The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture.

Haemonchus contortus eggs were cultured in intact fecal pellets at various temperatures (5-35 degrees C) for 22 days. Temperature and relative humidity were kept constant throughout the incubation period. Nl larval development occurred at 5 degrees C; peak third-stage larval recovery occurred at 20 degrees C. Egg mortality was an age-dependent phenomenon, whereas larval mortality remained constant irrespective of larval age. Development was characterized by a minimum development time followed by a transition to the next stage which occurred at a constant rate. All rates were temperature dependent. The minimum development times reported here are much less than those previously reported. Based on these results a mathematical model was used to describe the demography of the free-living stages of H. contortus at various temperatures.     

Characteristics of DNase activities in excretory/secretory products of infective larvae of Haemonchus contortus

While multiple DNase activities occur in the excretory/secretory products (ESPs) of the adult Haemonchus contortus, the DNase activities in ESPs of the infective larvae (L3) have not been studied. Thus, the DNase activities in ESPs of H. contortus L3 were investigated and compared to those of adults for developmental stage-specific analysis. The DNase activities had relative molecular masses (M rs) of 34 and 36 kDa upon zymographic analysis at pH 5.0 and 7.0 when the larvae were incubated for over 48 h. The 34 and 36 kDa DNases of L3 ESPs were also detected in adult ESPs with similar characteristics. However, the 37 and 38.5 kDa DNases of the adult ESPs were not detected in the L3 ESPs. Since the 37 and 38.5 kDa DNase activities were mainly detected in adult ESPs, these activities appear to be specific to the adult stage whereas the other ESP DNase activities appear to be expressed during multiple stages of the parasite's life cycle. While the difference in DNase activities of L3 and adults remains obscure, the role of DNase in larval development should be further clarified and the identification of stage-specific developmental markers will lead to the discovery of specific factors that stimulate larval development.     

A preliminary study of the effect of microclimate on third-stage larvae of Haemonchus contortus and Haemonchus placei on irrigated pasture.

Assessments were made on the influence of several microclimatic variables on the availability of third-stage larvae of Haemonchus contortus and Haemonchus placei on four strata of irrigated Kikuyu pasture. Three replicates of these pasture samples were collected on 18 sample days over 12 months and the log10 mean counts of the larvae recovered were analysed by a step-wise regression model. Predictors for the log counts of the four strata for the two nematode species included relative humidity, illumination, air temperature and windspeed. The effect of air temperature on larvae of both Haemonchus species was similar; as air temperature increased, the number of larvae on pasture increased. The inverse was true for windspeed; as windspeed increased larval counts decreased. For H. contortus, relative humidity increased as the number of larvae increased on all strata except upper herbage. The R2 values ranged from 0.11 to 0.21 for H. contortus and from 0.04 to 0.12 for H. placei. Under the conditions of this study, only 21% of the effect on H. contortus and 12% on H. placei third-stage larvae on pasture can be explained by microclimatic conditions.     

Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.     

Toxicity of Bacillus thuringiensis to parasitic and free-living life-stages of nematode parasites of livestock

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH相似文献   

Silencing of essential genes by RNA interference in Haemonchus contortus

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.     

The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.