| Porcine viral diarrhea is a common epidemic disease in pig industry,its main pathogens include epidemic diarrhea virus of porcine(PEDV),transmissible gastroenteritis Virus of porcine(TGEV),and porcine rotavirus(Po RV).Among them,the rate of PEDV infection and mortality were the highest.At present,the main prevention and control of porcine viral diarrhea is vaccine immunization,commercial vaccines have been commercialized without uniform standards,poor immunogenicity and high incidence of piglets.By consulting the relevant information and datas,Probiotics can be used as green environment-friendly addition,and combined application of nucleic acid vaccine can enhance its immune effect,it can promote the growth performance,cytokines and mucosal SIg A secretion of piglets,the development of immune organs and small intestinal villi.In this study,we screened candidate vaccine strains closely related to the prevalent strains of Guizhou province,gestation sow and fetal immunization,Study on the effect of compound probiotics on the immune effect of porcine viral diarrhea.The specific research contents are as follows:1.Molecular epidemiology of some strains of PEDV in Guizhou Province:Collected 120 intestinal tract contents and feces of newborn piglets in a large-scale pig farm in Guizhou,PEDV S and M gene sequences were obtained by RT-PCR amplification,sequencing and bioinformatics analysis.RESULTS : the nucleotide sequence of the S gene is 4 161 bp,encoding 1386 amino acids,it named GZFQ11-2-016,the M gene is 681 bp,encoding 226 amino acids,it named GZFQ06-2017.The S gene sequence was the highest with the reference strain CV777 and KH 86.6%,in the same branch of the phylogeny tree,M gene was the highes with Attenuated DR13,SC1402 and SQ2014 71.4%,in the same branch of the phylogeny tree,the conformance with Attenuated DR13,SC1402 and SQ2014 is 71.4%,in the same branch of the phylogen-y tree,the consistency with reference strain CV777 was 70.6%.GZFQ11-2016 and GZFQ06-2017 strains mutated or mutated compared with multiple reference strains,the homology of the reference strain CV777 is relatively high,it is suggested that the reference strain CV777 can be used as a candidate vaccine for PEDV vaccine in Guizhou province.2.Study on the growth and decline of antibody against porcine viral diarrhea vaccine: A total of 100 pregnant sows were screened for TGEV and Po RV antibodies and antigens.They were divided into 5 groups: A,B,C,D and control groups,Among them,group A was inoculated with PTR triple attenuated vaccine on both the first of 40 days and the second immunization of 20 days before birth(hereinafter referred to as PTR),group B was inoculated with PT combined inactivated vaccine on both one of 40 days and two times immunization of 20 days before birth(hereinafter referred to as PT),group C first immunized PTR on 40 days before birth and PT on 20 day before birth,group D first immunized PT on 40 days before birth and PTR on 20 days before birth,the control group was injected with saline 40 days and 20 days before birth.Separately,20 piglets were randomly selected from A,B,C and D groups,is divided into the first,second,third and fourth groups,the 28 day old piglets were first and 65 days of age were second immunized,immune program is the same as sows.Control group 60 piglets from sows were randomly divided into 3 groups,among them,the fifth group was inoculated with PTR for 3 days and 35 days old,the sixth group was inoculated with PTR for 1 and 3 days,and the control group for PTR for 28 days.The milk collected from sows after 0 d to 14 d after delivery was detected by ELISA method.The titer of PEDV SIg A antibody was detected,the supernatants were collected and centrifuged at the age of 7 days and 80 days old.The titers of PEDV,TGEV and Po RV Ig G antibody were detected by ELISA method.RESULTS : There was no protective effect on piglets by injecting PT or PTR only at 40 and 20 days postpartum for 7 to 10 days after delivery,injection of PTR and PT on day 14 of parturition had protective effect on piglets.It show that the effect of PTR and PT inoculation was better than that of single PTR or PT,the piglets were inoculated with PTR twice at 28 days of age and 3 times with PT at 65 days of age by nasal drip inoculation of the pig-lets at the age of 3 days,after 15 days,the titer of antibody was on the rise.In this study,the clinical effect of group C was the best.3.The effects of probiotics on growth performance,cellular and mucosal immunity of piglets:According to the experimental group C,the piglets were divided into 120 groups: the experimental group and the control group,Weaning at 21 old days,the piglets in the experimental group were divided into 60 groups: group I,II and III,adding 0.10%,0.20%,0.30% compound probiotics in the basal diet respectiv-ely,the control group was randomly selected 20 pigs as the control group to feed the basal diet(excluding compound probiotics).The piglets were evaluated for their growth performance,blood physiological and biochemical,serum cytokine,intestinal mucosal SIg A,MC distribution of immune organs and small intestinal villi form of piglets at different stages.RESULTS : Lactobacillus cases-saccharomyces cerevisiae-Bacillus subtilis 3 compound probiotics can significantly increase the average daily gain of suckling piglets(P < 0.05),the rate of diarrhea in suckling piglets was reduced(P <0.01).The number of white blood cells,lymphocytes,red blood cells and platelets in piglets were increased(P < 0.05).The levels of serum LDH,ALP and TP were all higher than those in the control group(P < 0.01),and the contents of ALT,AST and serum BUN were higher than those in the control group(P > 0.05).The secretion of IL-6,IL-1 beta and TNF-a was increased(P < 0.05);SIg A level in duodenum,ileum and cecum mucous membrane(P < 0.05)and secretion of SIg A level in jejunum mucosa were increased(P < 0.01).The level of MC secretion in the immune organs of suckling piglets was significantly higher than that in the control group(P < 0.01).The structure of duodenum and ileum villus in the lactation stage was complete and orderly,but the recess mean test group was significantly higher than that of the control group(P < 0.01),and the average villus test group was significantly higher than that of the control group.The mean values of empty villus and recess in the experimental group were significantly higher than those in the control group(P < 0.01).However,the ratio of villus recess in jejunum and ileum was significantly lower than that in the control group(P < 0.01),and the duodenum test group was significantly lower than that of the control group(P < 0.05).After treatment,The average daily gain and aver-age daily feed intake were significantly higher in the experimental group than in the control group(P <0.05),and the difference of decrease diarrhea rate were extremely significant(P<0.01).In test group,the number of biochemical indexes including Lactate dehydrog-enase(LDH),Alanine aminotransferase(ALT),alkaline phosphatase(ALP),aspartate aminotransferase(AST),serum urea nitrogen(BUN)and total protein(TP)were higher than the control group,but the difference were not significant(P>0.05),inside the test group,those biochemical indexes shown no difference(P>0.05).After treatment 0 day,the levels of IL-6,IL-1β and TNF-a in the test group were same as control group(P>0.05),after treatment 20 days,the levels of IL-1β,IL-6 and TNF-a in the experimental group were significantly higher than those in the control group(P <0.05),after treatment 40 days,in test group,the IL-1β level were higher than control group(P<0.05),the level of IL-6 and TNF-a were extremely higher than control group(P<0.01).The level of SIg A in duodenum,jejunum,ileum and cecum in the experimental groups were higher than the control group but the difference are not significant,and the level of SIg A in jejunum mucosa was shown significantly different(P <0.01).the MC secretion level test in the inguinal lymph node group II was higher than that of the experimental group I and the test group III(P <0.01),and the MC secretion level test in the thymus MC group was higher than that of the test group.Group I and group III(P < 0.01).The level of splenic MC secretion in group III was higher than that in group I and group II(P < 0.01).The mean values of duodenal,empty and ileum villi in group II and III were significantly higher than those in group I and control group(P < 0.01).For the mean of recess,duodenal and jejunal test group II was significantly higher than group I,III and control group(P <0.01),the mean value of ileum recess group was significantly higher than group II,group III and control group(P < 0.01).It show that the compound probiotics can improve the growth performance,cellular and mucosal immunity of piglets,and promote the development of immune organs and small intestine.In Summary:In this study,successful cloning of PEDV S and M genes from two strains of Guizhou Province,bioinformatics analysis shows that there are new epidemic strains of PEDV in Guizhou Province in recent years,the current reference strain CV777 can be used as a candidate strain for vaccine selection in Guizhou Province.Respectively,PTR and PT were injected into pregnant sows 40 days and 20 days before pregnancy,Intranasal inoculation of PTR in 3-day-old piglets,PTR was immunized twice at the age of 28 days and PT was immunized three times at the age of 65 days.In this immune program,the PEDV SIg A antibody in the milk and the titer of PEDV,TGEV and Po RV Ig G in the immunization procedure lasted longer.,the optimum proportion of compound probiotics is 0.30%,compound probiotics can improve the growth performance of suckling piglets and early weaned piglets,Enhancement of cellular and mucosal immunity in piglets,it is helpful for the secretion of immune system MC and the development of villi of small intestine in suckling and early weaning piglets. |
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