| Riemerella anatipestifer is a highly pathogenic and infectious pathogen that causes perihepatic inflammation,pericarditis and balloon inflammation in ducks and geese.This bacterium mainly infects ducklings aged from 10 to 35 days,the incidence rate can be more than 90%,and the mortality rate can be up to 100%.The pathogen is widely found in duck farms all over the world.Once the disease occurs in duck farms,it is difficult to completely eliminate the infection,which seriously affects the development of duck farming.Currently,the control of the disease mainly depends on antibiotics,but due to the bacteria’s increasing resistance to a variety of antibiotics and the need for food safety,Riemerella anatipestifer has been widely used.At present,the actual production of Riemerella anatipestifer is not ideal and has strong side effects,in order to improve the safety and efficacy of Riemerella anatipestifer,it is necessary to make further research breakthroughs on this kind of vaccine.Referring to the production process of pertussis vaccine,it was found that such problems existed in the vaccine.However,the inactivation of glutaraldehyde greatly improved the irritation and side effects of the vaccine,which provided a direction for the improvement of the vaccine against Riemerella anatipestifer.At present,the vast majority of Riemerella anatipestifer use formaldehyde as an inactivated agent,and although formaldehyde is currently the most widely used inactivated agent,its damage to the immunogenicity,irritation and carcinogenicity have long been reported.Therefore,by comparing the effects of different inactivation methods on the immunogenicity of Riemerella anatipestifer,it is of great significance to improve the vaccine of r Riemerella anatipestifer.The traditional inactivation process of the inactivated vaccine of r Riemerella anatipestifer is to inactivate the vaccine at 37 ℃ for more than 24 h by adding formaldehyde solution with the final concentration of 0.1-0.38%.After referring to relevant literature,in addition to formaldehyde inactivation,the inactivation effect was monitored repeatedly by four methods: thermal inactivation,glutaraldehyde inactivation and povidone iodine inactivation to explore the inactivation conditions of Riemerella anatipestifer.After the preliminary inactivation scheme was designed from three perspectives of different action concentration,action time and action temperature,the design of the inactivation scheme was improved through experiments,and four inactivation schemes that could completely inactivate Riemerella anatipestifer were selected through repeated experiments.Finally,it was concluded that the inactivation methods of R.anatipestifer bacteria liquid within the concentration range of this test(A525=2.0±0.05)were as follows: 56℃ water bath for 30min;Water bath at 52℃ for 75min;(3)the final concentration was 0.03%~0.2% formaldehyde inactivation(37℃,24h);The final concentration was 0.1%~0.2% glutaraldehyde inactivation(4~8 ℃,24h);The final concentration was 0.74% povidone iodine inactivation(37℃,24h).R.anatipestifer-SCinactivated bacterial solution was prepared according to the above inactivated methods,and the inactivated oil emulsion inactivated seedlings of the corresponding group were prepared after passing the inactivated test.A total of 49 and 90 one-day-old non-healthy ducklings were successively selected and inoculated with immunogen at the age of 4 days after feeding.The same dose(109CFU/mL,0.3ml/duck)of the same strain(ra-sc)at the age of 18 days was used for two immune challenge protection tests.The results of the first test showed that the protective effect was the best in the group with final concentration of 0.1% formaldehyde inactivated,the group with final concentration of 0.2% glutaraldehyde inactivated and the group with final concentration of 0.74% povidone iodine inactivated,and the protective rate was as high as 100%(7/7),followed by the group with 56℃,30 min inactivated and the group with final concentration of 0.03% formaldehyde inactivated,with a protective rate of 85.7%.Second immune attack poison protection test results show that: 56 ℃ water bath for 30 min,52 ℃ water bath 75 min,the final concentration of 0.1% formaldehyde to inactivate group and the final concentration of 0.2% glutaraldehyde inactivated group protection effect is best,the recovery was 66.6%(6/9),the final concentration of 0.065% formaldehyde to inactivate group protection ratio was 55.5%(5/9),and the final concentration of 0.03% formaldehyde to inactivate group,0.1% glutaraldehyde inactivated and the final concentration of 0.74% povidone iodine inactivated group which was only 44.4%(4/9).The results of the two experiments showed that the vaccine prepared by thermal inactivation at 56℃,30 min,52℃,75 min,0.1% formaldehyde inactivation and 0.2% glutaraldehyde inactivation was ideal,but the immunogenic protective effect of 0.74% povidone iodine inactivation was decreased and unstable.Therefore,the methods of thermal inactivation and glutaraldehyde inactivation are expected to replace the traditional formaldehyde inactivation process in preparing the inactivated vaccine of Riemerella anatipestifer,which provides a new direction for the improvement of the inactivated vaccine of Riemerella anatipestifer.According the immune attack poison protection test and set up 56 ℃,52 ℃,0.1% formaldehyde,0.2% glutaraldehyde and 0.74% povidone iodine five patients and a healthy immune group,each group has 5 test duck,use the second immune attack poison protection test preparation of the corresponding set of vaccination(0.2 mL/only),immune preparation in 14 days after the blood serum.The first batch was inoculated at 5 days of age,and the second batch was inoculated at 26 days of age.The serum samples were subjected to a microagglutination test using the standardized ra-sc staining antigen to detect the antibody level of the serum.The results showed that the MAT background value of non-healthy ducks was 0~1 log2.The average MAT titers of the first batch of 5-day-old immunized ducks in the groups of 56℃,52℃,0.1% formaldehyde,0.2% glutaraldehyde and 0.74% polyvidone iodine were 2.90±1.34、2.35±0.65、3.40±1.29、2.50±0.71 and 3.35±1.31,respectively,with no significant difference between the groups.In the second batch of experimental ducks immunized at 26 days old,the average MAT titer of the groups of 56 ℃,52 ℃,0.1% formaldehyde,0.2% glutaraldehyde and 0.74% povidone iodine were 5.90±0.80、6.35±0.74、5.80±0.59、7.35±0.60 and 6.40±0.45,respectively.The titer of the glutaraldehyde group was significantly higher than that of the other groups,and the difference of the other four groups was not significant.Therefore,the age of 5,immune,56 ℃,52 ℃,0.1%,0.2% glutaraldehyde and formaldehyde povidone iodine immunogen to the methods of five kinds of inactivated in the effectiveness of the inducing antibodies from the duck is no too much of a difference,but in the age of 26,immune,0.2% glutaraldehyde inactivated immunogen in inducing antibodies from the duck is inactivated effect will be better than the other group. |
