| Histomoniasis,also known as infectious enterohepatitis or "lackhead disease",is a type of protozoan parasitosis caused by Histomonas meleagridis(H.meleagridis),which can infect galliform birds.It is mainly characterized by liver necrosis,cecal enlargement and sulfur-like feces.Chemical medicine has played important roles in the control of this disease for a long time,however,it has been banned to use by many countries and regions in recent years because of its potential toxicity and carcinogenicity,which causes the spread of this disease and serious harm to poultry industry.The host protective immunity triggered by H.meleagridis mainly based on cellular immunity.This study carried out a series of work around the development of attenuated live vaccines of H.meleagridis in vitro,and a safe and effective attenuated live vaccine was preliminary developed successfully in this study,which will be expected to become a safe,efficient and convenient novel method for the control of the disease.1.Evaluation of the attenuate effect of H.meleagridis from different generations H.meleagridis JSYZ-A strain was isolated from chicken and was attenuated through serial passages in vitro.Different generations(A59,A163,A250)of H.meleagridis were used to infect 15-day-old Jinghai yellow chickens through the cloaca.Chickens were culled at 20 days after infection.Incidence,mortality,weight gain,lesion scores and PCR detection rate of viscera were analyzed and compared amongst the different generations to evaluat the attenuated effects of H.meleagridis.The results showed that the incidence rates of the chickens decreased gradually with the increase number of generations in vitro,while the weight gains gradually improved.The relative weight gain rate of A250 generation was the highest,reaching 88.42%.The score of liver lesions in A250 generation was zero,which was not significantly difference with the uninfected negative control group(p>0.05),and was significantly lower than that of the high virulent group and A59 generation(p<0.05).For the scores of caecum lesion,there was no significant difference among A250,A163 and A59 generations and between A250 and the negative control group,respectively(p>0.05),and a extremely significant difference was found between A250 and the high virulent group(p<0.01).The scores of liver and cecum gradually decreased with the increase of the number of generations.PCR results confirmed that the replication of the attenuated H.meleagridis in the target organs was not significantly affected.The results of this study suggests that the pathogenicity of H.meleagridis was weakened by serial passages in vitro,and was gradually decreased as the increase of the number of generations.The A250 generation of H.meleagridis had been basically completely weakened and safe,which laid a foundation for the study of the immune protection effect of the attenuated strain.2.Preliminary study on the immune protection effect of the attenuated strain of H.meleagridisIn order to understand the immune protection effect of the attenuated strain,the 15-day-old Jinghai yellow chickens were inoculated with attenuated strain A163 through the cloaca,and then was challenged with the virulent strain at 15 days post inoculation.At the same time,a non-immunized challenge group(positive control)and non-infected negative control were set up in this study.All chickens were culled at 20 days after challenge.The indicators containing incidence,mortality,weight gain,score of viscera lesions were used to preliminarily evaluate the immune protection effect of the attenuated strain.The results showed that morbidity of the immunization group decreased by 40%compared with the positive control group,and the symptoms were relatively mild.The relative weight gain rate of the immunization group was 88.57%,significantly higher than 62.46%of the positive control group(p<0.05),and no significant difference was found in relative weight gain between the immunization group and the negative control grouq(p>0.05).Compared to the postive control,the score of the liver lesion in the immunized group reduced by nearly 50%,which was not significantly different from the negative control(p>0.05).Compared to the positive control,less caecum injury was found in the immunized group,of which chickens with cecal injury decreased by 40%,and the average lesion score decreased significantly(p<0.05),however,the difference was still significant compared with the negative control(p<0.05).Above results showed that the pathogenicity of the A163 H.meleagridis gradually decreased with the increase of the number of generations,and A163 can have a certain immune protection effect on the chickens.A larger number of generations of attenuated H.meleagridis will be used for the further development of live vaccines3.Determination of the optimal immune dose of the attenuated strain of H.meleagridisTo explore the optimal immunization dose of attenuated strain,15-day-old Jinghai yellow chickens were immunized with three different immune dose(2×105/feather,105/feather,and 5×104/feather)of the attenuated strain A250 through cloacas,respectively.At 30 days post immunization,the virulent strains were used to performe the parasite challenge of chickens,which were killed at 15 days post challenge.Incidence,mortality,weight gain and score of viscera lesions were used as indicators to compare the immune effects among three immune dose groups.The results showed that morbidity the chickens was from high to low in the order of the positive control,low immune dose,medium immune dose,high immune dose and the negative control groups.The incidence rate of the high immune dose group was 11.11%,which was lower than the positive control(88.89%).No significant difference was found in the weight gain among these three immunized groups(p>0.05),which were significantly higher than the positive control group(p<0.05).The weight gain of the high immune dose group was similar to that of negative control group,and the relative weight gain rate reached 91.08%.There was no significant difference in the average liver lesion scores among the four groups:low immune dose group,medium immune dose group,high inunune dose group and negative control group(p>0.05),however,a significant difference was found between these four groups and the positive control group(p<0.05).The average of lesion score of high immune dose group is lowest(0.11).In terms of cecal lesion score,there was no significant difference among high immune dose,medium immune dose and the negative control groups(p>0.05),while there was a significant difference among high immune dose,low immune dose and postive groups(p<0.05),and the average of lesion score of the high immune dose group is the lowest There were significant differences between the positive control and the other groups(p>0.05),and the difference between the positive control and the negative control groups was extremely significant(p<0.01).The above results showed that the high immune dose had the best immune protection effect.Considering the total volume of the inoculated vaccine and the tolerance of chickens,a immune dose of 2×105/feather is recommended in clinic,and the effect of a higher immune dose was not verified.4.Determination of the best immunization program of the attenuated strain of H.meleagridisIn order to explore the best immunization program for the attenuated strains,two experiments were conducted:test one with twice immunizations at 3 and 9 days(group A)and once immunization at 3 days(group B),and test two with twice immunizations at 3 and 15 days(group C)and once immunization at 15 days(group D).In both experiments,a non-immunized challenge with a virulent strain(positive control)group and a non-immunized negative control group were established,respectively.The virulent strain challenge was carried out at 15 days after the second immunization.Then,chickens were killed at 18 and 24 days after the challenge.Incidence,mortality,weight gain,and lesion scores were used as indicators to evaluate the immune effects.The results showed that compared to the once immunization groups(groups B and D),the incidence rate was significantly lower,the weight gain effect was slightly better,and the target organ damage was less in chickens of the twice immunizations groups(groups A and C).Comparing the indexes of morbidity,weight gain and lesion scoring,group C was slightly better than that of group A.The results suggested that twice immunizations at 3 and 15 days were the better immunization procedures,which might be suitable for clinical application.The above research results indicate that the immune protection effects of the attenuated strain of H.meleagridis through serial passages in vitro obtained in this study is indeed,which is expected to become a green and safe novel method for the control of Histomonosis,and has certain significance for the steady development of poultry industry and human health. |
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