geographical position of the province of Trento......................................................33
• Fig. 5 . Altitude range in the province of Trento.................................................................34
• Fig. 6. Land cover types in the province of Trento............................................................34
• Fig. 7. Municipalities from which serum samples were collected......................................36
• Fig. 8. Ants in tetania (F. pratensis) on grass collected early in the morning in eastern
Brandenburg (Photo A. Broglia).........................................................................................37
• Fig. 9. Metacercariae distribution classes in ants collected in tetania.................................38
• Figure 10. Dissected gall bladder of sheep experimentally infected (infection dose: 2000
metacercariae) with D. dendriticum. Many D. dendriticum adults were present........... 39
• Figure 11. Liver of sheep experimentally infected (infection dose: 2000 metacercariae).
The gall bladder has been removed.....................................................................................40
• Figure 12. OD values of IGg antibodies against D. dendriticum
excretory/secretory antigen throughout the experimental infection....................................43
• Figure 13. OD values of IGg antibodies against D. dendriticum So. antigen
throughout the experimental infection................................................................................44
• Figure 14. OD values of sera from sheep infected with F. hepática and
Paramphistomum spp. with relative cut-off values.............................................................47
• Figure 15. OD values of antibody response of lambs infected with N. battus. T.
colubriformis, H. contortus against E/S and So. antigen of/), dendriticum.......................48
IV
Figure 16. OD values of antibody response of lambs infected with repeated dose of
different nematode species L3 against excretory/secretory and So. antigens of D.
dendriticum.........................................................................................................................49
Figure 17. Pattern of proteic fractions of E/S and So. antigen of D. dendriticum
bySDS-PAGE.....................................................................................................................50
Figure 18. Immunoblot of/), dendriticum E/S antigen with sera of experimentally
infected lambs (inf. dose: 2000) through the study period..................................................51
Figure 19. Immunoblot of D. dendriticum So. antigen with sera of experimentally
infected lambs (inf. dose: 2000) during the study period....................................................52
Figure 20. Fmrnunoblot of D. dendriticum E/S antigen with field sera of sheep
infected with F. hepática (lane 1-6) and sera of lambs infected with D. dendriticum...... 53
Figure 21. Immunoblot of D. dendriticum E/S and So. antigens with sera of lambs
infected with T. colubriformis (lane 1), N. battus (lane 2) and sera of lambs infected
withD. dendriticum.............................................................................................................54
Figure 22. Seroprevalence rates of dicrocoeliosis (E/S antigen) in sheep flocks
in province of Trento (northeastern Italy)...........................................................................56
Figure 23. Seroprevalence rates of dicrocoeliosis (So. antigen) in sheep flocks
in province of Trento (northeastern Italy)...........................................................................56
Figure 24. An adult exemplar of D. chinensis found in a
male red deer (province of Trento)......................................................................................57
Figure 25. Detail of the cranial end of D. chinensis (left) and D. dendriticum (right).......58
TABLE LIST
• Table 1. Serial dilution of antigen and sera in an ELISA plate...........................................24
• Table 2. Sheep flocks, municipality, district, and number of serum samples.....................35
• Table 3. Prevalence, intensity, infection range and total metacercariae in collected ants.. 37
• Table 4. Number of recovered flukes at necropsy and relationship with infection dose... 39
• Table 5. OD values in negative serum samples..................................................................42
• Table 6. OD values and coprological analysis results of sheep infected by
F. hepática and Paramphistomum spp................................................................................46
• Table 7. Seroprevalence rates of dicrocoeliosis (E/S and So. antigen) in sheep
flocks in the province of Trento (northeastern Italy)..........................................................55
• Table 8. OD values of experimentally infected lambs sera against E/S antigen.................89
• Table 9. OD values of experimentally infected lambs sera against So. antigen.................89
• Table 10. OD values of sera from sheep naturally infected by F. hepática
against E/S and So. D. dendriticum antigens......................................................................89
• Table 11. OD values of sera from experimentally infected sheep with
Nematodirus battus. Trichostrongylus colubriformis, Haemonchus contortus,
single infection dose....................................................................................................... 90
• Table 12. OD values of sera from experimentally infected sheep with
Dictyocaulus filaría, Oesophagostomum venulosum, Ostertagia ostertagi,
T. colubriformis, Cooperia curticei, N. battus, four repeated infection dose.....................90
|
| adam_txt |
TABLE OF CONTENTS
FIGURE LIST. iv
TABLE LIST. vi
1. INTRODUCTION.1
2. LITERATURE REVIEW.3
2.1. Taxonomy of Dicrocoelium spp.3
2.2. Species.3
2.3. Morphology. 4
2.4. Life cycle.4
2.5. Epidemiology.5
2.5.1. Environmental factors.5
2.5.2. Intermediate hosts.6
2.5.3. Definitive host.7
2.6. Pathogenesis, clinical findings and lesions.9
2.7. Diagnosis.11
2.7.1. Direct methods.11
2.7.2. Indirect methods.12
2.7.2.1. The indirect ELISA (Enzyme-Linked
Immunosorbent Assay). 13
2.7.2.2. Enzyme-Assisted Immunoelectroblotting.14
2.8. Treatment and control of dicrocoeliosis._17
3. MATERIALS AND METHODS._20
3.1. Study area.„.„._.20
3.2. Experimental infection_22
33. Enzyme -Linked Immunosorbent Assay (ELISA)_23
3.3.1. Antigen preparation.23
3.3.2. Setting ELISA parameter.24
3.3.2.1. Titration of antigen.24
3.3.2.2. Titration of serum.24
3.3.2.3. Conjugate and substrate.24
3.3.2.4. Implementation of ELISA test.25
3.3.2.5. Cut-off definition.26
3.3.3. Cross-reaction with other parasitic infections.26
3.3.3.1. Cross-reaction with Fasciola hepática and
Paramphistomum spp.26
3.3.3.2. Cross-reaction with nematodeparasite infection.27
3.4. Enzyme Immune Transfer Blotting (EITB).28
3.4.1. Sample treatment.28
3.4.2. SDSPAGE.29
3.4.3. Blotting.30
3.4.4. Immunoassay.31
3.4.5. Determination of protein molecular weight.32
3.5. Field serum samples of sheep flocks from Trentino (Italy). 33
3.5.1. Serum samples.35
4. RESULTS.37
4.1. Experimental infection._.37
4.1.1. Ants collection.38
4.1.2. Establishment rate.39
4.2. ELISA test._.41
4.2.1. Determination of protein concentration of antigen solution.41
4.2.2. Antigen and serum dilution.41
4.2.3. Cut off definition.41
4.2.4. Temporal dynamics of IGg response
of experimentally infected lambs.43
4.2.5. Cross reaction with other helminths.46
4.2.5.1. Cross -reaction with F. hepática and
Paramphistomum spp.46
4.2.5.2. Cross-reaction with nematode parasite infection.48
43. EITB test._50
4.3.1. Proteic fractions D. dendriticum antigens of by SDS-PAGE.50
4.3.2. Antibody response dynamics in experimentally infected lambs.51
4.3.3. Comparative immunoblot with sera of lambs infected with F.
hepática.53
4.3.4. Comparative immunoblot with sera of lambs infected with others
nematodes.54
4.4. Experimental screening of dicrocoeliosis in sheep flocks in
North East Italy (Trentino Province).55
4.4.1. Detection of D. chinensis in red deer (Cervus elaphus)\a province of
Trento.57
5. DISCUSSION._.59
6. CONCLUSIONS._._67
7. SUMMARY.69
8. ZUSAMMENFASSUNG.70
9. REFERENCES.71
10. APPENDIX.85
10.1. Reagents.85
10.1.1. Reagents for antigen preparation.85
10.1.2. Reagent for ELISA test.85
10.1.3. Reagents for SDS-PAGE.86
10.1.4. Reagents for Immunoblot.87
10.2. Tables_.89
11. ACKNOWLEDGEMENTS_.91
12. CURRICULUM VITAE_92
13. SELBSTANDIGKEITSERKLÄRUNG_.93
FIGURE LIST
• Figure 1. Localization of Brandenburg in Germany (left) and eastern Brandenburg
districts (right) (1: Uckermark; 2: Barnim; 3: Märkisch-Oderland; 4: Oder-Spree).20
• Figure 2. Typical D. dendriticum biotope in eastern Brandenburg (Photo; R.Schuster). 21
• Fig. 3. Scheme of the SemiDry blotting sandwich.30
• Fig.4. Geographical position of the province of Trento.33
• Fig. 5 . Altitude range in the province of Trento.34
• Fig. 6. Land cover types in the province of Trento.34
• Fig. 7. Municipalities from which serum samples were collected.36
• Fig. 8. Ants in tetania (F. pratensis) on grass collected early in the morning in eastern
Brandenburg (Photo A. Broglia).37
• Fig. 9. Metacercariae distribution classes in ants collected in tetania.38
• Figure 10. Dissected gall bladder of sheep experimentally infected (infection dose: 2000
metacercariae) with D. dendriticum. Many D. dendriticum adults were present. 39
• Figure 11. Liver of sheep experimentally infected (infection dose: 2000 metacercariae).
The gall bladder has been removed.40
• Figure 12. OD values of IGg antibodies against D. dendriticum
excretory/secretory antigen throughout the experimental infection.43
• Figure 13. OD values of IGg antibodies against D. dendriticum So. antigen
throughout the experimental infection.44
• Figure 14. OD values of sera from sheep infected with F. hepática and
Paramphistomum spp. with relative cut-off values.47
• Figure 15. OD values of antibody response of lambs infected with N. battus. T.
colubriformis, H. contortus against E/S and So. antigen of/), dendriticum.48
IV
Figure 16. OD values of antibody response of lambs infected with repeated dose of
different nematode species L3 against excretory/secretory and So. antigens of D.
dendriticum.49
Figure 17. Pattern of proteic fractions of E/S and So. antigen of D. dendriticum
bySDS-PAGE.50
Figure 18. Immunoblot of/), dendriticum E/S antigen with sera of experimentally
infected lambs (inf. dose: 2000) through the study period.51
Figure 19. Immunoblot of D. dendriticum So. antigen with sera of experimentally
infected lambs (inf. dose: 2000) during the study period.52
Figure 20. Fmrnunoblot of D. dendriticum E/S antigen with field sera of sheep
infected with F. hepática (lane 1-6) and sera of lambs infected with D. dendriticum. 53
Figure 21. Immunoblot of D. dendriticum E/S and So. antigens with sera of lambs
infected with T. colubriformis (lane 1), N. battus (lane 2) and sera of lambs infected
withD. dendriticum.54
Figure 22. Seroprevalence rates of dicrocoeliosis (E/S antigen) in sheep flocks
in province of Trento (northeastern Italy).56
Figure 23. Seroprevalence rates of dicrocoeliosis (So. antigen) in sheep flocks
in province of Trento (northeastern Italy).56
Figure 24. An adult exemplar of D. chinensis found in a
male red deer (province of Trento).57
Figure 25. Detail of the cranial end of D. chinensis (left) and D. dendriticum (right).58
TABLE LIST
• Table 1. Serial dilution of antigen and sera in an ELISA plate.24
• Table 2. Sheep flocks, municipality, district, and number of serum samples.35
• Table 3. Prevalence, intensity, infection range and total metacercariae in collected ants. 37
• Table 4. Number of recovered flukes at necropsy and relationship with infection dose. 39
• Table 5. OD values in negative serum samples.42
• Table 6. OD values and coprological analysis results of sheep infected by
F. hepática and Paramphistomum spp.46
• Table 7. Seroprevalence rates of dicrocoeliosis (E/S and So. antigen) in sheep
flocks in the province of Trento (northeastern Italy).55
• Table 8. OD values of experimentally infected lambs sera against E/S antigen.89
• Table 9. OD values of experimentally infected lambs sera against So. antigen.89
• Table 10. OD values of sera from sheep naturally infected by F. hepática
against E/S and So. D. dendriticum antigens.89
• Table 11. OD values of sera from experimentally infected sheep with
Nematodirus battus. Trichostrongylus colubriformis, Haemonchus contortus,
single infection dose. 90
• Table 12. OD values of sera from experimentally infected sheep with
Dictyocaulus filaría, Oesophagostomum venulosum, Ostertagia ostertagi,
T. colubriformis, Cooperia curticei, N. battus, four repeated infection dose.90 |
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| author | Broglia, Alessandro 1975- |
| author_GND | (DE-588)132498448 |
| author_facet | Broglia, Alessandro 1975- |
| author_role | aut |
| author_sort | Broglia, Alessandro 1975- |
| author_variant | a b ab |
| building | Verbundindex |
| bvnumber | BV022356114 |
| ctrlnum | (OCoLC)179548594 (DE-599)BVBBV022356114 |
| dewey-full | 636.308969630756 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 636 - Animal husbandry |
| dewey-raw | 636.308969630756 |
| dewey-search | 636.308969630756 |
| dewey-sort | 3636.308969630756 |
| dewey-tens | 630 - Agriculture and related technologies |
| discipline | Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
| discipline_str_mv | Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
| edition | 1. Aufl. |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV022356114 |
| illustrated | Illustrated |
| index_date | 2024-07-02T17:01:33Z |
| indexdate | 2024-07-09T20:55:50Z |
| institution | BVB |
| isbn | 3938026987 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-015565523 |
| oclc_num | 179548594 |
| open_access_boolean | |
| owner | DE-188 |
| owner_facet | DE-188 |
| physical | VI, 93 S. Ill., graph. Darst. 21 cm |
| publishDate | 2006 |
| publishDateSearch | 2006 |
| publishDateSort | 2006 |
| publisher | DVG |
| record_format | marc |
| spelling | Broglia, Alessandro 1975- Verfasser (DE-588)132498448 aut Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis von Alessandro Broglia 1. Aufl. Gießen DVG 2006 VI, 93 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Berlin, Freie Univ., Diss., 2006 ELISA cabt Sheep Disease cabt Parasitosis cabt Diagnostic Techniques cabt Immunodiagnosis cabt (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015565523&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Broglia, Alessandro 1975- Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis ELISA cabt Sheep Disease cabt Parasitosis cabt Diagnostic Techniques cabt Immunodiagnosis cabt |
| subject_GND | (DE-588)4113937-9 |
| title | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |
| title_auth | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |
| title_exact_search | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |
| title_exact_search_txtP | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |
| title_full | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis von Alessandro Broglia |
| title_fullStr | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis von Alessandro Broglia |
| title_full_unstemmed | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis von Alessandro Broglia |
| title_short | Development of an indirect ELISA for the diagnosis of ovine dicrocoeliosis |
| title_sort | development of an indirect elisa for the diagnosis of ovine dicrocoeliosis |
| topic | ELISA cabt Sheep Disease cabt Parasitosis cabt Diagnostic Techniques cabt Immunodiagnosis cabt |
| topic_facet | ELISA Sheep Disease Parasitosis Diagnostic Techniques Immunodiagnosis Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015565523&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT brogliaalessandro developmentofanindirectelisaforthediagnosisofovinedicrocoeliosis |