Doctor of Philosophy / Division of Biology / Kristin Michel / The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

This thesis deals with biting midges of the genus Culicoides, which are tiny nematoceran insects belonging to the Diptera, and their parasites. Biting midges partake in transmissions of several parasitical organisms of various groups. Most notably, they are the vectors of several pathogenic viruses which might have a serious impact on livestock. However, the thesis deals with detection of parasites belonging to Trypanosomatids and Filarioids related to two independent biting midges collections - from the Czech Republic and from the Central African region. Apart from testing biting midges, there were carried out the tests on the occurrence of the same group of parasites within ruminant hoofed games in the Czech Republic. Our goal was to find out whether the same parasites occur with biting midges and their hosts (hoofed games) and whether biting midges could play a role as vectors. Biting midges are relatively overlooked group of haematophagous insects. Until recently, they had not been paid much attention, which changed with the spread of Bluetongue virus over Europe. This stimulated a widespread monitoring of biting midges in several European countries, including the Czech Republic. This helped to gain a large amount of data about the occurrence of biting midges near livestock. Nevertheless, there is no...

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Die Therapiemöglichkeiten für Pferde, die an Culicoides-Hypersensitivität (Sommerekzem) leiden, sind heute noch oft unbefriedigend. Sie reduzieren sich größtenteils auf symptomatische Behand-lungen und auf die Vermeidung der allergieauslösenden Allergene. Es existieren Studien, bei denen rekombinante Proteine in ihrer Wirksamkeit auf die Culicoides-Hypersensitivität beim Pferd untersucht wurden. Für die spezifische Immuntherapie gibt es in der Veterinärmedizin bislang keine oralen Präparate für eine mögliche sublinguale Therapie. Am Beispiel der Culicoides-Hypersensitivität soll letztlich ein Verfahren für die spezifische Immuntherapie entwickelt werden, bei dem die Peptide sowohl zur Diagnostik als auch Therapie allergischer Erkrankungen in der Veterinärmedizin genutzt werden können. In der vorliegenden Studie wurden erste Schritte in diese Richtung unternommen. Dafür wurden eine intrakutan zu applizierende Darreichungsform und eine für das Pferd neuartige, sublingual zu applizierende Arzneiform eingesetzt. Ausgewählte, synthetisch hergestellte Peptide von Mücken der Gattung Culicoides wurden bezüglich ihres Potentials für die spezifische Immuntherapie hinsichtlich der Sicherheit bei ihrer Anwendung im Pferd und Nutzung in der serologischen Diagnostik untersucht. Sieben ausgewählte Peptide, bestehend aus 30-35 Aminosäuren, wurden für in-vitro und in-vivo-Untersuchungen an gesunden (n=6) und an Sommerekzem erkrankten (n=12) Pferden eingesetzt. Nach einer Testphase auf die Verträglichkeit an klinisch gesunden Pferden, wurden die an Culicoides-Hypersensitivität erkrankten Tiere randomisiert in zwei Gruppen (intradermale und sublinguale Arzneiform) eingeteilt. Sie bekamen im Sonner, bei möglicher natürlicher Culicoides-Exposition die jeweilige Arzneiform über 21 Wochen hinweg verabreicht. Während dieser Zeit fanden in regelmäßigen Abständen Kontrollen des Gesundheitszustandes der Tiere statt, bei denen insbesondere klinische Symptome, Blutbilder und die Bildung spezifischer Antikörper erfasst wurden. Das Hautbild der Tiere wurde in Woche 1, 13 und 25 mithilfe eines Sommerekzem-Scores beurteilt. Die zwölf häufigsten vom Ekzem betroffenen Stellen am Pferde-körper wurden hierbei unabhängig voneinander bewertet. Die Besitzer der Tiere wurden mithilfe eines Fragebogens nach ihrer Meinung zur Entwicklung bzw. Ausprägung der klinischen Symptome und dem Befinden ihres Pferdes befragt. Zur Detektion spezifischer Antikörper im Serum der Tiere, unter Verwendung rekombinanter Pepti-de als Antigen, konnten für IgE und die IgG-Isotypen IgG1, IgG3/5 und IgG4/7 direkte bzw. indirekte ELISA etabliert werden. Des Weiteren wurde ein kommerzielles ELISA-Kit verwendet, um den Verlauf der IgE-Gesamtkonzentration während der Studienlaufzeit zu untersuchen. Aufgrund der geringen Studienteilnehmerzahl und vieler nicht verwendbarer Daten aus den ELISA Untersuchungen wurde die statistische Auswertung auf deskriptive Analysen beschränkt. Beide Applikationsarten wurden von den Studienteilnehmern gut toleriert und vertragen. Die Verabreichung der Sublingualtablette konnte durch die zuvor geschulten Pferdebesitzer eigenständig durchgeführt werden. Zu Beginn der Studie betrug der Dermatitisgrad 14 Punkte in der intradermalen Gruppe bzw. 13,67 Punkte in der sublingualen Gruppe. Am Ende der Studienlaufzeit lag der durchschnittliche Dermatitisgrad bei 40,33 bzw. 38,50 Punkten. Damit verschlechterte er sich während der Studienlaufzeit. Der Gehalt an gesamtem IgE im Serum der Probanden sank während des Untersuchungszeitraumes über beide Gruppen hinweg von 2,7 U/l auf 2,1 U/l. Bei den Tieren der Intradermalen Applikationsgruppe reduzierte sich das freie IgE dabei um 0,8 U/l und in der Sublingualen Gruppe um 0,3 U/l. Die Peptid-basierten ELISA zur spezifischen Serologie verschiedener Serum-Antikörperklassen waren nicht valide auswertbar. Die Untersuchungen zu peptidspezifischen IgE ergaben individuelle Verlaufsformen. Da keine Kontrollgruppe ohne Peptid-Applikation eingeschlossen wurde und die Studie in der Saison mit unkontrollierter, natürlicher Culicoides-Exposition durchgeführt wurde, ist keine Aussage zur Wirksamkeit der Behandlung auf klinische oder serologische Parameter möglich. Die entwickelte Sublingualtablette war durch ihre Form, Stabilität und Handhabung gut anwendbar und kann als Applikationsmethode in der Veterinärmedizin in Erwägung gezogen werden. Die synthetischen Peptide waren für den Einsatz in-vivo unschädlich und einsetzbar. Es bleibt zukünftigen Untersuchungen vorbehalten, ihr diagnostisches und therapeutisches Potential zur An-wendung bei Culicoides-Hypersensitivität und gegebenenfalls weiteren allergisch bedingten Erkrankungen des Pferdes weiter zu evaluieren. Aufgrund von limitierenden Faktoren, wie der sehr kleinen Zahl an Studienteilnehmern, dem Fehlen einer Placebogruppe und der Durchführung während der Expositionszeit hat die vorliegende Arbeit insgesamt präliminären Charakter.:Inhaltsverzeichnis Verzeichnis der verwendeten Abkürzungen 1 Einleitung 2 Literaturübersicht 2.1 Allergie 2.1.1 Allergene 2.1.1.1 Charakteristika von Allergenen (Was macht eine Substanz zum Allergen) 2.1.1.2 Kreuzreaktivitäten 2.1.2 Typ-I-Allergie 2.1.2.1 Produktion sensibilisierender Antikörper bei Erstkontakt 2.1.2.2 Ausbildung allergischer Reaktionen bei Folgekontakt 2.1.3 Immunglobulin-System des Pferdes 2.1.4 Potenzielle Bedeutung der IgG-Isotypen bei verschiedenen Erkrankungen 2.2 Das Sommerekzem des Pferdes 2.2.1 Epidemiologie, Ätiologie und Pathogenese 2.2.2 Bedeutende Allergene 2.2.3 Diagnostische Ansätze 2.2.3.1 In-vivo-Test (Intradermal Test) 2.2.3.2 In-vitro-Tests 2.2.3.3 Nachweis von IgE 2.2.3.4 Funktionelle in-vitro-Tests 2.2.4 Therapeutische Ansätze 2.2.4.1 Vermeidung Allergenkontakte, medikamentöse Therapieversuche 2.2.4.2 Allergenspezifische Immuntherapie (ASIT) 2.2.4.3 Desensibilisierung mit nativen Allergenextrakten 2.3 Wechsel von Allergenextrakten auf die molekulare Ebene der Allergene - Bedeutung für Diagnostik und Therapie 2.4 Einsatz synthetischer Peptide in der ASIT 3 Geräte, Material und Methoden 3.1 Geräte 3.2 Material 3.2.1 Klinikbedarf 3.2.2 Laborbedarf 3.2.3 Reagenzien, Puffer und Lösungen 3.2.4 Allergene (Peptide) - Auswahl und Synthese 3.2.5 Antikörper 3.3 Tiere 3.3.1 Gesunde Kontrollgruppe 3.3.2 An Sommerekzem erkranke Studienteilnehmer 3.4 Methoden 3.4.1 Blutentnahme und weitere Verarbeitung 3.4.2 Herstellung der arzneilichen Formulierungen 3.4.2.1 Arzneiliche Formulierung für die intradermale Applikation 3.4.2.2 Arzneiliche Formulierung für die orale Applikation 3.4.3 Prüfung der Unbedenklichkeit an klinisch gesunden Pferden 3.4.3.1 Durchführung der Unbedenklichkeitsprüfung 3.4.3.2 Kontrollen des Gesundheitsstatus 3.4.4 Verabreichung der hergestellten Applikationslösungen an Patienten 3.4.4.1 In-Vivo-Test (Intradermaltest) 3.4.4.2 Intradermale Verabreichung der individuellen Peptidlösungen 3.4.4.3 Orale Verabreichung der individuellen Peptidlösungen 3.4.5 Klinische Veränderungen infolge der SIT 3.4.5.1 Dokumentation und klinische Beurteilung der Symptome 3.4.5.2 Fragebogen zur objektiven Einschätzung durch die Patientenbesitzer 3.4.6 In-Vitro-Test (ELISA) 3.4.6.1 Prinzip des ELISA (Enzyme-linked Immunosorbent Assay) 3.4.6.2 Methode des eingesetzten ELISA 3.4.7 Statistik 4 Ergebnisse 4.1 Auswahl und Einsatz synthetischer Peptide für die Diagnostik und Therapie des equinen Sommerekzems 4.1.1 Arzneiliche Formulierung für den Intrakutantest 4.1.2 Arzneiliche Formulierung für die Intradermale Applikation 4.1.3 Arzneiliche Formulierung für die orale Applikation 4.2 Klinische Veränderungen infolge der SIT 4.2.1 Dermatitisgrad– Beurteilung des Hautbildes 4.2.2 Veränderte Symptomatik in der Studienlaufzeit – Einschätzung der Besitzer 4.3 Labordiagnostische Veränderungen infolge der SIT 4.3.1 Differentialblutbild 4.3.2 Ergebnisse des IgE-ELISA 4.3.3 Ergebnisse des IgG-ELISA 4.4 Vergleich der Werte des IgE ELISA mit den klinischen Symptomen (Score) 5 Diskussion 5.1 Einsatz synthetisch hergestellter Peptide in der Diagnostik und Therapie 5.2 Therapeutischer Einsatz synthetisch hergestellter Peptide für die intradermale Applikation 5.3 Etablierung einer Arzneiform für die sublinguale Therapie beim Pferd 5.4 Klinische Veränderungen in Folge der SIT 5.5 Therapiebegleitende Labordiagnostik 6 Zusammenfassung 7 Summary 7 Literaturverzeichnis 8 Anhang 8.1 Tierkarte der Vorstudie mit klinisch gesunden Pferden 8.2 Besitzereinverständniserklärung und Datenschutzerklärung am Beispiel der Intradermalen Verabreichung 8.3 Tierkarte zur Erfassung des Gesundheitsstatus und Symptomentwicklung 8.4 Protokoll zur Durchführung des Intradermaltests 8.5 Protokoll zur Evaluierung des Dermatitisscores der an SE erkankten Pferde 8.6 Fragebogen zur Besitzerbefragung am Ende der Studie 8.7 Protokoll zur Durchführung eines ELISA am Beispiel der Untersuchung auf peptidspezifisches IgE im Patientenserum 9 Danksagung / The treatment options for horses suffering from Culicoides hypersensitivity are still often unsatisfac-tory. They are mainly reduced to symptomatic treatments and avoiding the allergy-triggering aller-gens. In various studies recombinant proteins were examined for their effectiveness on Culicoides hyper-sensitivity in horses. However, there are currently no oral preparations available in veterinary medicine for specific immunotherapy. Using Culicoides hypersensitivity as an example, a process for specific immunotherapy shall ulti-mately be developed in which the peptides can be used for both, the diagnosis and therapy od allergic diseases in veterinary medicine. The present study tool the first steps in this direction. For this purpose, an intracutaneously adminis-tered dosage form and a sublingually administered dosage form, which is new for horses, were used. Selected, synthetically produced peptides from mosquitoes of the genus Culicoides were ex-amined regarding their potential for specific immunotherapy in terms of their safety after application in horses in-vivo and their use in serology. Seven selected peptides, consisting of 30-35 amino acids, were used for in-vitro and in-vivo studies on healthy (n=6) and horses suffering from Culicoides hypersensitivity (n=12). After a test phase for tolerability in clinically healthy horses, the animals suffering from Culicoides hypersensitivity were randomly divided into two groups (intradermal and sublingual dosage forms). They were administered the respective dosage form for 21 weeks during the summer season with possible natural Culicoides exposure. During this time, the animals' health status was checked at regular intervals, in particular clinical symptoms, blood counts and the formation of specific antibodies were recorded. The skin appearance of the animals was assessed at weeks 1, 13 and 25 using a dermatitis score. The twelve most common areas on the horse's body affected by eczema were evaluated inde-pendently of each other. The owners of the animals were asked using a questionnaire about their opinion on the development or severity of their horse's clinical symptoms and well-being. To detect specific antibodies in the serum of the animals, using recombinant peptides as antigen, direct and indirect ELISAs were established for IgE and the IgG isotypes IgG1, IgG3/5 and IgG4/7. Furthermore, a commercial ELISA kit was used to examine the course of the total IgE concentration during the study period. Due to the small number of study participants and numerous invalid data from the ELISA studies, the statistical analysis was limited to descriptive analyses. Both types of application were well tolerated by the study participants. The administration of the sublingual tablet could be carried out by previously trained horse owners. At the start of the study, the dermatitis score was 14 points in the intradermal group and 13.67 points in the sublingual group. At the end of the study period, the average dermatitis grades were 40.33 and 38.50 points, respectively, and deteriorated during the course of this study. The owner survey revealed a slight improvement in symptoms compared to previous years. The level of total, free IgE in the test subjects' serum fell from 2.7 U/l to 2.1 U/l during the study period. In the animals in the intradermal application group, the free IgE was reduced by 0.8 U/l and in the sublingual group by 0.3 U/l. Peptide-based ELISA to determine different serum antibody isotypes binding the peptides could not be evaluated reliably. Peptide-specific IgE revealed individ-ual variations. Due to the lack of a control group without peptide application and conduction of the study during the season with uncontrolled, natural Culicoides exposure conclusions of effectiveness regarding clinical disease or serological effects cannot be drawn. The sublingual tablet developed was easy to use due to its shape, stability and handling and can be considered as an application method in veterinary medicine. The synthetic peptides were harmless and usable for in-vivo application. It remains reserved for future studies to further evaluate its diagnostic and therapeutic potential for use in equine Culicoides hypersensitivity and possibly other allergic diseases in horses. Due to limiting factors such as the very small number of study participants, the lack of a placebo group and the implementation during the exposure period, the present work is of a preliminary nature.:Inhaltsverzeichnis Verzeichnis der verwendeten Abkürzungen 1 Einleitung 2 Literaturübersicht 2.1 Allergie 2.1.1 Allergene 2.1.1.1 Charakteristika von Allergenen (Was macht eine Substanz zum Allergen) 2.1.1.2 Kreuzreaktivitäten 2.1.2 Typ-I-Allergie 2.1.2.1 Produktion sensibilisierender Antikörper bei Erstkontakt 2.1.2.2 Ausbildung allergischer Reaktionen bei Folgekontakt 2.1.3 Immunglobulin-System des Pferdes 2.1.4 Potenzielle Bedeutung der IgG-Isotypen bei verschiedenen Erkrankungen 2.2 Das Sommerekzem des Pferdes 2.2.1 Epidemiologie, Ätiologie und Pathogenese 2.2.2 Bedeutende Allergene 2.2.3 Diagnostische Ansätze 2.2.3.1 In-vivo-Test (Intradermal Test) 2.2.3.2 In-vitro-Tests 2.2.3.3 Nachweis von IgE 2.2.3.4 Funktionelle in-vitro-Tests 2.2.4 Therapeutische Ansätze 2.2.4.1 Vermeidung Allergenkontakte, medikamentöse Therapieversuche 2.2.4.2 Allergenspezifische Immuntherapie (ASIT) 2.2.4.3 Desensibilisierung mit nativen Allergenextrakten 2.3 Wechsel von Allergenextrakten auf die molekulare Ebene der Allergene - Bedeutung für Diagnostik und Therapie 2.4 Einsatz synthetischer Peptide in der ASIT 3 Geräte, Material und Methoden 3.1 Geräte 3.2 Material 3.2.1 Klinikbedarf 3.2.2 Laborbedarf 3.2.3 Reagenzien, Puffer und Lösungen 3.2.4 Allergene (Peptide) - Auswahl und Synthese 3.2.5 Antikörper 3.3 Tiere 3.3.1 Gesunde Kontrollgruppe 3.3.2 An Sommerekzem erkranke Studienteilnehmer 3.4 Methoden 3.4.1 Blutentnahme und weitere Verarbeitung 3.4.2 Herstellung der arzneilichen Formulierungen 3.4.2.1 Arzneiliche Formulierung für die intradermale Applikation 3.4.2.2 Arzneiliche Formulierung für die orale Applikation 3.4.3 Prüfung der Unbedenklichkeit an klinisch gesunden Pferden 3.4.3.1 Durchführung der Unbedenklichkeitsprüfung 3.4.3.2 Kontrollen des Gesundheitsstatus 3.4.4 Verabreichung der hergestellten Applikationslösungen an Patienten 3.4.4.1 In-Vivo-Test (Intradermaltest) 3.4.4.2 Intradermale Verabreichung der individuellen Peptidlösungen 3.4.4.3 Orale Verabreichung der individuellen Peptidlösungen 3.4.5 Klinische Veränderungen infolge der SIT 3.4.5.1 Dokumentation und klinische Beurteilung der Symptome 3.4.5.2 Fragebogen zur objektiven Einschätzung durch die Patientenbesitzer 3.4.6 In-Vitro-Test (ELISA) 3.4.6.1 Prinzip des ELISA (Enzyme-linked Immunosorbent Assay) 3.4.6.2 Methode des eingesetzten ELISA 3.4.7 Statistik 4 Ergebnisse 4.1 Auswahl und Einsatz synthetischer Peptide für die Diagnostik und Therapie des equinen Sommerekzems 4.1.1 Arzneiliche Formulierung für den Intrakutantest 4.1.2 Arzneiliche Formulierung für die Intradermale Applikation 4.1.3 Arzneiliche Formulierung für die orale Applikation 4.2 Klinische Veränderungen infolge der SIT 4.2.1 Dermatitisgrad– Beurteilung des Hautbildes 4.2.2 Veränderte Symptomatik in der Studienlaufzeit – Einschätzung der Besitzer 4.3 Labordiagnostische Veränderungen infolge der SIT 4.3.1 Differentialblutbild 4.3.2 Ergebnisse des IgE-ELISA 4.3.3 Ergebnisse des IgG-ELISA 4.4 Vergleich der Werte des IgE ELISA mit den klinischen Symptomen (Score) 5 Diskussion 5.1 Einsatz synthetisch hergestellter Peptide in der Diagnostik und Therapie 5.2 Therapeutischer Einsatz synthetisch hergestellter Peptide für die intradermale Applikation 5.3 Etablierung einer Arzneiform für die sublinguale Therapie beim Pferd 5.4 Klinische Veränderungen in Folge der SIT 5.5 Therapiebegleitende Labordiagnostik 6 Zusammenfassung 7 Summary 7 Literaturverzeichnis 8 Anhang 8.1 Tierkarte der Vorstudie mit klinisch gesunden Pferden 8.2 Besitzereinverständniserklärung und Datenschutzerklärung am Beispiel der Intradermalen Verabreichung 8.3 Tierkarte zur Erfassung des Gesundheitsstatus und Symptomentwicklung 8.4 Protokoll zur Durchführung des Intradermaltests 8.5 Protokoll zur Evaluierung des Dermatitisscores der an SE erkankten Pferde 8.6 Fragebogen zur Besitzerbefragung am Ende der Studie 8.7 Protokoll zur Durchführung eines ELISA am Beispiel der Untersuchung auf peptidspezifisches IgE im Patientenserum 9 Danksagung

A large epizootic of the vector-borne disease bluetongue occurred in northern Europe from 2006-2009, costing the economies of the infected countries several hundreds of millions of euros. During this time, the United Kingdom (UK) was exposed to the risk of bluetongue by windborne incursions of infected Culicoides biting midges from the northern coast of mainland Europe. The first outbreaks which occurred in the UK in 2007 were attributed to this cause. Although bluetongue virus (BTV) no longer appears to be circulating in northern Europe, it is widely suggested that it and other midge-borne diseases may emerge again in the future, particularly under a changing climate. Spread of BTV is strongly influenced by the weather and climate however limited use has been made of meteorologically based models to generate predictions of its spread to the UK. The extent to which windborne BTV spread can be modelled at timescales from days to decades ahead, to inform tactical and strategic decisions taken to limit its transmission, is therefore examined here. An early warning system has been developed to predict possible incursion events on a daily timescale, based on an atmospheric dispersion model adapted to incorporate flight characteristics of the Culicoides vectors. The system’s warning of the first UK outbreak in September 2007 was found to be greatly beneficial to the UK livestock industry. The dispersion model is also shown to be a useful post-outbreak epidemiological analysis tool. A novel approach has been developed to predict BTV spread into the UK on climate-change timescales as dispersion modelling is not practical over extended periods of time. Using a combination of principal component and cluster analyses the synoptic scale atmospheric circulations which control when local weather conditions are suitable for midge incursions were determined. Changes in the frequency and timing of these large scale circulations over the period 2000 to 2050 were then examined using an ensemble of regional climate model simulations. The results suggest areas of UK under the influence of easterly winds may face a slight increase in risk and the length of the season where temperatures are suitable for BTV replication is likely to increase by around 20 days by 2050. However a high level of uncertainty is associated with these predictions so a flexible decision making approach should be adopted to accommodate better information as it becomes available in the future.

This thesis focuses on the development of protozoan parasites from Leishmania donovani complex in their insect vectors and summarizes results of five parts of the project I participated in during my Ph.D. studies. Sand flies of genera Phlebotomus and Lutzomyia are the only proven vectors of leishmaniasis, however, the role of alternative vectors, like ticks, fleas and biting midges is frequently discussed in the literature. In this work, we showed that Eurasian species of biting midge Culicoides nubeculosus does not support late stage infections of L. major and L. infantum. We also demonstrated that microscopical observation of Leishmania promastigotes in the digestive tract of bloodfeeding arthropods remains a crucial method for any conclusion about the vector competence of the suspected insect. In the second part of our study were compared the life-cycle parameters and vector competence of two Ethiopian P. orientalis colonies for L. donovani. Marked differences between colonies were found in life-cycle parametes, however, molecular analyses did not reveal any genetic differences. Experimental infections showed that both P. orientalis colonies are very susceptible to L. donovani infection and even the lowest infective dose tested (2 × 103 promastigotes/ml; corresponding to 1-2 promastigotes) was...

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:34:20Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:42:38Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) / Made available in DSpace on 2017-04-13T19:42:38Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) Previous issue date: 2005-10-14 / Sem bolsa / Dermatite alérgica estacional foi estudada em um rebanho de ovinos Hampshire Down em um estabelecimento no município de Capão do Leão, Rio Grande do Sul. A epidemiologia, sinais clínicos e lesões macroscópicas foram observadas no estabelecimento verificando-se a época de ocorrência da enfermidade, a idade e sexo dos animais afetados e localização das lesões. A patologia microscópica foi estudada em biopsias de pele de ovinos afetados. Para a verificação da etiologia, insetos foram capturados com aspiradores entomológicos, semanalmente, das 16h00minh às 09h00minh, entre os meses de janeiro a abril de 2005. Os dados relativos à temperatura média, umidade relativa do ar e velocidade média dos ventos na região de ocorrência da doença foram obtidos na Estação Agro-climatológica da Universidade Federal de Pelotas. A prevalência da doença no período estudado foi de 40% e animais de qualquer idade e ambos os sexos foram afetados. A enfermidade ocorreu principalmente entre dezembro e março e alguns animais permaneceram com lesões durante todo o ano. Os sinais clínicos eram de inquietação e prurido e as lesões ocorriam principalmente nas orelhas, ao redor dos olhos e na região ventral do abdômen, caracterizando-se pela presença de eritema e pápulas, nos casos agudos, e espessamento da pele com rachaduras, exsudação, formação de crostas, e alopecia, nos casos crônicos; esses freqüentemente apresentavam infecções secundárias. As lesões histológicas eram principalmente de dermatite eosinofílica perivascular, observando-se, também, hiperqueratose, acantose e presença de pústulas sub corneais ou intra-epidérmicas nos casos crônicos. Os insetos capturados no período de estudo da doença foram identificados como Anopheles albitarsis e Culicoides insignis. Durante a captura observou-se que os animais apresentavam prurido intenso quando picados por Culicoides, mas não por Anopheles. As temperaturas média mínima e máxima no período, foram de 17ºC e 28ºC, respectivamente, e a velocidade média dos ventos foi de 12,84km/h. Ambas as espécies de insetos capturados têm hábitos noturnos aproximando-se dos animais para hematofagia em torno de 30min após o ocaso do sol.Testes intradérmicos realizados com antígenos obtidos a partir desses dípteros revelaram reação de hipersensibilidade aos antígenos de C. insignis. Os resultados obtidos indicam que a enfermidade estudada ocorre em conseqüência de hipersensibilidade imediata causada pela picada desse díptero. / A seasonal dermatitis was studied in a Hampshire Down flock in a farm at the minicipality of Capão do Leão, state of Rio Grande do Sul, southern Brazil. Frequency and seasonal distribution of the disease, breed, sex and age of affected sheep, and clinical signs, macroscopic pathology and distribution of the lesions were obtained by visiting the farm. Histological lesions were studied in skin biopsies of affected sheep. Biting insects were collected weekly between 4:00 p.m. to 9:00 a.m., from January to April of 2005 to identify the etiological agent of disease. Data about temperature, humidity and wind speed in the region where the disease occurs were obtained in the agro climatologic Station of Pelotas Federal University. The prevalence of disease during the study period was 40% and the age of affected animals was variable. The disease occurs between December and March, but some animals stayed with lesions during the whole year. Clinical signs were characterized by pruritus and skin lesions in the ears, around the eyes, and ventral abdomen. Erythema and papules were observed in acute cases. In chronic cases, the skin was thicken, exsudative, crusty and alopecic, frequently with secondary infections. Histological lesions of affected sheep were characterized by perivascular eosinofilic dermatitis. Hyperkeratosis, acanthosis and intraepidermal pustules were observed in chronic lesions. Anopheles albitarsis and Culicoides insignis were captured during the observation period. Sheep showed pruritus mainly during de Culicoides bites. The mean minimum and maximum temperature in the period were 17ºC e 28ºC, respectively, and the mean speed wind was 12,84km/h. Both insects were caught when they approached the sheep bait 30minutes after sunset. Intradermal tests performed with de antigens of the insect extracts showed hypersensitivity reaction to C.insignis antigens. Based on the results it was suggested that the disease occurs as a consequence of an immediate hypersensitivity caused by C. insignis bite.

Culicoides molestus (Skuse) is the major species of biting midge that plagues human comfort in the estuarine Gold Coast region of southeast Queensland. Local authorities have initiated a search for an effective, non-chemical means of control, that would minimize human-midge interaction. The effectiveness of a program to control an organism, such as a biting midge, is dependent upon knowledge of the biology of the particular organism of interest. This project revolved examines the lifecycle of C. molestus in detail. It addresses questions regarding the location, seasonal distribution, and dispersal of its juvenile stages in the sand of infested beaches, and their response to chemical treatment, the monthly and annual cycles of the adult midge, and the possibilities of achieving laboratory oviposition, as a first step to laboratory colonisation. The distribution of eggs, larvae and pupae of C. molestus was found to be mostly concentrated around, but below, mean tide level. They also occurred well below the mean tide level. Eggs and larvae have been recovered from as deep as 10 cm in the sand. A seasonal study of the juveniles of this species indicated that they were more strongly influenced by tides than seasons. After a routine pest-control larviciding treatment, a beach recolonisation study revealed that beaches become suitable for oviposition approximately two months after treatment. Large larvae invaded the sprayed areas within days of treatment, which suggests the existence of a refuge outside of the reach of the insecticide. Larvae found in clean (egg- and larva-free), isolated sand containers, placed on the study beach, indicated that larvae could swim in or on the water as a way of moving around the beach. Extended bite-rate studies highlighted the existence of four peaks in adult midge biting activity during the course of a year, around the mid seasons. The strongest peaks of activity were found to be in autumn and spring, but the data suggest that the species undergoes four generations in a year. Through a series of trial-and-error experiments, oviposition under laboratory conditions was achieved. Although the time from blood-feeding to egg maturation is not yet well determined, it occurs within an eight day mean survival period. Blood quality appears critical for adult blood-fed midge survival. Midges fed on the blood of a volunteer who was frequently exposed to midge bites do not live long enough to mature its eggs. The partial ovarial development of one unfed adult female, reared in the laboratory, indicates that C. molestus is facultatively anautogenous.

La découverte du rôle des insectes en tant que vecteurs de pathogènes, établi depuis plus d'un siècle, a été l'élément moteur de la discipline " entomologie médicale et vétérinaire ". Malgré le succès de nombreuses campagnes de prévention et de programmes de lutte, nous assistons depuis une trentaine d'années à l'émergence et à la recrudescence de maladies à transmission vectorielle. Le virus de la fièvre catarrhale ovine (FCO) (Reoviridae : Orbivirus) est un très bon exemple de virus émergent en Europe dont les mécanismes de transmission sont encore peu connus dans cette région. Ce virus est transmis par des moucherons hématophages du genre Culicoides (Diptera : Ceratopogonidae) aux ruminants sauvages et domestiques. En Europe, la FCO a été pendant longtemps considérée comme une maladie exotique. À partir de 1998, plusieurs incursions apparaissent dans l'ouest du bassin méditerranéen en lien avec la remontée vers le nord de populations de Culicoides imicola, le principal vecteur afrotropical. À partir d'août 2006, l'apparition et la transmission du sérotype 8 dans le nord de l'Europe, dans des zones où C. imicola est absent, révèle l'importance des espèces autochtones et la nécessité de comprendre leur rôle vecteur. Ce travail s'intéresse aux mécanismes de transmission du virus de la FCO en Europe non méditerranéenne, en i) présentant un état de l'art de la biologie et l'écologie des Culicoides adultes, ii) en évaluant les conditions possibles d'utilisation de pièges pour estimer le taux de piqûre et iii) en décrivant les comportements trophiques pour les espèces d'intérêt vétérinaire.

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-07-31T13:08:07Z No. of bitstreams: 1 Dissertação - Emanuelle de Sousa Farias.pdf: 2780340 bytes, checksum: bac6d0e67ce460607bfe6e587aa2068a (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-03T15:22:46Z (GMT) No. of bitstreams: 1 Dissertação - Emanuelle de Sousa Farias.pdf: 2780340 bytes, checksum: bac6d0e67ce460607bfe6e587aa2068a (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-03T15:26:45Z (GMT) No. of bitstreams: 1 Dissertação - Emanuelle de Sousa Farias.pdf: 2780340 bytes, checksum: bac6d0e67ce460607bfe6e587aa2068a (MD5) / Made available in DSpace on 2015-08-03T15:26:45Z (GMT). No. of bitstreams: 1 Dissertação - Emanuelle de Sousa Farias.pdf: 2780340 bytes, checksum: bac6d0e67ce460607bfe6e587aa2068a (MD5) Previous issue date: 2014-02-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The biting midges Culicoides are vectors of some etiological agents to humans and other mammals. There are just a few studies in Amazon basin about that insects and the diversity and richness of this genus is underestimated. The aims of this study were to describe the fauna of midges in a rural settlement in Amazon and to understand how the anthropic effects may affect the richness and abundance of these putative local vectors. Collections were done by CDC light traps in some environment as primary forest, forest borders, secondary forest, fruit garden and peridomiciliary area with animal shelters. We identified 6.093 individuals, distributed in 42 species, 39 morphotypes of seven subgenera and /or species groups and five new species of Reticulatus group. We found 13 new occurrence to Amazonas State C. aldomani, C. batesi, C. brownie, C. debilipalpis, C. flavivenula , C. franklini, C. glabellus, C. guamai, C. guttatus, C. ocumarensi, C. paramaruim, C. pusilloides and C. tidwelli, and two new occourence to the country, the species C. brownie and C. tidwelli. Our results show that different anthropic levels affect the abundance and diversity of midges. The diversity was high in less anthropized environments. The peridomiciliary areas had a high richness and abundance of Culicoides. / Os Culicoides são insetos transmissores de agentes patogênicos ao homem e outros animais. Na região amazônica existem poucos estudos sobre esses insetos e o número de espécies é subestimado. Este estudo objetivou conhecer a fauna de Culicoides de um assentamento rural na Amazônia e verificar se o efeito antrópico afeta a diversidade e abundância dos mesmos. As coletas foram feitas com armadilhas luminosas do tipo CDC em ambientes de floresta, borda de floresta, capoeira e peridomicílio. Foram identificados 6.093 indivíduos, distribuídos em 42 espécies, 39 morfótipos de sete subgênero/grupo e cinco espécies novas do grupo Reticulatus. Foram encontrados 13 novos registros de espécies de Culicoides para o estado do Amazonas: C. aldomani, C. batesi, C. brownie, C. debilipalpis, C. flavivenula , C. franklini, C. glabellus, C. guamai, C. guttatus, C. ocumarensi, C. paramaruim, C. pusilloides e C. tidwelli, sendo C. brownie e C. tidwelli novos registros para o Brasil. Os resultados indicam que diferentes graus de antropização do ambiente influenciaram a abundância e diversidade dos Culicoides, cuja diversidade foi alta em ambientes menos antropizados. Ambientes totalmente antropizados, como peridomicílio, apresentaram maior abundância e riqueza.