| The precocious line of Eimeria was first selected by Jeffers in1975. Compared with its parentsrtain, the precocious line has a reduced prepatend period for the missing or defect of the last generationschizont, markedly less pathogenicity, and much lower reproductive potential, but retained goodimmunogenicity and genetic stability. The attenuated live anti-coccidial vaccines, Paracox and Livacox,have been developed and widely used for the control of coccidiosis. Immunization with the attenuatedvaccines had been proved to be effective and is increasingly playing an important role in the control ofcoccidiosis in the poultry industry. The precocious lines of Eimeria acervulina, E. maxima and E.tenella have been selected successfully and their biological characteristics have been studied in our lab.The objective of the present study was to study the drug sensitivity and optimal immunizing dose of thethree precocious lines, and to evaluate the anti-coccidial efficacy of trivalent vaccine. The results fromthis study would help to develop the live, attenuated anticoccidial vaccine.1. Sensitivity of three precocious lines to eight anticoccidial drugIn order to test sensitivity of precocious line of E.maxima, E.acervulina and E.tenella toanticoccidial drugs, eight anticoccidial drugs, including of diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin, salinomycin and lasalocidt, were selected to use in the study.Based on the percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS),relative oocyst production (ROP) and anticoccidial index (ACI), the results showed that the sensitivityof the precocious line and parent strain of E.acervulina were basically similar. The two strains ofE.acervulina were completely sensitive to diclazuril, robenidine, decoquinate, nicarbazin, maduramicinand lasalocid, partly sensitive to salinomycin and not sensitive to dinitolmide.the precocious line ofE.maxima was completely sensitive to eight anticoccidial drugs, the precocious line of E. tenella waspartly sensitive to salinomycin and completely sensitive to diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin and lasalocidt. This study will provide experimental bases fordevelopment and application of the precocious lines vaccines of coccidia.2. Optimal immunization dose of three precocious linesIn order to test optimal immunizing doses of precocious lines of E. acervulina, E.maxima andE.tenella, dosages of100,200,400,600,800,1000and2000sporulated oocysts were administeredorally to7-day-old chickens for two times at a7-day interval.21-day-old chickens were challenged withthe sporulated oocysts of the parent strains. Weight gain, intestinal lesion scores and oocysts inhibitionratio were used to evaluate the immunizing effectiveness.2-4optimal immunizing doses obtained fromthe first trial was repeated. The results from the first trial showed that the600-1000E. acervulina,200-800E. maxima and600-2000E. tenella could induce sufficient immunological protection, whichthe reduction of oocysts ratios were over90%、95%and60%, respectively. The results from therepeated trial indicated that the optimal immunizing dose of the precocious line of E. acervulina, E.maxima and E. tenella was600,200and600ooysts/bird, respectively. 3. Protective efficacy of the trivalent attenuated live coccidiosis vaccineThe trivalent coccidiosis vaccine was prepared based on the optimal immunizing doses of theprecocious lines of E. maxima, E. acervulina and E. tenella, containing200,600and600oocysts ofeach line, respectively, per dose.0.25,0.5,1.0,1.5and2.0dose of vaccine were administered to4-day-old chicks, respectively. The protective efficacy of this vaccine was evaluated as comparison withthe commercial vaccine, Coccivac (1dose per chicken) and the unvaccinated birds. The oocyst per gramfecal (OPG) was accounted at4-24days after vaccination. The antibody level, the capability of splenicT lymphocyte proliferation and the weigh gains were investigated at the day of-1,7,14and21aftervaccination. On the day of14and21after vaccination, ten birds of each group was challenged with themixed oocysts of the parent and virulent strains of each precocious lines used in this vaccine,respectively. The results demonstrated that the reduction in weigh gains of the vaccinated chickens wasobserved at7and14days after immunization, while the weigh gains were similar at21days afterimmunization between the vaccinated chickens and unvaccinated ones. Three peaks of oocystsproduction were observed and the highest peak occurred at the day11-12after immunization. Theantibody level of the vaccinated group at the day of14after immunization was significant higher thanthe unvaccinated one, and maintained at the high level at the day of21after immunization. Thecapability of splenic T lymphocyte proliferation of immunized chickens was significantly higher thanthe he unvaccinated ones at the day of14after immunization, reached the highest at21days afterimmunization and maintained at the high level at the day of28after immunization. Results from the twochallenged trials showed that the protective efficacy of this trivalent coccidiosis vaccine was superior tothe Coccivac vaccine.4Identification and partial characterization of ATPase geneThe results showed that the ATPase gene can be amplified from three precocious lines of E.maxima, E. acervulina and E. tenella. Homology analysis revealed that the sequence similarity were upto100%, indicating that the ATPase were conserved genes in different species of Eimeria. Real-timequantitative PCR analysis revealed that the EtATPase gene was expressed at lower levels in the threeprecocious line than their parent strains, and at higher levels in sporozoites than in the otherdevelopmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites).The fusion protein of EtATPase gene is an inclusion body protein with the molecular weight of22kDa.Western-blot analysis showed that this protein had good antigenicity. Indirect immunofluorescencetechnique with specific antiserum generated against the recombinant protein localized the EtATPaseprotein in the refractive body of E. tenella sporozoites. Invasion inhibition assay showed that EtATPasemay not play an important role in invasion of the sporoziotes in the host. |
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