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

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 45 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 38-43).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1957. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 113-117).

Orientador: Alessandro Francisco Talamini do Amarante / Banca: Fernando de Almeida Borges / Banca: Renato de Oliveira Orsi / Resumo: Neste estudo avaliou-se a eficácia de uma vacina constituída de glicoproteínas obtidas da membrana do intestino de Haemonchus contortus em bezerros desafiados com H. contortus ou H. placei. Bezerros holandeses machos, criados livres de infecções por helmintos, foram distribuídos em quatro grupos com nove animais cada. Dois grupos foram vacinados com 50 μg do imunógeno diluído no adjuvante QuilA, enquanto os outros dois grupos foram os controles, receberam apenas adjuvante. A vacina foi administrada três vezes com intervalo de 21 dias entre as aplicações. Os bezerros foram artificialmente infectados sete dias após a última imunização e sacrificados para contagem dos vermes 43 dias depois. Os bezerros de um dos grupos vacinados receberam 8000 larvas infectantes (L3) de H. contortus enquanto os do outro grupo foram infectados com o mesmo número de L3 de H. placei. Os controles foram infectados na mesma ocasião com o mesmo número de L3 de H. contortus ou H. placei. A vacinação reduziu significativamente a contagem de ovos por grama de fezes (OPG) e a carga parasitária (P<0,01). A titulação de anticorpos nos animais controle permaneceu perto de zero enquanto nos animais vacinados verificou-se elevada titulação de anticorpos no soro. Os bezerros vacinados e desafiados com H. contortus não eliminaram ovos nas amostras de fezes, enquanto os controles apresentaram média máxima (± erro padrão) de 61,1 (±42,3) OPG 31 dias após a infecção. A partir deste dia, a contagem de OPG diminuiu progressivamente neste grupo controle com apenas um animal eliminando ovos nas fezes 42 dias após a infecção. Nos controles de H. placei, o ápice na contagem de OPG ocorreu 35 dias após a infecção (61,1 ± 21,7) e permaneceu relativamente constante até o final do estudo, enquanto no grupo vacinado apenas dois animais eliminaram ovos nas fezes, na última coleta. Os animais controle... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the efficacy of a vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus in calves challenged with H. contortus or Haemonchus placei. Males Holstein calves, raised worm free, were distributed into four groups with nine animals each. Two groups were vaccinated with 50 μg of antigen diluted on QuilA adjuvant, while the others two groups were the controls and received only adjuvant. The vaccine was administered three times 21 days apart. Either vaccinated or not and challenged with either 8,000 H. contortus or H. placei infective larvae. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Vaccination significantly reduced faecal egg counts (FEC) and worm burdens (P< 0.01). Antibody titres in the calves control stayed close to zero meanwhile in the calves vaccinated was observed high antibody titres in the serum. Calves vaccinated and challenged with H. contortus did not shed eggs in faecal samples, while the controls showed a maximum mean (± standard error) FEC of 61.1 (±42.3) at 31 days post infection. Then, FEC progressively declined in this group with only one animal shedding eggs in faeces 42 days post infection. With H. placei the controls FEC peaked at 35 days post infection (61.1 ±21.7) and remained relatively constant until the end of the study, while in the vaccinated group only two animals shed eggs and only on the last collection date. With H. placei the controls contained a mean of 551.1 (±93.7) parasites, while the vaccinates had 174.4 (±56.3) worms. The establishment rate of H. contortus was lower than that of H. placei (P< 0.01) with an average of 163.9 (±39.4) and 74.4 (±20.4) specimens in the control and vaccinated groups, respectively. It was concluded that vaccination of calves with antigens obtained from H. contortus conferred protection against both H. placei and H. contortus / Mestre

Resumo: 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 / Abstract: 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 / Orientador: Alessandro Francisco Talamini do Amarante / Coorientador: Ana Carolina de Souza Chagas / Coorientador: Jorge Freire da Silva Ferreira / Banca: Michiko Sakate / Banca: Helder Louvandini / Banca: Regina Kiomi Takahira / Banca: Cecília José Veríssimo / Doutor

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-04T12:35:24Z No. of bitstreams: 1 DissertaçãoFinal -Thaís Brito de Oliveira.pdf: 915122 bytes, checksum: 7a8d61b2e4d353771e4aadc74590debd (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-03T15:48:47Z (GMT) No. of bitstreams: 1 DissertaçãoFinal -Thaís Brito de Oliveira.pdf: 915122 bytes, checksum: 7a8d61b2e4d353771e4aadc74590debd (MD5) / Made available in DSpace on 2017-07-03T15:48:47Z (GMT). No. of bitstreams: 1 DissertaçãoFinal -Thaís Brito de Oliveira.pdf: 915122 bytes, checksum: 7a8d61b2e4d353771e4aadc74590debd (MD5) / CAPES / Haemonchus contortus é um nematoide gastrointestinal de ruminantes, principalmente de ovinos e caprinos, de grande importância mundial que causa diversas perdas econômicas. Este helminto tem apresentado resistência anti-helmintica, o que vem dificultando o tratamento dos pequenos ruminantes pelos seus criadores. Por isso novas alternativas estão sendo testadas a fim de combater o parasita, a exemplo do extrato aquoso da planta Poincinella pyramidalis. Foram utilizados onze animais, divididos em três grupos: GI (sem infecção); Grupo II (infectados) e GIII (infectados e tratados). Para a infecção experimental utilizou-se aproximadamente 20.000 larvas L3. Durante 90 dias amostras de fezes foram colhidas para análise parasitológica, e de sangue para análise da resposta imunológica através da dosagem do anticorpo sérico IgG e da citocina Interferon gama. A administração do extrato da planta ocorreu no GIII no tempo 45 dias. Obteve-se no tempo 60 dias em relação aos resultados parasitológicos uma redução carga parasitária de 79% no Grupo III e uma eficácia do extrato de 85%; em relação à resposta imune ocorreu uma baixa produção de interferon gama. A produção de anticorpos séricos IgG foi levemente estimulado após a administração do extrato. Os resultados indicam que esta planta possui um moderado efeito relacionado aos ovos encontrados nas fezes, assim como um leve potencial imunomodulador. Faz-se necessário, novos estudos para verificar novos parâmetros imunológicos relacionados à infecção e a administração deste extrato em estudo. / Haemonchus contortus is a gastrointestinal nematodes of ruminants, mainly sheep and goats , of great global importance that causes severe economic losses. This helminth has shown anthelmintic resistance, which is hampering the treatment of small ruminants by its creators. So new alternatives are being tested to combat the parasite , such as the plant Poincinella pyramidalis aqueous extract . Eleven animals were used, divided into three groups: GI ( without infection), Group II (infected ) and GIII ( infected and treated ). For experimental infection, we used about 20,000 L3 larvae. 90 days stool samples were collected for parasitological examination , and blood samples for analysis of the immune response through the measurement of serum IgG antibody and cytokine gamma interferon. The administration of the extract of the plant occurred in GIII time in 45 days. It was obtained in 60 days time in the parasitological reduced parasitic load of 79 % in Group III and efficacy of the extract of 85 %, compared to the immune response was a low production of interferon gamma. The production of serum IgG was slightly stimulated after administration of the extract. The results indicate that this plant has a moderate effect related to eggs found in the faeces, as a light immunomodulatory potential. Futher studies are necessary, to verify new immunological parameters related to infection and administration of this extract under study.