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Zugl.: Giessen, Univ., Diss., 2008

Zugl.: Giessen, Univ., Diss., 2008

Universiẗat, Diss., 2008--Giessen.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-20Bitstream added on 2014-06-13T21:02:23Z : No. of bitstreams: 1 katiki_lm_dr_botfmvz.pdf: 1503507 bytes, checksum: 7ad92e791989f524b8cad6907f21f758 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os óleos essenciais de Mentha piperita, Cymbopogon martinii e Cymbopogon schoenanthus foram avaliados in vitro sobre tricostrongilídeos de ovinos por meio dos testes de eclodibilidade, do desenvolvimento, da inibição da alimentação e da eliminação da cutícula larvar. Utilizou-se cromatografia gasosa acoplada ao espetrômetro de massas para identificação dos constituintes dos óleos. Os óleos apresentaram elevada atividade anti-helmíntica avaliada pela comparação de CL50, sendo o C. schoenanthus o mais ativo. Os mesmos óleos foram testados em ratos Wistar infectados artificialmente com o parasita intestinal Strongyloides venezuelensis nas dosagens de 1,5 mL/kg e 2,3 mL/kg cada um. Os óleos não apresentaram efeito anti-helmíntico significativo medido pela contagem de ovos nas fezes e contagem parasitária quando comparado ao albendazol. C. schoenanthus foi o óleo essencial que apresentou melhor atividade anti-parasitária in vitro sobre trichostrongilídeos de ovinos, portanto, sua atividade foi testada in vivo em cordeiros artificialmente infectados com Haemonchus contortus nas dosagens de 0,2 e 0,4 mL/kg. A redução parasitária (por meio de de contagem de ovos nas fezes e contagem parasitária) e a toxicidade (por meio de de perfis bioquímicos renal e hepático) foram avaliadas. O óleo essencial de C. schoenanthus não foi tóxico nas dosagens utilizadas e embora não tenha propiciado redução significativa no grau de infecção parasitária, proporcionou maior valor de hematócrito e proteína sérica total. Além desse efeito, causou discreta redução no desenvolvimento de larvas nas fezes. Uma metodologia aperfeiçoada de teste in vitro utilizando o nematoide de vida livre Caenorhabditis elegans, mantidos em cultura líquida estéril, também foi descrito, assim como os testes de sensibilidade destes nematoides aos principais solventes utilizados na preparação dos extratos de plantas / Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated in vitro against sheep trichostrongylids through eclodibility assay, larval development assay, larval feeding inhibition assay and larval exsheathment assay. Oils were analysed by chromatography coupled to mass spectrometry. The oils presented high anthelmintic activity by comparison of LC50, being C .schoenanthus is the most active. The oils were tested withWistar rats artificially infected with Strongyloides venezuelensis at 1.5 mL/kg and 2.3 mL/kg. The essential oils didn’t present a significant anthelmintic effect measured by fecal egg count and worm burden when compared to albendazole. The oil of C. schoenanthus had the best anthelmintic activity against sheep trichostrongylids and were evaluated in vivo in lambs artificially infected with Haemonchus contortus at doses of 0.2 and 0.4 mL/kg. Its activity were evaluated by fecal egg count and worm burden and the toxicity evaluated by kidney and liver profile. C. schoenanthus did not show toxic effects at the doses tested and although without significant reduction in parasite infection, it led to a higher packed cell volume and total serum protein and small reduction in larval development in feces. An improved methodology of in vitro test employing the free living nematode Caenorhabditis elegans raised in sterile liquid medium was described as well the toxicity to the major solvents used in preparation of plant extracts

Genetic control of resistance to H. contortus was assessed in 198 ewes and 386 lambs of 50% Dorset, 25% Rambouillet and 25% Finnsheep breeding in fall and spring over 2 yr. After deworming, lambs that were approximately 120 d old and ewes that had weaned their lambs at 60 d and dried off were individually dosed with approximately 10,000 infective larvae. After infection, body weight (BW), fecal egg counts (FEC) and packed cell volume (PCV) were measured weekly for 7 wk in lambs and fortnightly for eleven wk in ewes. Summary traits were defined as initial PCV, mean BW (MBW) across all times, and means for FEC, log-transformed FEC (MLFEC), and PCV (MPCV) at wk 3 to 7 post-infection for lambs and wk 3 to 11 post-infection for ewes. No consistent seasonal variation in FEC was observed. Younger ewes were more susceptible to infection than older ewes. Sex differences in FEC were not observed in lambs. Heritabilities for summary traits were estimated from a REML analysis that included fixed effects of year and season plus effects of either sex (for lambs) or age category (for ewes). Heritability estimates for MBW, MPCV, and MLFEC were 0.74, 0.57, and 0.27 respectively (all P < 0.01), in lambs, and 0.24 (P < 0.1), 0.25 (P < 0.05) and 0.55 (P < 0.01), respectively, in ewes. Across-year repeatability estimates in ewes for MBW, MPCV, and MLFEC were 0.83, 0.54 and 0.56, respectively (all P < 0.01). Resistance was antagonistically associated with estimated breeding values for growth in ewes but not in lambs. Fertility and prolificacy in ewes were not related to resistance. Breed differences in resistance to H. contortus were also evaluated in 4 to 6-mo-old crossbred Dorset and Dorper, straightbred Katahdin, and Barbados Blackbelly x St. Croix lambs. Dorpers were not more resistant than Dorsets but appeared to cope better, with higher PCV and similar BW during infection compared to Dorsets. Katahdin and Barbados Blackbelly x St. Croix lambs were more resistant with lower FEC. / Master of Science

The avermectins (ivermectin, doramectin etc) and milbemycins are effective anthelmintics used widely in animal and human medicine for the past twenty years. The actual site of action of the avermectins on the GluCl is unclear, but binding studies have concluded that it does not share a binding site with glutamate. The GluCl channels have been well characterised in Caenorhabditis elegans and are beginning to be characterised in parasitic nematode species such as Haemonchus contortus, Dirofilaria immitis and Cooperia oncophora. The aim of this project was to characterise the H. contortus GluClα3B subunit and its interactions with agonists, glutamate and ivermectin using electrophysiology to study Xenopus oocytes expressing GluClα3B homomeric channels and ligand binding studies on COS-7 cells expressing the subunits. Site–directed mutagenesis was used to introduce resistance associated candidate polymorphisms into the H. contortus GluClα3B subunit. The effects of these changes on the response to glutamate and ivermectin were assessed. One mutation found in IVR C. oncophora, L256F, confers a 3-fold loss of sensitivity to glutamate and a 6.5 fold loss of sensitivity to IVM. This mutation is found in the C-terminal area of the extracellular region of the channel and, from homology modelling, we know it lies in close proximity and possibly interferes with another candidate mutation V235A, and the Cysteine residue at position 192 which forms one side of the structurally significant disulphide bridge. Further introduction of different mutations at this position showed the larger the substituted amino acid, the greater the effect on IVM sensitivity. Another amino acid substitution (T300S) results in the prohibition of a functional channel. The protein is produced and is able to bind IVM with high affinity but does not create a functional channel. These data show that polymorphisms found in field isolates of parasites can have a significant effect on GluCl channels and may contribute to drug resistance.

Thesis (MSc. (Veterinary Tropical Diseases Veterinary Science))--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Resistance to ivermectin (IVM) in Haemonchus contortus has developed in many countries and its mechanism is still under investigation. P-glycoproteins (P-gp) are transmembrane proteins that can transport drugs out of cells. Researchers have found that there is polymorphism in a P-gp gene from H. contortus between IVM-selected and unselected worms. Three main P-gp polymorphs were identified, polymorph A was found to be related to IVM selection, while polymorphs B and X were associated with susceptibility. The purpose of this research is to investigate the genetic variations in P-glycoprotein that are associated with IVM selection or susceptibility in H. contortus. Total RNA and genomic DNA were extracted from individual male adult worms of IVM-selected and unselected strains of H. contortus. A fragment of the P-gp gene was amplified from the genomic DNA of individual worms and RFLP analysis was performed on the PCR product to genotype the corresponding worms. The homozygous worms that possessed polymorph A, B or X were identified.