Preparation And Immunological Characterization Of Two Combined Inactivated Vaccine Of Canine Sudden Death

Canine sudden death syndrome can lead to rapid death of young age dog,the sick animal died within hours of its onset,usually can cause gas gangrene,intestinal toxemia,hemorrhagic enteritis and other diseases.Death usually results from inability to be treated promptly after onset.Therefore,the prevention of canine sudden death should be based on prevention,but currently there is no clinical vaccine to prevent sudden death in dogs.The main pathogens causing sudden death in dogs are Clostridium perfringens and Clostridium botulinum.Clostridium perfringens is the most important pathogen.C.perfringens is Gram-positive bacterium widely distributed in nature,anaerobic and capable of forming spores.C.perfringens can be divided into type A,type B,type C,type D,type E and type F based on the genotypes of the four major exotoxins(α,β,ε,and ι)produced.Among them,type A is the most common type,and its main virulence factor is alpha toxin.Clostridium botulinum(C.botulinum)is gram-positive anaerobic bacterium,and the main virulence factor is botulinum toxin.Botulinum toxin can be divided into seven types: A,B,C,D,E,F,and G.Among them,botulinum toxin type A,which causes human disease.Botulinum toxin type C is a common cause of animal illness.In order to study the vaccine against canine sudden death,the research established models of infection of animals infected with C.perfringens and botulinum toxin,respectively(mice and dogs).Then use formaldehyde to detoxify the cultured bacteria or toxins to optimize the vaccine effective dose and adjuvant.Animal models were used to evaluate the safety and immunological characteristics of inactivated vaccines against C.perfringens and inactivated botulinum toxin vaccines,respectively.The proportion of the two vaccines was further optimized to prepare a double-inactivated vaccine for preventing sudden death in dogs.Study on Clostridium perfringens Inactivated VaccineDifferent doses of C.perfringens culture were used to inoculate the Kunming mice and Beagle dogs with intraperitoneal injection of toxin,and a mouse model of C.perfringens infection was established.C.perfringens Infected Mouse Model and Dog Model were established.The minimal lethal dose(MLD)of C.perfringens in mice and dogs are 200 μL per mouse(the number of viable cells is approximately 6×107 CFU)and 2,000μL per dog(the number of viable cells is approximately 6×108 CFU).All animals died within 1 day after infection.The main clinical symptoms were abdominal swelling of the animal,weakness in limb weakness,hemorrhage in the anus.Histopathological section showed large infiltration of inflammatory cells in the small intestine,thickening of the alveolar wall,and inflammation of the interstitial lung.Infiltration of cells with slight fibrosis around the central vein of the liver.The use of formaldehyde to inactivate C.perfringens cultures optimized the formaldehyde inactivation conditions for C.perfringens for a final concentration of 0.5% formaldehyde 48 h detoxification.After the detoxicated C.perfringens culture solution was mixed with oil adjuvant and aluminum adjuvant,the mice were immunized by intraperitoneal injection and subcutaneous injection respectively,and the serum antibody was detected by ELISA.The results showed that the antibody titer of immunized mice reached up to 105.The serum antibody levels in mice immunized with subcutaneous injections were significantly higher than those in mice immunized intraperitoneally.Serum antibodies produced by immunization with aluminum adjuvant were slightly higher than those produced by immunization with oil adjuvant vaccines.The mice were used to evaluate the immune-protective effect of the inactivated vaccine against C.perfringens.The mice were immunized twice at a dose of 200 μL per mouse and the immunization interval was 14 days.Fourteen days after strengthened immunization,the challenged mice were challenged and all mice survived after subcutaneous injection of aluminum adjuvant(10/10).All other immunized mice had different degrees of death,while the mice of control group were all died,indicating that the vaccine had a good immune protective effect.Carrying on the long-term immune level monitoring via mice,the results showed that the mouse serum antibody reached up to the highest at the 28 th day after immunization,and then slowly decreased until the 120 th day after immunization,the antibody level was still keep at a relatively high level.Evaluation of immune protection of inactivated vaccine against C.perfringens using original host animal canine.The dogs were immunized with doses of 0.5 m L/dog,1 m L/dog,2 m L/dog,and 4 m L/dog,respectively.The results showed that the use of double immunization methods(immunization interval was14 d);Once immunized,an immunization dose of 4 m L per dog could produce a complete immune protective effect in dogs(5/5).This research also prepared alpha toxin inactivated vaccine.After C.perfringens was cultured overnight in toxigenic medium,the supernatant was collected by centrifugation.After adding formaldehyde to detoxify the virus for a final concentration of 0.5% for 48 h,add saturated ammonium sulfate to a saturation of 50%,collected the precipitate after centrifugation at 4°C overnight.The precipitaite was dissolved in PBS with an original volume of 1/10 to obtain the crude α-toxin.Toxin protein was added to the aluminum adhesive adjuvant to prepare an inactivated toxin vaccine.The dogs were subjected to an immune-protective experiment,and the dogs were challenged intraperitoneally on the 14 th day after subcutaneous immunization of the dogs.As a result,a single immunization dose of 0.5 m L per dog produced a completely immuneprotective effect(5/5).Study on botulinum toxin inactivated vaccineClostridium botulinum is an obligate anaerobic bacterium,large-scale fermentation production is difficult and costly.Therefore,this study was based on the reported botulinum toxin gene sequence(Gen Bank accession number: D90210).According to the codon tropism of E.coli,the optimized botulinum toxin gene(Bo NT-C)was synthesized.The synthesized Bo NT-C gene was cloned into the temperature-controlled expression plasmid p BV220 between the Eco RI and Bam HI sites.A temperaturecontrolled expression recombinant plasmid p BV220-Bo NT-C carrying botulinum toxin type C was constructed.Recombinant strain DH5α(p BV220-Bo NT-C)expressing botulinum toxin type C was constructed.Intraperitoneal injections of Kunming mice and Beagle dogs were performed with different doses of botulinum toxin-producing strain DH5α cultures.The results showed that the minimum lethal dose(MLD)in mice and dogs were 20 μL(the number of viable cells about 6×106 CFU)and 1,000μL(the number of viable cells about 6×108 CFU),respectively.All animals died within 1 day after infection.The main clinical symptoms were shortness of breath,slow movement,and difficulty in looking up.Histopathological sections showed that there was a large number of inflammatory cell infiltrate in the lung interstitium,mainly mixed inflammatory cells and visible in the small intestine.Inflammatory cell infiltration.Using formaldehyde to inactivate the recombinant strain DH5α culture,optimization of formaldehyde detoxification of C.botulinum toxin producing E.coli in 0.5% formaldehyde concentration for 24 h.The botulinum toxin-producing Escherichia coli culture solution detoxified by formaldehyde was mixed with the aluminum adhesive adjuvant and the oil adjuvant in proportion,and the mice were immunized by subcutaneous injection and intraperitoneal injection,respectively.In the 28 th day,the level of antibody produced by the aluminum adjuvant group was significantly higher than that of the oil adjuvant group.Among them,the level of antibody produced by subcutaneous injection of aluminum adjuvant and intraperitoneal injection of aluminum adjuvant reached to the highest level,but there was no significant difference between the two groups.Combined with the ELISA results and the immune conditions determined by C.perfringens,the adjuvants for follow-up experiments in this study were all aluminum adhesive adjuvants.The immunization route was subcutaneous injection.The use of mice to evaluate the immune-protective effect of an inactivated E.coli botulinum toxin type C vaccine,the immunization dose was 200 μL,the mice were immunized twice and immunization interval was 14 days,challenged after 14 days of the strengthened immunization,the results showed that all mice survived after subcutaneous injection of aluminum adjuvant and intraperitoneal injection of aluminum adjuvant(10/10).All other immunized mice had different degrees of death,while all mice in the control group died,indicating that the aluminum adjuvant vaccine had a good immune protective effect.Carrying out a long-term monitoring of the immune level via mice,the results showed that the mouse serum antibody reached the highest level on the 35 th day after immunization,and then decreased slightly.From the 70 th day to the 120 th day,the antibody level decreased slightly but remained within a certain range.And the antibody still kept a high level.Immune protection of botulinum toxin type C of E.coli DH5α inactivated vaccine was evaluated using the original host animal canine.The dogs were immunized with 0.5 m L/dog,1 m L/dog,2 m L/dog,and 4 m L/dog,respectively.Two immunization methods(immunization interval was 14 d);Once immunized,immunization doses of 4 m L per dog can completely protect the dog(5/5).In this study,a botulinum toxin inactivated vaccine was prepared.After culturing botulinum toxin type C of Escherichia coli DH5α in LB medium overnight,the supernatant was collected by centrifugation,formaldehyde was added to a final concentration of 0.5%,detoxification was conducted for 48 h,and saturated ammonium sulfate was added until the saturation was 50% at 4°C.After overnight,the precipitate was collected by centrifugation.The botulinum toxin was obtained by dissolving it in an amount of 1/10 of the original volume of PBS.Toxin protein was added to the toxin protein to prepare an inactivated toxin vaccine.The dogs were subjected to an immuneprotective experiment.The dogs were challenged intraperitoneally on the 14 th day after subcutaneous immunization,the results showed that a single immunization dose of 1 m L per dog produced a completely immune protective effect(5/5).Study on Inactivated Vaccine of Canine Sudden Death Syndrome(Alpha Toxin and Botulinum Toxin)In order to prevent the occurrence of sudden canine death more comprehensively,the alpha-toxin-containing solution and the botulinum-toxin-containing solution were mixed in a ratio of 1:1,1:2,and 1:4,and an aluminum adjuvant was added to prepare a double-combined inactivated vaccine.Dogs were used to evaluate the immune effect of a double-inactivated vaccine.The dogs were immunized with subcutaneous injection at a dose of 2 m L for a single immunization.The C.perfringens and C.botulinum toxin recombinant E.coli cultures were injected on the 14 th day after the immunization,and the mixing ratio was 1: 1 could produce completely immune protective effects for both toxins at the same time,and all experimental dogs survived(5/5).For the non-immune control group,the dead dog collected tissues were examined by microscopy,the tissues were collected after the euthanasia of dogs and normal dogs after challenge with the immune group were observed under microscopy.As a result,the immunized group was similar to the normal dog tissue sections,and there were no obvious pathological changes.However,in the uninfected control dogs,a large number of inflammatory cell infiltrates appeared in the pathological sections of the small intestine after challenge with gas-producing genomic bacterium,and the alveolar wall was thickened significantly and there was a large amount of inflammatory cell infiltration in the lung interstitium.The hepatic venous fibrosis in the liver.Pathological sections of the lungs after injection of type C E.coli DH5α showed a large number of inflammatory cell infiltrate in the interstitial space,mainly inflammatory cells in the mixed intestine,and inflammatory cells infiltrated in the small intestine.Summarize the result of this study:(1)Clostridium perfringens and botulinum toxin-producing of E.coli animal models of infection(mouse,dog)were established;(2)The type A and C type toxin inactivated vaccines were prepared,optimized vaccine preparation methods and immunization methods,animal experiments confirmed that the vaccine had good safety and immune protection;(3)An inactivated combined vaccine against canine death(alpha toxin and botulinum toxin)was prepared,optimized distribution ratio of the vaccine group,animal experiments confirmed the vaccine has a good safety and immune protection,could be used to prevent sudden death in dogs.