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.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics, such as IVM, and the milbemycins, such as MOX. The objective of this thesis was to determine whether Haemonchus contortus GluCls are important targets for these anthelminthics. To begin to address this we cloned a full length GluCl alpha-subunit cDNA from H. contortus (HcGluCla). This subunit shares a high homology with GluCl subunits from Caenhorhabditis elegans that have been shown to be important targets for IVM, suggesting that HcGluCla is also an IVM target. However, if HcGluCla is an IVM receptor then it should contain an IVM binding site. To investigate this, the HcGluCla gene was expressed in COS-7 cells. The resulting subunit bound [3H]IVM and [ 3H]MOX with affinities sufficiently high enough to explain their high in vivo potency. Interestingly, glutamate was an allosteric modulator of [ 3H]MOX and [3H]IVM binding where it increased the affinity of these drugs to HcGluCla. To gain further insight into the potentiation of IVM, various glutamatergic and non-glutamatergic ligands were screened for their ability to enhance [3H]IVM binding to HcGluCla. Of the ligands tested, only the GluCl agonists glutamate and ibotenate potentiated [3H]IVM binding. It is possible therefore that if IVM interacts with GluCls in vivo then IVM efficacy may be enhanced by GluCl agonists. To examine this, we tested whether ibotenate could enhance IVM efficacy in gerbils infected with H. contortus. In in vivo efficacy studies, ibotenate (at 1 mg/kg) increased IVM efficacy by 15% (p = 0.048). The enhancement of IVM efficacy in vivo by a GluCl agonist suggests that one of the IVM targets in H. contortus is the GluCl. Finally, to determine the potential physiological response from an interaction between IVM and H. contortus GluCls, we expressed HcGluCla in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to

Glutamate binding studies on membrane preparations from unselected and ivermectin-selected strains of the parasitic nematode of ruminants, Haemonchus contortus, indicated a single class of saturable, high affinity binding sites which are sensitive to ivermectin and exhibit different pharmacological characteristics from any known mammalian glutamate receptor. These studies showed that H. contortus larvae possess substantially more glutamate binding sites with lower affinity than adults. Moreover, selection with ivermectin was associated with an increase in the number of glutamate binding sites in adults and an increase in the affinity for glutamate binding in larvae. When investigating the effects of ivermectin on glutamate binding kinetics, it was discovered that ivermectin decreased Bmax values in unselected, but not in ivermectin-selected, parasites. Inulin intake studies were performed in unselected and ivermectin-selected H. contortus worms to relate the glutamate binding results to the biological activity, pharyngeal pumping. These studies showed that glutamate, ivermectin and the structurally similar anthelmintic, moxidectin, inhibit pharyngeal pumping, and that glutamate influences the effects of ivermectin and of moxidectin, on pharyngeal pumping. Lastly, selection with ivermectin was associated with an alteration of the effects of ivermectin, but not moxidectin, on pharyngeal pumping. The results of this work suggest that a novel ivermectin-sensitive, glutamate receptor, which influences pharyngeal function, is involved in the development of ivermectin resistance in H. contortus.

A study on the epidemiological dynamics of gastrointestinal nematodes of small ruminants was conducted in a semi-arid area of Kenya over a period of two years. Three major trichostrongylid species were identified; Haemonchus, Trichostrongylus and Oesophagostomum. Trichuris and Strongyloides species were occasionally encountered. Of the major trichostrongylids, Haemonchus spp. was the most prevalent (90%) and accounted for about 80% of the total worm burden. This species was found to undergo hypobiosis at levels that varied with seasons: nil levels of hypobiosis were recorded during the wet months and as much as 80% was recorded during the dry months. Hypobiosis was not investigated in the other nematode species. Evaluation of the relationship between the faecal egg count and the worm burden showed that the two parameters were more highly correlated during the wet months than during the dry months. This was a desirable situation because it is during the wet season that livestock owners in this area need to closely monitor the worm burdens in their animals. Treatment with ivermectin before the onset of the rains not only delayed the onset of egg shedding but also controlled clinical helminthiasis. In addition, a temporal change in the pattern of the appearance of infective larvae on pasture was observed; the appearance was delayed for about a month after the onset of the rains. The effect of treatment administered during the rains was a temporary and a short-lived relief of infection as evidenced by a brief decline in egg output: it had no detectable impact on pasture infectivity. These results suggested that removal of hypobiotic larvae before they resumed development had the combined benefit of reducing both the severity of clinical helminthiasis and the level of pasture contamination. This impact was expressed in improved flock performance and particularly in the improvement of birth weights that subsequently enhanced kid and lamb survival rates. Probably due to

No description available.

The mechanism of benzimidazole (BZ) anthelmintic resistance in Haemonchus contortus was investigated. The total binding (TB), low-affinity binding (LAB) and high-affinity (specific) binding (HAB) of ($ sp3$H) BZs (mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ) and oxfendazole (OFZ)) in supernatants derived from BZ-susceptible (S) and BZ-resistant (R) strains were examined and compared. The TB of all ($ sp3$H) BZs was reduced for the R strain. The TB of OBZ, MBZ and ABZ was separated into LAB and HAB. However, OFZ bound with low-affinity. The binding affinity, K$ sb{ rm a},$ and maximum binding, B$ sb{ rm max},$ for the HAB of OBZ and MBZ were calculated using computer programs. Compared with the S strain, the B$ sb{ rm max}$ of the R strain was reduced but the K$ sb{ rm a}$ was not affected. LAB to parasite preparations devoid of tubulin was observed but HAB occurred to preparations containing tubulin only. The HAB per mg protein decreased from egg through larva to adult stage. It was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA) analysis that the tubulin content per mg protein decreased from egg, through larva to adult worm. The ability of various BZs--OBZ, MBZ, ABZ, OFZ, fenbendazole (FBZ), albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO$ sb2),$ and thiabendazole (TBZ)--to bind tubulin was compared by displacement analysis and their IC$ sb{50}$ ( (BZ) required to inhibit 50% of the ($ sp3$H) BZ binding) and K$ sb{ rm a}$ values were determined. The IC$ sb{50}$ and K$ sb{ rm a}$ values approximately correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZs except for OFZ and ABZSO. Tubulin bound BZs at 4$ sp circ$C with lower K$ sb{ rm a}$ than at 37$ sp circ$C. Western blot of tubulin separated by 2-dimensional electrophoresis showed that the $ beta$-tubulin isoform pattern of the S and R strains were dissimilar whil