CN1519311B - Lawsonia intracellularis cultivation, anti-lawsonia intracelluaris vaccines and diagnostic agents - Google Patents

Abstract

The invention discloses a method for large-scale cultivation and attenuation of the lower part of Lawsonia intracellulare, which comprises inoculating cells with Lawsonia intracellulare bacteria to infect the cells, and cultivating the infected cells at a lower oxygen concentration And maintain the infectious bacteria in suspension. Vaccines against Lawsonia intracellulare were prepared from cultures grown in suspension. Diagnostic reagents are also disclosed.

Description

Lawsonia intracellulare cultures, vaccines and diagnostics against it

This divisional application is a divisional application of the Chinese patent application CN 96114542.0 entitled "Cultivation of Lawsonia intracellulare, Vaccine and Diagnostic Reagent Against the Bacteria".

technical field

The present invention relates to vaccines against Lawsonia intracellularis and methods of preventing and diagnosing LI infections. The products and methods of the present invention are partly a result of our discovery of improved methods for the large-scale cultivation of L. intracellulare.

Background technique

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy (PPE), which affects almost all animals, including humans, rabbits, ferrets, hamsters, foxes, horses, and a variety of other animals such as Ostrich and rhea etc.

Lawsonia intracellulare is a particularly important cause of losses in pig herds. Estimates of the incidence and infection rates of PPE in the United States are that up to 20% of the herd becomes infected and costs $20 million per year.

A fixed feature of PPE is the presence of intracytoplasmic, non-membrane-bound Campylobacter species in the enterocytes of the infected intestinal portion. Bacteria associated with PPE have been termed "Campylobacter-like organismsims" S. McOrist et al., Vet. Pathol., Vol. 26, 260-64 (1989). Subsequently, the pathogenic bacterium was identified as a new taxonomic genus and species, called "Ilealsymbiont (IS) intracellularis" (Intracellular ileal symbiont, ISi) in the native language. C. Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol.43, No.3, 533-38 (1993). Recently, these new bacteria were given the taxonomic name: Lawsonia (L.) intracellularis. S. McOrist et al., Int'l J. of Systemic Bateriology, Vol.45, No.4, 820-25 (1995). These 3 names are used interchangeably and all refer to the same organism described and further identified here. We have endeavored to use the taxonomic name L. intracellularis in the discussion of the present invention.

Lawsonia intracellulare is an obligate, intracellular bacterium that cannot be cultured in conventional cell-free media by usual bacteriological methods and has long been thought to require attachment to epithelial cells for growth. S.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 ), cultured Lawsonia intracellulare in conventional tissue culture flasks using intestinal epithelial cell monolayers from IEC-18 rats. In addition, H.Stills in Infection and Immunity, Vol.59, No.9, 3227-36 (1991) discussed the use of intestinal 407 human embryonic intestinal cell monolayer and GPC-16 guinea pig colon adenocarcinoma in conventional tissue culture flasks Cell monolayer. These existing culture methods are laborious and not suitable for large-scale culture.

Current understanding of L. intracellulare infection, as well as treatment and effective control of the disease, are hampered by the harsh growth conditions required to culture L. intracellulare in vitro. There is therefore a need for an improved method of culturing L. intracellulare. There is also a need for vaccines against L. intracellularis and effective tools for diagnosing L. intracellulare infection.

Contents of the invention

It is an object of the present invention to provide a vaccine against Lawsonia intracellulare.

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

Another object of the present invention is to provide an improved culture method capable of culturing Lawsonia intracellulare on a large scale for the production of vaccines and diagnostic reagents.

To accomplish these and other objects, and consistent with the intent of the present invention as broadly described and embodied herein, the present invention provides a method of cultivating Lawsonia intracellulare and obtaining large quantities of bacterial material therewith. According to this method, Lawsonia intracellulare bacteria are incubated at an oxygen concentration of about 0-18% while agitating the bacteria to grow the Lawsonia intracellulare and maintain the bacteria in suspension.

According to another example, there is provided a method of culturing Lawsonia intracellulare bacteria by: inoculating a monolayer of HEp-2, McCoys or IEC-18 cells (which Confluency is about 30%), allowing the bacteria to infect the cells. The infected cells are then incubated at a temperature of about 36-38° C. with an oxygen concentration of about 0-8.0% until the cells are confluent. The infected cells and growth medium are then placed in a fermenter, bioreactor, spinner flask, or other vessel suitable for maintaining the cells in suspension. Infected cells should be incubated with agitation in order to grow L. intracellulare bacteria and maintain infected cells in suspension. A portion of the cultured L. intracellulare was subcultured to fresh cultured cells to increase the yield of L. intracellulare bacteria.

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

The present invention also provides a method for determining the presence or absence of antibodies specifically reactive with Lawsonia intracellulare bacteria in a biological sample by: harvesting at least a portion of the cultured Lawsonia intracellulare bacteria, using the harvested cells The Lawsonia intracellulare bacteria or components thereof are contacted with a biological sample from an animal under conditions under which antibodies present in the biological sample would react with the Lawsonia intracellulare bacteria or components, and it is determined whether an antibody-antigen reaction occurs.

Additional features and advantages of the invention will be set forth in the following description, and will be apparent from the description, or may be learned by practice of the invention.

Detailed ways

As used herein, the term "Lawsonia intracellulare" refers to the invention by C.Gebhart et al., Int'l.J.of SystemicBacteriology, Vol.43, No.3, 533-38 (1993) and S.McOrist et al. al., Int'l J.of Systemic Bateriology, Vol.45, No.4, 820-25 (1995) (the entire contents of these two documents are hereby cited as a reference) detailed description of intracellular, curved, genetic Lambert-negative bacteria, which include but are not limited to: ATCC 55672 bacteria deposited at the American Type Culture Collection (ATCC, Rockville, MD); NCTC 12656 deposited at the National Type Culture Collection (NCTC, Colindale, London) and 12657 bacteria; pathogenic bacteria that can be obtained from PPE-infected pigs or other animals worldwide, according to the knowledge in the art and the teachings herein; and spontaneous or artificially induced mutations of any of the above bacteria body or variant.

As used herein, the term "attenuated strain" refers to any strain of Lawsonia intracellulare which has been prepared by the cultivation and passage techniques taught herein so as to be rendered avirulent and when administered to a host animal while maintaining its immunogenic properties. As described below, according to the present invention, various L. intracellulare strains were cultured and attenuated to obtain attenuated immunogenic strains. These attenuated strains are effective as vaccines for pigs or other animals susceptible to L. intracellulare infection.

The attenuated strains of the present invention are expected to be used as immunogens in antimicrobial vaccines for animals including birds, fish, cattle, pigs, horses, mammals and primates in general, as well as humans. These vaccines can be prepared using techniques known to those of skill in the art having learned the teachings herein. Such vaccines may contain an immunologically effective amount of attenuated strains and a pharmaceutically acceptable carrier. Vaccines can be administered in single or multiple doses. Immunologically effective amounts can be determined by methods known in the art without undue experimentation based on the teachings herein. The number of avirulent bacteria should be sufficient to elicit an immune response in a susceptible animal while still being avirulent. It depends on the specific animal, bacteria, and disease involved. The suggested dose for administration to susceptible animals is preferably about 10 ³ -10 ⁹ bacteria/kg body weight, optimally about 10 ⁵ -10 ⁷ bacteria/kg 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 vaccines of the invention may be used in combination with other vaccines such as a dilution of another lyophilized vaccine, or in combination with other vaccines prior to lyophilization. Vaccine formulations may also be dried, eg, lyophilized, for storage or for later formulation into liquid vaccines.

Accordingly, the present invention also includes a method of inducing an immune response in an animal host against a virulent, wild-type Lawsonia intracellulare bacterium thereby protecting the host from the bacterium. This method comprises: administering an immunologically effective amount of the attenuated or killed bacteria of the present invention to a host, preferably a vaccine of the present invention.

As used herein, the term "large scale culture" refers to: Lawsonia intracellulare cultures at levels greater than about 2.0-3.0 liters and include 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 L. intracellulare.

In carrying out the culture method of the present invention, cultured cells are first inoculated with an inoculum containing Lawsonia intracellulare bacteria to infect the cells with the bacteria. A variety of cell lines can be used to practice the present invention, including but not limited to: IEC-18 (ATCC 1589)-mouse intestinal epithelial cells, HEp-2 (ATCC 23)-human epidermoid tumor cells, McCoys (ATCC 1696)-mouse (non-specific) cells, MDCK (ATCC 34) - Madin-Darby dog kidney cells, BGMK (Biowhittaker #71-176) - Buffalo green monkey kidney cells and porcine intestinal epithelial cells. Preferred cultured cells are HEp-2, McCoys or IEC-18 cells. Alternatively, bacteria can be cultured in a cell-free system as long as the bacteria are maintained at the appropriate dissolved oxygen concentrations as taught herein.

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

We found that the successful cultivation of L. intracellulare was better achieved by maintaining the cultured cells in a steady state of growth. Therefore, culture cell monolayers should be approximately 20-50% confluent at the time of seeding. More preferably, the cells should be about 30-40% confluent at the time of seeding, with an optimal confluence of about 30%.

The inoculum may be a pure Lawsonia intracellulare culture obtained, for example, from ATCC Deposit No. 55672, NCTC Deposit Nos. 12656 or 12657, or from infected pigs or other animals using the isolation and purification techniques described herein pure culture.

According to one example, the inoculum used to practice the invention is a homogenate of intestinal tissue prepared by scraping the ileal mucosa of a pig or other animal infected with PPE. When preparing intestinal homogenates, the portion of the ileum selected for culture should show severe damage with marked thickening. Due to the delicate nature of bacteria, samples should preferably be stored at -70°C as soon as possible after autopsy. Antibiotics to which L. intracellulare has developed resistance are preferably added to the inoculum to inhibit the contaminating bacteria while allowing the growth of L. intracellulare. Examples of such antibiotics include vancomycin, amphotericin Antibiotics B or members of the aminoglycoside antibiotic class such as gentamicin and neomycin. Regardless of whether the inoculum is a pure culture or a homogenate of intestinal tissue, inoculation of 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 reduced dissolved oxygen concentrations. When the dissolved oxygen concentration was greater than 18%, L. intracellulare grew less than optimal and eventually stopped growing at oxygen concentrations outside 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%, and optimally, an oxygen concentration of about 0-3.0%.

Proper concentration of carbon dioxide is also critical for proper growth of L. intracellulare. When the concentration of carbon dioxide is greater than 10% or less than 4%, non-optimal growth occurs and eventually growth stops when the carbon dioxide exceeds this range. Preferably, the concentration of carbon dioxide is about 6-9%, 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 can 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.

Inoculated cells can be incubated in a dual gas incubator or other gas chamber that contains appropriate concentrations of oxygen and carbon dioxide and allows cells to be suspended during incubation. The chamber shall contain means to maintain the seeded cells in suspension, a gas monitor, and a supply to provide and maintain the appropriate gas concentration. The incubation temperature should be 30-45°C, preferably 36-38°C. Optimally, the temperature is about 37°C. The equipment required for the cultivation and attenuation methods of the present invention will be readily available to one of ordinary skill in the art given the teachings herein. An example of an apparatus suitable for practicing the invention is a dual gas incubator, such as model 480 available from Lab-Line, Melrose Park, Illinois, combined with a spinner flask to maintain the cells in suspension. Presently preferred devices include fermenters, bioreactors, or rotary shakers that contain at least about 2 liters of medium and are capable of maintaining cultured cells in suspension by sparging appropriate concentrations of gas or by other means of mechanical agitation and capable of Continuously monitor the level of dissolved oxygen in the medium. New Brunswick, Braun and other companies manufacture suitable fermenters and bioreactors for this purpose.

By maintaining the inoculated cells in suspension during incubation, the maximum growth of the cells, and thus Lawsonia intracellularis, is achieved by increasing the exposure of each cell to the growth medium and the mixture of oxygen and carbon dioxide . Cells can be cultured with agitation and maintained in suspension by various methods known in the art, including, for example, culture flasks, roller bottles, membrane culture vessels, or spinner bottles. Cells can be kept in suspension during the incubation by incubating the cells in spinner flasks in a dual gas incubator or similar device. As used herein, the term "spinner bottle" refers to a bottle 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 inoculated cells are incubated until the cells reach confluence, and then placed in spinner flasks containing growth medium and incubated in a dual gas incubator while spinning the spinner flask. Preferably, the seeded cells are scraped into a spinner flask. This can be accomplished by various methods known in the art, such as using a cell scraper to dissociate the cells. Once the cells are introduced into the spinner flask, the paddles of the spinner flask are mechanically rotated, typically at a speed of about 30-60 rpm, to maintain the infected cells in suspension.

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

Passage of L. intracellulare in suspension cultures can be achieved by removing a portion of the original culture and adding it to a new bottle containing freshly cultured cells. If the original culture has a high number of bacteria/ml value, e.g. greater than about ¹⁰⁴ bacteria/ml, it is preferred to add about 1-10% (by volume) of the culture from the infected bottle to a new bottle containing fresh cells middle. This is best done when 50-100% of the cells are infected. If less than 50% of the cells are infected, subculture is best achieved by splitting the culture in half into new bottles, then adding new medium to the desired volume. In any case, cell lysis and other steps are not required, in contrast to prior art passaging of monolayer cultures.

At least a portion of the cultured L. intracellulare bacteria is harvested after the cultured cells have grown sufficiently and subsequently infected with L. intracellulare at a cell infection rate of greater than about 70% (as determined by IFA, _TCID50 , or other similar method). 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. intracellulare bacteria are harvested by centrifuging all or part of the contents of the suspension to pellet the cultured cells, resuspending the formed cell pellet, and lysing the infected cells. Typically, at least a portion of the contents are centrifuged at about 3000 xg for about 20 minutes to pellet the cells and bacteria. The pellet is resuspended, eg, in a sucrose-phosphate-glutamate (SPG) solution, and passed through a 25 gauge needle about 4 times to lyse the cells. If further purification is required, samples can be centrifuged at approximately 145 x g for approximately 5 minutes to remove nuclei and debris. The supernatant is centrifuged at about 3000 x g for about 20 minutes, and the resulting pellet is resuspended in a suitable diluent, such as SPG with fetal bovine serum (to prepare harvested bacteria suitable for refrigeration or use as an inoculant) or Growth medium (to prepare harvested bacteria more suitable for passaging to fresh cells).

As described above, efficient growth of L. intracellulare for large-scale production can be enhanced by actively growing tissue cells. For monolayers, the rate of cell division drops dramatically when the culture is confluent. Attempts to grow L. intracellulare on monolayer tissue culture have met with limited success, and large-scale production has not been possible. However, active cell growth is greatly facilitated by suspension culture and allows continuous culture growth and scale-up. With a fermenter and the aforementioned ~0-3% dissolved oxygen, we have been able to grow up to ¹⁰⁸ bacteria/ml. We have also grown cultured bacteria actively for many months and expect to continue indefinitely.

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

For HEp-2 cultures, it is best to remove 25-50% of the culture weekly and replace it with fresh medium for maintenance purposes. For cell culture with microcarriers or beads, preferably 1-2 times per week, 25-50% of the culture is removed and replaced with fresh microcarriers or beads and fresh medium. For scale-up, 25-50% additional medium or medium with microcarriers can be added to the culture.

Passaging to fresh cells is typically done about every 2-5 weeks, depending on how quickly the cultured cells are infected. Passaging is preferably performed at about 3-4 weeks, assuming at least 70% of the cultured cells are infected within 2-3 weeks.

The invention also provides a vaccine against Lawsonia intracellulare and a method for producing the vaccine. According to a particularly preferred example, after maintaining the infected cells in suspension for a prolonged period (eg 6-8 months), at least a portion of the cultured Lawsonia intracellulare bacteria is harvested and monitored for a potential attenuating effect. Such monitoring is preferably accomplished by immune challenge of host animals or animal models to select for attenuated strains. Such attenuated strains can be used in vaccines in the methods described herein. The attenuated L. intracellulare vaccines of the present invention have been shown to be effective against L. intracellulare infection in various animals and are expected to be effective in humans as well.

The present invention allows rapid culture expansion, such as a 100-1000-fold increase in yield, and reduces costs. As a result, sufficient L. intracellularis bacterial stock produced by the culture method of the present invention can be readily attenuated for use in vaccine production. Attenuation in monolayer cultures is difficult because of the low yields of bacteria produced using traditional monolayer growth techniques. In contrast, the method of growing L. intracellulare of the present invention increases the ease, speed, and number of bacteria available for attenuation. The more cells and cells divide, the higher the level of mutations that occur, which is beneficial for vaccine development. Growth in the suspension of the present invention increases the expression of important immunogens and their expression products controlled by environmentally regulated genes.

The resulting attenuated strains can be cultured in tissue culture monolayers as described in Example 1 below, but are preferably cultured in suspension cultures according to the invention. Other methods of attenuation include chemical attenuation such as by use of N-methylnitrosoguanidine and other attenuating substances known in the art. Whether by multiple passages or chemically, attenuated L. intracellulare can be produced and selected for vaccine preparation.

According to an example vaccine of the present invention, the antigen is harvested by centrifugation or microfiltration as described above. Antigens are then normalized to a level determined by dose potency in the host animal species based on the optimal host animal immune response. Killed vaccines can be prepared by inactivating the bacteria by exposing them to ambient oxygen concentrations for an extended period of time, such as a week, or by inactivating them with 0.3% formalin or other inactivating agents. The antigen is then incorporated into a suitable adjuvant such as aluminum hydroxide or mineral oil to enhance the immune response. Next, the antigen is used to immunize the host by intramuscular or subcutaneous injection, and for piglets of about 3-4 weeks, a booster dose can be used if necessary.

Alternatively, according to a particularly preferred example of a vaccine using the culture method described above, the bacteria are serially passaged to induce and select for attenuated, avirulent live cultures. The cultures are tested in host animals (preferably after at least 6-8 months of growth in suspension culture) for attenuation. Cultures were harvested and diluted as previously described. For example, pigs can be immunized by oral administration of 1 x 10 ⁵ -1 x 10 ⁶ bacteria. Twenty-eight days after immunization, pigs were inoculated orally with 1 x 10 ^<7> passages of lesser (approximately 30 to 40 days), virulent cultures of L. intracellularis. Infected animals were necropsied 21 days after immune challenge, and macrolesions and microlesions were observed in the small intestine. PCR and fluorescent antibody (FA) analysis should also be performed. About 80% of the control animals had macro or micro lesions and tested positive for the presence of L. intracellulare in the mucosal cells of the intestine by PCR or FA testing methods. Immunized animals had a normal mucosal surface as determined by histology and were negative in PCR testing.

Generally, an attenuated immunogenic L. intracellulare strain is produced by continuous culturing for at least about 150-250 days with passages of the culture at least about 7-12 times therebetween. Alternative attenuated cultures can also be generated by varying these parameters, as long as the monitoring and selection methods described herein are used.

A vaccine is then prepared comprising an immunologically effective amount of the attenuated L. intracellulare and a pharmaceutically acceptable carrier. The combined immunogen and carrier can be aqueous solutions, emulsions and suspensions. Immunologically effective amounts can be determined by methods known in the art without undue experimentation, given the teachings herein. Typically, the amount of immunogen is 50-500 µg, and when purified bacteria are used, 10 ⁷ -10 ⁹ TCID ₅₀ .

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

Effective diagnosis of PPE is also hampered by the time required to culture the causative bacteria. As a result of the present invention, it is now possible to develop diagnostic tools for the rapid and accurate analysis of biological samples from pigs or other animals at risk of PPE for the presence of Lawsonia intracellulare.

Lawsonia intracellulare bacteria grown according to the method of the present invention, or components derived from the bacteria, can be used as antigens in ELISA or other immunoassays such as immunofluorescent antibody assays (immunofluorescent antibody, IFA), to detect bacteria that may be affected by the bacteria. Antibodies against Lawsonia intracellulare in the serum or other body fluids of animals infected with the bacteria. A presently preferred immunoassay is IFA as described in the Examples below. Alternatively, bacteria grown by the method of the present invention are used for Western Blot analysis.

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

1. Add 0.1 ml/well of antigen diluted in coating buffer. Incubate at 4°C for 18 hours.

2. Wash 3 times with PBS.

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

4. Wash 3 times with wash buffer.

5. Dilute the serum in blocking buffer and add 0.1 ml to the first row of wells of the plate. Serial 1:2 dilutions were performed on the plate. Incubate for 1 hour at 37°C.

6. Wash 3-5 times with wash buffer.

7. Dilute the conjugate in blocking buffer and add 0.1 ml to each well of the plate. Incubate for 1 hour at 37°C.

8. Wash 3-5 times with wash buffer.

9. Add substrate.

12. Measure the absorbance with a spectrophotometer.

13. Wells with no added antigen were used as blank controls.

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

A preferred Western blotting protocol is as follows:

1. Electrophoresis of the antigen on 12% SDS-PAGE, and then transfer it to a nitrocellulose membrane.

2. Place the membrane in blocking buffer for 2 hours.

3. Remove blocking agent and rinse with PBS for 1 minute.

4. Dilute serum in blocking buffer and add to membrane. Incubate for 2 hours at room temperature.

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

6. Dilute the conjugate in blocking buffer and apply to the membrane. Incubate for 1 hour at room temperature.

7. Wash 3 times with wash buffer.

8. Add substrate for 10 minutes until strong binding occurs.

9. Rinse with PBS.

10. Air dry and store in the dark.

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

Example 1

Isolation of Lawsonia intracellulare from the gut of American pigs with porcine proliferative enteropathy

Materials and methods

Selection of Inoculation Samples

Obtaining sample N24912 in a herd on a farm in Iowa, 15 of 300 5-month-old finisher pigs were observed to have consistently bloody stools despite treatment with penicillin. After autopsy of the pig, the intestine (ileum) was found to have a thick mucosa. Histopathological examination with silver staining revealed the presence of curved, intracellular bacterial and crypt intestinal epithelial cell proliferation, confirming the diagnosis of PPE. Sample N72994 was obtained from a 1.5-year-old second-parity SPF sow on a farm in Minnesota. The herd size was 70-80 sows and the antibiotic treatment used was unknown. Autopsy revealed thickened and somewhat hemorrhagic ileal mucosa. Giminez staining of the mucosa revealed the presence of numerous Curlybacteria. Sample N101494 was obtained from pigs aged 12 weeks on a farm in Indiana (600 acres with sows). The pig was treated with Tylan injection after developing bloody dysentery, but the animal died shortly after treatment.

Prepare inoculum from pigs:

Intestinal samples were stored at -70°C. Intestines were dissected and washed with phosphate buffered saline (PBS). One gram of mucosa was scraped into sodium potassium glutamate (SPG) and then homogenized in SPG with 4.0 mL of 0.1% Trypsin (JRH Biosciences, Lenexa, KS) for 30 s. The suspension was incubated at 37°C for 35 minutes. 10 ml of SPG/10% fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS) was added, followed by grinding for 1 min on a tissue grinder. 10 mL of SPG/10% FCS was added and then filtered once with filter paper (Whatman 113V; Whatman Labsales, Hillsboro, OR) followed by 5.0, 1.0 and 0.65 micron filters. The filtrate was divided into 1.0 mL portions and stored at -70°C. Mucosa were spread on glass slides for Giminez staining. Each smear of the filtrate was passed by the IFA method with a specific monoclonal antibody against Lawsonia intracellulare (S.McOrist et al., Vet.Rec.121:421-422, the entire contents of which are hereby incorporated by reference ) for coloring.

cell culture

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

Infection of cell cultures

IEC-18 cells were seeded in ²⁵ cm flasks at 1.25 x ¹⁰⁵ cells and seeded on chamber slides (Nunc, Inc., Naperville, Il) at comparable ratios, incubated for 24 hours, and then removed from the culture. base. Thaw frozen bacterial isolates from porcine quickly and dilute in DMEM/7% FCS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml) to 1.0 ml tissue homogenate For 15 ml medium, then add to monolayer. The monolayer and bacterial suspension were centrifuged at 2000 g for 30 minutes and transferred to anaerobic jars. The tank was emptied and the air was replaced by hydrogen and carbon dioxide to give a mixture of 8.0% oxygen, 10% carbon dioxide and 82% hydrogen. The culture was incubated at 37°C for 3 hours, and then fed with DMEM/ 7% FCS. The cultures were returned to the anaerobic tank and incubated for 6 days with medium changes every 2 days.

Passaging of Lawsonia intracellulare

According to the method previously described in G.Lawson et al. in J.Clin.Microbiol., 31:1136-1142 (1993) (the entire content of this document is cited as a reference), the cells are lysed with potassium chloride to cause intracellular labor. Senia bacteria were passaged and added to fresh IEC-18 monolayers. The medium was poured off the monolayer, 0.1% potassium chloride was added, and the cells were incubated at 37°C for 10 minutes. KCl was removed, SPG/10% was added, and the monolayer was dissociated with a cell scraper. Cells were lysed 3 times through a syringe with a 21 gauge needle. 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

Infection was monitored by fixing cells on chamber slides with cold acetone/methanol for 5 min. Staining was performed using immunofluorescence and immunoperoxidase methods. Both methods employ a mouse monoclonal antibody (as described by S. McOrist et al. in Vet. Rec. 121:421422 (1987)) as the primary antibody, and an anti-mouse IgG-fluorophore conjugate (fluorescein isothiocyanate; Organon Teknika Corporation, Durham, NC) or peroxide conjugates (goat anti-mouse immunoglobulin G; Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD). Quantitative analysis of bacteria is accomplished by counting the number of specifically stained bacteria in cells on each slide.

polymerase chain reaction

The sample inoculum and subcultured bacteria were combined as template DNA for PCR using sample preparation methods, primers and cycle parameters as described in Jones et al. in J.Clin.Microbiol., 31:2611-2615 (1993) and McOrist et al. in Vet. Microbiol. 1-8 (1994) (the contents of both documents are incorporated by reference in their entirety). The first cycle in the cycle parameters was 93°C for 5 minutes; 55°C for 45 seconds; and 72°C for 45 seconds. Thirty-three cycles were performed at each of the above temperatures for 45 seconds, and one cycle of: 93°C, 45 seconds; 55°C, 45 seconds; and 72°C, 2 minutes. Positive inoculum was only used to inoculate IEC-18 cells. PCR was also performed to monitor passage material to confirm infection. DNA generated by PCR was sent to the Iowa State University Nucleic Acid Facility for sequencing. The sequencing results were compared to the sequence obtained by Gary F. Jones and reported in his doctoral dissertation (University of Minnesota, Minneapolis, MN (June 1993)).

result:

Select an inoculum sample:

Pig numbers N24912 and N72994 had severe PPE with bloody intestinal contents and thickened mucosa. N101494 suffered from severe PPE and severe diarrhea that resulted in the formation of large blood clots in the intestinal lumen. Giminez's staining of mucosal smears revealed numerous curved or S-shaped bacteria. IFA staining revealed the presence of a large number of strongly fluorescent bacteria in the inoculum from pigs.

To monitor infection of cell cultures:

The seeded monolayer was monitored by light microscopy during growth and slight changes in cell morphology were observed. Uninfected monolayers grown at lower oxygen concentrations (8% _O2 ) had similar morphology.

Infected cultures stained by immunofluorescence and immunoperoxidase showed a significant number of curved or S-shaped bacteria within the cells. Monolayers are free of confluent infection. Infected cells are often tightly connected, with 1-10 cells in the center. It was also seen that heavily infected cells (ie carrying 30 or more bacteria) were also associated with cells carrying less than 30 bacteria. The number of bacteria reached its peak around 6 days. Infection depends on specific growing conditions. Bacteria were successfully passaged by the lysis procedure described here. Centrifugation of freshly plated cells is not required, but doing so will increase the number of infected cells. Centrifugation also reduces the possibility of contamination by placing cells exposed to infection in antibiotic-free medium for 3 hours in antibiotic-containing medium. To reduce the growth rate of IEC-18 cells so that bacteria can multiply to higher numbers before the monolayer becomes confluent, it is necessary to reduce the FCS concentration in the medium from 10% to 7%.

Polymerase Chain Reaction:

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

Example 2

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

Prepare intestinal homogenate for inoculation:

Intestinal tissue homogenates were prepared by scraping the mucosa from 6.0-8.0 cm of the ileum in the intestinal samples of Example 1. Trypsin (1%) was added to the scraped mucosa and samples were briefly homogenized and then incubated at 37°C for 35 minutes. Another 10 ml of SPG/10% FBS was added and the samples were ground in a tissue grinder. Another 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 filter membranes. The filtrate was divided into 1.0 ml portions and stored at -70°C.

Infection of cell cultures

Method A:

Tissue cells were inoculated at 1×10 ⁷ cells in a 100 ml spinner flask containing 50 ml DMEM/10% FBS. Cultures were incubated for 24 hours before addition of vancomycin and amphotericin B. A vial of frozen intestinal homogenate was quickly thawed and diluted in 3.0 ml DMEM/5% FBS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). Samples were passed through a 0.65 μm filter and added to vials. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. Cultures were incubated at 37°C for 3 hours before neomycin and gentamicin were added. Then within 24 hours, the culture was fed with L-glutamine, vancomycin (100 μg/ml), neomycin (50 μg/L), gentamicin (50 μg/L) and amphotericin B (2.0 μg/ml) in DMEM/5% FBS.

Method B:

Seed HEp-2 cells at 1.25×10 ⁵ cells in a 25 cm ² regular flask containing DMEM/10% FBS, and incubate for 18-24 hours. Cells were 30% confluent at the time of seeding. Inoculum was diluted in DMEM/5% FBS. When the inoculum was from intestinal homogenate, the medium also contained vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. Cultures were incubated at 37°C for 3 hours before neomycin and gentamicin were added. Then within 24 hours, the culture was fed with L-glutamine, vancomycin (100 μg/ml), neomycin (50 μg/L), gentamicin (50 μg/L) and amphotericin B (2.0 μg/ml) in DMEM/5% FBS. Antibiotics are not required when the inoculum is a pure culture. Cultures were incubated for 6 days or until confluent. Cells were scraped from the flask and added to 100 mL spinner flasks containing 50 mL DMEM/10% FBS.

Cultures were diluted 1:2 weekly by harvesting half of the culture and adding fresh medium, or by transferring the culture to a larger spinner bottle and adding more medium.

Subculture of cultures:

Fresh HEp-2 cells were inoculated in DMEM/5% FBS at 1×10 ⁷ cells, and the culture was passaged to fresh HEp-2 cells. Fresh cultures were incubated overnight in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. New cultures were then inoculated with infection cultures and incubated at the lower oxygen concentrations described above. The number of inoculum depends on the degree of infection of the original culture.

Harvest and storage of cultures:

Harvest the culture by collecting the desired number of cultures and centrifuging at 3000 xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamate (SPG) solution and passed through a 25 gauge needle 4 times. The culture was aliquoted and frozen at -70°C. For further purification, samples can be centrifuged at 145 x g for approximately 5 minutes to remove nuclei and debris. The supernatant was centrifuged at 3000 xg for 20 minutes, and the pellet was resuspended in diluent.

Estimation of live Lawsonia intracellulare in tissue culture:

The quantification of live Lawsonia intracellulare was accomplished by determining the tissue culture infectious dose (50%) (TissueCultureInfectious Dose 50 percent, TCID ₅₀ ). This is done by removing 2.0 ml of the culture to be tested and lysing the cells 4 times through a 25 gauge needle. Samples were serially diluted 1:10 in DMEM/5% FBS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The dilution was added to a 96-well microtiter plate at 0.1 ml per well. Microtiter plates were seeded with HEp-2 cells at 1250 cells/well and grown for 18-24 hours prior to infection. Use 3-6 wells for each dilution. Plates were incubated for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. Fix the cells with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well anti-IS intracellularis monoclonal antibody (McOrist, 1994) diluted 1:2000 in PBS. Plates were incubated at 37°C for 30 minutes and then washed 3 times with PBS. Anti-mouse FITC diluted 1:30 was added at 0.03 ml/well, and incubated at 37°C for 30 minutes. Plates were washed 3 times with double distilled water and allowed to dry. Samples were observed with a fluorescence microscope to determine TCID ₅₀ /ml.

result:

_TCID50 results showed that the culture could contain up to 1 x ¹⁰⁶ bacteria/ml. This was achieved within 45 days. The culture volume was expanded to 3.0 liters during the same time period.

Example 3

Growth of Lawsonia intracellulare in suspension cultures of McCoys cells

Prepare intestinal homogenate for inoculation:

According to the method described in Example 2, intestinal tissue homogenate was prepared. The Lawsonia intracellulare sample cultivated by the method of the following examples was deposited in the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland U.S.A 20852) on May 19, 1995 according to the Budapest Treaty, and the deposit number is 55672.

Infection of cell cultures

McCoys cells were inoculated in two 25 cm ² regular flasks containing DMEM/10% FBS as 1.25×10 ⁵ cells and incubated for 18-24 hours. Cells were 30% confluent upon seeding. Inoculum was diluted in DMEM/5% FBS. When the inoculum was from intestinal homogenate, the medium also contained vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture of 8.0% oxygen, 10% carbon dioxide, and 82% hydrogen. Cultures were incubated at 37°C for 3 hours before neomycin and gentamicin were added. Then within 24 hours, add L-glutamine, vancomycin (100 μg/ml), neomycin (50 μg/L), gentamicin (50 μg/L) and amphotericin B (2.0 μg/ml). ml) of DMEM/5% FBS to feed the culture. Antibiotics are not required when the inoculum is a pure culture. Cultures were incubated for 6 days until confluent. Cells were scraped from the flask and added to a 100 ml spinner flask containing 50 ml DMEM/2% FBS and 0.05 g Cultisphere-G microcarriers. Stir the bottle at 40-50 rpm.

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

Subculture of cultures:

Cultures were passaged to fresh McCoys cells by inoculating fresh McCoys cells at 1×10 ⁷ cells in DMEM/5% FBS and 0.05 g of Cultisphere-G beads. Fresh cultures were incubated overnight in 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. New cultures were then inoculated with 25 ml of infection culture and incubated at the lower oxygen concentrations described above.

Harvest and storage of cultures:

Harvest the culture by collecting the desired number of cultures and centrifuging at 3000 xg for 20 minutes. The pellet was resuspended in SPG and passed 4 times through a 22 gauge needle. The culture was aliquoted and frozen at -70°C. For further purification, samples can be centrifuged at 145 xg for approximately 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.

Estimation of live Lawsonia intracellulare in tissue culture:

Quantification of viable Lawsonia intracellularis was performed as described in Example 2, except that the cells were lysed with a 22-gauge needle, and McCoys cells were used to inoculate 1250 cells/well in a microtiter plate.

result:

_TCID50 results showed that the culture contained up to 1 x ¹⁰⁶ bacteria/ml. This was achieved in less than 1 month. The culture volume was expanded to 3.0 liters during the same time period.

Example 4

Determining the infectious dose of a pure culture of Lawsonia intracellulare in host animals

summary:

The study of 31 pigs was done by infecting 6 week old normal pigs with a pure culture of L. intracellulare from sample N72994. The pigs were randomly divided into 4 groups and each group was individually penned. Group 1 contained 7 pigs and served as a negative control group (infected or not infected with uninfected tissue culture). Group 2 contained 8 pigs and the infection dose was 10 ⁷ bacteria/pig. Group 3 contained 8 pigs and the infection dose was 10 ⁶ bacteria/pig. Group 4 contained 8 pigs and the infection dose was 10 ⁵ bacteria/pig.

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

PCR tests on the ileal mucosa revealed the presence of Lawsonia intracellulare in 100% of the high dose group, 75% of the medium dose group and 50% of the low dose group. Histopathological analysis of the data showed that increased numbers of monocytes in the lamina propria and submucosa occurred in 88% of the high dose group, 75% of the middle dose group and 88% of the low dose group. Crypt hyperplasia was observed in 50% of the high dose group, 63% of the middle dose group and 50% of the low dose group. FA staining revealed the presence of Lawsonia intracellulare in 88% of the high dose group, 63% of the middle dose group and 63% of the low dose group of ileum, jejunum and colon tissue sections. Control animals were negative for the presence of L. intracellulare by PCR, FA and silver stain analysis.

Altogether, pure cultures were successfully used to infect and cause PPE damage. Koch's hypothesis was confirmed by isolating and characterizing L. intracellulare from infected animals.

Among the immune challenged animals, 100% of the animals in the high dose group were cured and species confirmed by silver staining, FA and PCR.

Materials and methods:

Growth of inoculum:

3.75×10 ⁵ HEp-2 cells were seeded in a 75 cm ² conventional flask containing DMEM/10% FBS, and incubated at 5% carbon dioxide and 37°C for 18-24 hours. (Cells were 30% confluent at the time of seeding). Dilute one vial of N72994 in 15 ml DMEM/5% FBS. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. Cultures were then fed DMEM/5% FBS within 24 hours.

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

Harvesting of cultures:

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

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

Quantification of Lawsonia intracellulare:

Quantification of viable L. intracellularis was accomplished by determining the 50% tissue culture infectious dose ( _TCID50 ). This is done by removing 2 ml of the culture to be tested and lysing the cells 4 times through a 22 gauge needle. Samples were serially diluted 1:10 in DMEM/5% FBS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The dilution was added to a 96-well microtiter plate at 0.1 ml per well. Microtiter plates were seeded with HEp-2 cells at 2500 cells/well and grown for 18-24 hours prior to infection. Twelve wells were used for each dilution. Plates were incubated for 6 days at gas concentrations of 8.0% oxygen, 8.8% carbon dioxide and 83.2% hydrogen. Fix the cells with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well anti-L. intracellularis monoclonal antibody (McOrist, 1987) diluted 1:2000 in PBS. Plates were incubated at 37°C for 30 minutes and then washed 3 times with PBS. Anti-mouse FITC diluted 1:30 was added at 0.03 ml/well, and incubated at 37°C for 30 minutes. Plates were washed 3 times with double distilled water and allowed to dry. Samples were observed with a fluorescence microscope to determine TCID ₅₀ /ml.

animal:

31 PIC x Lieske sows and Large White boars of all sexes at 6 weeks of age were provided by Dr Kent Schwartz. On day 0, pigs were randomly assigned by weight to 4 pens.

equipment:

Pigs were housed in 4 pens in a small nursing facility, spaced at least 3 feet apart. The pens have wire floors and solid partitions. Heating is provided by a stove and locally supplemented by exothermic bulbs. During the study, the temperature was maintained between 78-85.

feed and water

Antibiotic-free, ground corn-soybean feed with a protein content of 19% was supplied ad libitum via a stainless steel feeder. Water was supplied arbitrarily through water supply nozzles.

Infections in pigs:

On day 0, pigs were weighed and blood samples were collected via capillaries in the retroorbital sinus. Serum was collected and stored at -20°C. Fecal samples were also collected for PCR. Pigs were administered 10 ml of the inoculum intragastrically using a gastric tube.

Processing method number of pigs Control - uninfected cells 5 Control - untreated 2 high dose 8 medium dose 8 low dose 8

Pigs were weighed and blood samples collected on days 0, 10, 17 and 24.

Polymerase Chain Reaction:

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

Histopathology:

Sections of the ileum, jejunum, and colon were fixed in formalin, processed conventionally, stained with hematoxylin and eosin, and impregnated with silver, and evaluated. Sections were also stained with a monoclonal antibody specific for L. intracellulare.

result:

Clinical symptoms:

On day 3, clinical symptoms including loose stools were observed in the high dose group. It peaked at 14 days and then eased.

Weight gain:

The average daily weight gain was calculated and 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 body weight gain when comparing the groups.

PCR:

Fecal dripping was not observed until 14 days. At 21 days, 37.5% of the feces of pigs in the high dose group were PCR positive. After dissection, the ileal mucosa was checked by PCR, and the positive rate was: 100% in the high-dose group, 75% in the middle-dose group, 50% in the low-dose group and 0% in the control group.

Serious injury:

Two pigs were found to have severe lesions in the high dose group (#50 and #202). These 2 pigs had approximately 3 feet of thickening in the ileum and necrosis in #202.

Histopathology:

FA:

FA staining of sections of the ileum, jejunum and colon revealed the presence of L. intracellulare in 87.5% of the high dose group, 62.5% of the middle and low dose groups, and 0% of the control group.

Micro damage:

Injuries were observed in 100% of the high-dose group, 75% of the middle-dose group, 87.5% of the low-dose group and 14% of the control group. This was determined by observing increased numbers of monocytes in the lamina propria and submucosa, a condition often associated with Peyer's Patchers hyperplasia. Crypt hyperplasia was also observed.

Silver stain:

Sections were also silver stained for the presence of intracellular, curved bacteria. The results showed that bacteria were present in 87.5% of the high-dose group, 62.5% of the middle-dose group, 87.5% of the low-dose group and 0% of the control group.

discuss:

Pigs were successfully infected with pure L. intracellulare cultures. At ¹⁰⁷ doses of bacteria, 100% of the pigs were infected as determined by PCR and microscopic observation of the lesions. The severity of the injury and the number of bacteria in the tissue sections were quite low. This study is a satisfactory model for L. intracellulare immune challenge because of the presence of L. intracellulare and microlesions in pigs. Injuries were increased when a second dose was given 7 days after the first dose.

Example 5

Hamster Vaccine Effectiveness Test

Purpose:

Evaluation of a laboratory animal model to determine the safety and efficacy of an avirulent live Lawsonia intracellularis vaccine in hamsters.

summary:

A study of 40 hamsters was completed by inoculating 3-week-old hamsters with a pure culture of a high-passage Lawsonia intracellulare strain, followed by immune challenge with low-passage virulent material 22 days after inoculation . Hamsters were divided into 3 groups. Group A was inoculated on day 0 with 1 dose of L. intracellulare strain N72994. Group B served as a control group without the use of vaccine cultures. At 22 and 25 days after inoculation, the two groups were challenged with 2 doses of a pure culture of L. intracellulare strain N343. Panel C used challenger strain N101494 to compare relative virulence with strain N343. Groups A and B each had 15 hamsters, and Group C had 10 hamsters. The 50% tissue culture infectious dose ( _TCID50 ) data revealed that hamsters were inoculated with ¹⁰⁵ _TCID50 /dose. N343 immune challenge contains 10 ⁵ .5 TCID ₅₀ / dose. The challenge dose of group C was 10 ² .75 TCID ₅₀ /dose. Fecal samples were collected on days 0, 7, 14, 21, 29, 36 and 43 for PCR assays. On day 5, 5 animals from groups A and B were dissected for PCR examination of the mucosa and FA, hematoxylin and eosin staining of intestinal sections, and silver staining to determine bacterial colonization in the inoculated hamsters continuity. The remaining animals were dissected and similarly tested 21 days after challenge.

PCR data showed that Lawsonia intracellulare was present in 100% of the intestinal mucosa of group A hamsters 21 days after inoculation. Group B was negative 21 days after inoculation. Twenty-one days after challenge, 50% of the hamsters in group A were PCR positive and 100% of the hamsters in group B were positive. 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 B 21 days after challenge. No animals showed lesions 21 days after inoculation. Group C animals were uninjured 21 days after challenge. Neither FA nor silver staining could reveal the presence of L. intracellulare in the sections.

In conclusion, PCR showed that a 50% reduction in infection was observed when hamsters were inoculated with high passage L. intracellulare strains. The intestine was colonized by a small number of intracellular organisms (referred to as Lawsonia intracellularis), as this organism was not observed in FA and silver-stained sections. Group C hamsters showed no infection throughout the study, most likely due to the low bacterial dose.

Materials and methods:

For hamsters:

Forty 3-week-old female hamsters from Harlan Sprague Dawley were used.

Growth of inoculum:

Vaccine cultures:

Continuous cultures of L. intracellulare grown in HEp-2 cells for 29 weeks were used. The cultures were grown in a similar manner as described in the Challenge Cultures section, except that the cultures were passaged to new HEp-2 cells every 2-3 weeks.

To stimulate cultures:

Inoculate 3.75×10 ⁵ McCoys cells in 75 ^cm conventional tissue culture flasks in DMEM medium (Dulbecco's Modified Eagle's medium) containing 10% fetal bovine serum (FBS), and incubate at 37°C and 5% carbon dioxide for 18- 24 hours. The medium was separated from the cells, and a vial of N343 MSC X diluted in 14 ml DMEM/2% FBS was added to the vial. The culture was placed in an air tank, evacuated, and refilled with hydrogen and carbon dioxide to obtain a mixture of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen. Cultures were grown for 6 days and cells were scraped from flasks and added to 100 ml spinner flasks containing 90 ml DMEM/2% FBS and 0.01 g Cutispere-G beads. Cultures were grown at the gas concentrations mentioned above. The flask culture volume was expanded weekly by doubling the medium volume. Cultures were grown in spinner flasks for 25 days to a final volume of 250 ml.

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

To harvest the culture:

Vaccine cultures:

Cultures were harvested by centrifugation at 3000 xg for 20 minutes. The pellet was resuspended in a sucrose-phosphate-glutamate (SPG) solution containing 10% FBS and passed through a 25 gauge needle 4 times. The inoculum was diluted to final volume (15 mL) in SPG/10% FBS.

To stimulate cultures:

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

Dosage for hamsters:

vaccine:

On day 0, all hamsters in group A were orally inoculated with 1 ml of the prepared vaccine.

excitation:

21 days after inoculation, 0.5 ml of challenge culture strain N343 was orally administered to 10 hamsters of group A and 10 hamsters of group B. Group C was challenged with 0.5 ml of challenge culture strain N101494.

Quantification of ISi:

Quantification of viable ISi was accomplished by determining the 50% tissue culture infectious dose (Tissue culture InfectiousDose 50 percent, TCID ₅₀ ). This is done by removing 2 ml of the culture to be tested and lysing the cells 4 times through a 22 gauge needle. Samples were serially diluted 1:10 in DMEM/5% FBS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The dilution was added to a 96-well microtiter plate at 0.1 ml per well. Prior to infection, the plates had been seeded with McCoys cells at 1250 cells/well and grown at 37°C and 5% carbon dioxide for 18-24 hours. Twelve wells were used for each dilution. Plates were incubated for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Fix the cells with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well anti-ISi monoclonal antibody diluted 1:2000 in PBS. Plates were incubated at 37°C for 30 minutes and then washed 3 times with PBS. Anti-mouse FITC diluted 1:30 was added at 0.03 ml/well, and incubated at 37°C for 30 minutes. Plates were washed 3 times with double distilled water and allowed to dry. Samples were observed with a fluorescence microscope to determine TCID ₅₀ /ml.

Surveillance for infection in hamsters:

Infection in 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 after inoculation. After the hamsters were sacrificed, their intestinal mucosa was also examined by PCR.

Histopathology:

Sections of the ileum and colon were fixed in formalin, processed conventionally, stained with hematoxylin and eosin and impregnated with silver, and evaluated. Sections were also stained with a monoclonal antibody specific for L. intracellulare.

Average daily weight gain:

Hamsters were weighed at 21, 28, 35 and 42 days after inoculation to determine average daily body weight gain. result:

See table below.

TCID ₅₀ :

TCID ₅₀ data showed that the vaccine group (group A) received 10 ⁴ .86 TCID ₅₀ /hamster. Hamsters in group A and group B were challenged with strain N343, and the dose received was 10 ⁵ .5 TCID ₅₀ /hamster. Group C hamsters challenged with strain N101494 received 10 ² .75 TCID ₅₀ /hamster.

PCR:

PCR testing showed the presence of L. intracellulare in 100% of the vaccinated hamsters dissected 21 days after inoculation. Tests 43 days after inoculation showed that 100% of the control hamsters and 50% of the vaccinated hamsters were infected with L. intracellulare. None of the hamsters challenged with N101494 were PCR positive. Fecal dripping was not observed in this study with hamsters.

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 group had mild to severe lymphocytic enteritis, and 50% of the control group had mild lymphocytic enteritis. No damage was found in the N101494 challenge group.

FA staining failed to reveal the presence of L. intracellulare in hamsters 43 days post-inoculation.

discuss:

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

Excreta Excreta Excreta Excreta Excreta Excreta Excreta Mucosa Microtrauma

Mucosal PCR H&E staining FA

PCR PCR PCR PCR PCR PCR PCR Silver staining

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

A-1 - - - - - - - - - - - NA + Mild enteritis - - -

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

A-3 - - - - - - - - - - - NA + Mild enteritis - - -

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

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

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

A-7 - - - - - - - - - - - NA + Severe enteritis - - -

A-8 - - - - - - - - - - - NA + Moderate enteritis - - -

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

A-10 - - - - - - - - - - - NA - Mild enteritis - - -

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

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

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

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

A-15 - - - - - - NA 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 - 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

Porcine Vaccine Effectiveness Test

Purpose:

The aim of the study was to evaluate the safety, colonization persistence and efficacy of avirulent live and killed isolates of L. intracellularis in 2-3 week old pigs. A host animal study was performed in which 3-week-old pigs were vaccinated and then challenged virulently with Lawsonia intracellulare strain N343 to compare differences in protection between the vaccines.

method

On December 11, 1995, a total of 45 3-week-old pigs were purchased from H&K Farms. They were shipped to Veterinary Resources, Inc., a research facility in Cambridge, Iowa, where the pigs were tagged to identify each pig. Pigs were acclimatized at the facility for 2 days prior to the study and were fed antibiotic-free feed during the study.

On December 13, all pigs were weighed, bled for serum, clinically scored and rectal samples collected. Pigs were randomly divided into 5 groups and placed in pots. 20 pigs were housed in a separate room for control and strict control groups. Fifteen pigs were placed in a second room for the ISi-1 vaccine trial. The third room, with 10 pigs, was used for the ISi-2 vaccine trial.

The live vaccine was prepared at the NOBL Laboratories Research and Development facility and is known as the test series ISi-1. ISi-1 (strain N343) was isolated from pigs and continuously grown in pure culture for 29 weeks. Vaccines were grown on McCoys cells in spinner flasks at low oxygen concentrations until approximately 100% infection was observed. High passage N343 strain used for ISi-1 was further passaged for 11 weeks in a spinner bottle ("N343NP40wk"), deposited with the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland) on May 22, 1996 under the Budapest Treaty U.S.A 20 852), deposit number 55783. Cultures were harvested by centrifugation at 3000 xg for 20 minutes. The pellet was resuspended in sucrose-phosphate-glutamate (SPG) solution and passed through a 25 gauge needle 4 times. The lysate was pelleted by centrifugation at 500 x g for approximately 5 minutes to remove debris and microcarrier beads. Supernatants were collected and stored at -70°C until 1 hour before inoculation and kept on ice for administration.

The inactivated vaccine (ISi-2) was grown, passaged for 12.5 weeks and harvested in a similar manner as above, then purified using a percol gradient. The purified bacteria were then stored at -70°C until 1 hour before inoculation, when they were stored at 4°C and normal atmospheric oxygen concentrations, which are toxic to L. intracellulare. Aluminum hydroxide was added to the bacteria until the final mixture contained 10% aluminum hydroxide. Protein concentration was determined by the Biurett method.

Quantification of live ISi:

Quantification of viable L. intracellularis was accomplished by determining the 50% tissue culture infectious dose ( _TCID50 ). This was done by seeding 96-well microtiter plates with 1250 cells/well of McCoys cells prior to infection and growing for 18-24 hours. Samples were serially diluted 1:10 in DMEM/5% FBS containing vancomycin (100 μg/ml) and amphotericin B (2.0 μg/ml). The dilution was added to a 96-well microtiter plate at 0.1 ml per well. Twelve wells were used for each dilution. Plates were incubated at 37°C for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Fix the cells with cold 50% acetone and 50% methanol for 2 min. To each well was added 0.03 ml/well anti-L. intracellularis monoclonal antibody (developed by Dr. Steven McOrist) diluted 1:2000 in PBS. Plates were incubated at 37°C 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°C for 30 minutes. Plates were washed 3 times with double distilled water and allowed to dry. The samples were observed with a fluorescence microscope, and the TCID ₅₀ /ml was determined using the Reed-Meunsch calculation.

TCID ₅₀ data showed that ISi-1 had 1.8×10 ⁵ bacteria/ml. The fourth inoculum was a placebo, which came from tissue culture cells processed in the same way as the vaccine.

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

Pigs were vaccinated on December 13, 1995. The dose of live vaccine is 2 ml IN, 1 ml in each nostril. The ISi-2 (inactivated) vaccine was administered intramuscularly at 1.5 ml/pig and repeated after 14 days. All control animals received uninfected cells in the same manner as the live vaccines.

Observations and samples:

Fecal samples and serum were collected every 7 days during the study. A PCR test was performed on the fecal samples to amplify the DNA using the primer pair: 5’-TATGG CTGTC AAACA CTCCG-3’ and: 5’-TGAAG GTATT GGTATTCTCC-3’. The first cycle of the cycle was 93°C for 5 minutes; 55°C for 45 seconds; and 72°C for 45 seconds. 33 cycles were performed for 45 seconds at each of the above temperatures. The last cycle was: 93°C, 45 seconds; 55°C, 45 seconds; and 72°C, 2 minutes. Primers were determined by Jones et al.

immune challenge:

All animals, except stringent controls, were given a challenge culture consisting of low-passage cultures of Lawsonia intracellulare strains N343 and N72994 (grown continuously for 8-12 weeks) at 26 and 27 days after inoculation. )constitute. Cultures were harvested by centrifugation at 3000 xg for 20 minutes. The pellet was resuspended in a sucrose-phosphate-glutamate (SPG) solution containing 10% fetal bovine serum and passed through a 25 gauge needle 4 times. Some harvested cultures were stored at -70°C until used for challenge, while others continued to grow until the day of challenge and then harvested. The challenge inoculum was mixed and the _TCID50 of the culture was determined. Samples were stored on ice for administration.

The 1/8/96 challenge culture contained 4 x 10 ⁴ bacteria/ml and the 1/9/96 challenge culture contained 3 x 10 ⁴ bacteria/ml. Pigs were administered 15 ml of the challenge vaccine by gastric lavage on both days. Therefore, animals received 6 x ¹⁰⁵ bacteria/pig and 4.7 x ¹⁰⁵ bacteria/pig on 1/8/96 and 1/9/96, respectively.

result:

safety:

Excreta PCR Results: Detection of L. intracellulare by PCR revealed that no pigs excreted the bacteria at the start of the study. All pigs tested 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 revealed that none of the animals excreted bacteria 26 days after inoculation. 26 days after inoculation, 5 pigs in the ISi-1 group and 4 pigs in the ISi-2 group were dissected. Samples collected were ileum, colon, enteric lymph nodes and tonsils, and lung samples (from pigs with possible pneumonia lesions).

PCR tests were performed on each ileal and lung sample. Tonsils, colons and lymph nodes were pooled for each treatment group and PCR was performed. The PCR results are as follows.

Group 26 days after inoculation, ileal PCR mixed colon mixed tonsils Mixed intestinal lymph nodes mixed lung ISi-1 1 out of 5 + - - 1 of 1 ISi-2 0 out of 4 - - - 0 out of 1 control 0 out of 5 - - not tested strict control not tested not tested not tested not tested not tested

Sections of ileal tissue were stained using a monoclonal antibody specific for L. intracellulare as the primary antibody and an anti-mouse immunoglobulin G-fluorophore conjugate as the secondary antibody. L. intracellularis was observed in 3 out of 5 pigs from ISi-1. All other pigs were negative by fluorescent antibody staining.

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

Group 26 days after challenge, ileal PCR mixed colon mixed tonsils Mixed intestinal lymph nodes mixed lung ISi-1 0 out of 10 + - - - ISi-2 0 out of 6 - - - - control 4 out of 10 - - - strict control 0 out of 5 - - - -

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

Serum was also tested to determine the production of IgG antibodies produced by pigs exposed to L. intracellulare. The assay was carried out as follows: McCoys cells were seeded at 1250 cells/well on tissue culture treated Terasaki plates prior to infection and grown for 18-24 hours. Dilute the pure culture of Lawsonia intracellulare to 1000-3000 bacteria/ml in DMEM containing 5% fetal bovine serum, and add 0.1 ml to each well. Plates were incubated for 6 days in 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen. Fix the cells with cold 50% acetone and 50% methanol for 2 min. Pig serum was diluted 1:75 in sterile PBS. Add diluted serum to each well at 0.01 ml per well. Plates were incubated at 37°C for 30-60 minutes and then washed 5 times with sterile PBS. Anti-porcine IgG-fluorophore conjugate was added to each well at 0.01 ml/well, and the plate was incubated at 37°C for 30 minutes. Plates were washed 5 times with double distilled water and allowed to dry. The samples were washed 5 times with double distilled water and allowed to dry. Samples were observed with a fluorescence microscope, and wells where bacteria were observed were scored as positive, and wells where no bacteria were observed were scored as negative.

result:

Group Day 0 26 days after inoculation 47 days after inoculation, 21 days after challenge ISi-1 0 out of 15 6 out of 15 8 out of 10 ISi-2 0 out of 10 3 out of 10 5 out of 6 control 1 in 15 0 out of 15 9 out of 10 strict control 0 out of 5 0 out of 5 0 out of 5

Animals that tested positive on Day 0 were retested weekly. The results showed that the sera were all negative 14 days after inoculation. This was expected as pigs were only 3 weeks old on day 0 and positivity at this age was likely due to maternal antibodies.

Positive control sera used when testing sera were obtained by immunizing pigs with high doses of L. intracellulare grown on pure culture. The negative control serum used was collected from sterile pigs at South Dakota State University.

The above descriptions and embodiments are only used to illustrate preferred examples that can realize the objects, features and advantages of the present invention, and should not be construed as limiting the present invention thereto. Any modification of the invention which comes within the spirit and scope of the appended claims is considered part of the invention.

Claims (4)

1. one kind is used for inducing vaccine for the immunne response of Lawsonia intracellularis bacterium animal, it is characterized in that described vaccine contains Lawsonia intracellularis attenuated strain and pharmaceutically acceptable carrier, and wherein, described attenuated strain is ATCC 55783.

2. vaccine as claimed in claim 1 is characterized in that, described vaccine contains 10 ⁷-10 ⁹TCID ₅₀Immunogen.

3. vaccine as claimed in claim 1 is characterized in that, described carrier is selected from aqueous solution, emulsion or suspension.

4. vaccine as claimed in claim 1 is characterized in that, the quantity of described attenuated strain can produce immunne response effectively, and described vaccine contains the immunogen of 50-500 microgram, and uses the bacterium of purifying.