CN100392067C - Lawsonia intracellularis cultivation, anti-lawsonia intracellularis vaccines and diagnostic agents - Google Patents

Abstract

A method for large scale cultivation and attenuation of L. intracellularis bacteria by inoculating cells with L. intracellularis bacteria to infect the cells, incubating the infected cells in a reduced oxygen concentration and maintaining the infected cells in suspension. Anti-L. intracellularis vaccines are prepared from cultures grown in suspension. Diagnostic agents are also disclosed.

Description

The cultivation of Lawsonia intracellularis in the McCoy cell

The application of this division is the dividing an application of Chinese patent application CN 96114542.0 that is entitled as " Lawsonia intracellularis is cultivated, vaccine and the diagnostic reagent of anti-this bacterium ".

Technical field

The present invention relates to the method that anti-Lawsonia intracellularis (Lawsonia.intracellularis) vaccine and control and diagnosis LI infect.Product of the present invention and method partly are the results that modification method caused of the large scale culturing Lawsonia intracellularis found by us.

Background technology

Lawsonia intracellularis is pig proliferative enteropathy (porcine proliferative enteropathy, PPE) pathogenic former, it influences nearly all animal, comprises people, rabbit, ferret, hamster, fox, horse and other various different animals such as ostrich and rhea etc.

Lawsonia intracellularis is the reason of damnous particularly important in the swinery.In the U.S. be infected and annual loss $2000 ten thousand up to 20% swinery to the incidence of PPE and the estimation of infection rate.

The fixed character of PPE is to have intracytoplasmic, non-membrane-bound Campylobacter in the intestinal cells of infected intestines part.The bacterium relevant with PPE is called as " Campylobacter sample biology (Campylobacter-likeorgansims) " S.McOrist et al., Vet.Pathol., Vol.26,260-64 (1989).Subsequently, malignant bacteria is differentiated to be a kind of new branch generic and kind, be called as in the language home " Ilealsymbiont (IS) intracellularis " (ileum symbiote in the born of the same parents, ISi).C.Gebhart et al.，Int’l.J.of Systemic Bacteriology，Vol.43，No.3，533-38(1993)。Recently, these new bacterias are endowed systematic name: Lawsonia (L.) intracellularis.S.McOrist et al.，Int’lJ.ofSystemic Bateriology，Vol.45，No.4，820-25(1995)。These 3 kinds of titles are used interchangeably, the same organisms that all refers to describe herein and further differentiate.We use systematic name Lawsonia intracellularis (L.intracellularis) as far as possible in argumentation of the present invention.

Lawsonia intracellularis is a kind of obligate, intracellular bacterium, and it can not be cultivated with common bacteriological method in the acellular substratum of routine, and thinks always and need could grow attached to epithelial cell.S.McOrist etal. is at Infection and Immunity, Vol.61, No.10,4286-92 (1993) and G.Lawson et al. are at J.of Clinical Microbiology, Vol.31, No 5, discuss among the 1136-42 (1993), cultivate Lawsonia intracellularis with the intestinal epithelial cells individual layer of IEC-18 rat in the conventional organization culturing bottle.In addition, H.Stills is at Infection and Immunity, and Vol.59 discusses among the No.9,3227-36 (1991), uses intestines 407 people embryo intestinal cells individual layers and GPC-16 guinea pig colon adenocarcinoma cell individual layer in the conventional organization culturing bottle.These existing cultural methods are required great effort and are not suitable for large scale culturing.

Owing to need harsh growth conditions at the vitro culture Lawsonia intracellularis, thus at present the understanding that Lawsonia intracellularis is infected and to this treatment of diseases and effectively control all hindered.Therefore the method that needs a kind of cultivation Lawsonia intracellularis of improvement at present.The effective tool that also needs anti-Lawsonia intracellularis vaccine and diagnosis Lawsonia intracellularis to infect.

Summary of the invention

An object of the present invention is to provide anti-Lawsonia intracellularis vaccine.

Another object of the present invention provides and detect the method whether anti-Lawsonia intracellularis antibody exists in biological sample.

Another object of the present invention provides a kind of cultural method of improvement, and it can cultivate Lawsonia intracellularis on a large scale to be used to produce vaccine and diagnostic reagent.

In order to realize these and other purposes, and extensively describe with this paper and the invention is intended to of embodying consistent, the invention provides and a kind ofly cultivate the method for Lawsonia intracellularis and obtain a large amount of bacterium raw materials with this method.According to this method, the Lawsonia intracellularis bacterium hatches under about 0-18% at oxygen concn, stirs bacterium simultaneously to cultivate Lawsonia intracellularis and to keep bacterial suspension.

According to another example, a kind of method of cultivating the Lawsonia intracellularis bacterium is provided, promptly pass through: inoculum inoculation HEp-2, McCoys or IEC-18 cell monolayer (its degree of converging is about 30%) with containing the Lawsonia intracellularis bacterium make the infectation of bacteria cell.Then the cell that infects is in about 36-38 ℃ temperature, and oxygen concn is converged until cell for hatching under about 0-8.0%.Then cells infected and growth medium are placed fermentation container, bio-reactor, revolving bottle (spinner flask) or other to be fit to keep the container that cell is in suspended state.Cells infected should stir cell when hatching, so that cultivate the Lawsonia intracellularis bacterium and keep the cell suspension of infection.The Lawsonia intracellularis that a part is cultivated is passaged to fresh culturing cell again to increase the output of Lawsonia intracellularis bacterium.

The invention provides anti-Lawsonia intracellularis vaccine and generation method at the vaccine of Lawsonia intracellularis.By the Lawsonia intracellularis bacterium of cultivating being gone down to posterity abundant number of times and select attenuated strain, perhaps carry out chemical attenuation, thereby produce avirulent Lawsonia intracellularis bacterium by the bacterium that will cultivate.The Lawsonia intracellularis vaccine that also can prepare deactivation with cultural method of the present invention.According to a particularly preferred embodiment, bacterium is by cultured continuously 6-8 month at least, is passaged to few about attenuated strain that can be used as vaccine for 7-12 time with generation simultaneously.Then attenuated bacteria is mixed with pharmaceutically acceptable carrier, and be administered to animal to produce immunne response with significant quantity.We are deposited in U.S. typical culture preservation center (ATCC) with present preferred attenuated strain (N343NP40wk).

The present invention also provides a kind of method of determining whether to exist with the antibody of Lawsonia intracellularis bacterium specific reaction in biological sample, promptly pass through: the Lawsonia intracellularis bacterium that results at least a portion is cultivated, the antibody that in biological sample, exists with the Lawsonia intracellularis bacterium of results or its component and biological sample from animal can with condition that Lawsonia intracellularis or component react under contact, and determine whether to take place antibody-antigen-reactive.

Other features and advantages of the present invention will provide in the following description, they in description clearly or can comprehend by implementing the present invention.

Embodiment

As used herein, term " Lawsonia intracellularis " refers to the al. by C.Gebhart et, Int ' l.J.of SystemicBacteriology, Vol.43, No.3,533-38 (1993) and S.McOrist et al., Int ' l J.ofSystemicBateriology, Vol.45, No.4, it is intracellular that 820-25 (1995) (full content of these two pieces of documents is incorporated herein by reference) describes in detail, crooked, gram negative bacterium, it includes, but are not limited to: at American type culture collection (ATCC, Rockville, MD) ATCC 55672 bacteriums of preservation; (NCTC, Colindale, London) NCTC 12656 of preservation and 12657 bacteriums at state-run typical culture collection center; According to knowledge of this area and teachings herein, the malignant bacteria that obtains the pig that can in world wide, be infected or other animals by PPE; And mutant spontaneous or induced mutations or the varient of arbitrary above-mentioned bacterium.

As used herein, term " attenuated strain " refers to any such Lawsonia intracellularis bacterial strain, thereby cultivation that it is lectured by this place and the technology of going down to posterity prepare and make it reach avirulence, keep its immunogenicity feature simultaneously when it is administered to host animal again.As described below, according to the present invention, various Lawsonia intracellularis bacterial strain is cultivated and attenuation, thereby has obtained attenuation immunogenicity bacterial strain.These attenuated strains can be effective as pig or other easily suffer from the vaccine of Lawsonia intracellularis infected animals.

Attenuated strain of the present invention expection can be used as the immunogen in the antimicrobial vaccine of animal, and these animals comprise bird, fish, ox, pig, horse, Mammals and common primate and people.After obtaining the content of lecturing herein, these vaccines can be prepared with the known technology of those skilled in the art.This vaccine can contain the attenuated strain and the pharmaceutically acceptable carrier of immune significant quantity.Vaccine can single dose or multiple doses ground use.Herein on the basis of teachings, immune significant quantity can be determined and needn't be carried out too much experiment with methods known in the art.Avirulent number of bacteria should be enough to excite in easily ill animal immunne response to remain avirulent simultaneously.This depends on specific animal, bacterium and related disease.The recommended doses that is applied to susceptible animal is preferably about 10 ³-10 ⁹Bacterium/kg body weight, the best is about 10 ⁵-10 ⁷Bacterium/kg body weight.Carrier is well known to those skilled in the art, and comprises stablizer and thinner.This vaccine can also contain suitable adjuvant.Vaccine of the present invention can with other vaccines for example the diluent of another kind of freeze dried vaccine be used in combination, perhaps before freeze-drying with the combination of other vaccines.Vaccine preparation also can carry out drying, for example freeze-drying so that the storage or in order to be mixed with liquid vaccine later on.

Therefore, thus the present invention also comprises a kind of inducing at immunne response toxic, wild-type Lawsonia intracellularis bacterium in the animal host protect this host to avoid the method for this bacterium infringement.This method comprises: use the attenuated bacteria of the present invention of immune significant quantity or the bacterium that kills to the host, be preferably to the host and use vaccine of the present invention.

As used herein, term " large scale culturing " refers to: the cultivation level of Lawsonia intracellularis rises greater than about 2.0-3.0 and comprises that production scale is greater than 100 liters or bigger.As used herein, " cultivation " refers to promote the process of growth, regeneration and/or the propagation of Lawsonia intracellularis.

In implementing cultural method of the present invention, culturing cell is at first inoculated so that the infectation of bacteria cell with the inoculum that contains the Lawsonia intracellularis bacterium.Various kinds of cell system can be used for implementing the present invention, comprising but be not limited to: IEC-18 (ATCC 1589)-mouse intestinal epithelial cells, HEp-2 (ATCC 23)-people's epidermoid cell, McCoys (ATCC1696)-mouse (non-specific) cell, MDCK (ATCC 34)-Madin-Darby Madin-Darby canine kidney(cell line) (MDCK), BGMK (Biowhittaker#71-176)-Buffalo green monkey kidney cell and chitling epithelial cell.Preferred culturing cell is HEp-2, McCoys or IEC-18 cell.Perhaps, bacterium can be cultivated in acellular system, as long as bacterium is maintained under this place professor's the suitable dissolved oxygen concentration.

If the use culturing cell, cell best (but not necessarily) is in form of single sheet before inoculation so.In order to form individual layer, can be in the bottle of routine with cell inoculation.The inoculum size that each bottle is general is about 1 * 10 ⁵-10 * 10 ⁵Cell/25 square centimeter bottle, and mix with growth medium.Growth medium can be any medium that is used for cell cultures, and it contains nitrogenous source, selected required somatomedin and carbon source such as glucose or the lactose of culturing cell.Preferred substratum is the DMEM that adds the 2-5% foetal calf serum, although also can use various other commercially available substratum and obtain good result.

We find, by culturing cell being maintained under the stable growth conditions, can successfully cultivate Lawsonia intracellularis better.Therefore, the culturing cell individual layer should have 20-50% degree of converging approximately when inoculation.More preferably, cell degree of converging should be about 30-40% when inoculation, and the best degree of converging is about 30%.

Inoculum can be pure Lawsonia intracellularis culture, for example from ATCC preserving number 55672, NCTC preserving number 12656 or 12657 or the pure growth that obtains with separation described herein and purification technique on one's body from the pig that infects or other animals.

According to an example, be used to implement inoculum of the present invention and be the intestinal tissue homogenate for preparing by the ileal mucous membrane that scrapes the pig that infects PPE or other animals.During the homogenate of preparation intestinal tissue, the ileal segment that selection is used to cultivate should demonstrate serious damage and have significantly and thicken.Because bacterium nature fragility, thus preferably should be as early as possible after the necrotomy with sample preservation under-70 ℃.Be preferably in the inoculum and add Lawsonia intracellularis and produced the microbiotic of resistance to suppress the growth that polluted bacteria can allow Lawsonia intracellularis simultaneously again, in order to exemplify, this type of antibiosis have vancomycin, amphotericin B or aminoglycoside antibiotics member such as gentamicin and Xin Meisu.No matter inoculum is pure growth or intestinal tissue homogenate, provide teachings herein after, the various technology that culturing cell is inoculated in available this area are carried out.

Then, the culturing cell of bacterium and/or inoculation is hatched under minimizing dissolved oxygen concentration condition.When dissolved oxygen concentration greater than 18% the time, Lawsonia intracellularis growth fraction optimal cases is low and exceed under this scope at oxygen concn and finally to stop growing.Preferably, the culturing cell of inoculation is hatched under about 0-10% at dissolved oxygen concentration.More preferably, cell is being hatched under about 0-8% at dissolved oxygen concentration, and best, oxygen concn is about 0-3.0%.

The proper concn of carbonic acid gas also is vital to the proper growth of Lawsonia intracellularis.When concentration of carbon dioxide greater than 10% or less than 4% the time, being non-optimum growh and exceeding at carbonic acid gas that final growth stops under this scope situation of generation.Preferably, concentration of carbon dioxide is about 6-9%, and best, concentration of carbon dioxide is about 8.8%.

In addition, cell is preferably hatched under about 73-94% at density of hydrogen.Can use nitrogen to replace part or all of hydrogen.According to a more preferred example, cell is hatched under about 0-8.0% oxygen, about 8.8% carbonic acid gas and about 83.2% hydrogen.

Inoculating cell can contain proper concn oxygen and carbonic acid gas and allow cell hatching in the air chamber that suspends in the process at two gas couveuses (incubator) or other hatches.This chamber should be contained inoculating cell is maintained device, the gas monitor of suspended state and the source of supply that provides and keep suitable gas concentration.Incubation temperature should be 30-45 ℃, is preferably 36-38 ℃.Best, temperature is about 37 ℃.After the teachings that provides herein, the required equipment of cultivation of the present invention and method of attenuating can obtain for one of ordinary skill in the art easily.An example that is suitable for implementing equipment of the present invention is two gas couveuses, for example can be from Lab-Line, and Melrose Park, 480 types that Illinois obtains, and combine to keep cell with revolving bottle and to be in suspended state.At present preferred equipment comprises: fermentation container, bio-reactor or rotary shaker, they contain at least about 2 liters of substratum and gas that can be by spraying into proper concn or by other mechanical stirring means culturing cell are maintained at suspended state, and the level of dissolved oxygen in can the continuous monitoring substratum.New Brunswick, Braun and other companies make suitable fermentation container and the bio-reactor that is used for this purpose.

By in the process of hatching, inoculating cell being maintained at suspended state, just can be by increasing each cellular exposure in the degree of growth medium and oxygen and carbon dioxide mixture, realize the maximum growth of cell, and then realize the maximum growth of Lawsonia intracellularis.Can and be maintained at suspended state with the whole bag of tricks stir culture cell in this area, comprising for example, culturing bottle, bottle (a roller bottle), film incubator or revolving bottle roll.By incubated cell in two gas couveuses or similar devices inward turning rolling bottle, can make cell in the process of hatching, keep suspended state.As used herein, term " revolving bottle " refers to such bottle or other containers, and they adopt oar, water screw or other devices to come the stir culture thing and contained cell is maintained at suspended state.

In a more preferred example of the present invention, hatch inoculating cell and reach until cell and converge, then cell is placed the revolving bottle that contains growth medium and hatches to make the revolving bottle rotation simultaneously at two gas couveuses.Preferably, inoculating cell is scraped in the revolving bottle.This can realize with the whole bag of tricks as known in the art, for example use the cell beater to come dissociated cell.In case cell is introduced into revolving bottle, the oar of revolving bottle typically with the speed mechanical rotation of about 30-60rpm, is maintained at suspended state with cells infected.

The Lawsonia intracellularis that a part is cultivated then is passaged to fresh culturing cell, to increase the output of Lawsonia intracellularis bacterium.Term " goes down to posterity " or similar word refers to the Lawsonia intracellularis that a part is cultivated is transferred to the fresh culture cell so that with the process of the new fresh cell of this infectation of bacteria.As used herein, term " fresh " refers to the cell that do not infected by Lawsonia intracellularis.Preferably, this cell is on average to be no more than about 1 day cell.

The going down to posterity of Lawsonia intracellularis in suspension culture can be realized by taking out a part of primary culture and it being added in the new bottle that contains the fresh culture cell.If the number of bacteria of primary culture/milliliter value is higher, for example greater than about 10 ⁴Bacteria/milliliters is in then preferably that the culture adding of about 1-10% (volume ratio) in the infected bottle is the new bottle that contains new fresh cell.Be preferably in the 50-100% cell and carry out this operation when infected.If infected cells is lower than 50%, then preferably, culture adds new bottle by being divided into two, and add new substratum then and realize going down to posterity to volume required scale.Under any circumstance, do not need lysis and other steps, this with prior art in the monolayer culture go down to posterity just in time opposite.

Culturing cell fully grow and subsequently with Lawsonia intracellularis to infect (with IFA, TCID greater than about 70% cell infection rate ₅₀Or other similar approach are measured) afterwards, the Lawsonia intracellularis bacterium that results at least a portion is cultivated.After the teachings that provides herein, can use the known various technology of persons skilled in the art, gather in the crops step by separation of bacterial from suspension.Preferably, the results of Lawsonia intracellularis bacterium are by the contained material of centrifugal all or part of suspension, make the culturing cell precipitation, and then the cell precipitation that suspends and form, cracking cells infected again.Typically, with at least a portion inclusion under about 3000 * g centrifugal about 20 minutes, make cell and bacterium form precipitation.The precipitation resuspending in for example sucrose-phosphoric acid salt-L-glutamic acid (SPG) solution, then the syringe needle by 25 specifications (gauge) about 4 times so that lysis.If also need to be further purified, sample can be under about 145 * g centrifugal about 5 minutes, to remove nucleus and fragment.Supernatant liquor under about 3000 * g centrifugal about 20 minutes, the throw out resuspending that forms in suitable diluent, as contain foetal calf serum SPG (with preparation be suitable for refrigerating or as the results bacterium of Inoculant) or growth medium (being more suitable for being passaged to the results bacterium of new fresh cell with preparation).

As mentioned above, the effective growth that is used for the Lawsonia intracellularis of scale operation can improve by histocyte is grown actively.For individual layer, when culture converged, fissional speed significantly descended.The trial that Lawsonia intracellularis is grown on monolayer organization's culture can only obtain limited success, and can not carry out scale operation.Yet, can be convenient to make cell to grow actively greatly with suspension culture, and allow the increase of successive culture and scale to enlarge.With fermentation container and above-mentioned about 0-3% dissolved oxygen, we can make it grow to 10 ⁸Bacteria/milliliters.The bacterium of cultivation was grown many months actively for we and expection can unrestrictedly continue.

Before the present invention, generally believe that cell must be attached to the surface and could be infected by Lawsonia intracellularis.Cell suspending liquid of the present invention is unique, and opposite with this theory.When using McCoys or IEC-18 cell, preferably with growth medium add gelatin, agarose, collagen protein, acrylamide or silica beads for example HyCloneLaboratories (Logan, UT) Cultisphere-G of Zhi Zaoing (cultivates the porous microcarrier (microcarries) of ball-G).Yet according to the inventive method, HEp-2 cell and other cells do not need microcarrier.This provides particularly advantageous and economic approach for large scale culturing.

Cultivate for HEp-2,, preferably take out the 25-50% culture weekly and replace with fresh culture in order to keep the purpose of cultivation.For the cell cultures of band microcarrier or pearl, preferably take out the 25-50% culture weekly for 1-2 time and with fresh microcarrier or pearl and fresh culture replacement.For the expansion scale, can in culture, add the substratum of 25-50% substratum or band microcarrier again.

According to the infected speed of culturing cell, usually every carrying out once going down to posterity about 2-5 week to new fresh cell.Suppose at 2-3 at least 70% infectedly in the culturing cell in week, so preferably go down to posterity in about 3-4 week.

The present invention also provides the vaccine of anti-Lawsonia intracellularis and the method for producing this vaccine.According to a more preferred example, keep cells infected behind suspended state in long-time (for example 6-8 month), potential attenuation effect is gathered in the crops and monitored to the Lawsonia intracellularis bacterium that at least a portion is cultivated.This monitoring is preferably finished by the immune stimulating of host animal or animal model, to select attenuated strain.This attenuated strain can be used for the vaccine in the method described herein.The Lawsonia intracellularis vaccine of attenuation of the present invention has demonstrated can be in various animals anti-effectively Lawsonia intracellularis and has infected, and to be expected at philtrum also be effective.

The present invention allows to cultivate apace amplification, increases 100-1000 doubly as output, and has reduced cost.As a result, the competent Lawsonia intracellularis bacterium raw material that produces with cultural method of the present invention can be attenuated easily to be used for production of vaccine.It is difficult carrying out attenuation in the monolayer culture thing, because the bacterium that produces with traditional monolayer growth technology yields poorly.On the contrary, Lawsonia intracellularis growth method of the present invention has improved simple and easy degree, speed and has been used for the number of bacteria of attenuation.Cell and cell fission must be many more, and the sudden change level of generation is high more, and this is favourable to vaccine development.Growing in suspension of the present invention has increased the important immunogenic expression and the expression product thereof of the Gene Handling that is subjected to environment conditioning.

The attenuated strain that obtains can as below cultivate in the tissue culture individual layer described in the embodiment 1, but preferably in suspension culture of the present invention, cultivate.Other method of attenuating comprises that chemical attenuation is for example by using N-methyl nitrosoguanidine and other attenuation materials as known in the art to carry out attenuation.No matter be by repeatedly going down to posterity or use chemical process, all can producing the attenuation Lawsonia intracellularis and also be selected with the preparation vaccine.

According to a vaccine example of the present invention, by above-mentioned centrifugal or micro-filtration results antigen.Then according to best host animal immunne response, tire by the dosage in the host animal species and to determine a level, and on the level of this qualification, antigen is carried out stdn.Bacterium can be by for example a week being exposed to ambient oxygen concentration for a long time, perhaps the vaccine by with 0.3% formalin or other deactivators its inactivation is killed with preparation.Then, antigen being mixed suitable adjuvant for example replys with enhancing immunity in aluminium hydroxide or the mineral oil.Then,, antigen is used for immune host by intramuscular or subcutaneous injection, for the piggy in about 3-4 week, available if desired booster dose.

Perhaps, according to particularly preferred, as to use an above-mentioned cultural method vaccine example, bacterium is gone down to posterity continuously to induce and to select attenuation, avirulent culture alive.The attenuation situation of this culture of test (be preferably in grow in the suspension culture 6-8 after the month) at least in host animal.Collect culture and dilution as previously mentioned.For example, pig can be with oral 1 * 10 ⁵-1 * 10 ⁶Bacterium and carry out immunity.After immunity 28 days, pig uses 1 * 10 again ⁷Individual passage number less (about 30 to 40 days), the Lawsonia intracellularis culture oral vaccination of virulence is arranged.Infection animal carried out necrotomy in 21 days behind immune stimulating, observe big damage and small damage at small intestine.Also should carry out the analyses of PCR and fluorescence antibody (FA).About 80% control animal has big or small damage, and shows with PCR or FA testing method, the existence of the Lawsonia intracellularis test result that is positive in the mucomembranous cell of intestines.The animal of immunity determines to have normal mucomembranous surface by histological observation, and is negative in the PCR test.

Generally, the immunogenicity Lawsonia intracellularis bacterial strain of attenuation by cultured continuously at least about 150-250 days and therebetween culture go down to posterity and produce at least about 7-12 time.Also can change these parameters and produce other attenuation culture, as long as adopt monitoring and system of selection described herein.

Prepare vaccine then, it contains the attenuation Lawsonia intracellularis and the pharmaceutically acceptable carrier of immune significant quantity.Immunogen after the merging and carrier can be the aqueous solution, emulsifying agent and suspension.After the teachings that provides herein, immune significant quantity can be determined and not need undue experimentation with the currently known methods of this area.Generally, immunogenic quantity is the 50-500 microgram, and is 10 when using purification of bacterial ⁷-10 ⁹TCID ₅₀

Be administered to easy trouble animal (being preferably pig) general single dose of vaccine of the present invention or multiple doses.By 2 weekly intervals, can use vaccine 1 alive or deactivation or 2 times.For the living vaccine of attenuation, preferred single dose.The preferred route of administering of the attenuated strain of living is oral or intranasal administration, but also can pass through intramuscular or subcutaneous injection.For inactivated vaccine, intramuscular and subcutaneous injection approach are best.

The efficient diagnosis of PPE is also cultivated the required time of malignant bacteria and is hindered.As result of the present invention, can develop fast now and analyze exactly from pig or other and may suffer from the diagnostic tool that whether has Lawsonia intracellularis in the biological sample of animal of PPE.

According to the Lawsonia intracellularis bacterium of the inventive method growth or from the component of this bacterium, can be at ELISA or other immunoassays such as immunofluorescent antibody test (immunofluorescent antibody, IFA) be used as antigen in, to detect may be by the antibody of the anti-Lawsonia intracellularis in the serum of the animal of this infectation of bacteria or other body fluid.At present preferred immunoassay is the IFA that describes among the embodiment below.Perhaps, the bacterium with the inventive method growth is used to Western Blot analysis.

The preferred ELISA scheme of example is as follows according to the present invention:

1. adding 0.1 milliliter/hole is diluted in bag and is cushioned antigen in the liquid.Hatched 18 hours at 4 ℃.

2. with PBS washing 3 times.

3. every hole adds 0.25 milliliter of sealing (blocking) damping fluid on plate.Hatched 1-2 hour at 37 ℃.

4. with lavation buffer solution washing 3 times.

5. dilute serum in the sealing damping fluid, and in first round of plate, add 0.1 milliliter.Carry out a series of 1: 2 dilutions onboard.Hatched 1 hour at 37 ℃.

6. with lavation buffer solution washing 3-5 time.

7. in the sealing damping fluid, dilute conjugate, in each hole of plate, add 0.1 milliliter.Hatched 1 hour at 37 ℃.

8. with lavation buffer solution washing 3-5 time.

9. adding substrate.

12. use the spectrophotometer measurement absorbancy.

13. do not add antigenic hole as blank.

14. in each test, all should use the positive and negative control.

Preferred Western trace scheme is as follows:

1. at 12%SDS-PAGE antigen is carried out electrophoresis, then it is gone to nitrocellulose filter.

2. film was placed the sealing damping fluid 2 hours.

3. remove encapsulant and use PBS rinsing 1 minute.

4. dilute serum in the sealing damping fluid, and adding film.At room temperature hatched 2 hours.

5. with 3 times (each 5 minutes) of lavation buffer solution washing.

6. in the sealing damping fluid, dilute conjugate, and add on the film.At room temperature hatched 1 hour.

7. with lavation buffer solution washing 3 times.

8. add substrate 10 minutes until strong combination takes place.

9. use the PBS rinsing.

10. air drying and storage are in the dark.

The present invention also further describes in conjunction with the following example.Providing of these embodiment only is used for purposes of illustration, can not be interpreted as to provide constraints.

Embodiment 1

From the enteron aisle of the U.S. pig that suffers from pig proliferative enteropathy, separate Lawsonia intracellularis

Material and method

The selection of inoculation sample

Obtain sample N24912 in the herd on a farm of Iowa, observed 15 in 300 growing and fattening pigs in 5 months (finisher pig) has bloody stool always, although treat with penicillin.After pig carried out necrotomy, find that intestines (ileum) have thick mucous membrane.Dye with silver and to carry out histopathological examination and show, have bacterium and crypts (crypt) intestinal epithelial cells hyperplasia in crooked, the cell, thereby confirm the diagnosis of PPE.Sample N72994 obtains 1.5 years old the two tire SPF sows from a Minnesotan farm.The herd size is a 70-80 sow, and used antibiotic therapy the unknown.Necrotomy finds, ileal mucous membrane thickens and some is hemorrhage.Mucous membrane is carried out Giminez dyeing show, have many crooked bacteriums.Sample N1014 94 is to be to obtain the pig in 12 weeks at the age from farm, Indiana (tool 600 plough farmland feeding mother pigs).This pig is taking place to treat still just death of this animal soon after treatment after the bloody flux disease with the Tylan injected material.

Preparation is from the inoculum of pig:

The intestines sample is-70 ℃ of preservations.Cut intestines open, with phosphate buffered saline buffer (PBS) washing.With 1 gram mucous membrane scrape into Sodium Glutamate potassium (sodium potassium glutamate, SPG) in, use 4.0 milliliters of 0.1%Trypsin (JRHBioscience s, Lenexa, KS) homogenizing 30 seconds in SPG then.Suspension was hatched 35 minutes at 37 ℃.(JRH Biosciences, Lenexa KS), ground 1 minute in tissue grinder then to add 10 milliliters of SPG/10% foetal calf serums (FCS).Add 10 milliliters of SPG/10%FCS, use filter paper (Whatman 113V then; WhatmanLabsales, Hillsboro OR) filters once, filters on 5.0,1.0 and 0.65 microns filtering membranes subsequently.Filtrate is divided into several parts, is stored in-70 ℃ by 1.0 milliliters of portions.Mucous membrane is applied on the slide glass so that Giminez dyeing.Each smear of filtrate is by the IFA method, dyes with the monoclonal antibody specific (full content of the document is incorporated herein by reference for S.McOristet al., Vet.Rec.121:421-422) of anti-Lawsonia intracellularis.

Cell cultures

(JRH Biosciences, Lenexa make IEC-18 (mouse intestinal epithelial cells, ATCC CRL 1589) growth in KS), and carry out routine with trypsinase weekly and go down to posterity at the DMEM that contains L-glutaminate and 10%FCS.Cell monolayer is grown in the air that contains 5% carbonic acid gas at 37 ℃.

The infection of cell culture

The IEC-18 cell is with 1.25 * 10 ⁵Cell inoculation is in 25cm ²Bottle in, and (Naperville Il), was hatched 24 hours, removed substratum then for Nunc, Inc. to be inoculated in chamber slide (chamberslide) with comparable and ratio.Melt fast freezing bacterial isolates, and in the DMEM/7%FCS that contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml), dilute from pig, Dilution ratio be 1.0 milliliters of tissue homogenates to 15 milliliters of substratum, add to individual layer then.Individual layer and bacterial suspension at 2000g centrifugal 30 minutes are transferred in the anaerobic jar.Drain tank, thus air is replaced the mixture that obtains 8.0% oxygen, 10% carbonic acid gas and 82% hydrogen formation by hydrogen and carbonic acid gas.Culture was hatched 3 hours at 37 ℃, and then supplied with the DMEM/7%FCS that contains L-glutaminate, vancomycin (100 μ g/ml), Xin Meisu (50 μ g/L) and amphotericin B (2.0 μ g/ml).Culture is put back in the anaerobic jar again, hatches 6 days, changes a subculture in per 2 days.

Going down to posterity of Lawsonia intracellularis

Press former G.Lawson et al. at J.Clin.Microbiol., method described in the 31:1136-1142 (1993) (document full content is quoted as a reference), with the Repone K lysing cell Lawsonia intracellularis bacterium is gone down to posterity, then it is added to fresh IEC-18 individual layer.Substratum is outwelled from the individual layer updip, added 0.1% Repone K, cell was hatched 10 minutes at 37 ℃ then.Remove Repone K, add SPG/10%, with the cell beater individual layer is disintegrated down then.Make lysis 3 times by syringe with 21 specifications (gauge) syringe needle.Remove nucleus centrifugal 5 minutes of 100 * g, the bacterial suspension in the supernatant liquor is added to 1 day fresh IEC-18 cell monolayer.

The infection of monitoring cell culture

With cold acetone/methyl alcohol with cell fixation chamber slide last 5 minute, monitoring is infected.Dye with immunofluorescence and immunoperoxidase method.Two kinds of methods all adopt mouse monoclonal antibody (as S.McOrist et al. described in the Vet.Rec.121:421422 (1987)) as first antibody, and anti--rat immune globulin G-fluorophore conjugated thing (isothiocyanic acid fluorescin; Organon Teknika Corporation, Durham, NC) or superoxide conjugate (goat-anti-rat immune globulin G; Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD).By in the cell on each slide by the counting of the number of bacteria of specific stain, finish quantitative analysis to bacterium.

Polymerase chain reaction

The sample inoculum and the bacterium of going down to posterity are merged as template DNA, be used to use PCR, wherein used sample preparation methods, primer and loop parameter such as Jones et al. are at J.Clin.Microbiol., and 31:261 1-2615 (1993) and McOrist etal. are (these two pieces of literature contents are all quoted as a reference) described in the Vet.Microbiol.1-8 (1994).First circulation is 93 ℃ in the loop parameter, 5 minutes; 55 ℃, 45 seconds; With 72 ℃ 45 seconds.33 circulations are respectively to carry out 45 seconds at each temperature above-mentioned, and a following circulation: 93 ℃, and 45 seconds; 55 ℃, 45 seconds; With 72 ℃ 2 minutes.Positive inoculum only is used to inoculate the IEC-18 cell.Also carry out the PCR monitoring and go down to posterity material to confirm infection.The DNA that produces with PCR is sent to Iowa S tate University Nucleic AcidFacility and checks order.Sequencing result and Gary F.Jones sequence that obtain, that report in its Ph D dissertation (Universityof Minnesota, Minneapolis, MN (in June, 1993)) are compared.

The result:

Select the inoculum sample:

Pig numbering N24912 and N72994 suffer from serious PPE, and the intestines inclusion of band blood and the mucous membrane that thickens are arranged.N101494 suffers from serious PPE and serious diarrhoea, causes forming in the intestines inner chamber big clot.The bacterium that the dyeing of the Giminez of mucous membrane smear is shown a large amount of bendings or S shape.There is the bacterium that sends hyperfluorescence in a large number in the IFA announcement of dyeing in from the inoculum of pig.

The infection of monitoring cell culture:

In process of growth, monitor the individual layer of being inoculated, and observe the variation slightly of cellular form by opticmicroscope.Than low oxygen concentration (8%O ₂) descend the not infection individual layer of growth to have similar form.

Shown that by the infection culture of immunofluorescence and immunoperoxidase staining the bacterium of a large amount of bendings or S shape is obviously arranged in cell.Individual layer does not converge infection.Cells infected usually closely links to each other, and the center is a 1-10 cell.See that also the cell that double infection is dyed (promptly with 30 or more bacterium) also links to each other with the cell of number less than 30 that carry disease germs.Number of bacteria reaches peak value about 6 days.Specific growth conditions is depended in infection.Bacterium is successfully gone down to posterity by cracking program described herein.New inoculating cell is carried out centrifugally not needing, but do the number that can increase cells infected like this.Centrifugally also can reduce contamination of heavy, be about to be exposed to the cell that in the antibiotic-free substratum, infects and put into again and contain antibiotic substratum 3 hours.Can breed higher number for thereby the speed of growth that reduces the IEC-18 cell makes bacterium before individual layer converges, it is necessary that the F CS concentration in the substratum is reduced to 7% from 10%.

Polymerase chain reaction:

Chromosomal DNA is carried out PCR, all isolates are all produced the fragment (comprising primer) of a 319bp.Sizeable fragment is compared with the positive that PCR produces by people such as naked eyes and known McOrist (1994).PCR product to N24912, N72994 and N101494 carries out sequential analysis, confirms that the p78 sequence that records with Jones (1993) has close homology (97-99%).

Embodiment 2

The growth of Lawsonia intracellularis in HEp-2 suspension culture of cells thing

The intestinal tissue homogenate that preparation is used to inoculate:

Scrape mucous membrane by 6.0-8.0 centimetre ileum from the intestines sample of embodiment 1 and prepare intestinal tissue homogenate.Add trypsin 1% to the mucous membrane that scrapes), sample carries out homogenizing tout court, hatches 35 minutes at 37 ℃ then.Add 10 milliliters of SPG/10%FBS again, sample grinds in tissue grinder.Add 10 milliliters of SPG/10%FBS again.Homogenate is filtered with Whatman 113V filter paper, filters on 5.0,1.0 and 0.65 microns filtering membranes subsequently.Filtrate is divided into 1.0 milliliters every part, is stored in-70 ℃.

The infection of cell culture

Method A:

Histocyte is with 1 * 10 ⁷Cell inoculation is in 100 milliliters of revolving bottles that contain 50 milliliters of DMEM/10%FBS.Culture was hatched 24 hours, added vancomycin and amphotericin B then.The homogenate of one bottle of refrigerated intestines is melted fast dilution in 3.0 milliliters of DMEM/5%FBS (containing vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml)).Sample adds in the bottle then by 0.65 μ m strainer.Culture is placed air vessel, and emptying charges into hydrogen and carbonic acid gas again and obtains the mixture that is made of 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.Culture was hatched 3 hours at 37 ℃, and then added Xin Meisu and gentamicin.Then in 24 hours, supply with the DMEM/5%FBS that culture contains L-glutaminate, vancomycin (100 μ g/ml), Xin Meisu (50 μ g/L), gentamicin (50 μ g/L) and amphotericin B (2.0 μ g/ml) again.

Method B:

With the HEp-2 cell with 1.25 * 10 ⁵Cell inoculation is in the 25cm that contains DMEM/10%FBS ²In the conventional bottle, hatched 18-24 hour.Cell degree of converging is 30% during inoculation.Inoculum dilutes in DMEM/5%FBS.When inoculum during from intestines homogenate, substratum also contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Culture is placed air vessel, and emptying charges into hydrogen and carbonic acid gas again and obtains the mixture that is made of 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.Culture was hatched 3 hours at 37 ℃, and then added Xin Meisu and gentamicin.Then in 24 hours, supply with the DMEM/5%FBS that culture contains L-glutaminate, vancomycin (100 μ g/ml), Xin Meisu (50 μ g/L), gentamicin (50 μ g/L) and amphotericin B (2.0 μ g/ml) again.When being pure growth, inoculum do not need microbiotic.Culture was hatched 6 days or until converging.Scrape cell and add to 100 milliliters of revolving bottles that contain 50 milliliters of DMEM/10%FBS from bottle.

Culture dilutes in 1: 2 ratio weekly, and this can perhaps add more substratum by culture being moved to bigger revolving bottle then by collecting half culture and adding fresh culture.

Going down to posterity of culture:

Fresh HEp-2 cell is pressed 1 * 10 ⁷Cell inoculation is in DMEM/5%FBS, thereby culture is passaged to fresh HEp-2 cell.New culture overnight incubation in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.New culture is then with the inoculation of infection culture and in above-mentioned hatching than under the low oxygen concentration.The quantity of inoculum depends on the gradient of infection of primary culture.

The results of culture and storage:

Collect the culture of desired number, at 3000 * g last centrifugal 20 minutes, thus gather in the crops culture.To precipitate resuspending in sucrose-phosphoric acid salt-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications (gauge) is 4 times then.With the culture break into portions, freezing at-70 ℃.For being further purified, sample can be under 145 * g centrifugal about 5 minutes to remove nucleus and fragment.Supernatant liquor under 3000 * g centrifugal 20 minutes, the throw out resuspending is in thinner.

Estimation to the Lawsonia intracellularis of living in the tissue culture:

To what live Lawsonia intracellularis quantitatively is by determining TCID's (50%) (TissueCultureInfectious Dose 50 percent, TCID ₅₀) finish.Way is: take out 2.0 milliliters of cultures to be tested, the syringe needle by 25 specifications (gauge) makes lysis 4 times.Sample carries out a series of 1: 10 dilutions in the DMEM/5%FBS that contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Diluent is added to 96 hole microtiter plates by 0.1 milliliter in every hole.Be inoculated in microtiter plate with the HEp-2 cell by 1250 cells/well, and before infection, grew 18-24 hour.Each weaker concn is used the 3-6 hole.Plate was hatched 6 days in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.With 50% cold acetone and 50% methyl alcohol fixed cell 2 minutes.Add 0.03 milliliter/hole and resist-IS intrace llularis monoclonal antibody (McOrist, 1994) in each hole, this antibody was diluted among the PBS by 1: 2000.Plate was hatched 30 minutes at 37 ℃, then with PBS washing 3 times.Add the anti--mouse FITC of dilution in 1: 30 by 0.03 milliliter/hole, hatched 30 minutes at 37 ℃.Plate is with distilled water washing 3 times, allows its drying.Use the fluorescence microscope sample, determine TCID ₅₀/ ml.

The result:

TCID ₅₀The result shows that culture can contain up to 1 * 10 ⁶Bacteria/milliliters.This realized in 45 days.Volume of culture is enlarged to 3.0 liters in the same time.

Embodiment 3

The growth of Lawsonia intracellularis in McCoys suspension culture of cells thing

The intestinal tissue homogenate that preparation is used to inoculate:

Press embodiment 2 described methods, the homogenate of preparation intestinal tissue.The Lawsonia intracellularis sample of cultivating with following embodiment method is according to budapest treaty, be preserved in American type culture collection (ATCC, 12301 Parklawn Drive, Rockville May 19 nineteen ninety-five, Maryland U.S.A 20852), preserving number is 55672.

The infection of cell culture

With the McCoys cell with 1.25 * 10 ⁵Cell inoculation is in two 25cm that contain DMEM/10%FBS ²In the conventional bottle, hatched 18-24 hour.Cell reaches 30% degree of converging when inoculation.Inoculum dilutes in DMEM/5%FBS.When inoculum during from intestines homogenate, substratum also contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Culture is placed air vessel, and emptying charges into hydrogen and carbonic acid gas again and obtains the mixture that is made of 8.0% oxygen, 10% carbonic acid gas and 82% hydrogen.Culture was hatched 3 hours at 37 ℃, and then added Xin Meisu and gentamicin.The DMEM/5%FBS that then will contain L-glutaminate, vancomycin (100 μ g/ml), Xin Meisu (50 μ g/L), gentamicin (50 μ g/L) and amphotericin B (2.0 μ g/ml) again in 24 hours supplies with culture.When being pure growth, inoculum do not need microbiotic.Culture is hatched 6 days until converging.Scrape cell and add to 100 milliliters of revolving bottles that contain 50 milliliters of DMEM/2%FBS and 0.05 gram Cultisphere-G microcarrier from bottle.Press the 40-50rpm blender jar.

Culture diluted in 1: 2 ratio every 2-3 days, and this can perhaps add more substratum and Cultisphere-G pearl by culture being moved to bigger revolving bottle then by collecting half culture and adding fresh culture and the Cultisphere-G pearl.The about 0.001 gram pearl/milliliter of the ultimate density of pearl in the culture.

Going down to posterity of culture:

Fresh McCoys cell is pressed 1 * 10 ⁷Cell inoculation restrains in the Cultisphere-G pearl in DMEM/5%FBS and 0.05, thereby culture is passaged to fresh McCoys cell.New culture overnight incubation in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.New culture infects the culture inoculation with 25 milliliters then, and in above-mentioned hatching than under the low oxygen concentration.

The results of culture and storage:

Collect the culture of desired number, at 3000 * g last centrifugal 20 minutes, thus gather in the crops culture.To precipitate resuspending in SPG, the syringe needle by 22 specifications is 4 times then.With the culture break into portions, freezing at-70 ℃.For being further purified, sample can be under 145 * g centrifugal about 5 minutes to remove pearl, nucleus and fragment.Supernatant liquor under 3000 * g centrifugal 20 minutes, the throw out resuspending is in thinner.

Estimation to the Lawsonia intracellularis of living in the tissue culture:

To quantitatively being undertaken by embodiment 2 described methods of the Lawsonia intracellularis of living, difference is to make lysis with the syringe needle of 22 specifications, and is inoculated in microtiter plate with the McCoys cell by 1250 cells/well.

The result:

TCID ₅₀The result shows that culture contains up to 1 * 10 ⁶Bacteria/milliliters.This is to be less than 1 month interior realization of time.Volume of culture is enlarged to 3.0 liters in the same time.

Embodiment 4

Determine the infective dose of Lawsonia intracellularis pure growth in host animal

Summary:

By using Lawsonia intracellularis pure growth to infect big or small common pig of 6 weeks, finished research to 31 pigs from sample N72994.These pigs are divided into 4 groups randomly, and each group is fenced up individually.The 1st group contains 7 pigs, and as negative control group (infect or do not infect with the tissue culture that does not infect).The 2nd group contains 8 pigs, and infective dose is 10 ⁷Bacterium/pig.The 3rd group contains 8 pigs, and infective dose is 10 ⁶Bacterium/pig.The 4th group contains 8 pigs, and infective dose is 10 ⁵Bacterium/pig.

Collect faecal samples at the 0th, 7,14,21 and 24 day and be used for the PCR test.At the 24th day, pig is dissected, to collect ileum, jejunum and colon and be used for PCR test, histopathological analysis and FA dyeing, method is as mentioned above.

Test discloses to the PCR of ileal mucous membrane, in high dose group 100%, in the middle dosage group 75% and low dose group in 50% have Lawsonia intracellularis.The histopathological analysis data presentation, in high dose group 88%, in the middle dosage group 75% and low dose group in 88% take place that the monocyte number increases in proper mucous membrane and the submucosa.In high dose group 50%, in the middle dosage group 63% and low dose group in 50% observe the crypts hyperplasia.The FA announcement of dyeing, in high dose group 88%, in the middle dosage group 63% and low dose group in 63% ileum, jejunum and colon's section have Lawsonia intracellularis.Control animal is dyed the negative findings that analysis all presents the Lawsonia intracellularis existence to PCR, FA and silver.

Generally speaking, pure growth can be successfully used to infect and cause PPE to damage.The hypothesis of Koch confirms by separating Lawsonia intracellularis and identified from infection animal.

In the animal of immune stimulating, 100% animal confirms recovery from illness and kind by argentation, FA and PCR in the high dose group.

Material and method:

The growth of inoculum:

With 3.75 * 10 ⁵The HEp-2 cell inoculation is in a 75cm who contains DMEM/10%FBS ²In the conventional bottle, under 5% carbonic acid gas and 37 ℃, hatched 18-24 hour.(cell degree of converging when inoculation is 30%).One bottle of N72994 is diluted in 15 milliliters of DMEM/5%FBS.Culture is placed air vessel, and emptying charges into hydrogen and carbonic acid gas again and obtains the mixture that is made of 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.In 24 hours, DMEM/5%FBS is supplied with culture again.

Culture was hatched 6 days, scraped cell and added to 100 milliliters of revolving bottles that contain 50 milliliters of DMEM/5%FBS from bottle then.Weekly by making culture volume double to enlarge the scale of culture in the bottle.Culture was grown for 3 weeks in revolving bottle.

The results of culture:

By gathering in the crops culture in centrifugal 20 minutes at 3000 * g.To precipitate resuspending in containing 10%FBS sucrose-phosphoric acid salt-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications (gauge) is 4 times then.Inoculum is diluted to final volume in SPG/10%FBS, and does dilution in 1: 10.

The inoculum that is used to contrast is diluted into and is infected the not infection HEp-2 cellularity of culture same concentrations by viable cell concentrations.Press and cells infected same procedure harvested cell.The pig of control group is accepted in the suitable dosage of high dose group.

Lawsonia intracellularis quantitatively:

To what live Lawsonia intracellularis quantitatively is by determining the 50% (TCID of TCID ₅₀) finish.Way is: take out 2 milliliters of cultures to be tested, the syringe needle by 22 specifications (gauge) makes lysis 4 times.Sample carries out a series of 1: 10 dilutions in the DMEM/5%FBS that contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Diluent is added to 96 hole microtiter plates by 0.1 milliliter in every hole.Be inoculated in microtiter plate with the HEp-2 cell by 2500 cells/well, and before infection, grew 18-24 hour.Each weaker concn is used 12 holes.Plate was being hatched 6 days under the gas concentration of 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.With 50% cold acetone and 50% methyl alcohol fixed cell 2 minutes.Add 0.03 milliliter/hole and resist-Lawsonia intracellularis monoclonal antibody (McOrist, 1987) in each hole, this antibody was diluted among the PBS by 1: 2000.Plate was hatched 30 minutes at 37 ℃, then with PBS washing 3 times.Add the anti--mouse FITC that presses dilution in 1: 30 by 0.03 milliliter/hole, hatched 30 minutes at 37 ℃.Plate is with distilled water washing 3 times, allows its drying.Use the fluorescence microscope sample, determine TCID ₅₀/ ml.

Animal:

The PIC x Lieske sow of 31 6 all sizes, various sexes and big Baigong pig have Kent doctor Schwartz to provide.At the 0th day, pig was dispensed in 4 swinerys by weight randomly.

Equipment:

Use one look down upon protect 4 swinerys settle pig, each swinery separates 3 feet at least.Swinery has wire system floor and solid barrier.With stove heat supply and local with heat release bulb complementary heating.In research process, temperature maintenance is between 78-85.

Feed and water

With not containing microbiotic, protein content is levigated corn-bean feed of 19%, optionally supplies by the stainless steel loader.Water is optionally supplied with by water supply nozzle.

The infection of pig:

At the 0th day, pig is weighed, and by capillary vessel at eye retro-orbital sinus (retroorbital sinus) blood sample collection.Collect serum, be stored in-20 ℃.Also collect faecal samples and be used for PCR.Use stomach tube, take 10 milliliters of inoculums to pig by mode in the stomach.

Processing mode	The pig number
		Contrast-non-infected cells	5
Contrast-be untreated	2
		High dosage	8
Middle dosage	8
		Low dosage	8

At the 0th, 10,17 and 24 day, pig is weighed and blood sample collection.

Polymerase chain reaction:

With the PCR of described loop parameter of Jones (1993) and primer, the infection of monitoring pig.Faecal samples and the intestinal mucosa collected at the 0th, 7,14,21 and 24 day are checked with PCR.

Histopathology:

The section formalin fixed of ileum, jejunum and colon with ordinary method processing, with phenodin and eosin dyeing and silver dipping, and is assessed.The monoclonal antibody dyeing special to Lawsonia intracellularis is also used in section.

The result:

Clinical symptom:

At 3 days, observe clinical symptom such as comprising rare stool in high dose group.In the time of 14 days, peak, alleviate to some extent subsequently.

The body weight gain situation:

Calculate the mean body weight growth pattern of every day, result's demonstration is compared with control group, and the body weight gain of high dosage and middle dosage group reduces.When respectively organizing, the dosage effect of tiring is arranged in body weight gain.

PCR：

Observe dripping of ight soil up to 14 talentes.At 21 days, 37.5% ight soil was the PCR positive in the pig of high dose group.After the dissection, ileal mucous membrane checks that with PCR positive rate is: high dose group 100%, middle dosage group 75%, low dose group 50% and control group 0%.

Major injury:

Find that in high dose group 2 pigs have major injury (#50 and #202).In ileum, have an appointment 3 feet thickening of these 2 pigs, and in #202, occur downright bad.

Histopathology:

FA：

The FA dyeing of the section of ileum, jejunum and colon discloses: in high dose group 87.5%, have Lawsonia intracellularis in middle dosage group and low dose group 62.5% and the control group 0%.

Micro-damage:

In high dose group 100%, middle dosage group 75% is observed damage in low dose group 87.5% and the control group 14%.This increases by monocyte number in observation proper mucous membrane and the submucosa determines that this situation is usually relevant with Peyer ' s Patchers hyperplasia.Also observe the crypts hyperplasia.

Silver dyes:

Also silver has been carried out in section and dyed to check whether have intracellular, crooked bacterium.The result shows that in high dose group 87.5%, there is bacterium in middle dosage group 62.5% in low dose group 87.5% and the control group 0%.

Discuss:

With pure Lawsonia intracellularis culture infected pigs successfully.10 ⁷Under the bacterium of dosage, determine that with PCR and microscopic examination damage 100% pig is infected.Severity of damaging in tissue slice and bacterial number are quite lower.This research is gratifying Lawsonia intracellularis immune stimulating model, because have Lawsonia intracellularis and micro-damage is arranged in pig.Behind first time dosage, give second dosage again in 7 days, can increase damage.

Embodiment 5

Hamster vaccine efficiency assay

Purpose:

Assessment laboratory animal model is to determine security and the validity of avirulent Lawsonia intracellularis vaccine alive in hamster.

Summary:

With the Lawsonia intracellularis bacterial strain of high passage number 3 week of pure growth inoculation size hamsters, carry out immune stimulating at back 22 days materials that virulence is arranged of inoculation then, thereby finish research 40 hamsters with the generation number of passing at the low.Hamster is divided into 3 groups.The A group was inoculated with 1 dosage Lawsonia intracellularis bacterial strain N72994 at the 0th day.The B group is group in contrast, does not use the vaccine culture.After inoculation 22 and 25 days, excite these two groups with the Lawsonia intracellularis bacterial strain N343 pure growth of 2 dosage.The C group is used and is excited bacterial strain N101494 so that the virulence more relative with bacterial strain N343.A and B group respectively have 15 hamsters, and the C group has 10 hamsters.50% (the TCID of TCID ₅₀) the data announcement, with 10 ⁵TCID ₅₀/ potion inoculation hamster.The N343 immune stimulating contains 10 ⁵.5TCID ₅₀/ potion.The booster dose of C group is 10 ².75 TCID ₅₀/ potion.Collect faecal samples at the 0th, 7,14,21,29,36 and 43 day and be used for the PCR test.At the 5th day, A group and B group are dissected 5 animals, be used for FA, phenodin and eosin dyeing and silver that PCR checks mucous membrane and carry out the intestines section and dye, to determine the persistence of bacterial colonization in the hamster that inoculates.Similar test is dissected and carried out to the residue animal exciting back 21 days.

In inoculation back 21 days, there is Lawsonia intracellularis in the PCR data presentation on the intestinal mucosa of A group hamster 100%.The B group all was negative in inoculation in back 21 days.Exciting back 21 days, 50% is the PCR positive in the hamster of A group, and 100% is positive in the hamster of B group.The histopathological analysis of section shows, is exciting back 21 days, and slight extremely serious damage is arranged in 50% animal of A group, in 50% animal of B group the minor injury is arranged.There is not animal to show damage in 21 days in inoculation.The C treated animal is exciting back 21 days less than damage.FA and silver dye all can not be presented at and have Lawsonia intracellularis in the section.

In a word, PCR shows, during with the Lawsonia intracellularis inoculation hamster in high pass generation, can be observed to infect and descends 50%.Intestines are moved life by biological (referring to Lawsonia intracellularis) in a small amount of born of the same parents, do not observe this organism in the section because dye at FA and silver.C group hamster does not show infection in whole research process, this is likely because bacterium dosage is low causes.

Material and method:

The situation of hamster:

Use is from the female hamster of 40 3 all sizes of Harlan Sprague Dawley.

The growth of inoculum:

The vaccine culture:

In the HEp-2 cell, the grow Lawsonia intracellularis continuous culture in 29 weeks of use.This culture is by exciting the similar fashion described in the culture chapters and sections to grow, and difference is: every 2-3 week is passaged to new HEp-2 cell with culture.

Excite culture:

With 3.75 * 10 ⁵The McCoys cell inoculation in the 75cm of the DMEM substratum that contains 10% foetal calf serum (FBS) (Dulbecco ' s Modified Eagle ' s substratum) ²In the conventional organization culturing bottle, under 37 ℃ and 5% carbonic acid gas, hatched 18-24 hour.Substratum and cell are separated, be diluted in 14 milliliters of N343 MSC X among the DMEM/2%FBS with one bottle again and add in the bottle.Culture is placed air vessel, and emptying charges into hydrogen and carbonic acid gas again and obtains the mixture that is made of 8.0% oxygen, 8.8% carbonic acid gas and 83.2% hydrogen.Culture growth 6 days scrapes cell from bottle and adds to 100 milliliters of revolving bottles that contain 90 milliliters of DMEM/2%FBS and 0.01 gram Cultispere-G pearl.Culture is grown under above-mentioned gas concentration.Weekly by making culture volume double to enlarge the flask culture volume.Culture in revolving bottle, grow 25 days be 250 milliliters until final volume.

Bacterial strain N10494 is by growing with bacterial strain N343 same way as.

The results culture:

The vaccine culture:

By gathering in the crops culture in centrifugal 20 minutes at 3000 * g.Will the precipitation resuspending in the sucrose-phosphoric acid salt that contains 10%FBS-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications (gauge) is 4 times then.Inoculum is diluted to final volume (15 milliliters) in SPG/10%FBS.

Excite culture:

By gathering in the crops culture in centrifugal 20 minutes at 3000 * g.Will the precipitation resuspending in the sucrose-phosphoric acid salt that contains 10%FBS-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications (gauge) is 4 times then.Inoculum is diluted to final volume (the N343 bacterial strain is 20 milliliters, and the N101494 bacterial strain is 10 milliliters) in SPG/10%FBS.

The dosage of hamster:

Vaccine:

At the 0th day, all hamsters of A group are the vaccine of 1 milliliter of preparation of oral vaccination all.

Excite:

In inoculation back 21 days, 10 hamsters of A group and 10 hamsters of B group excited culture bacterial strain N343 for oral 0.5 milliliter.The C group excites culture bacterial strain N101494 to carry out immune stimulating with 0.5 milliliter.

ISi's is quantitative:

To what live ISi quantitatively is by determining 50% TCID (Tissue culture InfectiousDose 50 percent, TCID ₅₀) finish.Way is: take out 2 milliliters of cultures to be tested, the syringe needle by 22 specifications makes lysis 4 times.Sample carries out continuous 1: 10 dilution in the DMEM/5%FBS that contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Diluent is added to 96 hole microtiter plates by 0.1 milliliter in every hole.Before infection, this plate with the McCoys cell by the inoculation of 1250 cells/well, and under 37 ℃ and 5% carbonic acid gas, grew 18-24 hour.Each weaker concn is used 12 holes.Plate was hatched 6 days in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% nitrogen.With 50% cold acetone and 50% methyl alcohol fixed cell 2 minutes.Add 0.03 milliliter/hole and resist-the ISi monoclonal antibody in each hole, this antibody was diluted among the PBS by 1: 2000.Plate was hatched 30 minutes at 37 ℃, then with PBS washing 3 times.Add the anti--mouse FITC of dilution in 1: 30 by 0.03 milliliter/hole, hatched 30 minutes at 37 ℃.Plate is with distilled water washing 3 times, allows its drying.Use the fluorescence microscope sample, determine TCID ₅₀/ ml.

The monitoring that hamster infects:

With the PCR of described loop parameter of Gary Jones and primer, the infection of monitoring hamster.The the 0th, 7,14,21,29,36 and 43 day collection faecal samples after inoculation.After killing hamster, its intestinal mucosa is also checked with PCR.

Histopathology:

The section formalin fixed of ileum and colon with ordinary method processing, with phenodin and eosin dyeing and silver dipping, and is assessed.The monoclonal antibody dyeing special to Lawsonia intracellularis is also used in section.

Average every day the weight increase situation:

After inoculation, hamster was weighed in 21,28,35 and 42 days, to determine average every day of weight increase situation.

The result:

See table.

TCID ₅₀：

TCID ₅₀Data presentation, vaccine group (A group) accepts 10 ⁴.86 TCID ₅₀/ hamster.The hamster of A group and B group excites with bacterial strain N343, and receiving amount is 10 ⁵.5 TCID ₅₀/ hamster.The receiving amount of the C group hamster that excites with bacterial strain N101494 is 10 ².75 TCID ₅₀/ hamster.

PCR：

PCR tests demonstration, and 100% exists Lawsonia intracellularis in the hamster of the inoculation that inoculation was dissected in back 21 days.Show that in the test of inoculation after 43 days contrast hamster 100% and the hamster that inoculated 50% are infected by Lawsonia intracellularis.There is not one to be the PCR positive in the hamster that excites with N101494.Do not observe the situation of movement drippage in to the research of hamster at this.

Histopathology:

Phenodin and eosin dyeing (H﹠amp; E dyeing) disclose, in all sections of the hamster that inoculation was dissected in back 21 days, do not have tissue injury.In the section that inoculation is collected after back 43 days, 50% of vaccine group has slight to serious lymphocyte enteritis, and control group 50% has slight lymphocyte enteritis.Excite discovery damage in the group at N101494.

FA dyeing can not be presented in the back 43 days hamster of inoculation and have Lawsonia intracellularis.

Discuss:

PCR shows, in the hamster with the Lawsonia intracellularis inoculation in high pass generation, observes infection rate and descends 50%.

Movement movement movement movement movement movement movement mucous membrane micro-damage

Mucous membrane PCR H﹠amp; The E FA that dyes

PCR PCR PCR PCR PCR PCR PCR PCR silver dyes

ID 0 day 7 days 14 days 21 days 29 days 36 days 43 days 21 days 43 days

A-1-------NA+slight enteritis--

A-2 - - - - - - - NA - - - -

A-3-------NA+slight enteritis--

A-4 - - - - - - - NA - - - -

A-5 - - - - - - - NA + - - -

A-6 - - - - - - - NA - - - -

A-7-------NA+hyperenteritis--

A-8-------NA+moderate enteritis--

A-9 - - - - - - - NA - - - -

A-10-------NA-slight enteritis--

A-11 - - - - NA NA NA + NA - - -

A-12 - - - - NA NA NA + NA - - -

A-13 - - - - NA NA NA + NA - - -

A-14 - - - - NA NA NA + NA - - -

A-15 - - - - NA NA NA + NA - - -

B-1-------NA+minimum level enteritis--

B-2-------NA+minimum level enteritis--

B-3 - - - - - - - NA + - - -

B-4-------NA+minimum level enteritis--

B-5-------NA+minimum level enteritis--

B-6-------NA+minimum level enteritis--

B-7 - - - - - - - NA + - - -

B-8 - - - - - - - NA + - - -

B-9 - - - - - - - NA + - - -

B-10 - - - - - - - NA + - - -

B-11 - - - - NA NA NA - NA - - -

B-12 - - - - NA NA NA - NA - - -

B-13 - - - - NA NA NA - NA - - -

B-14 - - - - NA NA NA - NA - - -

B-15 - - - - NA NA NA - NA - - -

C-1 - - - - - - - NA - - - -

C-2 - - - - - - - NA - - - -

C-3 - - - - - - - NA - - - -

C-4 - - - - - - - NA - - - -

C-5 - - - - - - - NA - - - -

C-6 - - - - - - - NA - - - -

C-7 - - - - - - - NA - - - -

C-8 - - - - - - - NA - - - -

C-9 - - - - - - - NA - - - -

C-10 - - - - - - - NA - - - -

Embodiment 6

The pig vaccine efficiency assay

Purpose:

The purpose of this research is the security in the pig of 2-3 week size of the isolate avirulent alive of assessment Lawsonia intracellularis and the isolate killed, moves living persistence and validity.Carry out host animal research, in this research, the pig in 3 ages in week of inoculation has the immune stimulating of virulence with it with Lawsonia intracellularis bacterial strain N343, then to compare the difference of protecting between vaccine.

Method

In December 11 nineteen ninety-five, from H﹠amp; K buys on the farm pig that amounts to 45 3 ages in week.They are transported to Veterinary Resources, Inc., and this is one and is positioned at Cambridge, the research institution of Iowa, here pig is marked to distinguish every pig.Before studying, pig is closed 2 days so that conform in this mechanism, and to the pig nursing in research process is the feed of antibiotic-free.

December 13, all pigs were all weighed, draw blood and gather serum, clinical marking and collect the rectum sample for testing.Pig is divided into 5 groups randomly, and places basin.20 pigs are placed in an independent room, with comparing and strict control group.15 pigs are placed in second room, carry out the ISi-1 vaccine test.10 pigs are arranged between three rooms, be used for the ISi-2 vaccine test.

Living vaccine is prepared in NOBL Laboratories Research and Development mechanism, is called as series of trials ISi-1.ISi-1 (bacterial strain N343) isolates from pig and 29 weeks of continuous growth in pure growth.On the McCoys cell of vaccine in revolving bottle, grow down until observing about 100% infection in low oxygen concentration.The high pass that is used for ISi-1 is for N343 bacterial strain revolving bottle went down to posterity for 11 weeks (" N343NP40wk ") again, according to budapest treaty, be preserved in American type culture collection (ATCC on May 22nd, 1996,12301 ParklawnDrive, Rockville, Maryland U.S.A 20 852), preserving number is 55783.By gathering in the crops culture in last centrifugal 20 minutes at 3000 * g.To precipitate resuspending in sucrose-phosphoric acid salt-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications is 4 times then.Lysate formation in centrifugal about 5 minutes under 500 * g precipitates to remove fragment and microcarrier bead.Collect supernatant liquor, be stored in-70 ℃, just took out in preceding 1 hour, place on ice for using up to inoculation.

The vaccine of deactivation (ISi-2) is grown, is gone down to posterity by above-mentioned similar fashion and also gathered in the crops in 12.5 weeks, carries out purifying with the percol gradient then.The bacterium of purifying then is stored in-70 ℃, just took out in preceding 1 hour up to inoculation, with its be stored at 4 ℃ with general atmosphericoxygen concentration under, this oxygen concn is virose to Lawsonia intracellularis.Aluminium hydroxide is added bacterium to final mixture contain 10% aluminium hydroxide.Measure protein concn with the Biurett method.

The quantitative of ISi lives:

To what live Lawsonia intracellularis quantitatively is by determining the 50% (TCID of TCID ₅₀) finish.Way is: before infection, 96 hole microtiter plates, were grown 18-24 hour by the inoculation of 1250 cells/well then with the McCoys cell.Sample carries out continuous 1: 10 dilution in the DMEM/5%FBS that contains vancomycin (100 μ g/ml) and amphotericin B (2.0 μ g/ml).Diluent is added to 96 hole microtiter plates by 0.1 milliliter in every hole.Each weaker concn is used 12 holes.Plate was hatched 6 days in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% nitrogen in 37 ℃.With 50% cold acetone and 50% methyl alcohol fixed cell 2 minutes.Add 0.03 milliliter/hole and resist-Lawsonia intracellularis monoclonal antibody (by Dr.Steven McOrist exploitation) in each hole, this antibody was diluted among the PBS by 1: 2000.Plate was hatched 30 minutes at 37 ℃, then with PBS washing 3 times.Add the anti--rat immune globulin G-fluorophore conjugated thing (FITC) of dilution in 1: 30 by 0.03 milliliter/hole, hatched 30 minutes at 37 ℃.Plate is with distilled water washing 3 times, allows its drying.Use the fluorescence microscope sample, determine TCID with the Reed-Meunsch computing method ₅₀/ ml.

TCID ₅₀Data presentation, ISi-1 has 1.8 * 10 ⁵Bacteria/milliliters.The 4th kind of inoculum is placebo, the tissue culture cells that it is used by oneself and the vaccine same way as is processed.

Measuring the total protein content of the vaccine of deactivation with the Biurret method, is 0.311 mg/ml.

Inoculate to pig in December 13 nineteen ninety-five.The dosage of living vaccine is 2 milliliters of IN, 1 milliliter in each nostril.ISi-2 (deactivation) vaccine is used with muscle injection mode, is 1.5 milliliters/every pig, and carries out once after 14 days again.All control animals are all pressed with the living vaccine same way as and use the cell that does not infect.

Observe and sample:

In research process, collected faecal samples and serum every 7 days.Faecal samples is carried out PCR test with DNA amplification, and the primer is to being: 5 '-TATGG CTGTC AAACA CTCCG-3 ' and be: 5 '-TGAAG GTATT GGTATTCTCC-3 '.First circulation of round-robin is 93 ℃, 5 minutes; 55 ℃, 45 seconds; With 72 ℃ 45 seconds.33 circulations are respectively to carry out 45 seconds at each temperature above-mentioned.Last circulation is: 93 ℃, and 45 seconds; 55 ℃, 45 seconds; With 72 ℃ 2 minutes.Primer is determined by people such as Jones.

Immune stimulating:

All animals except strictness contrast, all excited culture in 26 and 27 days after inoculation, this excites culture to be made of the low subculture (continuous growth 8-12 week) of Lawsonia intracellularis bacterial strain N343 and N72994.By gathering in the crops culture in last centrifugal 20 minutes at 3000 * g.Will the precipitation resuspending in the sucrose-phosphoric acid salt that contains 10% foetal calf serum-L-glutamic acid (SPG) solution, the syringe needle by 25 specifications is 4 times then.The culture of part results is stored in-70 ℃ and excites up to being used to, and other continued growths are up to immune stimulating same day, results then.The mixed activation inoculum, the TCID of mensuration culture ₅₀Sample storage is on ice for using.

The culture that excites on January 8th, 96 contains 4 * 10 ⁴Bacteria/milliliters, and the culture that excites on January 9th, 96 contains 3 * 10 ⁴Bacteria/milliliters.All used 15 milliliters to pig in these two days and excite vaccine by gastric lavage.Therefore, animal accepts 6 * 10 respectively on January 8th, 96 and on January 9th, 96 ⁵Bacterium/pig and 4.7 * 10 ⁵Bacterium/pig.

The result:

Security:

Movement PCR result: detect Lawsonia intracellularis with PCR and show, do not have pig when the research beginning, to drain bacterium.In inoculation back 7 days, all pigs were negative.In inoculation back 14 days, 3 pigs were positive in the ISi-1 group.In inoculation back 21 days, 2 pigs were positive in the ISi-1 group, and other all pigs are negative.Detect demonstration with PCR, do not have animal to drain bacterium in back 26 days in inoculation.In inoculation back 26 days, dissect 5 pigs in the ISi-1 group and 4 pigs in the ISi-2 group.The sample of collecting is that ileum, colon, intestines are lymphoglandula and tonsilla and lung sample (from the pig that has the pneumonia damage).

Each ileum and lung sample are carried out the PCR test.Mix tonsilla, colon and lymphoglandula, the performing PCR of going forward side by side by each treatment group.PCR result is as follows.

Group	Inoculate after 26 days ileum PCR	The blended colon	The blended tonsilla	The blended intestines are lymphoglandula	The blended lung
						ISi-1	In 51	+	-	-	In 11
ISi-2	In 40	-	-	-	In 10
						Contrast	In 50	-	-		Not test
Strict contrast	Not test	Not test	Not test	Not test	Not test

The ileum tissue slice is dyeed, wherein use the special monoclonal antibody of Lawsonia intracellularis, use anti--rat immune globulin G-fluorophore conjugated thing as second antibody as first antibody.In 5 pigs, there are 3 to observe Lawsonia intracellularis from ISi-1.Detect with fluorescent antibody staining, other all pigs all are negative.

Remaining pig is exciting dissection in back 21 days, collects identical sample and is used for assessment.PCR result is as follows.

Group	After exciting 26 days, ileum PCR	The blended colon	The blended tonsilla	The blended intestines are lymphoglandula	The blended lung
						ISi-1	In 10 0	+	-	-	-
ISi-2	In 60	-	-	-	-
						Contrast	In 10 4	-	-		-
Strict contrast	In 50	-	-	-	-

As above ileum is carried out FA dyeing, in 10 animals of control group, have 7 positive.For whether having Lawsonia intracellularis, other all animals all are negative.

Also tested serum, to determine that pig is exposed to the IgG antibody production that produces behind the Lawsonia intracellularis.This test is performed such: before infection, the McCoys cell is inoculated in the Terasak i plate of having handled with tissue culture by 1250 cells/well, grew then 18-24 hour.The Lawsonia intracellularis pure growth is diluted to the 1000-3000 bacteria/milliliters in the DMEM that contains 5% foetal calf serum, adds in each hole for 0.1 milliliter by every hole.Plate was hatched 6 days in 8.0% oxygen, 8.8% carbonic acid gas and 83.2% nitrogen.With 50% cold acetone and 50% methyl alcohol fixed cell 2 minutes.Porcine blood serum is by being diluted in aseptic P BS at 1: 75.The serum of dilution is added in each hole for 0.01 milliliter by every hole.Plate was hatched 30-60 minute at 37 ℃, then with aseptic PBS washing 5 times.Add anti--PIG-fluorophore conjugated thing by 0.01 milliliter/hole to each hole, plate was hatched 30 minutes at 37 ℃.Plate is with distilled water washing 5 times, allows its drying.Sample is with distilled water washing 5 times, allows its drying.Use the fluorescence microscope sample, the hole of observing bacterium is designated as the positive, and the hole of not observing bacterium is designated as feminine gender.

The result:

Group	The 0th day	Inoculate back 26 days	Inoculate back 47 days, excite back 21 days
				ISi-1	In 15 0	In 15 6	In 10 8
ISi-2	In 10 0	In 10 3	In 65
				Contrast	In 15 1	In 15 0	In 10 9
Strict contrast	In 50	In 50	In 50

The animal of being positive at the 0th day is test once more weekly.The result shows, is negative at back 14 days serum of inoculation.This is among expecting because in the time of the 0th day pig only 3 week size, the positive under this age may be that the antibody by parent causes.

Used positive control serum is to obtain by being used in the Lawsonia intracellularis high dosage ground immune swine of growing on the pure growth during test sera.The negative control sera that uses is to gather from the aseptic pig of South Dakota State University.

Foregoing description and embodiment only are used to set forth the preferred example that can realize the object of the invention, feature and advantage, unintelligiblely are confined to this for the present invention.Of the present invention any change form within appended claims purport and scope all is considered to a part of the present invention.

Claims (10)

1. in the McCoy cell, cultivate the method for Lawsonia intracellularis (L.intracellularis) bacterium, it is characterized in that described method comprises:

A) infect the McCoy cell with Lawsonia intracellularis;

B) be less than the McCoy cell of cultivating described infection under 18% the condition at oxygen concn.

2. the method for claim 1 is characterized in that, cultivates described McCoy cell with adherent form.

3. method as claimed in claim 2 is characterized in that, is to infect described McCoy cell with Lawsonia intracellularis at 30% o'clock at the degree of converging of described McCoy cell.

4. the method for claim 1 is characterized in that, with described McCoy cell suspension culture.

5. as each described method among the claim 1-4, it is characterized in that, in oxygen concn is the scope of 0-8%, cultivate described McCoy cell.

6. as each described method among the claim 1-4, it is characterized in that, in the mixture of 8% oxygen, 10% carbonic acid gas and 82% hydrogen, cultivate the cell of described infection.

7. as each described method among the claim 1-4, it is characterized in that described method also comprises the McCoy cell of the infection of collecting the Lawsonia intracellularis form.

The purposes during 8.McCoy cell is cultivated in the born of the same parents of Lawsonia intracellularis (L.intracellularis).

9. purposes as claimed in claim 8 is characterized in that, infects described McCoy cell with Lawsonia intracellularis, is less than the McCoy cell of cultivating described infection under 18% the condition at oxygen concn.

10. produce the method for the vaccine that contains Lawsonia intracellularis (L.intracellularis) and pharmaceutically acceptable carrier, wherein said Lawsonia intracellularis is cultivated according to each described method among the claim 1-7.