Japanese yellowtail Seriola quinqueradiata has been commercially farmed in Japan since the 1940s. In comparison, sea-cage farming of yellowtail kingfish Seriola lalandi in Australia is still developing, with commercial production commencing in 1998. In Australia, S. lalandi is parasitised by Zeuxapta seriolae and Benedenia seriolae. In Japan, S. quinqueradiata is parasitised by Heteraxine heterocerca and B. seriolae. These monogeneans affect industries in both countries and management of these parasites is required to prevent impacts on fish health and commercial losses. I investigated efficacy (% reduction of mean parasite abundance) for orally administered praziquantel, fenbendzole and oxfendazole against Z. seriolae and B. seriolae on S. lalandi and the efficacy of orally administered praziquantel and febantel against H. heterocerca and B. seriolae on S. quinqueradiata. Medications were administered to fish by surface coating feed pellets or via direct intubation of the stomach. Seriola lalandi administered fenbendazole and oxfendazole by surface coating of feed had lower abundance of the gill parasite Z. seriolae. Seriola quinqueradiata intubated with febantel had lower abundance of the gill parasite H. heterocerca. Neither fenbendazole nor oxfendazole administered to S. lalandi in Australia, nor febantel administered to S. quinqueradiata in Japan resulted in a lower abundance of the skin parasite B. seriolae. Praziquantel was first administered to S. lalandi by surface coating of feed. Fish rejected medicated feed, suggesting praziquantel affected its palatability. Fish treated with feed medicated with praziquantel had fewer Z. seriolae and B. seriolae than untreated fish. Praziquantel administered to S. lalandi by intubation allowed a more accurate dose to be tested without differential feeding or reduced palatability obstructing results, and resulted in fewer Z. seriolae (99.5-100 % reduction) and B. seriolae (91 – 97.7 % reduction). Intubated praziquantel also led to fewer recruitment life stages of Z. seriolae and B. seriolae, even at low doses, but did not completely eliminate them from S. lalandi. Praziquantel administered to S. lalandi alone and combined with cimetidine had high efficacy (>99%) against Z. seriolae. In comparison, praziquantel administered alone resulted in fewer B. seriolae (68.3 – 69.7 % reduction) than the same doses of praziquantel combined with cimetidine (36.9 – 40.9 % reduction). A 90.4 -100% reduction in H. heterocerca was achieved when praziquantel was administered by intubation to S. quinqueradiata in Japan but there was only a 22-77.8 % reduction in B. seriolae. The dose of PZQ (150 mg kg⁻¹ body weight day⁻¹ for 3 days) on the label of a commercially available product used to treat B. seriolae in Japanese aquaculture resulted in a 50.9% reduction against B. seriolae, but completely eliminated H. heterocerca. In trials against Z. seriolae and B. seriolae on S. lalandi in South Australia, I also screened 27 other anthelmintics and antiparasitics from the chemical groups: amprolium derivatives, benzimidazoles, benzyl ureas, diphosphate salts, imidazothiazoles, macrocyclic lactones, nitromidazoles, organophosphates, piperazines, salicylanilides, substituted phenols and tetrahydropyrimidines. Of these, only the benzimidazole, albendazole, was effective against Z. seriolae and none appeared to have an effect against B. seriolae. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1459172 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2010

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infections by gastrointestinal nematodes are among the main diseases of the ruminants around the world. These infections cause - in their clinical or subclinical courses a decrease of the productivity of the herds, thus, the economic viability of the ruminant farms is directly related to an adequate control of these parasites. Although the benefits of the use of several practices of alternative control of parasites are proven and, despite of that these practices have been improved through the years, in general, the use of efficacious antiparasitic drugs is imperative to control satisfactorily the helminths of the ruminants. However, the development of parasite resistance to these drugs has become common and, in some cases, parasitic populations are multiple resistant to several classes of anthelmintics and endectocides, and it makes very difficult to control the helminthosis adequately. In this study are shown some experiments conducted with the aim of to find ways to restore the efficacy of the treatments, for recovering the usefulness of active ingredients for which the gastrointestinal nematodes of ruminants became resistant. Efficacy tests of the drugs were made using Fecal Egg Count Reduction Tests (FECRT) and larvae cultures, for the identification of the genus of nematodes existing, before and after the treatments. We evaluated the anthelmintic action of high concentrated (long acting) drugs, overdoses (high dosages), commercial combinations of drugs and combinations of chemicals, formulated with basis in previous efficacy tests for each active ingredient alone. These combinations, done from drugs sold separately, gave us the best results. Based in these observations, we are proposing a new approach regarding helminth control in multi-resistant populations, by using combinations of drugs with different mechanisms of action on the parasites, and taking advantage of the efficacy tests, performed before the choose of drugs, and then, to prove the results obtained with the combinations done particularly for each herd. / As infecções por nematódeos do trato gastrintestinal estão entre as principais enfermidades de ruminantes ao redor do mundo. Essas infecções, clínicas ou subclínicas, impactam negativamente a produtividade dos rebanhos, e a viabilidade econômica dos mesmos está diretamente relacionada a um controle adequado desses parasitas. Embora estejam comprovados os benefícios de utilizarem-se diversas práticas de controle alternativo de parasitas, as quais vêm sendo aprimoradas ao longo dos anos, em geral, a utilização de fármacos antiparasitários eficazes sobre os helmintos que infectam os ruminantes ainda é essencial para que se obtenham resultados satisfatórios. No entanto, o desenvolvimento de resistência parasitária a tais fármacos tem se tornado cada vez mais comum e, em alguns casos, as populações de parasitas já apresentam resistência múltipla a várias classes de anti-helmínticos e endectocidas, comprometendo o adequado controle das helmintoses. Neste estudo apresentam-se experimentos que foram conduzidos com o objetivo de encontrarem-se meios de restabelecer a eficácia dos tratamentos, recuperando-se a utilidade de princípios ativos para os quais os nematódeos gastrintestinais de rebanhos de ruminantes já tenham desenvolvido resistência. Foram utilizados testes de eficácia com base na redução da contagem de ovos nas fezes dos animais após os tratamentos (FECRT), e em culturas de larvas para a identificação dos gêneros de nematódeos presentes nessas amostras. Avaliaram-se os efeitos anti-helmínticos de fármacos de alta concentração (longa ação), de superdosagens, de combinações comerciais e de combinações de drogas elaboradas com base em estudos prévios de eficácia das mesmas (isoladamente), utilizando-se para isso, fármacos comercializados separadamente. Os melhores resultados foram obtidos, justamente, com estas últimas. Propõe-se, a partir destas constatações, uma nova abordagem em relação ao controle antiparasitário em populações de helmintos multi-resistentes, utilizando-se a combinação de diferentes fármacos (com princípios ativos e mecanismos de ação distintos), e valendo-se de testes de eficácia para a escolha dos fármacos e para a comprovação dos resultados obtidos com suas combinações especificamente em cada rebanho.

Σκοπός: Ο προσδιορισμός των επιπέδων συγκέντρωσης καταλοίπων κτηνιατρικών παρασιτοκτόνων ουσιών (ανθελμινθικών), σε νωπό γάλα μηρυκαστικών από περιοχές της Νοτίου Ελλάδος και η εκτίμηση της πρόσληψής τους από τον άνθρωπο. Συγκεκριμένα εξετάσθηκαν οι ουσίες, Albendazole, Febantel, Fenbendazole, Mebendazole και κάποιοι μεταβολίτες τους (Albendazole sulfoxide, Albendazole sulfone, Fenbendazole sulfone) στο νωπό γάλα προβάτων, αιγών και βοοειδών. Μέθοδοι: Χημική ανάλυση δειγμάτων γάλακτος, με τη βοήθεια Υγρής Χρωματογραφίας Υψηλής Απόδοσης και ανιχνευτή Συστοιχίας Διόδων, Υπεριώδους (UV). Χρήση βάσης δεδομένων σχετικά με την εκτίμηση της ημερήσιας κατανάλωσης γάλακτος και Φέτας (λήψη προσωπικών συνεντεύξεων, συμπλήρωση ερωτηματολογίων, συχνοτήτων κατανάλωσης τροφίμων, από δείγμα 723 μαθητών ηλικίας 10-12 ετών, από τη Νότια Ελλάδα). Αποτελέσματα: 34 από τα 123 δείγματα γάλακτος βρέθηκαν να περιέχουν κατάλοιπα των διερευνώμενων ουσιών, εκ των οποίων τα 11 υπερέβαιναν τα θεσπισμένα από την Ε.Ε. ανώτατα επιτρεπτά όρια καταλοίπων. Η Εκτιμώμενη Ημερήσια Πρόσληψη των ουσιών αυτών, από το εξετασθέν δείγμα πληθυσμού, κυμαίνεται από 0,4-15,9% της Αποδεκτής Ημερήσιας Πρόσληψης, ανάλογα με την ουσία και το τρόφιμο (νωπό αγελαδινό γάλα ή τυρί Φέτα). Υπάρχει γεωγραφική διακύμανση στη συχνότητα εμφάνισης των καταλοίπων η οποία είναι ιδιαίτερα αυξημένη σε περιοχές οι οποίες στερούνται επαρκών υπηρεσιών ελέγχου τροφίμων. Συμπεράσματα: Από την παρούσα εργασία προκύπτει ότι 11.4% των δειγμάτων γάλακτος, περιέχει κατάλοιπα ανθελμινθικών ουσιών, σε επίπεδα συγκεντρώσεων που υπερβαίνουν το ανώτατο επιτρεπτό όριο (έως και 7 φορές για τη Febantel). Το εύρημα αυτό, εγείρει ερωτήματα για την πλήρη εφαρμογή της Ο.Κ.Π.. Ωστόσο η κατανάλωση νωπού γάλακτος το οποίο περιέχει κατάλοιπα των υπό διερεύνηση ενώσεων, στα προαναφερθέντα επίπεδα συγκεντρώσεων, δεν οδηγεί σε υπέρβαση της Ανώτερης Ημερήσιας Πρόσληψης. Από την άλλη πλευρά, είναι απαραίτητο να τονιστεί ότι οι ουσίες αυτές είναι δυνατό να ανιχνευθούν και σε άλλα τρόφιμα ζωικής προέλευσης για τα οποία δεν έχουν θεσπιστεί MRLs και συνεπώς δε διεξάγονται οι αντίστοιχοι έλεγχοι. Κατά συνέπεια, είναι απαραίτητη η εκπαίδευση των κτηνοτρόφων σε θέματα Ο.Κ.Π., η εντατικοποίηση των ελέγχων και η στελέχωση των κρατικών υπηρεσιών, έτσι ώστε να αντισταθμίζονται οι ελλείψεις των νόμων και να αποφευγονται πιθανές δυσμενείς συνέπειες για τη δημόσια υγεία. / Aim: The determination of the concentration levels, of veterinary parasiticide drug (anthelmintics) residues, in ruminants’ raw milk, from regions of Southern Greece, and the residues’ intake estimation for humans. In particular, the investigated, residues were Albendazole, Febantel, Fenbendazole, Mebendazole and some of their metabolites (Albendazole sulfoxide, Albendazole sulfone, Fenbendazole sulfone), in sheep, goat and bovine raw milk. Methods: Chemical analysis of raw milk samples with High Performance Liquid Chromatography and UV Diode Array Detector. A databank concerning the daily consumption of milk and feta cheese was utilized (personal interviews and filling in of food frequency questionnaires, from a population sample of 723 pupils aged from 10-12 years old, in Southern Greece). Results: 34 out of the 123 milk samples, were found to contain residues of the investigated compounds and 11 of the contaminated samples, contained residues, exceeding the EU established MRLs. The Estimated Daily Intake for these residues resulting from the interviewed population sample, was ranging from 0,4 to 15,9% of the Acceptable Daily Intake, depending on the substance and the kind of food (raw bovine milk or feta cheese). There is a geographical variation concerning the residue occurrence, which is high in regions where food control agencies are poorly staffed. Conclusions: The present study indicates that 11.4% of milk samples analysed, contains concentration levels of anthelmintic residues above the maximum residue limit (up to 7 times for Febantel). This finding raises questions about the full implementation of Good Veterinary Practice. However, consumption of raw milk containing the aforementioned concentration levels of the compounds under investigation, does not result to exceedance of the Acceptable Daily Intake. On the other hand, it is necessary to emphasize that these substances are possible to be detected in other foods of animal origin for which no MRLs have been established and therefore the respective controls are not carried out. Consequently, training of stock-farmers on GVP, intensification of controls and staffing of government agencies, are needed to counterbalance deficiencies of laws and avoid potential adverse effects on public health.

Traditional medicine in South Africa is part of the culture of the people and has been in existence for a long-time. Although animal components form part of the ingredients used, plant material constitutes the major component. South Africa is endowed with vast resources of medicinal and aromatic plants which have been employed for treatment against various diseases for decades. A large number of South Africans still depend on traditional medicine for their healthcare needs due to its affordability, accessibility and cultural importance. Helminth infections are among the variety of diseases treated by traditional healers. These infections are regarded as neglected tropical diseases (NTDs) due to their high prevalence among the economically disadvantaged living in rural areas in different regions of the world. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2016-06-02T20:21:35Z (GMT). No. of bitstreams: 1 6279.pdf: 1318446 bytes, checksum: 3ea93c315d61578a7b50605db41ae915 (MD5) Previous issue date: 2014-02-28 / Financiadora de Estudos e Projetos / Among the sheep parasites, Haemonchus contortus is the most prevalent and pathogenic nematode in tropical areas, causing huge economic losses. Alterations in the gene encoding the membrane P-glycoprotein (PgP) have been associated with multidrug resistance. The goal of this study was to identify single nucleotide polymorphisms (SNPs) on the PgP gene in H. contortus by comparing two isolates with different status of anthelmintic resistance. For this purpose, two ewes were experimentally infected, one with a susceptible H. contortus isolate (McMaster, Australia) and the other with a multidrug-resistant isolate (Embrapa2010, Brazil). Feces from infected animals were submitted to coproculture in order to obtain larvae and, after the slaughter of the ewes, adults H. contortus were collected from abomasum and individually submitted to DNA extraction. For the evaluation of potential molecular markers of geographic isolation, larvae originating from coprocultures of two other Brazilian isolates (Bahia and Pernambuco), with no determined resistance status, were also submitted to DNA extraction. After PCR amplification of DNA extracted from adults H. contortus, a fragment of the PgP gene was sequenced and six SNPs (position described in relation to the sequence of contig 004690 from the Wellcome Trust Sanger Institute) were identified : 508 (A> G in exon), 580 (G> A), 601 (G> C), 800 (G> A), 845 (G> T), and 878 (G> T), the last five in introns. The Fisher's exact test, applying the Bonferroni correction, revealed a significant association (P<0.01) between 508, 580, 601, 845 and 878 SNPs and the state of resistance to anthelmintics. Among these SNPs, 601 and 878 are in linkage disequilibrium and form the CC haplotype associated (P < 0.05) to resistance and the GG haplotype associated to susceptibility. Considering that there is considerable genetic differentiation between different continental areas in H. contortus, the frequencies of the SNPs were compared between the Brazilian isolates and the susceptible Australian one. It was found that the SNPs 580 and 845 may be associated with geographic isolation. We conclude that the 508, 601 and 878 SNPs in the P-glycoprotein gene can be molecular markers for multiple resistance to anthelmintics, and that the 580 and 845 SNPs can be candidate markers of geographical isolation in H. contortus. We developed software for Risk Analysis Development of Parasitic Resistance to Anthelmintics in Sheep (SARA) which will provide information that may guide the rational use of anthelmintics. / Among the sheep parasites, Haemonchus contortus is the most prevalent and pathogenic nematode in tropical areas, causing huge economic losses. Alterations in the gene encoding the membrane P-glycoprotein (PgP) have been associated with multidrug resistance. The goal of this study was to identify single nucleotide polymorphisms (SNPs) on the PgP gene in H. contortus by comparing two isolates with different status of anthelmintic resistance. For this purpose, two ewes were experimentally infected, one with a susceptible H. contortus isolate (McMaster, Australia) and the other with a multidrug-resistant isolate (Embrapa2010, Brazil). Feces from infected animals were submitted to coproculture in order to obtain larvae and, after the slaughter of the ewes, adults H. contortus were collected from abomasum and individually submitted to DNA extraction. For the evaluation of potential molecular markers of geographic isolation, larvae originating from coprocultures of two other Brazilian isolates (Bahia and Pernambuco), with no determined resistance status, were also submitted to DNA extraction. After PCR amplification of DNA extracted from adults H. contortus, a fragment of the PgP gene was sequenced and six SNPs (position described in relation to the sequence of contig 004690 from the Wellcome Trust Sanger Institute) were identified : 508 (A> G in exon), 580 (G> A), 601 (G> C), 800 (G> A), 845 (G> T), and 878 (G> T), the last five in introns. The Fisher's exact test, applying the Bonferroni correction, revealed a significant association (P<0.01) between 508, 580, 601, 845 and 878 SNPs and the state of resistance to anthelmintics. Among these SNPs, 601 and 878 are in linkage disequilibrium and form the CC haplotype associated (P < 0.05) to resistance and the GG haplotype associated to susceptibility. Considering that there is considerable genetic differentiation between different continental areas in H. contortus, the frequencies of the SNPs were compared between the Brazilian isolates and the susceptible Australian one. It was found that the SNPs 580 and 845 may be associated with geographic isolation. We conclude that the 508, 601 and 878 SNPs in the P-glycoprotein gene can be molecular markers for multiple resistance to anthelmintics, and that the 580 and 845 SNPs can be candidate markers of geographical isolation in H. contortus. We developed software for Risk Analysis Development of Parasitic Resistance to Anthelmintics in Sheep (SARA) which will provide information that may guide the rational use of anthelmintics. / Entre os parasitas de ovinos, Haemonchus contortus é o nematoide mais prevalente e patogênico em áreas tropicais, causando grandes perdas econômicas. As alterações no gene que codifica a glicoproteína-P de membrana (PgP) foram associadas com a resistência a múltiplas drogas. Assim, o objetivo deste estudo foi identificar polimorfismos de nucleotídeo único (SNPs) no gene PgP em H. contortus por comparação de dois isolados com diferentes status de resistência a anti-helmínticos. Para esse fim, uma ovelha foi infectada experimentalmente com o isolado suscetível (McMaster, Austrália) e outra ovelha com o isolado multirresistente (Embrapa2010, Brasil) de H. contortus. Fezes dos animais infectados foram submetidas à coprocultura para a obtenção de larvas e, após o abate das ovelhas, H. contortus adultos foram colhidos do abomaso e individualmente submetidos à extração de DNA. Para a avaliação de possíveis marcadores moleculares de isolamento geográfico, larvas oriundas de coprocultura de dois outros isolados brasileiros (Bahia e Pernambuco), com status de resistência não determinado, também foram submetidas à extração de DNA. Após amplificação por PCR do DNA extraído de H. contortus adultos, um fragmento do gene PgP foi sequenciado e foram identificados seis SNPs (posição descrita em relação à sequência do contig 004690 do Wellcome Trust Sanger Institute): 508 (A>G, em éxon), 580 (G>A), 601 (G>C), 800 (G>A), 845 (G>T) e 878 (G>T), os cinco últimos em íntrons. O teste exato de Fisher, por meio da correção de Bonferroni, revelou associação significativa (P<0,01) entre os SNPs 508, 580, 601, 845 e 878 e o estado de resistência a anti-helmínticos. Desses SNPs, o 601 e o 878 estão em desequilíbrio de ligação e formam os haplótipos CC, que está associado (P<0,05) à resistência, e GG, à suscetibilidade. Levando em consideração que em H. contortus há considerável diferenciação genética entre áreas continentais distintas, as frequências dos SNPs foram comparadas entre os isolados brasileiros e o isolado suscetível australiano e foi verificado que os SNPs 580 e 845 podem estar associados ao isolamento geográfico. Conclui-se que os SNPs 508, 601 e 878 no gene de glicoproteína-P podem ser marcadores moleculares para a resistência múltipla a anti-helmínticos, e os SNPs 580 e 845 candidatos a marcadores de isolamento geográfico em H. contortus. Nós desenvolvemos um software para Análise de Risco de Desenvolvimento de Resistência Parasitária a Anti- Helmínticos em Ovinos (SARA) que fornecerá informações que poderão orientar a utilização racional de anti-helmínticos. / Entre os parasitas de ovinos, Haemonchus contortus é o nematoide mais prevalente e patogênico em áreas tropicais, causando grandes perdas econômicas. As alterações no gene que codifica a glicoproteína-P de membrana (PgP) foram associadas com a resistência a múltiplas drogas. Assim, o objetivo deste estudo foi identificar polimorfismos de nucleotídeo único (SNPs) no gene PgP em H. contortus por comparação de dois isolados com diferentes status de resistência a anti-helmínticos. Para esse fim, uma ovelha foi infectada experimentalmente com o isolado suscetível (McMaster, Austrália) e outra ovelha com o isolado multirresistente (Embrapa2010, Brasil) de H. contortus. Fezes dos animais infectados foram submetidas à coprocultura para a obtenção de larvas e, após o abate das ovelhas, H. contortus adultos foram colhidos do abomaso e individualmente submetidos à extração de DNA. Para a avaliação de possíveis marcadores moleculares de isolamento geográfico, larvas oriundas de coprocultura de dois outros isolados brasileiros (Bahia e Pernambuco), com status de resistência não determinado, também foram submetidas à extração de DNA. Após amplificação por PCR do DNA extraído de H. contortus adultos, um fragmento do gene PgP foi sequenciado e foram identificados seis SNPs (posição descrita em relação à sequência do contig 004690 do Wellcome Trust Sanger Institute): 508 (A>G, em éxon), 580 (G>A), 601 (G>C), 800 (G>A), 845 (G>T) e 878 (G>T), os cinco últimos em íntrons. O teste exato de Fisher, por meio da correção de Bonferroni, revelou associação significativa (P<0,01) entre os SNPs 508, 580, 601, 845 e 878 e o estado de resistência a anti-helmínticos. Desses SNPs, o 601 e o 878 estão em desequilíbrio de ligação e formam os haplótipos CC, que está associado (P<0,05) à resistência, e GG, à suscetibilidade. Levando em consideração que em H. contortus há considerável diferenciação genética entre áreas continentais distintas, as frequências dos SNPs foram comparadas entre os isolados brasileiros e o isolado suscetível australiano e foi verificado que os SNPs 580 e 845 podem estar associados ao isolamento geográfico. Conclui-se que os SNPs 508, 601 e 878 no gene de glicoproteína-P podem ser marcadores moleculares para a resistência múltipla a anti-helmínticos, e os SNPs 580 e 845 candidatos a marcadores de isolamento geográfico em H. contortus. Nós desenvolvemos um software para Análise de Risco de Desenvolvimento de Resistência Parasitária a Anti- Helmínticos em Ovinos (SARA) que fornecerá informações que poderão orientar a utilização racional de anti-helmínticos.

Le latex de Carica papaya est un milieu riche en cystéine protéinases. Celles-ci ont été régulièrement utilisées en cosmétique ou pour l’attendrissement de la viande. Mais ces protéines ont aussi un intérêt pharmaceutique. En effet, le latex est bien connu pour posséder une activité antifongique mais aussi une activité anthelminthique. Ces effets sont régulièrement attribués aux cystéine protéinases qui se trouvent en concentration importante dans le latex. Malgré ces concentrations importantes en protéinases, d’autres protéines restent actives dans ce milieu. C’est le cas de la glutamine cyclase, qui a été extraite intacte de ce milieu et cristallisée. Sa structure nous a révélé une architecture particulière en ‘’&61538;-propeller‘’ à cinq pales avec double fermeture. Cette structure lui confère sa très grande stabilité. <p>Les industries pharmaceutiques sont aussi à la recherche de protéines très stables et résistantes aux protéinases endogènes. Nous avons donc entrepris l’étude du protéome de Carica papaya afin de mettre en évidence d’autres protéines minoritaires relativement stables pouvant conférer au latex son activité anthelminthique. Cette analyse a permis la mise en évidence de différentes protéines appartenant à diverses familles des « pathogenesis related protéins » (PR-proteins): une &61538;-1,3 glucosidase, une analogue à la barwin, une thaumatine et deux chitinases.<p>Nous nous sommes particulièrement intéressés à ces deux dernières au cours de cette thèse. Une caractérisation de ces deux protéines a permis de montrer que celles-ci étaient bien deux protéines distinctes, identifiées comme chitinases majeure et mineure selon leur abondance dans le latex. Elles sont relativement stables et résistantes à la protéolyse. Une analyse de la séquence de la chitinase majeure a montré que celle-ci était homologue à la chitinase issue de l’orge et une analyse de sa structure révèle la présence d’une grande concentration en prolines localisées principalement dans les neuf boucles de sa structure. Cela pourrait expliquer sa grande résistance vis à vis des cystéine protéinases.<p>La cristallisation de cette même chitinase en présence de N-acétyl-glucosamine comme additif, a conduit à une structure contenant trois molécules de GlcNac, deux dans le centre actif de notre protéine et une participant au réseau cristallin. Aucune structure de chitinase n’avait encore pu être obtenue en co-cristallisation avec un substrat. A partir des deux GlcNac observés dans le centre actif, nous avons reconstruit un complexe chitinase/(GlcNac)4. L’analyse de ce complexe a permis de mettre en évidence de nouvelles interactions entre (GlcNac)4 et les acides aminés du centre actif ainsi que de confirmer le mécanisme de la famille GH 19.<p> Des tests préliminaires sur nématodes ont finalement confirmé l’activité anthelminthique du latex et montré que la chitinase pouvait aussi être un bon nématocide<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les nématodes parasites du tube digestif demeurent une contrainte majeure pesant sur la santé, le bien être et les productions des petits ruminants élevés à l’herbe. Le mode usuel de maîtrise de ce parasitisme repose sur l’emploi répété de molécules chimiques anthelminthiques. Toutefois, le développement et la diffusion généralisée de résistances à ces molécules dans les populations de vers imposent désormais d’utiliser ces traitements avec discernement et de trouver des solutions complémentaires ou alternatives. L’incorporation dans la conduite d’élevage (ration des moutons ou des chèvres) de légumineuses fourragères riches en tannins condensés dotées de propriétés anthelminthiques s’est avérée une option prometteuse pour réduire le recours aux molécules chimiques. Toutefois, une des difficultés d’application de ces plantes tient à la variabilité des résultats observés. En prenant le sainfoin comme modèle de légumineuse contenant des tannins et en s’appuyant essentiellement sur des méthodes in vitro basées sur les larves 3 infestantes, l’objectif général de cette thèse est d’analyser le rôle respectif de facteurs liés à l’environnement, aux variétés génétiques ou aux modes de préservation technologiques sur les propriétés anthelminthiques. Quelque soit le critère envisagé, une forte variabilité a été observée. Les principales variations liées à l’environnement dépendaient de l’année et du cycle de coupe, ainsi que du site d’exploitation. Parmi les 38 variétés testées, 9 se sont avérées à forte activité AH, alors que 22 étaient à très faible activité. Enfin, les résultats ont aussi surtout souligné une plus forte activité antiparasitaire dans des formes séchées ou ensilées par comparaison à des échantillons frais. La comparaison des profils biochimiques liés à cette variabilité a permis une exploration des composés phénoliques expliquant l’activité anthelminthique. L’existence d’une relation dose-réponse a été précisée. Le rôle des tannins condensés a été confirmé, notamment ceux à faible degré de polymérisation et à faible poids moléculaire. L’importance des prodelphinidines qui seraient plus actives que les procyanidines reste à confirmer. De plus, il a été montré que d’autres flavonoides peuvent jouer un rôle, notamment les flavan-3-ols et les flavonols. La différence d’activité entre les échantillons de sainfoin ensilés ou fanés par comparaison aux échantillons frais s‟expliquerait en partie par la présence de formes non glycosidés de flavonols. Ces résultats devraient conduire à développer des méthodes de dosage pour identifier les échantillons de sainfoin, et plus généralement de légumineuses riches en tannins, dotés de propriétés anthelminthiques significatives. / Gastrointestinal nematodes remain a major constraint on the health, welfare and production of small ruminants. Over the past decades, the usual mode of control of this parasitism has mainly relied on the repeated use of chemical anthelmintics. However these treatments are nowadaysfacing some limits among which the most important is the development and widespread diffusion of resistance to these chemical molecules within worm populations. Consequently, the need to find complementary or alternative solutions is becoming urgent. The possible exploitation of forage legumes, rich in condensed tannins, with anthelmintic properties, by incorporation in the diet of sheep or goats, seems a promising option to reduce the reliance on chemical molecules. However, one of the main difficulties to use these plants as nutraceuticals relates to the variations in results. By using sainfoin (Onobrychis viciifoliae) as a model of tannin-containing legume and based on in vitro methods on the infective third stage larvae, the main objectives of this PhD were i) to examine the influence of environmental, genetic (40 different varieties) and technological (mode of preservation) factors on the anthelmintic properties of sainfoin and ii) to analyse whether differences in phenolic compounds might explain the variations. Whatever the factor considered, a high variability in results was observed. The main variations due to the environmental factors depended on the year and the cycle of cutting, as well as on the site of cultivation. Among the 38 varieties tested, only 9 have shown AH activity over 50 %. Last, a higher antiparasitic activity was found in the dried or ensiled forms compared to the fresh samples. A comparison of the biochemical profiles associated with these variations indicated a role of proanthocyanidins plus other phenolic compounds in the anthelmintic properties. The dose-response relationship between the AH activity and the ability to form complex with proteins was defined. The role of condensed tannins was confirmed, particularly those with a low degree of polymerization. The respective importance of prodelphinidins vs procyanidins remains to be further investigated. Moreover, the possible role of other flavonoids, in particular of flavan-3-ols and flavonols was also confirmed. The difference in activity between dried or ensiled forms compared to fresh sainfoin samples was partly explained by the presence of flavonol aglycosides. These results should favour the development of measurements in order to identify sainfoin samples with higher anthelmintic properties.

Les strongyloses vétérinaires constituent l’une des principales causes de gastroentérites et d’anémie des ovins et caprins à l’origine de lourdes pertes économiques. Cependant, leurs prise en charge par les anthelminthiques existants, est confrontée à la prolifération d’une pharmacorésistance des nématodes incriminés. La première partie de ce travail de thèse présente l’ampleur du problème que pose la résistance des helminthes impliqués dans les strongyloses des petits ruminants. Elle révèle également le profil novateur des imidazo[1,2-a]pyridinyl-phénylacrylonitriles qui est proposé pour parer à l’inefficacité croissante des anthelminthiques. / Veterinary strongylosis is one of the main causes of gastroenteritis and anemia in sheep and goats causing major economic losses. However, their management by existing anthelmintics, is confronted with the proliferation of drug resistance of the nematodes incriminated. The first part of this thesis presents the magnitude of the problem posed by the resistance of helminths involved in strongylosis of small ruminants. It also reveals the innovative profile of imidazo[1,2-a]pyridinyl-phenylacrylonitriles that are proposed to counter the increasing ineffectiveness of anthelmintics.