WO2009088878A1

WO2009088878A1 - Improved culture media and growth-enhancing nutrients

Info

Publication number: WO2009088878A1
Application number: PCT/US2008/088566
Authority: WO
Inventors: Merrill Schaeffer; Michael Roof
Original assignee: Boehringer Ingelheim Vetmedica Inc.
Priority date: 2007-12-31
Filing date: 2008-12-30
Publication date: 2009-07-16

Abstract

A method of enhancing the growth and maintenance of bacteria, such as Lawsonia intracellularis is provided. Additionally, enhanced maintenance and growth mediums and conditions are provided herein.

Description

IMPROVED CULTURE MEDIA AND GROWTH-ENHANCING NUTRIENTS

BACKGROUND OF THE INVENTION

Field of the invention

The present invention is concerned with culture media used for the growth and maintenance of bacteria. More particularly, the present invention is concerned with such cultures and their use with obligate intracellular bacteria. Still more particularly, the present invention is concerned with such cultures and their use with Lawsonia intracellularis . Most particularly, the present invention is concerned with culture media that promote the enhancement of growth, maintenance and continued viability of Lawsonia intracellularis.

History of the prior art

Bacteria has been grown and maintained in various types of culture. Most cultures are designed to optimize conditions in which the bacteria can be maintained, grow, and remain viable. Culture ingredients are selected for their ability to provide the bacteria, as well as any cells that may also be required for the maintenance of the bacteria, with components necessary to be maintained and grown, as well as remain viable. Lawsonia intracellularis ^Lawsonia" or "Z. intracellularis^'") requires a cell-based system for growth and maintenance of growth. Prior art has shown it to be challenging to grow and maintain these bacterial cultures. A method for cultivating Lawsonia intracellularis in the absence of molecular hydrogen is disclosed in WO 2006/056853. Conditions under which molecular hydrogen is not present are thought to provide a safer environment for culturing Lawsonia. This method also includes the use of a culture medium supplemented with cysteine and/or a reducing agent.

Given the stringent conditions under which Lawsonia must be cultured and the difficulty of acquiring a strong yield from cultivation, what is needed in the art is an improved cell culture medium that will permit Lawsonia intracellularis to be efficiently maintained and/or grown, yet still remain viable for inclusion in other products, including vaccines and immunogenic compositions. SUMMARY OF THE INVENTION

The present invention overcomes the problems inherent in the prior art and provides for improved culture media for growth and maintenance of a bacteria as well as methods of producing and using the same. In some preferred forms, the cultures are designed for optimizing the maintenance and growth of obligate intracellular bacteria such as Lawsonia intracellularis . Specifically, in the context of Lawsonia, the invention provides for growth-enhancing nutrients that provide improved conditions under which Lawsonia can grow and be maintained, making this process technically easier, cheaper, and thereby making future vaccine and diagnostic technology more accessible to the industry. The growth-enhancing nutrients can be added to any basal cell culture medium, including, but not limited to, Agar, Blood Agar, Ames medium (Sigma Aldrich, St. Louis, Missouri), Alpha Minimum Essential Medium ("MEM") (Cambrex, Baltimore, Maryland), Basal Medium Eagle (Sigma), Basal Medium Eagle with Earle's BSS (Cambrex), Cyroprotective Medium(Cambrex), Click's Medium (Sigma), Dulbecco's Modified Eagle Medium ("DMEM") (JRH Biosciences, Inc., Lenexa, Kansas), Dulbecco's Media (Sigma), GMEM(Sigma), Grace's Insect Medium (Sigma), Ham's F-lO(Cambrex), Iham's F-12 (Sigma), IPL-41 Insect Medium(Sigma), L-15 Media (Sigma), Iscore's Modified Dulbecco's Medium(Sigma), McCoy's 5A Modified Medium(Sigma), L-15 Medium (Cambrex), MCDB medium(Sigma), Medium 199 (Sigma), Minimum Essential Media Eagle (Sigma), NCTC Medium (Sigma), NCTC- 109 (Cambrex), RPMI- 1640 Medium (Sigma), Richter's CM Medium (Cambrex), RPMI 1640 (Cambrex), Schneider's Insect Medium (Sigma), Shields and Sang M3 Insect Medium (Sigma), Waymouth Medium (Sigma), TC-100 Insect Medium (Sigma), TNM- FH Insect Medium(Sigma), and Williams Medium E (Sigma). For purposes of the present invention, the culture media may be supplemented with one or more additives. In a preferred embodiment, additives are selected from, but not limited to, the following additives Balanced Salt Solution ("BSS") (Sigma), Hank's Balanced Salt Solution ("HBSS") (Innovative), F12K Nutrient Mixture (Innovative Research, Southfield, Illinois), Dulbecco's Phosphate Buffered Saline ("DPBS") (Innovative), Phosphate Buffered Saline ("PBS") (Innovative), Glasgow Minimum Essential Medium ("GMEM") (Cambrex), Fetal Calf Serum (Cambrex), Porcine Serum (Sigma), Bovine Serum (Sigma), Fetal Bovine Serum, Irradiated Fetal Bovine Serum ("IFBS"), Nutrient Mixtures (Sigma), and combinations thereof. In the context of Lawsonia, the preferred media is DMEM, supplemented with Fetal Calf Serum, IFBS, and combinations thereof. In a most preferred embodiment, the media is DMEM supplemented with 5% IFBS.

In one preferred embodiment of the present invention, there is provided an improved culture media that includes a basal cell culture media, such as those noted above, cells (preferably McCoy cells), and at least one growth-enhancing nutrient. In a preferred embodiment, the basal cell culture media additionally comprises one or more additives, such as those noted above. In preferred forms, the growth-enhancing nutrient(s) are selected from the group consisting of sulfates, preferably iron sulfate, calcium sulfate, sodium sulfate; ATP; sulfites, preferably bisulfites, disulfites or hydrogensulfites such as sodium bisulfite, potassium disulfite, sodium hydrogensulfite and the like; and combinations thereof.

In an additionally preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, and sulfate. In another preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, a sulfate and an energy source. One preferred energy source is a nucleoside triphosphate. In a preferred embodiment, the basal media is supplemented with one or more additives as described above. Preferably the sulfate is selected from the group consisting of iron sulfate, calcium sulfate, sodium sulfate, and combinations thereof. The nucleoside triphosphate can be any nucleoside triphosphate, including adenine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), uridine triphosphate (UTP), and combinations thereof. Preferably, the nucleoside triphosphate is ATP.

In another preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, and a sulfate in an environment that is free of molecular hydrogen. In yet another preferred embodiment, there is provided an improved culture media including basal cell culture media, cells, a sulfate and an energy source, preferably a nucleoside triphospate, in an environment that is free of molecular hydrogen. In a preferred embodiment, the basal media is supplemented with one or more additives, preferably as described above. Preferably, the sulfate is selected from the group consisting of iron sulfate, calcium sulfate, sodium sulfate, and combinations thereof. The nucleoside triphosphate is preferably ATP. In yet another embodiment, there is provided an improved culture media that includes basal cell culture media, cells, and a sulfate in the presence of molecular hydrogen. In a still further preferred embodiment, there is provided an improved culture media including basal cell culture media, cells, a sulfate and an energy source, preferably a nucleoside triphosphate, in the presence of molecular hydrogen. In one preferred embodiment, the basal cell culture media is supplemented with one or more additives, preferably as described above. Preferably, the sulfate is selected from the group consisting of iron sulfate, calcium sulfate, sodium sulfate, and combinations thereof. The nucleoside triphosphate is preferably ATP.

In yet another preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, a sulfite, and an energy source, preferably a nucleoside triphosphate. Preferably, the sulfite is a bisulfite disulfϊte or hydrogen sulfite and, even more preferably, the sulfite is sodium bisulfite. The nucleoside triphosphate is preferably ATP. Preferably, the basal cell culture media is supplemented with one or more additives, preferably as described herein. In a most preferred embodiment, there is provided an improved culture media that includes DMEM supplemented with 5% IFBS, McCoy cells, sodium bisulfite and ATP.

In yet another preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, a sulfite, and an energy source, preferably a nucleoside triphosphate, wherein the environment is free of molecular hydrogen. In a preferred embodiment, the basal cell culture media is supplemented with one or more additives, preferably as described herein. Preferably, the sulfite is a bisulfites, disulfites or hydrogensulfites such as sodium bisulfite, potassium disulfite, sodium hydrogensulfite and the like and, even most preferred, the sulfite is sodium bisulfite. The nucleoside triphosphate is preferably ATP.

In yet another preferred embodiment, there is provided an improved culture media that includes basal cell culture media, cells, a sulfite, and a nucleoside triphosphate in the presence of molecular hydrogen. In a preferred embodiment, the basal cell culture media is supplemented with one or more additives. Preferably, the sulfite is bisulfites, disulfites or hydrogensulfites such as sodium bisulfite, potassium disulfite, sodium hydrogensulfite and the like and, even most preferred, the sulfite is sodium bisulfite. The nucleoside triphosphate is preferably ATP.

In another aspect, the present invention provides for a cell culture medium, comprising a basal cell culture medium and comprising or including a growth-enhancing nutrient selected from the group consisting of sulfates, nucleoside triphosphates, and combinations thereof. The present invention also provides for a method of enhancing cell culture medium comprising the step of adding a growth enhancing nutrient, preferably selected from the group consisting of an energy source, preferably a nucleoside triphosphate, and at least one sulfate, singularly or in combination, to basal cell culture medium.

The present invention additionally provides for a method of creating a cell culture medium comprising the steps of adding an energy source, such as a nucleoside triphosphate, or at least one sulfate, singularly or in combination, to basal cell culture medium, thereby enhancing cell growth and maintenance.

The present invention also provides for a method of enhancing the growth of Lawsonia intracellularis comprising the step of adding an ingredient selected from the group consisting of a nucleoside triphosphate, sulfates, and combinations thereof, to basal cell culture medium.

One embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said basal cell culture medium is Dulbecco's Minimum Eagle Medium ("DMEM").

Another embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said basal medium is supplemented with one or more additives, preferably as described above.

A further embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said additive is serum, preferably bovine or calf serum, more preferably Irradiated Fetal Bovine Serum ("IFBS").

An additional embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said additive is Fetal Calf Serum.

Another embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said sulfate is selected from the group consisting of iron sulfate, sodium sulfate, calcium sulfate, and combinations thereof.

Still another embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, wherein said energy source is a nucleoside triphosphate and is preferably ATP.

A further embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, further comprising the addition of McCoy cells to said cell culture medium for the growth of Lawsonia intracellularis. An additional embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, further comprising the step of growing said cells or growing Lawsonia intracellularis wherein such growing is performed under reduced oxygen conditions.

Another embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, further comprising the step of growing said cells or growing Lawsonia intracellularis wherein such growing is performed in the absence of molecular hydrogen.

A further embodiment of the present invention includes the cell culture medium or method of any of the paragraphs described above, further comprising the step of growing said cells or growing Lawsonia intracellularis wherein such growing is in the presence of molecular hydrogen.

Values for the preferred amount of the growth-enhancing nutrients including sulfates, preferably, sodium sulfate, calcium sulfate, and iron sulfate and energy source, preferably a nucleoside triphosphate, and still more preferably, ATP are those that are detailed further in the application.

The present invention further provides for a cell culture medium comprising a basal cell culture medium, a sulfite, and a nucleoside triphosphate.

The present invention additionally provides for a method of enhancing cell culture medium comprising the step of adding nucleoside triphosphate and a sulfite to basal cell culture medium.

The present invention also provides for a method of creating a cell culture medium comprising the steps of adding a nucleoside triphosphate and a sulfite to basal cell culture medium, thereby enhancing the growth and maintenance.

The present invention further provides for a method of enhancing the growth of Lawsonia intracellularis comprising the step of adding a nucleoside triphosphate and a sulfite, to basal cell culture medium

One embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said basal cell culture medium is DMEM.

Another embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said basal cell culture medium is supplemented with one or more additives, preferably those that are described herein.

An additional embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said additive is a serum, preferably a bovine or calf serum, most preferably IFBS.

A further embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said additive is Fetal Calf Serum.

Another embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, further comprising the addition of McCoy cells to said cell culture medium for the growth of Lawsonia intracellularis .

An additional embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said sulfite is a bisulfite, disulfϊte or hydrogensulfite such as sodium bisulfite, potassium disulfite, sodium hydrogensulfϊte and the like and, even most preferred, the sulfite is sodium bisulfite

A further embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said bisulfite is a bisulfites, disulfites or hydrogensulfite such as sodium bisulfite, potassium disulfite, sodium hydrogensulfite and the like and, even most preferred, the sulfite is sodium bisulfite.

An additional embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, wherein said nucleoside triphosphate is ATP.

Another embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, further comprising the step of growing said cells or Lawsonia intracellularis under reduced oxygen conditions.

An additional embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, further comprising the step of growing said cells or Lawsonia intracellularis in the absence of molecular hydrogen.

A further embodiment of the present invention includes the cell culture medium or method described in the paragraphs above, further comprising the step of growing said cells or Lawsonia intracellularis in the presence of molecular hydrogen.

Values for the preferred amount of sulfates, preferably, sodium sulfate, calcium sulfate, and iron sulfate and nucleoside triphosphates, preferably, ATP are detailed further in the application. In particularly preferred embodiments, the growth enhancing nutrient is selected from the group consisting of: iron sulfate only; calcium sulfate only; sodium sulfate only; iron sulfate and calcium sulfate; iron sulfate and sodium sulfate; calcium sulfate and sodium sulfate; iron sulfate, calcium sulfate, and sodium sulfate; iron sulfate and ATP; calcium sulfate and ATP; sodium sulfate and ATP; iron sulfate, calcium sulfate, and ATP; iron sulfate, sodium sulfate and ATP; calcium sulfate, sodium sulfate, and ATP; iron sulfate, calcium sulfate, sodium sulfate, and ATP; and, sodium bisulfite and ATP. The media with the growth enhancing nutrients can be cultured in the presence or absence of molecular hydrogen.

For purposes of the present invention, the preferred amount of sulfate in the final cell culture medium is from about 0.00 lμM to about 30OmM, more preferably the amount of sulfate is from about 0.1 μM to about 25OmM, even more preferably the amount of sulfate is from about lμM to about 225mM, still more preferably the amount of sulfate is from about lOμM to about 20OmM.

For purposes of the present invention, the preferred amount of iron sulfate in the final cell culture medium is from about 30μM to about 500μM, more preferably the amount of iron sulfate is from about 50μM to about 400μM, even more preferably the amount of iron sulfate is from about 80μM to about 300μM, still more preferably the amount of iron sulfate is from about 90μM to about 200μM, and most preferably about lOOμM.

For purposes of the present invention, the preferred amount of sodium sulfate in the final cell culture medium is from about 1OmM to about 30OmM, preferably about 10OmM, more preferably the amount of sodium sulfate is from about 25mM to about 20OmM, preferably about 10OmM, and most preferably the amount of sodium sulfate is from about 5OmM to about 15OmM, preferably about 10OmM.

For purposes of the present invention, the preferred amount of calcium sulfate in the final cell culture medium is from about 0.00 lμM to about lμM, preferably about O.OOβμM, more preferably the amount of calcium sulfate is a from about O.OlOμM to about 0.9μM, preferably about 0.092μM, and most preferably from about O.OOβμM to about 0.8μM, preferably about 0.734μM.

For purposes of the present invention, the preferred amount of sodium bisulfite is from about 0.ImM to about 22OmM, preferably, about 0.7mM, more preferably the amount of sodium bisulfite is from about ImM to about 21OmM, preferably about 5.63mM, even more preferably the amount of sodium bisulfite is from about 5mM to about 20OmM, preferably about 11.3mM, still more preferably the amount of sodium bisulfite is from about 1OmM to about 195mM, preferably about 22.5mM, more preferably the amount of sodium bisulfite is from about 2OmM to about 19OmM, preferably about 45mM, even more preferably the amount of sodium bisulfite is from about 5OmM to about 185mM, preferably about 9OmM, and most preferably the amount of sodium bisulfate is from about 6OmM to about 182mM, preferably about 10OmM.

For purposes of the present invention, the preferred amount of nucleoside triphosphate in the final cell culture medium is from about O.lmM to about 5mM, preferably about 0.39mM, more preferably the amount of nucleoside triphosphate is from about 0.2mM to about 4mM, preferably about 3mM, even more preferably the amount of nucleoside triphosphate is from about 0.3mM to about 3.8mM, preferably about 2.OmM, and most preferably the amount of nucleoside triphosphate is from about 0.35mM to about 3.5mM, preferably about 1.5mM. Preferably the nucleoside triphosphate is ATP.

In another preferred embodiment, the present invention provides a method for producing improved culture media for the maintenance and growth of Lawsonia using sulfates and/or a nucleoside triphosphate as growth-enhancing nutrients. Preferably, the nucleoside triphosphate is ATP. The method generally comprises maintaining and growing Lawsonia in a basal cell culture medium which includes cells, preferably McCoy cells, and a growth-enhancing nutrient that must be added to the medium as is not normally found in the medium. In a preferred embodiment the basal cell medium contains one or more additives. Preferably the basal medium is DMEM supplemented with the additives IFBS and Fetal Calf Serum. Preferably, the growth-enhancing nutrient is selected from the group consisting of iron sulfate, calcium sulfate, iron sulfate, sodium sulfate, sodium bisulfite, nucleoside triphosphates, and combinations thereof. Even more preferably, the growth enhancing nutrient is selected from the group consisting of: iron sulfate only; calcium sulfate only; sodium sulfate only; iron sulfate and calcium sulfate; iron sulfate and sodium sulfate; calcium sulfate and sodium sulfate; iron sulfate, calcium sulfate, and sodium sulfate; iron sulfate and ATP; calcium sulfate and ATP; sodium sulfate and ATP; iron sulfate, calcium sulfate, and ATP; iron sulfate, sodium sulfate and ATP; calcium sulfate, sodium sulfate, and ATP; iron sulfate, calcium sulfate, sodium sulfate, and ATP; and, sodium bisulfite and ATP. Preferably, the sulfate used is selected from the group of stock solutions consisting of iron sulfate (preferably 18OmM), calcium sulfate (preferably saturated), sodium sulfate (preferably IM), and combinations thereof. More preferably, the sulfate used is sodium sulfate.

In another embodiment, the present invention provides for improved methods for growing and maintaining Lawsonia. Generally, the method includes the steps of adding Lawsonia to a basal cell culture medium, which has been supplemented with at least one growth- enhancing nutrient. Preferably the basal media includes one or more additives. In a preferred embodiment the basal media is DMEM supplemented with the additives IFBS and Fetal Calf Serum. In some forms of this embodiment, the Lawsonia is grown in the absence of molecular hydrogen, and in other embodiments, the Lawsonia is grown in the presence of molecular hydrogen. The growth-enhancing nutrient is preferably selected from the group consisting of iron sulfate, calcium sulfate, iron sulfate, sodium sulfate, sodium bisulfite, and a nucleoside triphosphate, preferably ATP. Even more preferably, the growth enhancing nutrient is selected from the group consisting of: iron sulfate only; calcium sulfate only; sodium sulfate only; iron sulfate and calcium sulfate; iron sulfate and sodium sulfate; calcium sulfate and sodium sulfate; iron sulfate, calcium sulfate, and sodium sulfate; iron sulfate and ATP; calcium sulfate and ATP; sodium sulfate and ATP; iron sulfate, calcium sulfate, and ATP; iron sulfate, sodium sulfate and ATP; calcium sulfate, sodium sulfate, and ATP; iron sulfate, calcium sulfate, sodium sulfate, and ATP; and, sodium bisulfite and ATP.

One preferred growth medium is a solution of Dulbecco's Modified Eagle's Medium ("DMEM") and 5% serum, preferably IFBS. If ATP is used as the growth-enhancing nutrient, preferably, it is between 0.25M to 0.75M, and more preferably 0.5M, pH 7.3, 0.2μm filtered and is diluted in DMEM + 5% serum such that the resulting concentrations are between 1 :16 to 1 :128 (0.03M to 0.004M), more preferably about 1:32 to 1 :128 (0.016M to 0.004M) and, most preferably, about 1 :64 (0.008M). The sulfates can be neat solutions or can be titrated such that resulting concentrations are about 1 :2 to 1 :256. Preferred concentrations for each sulfate will depend upon the corresponding concentration of ATP used (or not) in combination as a growth- enhancing nutrient. In the preferred embodiment, a neat solution of saturated sodium sulfate is used, without ATP. A neat solution of calcium sulfate may also be used without any ATP. Another favorable nutrient combination is either a neat (-10OmM) solution or a 1 :2 (9OmM) dilution of sodium sulfate combined with a 1 :64 (7.8mM) dilution of ATP. The Lawsonia infected medium is then incubated under reduced oxygen conditions, meaning that the atmospheric oxygen in the flask is removed by vacuum, leaving only a small amount of oxygen in the flask. Preferably, the air in the flask removed by the vacuum is replaced by molecular hydrogen with a nitrogen balance. Preferably the flask is incubated for 2 to 10 days, preferably, about 3 to 8 days and more preferably, between 4 and 7 days. Any Lawsonia isolate can be used with the present invention. One representative Lawsonia stock is reference strain N343 or a high titer stock such as EU Lawsonia seed. Preferably, the high titer stock solution has a concentration of at least 4 logs/ml. In a preferred embodiment, the high titer stock solution has a concentration of about 4 -9 logs/ml, preferably at least 5 logs/ml, more preferably the concentration is 6-8 logs/ml, and most preferably at least 7 logs/ml. The reduced oxygen conditions can be obtained preferably by using a specialty gas mixture of either Linweld-type 4% hydrogen with a nitrogen balance (96% nitrogen) or new-Linweld-type 4% hydrogen and 10% carbon dioxide with a nitrogen balance (86% nitrogen). In the preferred embodiment, the reference strain is grown in the Linweld-type gas and the high titer stock is grown in the new-Linweld-type gas. It was also found that the high titer seed grew best in the neat solution sodium sulfate, and the reference strain grew well in the 1 :2 (~50mM) solution of sodium sulfate with 1 :64 (7.8mM) solution ATP.

In another embodiment of the present invention, there is provided a method for growing and maintaining Lawsonia using a basal cell culture media supplemented with growth-enhancing nutrients. In preferred forms, the basal cell culture media is supplemented with one or more additives. Preferably, the basal media is DMEM supplemented with the additives IFBS and Fetal Calf Serum. Preferably, the growth-enhancing nutrients consist of or consist essentially of at least one of sodium sulfate, calcium sulfate, and ATP. The method generally comprises the steps of adding Lawsonia to a culture medium that has been supplemented with at least one growth-enhancing nutrient. The growing and maintenance steps then proceed as they would with Lawsonia in a basal cell culture medium without the growth enhancing nutrients. In one preferred example of this method, a solution of DMEM and 5% IFBS was added to cell culture plates containing McCoy cells prepared for Lawsonia culture. 0.5 M ATP at a pH of 7.3 was diluted in the DMEM + 5% IFBS to make concentrations of 1 :32 (15.6mM), 1 :64 (7.8mM), and 1 :128 (3.9mM). Each dilution was added to the plate containing the media along with a control of just DMEM + 5% IFBS. Calcium sulfate and sodium sulfate were titrated 2-fold from left to right across the plates. Lawsonia stock was then added to each well in the cell culture plate. Final concentrations for sodium sulfate were 50OmM (1 :2 dilution wells), 62.5mM (1 :16 dilution wells), and 3.9mM (1 :256 dilution wells). Final concentrations for calcium sulfate were approximately 0.734μM (1 :2 dilution wells), 0.092μM (1 :16 dilution wells), and 0.006μM (1 :256 dilution wells). Final concentrations for ATP were 1.56mM (1 :32 dilution wells), 0.78mM (1 :64 dilution wells), and 0.39mM (1 : 128 dilution wells). The plate was then placed in a GasPak jar and the air was removed and replaced with 4% hydrogen with a nitrogen balance. The plate was incubated for 7 days and then the wells were IFA stained. The volumes of the wells were disturbed and collected. The volumes were then centrifuged at 20,000 x g for 5 minutes. The resulting supernate was then removed and the pellet suspended in PBS. The volume was placed on a microscope slide, stained, and then accessed by microscopy and 4-fϊelds counted for individual bacteria.

In another embodiment of the present invention, there is provided a method for growing and maintaining Lawsonia in an improved culture media, wherein the improved culture media is a basal culture media supplemented with at least one growth-enhancing nutrient, preferably sodium bisulfite and ATP. In some preferred forms, the basal cell culture media may contain one or more additives. Preferably, the basal cell culture media is DMEM supplemented with the additives IFBS and Fetal Calf Serum As a representative embodiment of this general method, DMEM + 5% IFBS was added to the wells of a cell culture plate containing McCoy cells. Two dilution plates were used to titrate 35% sodium bisulfite (1.8 M) and ATP (0.5 M). ATP was added resulting in the following dilutions: 1 :16 (31.3 mM) 1 :32 (15.6mM), and 1 :64 (7.8mM). Sodium bisulfite titrations were then transferred from the dilution plates to the culture plate. 1 :2 (90OmM) dilutions and 1 :256 (7mM) dilutions were used. Final concentrations of ATP were 3.13mM (1 :16 dilution wells), 1.56mM (1 :32 dilution wells), and 0.78mM (1 :64 dilution wells). Final concentrations of sodium bisulfite were 9OmM (1 :2 dilution wells) and 0.7mM (1 :256 dilution wells). The Lawsonia stock was then added to the wells, the plate placed in a GasPak jar, and the air replaced as in the embodiment above. The plate was incubated for 7 days, after which, the well contents were disrupted, pooled, and centrifuged. The pellets were then suspended, placed on a microscope slide, stained, and counted.

A "neat solution" as used herein, refers to a solution that is undiluted and relatively pure. The concentration of a neat solution can depend on the initial amount of compound added to the initial substrate.

In the "presence of molecular hydrogen" refers to the presence of hydrogen in the atmospheric air inside the flask. Preferably hydrogen is present in an amount of about 2% to 15%, more preferably hydrogen is present in an amount of about 3-12%, and most preferably hydrogen is present in an amount of about 4-10%.

In the "absence of molecular hydrogen" refers to the absence of hydrogen in the atmospheric air inside the flask. Preferably, there is less than 1% hydrogen present.

A "basal medium" or "basal media", as used herein, refers to a basic cell culture medium capable of supporting cell growth.

An "additive", as used herein, refers to a supplemental material, which can be added to a basal medium to form a medium specific for specific growth requirements or to add additional growth support for the basal medium.

A "nitrogen balance", as used herein, refers to an amount of nitrogen which is added to make the percentage of gas 100%. For example, if there is 5% oxygen present, a nitrogen balance would be 95%.

"Reduced oxygen conditions", as used herein, refers to conditions in which less oxygen is present than is normally present in the Earth's atmosphere. Preferably, reduced oxygen means that there is a concentration of less than about 21% present, more preferably there is less than about 10% present, even more preferably there is less than 5% present, and most preferably there is less than 1% present.

"Final cell culture medium" as used herein, refers to the cell culture medium, containing any additives, growth-enhancing nutrients, cells, etc. in which the Lawsonia or other pathogen is grown. The final cell culture medium is determined after all dilutions have been made and is the resulting culture medium after the additions mentioned above.

"Saturated", referring to chemical solutions and as used herein, refers to the point at which a solution of a compound or substance cannot dissolve any more of the substance in the substrate. At the point of saturation, any additional amounts of the compound or substance will appear as a precipitate. The saturation point, or point of maximum concentration, depends on the chemical nature of the substances and substrates used, as well as the temperature and pressure of the surrounding atmosphere. BRIEF DESCRIPTION OF FIGURES

Figure 1 : Average bacteria per field for media supplemented with calcium sulfate, sodium sulfate, and/or ATP;

Fig. 2: Average bacteria per field for sodium sulfate, calcium sulfate, and ATP (duplicate experiment);

Fig. 3: Titration results for sodium bisulfite and ATP;

Fig. 4: Bacteria per field for sodium bisulfite and ATP;

Fig. 5: Viable cells/ml for FeSO₄; and

Fig. 6: Titration results (logioTCID 50/ml) for FeSO₄.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred materials and procedures in accordance with the present invention. It is to be understood, however, that these examples are provided by way of illustration only, and nothing therein should be deemed a limitation upon the overall scope of the invention.

EXAMPLE 1

This method of practicing the present invention involves using sulfates and ATP as nutrients to aid in the maintenance and growth of Lawsonia intracellularis in cell culture.

Materials and Methods

6 spinner flasks, 500 ml with 200 ml culture volume were seeded with McCoy cells at 2 x 10⁵ cells/ml and infected with 0.1 MOI of Lawsonia intracellularis at the time of cell planting. 2 spinner flasks were used as controls, 2 spinner flasks were treated with lOOμM FeSO₄ (final concentration) on days 0, 2, and 4, and 2 spinner flasks were treated with lOOμM FeSO₄ on days 0, 2, 4, and 6. On day 0, lOOμM FeSO₄ was present in the flask. On day 2, additional FeSO₄ was added to the flask (lOOμM). On day 4, additional FeSO₄ was added to the flask (lOOμM). On day 6, additional FeSO₄ was added to the flask (lOOμM). Spinner cultures were incubated for 8 days and samples were taken daily for titration. Results and Conclusions

FeSO₄ had a positive effect on all the viable cell numbers. Values for "All FeSO₄" were obtained by averaging all the data for each respective day for cultures that were treated with FeSO₄. The results are shown in the table below:

EXAMPLE 2

This method of practicing the present invention involves using sulfates and ATP as nutrients to aid in the maintenance and growth of Lawsonia intracellularis . Materials and Methods

5ml of frozen Lawsonia intracellularis stock 063004 were thawed at about 37⁰C in a water bath. The outside of the tube was then sterilized by spraying with Septihol (Steris, Mentor, OH). A solution of Dulbecco's Modified Eagle's Medium ("DMEM") (JRH Biosciences, Inc., Lenexa, KS) and 5% IFBS was added to the 96 well cell culture plates (Corning Inc., Corning, NY) containing McCoy cells that were prepared for the Lawsonia culture. The plates were then warmed in an incubator at 37⁰C. 0.5M ATP at pH 7.3 was then diluted in the DMEM+5% serum to make final concentrations of 1 :32 (15.6mM), 1 :64 (7.8mM), and 1 :128 (3.9mM). 20μl of each dilution was then added to the plate containing the media. Final concentrations for ATP were 1.56mM (1 :32 dilution wells), 0.78mM (1 :64 dilution wells), and 0.39mM (1 :128 dilution wells). A control consisting solely of 20μl of the DMEM+5% serum was also added to the plate. Calcium sulfate and sodium sulfate were titrated 2-fold from left to right. Calcium sulfate was titrated in the top 4 rows and sodium sulfate in the bottom 4 rows. The first dilution was neat material IM sodium sulfate (stock concentration) or a saturated solution of calcium sulfate. The next dilution was a 1 :2 dilution in the DMEM+5% serum containing 50OmM sodium sulfate or ~7.34μM calcium sulfate and 15.6mM ATP. The next dilution was a 1 :16 dilution containing 62.5mM sodium sulfate or 0.918μM calcium sulfate and 15.6mM ATP. The next dilution was a 1 :256 dilution containing 3.9OmM sodium sulfate or 0.057μM calcium sulfate and 15.6mM ATP. 20μl of each dilution is transferred to the cell culture (200μL final volume). Final concentrations for sodium sulfate were 5OmM (1 :2 dilution wells), 6.25mM (1 :16 dilution wells), and 0.39mM (1 :256 dilution wells). Final concentrations for calcium sulfate were 0.734μM (1 :2 dilution wells), 0.092μM (1 :16 dilution wells), and 0.006μM (1 :256 dilution wells). Column 12 of the plate received 20μl of the DMEM+5% serum to serve as a control. Following this, 50μl of the Lawsonia stock was added to each well in the cell culture plate. The plate was then placed into a GasPak jar (BD, Franklin Lakes, NJ) and the air was removed from the jar with a pump. The jar was then filled with a specialty gas mixture of 4% hydrogen with a nitrogen balance (Linweld, Inc., Lincoln, NE). Fresh gas was added every other day into the jar. The plate was incubated for a total of 7 days. On the seventh day, specific wells were selected to look at using IFA staining. For these wells, the volume of the well was disturbed and collected. Next, the volume was centrifuged at 20,000 x g for 5 minutes. The resulting supernatant was then removed and the pellet suspended in lOμl PBS (Invitrogen, Carlsbad, CA). The volume was then placed on a microscope slide and allowed to air dry. Once dried, the slides were fixed using a 50/50 acetone/methanol solution, then stained using the anti-Lawsonia monoclonal VPM53 and goat anti-mouse FITC (KPL, Gaithersburg, MD). The slides were then assessed by microscopy and 4-fields are counted for individual bacteria. These numbers were recorded for each well and compared. Results and Conclusions

There was a positive effect on Lawsonia maintenance and growth with the addition of nutrients such as sulfates and ATP.

The average bacterium per field is summarized in Figure 1 above. The calcium sulfate dilution no ATP/1 :32 had the greatest number of bacteria per field of the dilutions using calcium sulfate, with almost 40 bacteria per field. The lowest number of bacteria per field for calcium sulfate was 1 :256/1 :64 (ATP/calcium sulfate) at less than 20 bacteria per field. The sodium sulfate dilution with the greatest number of bacteria per field was the 1 :2/1 :64 (ATP/sodium sulfate) dilution with over 50 bacteria per field. The lowest number of bacteria per field for the sodium sulfate field was the no/l :32 with less than 5 bacteria per field. Overall, the wells with calcium sulfate (either alone or in combination with ATP) exhibited the highest number of bacteria per field, when compared to the wells with sodium sulfate (either alone or in combination with ATP).

The experiment was completed in duplicate and the results from the two investigations compared. The main difference between the two experiments was that only two dilutions were used for the sulfates and ATP (high and low): Sodium sulfate H-neat (100 mM) and L- 1 :16; Calcium sulfate H-neat (saturated) and L-1 : 16; ATP H-1 :32 and L-1 :64. The results of the duplicate experiment are shown in Figure 2.

The results of the second experiment yielded similar results. The highest number of bacteria per field was the sodium sulfate high dilution/no group with about 200 bacteria per field. The second highest number of bacteria per field was the calcium sulfate high dilution/no group with about 180-190 bacteria per field. The lowest number of bacteria per field was the sodium sulfate low dilution/no group with about 10 or less bacteria per field. Overall, the sodium sulfate groups yielded a greater number of bacteria per field.

EXAMPLE 3

This example illustrates the effect of sodium bisulfite and ATP on the maintenance, growth and viability of Lawsonia intracellularis. Materials and Methods

First, frozen Lawsonia stock was removed from a freezer and allowed to thaw at room temperature. While the frozen Lawsonia is being thawed, lOOμL of a DMEM + 5% IFBS solution was added to each well of a 96-well cell culture plate containing McCoy cells. Two dilution plates were used to titrate 35% sodium bisulfite (1.8M) (2-fold) and ATP (0.5M) (2- fold) across and down the plate, respectively. 20μL of the ATP was added to the desired wells (1 :16 (31.3mM) dilutions were added to wells C3-C10; 1 :32 (15.6mM) dilutions were added to wells D3-D10; and the 1 :64 (7.8mM) dilutions were added to wells E3-E10). 20μL of the sodium bisulfite titrations were then transferred from the dilution plate to the culture plate (1 :2 (90OmM) dilutions in wells C3-C6, D3-D6, and E3-E6; 1 :256 (7mM) dilutions in wells C7-C10, D7-D10, and E7-E10. The method of Example 2 was also completed at this stage, but the steps not repeated herein. Finally, once the Lawsonia stock was thawed, 50μL of the stock was added to each well. Final concentrations of ATP were 3.13mM (1 :16 dilution wells), 1.56mM (1 :32 dilution wells), and 0.78mM (1 :64 dilution wells). Final concentrations of sodium bisulfite were 9OmM (1 :2 dilution wells) and 0.7mM (1 :256 dilution wells). The plate was then placed into a GasPak jar, and the air was evacuated and replaced with the same specialty gas as in Example 2. The jar was then placed in an incubator and incubated for 7 days at 37⁰C. The gas was exchanged every other day. After incubation, the plate was divided into 7 groups, as represented in Table 2:

Table 2

The contents of the wells in each group were then disrupted, pooled, and then centrifuged. The resulting supernatants were discarded and the resulting pellets each suspended in 50μL DMEM. 10μL of each group was then placed on a microscope slide and IFA stained for a direct count as in Examples 1 and 2.

30μL of the remaining pool was then added to 270μL of the DMEM + 5% IFBS serum for a 1 :10 dilution. Then, lOOμL of the resulting dilution was added to 900μL of DMEM + 5% IFBS serum. The dilution plate was then used to make the rest of the dilutions, which continued down to the bottom of the plate (for a final dilution of 1 :1 x 10^~9). On day 6, two cell culture plates were planted with 15,000 cells/ml and allowed to incubate overnight until they reached a confluency of approximately 85%. The plates were then examined for confluency. After this examination, the dilutions were then transferred onto the McCoy cells on the plates, and the two plates were then placed into a GasPak jar. The jar then had its air evacuated and replaced with the specialty Linweld gas (see above). The plates were then incubated at 37⁰C for 4 days, and the gas exchanged every other day. The plates were then IFA stained and the titer for each sample group calculated.

Results/Conclusions

The bisulfite plus ATP was shown to enhance growth and maintenance of Lawsonia.

EXAMPLE 4

This example examined the effect of ATP on the maintenance and growth of Lawsonia intracellularis .

Materials and Methods

First, McCoy cells were counted using a ViCeIl counter (Beckman Coulter, Fullerton, CA). The cells were counted to determine the starting volume of DMEM for the three 125mL spinner flasks to be used for incubation. The volumes were selected such that there were 200,000 cells per mL. Next, appropriate volumes of DMEM were added to each of the three spinner flasks. Then, the cells were added to the spinner flasks. Next, thawed Lawsonia stock was added to spinner flask 1. Then, thawed Lawsonia stock to which 75 μL of 0.5M ATP had been added three hours earlier was added to spinner flask 2. Finally, thawed Lawsonia stock to which 75 μL of 0.5M ATP had been added six hours earlier was added to spinner flask 3. Just prior to inoculation in the spinner flasks, and 750μL sample from each of the Lawsonia stocks was taken. The three flasks were then placed into GasPak jars, where the air was removed and then replaced with the same specialty gas that was used in Example 1. The cultures were then incubated for four days at 37⁰C, and the gas was changed every other day. On the fourth day, the cultures in each flask were re-fed with 65mL of fresh DMEM. Just prior to the re-feed, a ImL sample was taken from each of the three spinner flasks. After the re-feed, the cultures were placed back into the jars and allowed to incubate for an additional 3 days. After incubation, a 1.5mL sample was taken from each of the spinner flasks.

Each of the samples removed in the procedure above were subsequently prepared for microscopy examination according to the procedure described above in Example 1, and then examined according to the procedure described in Example 1, with the exception that the pre- inoculation sample was divided into 8, rather than 4, fields. Results and Conclusions

The results of the microscopy may be seen in Tables 3 and 4, below. Table 3

Table 4

In the pre -inoculation stocks, the addition of ATP at both 3 and 6 hours before inoculation seems to have resulted in an approximately twofold increase in the amount of Lawsonia present as compared to stock that contained no ATP.

In the pre-refeed samples, the actual bacterial field was much lower, due to the dilution that occurred when the inoculum was added to approximately 125mL of media and fresh McCoy cells. Even with this dilution, there is observed a significant difference in the number of bacteria present in comparing the Lawsonia stock with the 6-hour ATP as compared to the stock with no ATP.

With the post-refeed samples, there appears to be no significant differences in the bacterial fields in any of the flasks, although flasks 2 and 3 did show slightly higher numbers of bacteria than did flask 1.

EXAMPLE 5

Materials and Methods

This example used 8, 50OmL spinner flasks, with each flask containing 200 ml volume of cell culture. The spinner flasks were seeded with McCoy cells at about 2 x 105 cells/ml. Next, the spinner flasks were infected with about 0.1 MOL L. intracellularis . Three spinner flasks were made up as control flasks. One of the flasks was very magenta in color and the pH was not correct, so it was removed from the study. Three (3) of the flasks were treated with 10OmM FeSO₄ on day O, 2, and 4 post infection. Two (2) of the spinner flasks were treated with 10OmM FeSO₄ on day 0, 2, 4 and 6 post infection. All spinner flasks were incubated for 8 days and samples were taken daily for titration.

Results and Discussion

FeSO₄ had a positive effect on the viable cell numbers. The values for 'All FeSO₄' were obtained by averaging all the data for each respective day for cultures that were treated with FeSO₄. The results are illustrated in Figure 5.

Titration results included titers, which were obtained via the TCID50 method. Values for 'All FeSO₄' were obtained by averaging all the data for each respective day for cultures that were treated with FeSO₄. Peak titers of the Control (~5.6 log₁₀ TCID50/ml) were within ~0.5 log₁₀ of the historical average for this seed at this MOI. Relative to the Control, the FeSO₄- treated flasks averaged -0.5-1.0 logs higher than the Control from Day 4 onward. The titration results are summarized in Figure 6.

In conclusion, based on the data from this study, FeSO₄ appears to have a positive effect on Lawsonia growth in spinner flasks. Two additional spinner flasks got contaminated and were removed from the analysis. The average values for viable cells and titer are presented above. The results show that FeSO₄ acts as a growth enhancing nutrient for L.intracellularis when added to cell culture. EXAMPLE 6

This example illustrates the effect of sodium bisulfite and ATP on the growth, maintenance, and viability of Lawsonia. Materials and Methods

First, frozen Lawsonia stock was removed from a freezer and allowed to thaw at room temperature. While the frozen Lawsonia is being thawed, lOOμL of a DMEM + 5% IFBS solution was added to each well of a 96-well cell culture plate containing McCoy cells. The wells were filled with one of 5 dilutions as well as culture wells filled with the sodium bisulfite and ATP solution which was not diluted and a negative and positive control, for a total of 8 groups. A summary of the amount of sodium bisulfite and ATP in each group is illustrated in the table below.

20μL of the sodium bisulfite titrations were then transferred from the dilution plate to the culture plate. The method of Example 2 was also completed at this stage, but the steps not repeated herein. Finally, once the Lawsonia stock was thawed, 50μL of the stock was added to each well. The plate was then placed into a GasPak jar, and the air was evacuated and replaced with the same specialty gas as in Example 2. The jar was then placed in an incubator and incubated for 7 days at 37⁰C. The gas was exchanged every other day. The cell culture wells were then evaluated for titrations and bacteria per field. Results

Sodium bisulfite and ATP had a positive effect on the growth, maintenance, and viability of Lawsonia. Figure 3 illustrates the titer results for each group. Figure 4 illustrates the bacteria per field for sodium bisulfite and ATP. The highest bacteria per field was Group3/1.5mM ATP, with about 460-480 bacteria per field. All bacteria per field values were higher than the negative control indicating a positive effect on the growth of Lawsonia. The lowest bacteria per field, other than the control, was Group2/3mM ATP, with a little over 100 bacteria per field. The titers for each group can be found in Figure 3 and the accompanying table. The highest titer was in Group 4 and the lowest titer was in Group 5. All titer values were higher than the negative control indicating a positive effect on the growth of Lawsonia.

Claims

1. A cell culture medium, comprising a basal cell culture medium and growth-enhancing nutrient(s) selected from the group consisting of nucleoside triphosphate, sulfates, sulfites and combinations thereof.

2. The cell culture medium of claim 1, wherein said sulfate is selected from the group consisting of calcium sulfate, iron sulfate, sodium sulfate, and combinations thereof.

3. The cell culture of claim 1 or 2, wherein said nucleoside triphosphate is ATP.

4. The cell culture medium of any of the claims 1 to 3, wherein said ATP is present in an amount of about O.lmM to about 5mM.

5. The cell culture medium of any one of claims 1 to 4, wherein said nucleoside triphosphate sulfates, sulfites or combinations thereof, enhance the growth and maintenance of the bacteria Lawsonia intracellularis .

6. The cell culture medium of any one of claims 1 to 5, wherein said basal cell culture medium is supplemented with one or more additives.

7. The cell culture medium of any one of claims 1 to 6, wherein said basal cell culture medium is Dulbecco's Minimum Eagle Medium.

8. The cell culture medium of any one of claims 6 or 7, wherein said additive is serum.

9. The cell culture medium of claim 8, wherein said serum is Fetal Calf Serum.

10. The cell culture medium of any one of claims 1 to 9, wherein said sulfate is present in an amount of about 0.00 lμM to about 30OmM.

11. The cell culture medium of any one of claims 2 to 10, wherein said calcium sulfate is present in an amount of about 0.00 lμM to about lμM.

12. The cell culture medium of any one of claims 2 to 11, wherein said sodium sulfate is present in an amount of about 1OmM to about 30OmM.

13. The cell culture medium of any one of claims 2 to 12, wherein said iron sulfate is present in an amount of about 30μM to about 500μM.

14. The cell culture medium of claim 1, wherein said sulfite is a bisulfite.

15. The cell culture medium of claim 14, wherein said bisulfite is sodium bisulfite.

16. The cell culture medium of any one of claims 1 to 15, wherein said sulfite is present in an amount of 0.ImM to about 22OmM.

17. The cell culture medium of any one of claims 1 to 16, further comprising McCoy cells.

18. A method of enhancing cell culture medium comprising the step of adding nucleoside triphosphate or at least one sulfate, at least one sulfite, or a combination thereof, to a basal cell culture medium.

19. The method of claim 18, wherein said basal cell culture medium is supplemented with one or more additives.

20. The method of claims 18 or 19, wherein said basal cell culture medium is Dulbecco's Minimum Eagle Medium.

21. The method of claims 19 or 20, wherein said additive is serum.

22. The method of claim 21 , wherein said serum is Fetal Calf Serum.

23. The method of any one of claims 18 to 22, wherein said basal cell culture medium contains McCoy cells.

24. The method of any one of claims 18 to 23, wherein said sulfate is selected from the group consisting of calcium sulfate, iron sulfate, sodium sulfate, and combinations thereof.

25. The method of any one of claims 18 to 24, wherein said sulfate is present in an amount of about 0.00 lμM to about 30OmM.

26. The method of claims 24 or 25, wherein said calcium sulfate is present in an amount of about 0.00 lμM to about lμM.

27. The method of any one claims 24 to 26, wherein said sodium sulfate is present in an amount of about 1OmM to about 30OmM.

28. The method of any one of claims 24 to 27, wherein said iron sulfate is present in an amount of about 30μM to about 500μM.

29. The method of any one of claims 18 to 28, wherein said nucleoside triphosphate is ATP.

30. The method of claim 29, wherein said ATP is present in an amount of about 0.ImM to about 5mM.

31. The method of any one of claims 18 to 30, wherein said sulfite is a bisulfite.

32. The method of claim 31 , wherein said bisulfite is sodium bisulfite.

33. The method of any one of claims 18 to 32, wherein said sulfite is present in an amount of O.lmM to about 22OmM.

34. A method of creating any of cell culture medium of claims 1 to 17 comprising the steps of adding a nucleoside triphosphate, sulfates, sulfites, or combinations thereof, to basal cell culture medium.

35. The method of claim 34, wherein said culture medium enhances the growth and maintenance of Lawsonia intracellularis .

36. A method of enhancing the growth of Lawsonia intracellularis comprising the step of cultivating/growing said Lawsonia intracellularis in any of the cell culture medium of claims 1 to 17.

37. The method of claim 36, wherein said Lawsonia intracellularis being cultivated under reduced oxygen conditions.

38. The method of claims 36 or 37, wherein said Lawsonia intracellularis being cultivated under in the absence of molecular hydrogen.

39. The method of claims 36 or 37, wherein said Lawsonia intracellularis being cultivated in the presence of molecular hydrogen.