| Avian hepatitis E is an acute hepatitis disease caused by Avian Hepatitis E virus,aHEV).It is reported that pathogenic aHEV mainly infects adult hens and is often transmitted through faecal-oral route.The presence of aHEV can be found during the immunohistochemical detection of intestinal tract,which indicates the importance of protecting intestinal tract in preventing and controlling the disease.Since 2016,severe hepatosplenomegaly and hepatorrhagia syndrome have appeared in many large farms in China.The comprehensive mortality rate of adult chickens has increased significantly and seriously affected the hatching and breeding of chickens.aHEV has been detected from many sick chicken flocks,and the overall onset age has a tendency to move forward gradually.However,there is little research on chickens infected with aHEV at present.Bacillus subtilis is a common intestinal probiotic.It can quickly adhere,colonize and reproduce in the intestinal tract of animal body,restore the unbalanced microecology to a balanced state,enhance the immune function of the body,and has certain antiviral effect.In this study,the pathogenesis model of aHEV infected SPF chicks was established.Based on the identification and domestication of Bacillus subtilis,the antagonistic effect of Bacillus subtilis on assisting chicks in resisting aHEV infection was observed.One-day-old SPF hens were randomly divided into two groups,20 in each group.The infected group was orally administered VaHEV-HB strain at the age of 1 week,while the control group was orally administered PBS to observe the death and morbidity.Body weight was measured at 2 weeks of age and 10 chickens in each group were euthanized.Liver,thymus,spleen and bursa of Fabricius were collected and weighed to calculate immune organ index.RNA was extracted from each organ and HEV viral load was detected by qRT-PCR.The cecum contents of dissected chickens were taken under aseptic conditions,and theV3+V4 region of 16 S r RNA was amplified by extracting genomic DNA from the samples with hipure stol DNA kit b kit,and then analyzed on the computer to detect intestinal microbial flora.The results showed that the death rate in the infected group was 20%,while there was no death in the control group.The weight of chickens in the attack group was significantly lower than that in the control group,with a difference of about 20 g.Infection with VaHEV-HB can cause enlargement of liver and spleen.The proportion of liver in infected group is between 7% and 13%,while that in control group is between 4% and 8%.The proportion of spleen in infected group was about 2.7%,while that in control group was about 1.8%,with significant difference.The analysis results of intestinal microflora showed that the difference between the avian HEV infection group and the control group was obvious,while the difference between different samples in the group was small,which indicated that the avian HEV infection could cause the same common and significant changes in different individuals.The abundance of Firmicutes in aHEV infection group was significantly higher than that in control group,but the abundance of Proteobacteria was significantly lower than that in control group.Body weight is related to the distribution of intestinal flora,and the increase in the number of sclerenchyma in the intestine leads to more effective absorption of heat in food,thus resulting in weight gain.Bacillus subtilis liquid isolated and cultured from soil was identified by 16 S r RNA universal primer(27f-1492r)on the basis of Gram staining and microscopic examination.The identified single colony was acclimated to acid and bile salt in LB liquid medium with PH value of 2,3,4,5,6,7,8 and bile salt concentration of 0.5g/L,1 g/L,1.5 g/L,2 g/L,2.5 g/L,3 g/L,3.5 g/L and 4 g/L respectively.One-day-old SPF hens were randomly divided into four groups with 20 hens in each group.The blank control group was given sterile LB;VaHEV-HB strain was administrated to the drug attack group at the age of one week.The third group was treated with domesticated Bacillus subtilis continuously for 1 week after 1 week of attack.In the fourth group,only domesticated Bacillus subtilis was administrated for one week at the age of 1 day.The mortality rate of chickens in each group was recorded,the body weight and organ weight were weighed and the immune organ index was calculated.Anti-coagulation RNA was extracted and the HEV viral load was detected by qRT-PCR.After necropsy,thececum of each chicken was collected aseptically and the cecum contents were collected for microbial flora analysis.The results showed that there was no death in the control group and the group only treated with Bacillus subtilis.The death rate in the attack group was 20%.The death rate in the group treated with domesticated bacteria was not only reduced to 10%,but also its body weight was higher than that in the control group and the attack group.The proportion of liver was significantly reduced to 4%-6% compared with 9%-11% in the attack group.The analysis results of intestinal microflora showed that the difference between the Bacillus subtilis treatment group and the control group was obvious,while the difference between different samples in the group was small.The intestinal microflora in the treatment group tended to be positive and the intestinal microecology recovered stably,indicating that Bacillus subtilis could cause changes in the intestinal microflora.In this experiment,the pathogenesis model of chickens infected with aHEV was established.On the basis of observing the pathogenicity of the virus,the dysbacteriosis of intestinal normal flora induced by aHEV infection was analyzed.Furthermore,the isolated and domesticated Bacillus subtilis proves in animal experiments that it can reduce the damage of avian hepatitis E virus to intestinal tract,reduce the mortality rate induced by aHEV infection and inhibit the proliferation and invasion of virus in intestinal tract to a certain extent.It provides more reference data for understanding the pathogenicity of aHEV and puts forward possible auxiliary prevention and control measures. |
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