CN1031039C - Swine dysentery vaccine - Google Patents
Swine dysentery vaccine Download PDFInfo
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- CN1031039C CN1031039C CN 88101481 CN88101481A CN1031039C CN 1031039 C CN1031039 C CN 1031039C CN 88101481 CN88101481 CN 88101481 CN 88101481 A CN88101481 A CN 88101481A CN 1031039 C CN1031039 C CN 1031039C
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Abstract
Provided is a vaccine for a pig against swine dysentery characterised by administration to the pigs of a live strain or of an oxygen-treated, non-viable strain of T. hyodysenteriae.
Description
The present invention is relevant to the immunity of swine dysentery with pig, especially with relevant for this sick vaccine of control.A kind of anaerobic spirillum, promptly the swine dysentery treponema is separated, and proves that it is the main pathogens of swine dysentery.Swine dysentery shows as the mucus hemorrhagic diarrhea, is distributed widely in all over the world.This kind microorganism is bred well in the Radix Polygalae Crotalarioidis that has other anaerobe, and produces widely the mucus hemorrhagic so that the gangrenosum acne degeneration in this.The extensive infringement of visible tissue epithelium and lamina propria under optical microscope or electron micrograph, spirillum clearly is distributed in slough.
Though proved that suffering from the pig of fully recovering after the dysentery has resistance to infecting again, just in fact relevant pig there is no the immunoreation of swine dysentery treponema infection can be for the material that utilizes.
People attempt to set about setting up effective swine dysentery vaccine from several respects.The U.S. 4,100, No. 272 patents have been introduced a kind of vaccine of injection, it contains the pathogenic swine dysentery treponema cell that is obtained by cultivation that chemically kills, and the similar vaccine of 4,152, No. 413 patent introductions of the U.S. contains for oral killed pathogenic bacterium cell.The U.S. 4,152,415 and 4,469, No. 672 patents are introduced the improvement of oral vaccine inoculation step, and it comprises the step of taking vaccinate before oral.
International publication number WO85/03875 discloses an improvement vaccine program, and it comprises killed vaccine as the once injection of the amount of inducing, at the same time or afterwards oral work but the swine dysentery treponema of no pathogenicity.
Obviously, used killed pathogenic swine dysentery treponema in the trial of these former vaccinations, and cell is to use such as the stationarity chemical substance of formaldehyde to handle.All these, the application of killed vaccine can be separately orally also can inject separately, or in injection the oral killed pathogenic bacterial strains or the non-pathogenic bacterial strains of living.The reason of oral non-pathogenic bacterial strains is the local immunity that irritates large intestine, is likely to irritate the IgA reaction.
The intramuscular injection that has now found that the spirillum live strain can reach the effective immunity of pig to the swine dysentery treponema infection.And find that intramuscular injection can obtain pig to the treponemal effective immunity of swine dysentery through the swine dysentery treponema bacterial strain of the deactivation of peroxide processing.Both comprised pathogenicly in addition according to the applicable bacterial strain of present invention, also comprised attenuation.
According to first of the present invention, here provide and make swine dysentery that pig antagonism swine dysentery treponema causes and vaccinated method, be characterized in by injection preferably intramuscular injection, being swine dysentery treponema live strain or handling and the bacterial strain of deactivation of injection through peroxide.As previously mentioned, the both available pathogenic swine dysentery treponema bacterial strain of this law, the also bacterial strain of available attenuation, and the following introduction of picture, adaptable representational bacterial strain comprises and leaves U.S. typical case culture in and No. 31212 benchmark pathogenic bacterial strains and ATCC2 in integrated ATCC31287 number according to the present invention, No. 7167 benchmark non-pathogenic bacterial strains (referring to patent specification 4,100, No. 272).
According to this patent, can be for injection, particularly the bacterial strain of intramuscular injection comprises those protein scattergrams and pathogenic swine dysentery treponema bacterial strain 5380 or the identical bacterial strain of attenuated strain 70A, comprises above-mentioned benchmark bacterial strain 31287,31212 and 27164.As what introduce in more detail below, minute differences on the protein scattergram there is no obvious influence for the immunogenicity of various pathogenic bacterial strains or attenuated strain, and the utilization of pathogenic strain of introducing in detail 5380 and attenuated strain 70A is as the representative of utilizable pathogenic bacterial strains of the present invention and attenuated strain separator herein.The external membrane protein scattergram shows the significantly identical of all swine dysentery treponema separators, and the Wei Sideng blotting (Westernblot) of immunogenic protein the analysis showed that the antiserum that is caused by pathogenic swine dysentery treponema separator 5380 can discern the identical immunogenic protein of molecular weight ranges in all swine dysentery treponema separators.
Compare and indistinction with the swine dysentery treponema without the oxygen processing of living through the treponemal protein scattergram of swine dysentery that peroxide is handled.Similarly, the Wei Sideng blotting analysis of immunogenic protein demonstration can be discerned identical immunogenic protein by the antiserum that pathogenic swine dysentery treponema separator 5380 causes, no matter is that cell or the living cells of handling through oxygen all is like this.Because handling, oxygen do not change the albumen scattergram, therefore also no change on antigenicity, and this is true like clear and definite.The vaccine that kills as for former chemistry (for example Formalin kill vaccine) has confirmed that this chemical treatment can destroy some antigens, comprises the more treponemal surface antigens of swine dysentery.
The present invention provides a kind of vaccine composition on the other hand, and it is by injection, and especially intramuscular injection can make pig that the swine dysentery treponema infection is produced effectively immunity.This kind composition comprises that swine dysentery is treponemal and is present in certain a kind of live strain or a kind of inactivated strain of handling through oxygen in can received carrier, and adds optional adjuvant
The present invention prepare the used swine dysentery treponema separator of vaccine can be in class pancreatic enzyme Semen Glycines meat soup in 37-38 ℃ and oxygen free air (50% H for example
2/ 50% CO
2, or contain 10% CO
2N
2) in hatch growth.In culture medium, add cystine or other reducing substances to keep anaerobiosis, when culture is when growing in the first liquid medium within, hyclone (reaching 10%) may must be added in culture medium, glucose, citrate salt, pyruvate, and ferrum (every milliliter culture medium 2 micrograms, crucial concentration).Fluid medium also can add spectinomycin (Spectinomycin) does not change isolated cell to suppress possible pollution protein component by the concentration of 400 micrograms/ml.When needing, handle by the oxygen of introducing below then and make cell inactivation.
On the other hand, swine dysentery treponema cell can be grown on the blood agar flat board under oxygen free condition.When cell is when preparing by this kind mode, then must in phosphate buffered saline (PBS) or other isosmotic solution, wash, so that before using, remove the impurity in the agar, then, if necessary, be positioned over and make their inactivations in the oxygen saturation solution.
The treponemal culture of swine dysentery can be grown the protein component of the bacterial strain of growing and indifference in this temperature range in 35 °-42 ℃ temperature.
After the swine dysentery treponema grows out in fermentor, can be by collecting and be suspended in the normal saline (0.1M) of phosphate buffered saline (PBS) (0.1M) or pH7.0-7.4 from anti-precipitation, and under-70 ℃ or-20 ℃, preserved with spissated form.These cells also can be divided into plurality of small-capacity and be frozen the universe on the other hand.
Desire to kill the swine dysentery treponema cell that grows out in fermentor, the most handy oxygen is handled them, oxygen bubbles can be blown into culture medium and make it saturated.The oxygen processing procedure preferably is no less than 3-4 hour, preferably can reach six hours, so that all the swine dysentery treponema inactivations in the culture medium.These cells can directly be utilized after the oxidation through combining with adjuvant, or collect by centrifugation, suspend, and are stored with conc forms as stated above.Also these cells can be divided into less volume and lyophilizing on the other hand.Cell density for immunity inoculation must be near 10
9/ ml.Vaccine is preferably by suitable adjuvant, for example one milliliter of Freunds Freund (CSL) with one milliliter 1 * 10
9Antibacterial is formed.Vaccine also can comprise antiseptic, and for example sulfur hydrargyrum is removed (thimerosal).Vaccine is preferably done the deep intramuscular injection, so method can obtain maximum immunological effect, but the injection of vaccine also can be passed through other approach, subcutaneous injection for example, but do not advocate.
Increase the opposing of pig to the swine dysentery treponema infection, preferably give two vaccinating agents, every dose contains the swine dysentery treponema and is about 1 * 10
9, be separated by 7-14 days for two doses.After second dose,, can do the injection of the 3rd vaccinating agent if find that by enzyme-linked immunosorbent assay or Serological testing of equal value the immunoreation degree of the serum of 1/800 dilution does not reach the level of 1.0OD unit.
In the domestic animal of raising, preferably annually give inoculating of a booster dose of animal, with the IgG level of the serum that keeps its 1/800 dilution more than 1.00OD unit.
Accompanying drawing 1-5 explanation more treponemal clinical isolates of swine dysentery and benchmark strains A TCC31287, the result that the sodium dodecyl sulfate-polyacrylamide gel electrophoresis curve chart (SDS-PAGE) of 31212 and 27164 full cell and adventitia (OM) concentrating part is checked.
In these figure:
Fig. 1 represents the sodium dodecyl sulfate-polyacrylamide gel electrophoresis figure of treponemal separator of swine dysentery and benchmark strain whole-cell lysate.Road 1 and road 17 separator bacterial strains, 70A; Road 2, separator bacterial strain 5380; Road 3, separator bacterial strain 5541; Road 4, separator bacterial strain 32386; Road 5, separator bacterial strain 32486A; Road 6, separator bacterial strain 32486B; Road 7, benchmark strains A TCC31212; Road 8, benchmark strains A TCC27164; Road 9, benchmark strains A TCC31287; Road 10, separator bacterial strain 1545; Road 11, separator bacterial strain 9690; Road 12, separator 8841; Road 13,8441; Road 14, separator bacterial strain 508; Road 15, separator bacterial strain 1059; Road 16, separator bacterial strain 2549.
Fig. 2 represents to be insoluble to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis figure of the OM goods of Sarkosyl.The A group is from the OM protein 10 microgram roads 1 of swine dysentery treponema separator, 70A; Road 2, ATCC31287; Road 3, ATCC27164; Road 4,2549; Road 5,1545; Road 6,5380; Road 7,9690; Road 8,8841; Road 9,8441; Road 10, Treponema innocens 9509; Road 11, Treponema innocens 9510; Road 12,1039; Road 13, ATCC31212; Road 14,32486B; Road 15,508; Road 16 repeats 70A; Road 17,5380 full cytolysates.The B group, from the swine dysentery treponema, escherichia coli, Salmonella typhimurium, yersinia genus enterocolitis bacillus, campylobacter jejuni, and OM protein 10 micrograms of campylobacter fetus.Road 1,2 and 3 is respectively swine dysentery treponema separator 70A, ATCC31287,5380; Road 4, escherichia coli JP777; Road 5, Salmonella typhimurium V279; Road 6, yersinia genus enterocolitis bacillus 430-1; Road 7, campylobacter jejuni F1; Road 8, campylobacter jejuni F14, road 9, campylobacter fetus, fetus subspecies; Road 10 repeats 5380.
Fig. 3 represents to use the proteinic Wei Sideng immunoblotting assay of full cytolysis of the swine dysentery treponema of special porcine blood serum.With sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated cell lysate and give the Z probe for the Wei Sideng engram analysis.Detection of antigens is with control serum (A group) and the anti-swine dysentery treponema of pig (isolate 5380) polyvalent serum (B group) before the immunity, dilutes 1: 100.Road 1 and 5,70A; Road 2 and 6,5380; Road 3 and 7, ATCC31287; Road 4 and 8,32386; Road 9,1545; Road 10, ATCC31212; Road 11,1059; Road 12 repeats 5380; Road 13,5541; Road 14,32486B; Road 15 repeats ATCC31287; Road 16, ATCC27164; Road 17,9690; Road 18,8841; Road 19, harmless spirillum 9510; Road 20, harmless spirillum 9509.Bonded antibody is with the determination of autoradiography after (35S) protein and the Sui.
Fig. 4 represents to use the proteinic Wei Sideng trace of the OM figure of the swine dysentery treponema isolate of the anti-swine dysentery treponema of pig serum.OM protein prepares by Sarkosyl solubilization method.The A group is with the immunoblotting (Immuneblo) of the control serum before the immunity); B group, diluting with the immunoblotting serum of hyper-immuneserum is 1: 100.Road 1 and road 5, swine dysentery treponema 5380 cell extractum; Road 2 and 6,5380OMP; Road 3 and 7, ATCC31287OMP; Road 4 and 8,70AOMP; Road 9 2549OMP; Road 10 1545MP; Road 11, ATCC27164OMP; Road 12,8841OMP; Road 13, Treponema innocens 9510OMP; Cultivation and autoradiography Sui after the detection of the detection of antibodies with (35S).
Fig. 5 represents the Digestion of protease to swine dysentery treponema isolate 5380 full cells.Complete cell is cultivated mutually with trypsin 50 micrograms (A road) or with PBS buffer agent (B road).After 30 minutes, add PMSF reaction stopped, before the sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytolysis in sodium lauryl sulphate specimen buffer agent.The arrow indication is to the proteinic position of the Coomassie blue stain of protein decomposition sensitivity.
The result
Be presented at 4 to 7 kinds of main richs in protein in the molecular weight of 30kDa to 40kDa scope by the SDS-PAGE curve chart of the polypeptide of the full cytolysate of clinical strain separated and swine dysentery treponema typical strain, and be presented at SDS-PAGE and go up that each bacterial strain is distinguishable at least to go out the lower 30 kinds of other protein of Coomassie blue stain degree.On electrophoretic image, can't see high-molecular weight polypeptide (＞200kDa).Can find that in most of isolated strains 36kDa and 39kDa protein are the hyperbola that closely moves.In addition, according to silver dyeing and the bonded feature of nitrocellulose, be the district's band (not enumerating data) of being proved to be that disperse all appears in regional all bacterial strains of 22-25kDa for lipopolysaccharide (LPS) at molecular weight.
The proteinic contrast of the Coomassie blue stain of the full cytolysate of swine dysentery treponema has shown removing big in molecular weight is 30kDa to 40kDa scope on the SDS-PAGE glue rich in protein difference in quality on tangible electrophoretic mobility.Variation between bacterial strain on the band diagram and bacterial strain can be examined two electrophoretic pattens between bacterium in order to discriminating.Inspected spirillum show mostly its protein band diagram similar to the band diagram of bent type strains A TCC31212 and ATCC31287 (Fig. 1, be respectively 7 with road 9); And be referred to as A group, having the special molecular weight that contains is the proteinic big and rich in protein of 39kDa37kDa and 36kDa.Benchmark strains A TCC27164 (Fig. 1, road 8) illustrates that the polypeptide curve chart is identical with strains A TCC31287, but does not express 39kDa protein hyperbola (also being decided to be the A group).In isolated strains J960,8841 and 8441 (Fig. 1 is respectively 11,12 and 13) show a kind of different 40kDa protein, and these isolated strains are decided to be the B group.
By after the growth medium, four kinds of different full cytolysates that prepare by same kind of swine dysentery treponema bacterial strain (95OA, 70A, 31287,31212) have been checked repeatedly with SDS-PAGE.All bacterial strains all show similar polypeptide curve chart, and these figure illustrate that the protein component of isolated strains is not because of friendshipization (not introducing data) takes place by culture medium.And, challenge and after separating more subsequently in the process living body of some pigs, the protein curve chart of bacterial strain 5380 (A group, Fig. 1, road 2) still keeps stablizing (not introducing data).
Cell envelope by the isolated strains of clinical isolating swine dysentery treponema and typical strain and two harmless breechblocks partly extracts with Sarkolyl, and the goods that concentrated by OMP that are insoluble to Sarkolyl are resolved (Fig. 2 A) by SDS-PAGE.Big and rich in protein (see figure 1) that the big OM protein that exists in the swine dysentery treponema is equivalent to move in 30kDa to 40kDa molecular weight ranges in the cell protein goods.The curve chart of OMP shows that the isolated strains of all A groups and most of B group shows common 36kDa, 34kDa, 33kDa, 31.5kDa and 30kDa OM protein.In inspected 10 A group bacterial strain, 6 show a kind of big 37kDa OMP, and 4 show big 37.5kDa OMP.These isolated strains are decided to be subgroup A1 and A2 respectively.In B group OMPs, there is no the proteinic performance of the 37kDa that makes a variation with bacterium.The goods that concentrated by OMP that are insoluble to Sarkolyl of the OMP protein of swine dysentery treponema isolated strains and other gram negative bacilli have been done contrast (Fig. 2 B).The sign of the OM of swine dysentery treponema isolated strains is easily and those escherichia coli, Salmonella typhimurium, yersinia genus enterocolitis bacillus, campylobacter jejuni and campylobacter fetus embryo subgenus phase region other.
The full cytolysate specimen of treponema is isolating with SDS-PAGE, gives the Z probe then and makes immune detection.The hyperimmune porcine blood serum of anti-swine dysentery treponema bacterial strain 5380 full cells (A group) is discerned protein that molecular weight obviously surpasses 24kDa more than 20 kinds at least, and the variation (Fig. 3) on some bacterial strains is arranged.Control serum before the immunity then can not identification of protein.Hyperimmune porcine blood serum and the number molecular weight proteins react in 30kDa to 40kDa scope is strong, this protein almost is that all isolates (A group and B group) are common, but then inequality with the immune-reactive protein matter of discerning in harmless spirillum isolated strains.
When being separated by SDS-PAGE with the OM goods that are insoluble to Sarkosyl of a harmless spirillum isolated strains preparation by seven isolated strains of swine dysentery treponema (one of six B group of A group), and give the Z probe through immunoblotting, again when anti-swine dysentery treponema bacterial strain 5380 hyperimmune porcine blood serums are detected, the equal symbolic animal of the birth year of the reactive mode of nearly all isolated strains with, only variation (Fig. 4) between small bacterial strain is arranged at the protein of 38kDa to 40kDa dalton molecule weight range.In order to contrast, in also being included in the reactive mode of isolated strains 5380 full cytolysates (Fig. 4, road 1 and 5).From A group and B group and the OM protein of harmless spirillum isolated strains and the antibody response in the hyper-immuneserum, with the preceding control serum of immunity then do not react (Fig. 4, road 1 to 4).In all swine dysentery treponema isolated strains, the total OM protein that is the strong immunization reaction is observed at 34kDa and 30kDa.In harmless spirillum isolated strains 34kDa district band also be respond [Fig. 4, road 13).The 39kDa district band (Fig. 4, road 6-11) of A component in bacterial strain, B component in bacterial strain 8841 big 40kDa (Fig. 4, road 12) and the 40kDa of Treponema innocens isolated strains 9510 and 38.5kDa protein band also be have immunocompetent.When surveying with the anti-swine dysentery treponema of rabbit hyper-immuneserum, OM protein also sees same antibody response (not proposing data).When the OM of other gram negative bacteria protein is surveyed by the anti-swine dysentery treponema of same pig hyper-immuneserum, then can not measure antibody cross reaction (not proposing data).
In order to further specify the antigenic character of swine dysentery treponema important on immunological response, allow the OM protein of identification in the Wei Sideng trace detects do relevant contrast with the treponemal surface protein of swine dysentery.Swine dysentery treponema bacterial strain 5380 complete full cells stop the protein decomposition through trypsin treatment and with PMSF.Swine dysentery treponema 5380 cells with contrast by Protease Treatment all are dissolved in the SDS specimen buffer agent and are SDS-PAGE (Fig. 5).It is 39kDa that trypsin protein decomposition causes molecular weight values, 36kDa, and 34kDa (hyperbola) and 30kDa protein band are optionally lost, and illustrate that these protein pass OM and expand to the spirillum surface.When handling swine dysentery treponema 5380 cells with protease k, seen the proteolysis (not proposing data) of the same manner without trypsin.
Following experiment can further specify characteristics of the present invention for example.
Example 1
A. the pig of age, no swine dysentery medical history was put into isolation unit around laboratory animal was got, and raised not contain the cultivation feedstuff of antibiotics.Procto swab is made dark-field microscopy, and is inoculated into and adds and do not add in the cattle of spectinomycin (Spect inomycin) and the horse blood agar and in the selenite broth.Under microscopy or in the substrate of separation and Culture, can't see and also can not separate the swine dysentery treponema.Feces also can not find out Salmonella and Campylobacter.The treponemal enzyme linked immunological absorption of all animal via swine dysenteries checks that (revealing) is negative in Austria big Leah number of patent application PH09631/86.
B. the culture of vaccine production swine dysentery treponema bacterial strain 5380 or 70A was grown in culture medium 48 hours, culture medium is the class pancreatic enzyme Semen Glycines broth bouillon for preparing under oxygen free condition, and additional cystine, glucose, sodium citrate, Sodium Pyruvate and ferrum are adjusted to 6.8 with pH, bacterium is filled to contain 10%CO
2Nitrogen mixture gas.In order to promote growth, can add hyclone, best 1%, but can be to 10%.
With the culture resuspending collected in phosphate buffered saline (PBS).Before using, bacterial concentration is diluted to is about 1 * 10
9/ ml.
C. the provocative inoculation thing excites and connects thing by the identical swine dysentery treponema of growing in the liquid medium within, and (as mentioned above) or the antibacterial on a blood agar flat board of 48 hours of 37 ℃ of cultivations are formed.
The provocative inoculation thing is positioned over by in 200 milliliters of the homogenates that can milk powder be made into, and raises then to 24 hours pig of fasting before the inoculation.
D. toxicity test
Four of pigs, the blood agar culture of oral vaccination swine dysentery treponema 5380.The diet of pig keeps no antibiotics, and observes the dysentery symptom every day.
The clinical response of the pig of these inoculation swine dysentery treponemas 5380 is as follows:
Average onset the 10th day
Average course of disease the 3rd day
Dead 4/4
Originally it is pathogenic to experimental results show that No. 5380 bacterial strains have pig, and all suffer from all separable swine dysentery treponema of pig of dysentery.
These two experiments all are six repeated experiments 1 of pig in three weeks with the age, and all pigs in inoculation diarrhoea and dysentery took place in back 11 days.Two pigs in the experiment 2 were in death in the 12nd day, and all the other have the pig of typical swine dysentery treponema symptom to use antibiotics with control dysentery.The result of experiment 3 is identical with experiment 2.
By making provocative inoculation for 8 pigs with experiment 1,2,3 identical inoculation steps, but need not 5380 and with the culture of 70A, all each pigs all do not die from swine dysentery.
4 age in week totally 6 of pigs, 4 inoculation 5380 bacterial strains wherein, 2 inoculation 70A bacterial strains, vaccination ways is oral swine dysentery treponema concentrated inoculum (1 * 10
11) and the fresh superficial growth thing collected by agar plate of internal rectum inoculation.Allow these pigs stay among the isolated plate case within doors, raise with identical feedstuff.Four pigs that inoculate 5380 groups show clinical swine dysentery in 10 days behind provocative inoculation, 1/4 death was arranged in the 12nd day.In 5380 groups other pig through with antibiotic therapy to control its dysentery.When experiment finishes, reach 30 day for, any dysentery symptom all do not take place after the inoculation with the pig of 70A inoculation.
8 pigs, culture with 70A excites, vaccination ways is that an agar plate with swine dysentery treponema 70A group activity growth places Lac Bovis seu Bubali oral, and is suspended in one milliliter of the swine dysentery treponema culture broth with the superficial growth thing of the movable growth of swine dysentery treponema 70A and does the internal rectum inoculation.These pigs all do not show any symptom of swine dysentery.But they have shown the rising to swine dysentery treponema antibody titer.Inoculate back 24 hours and can from swine excrement, isolate the swine dysentery treponema.From inoculating back the 5th day, from the feces of pig, promptly no longer can isolate the swine dysentery treponema.
E. vaccination experiment
6 pigs were used the swine dysentery treponema 5380 (2 * 10 that adjuvant is arranged in the time of zero day
9) do intramuscular injection vaccination, remake a vaccination in the time of the 11st day.6 pigs of matched group and they separate, but do not do above processing.
These pigs excited with 5380 bacterial strains in the 22nd day, as experiment 1 excimer were placed in the milk and fed.All the pig of matched group suffers from typical swine dysentery and is dead in 17 days.And equal can the antagonism of the pig that is subjected to vaccination excites and the sign of apparent swine dysentery infection.
5 pigs do vaccination by experiment 7, but just vaccination is for the second time accepted in the 10th day after first vaccination.Excite with swine dysentery treponema 5380 at the 26th day (for the second time vaccination after the 16th day).The pig of not accepting vaccination shows the swine dysentery symptom in after exciting 9 days, begins to die from acute swine dysentery exciting in back 10 days.
None shows any marquis of levying that swine dysentery infects to vaccinated pig in exciting back 45.All isolate the swine dysentery treponema in all infected and dead pigs, and after the pig of vaccination is exciting, promptly can not separate the swine dysentery treponema on the 12nd.
In experiment 8, the clinical response of pig is as follows:
(pig of * dies from escherichia coli peritonitis.In intestinal, there is no swine dysentery treponema infection evidence, in intestinal tissue, also do not isolate the swine dysentery treponema.)
All pigs are all drawn blood and have made the mensuration of serum titer (IgG level) in experiment, the results are shown in accompanying drawing (Fig. 4).
Because being presented at the protein composition by the assay determination of wesbem blotting, the soluble protein of swine dysentery treponema separator 5380 and 70A cell and the polyacrylamide gel electrophoresis analysis of external membrane protein upward or on the antigenicity do not have any difference (to see Fig. 1,2 and 3), 7 pigs are in zero daily swine dysentery treponema 70A2 * 10
9Do vaccination, on 17th with 70A2 * 10
9Made revaccination, in contrast with 6 pigs separating.
All excite in the 24th day (for the second time vaccination after the 9th day) all pig with swine dysentery treponema 5380.All the pig of matched groups shows that all diarrhoea and dysentery levies the marquis, all dies from acute swine dysentery in exciting in back 28 days.
The vaccination pig does not all show any marquis of levying of swine dysentery.A vaccinated pig promptly excites back the 12nd day, because of acute peritonitis death experiment the 36th day.Intestinal inspection is not found any evidence of swine dysentery, and does not also isolate the swine dysentery spirillum in the feces.Vaccination pig not, microscopy, histological examination and electronic microscope photos find all to have the features of acute swine dysentery, and the microscopical or histological variation that caused by the swine dysentery treponema infection was not then found in above-mentioned inspection when the vaccination pig was finished in experiment.
The vaccination pig shows that with the serological reaction of vaccination pig not pig that infect or uninfection has tangible difference to the treponemal IgG titre of dysentery.When serum is 1/800 dilution, all vaccination pigs to the treponemal serum titer of dysentery (IgG) all greater than 1,0OD unit.These experimental results show that the pig that does vaccination with swine dysentery treponema pathogenic strain (5380) or non-pathogenic bacteria strain (70A) all can obtain to be enough to the antibody titer (surpassing 1.0 at 1/800 o'clock) of protecting their antagonism swine dysentery treponema pathogenic strains (5380) to excite.
Example 2
A. laboratory animal is selected the pig in age all around of no swine dysentery history to place in the isolation room and is raised not contain the cultivation grain ration of antibiotics.Cut-off intestinal swab is made dark-field microscopy, and is inoculated into and adds and do not add in the cattle and horse blood agar and selenite broth of spectinomycin.Microscopy can not find out the swine dysentery treponema, does not also isolate this pathogen in isolation medium.Feces is not also found Salmonella and Campylobacter.The enzyme connection radioimmunity determination of adsorption method swine dysentery treponema that all animals are all introduced by Australian patent application PH09631/86, the result is all negative.
B. grew 48 hours in the class pancreatic enzyme soybean broth culture medium that the culture of the vaccine product swine dysentery treponema 70A bacterial strain of oxygen processing prepares under oxygen free condition.This culture medium is also added Guang chloric acid, glucose, citric acid and ferrum.Regulate its PH to 6.8, sterilizing and charging into contains 10%CO
2N
2In order to promote growth, also can add hyclone, but as many as 10% is preferably 1%.
110 pigs, handle with oxygen, inactivation, the swine dysentery treponema vaccine that adds adjuvant was respectively at 0 day and the 10th heaven-made vaccination, and other is with 110 pigs in contrast.The equal Su Yu of these pigs has in the pig house that swine dysentery distributes.Do not show the acute dysentery symptom in vaccination pig 7 days after vaccine experiment beginning.Clinical response in this vaccination experiment is as follows:
* once finding swine dysentery, all performances have the pig of acute dysentery symptom all to use female woods (Tiamulin Hydrochloride) the 10mg/kg treatment of hydrochloric acid platform, and proof can reduce death, but if do not treat, then dead inevitable.
Anxious living dysentery symptom only appearred after the pig vaccination of+vaccination group in three days, and dead in a few hours after being ill.
It is believed that after the vaccination and still be not enough to produce enough immunoreation in three days.The seroreaction of vaccination pig and vaccination pig not show infected pigs and not infected pigs swine dysentery is being had tangible difference on the treponemal IgG titre.In all vaccination pigs, the dilution in 1/800 is 1 to the treponemal serology titre of swine dysentery, 0OD unit.These immunity inoculations experimental results show that the pig of inoculation oxygen processing deactivation swine dysentery treponema (70A) bacterial strain vaccine can obtain to surpass 1,0 antibody titer (1/800), and this can make its infringement that not excited by swine dysentery treponema pathogenic bacterial strains.
D. immunogenicity contrast
Produce the ability of IgG antibody in order to observe pig irritated by the swine dysentery treponema, to the great-hearted swine dysentery treponema 70A that adds adjuvant with add deactivation swine dysentery treponema 70A that the oxygen of adjuvant handles as a comparison.Totally two doses of vaccines, every dose contains the swine dysentery treponema 1.5 * 10 that adds adjuvant
9, do intramuscular injection for two groups of pigs (4 every group), 10 days at interval.4 pigs of the 3rd group in contrast.Extract the serum of all pigs, weekly, in totally three weeks, write down it to swine dysentery treponema IgG antibody titer (calculating absorptance).It the results are shown in Fig. 6.No matter be great-hearted as can be seen from Figure 6 or the vaccine of oxygenation, its absorptance that causes there is no significant difference.
Claims (13)
1. one kind prepares and is used for through the parenteral administration pig being carried out the method for vaccination with the vaccine of the swine dysentery that caused by the swine dysentery treponema infection of opposing, and this method comprises with oxygen handles swine dysentery treponema bacterial strain so that the step of said bacterial strain deactivation.
2. according to the process of claim 1 wherein that said administration is the intramuscular administration.
3. according to the process of claim 1 wherein that said swine dysentery treponema bacterial strain is a pathogenic bacterial strains.
4. according to the process of claim 1 wherein that said swine dysentery treponema bacterial strain is the bacterial strain of attenuation.
5. according to the process of claim 1 wherein that said swine dysentery treponema bacterial strain has been placed in the oxygen-saturated solution so that said bacterial strain deactivation.
6. according to each method of claim 1-5, wherein said swine dysentery treponema bacterial strain is an A group bacterial strain.
7. according to the method for claim 1, this method further comprises the step that the treponemal inactivated strain of swine dysentery is contacted with adjuvant.
8. one kind prepares and is used for effectively making the method for pig to the immunifacient vaccine combination of swine dysentery treponema through the parenteral administration, this method comprise make the treponemal inactivated strain of handling through oxygen of swine dysentery selectively with adjuvant, the step that contacts with its acceptable carrier.
9. method according to Claim 8, wherein said swine dysentery treponema bacterial strain is a pathogenic bacterial strains.
10. method according to Claim 8, wherein said swine dysentery treponema bacterial strain is the attenuation bacterial strain.
11. according to Claim 8 or the method for claim 9, wherein said swine dysentery treponema bacterial strain has been placed in the oxygen-saturated solution so that said bacterial strain deactivation.
12. each method according to Claim 8-11, wherein said swine dysentery treponema bacterial strain are A group bacterial strains.
13. each method according to Claim 8-12, this method further comprises the step that the treponemal inactivated strain of swine dysentery is contacted with incomplete Freund.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPI3940 | 1987-08-24 | ||
| AUPI394087 | 1987-08-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1031479A CN1031479A (en) | 1989-03-08 |
| CN1031039C true CN1031039C (en) | 1996-02-21 |
Family
ID=3772407
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 88101481 Expired - Lifetime CN1031039C (en) | 1987-08-24 | 1988-03-21 | Swine dysentery vaccine |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1031039C (en) |
| PH (1) | PH31030A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10821165B2 (en) * | 2016-01-07 | 2020-11-03 | Universiteit Gent | Vaccine strains of brachyspira hyodysenteriae |
-
1988
- 1988-01-08 PH PH36334A patent/PH31030A/en unknown
- 1988-03-21 CN CN 88101481 patent/CN1031039C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| PH31030A (en) | 1997-12-29 |
| CN1031479A (en) | 1989-03-08 |
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