| Haemonchus contortus is a blood-sucking nematode that primarily infects the abomasum of sheep and goats worldwide. Infections with this parasite can cause anaemia, weight loss and death, especially in lambs. The current methods for the control of gastrointestinal nematodes rely heavily on the use of chemicals, but this has led to an increase of anthelmintic-resistant parasites. Together with the growing concern over residual chemicals in the environment and food chain, the situation has resulted in the need to search for the development of alternative or supplementary means of control, including the molecular vaccines.In this study,we focused on the immunogenicity of HcACT and the clone of HcALD,did the experiments as follows:1. Construction and identification of its expression in vivo of DNA Vaccine pVAX1-ACTpVAX1-ACT DNA vectors were constructed by the DNA recombinant technique. After identification by restriction enzyme digestion, the recombinant DNA plasmids were then injected into leg muscle of mice respectively. In order to analyze the expression of the immune regulatory DNA vaccines in vivo, mice were administered with the mixture of pVAX1-ACT. Mice muscles were taken from the injected site, the transcriptions and translations of vaccines were tested by RT-PCR and Western Blot analysis, seven days post primary immunization and seven days after boosting. The result indicated that DNA vaccines could be well transcripted and expressed in injected tissues.2. Protective effect of ACT DNA vaccine on goats challenged with H. contortus L3larvaeFifteen locally bred goats (9-10months) were raised indoors under nematode-free conditions. The goats were allocated into3experimental groups, and balanced as far as possible for sex and weight. The H. contortus DNA vaccine was administered100μg each of pVAXl-ACT dissolved in lml of PBS (pH7.4).The lml injection volume was equally divided between two injections sites, alternating between the thigh and shoulder muscle. Unchallenged group served as the unvaccinated control animals that were not challenged with L3, but sham-vaccinated with1ml of PBS. Challenged group were the unvaccinated control, challenged with L3infection and sham-vaccinated with lml of PBS. Vaccinated animals received two vaccinations at2week intervals.5000infective H. contortus L3were given two weeks after the final injection. Faecal egg counts were performed once every two days from day22after challenge until the end of the experiment. Goats were killed35days after challenge. The abomasum was opened immediately, and worm counts were performed. In all goats, serum HcACT specific immunoglobulin IgG titres, IgA titres, mucosal surface (MS) IgA and mucosal homogenate (MH) IgA titres were determined with ELISA. In order to investigate protective mechanism of acquired immunity in goat, the peripheral CD4+CD8+T and B lymphocytes were determined by flow cytometry. The effects of pVAXl-ACT on the protein levels of IL-4, IL-22, interferon-γ(IFN-γ) and transforming growth factor-β(TGF-β) of goat peripheral blood were observed in this study. The peripheral blood eosinophil, basophil, neutrophil, monocyte counts and hemoglobin concentration were determined.Before L3challenge infection, the vaccinated groups had significantly higher HcACT-specific IgG titres and IgA titres than the unchallenged or challenged control goats. Following L3challenge, the vaccinated and challenged control groups had significantly higher serum HcACT-specific IgG titres and IgA titre compared to the unchallenged controls. There was a significant difference in the MH HcACT-specific IgA levels between the vaccinated group and unchallenged control. These findings suggested that the HcACT-specific IgG and MH IgA could play a role in protection against H. contortus parasite.It was found that prior to L3challenge infection, the%CD4+,%CD8+T lymphocytes and%B lymphocytes were significantly higher in the vaccinated group as compared to the unchallenged control. After L3challenge infection, the vaccinated and challenged control groups had significantly higher%CD4+T lymphocytes than the unchallenged control group. There was significant difference in%CD8+T lymphocyte between the challenged control group and vaccinated group. The challenged control group had significantly higher%B lymphocytes than the vaccinated group. The concentration changes of IL-4, IL-22, IFN-γ and TGF-β showed that the concentration of IL-4was significantly higher following DNA vaccination. This indicated that DNA vaccines constructed in this study could promote the secretion of cytokines. The mean number of blood eosinophils, neutrophil and monocyte increased after immunization coincident with low total worm burdens in the pVAXl-ACT protected goats. No significant difference was found in the basophil level between all groups. Following a single L3larvae challenge of group1(pVAX1-ACT) immunized goats, cumulative mean EPG (eggs per gram of feces) and worm burdens were reduced by34.4%and33.1%, respectively at the end of the experiment. These findings suggested that pVAXl-ACT against adult H. contortus offers partial immuno-protective effects against homologous infection in the goat.3.Molecular cloning, expression and characterization of aldolase from adult Haemonchus contortusThe aldolase gene of Haemonchus contortus (HcALD) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.AY594283) to amplify the3’-and5’-ends of HcALD. The full length of cDNA from this gene was obtained by overlapping the sequences of3’-and5’-extremities and amplification by reverse transcription PCR. The biochemical activities of the recombinant protein HcALD, which was expressed in prokaryotic cells and purified by affinity chromatography, were analyzed by assays of enzymatic activity. The results showed that the cloned full length cDNA comprised1235bp. The biochemical assay showed that the protein encoded exhibited enzymatic activity, whilst the HcALD was stable between pH6and8. The recombinant HcALD was recognized strongly by serum from naturally infected goats. These results suggested that the enzyme might play potential roles in the infection of H. contortus. |
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