CN1371748A - Zhonghua soft-shelled turtle aeromonad oral slow-releasing microsphere vaccinum and preparation process thereof - Google Patents

Abstract

The present invention discloses an aeromonad oral slow-released microsphere vacine for Chinese terrapin and its preparation method. It is microsphere vaccine made up by utilizing inactivated bacterial solution of Chinese terrapin common disease source-temperate aeromonad, using bio-degradable polymer material as carrier and adopting spray-drying method to make covering process. Its preparation method includes seven steps: preparing bacteria seed, cultivation, counting, inactivation, microsphere preparation, checking and analysis of immune effect. It can effectively prevent disease due to aeromonad.

Description

Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum and preparation method thereof

Technical field

The present invention relates to a kind of Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum and preparation method thereof.

Background technology

Because China's " excellent two-supremes " Agricultural Development, famous-particular-excellent aquacultures such as Trionyx sinensis Wiegmann already develop rapidly, but along with the expansion of Trionyx sinensis Wiegmann artificial cultivation scale and the raising of intensification degree, trionyx disease increases day by day.Aeromonas (Aeromonas) disease is to culture one of main disease of Trionyx sinensis Wiegmann at present, and its each growth stage Trionyx sinensis Wiegmann all can take place, and sickness rate makes foster Trionyx sinensis Wiegmann already suffer enormous economic loss more than 50%.Since relatively lagging behind of physiological characteristics, ecological habit and trionyx disease study on prevention that Trionyx sinensis Wiegmann is special, main antimicrobial drug and the chemosterilant of relying on of trionyx disease control at present.These medicines are abused in a large number for a long time, abused, and not only produce little effect, and produce a series of serious consequences: drug resistance strain increases, and the trionyx disease difficulty of prevention and cure strengthens, environmental pollution, and drug residue, Carnis Trionycis matter descends, and constitutes greatly to human health to threaten.Use vaccine to carry out the means of prevention that immunoprophylaxis is a kind of cheapness, the generation of control disease effectively overcomes the difficulty of trionyx disease administration and treatment, and quality that can also the retaining ring border and the quality of Trionyx sinensis Wiegmann.Therefore, novel, the efficient Aeromonas vaccine of development becomes the focus of current trionyx disease study on prevention.

Trionyx sinensis (Wiegmann) Aeromonas vaccine research is existing at present much reports, immunization route has to be injected and oral two kinds.Though the injection inoculation immune effect is better, but can damage the Trionyx sinensis Wiegmann body, and troublesome poeration, implement very difficultly in actual production, and oral immunization is simple, and body is not produced stress, but Yan Zhi vaccine so far, because antigen can be by digestive enzyme and stomach acids destroy in the body digestive tract, immune effect is very poor, and the vaccine consumption is big.So up to now, on producing, do not have vaccine formally to be used for the trionyx disease control as yet.The commercialization of Trionyx sinensis Wiegmann Aeromonas vaccine has subject matter to be solved: (1) seeks easy, economical, vaccination approach efficiently; (2) the Aeromonas disease all can take place in each growth stage of Trionyx sinensis Wiegmann, and it is particularly important to successfully preventing this disease to manage to prolong immune duration.Adopt the biodegradable polymer coating antigen to make novel microspheres vaccine; can reach and realize that (microsphere does not decompose oral immunity under one's belt; and in intestinal, can discharge antigen; pass through intestinal absorption), the enhance immunity effect, prolong the immunoprotection phase (microspheres vaccine can be in the quite a while slow released antigen; the long period stimulating immune system produces immunne response; have adjuvant effect, can strengthen and prolong immune response) and reduce purposes such as dosage of inoculation and number of times.Yet, do not see the bibliographical information that relevant soft-shelled turtle aeromonad oral slow-releasing microsphere vaccinum is studied so far.

Summary of the invention

The objective of the invention is to overcome the defective of prior art, a kind of Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum and preparation method thereof is provided.

The Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum is by the Trionyx sinensis (Wiegmann) encountered pathogenic---the full bacterium liquid of deactivation of Aeromonas sobria, with sodium alginate and polyvinylpyrrolidone biodegradable polymer is carrier, the oral slow-releasing microsphere vaccine that the spray drying method parcel is made.The step of preparation method is as follows:

(1) strain preparation: from the Trionyx sinensis (Wiegmann) liver that suffers from hemolytic ascites disease, painstaking effort, ascites thing, isolate Aeromonas sobria;

(2) amplification culture: add 5.0～10.0ppm copper sulfate, 100～200ppm potassium dihydrogen phosphate, 2.5～5.0ppm manganese sulfate and 2500～5000ppm ammonium sulfate in nutrient broth, the seed liquor of inoculation 1～2% is at 20～37 ℃ of shaken cultivation 24～48h;

(3) counting: carry out count plate with colony counting method, it is 6.0～7.5 * 10 that adjustment bacterium liquid contains Aeromonas sobria ⁹CFU/ml;

(4) deactivation: the full bacterium liquid (6.0～7.5 * 10 of Aeromonas sobria ⁹CFU/ml) with 28～37 ℃, 0.2～0.4% formalin effect, 36～48h;

(5) microsphere preparation: the full bacterium liquid of Aeromonas sobria deactivation, adopting sodium alginate and polyvinylpyrrolidone biodegradable polymer is carrier, makes microspheres vaccine with spray drying method.

Advantage of the present invention: (1) easy to use, safety, better tolerance to Trionyx sinensis Wiegmann body not damaged, is convenient to extensive use; (2) microspheres vaccine can be in the quite a while slow released antigen, the long period stimulating immune system produces immunne response, has adjuvant effect, can strengthen and prolong immune response, minimizing dosage of inoculation and number of times etc.; (3) requirement of the purity of oral vaccine and quality standard is low than vaccinate, and production cost is low; (4) microspheres vaccine is a dried powder, and character is more stable, and the storing cost is lower; (5) production technology is easy, is easy to batch production production.

This achievement has important practical to be worth and favorable social and economic benefits in the production development of foster Trionyx sinensis Wiegmann industry.The slow-releasing microcapsule The Application of Technology has important reference to the development of other aquatic animal oral vaccines.

Description of drawings

Fig. 1 is the microscope figure of Aeromonas sobria oral slow-releasing microsphere vaccine;

Fig. 2 be exempted from control mice serum in agglutinating antibody tire variation diagram (-◆-: inactivated bacterial liquid injection group;-■-: oral group of microspheres vaccine;-▲-: oral group of inactivated bacterial liquid);

Fig. 3 be exempted from and contrast in the Trionyx sinensis (Wiegmann) serum agglutinating antibody tire variation diagram (-◆-: inactivated bacterial liquid injection group;-■-: oral group of microspheres vaccine;-▲-: matched group).

The specific embodiment

Concrete steps of the present invention and process are described in detail below in detail: the preparation screening of 1 strain

From the Trionyx sinensis (Wiegmann) liver that suffers from hemolytic ascites disease, painstaking effort, ascites thing, isolate Aeromonas sobria,, detect cultural character, somatic antigen property and the virulence of different generation bacterial strains respectively its 35 generations of continuous passage to the on nutrient agar.In Aeromonas sobria continuous passage to 35 generation on nutrient agar,, its biochemical characteristic does not change.The somatic antigen property for preparing as basic seed with different generation bacterial strains after haemolysis valency, virulence and the deactivation of bacterial number, extracellular products of bacterium liquid does not have significant difference.Show that Aeromonas sobria can be used as good production of vaccine strain.2 production strain culturing conditions

To cultivate bacterium liquid bacteria containing amount and extracellular products hemolytic is index, whether investigate cultivation temperature, incubation time, vibration, factor such as medium pH is to the Aeromonas sobria breeding and the influence of producing poison, and the employing orthogonal test method, culture medium prescription is optimized test.Cultivation temperature, incubation time and the optimum culture medium prescription of Aeromonas sobria the best have been determined.2.1 temperature is to the influence of bacterial growth, temperature (4,20,28,37 ℃) has tangible influence to cultivating bacterium liquid bacteria containing amount and hemolytic, and wherein 28 ℃ of OD value and haemolysis valencys of cultivating bacterium liquid are all the highest.2.2 medium pH influences medium pH between 6～9 to bacterial growth, bacterium liquid OD value and haemolysis valency are basicly stable; When pH less than 6 the time, along with the decline of pH, bacterial reproduction and produce malicious ability drop, during pH4.5, antibacterial does not grow substantially.2.3 time and whether vibrating to the influencing under the oscillating condition of bacterial growth is cultivated 24h, the OD value has reached higher level, and is maintained until 36h; Extracellular products haemolysis valency 48h reaches peak value (28).Under the static condition, OD value 36h arrives the peak, and is maintained until 96 hours; Extracellular products haemolysis valency 60h reaches peak (27).This shows that shaken cultivation can obviously promote bacterial reproduction and produce poison, shorten OD value and haemolysis valency peak time; Shaken cultivation with leave standstill cultivation, the peak time of both bacterium liquid OD values and haemolysis valency is all inconsistent, OD value peak time all shifts to an earlier date 24h than haemolysis valency peak time.2.4 the culture medium prescription screening test is 2.4.1 influencing copper sulfate (P＜0.05), dipotassium hydrogen phosphate (P＜0.05), ammonium sulfate (P＜0.10) and manganese sulfate (P＜0.05) and all can promote bacterial reproduction successively to some extent bacterium liquid OD value as a result; Calcium chloride (P＜0.10) and cobaltous sulfate (P＜0.10) can suppress bacterial reproduction; Other composition influence is not remarkable.((P＜0.05), calcium chloride (P＜0.05) and manganese sulfate (P＜0.10) have in various degree the facilitation that has successively to extracellular products secretion 2.4.2 to the copper sulfate that influences of extracellular products hemolytic; Zinc chloride (P＜0.01) presents utmost point inhibitory action significantly; Other composition influence is not remarkable.2.4.3 the culture medium optimization formula is determined to carry out demonstration test by the preferable culture medium prescription that above-mentioned screening is obtained, the result shows: the culture medium optimization formula is that basic nutrient broth adds 5.0～10.0ppm copper sulfate, 100～200ppm potassium dihydrogen phosphate, 2.5～5.0ppm manganese sulfate and 2500～5000ppm.

Temperature, pH, time, trace element and other nutritional labeling etc. are the expression of scalable antibacterial related gene all, thereby influences bacterial reproduction and the ability of producing poison.Different Aeromonass and the required condition of culture of bacterial strain thereof have difference.The Aeromonas sobria optimal culture condition is: add 5.0～10.0ppm copper sulfate, 100～200ppm potassium dihydrogen phosphate, 2.5～5.0ppm manganese sulfate and 2500～5000ppm ammonium sulfate in nutrient broth, the seed liquor of inoculation 1～2% is at 20～28～37 ℃ of shaken cultivation 24～36～48h.3 countings

Carry out count plate with colony counting method, it is 6.0～7.5 * 10 that adjustment bacterium liquid contains Aeromonas sobria ⁹CFU/ml.4 produce the bacterial strain ablation method

With antibacterial extant number, extracellular products hemolytic and somatic antigen property after the deactivation is index, investigate the influence of different formalin concentration, different temperature, different inactivation time, determine the ideal conditions of Aeromonas sobria deactivation the deactivation of Trionyx sinensis (Wiegmann) Aeromonas sobria.4.1 the deactivation of thalline is at 4 ℃, 0.05%, 0.1%, 0.2% and 0.4% formalin needs 144h, 120h, 96h and 72h just can make the thalline complete deactivation respectively; In the time of 28 ℃, inactivation time foreshortens to 96h, 72h, 48h and 36h; 37 ℃ then are respectively 72h, 48h, 36h and 36h.4.2 the deactivation of extracellular products (ECP) is at 37 ℃, 0.05%, 0.1%, 0.2% and 0.4% formalin needs 13d, 9d, 6d and 3d to make the extracellular products complete deactivation respectively; In the time of 28 ℃, 0.1%, 0.2% and 0.4% formalin needs 13d, 9d and 6d respectively, and 0.05% formalin deactivation 15d, its extracellular products still has hemolytic (the haemolysis valency is 21); In the time of 4 ℃, in 15 days only 0.2% and 0.4% formalin can make extracellular products deactivation (inactivation time is 14d and 9d respectively).4.3 the deactivation condition all can cause certain loss to somatic antigen property to the temperature that influences deactivation, formalin concentration and the time of somatic antigen property; Especially under the situation that temperature raises, formalin concentration increases or prolong its action time, its antigenicity is all corresponding to be weakened.In the various deactivation modes, be the best with 28 ℃, the somatic antigen property of 0.2% formalin effect 2d.4.4 the safety testing safety testing shows that 5 Trionyx sinensis (Wiegmann) of lumbar injection inactivated bacterial liquid are all strong alive, no abnormal reaction.The comparatively ideal ablation method of Aeromonas sobria: full bacterium liquid (6.0～7.5 * 10 ⁹CFU/ml) with 28～37 ℃, the preparation of 0.2～0.4% formalin effect, 36～48h, 5 Trionyx sinensis (Wiegmann) Aeromonas sobria oral slow-releasing microsphere vaccines

The Aeromonas sobria inactivated bacterial liquid is after distinct methods (ultrasonic, freeze thawing) is handled; the employing biodegradable polymer is a carrier; make microspheres vaccine; oral immunity ICR mice is respectively tired by measuring agglutinating antibody in the serum, the immune effect of blood middle leukocytes sterilization percentage rate and three kinds of vaccines of immune protective efficiency comparative evaluation that viable bacteria is attacked.5.1 the particle diameter of three kinds of microspheres of preparation of vaccine is all less than 10 μ m, the about 3 μ m of mean diameter; The microscopic examination result shows microsphere features smooth surface, is the rounding sphere, narrow diameter distribution, good dispersion (Fig. 1).Measure through microscope count method, the thalline envelop rate is about 70～80% in the microsphere.The microspheres vaccine safety testing shows that 5 white mice of filling stomach are all strong alive, no abnormal reaction.5.2 agglutinating antibody is tired and is exempted from that the agglutinating antibody titration the results are shown in Table 1 in the mice serum.As seen from Table 1, in the 4th week behind the mice oral immunity, three kinds of microspheres vaccines all can produce certain agglutinating antibody, and it is the highest wherein directly to prepare the antibody titer of microspheres vaccine group with inactivated bacterial liquid, inactivated bacterial liquid freeze thawing treatment microspheres vaccine group is taken second place, and inactivated bacterial liquid supersound process microspheres vaccine group is minimum.Detect less than agglutinating antibody in the control group mice serum.As seen inactivated bacterial liquid all can to a certain degree reduce the immunogenicity of vaccine through ultrasonic or freeze thawing treatment.

Table 1 exempted from the mice serum agglutinating antibody tire Tab.1 The agglutinating antibody titers in the serum of immunized mice (n=6, X ±

SD)

Group group A B C agglutinating antibody (log2) 8.67 ± 0.52 7.83 ± 0.75 6.33 ± 0.82agglutinating antibody titers that tires

Annotate: A group and B group difference remarkable (P＜0.05), C group and A, B group difference be (P＜0.01) extremely significantly.5.3 being exempted from, leukocytic bactericidal activity sees Table 2 with the bactericidal activity measurement result of control mice blood middle leukocytes.As seen from Table 2, the leukocyte sterilization percentage rate of being exempted from mice is significantly higher than matched group (P＜0.01), it is the highest wherein directly to prepare the microspheres vaccine group with inactivated bacterial liquid, and inactivated bacterial liquid freeze thawing treatment microspheres vaccine group is taken second place, and inactivated bacterial liquid supersound process microspheres vaccine group is minimum.The bactericidal activity that all can significantly improve murine interleukin behind three kinds of microspheres vaccine oral immunities is described.5.4 immunoprotection experimental control and exempted from the death condition of mice after the Aeromonas sobria viable bacteria is attacked and see Table 3.By table 3 as seen, three kinds of microspheres vaccine oral immunities are attacked the Aeromonas sobria viable bacteria all the certain protection effect, and it is the highest wherein directly to prepare the immune protective efficiency of microspheres vaccine group with inactivated bacterial liquid, reaches 85.7%; Next inactivated bacterial liquid freeze thawing treatment microspheres vaccine group, its immune protective efficiency is 71.4%, inactivated bacterial liquid supersound process microspheres vaccine group is minimum, only is 50.0%.Be separated to and attack used identical antibacterial from attacking lethal liver of being exempted from mice, kidney, heart etc.

Table 2 is exempted from the bactericidal activity Tab.2 Bactericidal acticity of the blood leucocytes of immunized and control with the control mice blood leucocyte

Mice (the group of X ± SD) sterilization percentage rate (the %) (A 66.27,64.73,63.31,64.50,63.67,62.96 64.24 ± 1.20 of X ± SD) ^cB 61.42,62.13, and 64.73,61.90,61.78,61.3 62.21 ± 1.27 ^BcC 60.24,61.30, and 63.67,59.41,60.59,62.72 61.32 ± 1.60 ^bD 35.74,36.10, and 32.90,35.98,34.32,32.9 34.66 ± 1.50 ^aAnnotate: A, inactivated bacterial liquid directly prepare the microspheres vaccine group; B, inactivated bacterial liquid freeze thawing treatment microspheres vaccine group; C, inactivated bacterial liquid supersound process microspheres vaccine group; D, matched group; A, b represents difference extremely significantly (P＜0.01) between the different letters of c; A, b represents difference not remarkable (P＞0.05) between the c same letter.

Table 3 contrast and exempted from mice behind As Z-1 strain viable bacteria counteracting toxic substances immune protective efficiency Table 3 Relative percent survival of control and immunized mice against challenge

With live Aeromonas sobria Z-1 strain group is counted mortality rate immunoprotection group number of number of survival survival/% power RPS for examination Mus number survival Mus ^*/ %

Challenged mice mice A 14 12 14.3 85.7 B 14 10 28.6 71.4 C 14 7 50.0 50.0 control groups 14 0 0.0 notes: A, inactivated bacterial liquid directly prepare the microspheres vaccine group; B, inactivated bacterial liquid freeze thawing treatment microspheres vaccine group; C, inactivated bacterial liquid supersound process microspheres vaccine group; D, matched group;

$\×100\%\mathrm{Relative\; percent\; survival}=(1-\frac{\mathrm{mortality\; of\; immunized\; mice}}{\mathrm{mortality\; of\; controls}})\×100\%$ The immune effect of three kinds of microspheres vaccines prepares microsphere group the best with the direct encapsulation of inactivated bacterial liquid, is inactivated bacterial liquid freeze thawing treatment microspheres vaccine group secondly, and inactivated bacterial liquid supersound process microspheres vaccine group is the poorest.As seen Trionyx sinensis (Wiegmann) Aeromonas sobria oral slow-releasing microsphere vaccine is prepared as good with the direct encapsulation of inactivated bacterial liquid.6 checks

Check comprises steriling test and safety examination.Steriling test is that draw samples is inoculated on the nutrient agar, cultivates 48h at 28 ℃, and whether observe has bacterium colony to generate.Safety examination is to irritate 5 Trionyx sinensis (Wiegmann) of stomach and white mice with 5 times of microspheres vaccines to using dosage, and whether observe 10d has abnormal response.7 immune effects analyze 7.1 mices to the immunne response research of Aeromonas sobria oral slow-releasing microsphere vaccine with the full bacterium liquid of Trionyx sinensis (Wiegmann) Aeromonas sobria deactivation; the employing biodegradable polymer is a carrier; make microspheres vaccine; oral immunity ICR mice; measure agglutinating antibody in the serum; the activate the phagocytic capacity of monokaryon-macrophage and to the immune protective efficiency that viable bacteria is attacked in the blood is studied its immune response.7.1.1 the agglutinating antibody titration as a result behind the mouse immune titration of different time serum agglutinating antibody the results are shown in Figure 2 (-◆-: inactivated bacterial liquid injection group;-■-: oral group of microspheres vaccine;-▲-: oral group of inactivated bacterial liquid)).From Fig. 2 as seen, in the 1st～3 week after the Mus immunity, agglutinating antibody is tired and is higher than oral group of microspheres vaccine in the inactivated bacterial liquid injection group mice serum, and there were significant differences (P＜0.05); The 4th～5 week of immunity back, exempted from the mice serum agglutinating antibody for two groups and tire and all tend towards stability, both do not have significant difference (P＞0.05); And immunity the 7th～12 week of back, agglutinating antibody is tired and is significantly higher than inactivated bacterial liquid injection group (P＜0.05) in oral group of mice serum of microspheres vaccine; Agglutinating antibody is tired and is kept reduced levels always in oral group of mice serum of inactivated bacterial liquid, compares for oral group with inactivated bacterial liquid injection group, microspheres vaccine, and difference is (P＜0.01) extremely significantly.Detect less than agglutinating antibody in the control group mice serum.7.1.2 carbon clearance test result contrast and the phagocytic index measurement result of being exempted from monokaryon-macrophage in the mouse blood see Table 4.In the 4th week behind the mouse immune, the phagocytic index of monokaryon-macrophage is higher than oral group of inactivated bacterial liquid and matched group in oral group of mouse blood of inactivated bacterial liquid injection group and microspheres vaccine, and difference is (P＜0.01) extremely significantly; The inactivated bacterial liquid injection exempts to compare difference nonsignificance (P＞0.05) with oral group of microspheres vaccine, oral group of inactivated bacterial liquid and matched group.As seen, microspheres vaccine can significantly improve the activate the phagocytic capacity of monokaryon-macrophage after oral.7.1.3 immunoprotection experimental control and exempted from the death condition that mice attacks through the Aeromonas sobria viable bacteria and see Table 5.By table 5 as seen; the immune protective efficiency that oral group of mice of inactivated bacterial liquid injection group and microspheres vaccine attacked the Aeromonas sobria viable bacteria is 87.5%; and oral group of inactivated bacterial liquid is attacked lumbar injection effective protection can not be provided; the same whole death with matched group, but the death time is more late than matched group.Be separated to and attack used identical antibacterial from attacking lethal liver of being exempted from mice, kidney, heart etc.

Table 4 is exempted from the phagocytic index Table 4 Phagocytic index of monocytes and macrophages in immunized and control with control mice monokaryon-macrophage

Mice group phagocytic index X ± SDA 4.0437,4.4576,4.4634,4.0386,3.8598,4.5665 4.2383 ±

0.2924 ^bB 4.2195，4.2971，4.7380，3.9859，4.2394，3.6565 4.1894±

0.3582 ^bC 3.2270，2.5420，3.0381，3.0383，3.9716，3.4010 3.2030±

0.4737 ^aD 2.9832，3.1796，3.0893，2.9542，2.5175，2.8975 2.9369±

0.2289 ^aAnnotate: A, inactivated bacterial liquid injection group; Oral group of B, microspheres vaccine; Oral group of C, inactivated bacterial liquid; D, matched group.A represents difference extremely significantly (P＜0.01) between the different letters of b; A represents difference not remarkable (P＞0.05) between the b same letter.

Table 5 contrast and exempted from mice behind As Z-1 strain viable bacteria counteracting toxic substances immune protective efficiency Table 5 Relative percent survival of control and immunized mice against challenge

With live Aeromonas sobria Z-1 strain group is counted immune protective efficiency group number of challenged mice number of survival mice RPS for examination Mus number survival Mus ^*/ %A 87 87.5B 87 87.5C 80 0.0D 80 annotate: A, inactivated bacterial liquid injection group; Oral group of B, microspheres vaccine; Oral group of C, inactivated bacterial liquid; D, matched group; $\underset{\mathrm{mortality\; of\; controls}}{\mathrm{mortality\; of\; immunized\; mice}})\×100\%$

The 4th week behind the microspheres vaccine mice oral immunity; not only agglutinating antibody is tired in the serum, the phagocytic index of monokaryon-macrophage and the immune protective efficiency that viable bacteria is attacked all can reach the suitable level of inactivated bacterial liquid injecting immune (P＞0.05) in the blood; apparently higher than oral group of inactivated bacterial liquid (P＜0.01), and can in the long term, keep higher agglutinating antibody and tire.The oral immune effect that reaches the injection conventional vaccine of microspheres vaccine.7.2 Trionyx sinensis (Wiegmann) is to the immunne response research of Aeromonas sobria oral microsphere slow release vaccine

With the full bacterium liquid of Trionyx sinensis (Wiegmann) Aeromonas sobria deactivation; the employing biodegradable polymer is a carrier; make microspheres vaccine; the oral immunity Trionyx sinensis (Wiegmann); by measuring agglutinating antibody in the serum, blood middle leukocytes sterilization percentage rate and immune protective efficiency research Trionyx sinensis (Wiegmann) that viable bacteria is attacked are to the immune response of Aeromonas sobria oral slow-releasing microsphere vaccine.7.2.1 agglutinating antibody titration result exempted from and contrast agglutinating antibody titration in the turtle serum the results are shown in Figure 3 (-◆-: inactivated bacterial liquid injection group;-■-: oral group of microspheres vaccine;-▲-: matched group).From Fig. 3 as seen, behind the Trionyx sinensis (Wiegmann) microspheres vaccine oral immunity, agglutinating antibody is tired quite with inactivated bacterial liquid injection group in the serum, and no significant difference (P＞0.05), the utmost point are significantly higher than matched group (P＜0.01).7.2.2 leukocytic bactericidal activity is exempted from and the bactericidal activity measurement result that contrasts the Trionyx sinensis (Wiegmann) blood middle leukocytes sees Table 6.As known from Table 6, behind the Trionyx sinensis (Wiegmann) microspheres vaccine oral immunity, the sterilization percentage rate utmost point of blood middle leukocytes is significantly higher than matched group (P＜0.01), as seen, can significantly improve the bactericidal activity of blood middle leukocytes after microspheres vaccine is oral.Oral group of microspheres vaccine and inactivated bacterial liquid injection group compare, the two difference nonsignificance (P＞0.05).

The bactericidal activity Tab.6 Bactericidal acticity of the blood leucocytes in immunized and control soft-of Trionyx sinensis (Wiegmann) blood middle leukocytes is exempted from and contrasted to table 6

(X ± SD) group is exempted from Later Zhou Dynasty, one of the Five Dynasties's number and sterilization percentage rate weeks and Bactericidal percentgroup 123 4A 39.19 ± 1.30 to shelled turtle (Trionyx sinensis) ^b48.65 ± 1.32 ^b56.81 ± 1.85 ^b62.66 ± 2.09 ^bB 37.12 ± 1.46 ^b45.78 ± 1.96 ^b53.73 ± 1.82 ^b60.94 ± 1.72 ^bC 29.91 ± 3.26 ^a31.36 ± 2.00 ^a31.38 ± 1.25 ^a30.60 ± 2.35 ^aAnnotate: A, inactivated bacterial liquid injection group; Oral group of B, microspheres vaccine; C, matched group; A represents difference extremely significantly (P＜0.01) between the different letters of b; Represent difference not remarkable (P＞0.05) between the b same letter.7.2.3 immunoprotection experimental control and exempted from the death condition that Trionyx sinensis (Wiegmann) attacks through the Aeromonas sobria viable bacteria and see Table 7.By table 7 as seen, the immunoprotection power rate that oral group of microspheres vaccine and inactivated bacterial liquid injection group Trionyx sinensis (Wiegmann) are attacked the Aeromonas sobria viable bacteria is respectively 94.7% and 89.5%, and both differences are remarkable (P＞0.05) not, and control group mice 100% death.Be separated to and attack used identical antibacterial from attacking lethal liver of being exempted from Trionyx sinensis (Wiegmann), kidney, heart etc.

Table 7 contrast and exempted from Trionyx sinensis (Wiegmann) behind As Z-1 strain viable bacteria counteracting toxic substances immune protective efficiency

Tab.7 Relative percent survival of control and immunized soft-shelled turtle (Trionyx sinensis) against challenge with live Aeromonas sobria Z-1 strain group is counted immune protective efficiency group number of challenged turtle number of survival turtle RPS/%A 20 19 94.7B 20 18 89.5C 20 1 notes for examination soft-shelled turtle number survival soft-shelled turtle: A, inactivated bacterial liquid injection group; Oral group of B, microspheres vaccine; C, matched group; A represents difference extremely significantly (P＜0.01) between the different letters of b; Represent difference not remarkable (P＞0.05) between the b same letter; Immune protective efficiency=

$\underset{\mathrm{mortality\; of\; controls}}{\mathrm{mortality\; of\; immunized\; mice}})\×100\%$ Behind the microspheres vaccine Trionyx sinensis (Wiegmann) oral immunity, agglutinating antibody is tired in the serum, blood middle leukocytes sterilization percentage rate and immune protective efficiency that viable bacteria is attacked all can reach the suitable level of inactivated bacterial liquid injection group (P＞0.05), and the utmost point is significantly higher than matched group (P＜0.01); The oral immune effect that reaches the injection conventional vaccine of microspheres vaccine is described.7.3 the prophylactic tria of Trionyx sinensis (Wiegmann) Aeromonas sobria oral microsphere slow release vaccine

Trionyx sinensis (Wiegmann) Aeromonas sobria oral slow-releasing microsphere vaccine prevention result of the test sees Table 8.Trionyx sinensis (Wiegmann) Aeromonas sobria oral slow-releasing microsphere vaccine immunity Trionyx sinensis Wiegmann and not immune Trionyx sinensis Wiegmann mortality rate are respectively 4.285% and 15.49%, and the two difference is (P＜0.01) extremely significantly.The immunity Trionyx sinensis Wiegmann descends 72.37% than not immune Trionyx sinensis Wiegmann average mortality.

Table 8 contrast and survival rate and mortality rate Tab.8 Mortality of control and immunized soft-shelled turtle (Trionyx sinensis) group of being exempted from Trionyx sinensis Wiegmann are counted mortality rate average mortality group number of turtle number of survival mortality/% average for examination Trionyx sinensis Wiegmann number survival Trionyx sinensis Wiegmann

Turtle mortality/% tries 1,428 1,359 4.83 and tests 430 413 3.95 4.28 groups 734 709 3.41 pairs 836 691 17.34 according to 240 207 13.75 15.49 group 758 652 13.98

Claims (2)

1, a kind of Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum, it is characterized in that it is by the Trionyx sinensis (Wiegmann) encountered pathogenic---the full bacterium liquid of deactivation of Aeromonas sobria, with sodium alginate and polyvinylpyrrolidone biodegradable polymer is carrier, the oral slow-releasing microsphere vaccine that the spray drying method parcel is made.

2, a kind of preparation method of Trionyx sinensis (Wiegmann) aeromonad oral slow-releasing microsphere vaccinum is characterized in that its step is as follows:

(1) strain preparation: from the Trionyx sinensis (Wiegmann) liver that suffers from hemolytic ascites disease, painstaking effort, ascites thing, isolate Aeromonas sobria;

(2) amplification culture: add 5.0～10.0ppm copper sulfate, 100～200ppm potassium dihydrogen phosphate, 2.5～5.0ppm manganese sulfate and 2500～5000ppm ammonium sulfate in nutrient broth, the seed liquor of inoculation 1～2% is at 20～37 ℃ of shaken cultivation 24～48h;

(3) counting: carry out count plate with colony counting method, it is 6.0～7.5 * 10 that adjustment bacterium liquid contains Aeromonas sobria ⁹CFU/ml;

(4) deactivation: the full bacterium liquid (6.0～7.5 * 10 of Aeromonas sobria ⁹CFU/ml) with 28～37 ℃, 0.2～0.4% formalin effect, 36～48h;

(5) microsphere preparation: the full bacterium liquid of Aeromonas sobria deactivation, adopting sodium alginate and polyvinylpyrrolidone biodegradable polymer is carrier, makes microspheres vaccine with spray drying method.

Images