Compared to mammals, nematodes appear to exhibit a unique GABAergic nervous system. Haemonchus controtus is a parasitic nematode that infects ruminants worldwide. Hco-UNC-49 is a H. contortus GABA-gated chloride channel and is an orthologue to the UNC-49 channel from the free-living nematode Caenorhabditis elegans. Previous research by our group has shown that while the UNC-49 channels from the two nematodes share similar sequence homology they do not share identical sensitivity to GABA. To further investigate the characteristics of the Hco-UNC-49 channel, this study tested the effects of various modulators, insecticides and anti-parasitic drugs on channel function. Most notably, the molecules penicillin G, propofol and pregnenolone sulfate all had similar effects on Hco-UNC-49 as reported previously for Cel-UNC-49. On the other hand, Hco-UNC-49 appears to be less sensitive to picrotoxin inhibition compared to what has been reported for Cel-UNC-49. Novel effects of a number of anthelmintics were also observed. For example, the anthelmintics ivermectin and moxidectin both enhanced Hco-UNC-49 GABA responses, while piperazine was able to directly activate Hco-UNC-49 at high concentrations. These results suggest that Hco-UNC-49 is likely an in vivo target for these anthelmintics. / UOIT

The gastrointestinal parasite, Haemonchus contortus, is of major concern in the sheep and goat industry as well as in zoological settings. Over the years this parasite has developed resistance to the three classes of anthelmintics, benzimidazoles, imidazothiazoles and macrocyclic lactones, that are currently used for treatment. One of the mechanisms proposed to be involved in this resistance is the efflux transporter P-glycoprotein (Pgp). In this study, the resistance status of several strains of H. contortus was evaluated using the larval development assay DrenchRite®. After documenting the resistance status of these strains, transcription of Pgp in L3 larvae after exposure to anthelmintics was quantitated using polymerase chain reaction (PCR). Of the strains analyzed, only one was determined to be susceptible to all of the anthelmintics tested, while the others showed variable levels of resistance to one or more. A Haemonchus strain acquired from a giraffe at a zoo in Florida was the most resistant, showing extremely high levels of resistance to benzimidazoles and levamisole. Molecular characterization of the 18S rRNA gene and the internal transcriber spacer region (ITS) were performed on the giraffe strain to identify the species. Although there were variations in the isolate sequences, the most likely species for the giraffe strain was H. contortus. No transcription of Pgp was identified in H. contortus L3 larvae under the conditions of this study. Thus, increased Pgp does not appear to be a primary mechanism of drug resistance in this stage of the worm.

The objectives of this study are to estimate the degree to which a glutamate-gated chloride channel gene (HcGluCla) contributes to survival of moxidectin treatment and to study the relative dominance of those alleles. The phenotype of individual adult H. contortus with respect to feeding was determined using an inulin uptake assay. Genotype was determined using a diagnostic PCR assay. In the absence of moxidectin, homozygous susceptible genotypes fed significantly more than homozygous resistant genotypes. The effect of the susceptible allele was dominant. In the presence of moxidectin, feeding in the susceptible homozygotes was reduced to the level found in the resistant homozygotes, which were unaffected by the drug. These results suggest that the function of the two alleles is different and that they also respond differently to the drug, the resistant allele being unaffected by the drug. / The selection coefficient, s, is the selective difference between the resistant and susceptible genotypes with regard to feeding. Parasites with the resistant allele were seen to feed less in the absence of the drug, i.e., the effect is recessive. In the presence of the drug, there was no difference between resistant and susceptible parasite feeding. These results suggest that resistance may have hidden complexities. (Abstract shortened by UMI.)

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen

Anthelmintics, antiparasitic agents, have been developed as a main weapon to control parasitic nematodes of domestic ruminants. Unfortunately, the intensive use of anthelmintics leads to the development of drug resistance in parasite populations. Anthelmintic resistance has compromised the control of nematode parasites and has become a major problem in many countries of the world. Resistance to the newest anthelmintics such as ivermectin (IVM) and related anthelmintics in Haemonchus contortus in sheep has been developing rapidly in recent years. The development of drug resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. However, the mechanism of ivermectin resistance in nematode parasites is unknown. P-glycoprotein (Pgp) has been well documented in mammalian cells as a membrane transporter by actively extruding a variety of structurally and functionally unrelated hydrophobic cytotoxic drugs out of the cell. This study was to determine whether there is an association between specific alleles at the Pgp locus and IVM or moxidectin (MOX) selection in H. contortus, by investigating the genetic variation of the Pgp homologue in unselected and IVM- and MOX-selected strains of H. contortus. (Abstract shortened by UMI.)

Avermectins and milbemycins are believed to exert their anthelmintic effects by binding to glutamate-gated chloride channels (GluCls). Two GluCl subunits have been localized in the pharynx in Caenorhabditis elegans , and the pharynx has been implicated as a major target for avermectins in C. elegans. The HcGluCla gene encoding an alpha-type GluCl subunit has been cloned from Haemonchus contortus previously, however the localization of this gene has not been identified. To begin to investigate the expression site of this HcGluCla gene we have isolated a 1439bp 5'-flanking region and the entire genomic organization of this gene. The 1439bp 5'-flanking region and the first exon and intron and part of the second exon of the HcGluCla gene were fused to the green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. After microinjection of the construct into C. elegans, four stable transformed lines were established and assayed for GFP expression. The transformed animals exhibited fluorescence in the two pairs of MC and M2 pharyngeal neurons, but no expression was detected in the muscle cells. This result provides evidence that the pharynx is a major site for the mode of action of avermectins and milbemycins on parasitic nematodes, such as H. contortus.

Sustainable parasite control in livestock depends on anthelmintic drugs. The nematode Haemonchus contortus, the most important intestinal parasite of sheep and goats has developed resistance to all classes of anthelmintics including moxidectin, the most potent of the macrocyclic lactones. Pyrosequencing was used to screen H. contortus laboratory and field strains for single nucleotide polymorphisms (SNPs) associated with resistance in three genes, and determine their involvement in field resistance to macrocyclic lactones. Specific SNPs increased in frequency in ivermectin/moxidectin laboratory selected strains for all three genes. These did not protect a resistant field strain from a field dose of ivermectin and were not the major mechanism of resistance in the field strain. A gamma-aminobutyric acid chloride receptor SNP may be a potential marker for moxidectin resistance in the field. This study indicates results obtained from laboratory strains selected with sub-therapeutic doses of drug may not reflect the situation in the field.

Tierärztl. Hochsch., Diss., 2004--Hannover.

Orientador: Alessandro Francisco Talamini do Amarante / Resumo: Dois experimentos foram realizados no período de 2013 a 2015, em cordeiros da raça Suffolk, com objetivo de avaliar a dinâmica da infecção mista por Haemonchus contortus e Haemonchus placei em ovinos, bem como a competição e o cruzamento interespecífico com a formação de híbridos. Um cordeiro doador, livre de infecções helmínticas, recebeu infecção artificial oral única com 25.000 larvas infectantes (L3) de H. contortus, isolado de laboratório SpHco2, com múltipla resistência aos anti-helmínticos, enquanto um bezerro doador foi infectado via oral, em dose única, com 25.000 L3 de H. placei, isolado de laboratório SpHpl1, susceptível à ação do levamisol. No primeiro experimento, realizado no ano de 2013, cordeiros mantidos confinados e livres de infecções helmínticas foram inicialmente infectados com 2.000 L3 de H. placei no dia zero e 11 dias depois, os mesmos animais receberam 2.000 L3 de H. contortus (grupo parental, n=6). Coproculturas individuais dos animais do grupo parental foram utilizadas para a produção de L3, as quais foram destinadas à infecção dos animais dos grupos F1 (primeira geração filial), grupos F1-42 DPI (n=6) e F1-84 DPI (n=6), o que permitiu avaliar a produção de híbridos. Todos os animais pertencentes aos grupos: parental, F1-42 DPI e F1-84 DPI, foram eutanasiados aos 50, 42 e 84 dias pós-infecção (DPI), respectivamente, para a identificação e quantificação dos parasitas, a fim de determinar a competição interespecífica e a hibridização. ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor