HK1067662A1 - 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

Lawsonia intracellularis culture, vaccine and diagnostic reagent against the same

This divisional application is a divisional application of Chinese patent application CN 96114542.0 entitled "Lawsonia intracellularis culture, vaccine and diagnostic reagent against the same".

Technical Field

The present invention relates to vaccines against lawsonia intracellularis (lawsonia intracellularis) and methods for preventing and treating and diagnosing LI infection. The products and methods of the present invention are in part the result of our discovery of improved methods for large scale culture of L.intracellularis.

Background

Lawsonia intracellularis is a causative agent of porcine proliferative bowel disease (PPE) and affects almost all animals, including humans, rabbits, ferrets, hamsters, foxes, horses, and a variety of other different animals such as ostriches and llamas, among others.

Lawsonia intracellularis is a particularly important cause of loss in swine herds. Estimates of incidence and infection rates for PPE in the united states are that up to 20% of swine herds are infected and lose $2000 million per year.

One fixed feature of PPE is the presence of cytoplasmic, non-membrane bound campylobacter bacteria in the intestinal cells of the infected intestinal part. The PPE related bacteria have been called "Campylobacter-like organisms" (S.McOrist et al, vet.Pathol., Vol.26, 260-64 (1989)). Subsequently, the pathogenic bacteria were identified as a new taxonomic genus and species, known in the national language as "ilealsymbiont (is) intracellularis" (ISi). Gebhart et al, Int' l.J.of systematic Bacteriology, Vol.43, No.3, 533-38 (1993). Recently, these new bacteria were assigned taxonomic names: lawsonia (L.) intracellularis. McOrist et al, Int' lJ. of systematic Bateriology, Vol.45, No.4, 820-25 (1995). These 3 names are used interchangeably and all refer to the same organism as described and further identified herein. We endeavored to use the taxonomic name lawsonia intracellularis (l.

Lawsonia intracellularis is an obligate, intracellular bacterium that cannot be cultured in conventional cell-free media using conventional bacteriological methods and has been thought to require attachment to epithelial cells for growth. McOrist et al, in Infection and Immunity, Vol.61, No.10, 4286-92(1993) and G.Lawson et al, in J.of Clinical Microbiology, Vol.31, No 5, 1136-42(1993), culture of L.intracellularis in conventional tissue culture flasks with intestinal epithelial cell monolayers from IEC-18 rats. Stills, in Infection and Immunity, Vol.59, No.9, 3227-36(1991), also discussed using intestinal 407 human embryonic intestinal cell monolayers and GPC-16 guinea pig colon adenocarcinoma cell monolayers in conventional tissue culture flasks. These existing culture methods are laborious and not suitable for large-scale culture.

Because of the harsh growth conditions required to culture L.intracellularis in vitro, current knowledge of L.intracellularis infection, as well as treatment and effective control of the disease, is hampered. There is therefore a need for an improved method of culturing L.intracellularis. There is also a need for a vaccine against L.intracellularis and an effective tool for diagnosing L.intracellularis infections.

Disclosure of Invention

It is an object of the present invention to provide a vaccine against L.intracellularis.

It is another object of the present invention to provide a method for detecting the presence or absence of anti-L.intracellularis antibodies in a biological sample.

It is another object of the present invention to provide an improved culture process which enables the large-scale culture of L.intracellularis for the production of vaccines and diagnostic reagents.

To achieve these and other objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention provides a method of culturing lawsonia intracellularis and a large amount of bacterial feedstock obtained by the method. According to the method, the L.intracellularis bacteria are incubated at an oxygen concentration of about 0-18% while agitating the bacteria to culture the L.intracellularis and maintain the bacteria in suspension.

According to another example, there is provided a method of culturing a Lawsonia intracellularis bacterium by: HEp-2, McCoys or IEC-18 cell monolayers (with a confluency of approximately 30%) were inoculated with an inoculum containing L.intracellularis bacteria to infect the cells. The infected cells are then incubated at a temperature of about 36-38 ℃ and an oxygen concentration of about 0-8.0% until the cells are confluent. The infected cells and growth medium are then placed in a fermentor, bioreactor, spinner flask (spinner flash) or other vessel suitable for maintaining the cells in suspension. Infected cells the cells should be agitated during incubation in order to culture the L.intracellularis bacteria and maintain the infected cells in suspension. A portion of the cultured L.intracellularis is then passaged to fresh cultured cells to increase the yield of L.intracellularis bacteria.

The present invention provides vaccines against L.intracellularis and methods of producing vaccines against L.intracellularis. The avirulent L.intracellularis bacterium is produced by passaging the cultured L.intracellularis bacterium a sufficient number of times and selecting an attenuated strain, or by chemically attenuating the cultured bacterium. Inactivated lawsonia intracellularis vaccines can also be prepared using the culture method of the invention. According to a particularly preferred embodiment, the bacteria are cultured continuously for at least 6-8 months, while being passaged at least about 7-12 times to produce attenuated strains useful as vaccines. The attenuated bacteria are then mixed with a pharmaceutically acceptable carrier and administered to an animal in an amount effective to generate an immune response. We have deposited the presently preferred attenuated strain (N343NP40wk) at the American Type Culture Collection (ATCC).

The present invention also provides a method of determining the presence or absence of an antibody specifically reactive with a lawsonia intracellularis bacterium in a biological sample by: harvesting at least a portion of the cultured L.intracellularis bacteria, contacting the harvested L.intracellularis bacteria or components thereof with a biological sample from the animal under conditions in which antibodies present in the biological sample will react with the L.intracellularis bacteria or components, and determining whether an antibody-antigen reaction has occurred.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

As used herein, the term "L.intracellularis" refers to intracellular, curved, gram-negative bacteria as described in detail by C.Gebhart et al, Int 'l.J.of SystemicBacteriology, Vol.43, No.3, 533-38(1993) and S.McOrist et al, Int' l J.of systemicBateriology, Vol.45, No.4, 820-25(1995) (the entire contents of both of which are incorporated herein by reference), including but not limited to: ATCC 55672 bacteria deposited at the American type culture Collection (ATCC, Rockville, Md.); NCTC 12656 and 12657 bacteria deposited at the national center for type culture Collection (NCTC, Colindale, London); pathogenic bacteria that may be obtained from pigs or other animals infected worldwide with PPE, in accordance with the knowledge of the art and the teachings herein; and spontaneous or artificially mutagenized mutants or variants of any of the above bacteria.

As used herein, the term "attenuated strain" refers to any strain of lawsonia intracellularis that has been prepared by the culture and passaging techniques taught herein so as to be avirulent while maintaining its immunogenic characteristics when administered to a host animal. As described below, according to the present invention, various L.intracellularis strains are cultured and attenuated, thereby obtaining attenuated immunogenic strains. These attenuated strains are effective as vaccines for swine or other animals susceptible to lawsonia intracellularis infection.

The attenuated strains of the present invention are expected to be useful as immunogens in antimicrobial vaccines for animals, including birds, fish, cattle, pigs, horses, mammals, and primates in general, as well as humans. After obtaining the teachings herein, these vaccines can be prepared using techniques known to those skilled in the art. Such vaccines can comprise an immunologically effective amount of the attenuated strain and a pharmaceutically acceptable carrier. The vaccine may be administered in a single dose or in multiple doses. Based on the teachings herein, an immunologically effective amount can be determined by methods known in the art without undue experimentation. The number of avirulent bacteria should be sufficient to elicit an immune response in a susceptible animal while still being avirulent. Depending on the particular animal, bacteria and disease involved. The recommended dosage for administration to susceptible animals is preferably about 10³-10⁹Bacteria/kg body weight, most preferably about 10⁵-10⁷Bacteria per kilogram body weight. Carriers are well known to those skilled in the art and include stabilizers and diluents. Such vaccines may also contain suitable adjuvants. The vaccine of the invention may be used in combination with other vaccines, for example a diluent of another freeze-dried vaccine, or with other vaccines prior to freeze-drying. Vaccine formulations may also be dried, e.g., lyophilized, for storage or for later formulation into a liquid vaccine.

Accordingly, the present invention also includes a method of inducing an immune response in an animal host against a virulent, wild-type L.intracellularis bacterium, thereby protecting the host from the bacterium. Such a method comprises: an immunologically effective amount of an attenuated bacterium or killed bacterium of the invention is administered to a host, preferably a vaccine of the invention is administered to a host.

As used herein, the term "large-scale culture" refers to: l.intracellularis is cultured at levels greater than about 2.0-3.0 liters and includes production scales greater than 100 liters or greater. As used herein, "culturing" refers to a process that promotes the growth, regeneration, and/or proliferation of lawsonia intracellularis.

In practicing the culture method of the present invention, the cultured cells are first inoculated with an inoculum containing L.intracellularis bacteria to allow the bacteria to infect the cells. A variety of cell lines can be used in the practice of the present invention, including but not limited to: IEC-18(ATCC 1589) -murine intestinal epithelial cells, HEp-2(ATCC 23) -human epidermoid tumor cells, McCoys (ATCC1696) -murine (non-specific) cells, MDCK (ATCC 34) -Madin-Darby dog kidney cells, BGMK (Biowhittaker #71-176) -Bufaro green monkey kidney cells and porcine intestinal epithelial cells. Preferred cultured cells are HEp-2, McCoys or IEC-18 cells. Alternatively, the bacteria may be cultured in a cell-free system, so long as the bacteria are maintained at a suitable dissolved oxygen concentration as taught herein.

If cultured cells are used, the cells are preferably (but not necessarily) in monolayer form prior to seeding. To form a monolayer, the cells can be seeded into conventional flasks. The typical inoculum size per vial was about 1X 10⁵-10×10⁵Cells/25 cm square flask and mixed with growth medium. The growth medium may be any medium used in cell culture and contains a nitrogen source, selected growth factors required to culture the cells, and a carbon source such as glucose or lactose. The preferred medium is DMEM supplemented with 2-5% fetal bovine serum, although various other commercially available media may be used with good results.

We have found that L.intracellularis can be cultured more successfully by maintaining the cultured cells in a stable growth state. Therefore, the culture cell monolayer should have about 20-50% confluency at the time of seeding. More preferably, the cell confluence at the time of seeding should be about 30-40%, with about 30% being the most preferred confluence.

The inoculum may be a pure L.intracellularis culture, such as from ATCC accession No. 55672, NCTC accession No. 12656 or 12657, or a pure culture obtained from an infected pig or other animal using the isolation and purification techniques described herein.

According to one example, the inoculum for use in the practice of the invention is a homogenate of intestinal tissue prepared by scraping the mucosa of the ileum of a PPE-infected pig or other animal. When preparing intestinal homogenates, the ileal portion selected for culture should show severe lesions with significant thickening. Because bacteria are inherently fragile, it is desirable to preserve the samples at-70 ℃ as soon as possible after autopsy. It is preferred to add to the inoculum an antibiotic which is already resistant to L.intracellularis to inhibit contaminating bacteria while permitting growth of L.intracellularis, by way of example, such antibiotics are vancomycin, amphotericin B or members of the aminoglycoside antibiotics such as gentamicin and neomycin. Whether the inoculum is a pure culture or a homogenate of intestinal tissue, inoculation of the cultured cells can be performed using various techniques in the art, given the teachings herein.

The bacteria and/or inoculated cultured cells are then incubated under conditions of reduced dissolved oxygen concentration. When the dissolved oxygen concentration is more than 18%, the L.intracellularis growth is lower than optimum and growth is eventually stopped when the oxygen concentration is out of this range. Preferably, the inoculated cultured cells are incubated at a dissolved oxygen concentration of about 0-10%. More preferably, the cells are incubated at a dissolved oxygen concentration of about 0-8%, most preferably at an oxygen concentration of about 0-3.0%.

The proper concentration of carbon dioxide is also critical to the proper growth of L.intracellularis. When the concentration of carbon dioxide is greater than 10% or less than 4%, non-optimal growth occurs and the final growth stops if the carbon dioxide is outside this range. Preferably, the concentration of carbon dioxide is about 6-9%, and most preferably, the concentration of carbon dioxide is about 8.8%.

In addition, the cells are preferably incubated at a hydrogen concentration of about 73-94%. Nitrogen may be used to replace some or all of the hydrogen. According to a particularly preferred example, the cells are incubated at about 0-8.0% oxygen, about 8.8% carbon dioxide and about 83.2% hydrogen.

The seeded cells may be incubated in a dual gas incubator (incubator) or other gas chamber containing appropriate concentrations of oxygen and carbon dioxide and allowing the cells to suspend during incubation. The chamber should contain a means to maintain the seeded cells in suspension, a gas monitor, and a supply source to provide and maintain the appropriate gas concentration. The incubation temperature should be 30-45 deg.C, preferably 36-38 deg.C. Most preferably, the temperature is about 37 ℃. The equipment required for the culture and attenuation methods of the present invention is readily available to those of ordinary skill in the art given the teachings herein. An example of a device suitable for practicing the invention is a dual gas incubator, such as model 480 available from Lab-Line, Melrosepark, Illinois, and combined with a spinner flask to maintain cells in suspension. The presently preferred apparatus comprises: fermentors, bioreactors or rotary shakers containing at least about 2 liters of culture medium and capable of maintaining the cultured cells in suspension by sparging with a gas of appropriate concentration or by other means of mechanical agitation, and capable of continuously monitoring the level of dissolved oxygen in the culture medium. New Brunswick, Braun and others make suitable fermenters and bioreactors for this purpose.

By maintaining the inoculated cells in suspension during incubation, maximum growth of the cells, and thus of the L.intracellularis, can be achieved by increasing the exposure of each cell to the growth medium and the oxygen and carbon dioxide mixture. The cells can be cultured with agitation and maintained in a suspended state by various methods in the art, including, for example, a culture flask, a roller bottle (spinner flask), a membrane incubator, or a spinner flask. Cells can be maintained in suspension during incubation by incubating the cells in spinner flasks in a dual gas incubator or similar device. As used herein, the term "spinner flask" refers to a flask or other container that employs paddles, propellers, or other devices to agitate the culture and maintain the contained cells in suspension.

In a particularly preferred embodiment of the invention, the seeded cells are incubated until the cells reach confluence, then the cells are placed in a spinner flask containing growth medium and incubated in a dual gas incubator while the spinner flask is rotated. Preferably, the seeded cells are scraped into spinner flasks. This can be achieved in various ways known in the art, for example using a cell scraper to dissociate the cells. Once the cells are introduced into the spinner flask, the paddles of the spinner flask are typically mechanically rotated at a speed of about 30-60rpm, maintaining the infected cells in suspension.

A portion of the cultured L.intracellularis is then passaged to fresh cultured cells to increase the yield of L.intracellularis bacteria. The term "passaging" or the like refers to the process of transferring a portion of the cultured L.intracellularis to freshly cultured cells in order to infect the freshly cultured cells with the bacteria. As used herein, the term "fresh" refers to cells that have not been infected with L.intracellularis. Preferably, such cells are on average no more than about 1 day.

Passage of L.intracellularis in suspension culture can be achieved by removing a portion of the original culture and adding it to a new flask containing freshly cultured cells. If the bacterial count/ml value of the original culture is high, e.g., greater than about 10⁴Bacteria/ml, then preferably about 1-10% (by volume) of the culture in the infected bottle is added to a fresh bottle containing fresh cells. This is preferably done when 50-100% of the cells are infected. If the infected cells are below 50%, passaging is best achieved by adding the culture in two portions to a new flask and then adding new medium to the desired volume scale. In any case, cell lysis and other steps are not required, as opposed to passaging a single cell layer culture as in the prior art.

Growing well in cultured cells and subsequently growing with L.intracellularis to greater than aboutInfection with 70% cell infection rate (with IFA, TCID)₅₀Or other similar method), at least a portion of the cultured L.intracellularis bacteria are harvested. Given the teachings herein, the harvesting step can be performed by isolating the bacteria from the suspension using various techniques known to those of ordinary skill in the art. Preferably, the L.intracellularis bacteria are harvested by centrifuging all or a portion of the contents of the suspension to pellet the cultured cells, then resuspending the resulting cell pellet, and lysing the infected cells. Typically, at least a portion of the contents is centrifuged at about 3000 Xg for about 20 minutes to pellet the cells and bacteria. The pellet is resuspended in, for example, sucrose-phosphate-glutamic acid (SPG) solution and then passed through a 25 gauge (gauge) needle approximately 4 times to lyse the cells. If further purification is required, the sample can be centrifuged at about 145 Xg for about 5 minutes to remove nuclei and debris. The supernatant is centrifuged at about 3000 Xg for about 20 minutes and the resulting pellet is resuspended in a suitable diluent such as SPG containing fetal bovine serum (to prepare harvested bacteria suitable for refrigeration or for use as an inoculant) or growth medium (to prepare harvested bacteria more suitable for passage to fresh cells).

As described above, the efficient growth of L.intracellularis for large scale production can be improved by actively growing tissue cells. For monolayers, the rate of cell division decreases significantly when the cultures meet. Attempts to grow L.intracellularis on monolayer tissue cultures have met with limited success and have not been possible for large scale production. However, the use of suspension culture greatly facilitates the active growth of cells and allows for the addition and scale-up of continuous cultures. We have now been able to grow it to 10 ℃ using a fermenter and about 0-3% dissolved oxygen as described above⁸Bacteria/ml. We have also actively grown the cultured bacteria for many months and expect to continue indefinitely.

Prior to the present invention, it was generally believed that cells had to attach to a surface in order to be infected by L.intracellularis. The cell suspension of the present invention is unique and contrary to this theory. When McCoys or IEC-18 cells are used, gelatin, agarose, collagen, acrylamide, or silica beads such as Cultisphere-G porous microcarriers (microcarriers) manufactured by HyClonelaboratories (Logan, UT) are preferably added with the growth medium. However, according to the methods of the invention, HEp-2 cells and other cells do not require microcarriers. This provides a particularly advantageous and economical route to large scale cultivation.

For HEp-2 culture, it is desirable to remove 25-50% of the culture weekly and replace it with fresh medium for maintenance purposes. For microcarrier or bead-bearing cell cultures, it is preferred to remove 25-50% of the culture and replace it with fresh microcarriers or beads and fresh medium 1-2 times per week. For scale-up, additional 25-50% medium or microcarrier-containing medium may be added to the culture.

Depending on the rate at which the cultured cells are infected, passage to fresh cells is usually performed every about 2-5 weeks. Assuming that at least 70% of the cultured cells are infected within 2-3 weeks, it is preferred that the passaging is performed for about 3-4 weeks.

The invention also provides vaccines against L.intracellularis and methods for producing the vaccines. According to a particularly preferred embodiment, at least a portion of the cultured L.intracellularis bacteria is harvested and monitored for potential attenuation after maintaining the infected cells in suspension for an extended period of time (e.g., 6-8 months). Such monitoring is preferably accomplished by immune challenge of the host animal or animal model to select for attenuated strains. Such attenuated strains may be used as vaccines in the methods described herein. The attenuated lawsonia intracellularis vaccines of the invention have been shown to be effective against lawsonia intracellularis infection in various animals and are expected to be effective in humans as well.

The present invention allows for rapid culture amplification, such as a 100-fold increase in yield, and reduced cost. As a result, sufficient L.intracellularis bacterial material produced by the cultivation method of the invention can be easily attenuated for vaccine production. Attenuation in monolayer cultures is difficult because of the low yield of bacteria produced using conventional monolayer growth techniques. In contrast, the L.intracellularis growth method of the invention improves the ease, speed and number of bacteria used for attenuation. The more cells and cells divide, the higher the level of mutation that occurs, which is advantageous for vaccine development. Growth in the suspension of the invention increases the expression of important immunogens under the control of environmentally regulated genes and their expression products.

The resulting attenuated strain can be cultured in a tissue culture monolayer as described in example 1 below, but is preferably cultured in a suspension culture of the present invention. Other methods of attenuation include chemical attenuation such as by using N-methylnitrosoguanidine and other attenuating agents known in the art. Attenuated L.intracellularis can be produced and selected for vaccine production, whether by multiple passages or chemically.

According to one example of a vaccine of the invention, the antigen is harvested by centrifugation or microfiltration as described above. A level is then determined by dose titer in the host animal species according to the optimal host animal immune response, and the antigen is normalized at that defined level. Bacteria can be inactivated by exposure to ambient oxygen concentrations for extended periods of time, e.g., a week, or by use of 0.3% formalin or other inactivating agent to produce a killed vaccine. The antigen is then incorporated into a suitable adjuvant such as aluminium hydroxide or mineral oil to enhance the immune response. The antigen is then used to immunize the host by intramuscular or subcutaneous injection, with booster doses if necessary for piglets of about 3-4 weeks.

Alternatively, according to a particularly preferred example of a vaccine using the above described culture method, the bacteria are serially passaged to induce and select attenuated, avirulent live cultures. The cultures (preferably after at least 6-8 months of growth in suspension culture) are tested for attenuation in the host animal. Cultures were collected and diluted as described previously. For example, pigs can be orally administered 1X 10⁵-1×10⁶Bacteria to immunize. After 28 days of immunization, pigs were re-used at 1X 10⁷A small number of passages (about 30 to 40 days), virulent intracellularLawsonia cultures were inoculated orally. Infected animals were necropsied 21 days after immune challenge and large and small lesions were observed in the small intestine. PCR and Fluorescent Antibody (FA) analysis should also be performed. About 80% of the control animals had major or minor lesions and showed a positive test result for the presence of L.intracellularis in the mucosal cells of the intestine using PCR or FA test methods. The immunized animals were confirmed by histological observation to have a normal mucosal surface and were negative in the PCR test.

In general, attenuated immunogenic L.intracellularis strains are produced by continuous culture for at least about 150-250 days with at least about 7-12 passages in the culture. These parameters can also be varied to produce other attenuated cultures, so long as the monitoring and selection methods described herein are employed.

A vaccine is then prepared comprising an immunologically effective amount of the attenuated L.intracellularis and a pharmaceutically acceptable carrier. The combined immunogen and carrier may be an aqueous solution, an emulsifier, or a suspension. Given the teachings herein, an immunologically effective amount can be determined by methods known in the art without undue experimentation. Generally, the amount of immunogen will be between 50 and 500 micrograms and 10 when purified bacteria are used⁷-10⁹TCID₅₀。

The vaccines of the present invention are typically administered to susceptible animals (preferably pigs) in a single dose or in multiple doses. Live or inactivated vaccine may be administered 1 or 2 times at 2 week intervals. For a live attenuated vaccine, a single dose is preferred. The preferred route of administration of the live attenuated strain is oral or intranasal administration, but may also be by intramuscular or subcutaneous injection. For inactivated vaccines, intramuscular and subcutaneous routes of injection are optimal.

Effective diagnosis of PPE is also hampered by the time required to culture pathogenic bacteria. As a result of the present invention, diagnostic tools can now be developed for rapidly and accurately analyzing biological samples from pigs or other animals likely to be PPE for the presence of L.intracellularis.

Lawsonia intracellularis bacteria grown according to the methods of the invention, or fractions derived from such bacteria, can be used as antigens in ELISA or other immunoassays such as the immunofluorescent antibody assay (IFA) to detect antibodies against lawsonia intracellularis in the serum or other body fluids of animals that may be infected with the bacteria. The presently preferred immunoassay is IFA as described in the examples below. Alternatively, bacteria grown by the method of the present invention were used for Western Blot analysis.

A preferred ELISA protocol according to an example of the invention is as follows:

1. antigen diluted in coating buffer was added at 0.1 ml/well. Incubate at 4 ℃ for 18 hours.

2. Wash 3 times with PBS.

3. Add 0.25 ml blocking buffer to each well of the plate. Incubate at 37 ℃ for 1-2 hours.

4. Wash 3 times with wash buffer.

5. Serum was diluted in blocking buffer and 0.1 ml was added to the first row of wells of the plate. A series of 1: 2 dilutions were made on the plates. Incubate at 37 ℃ for 1 hour.

6. Washing with washing buffer 3-5 times.

7. The conjugate was diluted in blocking buffer and 0.1 ml was added to each well of the plate. Incubate at 37 ℃ for 1 hour.

8. Washing with washing buffer 3-5 times.

9. The substrate is added.

12. The absorbance was measured with a spectrophotometer.

13. Wells without added antigen served as blanks.

14. Positive and negative controls should be used in each experiment.

The preferred Western blot protocol is as follows:

1. the antigen was electrophoresed on 12% SDS-PAGE and then transferred to nitrocellulose membrane.

2. The membrane was placed in blocking buffer for 2 hours.

3. The blocking agent was removed and rinsed with PBS for 1 minute.

4. Serum was diluted in blocking buffer and added to the membrane. Incubate at room temperature for 2 hours.

5. Wash 3 times with wash buffer (5 min each).

6. The conjugate was diluted in blocking buffer and added to the membrane. Incubate at room temperature for 1 hour.

7. Wash 3 times with wash buffer.

8. Substrate was added for 10 minutes until strong binding occurred.

9. Rinse with PBS.

10. Dried in air and stored in the dark.

The invention is further described in connection with the following examples. These examples are provided for illustrative purposes only and are not to be construed as limiting.

Example 1

Isolation of L.intracellularis from the intestinal tract of U.S. pigs suffering from porcine proliferative bowel disease

Materials and methods

Selection of inoculated samples

Sample N24912 was obtained in herds of one farm in state, i.e., 15 consistently bloody stools were observed in 300 fattening pigs (finisher pig) for 5 months, despite treatment with penicillin. After autopsy of the pigs, the intestine (ileum) was found to have thick mucosa. Histopathological examination with silver staining showed the presence of tortuous, intracellular bacteria and proliferation of crypt (crypt) intestinal epithelial cells, confirming the diagnosis of PPE. Sample N72994 was obtained from a two-litter SPF sow 1.5 years old on one farm in minnesota. Herds were 70-80 sows in size, and antibiotic treatment was unknown. The ileal mucosa was found to thicken and bleed somewhat at necropsy. Giminez staining of the mucosa showed the presence of many campylobacter bacteria. Sample N101494 was obtained from pigs aged 12 weeks on a farm (sows raised at 600 ploughs) in indiana. The swine was treated with Tylan injection after bloody diarrhea, but the animal died soon after treatment.

Preparation of inoculum from pigs:

the intestinal samples were stored at-70 ℃. The intestine was dissected and washed with Phosphate Buffered Saline (PBS). 1 g of mucosa was scraped into Sodium Potassium Glutamate (SPG) and then homogenized in SPG for 30 seconds with 4.0 ml of 0.1% Trypsin (JRHBOSciences, Lenexa, KS). The suspension was incubated at 37 ℃ for 35 minutes. 10 ml of SPG/10% Fetal Calf Serum (FCS) (JRH Biosciences, Lenexa, KS) was added and then ground on a tissue grinder for 1 minute. 10 ml of SPG/10% FCS were added and then filtered once with filter paper (Whatman 113V; Whatman Labsales, Hillsboro, 0R) followed by filtration on 5.0, 1.0 and 0.65 micron filter membranes. The filtrate was divided into portions and stored in 1.0 ml portions at-70 ℃. The mucosa was smeared onto a glass slide for Giminez staining. Each smear of the filtrate was stained by IFA with a specific monoclonal antibody against L.intracellularis (S.McOristet al, vet.Rec.121: 421-.

Cell culture

IEC-18 (murine intestinal epithelial cells, ATCC CRL 1589) was grown in DMEM (JRH Biosciences, Lenexa, KS) containing L-glutamine and 10% FCS and routinely passaged weekly with trypsin. Cell monolayers were grown at 37 ℃ in air containing 5% carbon dioxide.

Infection of cell cultures

IEC-18 cells at 1.25X 10⁵Cells were seeded at 25cm²And inoculated in chamber slides (chamberslide) (Nunc, inc., Naperville, Il) at comparable and comparable rates, incubated for 24 hours, and the medium removed. Frozen bacterial isolates from pigs were flash thawed and diluted in DMEM/7% FCS containing vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml) at a dilution ratio of 1.0 ml tissue homogenate to 15 ml medium before addition to the monolayer. The monolayer and bacterial suspension were centrifuged at 2000g for 30 minutes and transferred to an anaerobic jar. The tank was emptied and air was replaced with hydrogen and carbon dioxide to give a mixture of 8.0% oxygen, 10% carbon dioxide and 82% hydrogen. The culture was incubated at 37 ℃ for 3 hours, and then supplied with DMEM/7% FCS containing L-glutamine, vancomycin (100. mu.g/ml), neomycin (50. mu.g/L) and amphotericin B (2.0. mu.g/ml). The culture was returned to the anaerobic tank and incubated for 6 days with medium change every 2 days.

Passage of L.intracellularis

The procedure was as described previously in g.lawson et al, j.clin.microbiol., 31: 1136-1142(1993), which is incorporated herein by reference in its entirety, L.intracellularis bacteria are passaged by lysing the cells with potassium chloride and then added to a fresh IEC-18 monolayer. The medium was decanted from the monolayer, 0.1% potassium chloride was added, and the cells were incubated at 37 ℃ for 10 minutes. The potassium chloride was removed, SPG/10% was added, and the monolayer was then detached with a cell scraper. Cells were lysed 3 times by syringe with 21 gauge (gauge) needle. The cell nuclei were removed by centrifugation at 100 Xg for 5 minutes and the bacterial suspension in the supernatant was added to a fresh 1 day IEC-18 cell monolayer.

Monitoring infection of cell cultures

The infection was monitored by fixing the cells on a chamber slide with cold acetone/methanol for 5 minutes. Staining was performed by immunofluorescence and immunoperoxidase methods. Both methods employ a murine monoclonal antibody (as described in S.McOrist et al, at vet.Rec.121: 421422 (1987)) as the primary antibody, and either an anti-murine immunoglobulin G-fluorophore conjugate (fluorescein isothiocyanate fluorofluorescein; Organon Teknika Corporation, Durham, NC) or a peroxide conjugate (goat anti-murine immunoglobulin G; Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.). Quantitative analysis of bacteria was done by counting the number of specifically stained bacteria in the cells on each slide.

Polymerase chain reaction

Sample inocula and passaged bacteria were combined as template DNA for use in PCR, with sample preparation methods, primers and cycling parameters used as described in Jones et al, j.clin.microbiol., 31: 2611-. The first cycle of the cycle parameters was 93 ℃ for 5 minutes; 45 seconds at 55 ℃; and 72 ℃ for 45 seconds. 33 cycles were carried out for 45 seconds each at the above-mentioned temperatures, and one cycle was as follows: 93 ℃ for 45 seconds; 45 seconds at 55 ℃; and 72 ℃ for 2 minutes. Positive inoculum was used only to inoculate IEC-18 cells. PCR was also performed to monitor passage material to confirm infection. The DNA generated by PCR was sent to Iowa S site University Nucleic acid facility for sequencing. The sequencing results were compared with the sequence obtained by Gary f.jones reported in his doctor paper (university of Minnesota, Minneapolis, MN (6 months 1993)).

As a result:

selecting an inoculum sample:

pig nos. N24912 and N72994 suffered from severe PPE with bloody intestinal content and thickened mucosa. N101494 suffers from severe PPE and severe diarrhea, resulting in the formation of large blood clots in the intestinal lumen. Giminez staining of mucosal smears revealed a large number of curved or S-shaped bacteria. IFA staining revealed the presence of a large number of strongly fluorescent bacteria in the inoculum from the pigs.

Monitoring of infection of cell cultures:

the seeded monolayer was monitored by light microscopy during growth and slight changes in cell morphology were observed. At a lower oxygen concentration (8% O)₂) Growth underHave a similar morphology.

Infected cultures stained with immunofluorescence and immunoperoxidase showed a significant number of bent or sigmoid bacteria in the cells. The monolayer was free of confluent infection. Infected cells are often tightly connected, centered at 1-10 cells. It is also seen that heavily infected cells (i.e., with 30 or more bacteria) are also associated with cells with a bacteria count of less than 30. The bacterial number peaked around 6 days. Infection depends on the particular growth conditions. The bacteria were successfully passaged through the lysis procedure described herein. Centrifugation of the freshly seeded cells is not required, but in so doing increases the number of infected cells. Centrifugation also reduces the possibility of contamination, i.e., the cells exposed to infection in antibiotic-free medium are placed in antibiotic-containing medium for 3 hours. In order to reduce the growth rate of IEC-18 cells so that bacteria can multiply to higher numbers before monolayer confluence, it is necessary to reduce the concentration of FCS in the medium from 10% to 7%.

Polymerase chain reaction:

PCR was performed on chromosomal DNA to generate a 319bp fragment (including primers) for all isolates. Fragments of appropriate size were visually compared to positive samples generated by PCR by known McOrist et al (1994). Sequence analysis of the PCR products of N24912, N72994 and N101494 demonstrated close homology (97-99%) with the p78 sequence determined by Jones (1993).

Example 2

Growth of Lawsonia intracellularis in suspension cultures of HEp-2 cells

Preparation of intestinal homogenate for inoculation:

intestinal homogenates were prepared by scraping mucosa from 6.0-8.0 cm of ileum in the intestinal samples of example 1. Trypsin (1%) was added to the scraped mucosa and the samples were briefly homogenized and then incubated at 37 ℃ for 35 minutes. A further 10 ml of SPG/10% FBS was added and the sample ground in a tissue grinder. 10 ml of SPG/10% FBS was added. The homogenate was filtered through Whatman 113V filter paper, followed by 5.0, 1.0 and 0.65 micron filtration membranes. The filtrate was divided into 1.0 ml portions and stored at-70 ℃.

Infection of cell cultures

The method A comprises the following steps:

tissue cells at 1X 10⁷Cells were seeded in 100 ml spinner flasks containing 50 ml DMEM/10% FBS. Cultures were incubated for 24 hours, then vancomycin and amphotericin B were added. A vial of frozen intestinal homogenate was rapidly thawed and diluted in 3.0 ml DMEM/5% FBS containing vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The sample was passed through a 0.65 μm filter and then added to the bottle. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture consisting of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. The cultures were incubated at 37 ℃ for 3 hours and then neomycin and gentamicin were added. Then, the culture was supplied with DMEM/5% FBS containing L-glutamine, vancomycin (100. mu.g/ml), neomycin (50. mu.g/L), gentamicin (50. mu.g/L) and amphotericin B (2.0. mu.g/ml) over 24 hours.

The method B comprises the following steps:

HEp-2 cells were cultured at 1.25X 10⁵Cells were seeded at 25cm with DMEM/10% FBS²Incubate for 18-24 hours in conventional flasks. The confluency of cells at the time of inoculation was 30%. The inoculum was diluted in DMEM/5% FBS. When the inoculum was derived from an intestinal homogenate, the medium also contained vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture consisting of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. The cultures were incubated at 37 ℃ for 3 hours and then neomycin and gentamicin were added. Then, the culture was supplied with DMEM/5% FBS containing L-glutamine, vancomycin (100. mu.g/ml), neomycin (50. mu.g/L), gentamicin (50. mu.g/L) and amphotericin B (2.0. mu.g/ml) over 24 hours. Antibiotics are not required when the inoculum is a pure culture. Cultures were incubated for 6 days or until confluence. Scraping cells from the vial andadded to a 100 ml spinner flask containing 50 ml of DMEM/10% FBS.

Cultures were diluted 1: 2 weekly by collecting half of the culture and adding fresh medium, or by moving the culture to a larger spinner flask and then adding more medium.

Passage of the culture:

fresh HEp-2 cells were plated at 1X 10⁷Cells were seeded in DMEM/5% FBS, and cultures were passaged to fresh HEp-2 cells. The new cultures were incubated overnight in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. The new culture was then inoculated with the infected culture and incubated at the lower oxygen concentrations described above. The number of inocula depends on the degree of infection of the original culture.

Harvest and storage of the culture:

the desired number of cultures was collected and centrifuged at 3000 Xg for 20 minutes to harvest the cultures. The pellet was resuspended in sucrose-phosphate-glutamic acid (SPG) solution and then passed through a 25 gauge (gauge) needle 4 times. The culture was divided into several portions and frozen at-70 ℃. For further purification, the sample may be centrifuged at 145 × g for about 5 minutes to remove nuclei and debris. The supernatant was centrifuged at 3000 Xg for 20 minutes and the pellet was resuspended in diluent.

Evaluation of live Lawsonia intracellularis in tissue culture:

the amount of L.intracellularis viable was quantified by determining the tissue culture infection Dose (50%) (tissue culture Current infection Dose 50 percent, TCID₅₀) And then the process is completed. The method comprises the following steps: 2.0 ml of the culture to be tested was removed and the cells were lysed 4 times through a 25 gauge (gauge) needle. Samples were diluted serially 1: 10 in DMEM/5% FBS with vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The dilutions were added to a 96-well microtiter plate at 0.1 ml per well. HEp-2 cells were seeded at 1250 cells/well in microtiter plates and grown 18-24 hours before infection. 3-6 wells were used for each dilution concentration. The plates were incubated in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen for 6 days. Cells were fixed with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well of anti-IS intraspecific LLULAris monoclonal antibody (McOrist, 1994) diluted 1: 2000 in PBS. The plates were incubated at 37 ℃ for 30 minutes and then washed 3 times with PBS. Anti-murine FITC, diluted 1: 30, was added at 0.03 ml/well and incubated at 37 ℃ for 30 minutes. The plates were washed 3 times with double distilled water and allowed to dry. The TCID was determined by observing the sample with a fluorescence microscope₅₀/ml。

As a result:

TCID₅₀the results show that the culture can contain up to 1X 10⁶Bacteria/ml. This was achieved within 45 days. The culture volume was enlarged to 3.0 liters in the same time.

Example 3

Growth of Lawsonia intracellularis in suspension cultures of McCoys cells

Preparation of intestinal homogenate for inoculation:

an intestinal homogenate was prepared as described in example 2. Lawsonia intracellularis samples grown by the methods of the examples below were deposited under the Budapest treaty at the American type culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland U.S. A20852) at 19.5.1995 under number 55672.

Infection of cell cultures

McCoys cells were cultured at 1.25X 10⁵Cells were seeded at 25cm in two DMEM/10% FBS-containing cells²Incubate for 18-24 hours in conventional flasks. Cells reached 30% confluence at the time of seeding. The inoculum was diluted in DMEM/5% FBS. When the inoculum was derived from an intestinal homogenate, the medium also contained vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture consisting of 8.0% oxygen, 10% carbon dioxide and 82% hydrogen. The culture was incubated at 37 ℃ for 3 hours,then neomycin and gentamicin are added. Then, DMEM/5% FBS containing L-glutamine, vancomycin (100. mu.g/ml), neomycin (50. mu.g/L), gentamicin (50. mu.g/L) and amphotericin B (2.0. mu.g/ml) was supplied to the culture over a further 24 hours. Antibiotics are not required when the inoculum is a pure culture. Cultures were incubated for 6 days until confluence. Cells were scraped from the flask and added to a 100 ml spinner flask containing 50 ml DMEM/2% FBS and 0.05G Cultisphere-G microcarriers. The flask was stirred at 40-50 rpm.

Cultures were diluted 1: 2 every 2-3 days by harvesting half of the culture and adding fresh medium and Cultisphere-G beads, or by moving the culture to a larger spinner flask and then adding more medium and Cultisphere-G beads. The final concentration of beads in the culture was about 0.001 g beads/ml.

Passage of the culture:

fresh McCoys cells were plated at 1X 10⁷The cells were seeded in DMEM/5% FBS and 0.05G Cultisphere-G beads to passage the cultures to fresh McCoys cells. The new cultures were incubated overnight in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. The new culture was then inoculated with 25 ml of the infected culture and incubated at the lower oxygen concentration described above.

Harvest and storage of the culture:

the desired number of cultures was collected and centrifuged at 3000 Xg for 20 minutes to harvest the cultures. The pellet was resuspended in SPG and then passed through a 22 gauge needle 4 times. The culture was divided into several portions and frozen at-70 ℃. For further purification, the sample may be centrifuged at 145 × g for about 5 minutes to remove beads, nuclei and debris. The supernatant was centrifuged at 3000 Xg for 20 minutes and the pellet was resuspended in diluent.

Evaluation of live Lawsonia intracellularis in tissue culture:

quantification of L.intracellularis was performed as described in example 2, except that 22 gauge needles were used to lyse the cells and McCoys cells were used to inoculate the microtiter plates at 1250 cells/well.

As a result:

TCID₅₀the results show that the culture contains up to 1X 10⁶Bacteria/ml. This is achieved in less than 1 month. The culture volume was enlarged to 3.0 liters in the same time.

Example 4

Determining the infectious dose of Lawsonia intracellularis pure cultures in a host animal

Summary:

the study on 31 pigs was completed by infecting 6 week-sized plain pigs with a pure culture of L.intracellularis from sample N72994. The pigs were randomly divided into 4 groups, each group being individually enclosed. Group 1 contained 7 pigs and served as a negative control group (infected or not with uninfected tissue culture). Group 2 contains 8 pigs, and the infection dose is 10⁷Bacteria/pig. Group 3 contains 8 pigs, infected at a dose of 10⁶Bacteria/pig. Group 4 contained 8 pigs, infected at a dose of 10⁵Bacteria/pig.

Fecal samples were collected on days 0, 7, 14, 21 and 24 for PCR testing. On day 24, pigs were dissected and ileum, jejunum and colon collected for PCR testing, histopathological analysis and FA staining, as described above.

PCR experiments on the ileal mucosa revealed the presence of lawsonia intracellularis in 100% of the high dose group, 75% of the medium dose group and 50% of the low dose group. Histopathological analysis data showed that an increase in the number of monocytes in the mucosal lamina propria and submucosa occurred in 88% in the high dose group, 75% in the medium dose group, and 88% in the low dose group. Crypt hyperplasia was observed in 50% of the high dose groups, 63% of the medium dose groups, and 50% of the low dose groups. FA staining revealed the presence of lawsonia intracellularis in 88% of the high dose group, 63% of the medium dose group, and 63% of ileal, jejunal, and colon tissue sections in the low dose group. Control animals showed negative results for the presence of L.intracellularis for PCR, FA and silver stain analyses.

In summary, pure cultures can be successfully used for infection and cause PPE damage. The hypothesis of Koch was confirmed by isolating and identifying L.intracellularis from infected animals.

In the immune-challenged animals, 100% of the animals in the high dose group demonstrated recovery and species by silver staining, FA and PCR.

Materials and methods:

growth of inoculum:

mixing 3.75X 10⁵HEp-2 cells were seeded at 75cm in DMEM/10% FBS²Incubate in conventional flasks at 37 ℃ for 18-24 hours at 5% carbon dioxide. (cell confluence at seeding was 30%). A vial of N72994 was diluted in 15 ml of DMEM/5% FBS. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture consisting of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. DMEM/5% FBS was supplied to the culture for another 24 hours.

The cultures were incubated for 6 days, then the cells were scraped from the flask and added to a 100 ml spinner flask containing 50 ml of DMEM/5% FBS. The culture in the flask was scaled up weekly by doubling the volume of the medium. Cultures were grown in spinner flasks for 3 weeks.

Harvesting of the culture:

the culture was harvested by centrifugation at 3000 Xg for 20 minutes. The pellet was resuspended in a sucrose-phosphate-glutamic acid (SPG) solution containing 10% FBS and then passed through a 25 gauge (gauge) needle 4 times. The inoculum was diluted to final volume in SPG/10% FBS and diluted 1: 10.

The inoculum for the control consisted of uninfected HEp-2 cells diluted to the same concentration as the infected culture in viable cell concentration. Cells were harvested in the same manner as infected cells. Pigs in the control group received a dose equivalent to the high dose group.

Quantification of L.intracellularis:

quantification of L.intracellularis viable was achieved by determining the 50% Tissue Culture Infectious Dose (TCID)₅₀) And then the process is completed. The method comprises the following steps: 2 ml of the culture to be tested are removed and the cells are lysed 4 times through a 22 gauge (gauge) needle. Samples were diluted serially 1: 10 in DMEM/5% FBS with vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The dilutions were added to a 96-well microtiter plate at 0.1 ml per well. The microtiter plates were seeded with HEp-2 cells at 2500 cells/well and grown 18-24 hours before infection. 12 wells were used for each dilution concentration. The plates were incubated for 6 days at a gas concentration of 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. Cells were fixed with cold 50% acetone and 50% methanol for 2 min. 0.03 ml/well of anti-L.intracellularis monoclonal antibody (McOrist, 1987) diluted 1: 2000 in PBS was added to each well. The plates were incubated at 37 ℃ for 30 minutes and then washed 3 times with PBS. Anti-murine FITC, diluted 1: 30, was added at 0.03 ml/well and incubated at 37 ℃ for 30 minutes. The plates were washed 3 times with double distilled water and allowed to dry. The TCID was determined by observing the sample with a fluorescence microscope₅₀/ml。

Animals:

31 PIC x Lieske sows and large white boars of 6 weeks size and sex were provided by doctor Kent Schwartz. On day 0, pigs were randomly allocated by weight into 4 swineries.

Equipment:

pigs were housed using 4 pigpens in a small nursing facility, each pigpen being spaced at least 3 feet apart. The pigsty is provided with a metal wire floor and a solid separator. Heat is supplied by a furnace and is supplemented locally by heat-emitting bulbs. During the study, the temperature was maintained between 78-85 deg.f.

Feed and water

Milled corn-soybean feed, containing no antibiotics and a protein content of 19%, was supplied ad libitum through a stainless steel feeder. Water is supplied at will through the water supply nozzle.

Infection of pigs:

on day 0, pigs were weighed and blood samples were collected via capillary in the retro-orbital sinus (retroorbital sinus). Serum was collected and stored at-20 ℃. Fecal samples were also collected for PCR. 10 ml of inoculum was administered intragastrically to the pigs using a gastric tube.

Treatment method	Number of pigs
		Control-uninfected cells	5
Control-untreated	2
		High dose	8
Middle dose	8
		Low dose	8

On days 0, 10, 17 and 24, pigs were weighed and blood samples were collected.

Polymerase chain reaction:

infection of pigs was monitored by PCR using cycling parameters and primers as described by Jones (1993). Fecal samples collected on days 0, 7, 14, 21 and 24 and intestinal mucosa were examined by PCR.

Histopathology:

sections of ileum, jejunum and colon were formalin-fixed, processed by conventional methods, stained with hematoxylin and eosin and silver-impregnated, and evaluated. Sections were also stained with monoclonal antibodies specific for L.intracellularis.

As a result:

clinical symptoms:

on day 3, clinical symptoms including loose stools were observed in the high dose group. Peak was reached at 14 days, followed by some relief.

Weight gain condition:

the daily average weight gain was calculated and the results showed a reduction in weight gain in the high and medium dose groups compared to the control group. There was a dose titer effect in weight gain when comparing the groups.

PCR：

Dripping of the feces was not observed until 14 days. 37.5% of the feces in the high dose group of pigs were PCR positive on day 21. After dissection, ileal mucosa was examined by PCR with a positive rate of: the high dose group was 100%, the medium dose group was 75%, the low dose group was 50% and the control group was 0%.

Severe damage:

severe lesions were found in 2 pigs in the high dose group (#50 and # 202). These 2 pigs had a thickening of about 3 feet in the ileum and necrosis occurred in # 202.

Histopathology:

FA：

FA staining of sections of ileum, jejunum and colon revealed: lawsonia intracellularis is present in 87.5% of the high dose group, 62.5% of the medium and low dose groups, and 0% of the control group.

Micro-damage:

lesions were observed in 100% of the high dose group, 75% of the medium dose group, 87.5% of the low dose group and 14% of the control group. This is determined by observing an increase in the number of monocytes in the lamina propria and submucosa, a condition often associated with proliferation of the Peyer's Patchers. Crypt hyperplasia was also observed.

Silver staining:

the sections were also silver stained to check for the presence of intracellular, tortuous bacteria. The results showed that bacteria were present in 87.5% of the high dose group, 62.5% of the medium dose group, 87.5% of the low dose group and 0% of the control group.

Discussion:

pigs can be successfully infected with pure L.intracellularis cultures. At 10⁷At doses of bacteria, 100% of pigs were infected as determined by PCR and microscopic lesions. The severity of the lesions and the number of bacteria in the tissue sections was considerably lower. This study is a satisfactory immuno-priming model for L.intracellularis, as L.intracellularis is present in pigs and is minimally invasive. If a second dose is given 7 days after the first dose, the damage may increase.

Example 5

Hamster vaccine efficacy test

The purpose is as follows:

laboratory animal models were evaluated to determine the safety and effectiveness of an avirulent live lawsonia intracellularis vaccine in hamsters.

Summary:

inoculation of hamsters of 3 weeks size with pure cultures of Lawsonia intracellularis strains at high passage times, followed by immune challenge with virulent material at low passage times 22 days after inoculation, to complete 40 hamstersStudy of hamster. Hamsters were divided into 3 groups. Group A was inoculated with 1 dose of L.intracellularis strain N72994 on day 0. Group B served as a control group, and no vaccine culture was used. Both groups were challenged with 2 doses of a pure culture of L.intracellularis strain N343 at 22 and 25 days post inoculation. Group C used challenge strain N101494 for comparison of relative virulence with strain N343. Groups A and B each had 15 hamsters, and group C had 10 hamsters. 50% Tissue Culture Infectious Dose (TCID)₅₀) Data disclosure, using 10⁵TCID₅₀One dose was inoculated into hamsters. N343 immune challenge contains 10⁵.5TCID₅₀One dose. Group C had a challenge dose of 10².75 TCID₅₀One dose. Fecal samples were collected on days 0, 7, 14, 21, 29, 36, and 43 for PCR assays. On day 5, 5 animals were dissected from group a and group B for PCR examination of the mucosa and for FA, hematoxylin and eosin staining of intestinal sections, and silver staining to determine the persistence of bacterial colonization in vaccinated hamsters. The remaining animals were dissected 21 days after challenge and subjected to similar tests.

The PCR data showed that there was lawsonia intracellularis on 100% of the intestinal mucosa in group a hamsters 21 days after inoculation. Group B was negative 21 days after inoculation. At 21 days post challenge, 50% were PCR positive in group a hamsters and 100% in group B hamsters. Histopathological analysis of the sections showed mild to severe lesions in 50% of the animals in group a and mild lesions in 50% of the animals in group B21 days after challenge. None of the animals showed lesions 21 days after inoculation. Group C animals had no lesions 21 days after challenge. Neither FA nor silver staining revealed the presence of L.intracellularis in the sections.

In summary, PCR showed that a 50% reduction in infection was observed when hamsters were inoculated with high passage lawsonia intracellularis strains. The intestine was colonized by a small number of intracellular organisms (Lawsonia intracellularis) as this organism was not observed in FA and silver stained sections. The group C hamsters did not show infection throughout the study, most likely due to the low bacterial dose.

Materials and methods:

hamster status:

40 female hamsters of 3 weeks size from Harlan Sprague Dawley were used.

Growth of inoculum:

vaccine cultures:

a continuous culture of L.intracellularis grown in HEp-2 cells for 29 weeks was used. This culture was grown in a similar manner as described in the section for the stimulated culture, except that: cultures were passaged to new HEp-2 cells every 2-3 weeks.

The culture was stimulated:

mixing 3.75X 10⁵The McCoys cells of (2) were seeded in a 75cm DMEM medium (Dulbecco's Modified Eagle's medium) containing 10% Fetal Bovine Serum (FBS)²Incubate at 37 ℃ and 5% carbon dioxide for 18-24 hours in a conventional tissue culture flask. The medium was separated from the cells and a bottle of N343 MSC X diluted in 14 ml DMEM/2% FBS was added to the bottle. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture consisting of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. Cultures were grown for 6 days and cells scraped from the flasks were added to 100 ml spinner flasks containing 90 ml DMEM/2% FBS and 0.01G Cultispere-G beads. The cultures were grown at the gas concentrations described above. Flask culture volume was expanded weekly by doubling the volume of the medium. The cultures were grown in spinner flasks for 25 days until a final volume of 250 ml.

Strain N10494 was grown in the same manner as strain N343.

Harvesting the culture:

vaccine cultures:

the culture was harvested by centrifugation at 3000 Xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamic acid (SPG) solution containing 10% FBS and then passed through a 25 gauge (gauge) needle 4 times. The inoculum was diluted to final volume (15 ml) in SPG/10% FBS.

The culture was stimulated:

the culture was harvested by centrifugation at 3000 Xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamic acid (SPG) solution containing 10% FBS and then passed through a 25 gauge (gauge) needle 4 times. The inoculum was diluted to final volume in SPG/10% FBS (20 ml for the N343 strain and 10 ml for the N101494 strain).

Hamster dose:

vaccine:

on day 0, all hamsters in group a were orally vaccinated with 1 ml of the prepared vaccine.

Excitation:

at 21 days post inoculation, 0.5 ml of challenge culture strain N343 was orally administered to 10 hamsters from group a and 10 hamsters from group B. Group C was immune challenged with 0.5 ml of challenge culture strain N101494.

Quantification of ISi:

quantification of live ISI was performed by determining a 50% Tissue culture infectious dose (TCID 50 percent, TCID)₅₀) And then the process is completed. The method comprises the following steps: 2 ml of the culture to be tested was removed and the cells were lysed 4 times through a 22 gauge needle. Samples were serially diluted 1: 10 in DMEM/5% FBS containing vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The dilutions were added to a 96-well microtiter plate at 0.1 ml per well. Prior to infection, the plates had been seeded with mccosys cells at 1250 cells/well and grown at 37 ℃ and 5% carbon dioxide for 18-24 hours. 12 wells were used for each dilution concentration. The plates were incubated for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Cells were fixed with cold 50% acetone and 50% methanol for 2 min. 0.03 ml/well of anti-ISI monoclonal antibody diluted 1: 2000 in PBS was added to each well. The plates were incubated at 37 ℃ for 30 minutes and then washed 3 times with PBS. Anti-murine FITC, diluted 1: 30, was added at 0.03 ml/well and incubated at 37 ℃ for 30 minutes. The plates were washed 3 times with double distilled water and allowed to dry. The TCID was determined by observing the sample with a fluorescence microscope₅₀/ml。

Monitoring of hamster infection:

infection of hamsters was monitored by PCR using cycling parameters and primers as described by Gary Jones. Fecal samples were collected on days 0, 7, 14, 21, 29, 36, and 43 post inoculation. After killing the hamsters, their intestinal mucosa was also examined by PCR.

Histopathology:

sections of the ileum and colon were formalin fixed, processed by conventional methods, stained with hematoxylin and eosin and silver impregnated, and evaluated. Sections were also stained with monoclonal antibodies specific for L.intracellularis.

Average daily weight gain:

hamsters were weighed 21, 28, 35 and 42 days after inoculation to determine the average daily weight gain. As a result:

see table below.

TCID₅₀：

TCID₅₀Data show that vaccine group (group A) received 10⁴.86 TCID₅₀A/hamster. The hamsters in groups A and B were challenged with strain N343 at an inoculation amount of 10⁵.5TCID₅₀A/hamster. The group C hamster challenged with the strain N101494 received an amount of 10².75 TCID₅₀A/hamster.

PCR：

PCR testing showed 100% presence of lawsonia intracellularis in vaccinated hamsters dissected 21 days post-vaccination. Experiments 43 days after inoculation showed that 100% of control hamsters and 50% of inoculated hamsters were infected with lawsonia intracellularis. None of the hamsters challenged with N101494 was PCR positive. In this hamster study, no dripping of excrement was observed.

Histopathology:

hematoxylin and eosin staining (H & E staining) revealed no tissue damage in all sections of hamsters dissected 21 days after inoculation. In sections collected 43 days after vaccination, 50% of the vaccine groups had mild to severe lymphocytic enteritis, and 50% of the control groups had mild lymphocytic enteritis. No damage was found in the N101494 challenge group.

FA staining failed to show the presence of lawsonia intracellularis in hamsters 43 days after inoculation.

Discussion:

PCR showed that a 50% reduction in infection rate was observed in hamsters inoculated with the high passage lawsonia intracellularis strain.

Fecal Excreta mucosa micro-damage

Mucosal PCR H & E staining for FA

PCR PCR PCR PCR PCR PCR PCR PCR silver staining

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

A-1- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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

A-3- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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

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

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

A-7-NA + severe enteritis- -

A-8-NA + enteritis- -

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

A-10-NA-mild 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 + minimal enteritis- -

B-2- -NA + minimal enteritis- -

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

B-4- -NA + minimal enteritis- -

B-5- -NA + minimal enteritis- -

B-6-NA + minimal 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 - - - -

Example 6

Swine vaccine effectiveness test

The purpose is as follows:

the objective of this study was to evaluate the safety, duration of colonization and effectiveness of live avirulent and killed isolates of L.intracellularis in 2-3 week-sized pigs. Host animal studies were conducted in which 3 week old pigs were vaccinated and then subjected to virulent immune challenge with L.intracellularis strain N343 to compare the differences in protection between vaccines.

Method of producing a composite material

A total of 45 pigs at 3 weeks of age were purchased from H & K farms on 11 days 12 months 1995. They were shipped to market Resources, inc, a research facility in Cambridge, Iowa, where pigs were marked to distinguish each pig. Pigs were kept in the facility for 2 days prior to the study to acclimatize, and were fed antibiotic-free feed during the study.

On day 13 of 12 months, all pigs were weighed, bled for serum, clinically scored and rectal test samples collected. Pigs were randomly divided into 5 groups and placed in pots. 20 pigs were placed in a separate room and used as control and stringent control groups. 15 pigs were placed in the second room and tested for the ISI-1 vaccine. The third room had 10 pigs for the ISi-2 vaccine trial.

Live vaccines were prepared at the NOBL Laboratories Research and Development institute and are referred to as test series ISI-1. ISI-1 (strain N343) was isolated from swine and grown in pure culture for 29 consecutive weeks. The vaccine was grown on mccosys cells in spinner flasks at low oxygen concentrations until approximately 100% infection was observed. The high passage N343 strain used for ISI-1 was spinner flasks for an additional 11 weeks ("N343 NP40 wk") and was deposited at the American type culture Collection (ATCC, 12301 ParklawnDrive, Rockville, Maryland U.S. A20852) at 5 months and 22 days 1996 under the Budapest treaty with a deposit number of 55783. The culture was harvested by centrifugation at 3000 Xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamic acid (SPG) solution and then passed through a 25 gauge needle 4 times. The lysate was centrifuged at 500 Xg for about 5 minutes to form a pellet to remove debris and microcarrier beads. The supernatant was collected, stored at-70 ℃ and removed until 1 hour prior to inoculation and placed on ice for administration.

Inactivated vaccine (ISI-2) was grown, passaged for 12.5 weeks, harvested and then purified using a percol gradient in a similar manner as described above. The purified bacteria were then stored at-70 ℃ until 1 hour prior to inoculation and were stored at 4 ℃ and normal atmospheric oxygen concentrations which were toxic to L.intracellularis. Aluminum hydroxide was added to the bacteria until the final mixture contained 10% aluminum hydroxide. Protein concentration was determined by the biuret method.

Quantification of viable ISi:

quantification of L.intracellularis viable was achieved by determining the 50% Tissue Culture Infectious Dose (TCID)₅₀) And then the process is completed. The method comprises the following steps: prior to infection, 96-well microtiter plates were seeded with McCoys cells at 1250 cells/well and then grown18-24 hours. Samples were serially diluted 1: 10 in DMEM/5% FBS containing vancomycin (100. mu.g/ml) and amphotericin B (2.0. mu.g/ml). The dilutions were added to a 96-well microtiter plate at 0.1 ml per well. 12 wells were used for each dilution concentration. Plates were incubated at 37 ℃ for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Cells were fixed with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well of an anti-L.intracellularis monoclonal antibody (developed by Dr. Steven McOrist) diluted 1: 2000 in PBS. The plates were incubated at 37 ℃ for 30 minutes and then washed 3 times with PBS. Anti-mouse immunoglobulin G-fluorophore conjugate (FITC) diluted 1: 30 was added at 0.03 ml/well and incubated at 37 ℃ for 30 minutes. The plates were washed 3 times with double distilled water and allowed to dry. The samples were observed with a fluorescence microscope and the TCID was determined by Reed-Meunsch calculation₅₀/ml。

TCID₅₀The data show that the ISi-1 has a value of 1.8X 10⁵Bacteria/ml. The fourth inoculum was a placebo derived from tissue culture cells processed in the same manner as the vaccine.

The total protein content of the inactivated vaccine was determined by the biuret method to be 0.311 mg/ml.

Pigs were vaccinated 12 months and 13 days 1995. The dose of live vaccine was 2 ml IN, 1 ml per nostril. The ISi-2 (inactivated) vaccine was administered intramuscularly at 1.5 ml per pig and again after 14 days. All control animals used uninfected cells in the same manner as the live vaccine.

Observation and sample:

during the study, fecal samples and serum were collected every 7 days. PCR experiments were performed on fecal samples to amplify DNA using primer pairs: 5'-TATGG CTGTC AAACA CTCCG-3' and is: 5'-TGAAG GTATT GGTATTCTCC-3' are provided. The first cycle of the cycle was 93 ℃ for 5 minutes; 45 seconds at 55 ℃; and 72 ℃ for 45 seconds. The 33 cycles were carried out at the above-mentioned temperatures for 45 seconds each. The last cycle is: 93 ℃ for 45 seconds; 45 seconds at 55 ℃; and 72 ℃ for 2 minutes. Primers were determined by Jones et al.

And (3) immune stimulation:

all animals, except the stringent controls, were given a challenge culture consisting of low passage cultures (8-12 weeks of continuous growth) of L.intracellularis strains N343 and N72994 at 26 and 27 days post inoculation. The culture was harvested by centrifugation at 3000 Xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamic acid (SPG) solution containing 10% fetal bovine serum and then passed through a 25 gauge needle 4 times. Part of the harvested cultures were stored at-70 ℃ until used for priming, while others continued to grow until the day of immune priming and then harvested. Mixed challenge inoculum, determination of culture TCID₅₀. Samples were stored on ice for administration.

96 years 1 month 8 day challenge cultures contained 4X 10⁴Bacteria/ml, whereas 96 years of 1 month, 9 days of challenge cultures contained 3X 10⁴Bacteria/ml. Both days 15 ml challenge vaccine was administered to pigs by gastric lavage. Thus, animals received 6X 10 on day 8 of month 1 of 96 and day 9 of month 1 of 96 respectively⁵Bacteria/pig and 4.7X 10⁵Bacteria/pig.

As a result:

safety:

excrement PCR results: detection of L.intracellularis by PCR showed that no pigs excreted bacteria at the beginning of the study. All pigs were negative 7 days after inoculation. At 14 days post inoculation, 3 pigs were positive in the ISI-1 group. At 21 days post inoculation, 2 pigs were positive in the ISI-1 group, and all other pigs were negative. Detection by PCR showed that no animals excreted bacteria 26 days after inoculation. At 26 days post inoculation, 5 pigs in the ISI-1 group and 4 pigs in the ISI-2 group were dissected. Samples collected were ileum, colon, mesenteric lymph nodes and tonsils and lung samples (from pigs with possible pneumonia lesions).

PCR assays were performed on each ileum and lung sample. Tonsils, colon and lymph nodes were mixed by treatment group and PCR was performed. The PCR results are as follows.

Group of	Ileal PCR 26 days after inoculation	Mixed colon	Mixed tonsils	Mixed mesenteric lymph nodes	Mixed lung
						ISi-1	1 in 5	+	-	-	1 in 1
ISi-2	0 of 4	-	-	-	0 in 1
						Control	0 of 5	-	-		Not tested
Stringent controls	Not tested	Not tested	Not tested	Not tested	Not tested

Ileal tissue sections were stained using a monoclonal antibody specific for L.intracellularis as the primary antibody and an anti-murine immunoglobulin G-fluorophore conjugate as the secondary antibody. Lawsonia intracellularis was observed in 3 out of 5 pigs derived from ISI-1. All other pigs were negative as detected by staining with fluorescent antibody.

The remaining pigs were dissected 21 days after challenge and the same samples were collected for evaluation. The PCR results are as follows.

Group of	Ileal PCR 26 days after challenge	Mixed colon	Mixed tonsils	Mixed mesenteric lymph nodes	Mixed lung
						ISi-1	0 of 10	+	-	-	-
ISi-2	0 in 6	-	-	-	-
						Control	4 of 10	-	-		-
Stringent controls	0 of 5	-	-	-	-

The ileum was FA stained as above and 7 of the 10 animals in the control group were positive. All other animals were negative for the presence of L.intracellularis.

Sera were also tested to determine the production of IgG antibodies produced in pigs after exposure to lawsonia intracellularis. The test was carried out as follows: McCoys cells were plated at 1250 cells/well on Terasak i plates that had been treated with tissue culture prior to infection and then grown for 18-24 hours. Lawsonia intracellularis pure cultures were diluted to 1000-. The plates were incubated for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Cells were fixed with cold 50% acetone and 50% methanol for 2 min. Porcine serum was diluted 1: 75 in sterile pbs. The diluted serum was added to each well at 0.01 ml per well. Plates were incubated at 37 ℃ for 30-60 minutes and then washed 5 times with sterile PBS. Anti-porcine immunoglobulin G-fluorophore conjugate was added to each well at 0.01 ml/well and the plates were incubated for 30 min at 37 ℃. The plates were washed 5 times with double distilled water and allowed to dry. The sample was washed 5 times with double distilled water and allowed to dry. When the sample was observed with a fluorescence microscope, the wells in which bacteria were observed were scored as positive, and the wells in which bacteria were not observed were scored as negative.

As a result:

group of	Day 0	26 days after inoculation	47 days after inoculation, 21 days after challenge
				ISi-1	0 in 15	15 of 6	8 in 10
ISi-2	0 of 10	3 in 10	5 in 6
				Control	1 in 15	0 in 15	9 of 10
Stringent controls	0 of 5	0 of 5	0 of 5

Animals positive on day 0 were tested again weekly. The results showed that the sera were negative 14 days after inoculation. This was expected since pigs were only 3 weeks in size at day 0, and positives at this age were likely due to maternal antibodies.

The positive control sera used when testing sera were obtained by immunizing pigs with high doses of L.intracellularis grown on pure cultures. The negative control sera used were collected from sterile pigs at South Dakota State University.

The above description and examples are intended only to illustrate preferred examples that may achieve the objects, features and advantages of the present invention, and are not to be construed as limiting the invention thereto. Any variations of the invention which fall within the spirit and scope of the appended claims are considered part of this invention.

Claims (4)

1. A vaccine for inducing an immune response in an animal against a lawsonia intracellularis bacterium, said vaccine comprising an attenuated strain of lawsonia intracellularis and a pharmaceutically acceptable carrier, wherein said attenuated strain is ATCC 55783.

2. The vaccine of claim 1, wherein said vaccine comprises 10⁷-10⁹TCID₅₀The immunogen of (1).

3. The vaccine of claim 1, wherein said carrier is selected from the group consisting of an aqueous solution, emulsion, and suspension.

4. The vaccine of claim 1, wherein the attenuated strain is present in an amount effective to generate an immune response, wherein the vaccine comprises 50-500 micrograms of immunogen, and wherein purified bacteria are used.