Immunization Program Optimization And Safety Study In Buffalo Calves Inoculated With SS And CST Double Expression DNA Vaccine

The buffaloes suffer from slow rates of growth and late maturity.Calves that grow faster show a short prepubertal period and calve at a younger age,shorter slaughter cycles,higher fecundity and lower cost of rearing.Therefore,how to promote the growth of buffalo calves has always been a hot spot for researchers over the globe.The somatostatin（SS）and cortistatin（CST）double-expression DNA vaccine without antibiotic resistance gene previously constructed by our laboratory can significantly improve the growth rate of mice,and explored the dose of intranasal immunization of buffalo calves,but the weight gain effect is not significant.Therefore,it is necessary to carry out further experiments to explore and optimize the immunization procedure of calves vaccinated with this vaccine.Which will provide a reasonable immunization program for the large-scale breeding of buffalo farms,and provide a theoretical basis for the popularization and application of this DNA vaccine.This experiment would discuss the immune response,growth-promoting effect and safety of calves vaccinated with double-expression DNA vaccine from the aspects of immunization routes and immunization dose.The main research contents and results are as follows:1.Identification and genetic stability of engineered bacteriaThe genetically engineered bacteria C500（pVGS/2SS-2A-S/CST-asd）stored in the laboratory was resuscitated and cultured,and the plasmid was extracted.After the correctly identifying of the plasmid by enzyme digestion and sequencing,it was continuously cultured for 50 generations.The bacteria of the 2^nd,10^th,20^th,30^th,40^thand50^thgenerations were cultured for PCR amplification.The target gene 2A-S/CST,the resistance screening gene asd,the attenuating gene crp and the Salmonella specific sequence inv A gene could be amplified,which indicated that the recombinant bacteria had no plasmid loss and reversion to virulence during the passage;Plot the growth curve of recombinant bacteria and control bacteria showed that the growth trend of recombinant bacteria containing target plasmid tended to be the same as that of control bacteria without target plasmid,indicating that the introduction of the dual-expression plasmid did not affect the normal growth of the attenuated Salmonella.2.Influence of the immunization route of double-expressed DNA vaccine on the immune response and growth of calves27 buffalo calves aged 2～6 months were randomly divided into four groups.The groups were orally（group O）,nasally（group N）,and orally plus nasally（group ON）vaccinated with dual-expression DNA vaccine at a dose of 3×10⁷CFU/mL,respectively.The control group（C）was orally plus nasally immunized with PBS,and boosted immunization with the same procedure after two weeks.The results showed that all three immunization routes could induce calves to produce antibody of somatostatin and cortistatin.After two weeks of primary immunization,the positive rates in N and ON groups were the highest,both at 85.71%.The end of test,the positive rates in ON group decreased to 14.29%,and no antibodies were detected in the O and N groups.With respect to weight gain,at 2^ndweek,the average daily gain in the ON group was significantly higher（P<0.05）than that in the O,N,and C groups.0～10 weeks,compared with the C group,the O,N and ON groups were respectively increased by 5.26%,10.53%,and 15.79%.After 10 weeks of primary immunization,the chest girth and leg-hip circumference in the ON group were significantly higher（P<0.05）than those in the C group.The hormone measurement results showed that the concentration of GH,IGF-1,and T₃in each group was as ON>N>O>C group.The IGF-1 concentration of O,N and ON group was significantly higher（P<0.05）than that of C group at 2^ndweek.At 6^thweek,the concentrations of T₃and T₄in the ON group were significantly higher（P<0.05）than those in the O and C groups.The cytokine test results showed that the contents of IL-4and INF-γin the three experimental groups were higher than those in the C group,indicating the three immune routes could stimulate the body to produce humoral and cellular immunity.The detection of blood biochemical indexes showed that there was no significant difference（P>0.05）in the contents of total protein,glucose,non-esterified fatty and urea nitrogen in the serum of calves between the groups.3.Effects of double expression DNA vaccine dose on the immunity of calves24 buffalo calves aged 2-6 months were randomly divided into 4 groups.They were immunized with nasal 3×10⁷CFU/mL+oral 3×10¹⁰CFU/mL(NO₁₀group),nasal 3×10⁷CFU/mL+oral 3×10⁹CFU/mL（NO₉group）,nasal 3×10⁷CFU/mL+oral 3×10⁸CFU/mL（NO₈group）.The control group（C1）were immunized orally+nasally with PBS,and boosed immunization once two weeks later.The results showed that the positive rates of NO₁₀,NO₉and NO₈groups were 50.00%,83.33%and 83.33%after two weeks of primary immunization,respectively.After booster immunization,the positive rate of calves in each group did not increase,but decreased;There was no significant difference in the serum SS antibody level of calves in each group at each period（P>0.05）.Throughout the whole test cycle,the antibody level and positive rate of NO₈group were the highest in both somatostatin antibody and cortistatin antibody.The average daily gain of calves in the NO₈group was the highest in different periods.After 2 weeks of first immunization,the average daily gain of calves in NO₁₀,NO₉and NO₈groups were increased by 13.04%,13.04%,and 17.39%,respectively.From the change of body size,the chest girth and leg-hip circumference of the NO₈group were significantly higher than those of the control group at ten weeks（P<0.05）.The results of serum hormone detection showed that the contents of GH and IGF-1 in serum of calves in NO₈group were higher than those of the other three groups at each period,and at 6^thweek,the content of IGF-1in NO₈group was significantly higher than that in the control group（P<0.05）.4.Safety assessmentThe target gene was not detected by PCR amplification of the enviromental water,soil,feces samples,and the blood genomic DNA extracted from immunized calves,indicating that the recombinant bacteria are safe for the environment and calves.At 2th week and 6th week after the initial immunization,the blood components（WBC,RBC,HGB,HCT,MCV,MCH,MCHC,RDW）of the calves in each group were detected,and it was found that the three immunization routes had no significant effect on the blood components of calves,indicating that the double-expression DNA vaccine did not effect the blood circulation system and immune cells of calves,preliminarily demonstrating that the vaccine was also safe to immunize large animals.In conclusion,the SS and CST double-expression DNA vaccine was safe for calves and the environment,and the optimal immunization procedure was:oral 3×10⁸CFU/mL+nasal 3×10⁷CFU/mL,with a better weight gain effect in buffalo calves.