Inventors Frederick R. Bethke, Eric Ellis, and Amy Yanega Assignee Akzo Nobel NV, Arnhem, The Netherlands Background of the Invention Bartonella henselae is an agent of human cat scratch disease (CSD) and has been associated with bacillary angiomatosis, bacillary peliosis, recurrent bacterimia, and endocarditis. (cat scratch disease, bacillary angiomatosis, and other infections due to Rochalimaea N. , et al. , New England Journal of Medicine, 1994,330 : pages 1509-1515).

While cats have been shown through evidence to serve as vectors for the transmission of Bartonella henselae to people, cats appear to be asymptomatic to natural infection.

(Bartonella R. Bacteriamia and Three Feline Populations, Kordick et al. , abstracts of the 34 Inter Science Conference on Anti-Microbial Agents and Chemotherapy, American Society for Microbiology Washington DC, 1994). However, some recent studies have indicated that experimentally infected cats may develop clinical signs such as fever, anorexia, lethargy, and peripheral lymphadenopathy. (Experimental and Natural Infection with Bartonella henselae in domestic cats, et al. , Comp. Immuno. Microbial. In Fact. Dis. , 1997,20 : pages 41-51). These clinical signs dissipate within a short time and may not even be noticed by the cat owner. However, infections are prone to relapse.

It has been well documented in the literature that there is a strong immune response to infection with Bartonella henselae. (Identification of Bartonella-specific imunodominant antigens recognized by the feline humoral immune system, Freeland et al, Clinical and Diagnostic Immunology, July 1999, pages 558-566). However, the parthógenesis of Bartonella henselae in cats is not clearly understood. A complicating factor in the detection of Bartonella henselae is that cats naturally infected with Bartonella henselae commonly have periods of recurring bacteremia that may last months to years without causing clinical disease during those periods. (Clinical disease in kittens inoculated with the pathogenic strain of Bartonella henselae, Mikolajczyk et al. , AJVR, volume 61, Number 4, April 2000, page 375).

There are conflicting reports with regard to clinical signs of experimentally infected cats. (Experimental of Natural infection with Bartonella henselae in domestic cats, Abbott et al. , Comp. Immunol. Microbol. Infect. Dis. , 1997 20: pages 41-51).

Various studies have reported absence of clinical signs in experimentally infected cats while others have reported mild clinical signs, including mild fever, as well as histopathological lesions in some cats up to 8 weeks post infection. (Relapsing bacteremia after blood transmission of Bartonella henselae infected cats, Kordick et al., American Journal of Veterinary Research, 1997,58 : pages 492-497). Other clinical signs in kittens experimentally infected with Bartonella henselae have included lethargy and anorexia. (Clinical disease in kittens inoculated with the pathogenic strain of Bartonella henselae, Mikolajczyk et al. , AJVR, volume 61, Number 4, April 2000, page 378).

Another interesting observation is that kittens infected with Bartonella henselae have experienced two episodes of clinical signs as opposed to adult cats infected with Bartonella henselae having experienced only one episode of clinical signs. (Id).

Pet cats are not normally screened for Bartonella infections or for antibodies to B. henselae. However, serological screening could be beneficial to owners who are immunocompromised or to owners having young children by safeguarding against the adoption of potentially infected cats. Cat scratch disease can lead to potentially serious diseases in humans, particularly in young children and immunocompromised individuals.

Therefore, screening of cats for Bartonella infections is desirable.

Fermentation is a common method of growing cultures. Typically, fermentations are characterized by the following phases, as illustrated in Figure 1: 1) The Lag phase: Characterized by the physicochemical equilibration between microorganism and the environment following inoculation. There is typically very little growth during lag phase.

2) The Log growth phase: Characterized by adaptation of the cells to the new conditions of growth. Growth of the cell mass can now be described quantitatively as a doubling of cell number per unit time.

3) The Stationary phase: Characterized by slowing down or stopping of growth. The biomass increases only gradually or remains constant.

4) The Death phase: Characterized by exhaustion of the energy reserves of the bacterial cells. The cells in this phase are dying at an exponential rate.

Fermentation of Bartonella henselae for production of quantities sufficient for vaccine production is not a procedure known in the art. This organism is commonly thought of as fastidious and slow-growing. Further, most descriptions for growth of Bartonella henselae in the scientific literature refer to growth on solid media (agar) (Clinical disease in kittens inoculated with the pathogenic strain of Bartonella henselae, Mikolajczyk et al. , AJVR, volume 61, Number 4, April 2000, page 375). Although there are several descriptions in the literature for growth of Bartonella in broth media, these were been described for small-scale research growth and did not include multiple passages or subcultures which are necessary for commercial scale-up for commercial production. (Clinical disease in kittens inoculated with the pathogenic strain of Bartotaella henselae, Mikolajczyk et al. , AJVR, volume 61, Number 4, April 2000, page 375).

Accordingly, the art field is in search of a method of growing bacteria, in general, and this organism in a controlled condition and in a repeatable manner such that it is useful for generating large quantities suitable for vaccine production.

Summary of the Invention Embodiments of the present invention generally provide for a novel and non- obvious process for scale-up and growth in a fermentor of a Gram-negative bacteria.

Further embodiments relate to apparatuses for conducting the fermentation.

In an embodiment, the bacteria is Bartonella henselae. In an embodiment, it is an object of the invention that from a single vial (starting point), Bartonella henselae can now be scaled-up and grown in a controlled fermentor to quantities useful for commercial preparation of vaccine antigen in as few as 11 days.

It has been found, surprisingly, that growth of bacteria conducted at low dissolved oxygen (DO) levels achieves a higher growth rate and better culture. In an embodiment, the DO is maintained below about 5.0%. Further, and surprisingly, it has been found that the combination of scale-up cultures to get to large scale, certain passage ratio for the scale-up cultures, initiation of a first broth culture from an agar culture, inclusion of hemin in the broth medium, and the use of optical density (OD) to determine harvest time coupled with certain passage times for the scale-up cultures (+/-8hours) operate to achieve a previously unknown and highly desirable process for fermentation of Bartonella henselae into quantities sufficient for vaccine production.

Brief Description of the Figures Figure 1 is an illustration of the phases of a typical fermentation.

Figure 2 is an illustration of a fermentation.

Figure 3 is an illustration of an alternate fermentation.

Figure 4 is an illustration of an alternate fermentation.

Figure 5 is an illustration of an alternate fermentation.

Figure 6 is an illustration of an experiment comparing OD of a fermentation culture with CFU titer. The data illustrate that the approach of an OD of 0.9 correlates with the peak titer in CFU/ml.

Figure 7 is an illustration of an alternate experiment comparing OD of a fermentation culture with CFU titer. These data also illustrate that the approach of an OD of 0.9 correlates with the peak titer in CFU/ml.

Detailed Description of the Invention For purposes of this disclosure, and any related or dependant disclosure, the term "bacteria"means and refers to a microorganism that does not have any internal cell membranes. For purposes of this disclosure, and any related dependent disclosure, the term"about"means and refers to a variance of 10% both above and below the number or numbers modified by the term"about, "unless specifically stated otherwise.

Further, for purposes of this disclosure, and any related or dependant disclosure, the term"scale-up" ;"scale up" ; and, any conjugation thereof, means and refers to a proportional increase, such as when taking a cell manufacturing process from a relatively small volume fermentor to a larger fermentor, to produce more cells. Examples of ferementors and methods of fermentation useful with various embodiments of the present invention comprise fermentators found and/or discussed within US Pat No. 6,631, 732 and US Pat Application Nos. 20020164653A1 ; 20030054546A1 ; 20030194692A1 ; 20020045347A1 ; 20030039731A1 ; 20030153059A1 ; 20020117445A1 ; 20030051396A1 ; and, 20030190742A1. However, any fermentor may be used in varying embodiments of the present invention.

In general, in an embodiment, the invention provides for a novel and otherwise undocumented process for scale-up and growth in a fermentor of a bacteria. In an embodiment, the bacteria is Bartonella henselae (B. henselae). However, embodiments of the invention are contemplated to function/effectively grow other forms of bacteria and other organisms. Specifically contemplated organisms of Bartonella comprise Bartonella quititana, Bartonella bacilliformis, Bartonella vinsonii, Bartonella claz7-idgeiae and the like.

In an embodiment, an organism grown according to the methods of the present invention can be scaled-up and grown in a fermentor to commercial quantities in approximately 11 days. In an alternate embodiment, scale-up is performed from about 7 days to about 20 days. In an alternate embodiment, scale-up is performed from about 5 days to about 35 days. In another alternate embodiment, scale-up is performed from about 3 days to about 50 days. However, the period of time for scale-up may vary, as one skilled in the art will appreciate for each individual fermentor and/or bacterium.

Scale-up procedures may be designed to, for example, and not by way of limitation, take a laboratory scale fermentation to a production scale fermentation. In an embodiment, the scale-up may be from about 1 liter to about 20 liters to a production scale of about 2000 liters. In an alternate embodiment, the scale-up may be from less than 1 liter to greater than 2000 liters. However, the scale-up size may vary according to the desired amount of product and the many embodiments of the present invention are intended to cover any size scale-up.

In a preferred embodiment, the scope of the invention comprises optimized conditions for the scale-up and fermentation procedures useful in obtaining commercial quantities of an organism. In a most preferred embodiment, the organism is B. henselae.

In general, embodiments of the invention may be carried out as such: Processes for preparing suitable quantities of antigen for vaccine production comprising the steps of : preparing a bacteria culture; fermenting the bacteria culture; and, harvesting the desired product.

In varying embodiments, the step of preparing the bacteria culture further comprises at least one of preparing solutions, scaling-up the bacteria cultures, setting fermentor preferences, growing the bacteria on an agar plate prior to transferring to a broth, and the like. Solutions that may be utilized with various embodiments of the present invention are well-known in the art. Common examples include"Brucella Broth, ""Hemin solutions, "Blood Agar, hydrochloric acid, sodium hydroxide, anti-foam, and the like. There are many manufacturers of such solutions, as is understood by one of ordinary skill in the art, such as Difco and BD.

Various embodiments of the present invention may scale-up cultures of bacteria.

However, in other embodiments, no scale-up is required. In embodiments utilizing a scale-up, any number of scale-ups may be done. In a preferred embodiment, four scale- up procedures are conducted. In an alternate embodiment, between about two and about ten scale-ups are performed. In an alternate embodiment, between about one and about fifty scale-ups are performed. However, the number of scale-ups may be any number.

However, it is preferred to have fewer scale-ups so that production may begin sooner rather than later.

In embodiments growing the bacteria on an agar plate, any agar medium may be utilized. Common agar mediums in the art include blood agars and the like. However, each organism grown may have a particular agar medium best suited for its growth. Such will be readily apparent to those of ordinary skill in the art and easily discernable from the literature, such as Bergey's Manual of Determinative Bacteriology.

In various embodiments, a desired product is a vaccine antigen. However, other embodiments are for diagnostic antigens for use in immunoassays (such as serological assays and antigen-detection assays) and immunogenic composition antigens.

The inventors of the claims appended hereto have discovered that, contrary to accepted fermentation methodology, certain bacteria cultures grow to sufficient optical densities at a relatively low DO level. In an embodiment, the DO level is less than about 5. 0% +/-3.0%. In another embodiment, the DO level is less than about 10.0% +/-3.0%.

In an alternate embodiment, the DO level is less than about 25.0% +/-5. 0%.

Further, and surprisingly, it has been found that the combination of scale-up cultures to get to large scale, certain passage ratio for the scale-up cultures, initiation of a first broth culture from an agar culture, inclusion of hemin in the broth medium, and the use of optical density (OD) to determine harvest time coupled with certain passage times for the scale-up cultures (+/-8hours) operate to achieve a previously unknown and highly desirable process for fermentation of bacterium, such as Bartonella Itenselae, into quantities sufficient for vaccine production.

Processes and methods used for fermentations are known. However, the inventors of the present process have discovered that acceptable growth may be realized when the OD at 500 nm becomes greater than 0.9 but before it reaches 1.0. Processes for measuring the OD may be by any method common the art, such as by spectrophotometer. In other embodiments, acceptable growth may be realized when the OD at 500 nm becomes greater than 0.7 but before it reaches 1.2.

For a further understanding of the scope of the invention, attention should be had to the appended claims and example.

Example: Materials and Methods: The following procedure can be utilized with various embodiments of the present invention. However, the scope of the invention is intended to cover variations of the following methods.

Media and Components Preparation of Columbia Blood Agar: Dissolve 44 g of Columbia Blood Agar Base per liter of deionized water while stirring on a heated hot plate (do not boil) for 3 hours. Autoclave at 121°C for 30 minutes.

Place agar in 50°C waterbath for 1-2 hours. Add 50 ml fresh defibrinated rabbit blood per liter of agar and gently swirl to mix. Add 80 ml of agar with rabbit blood to each 175 cm2 tissue culture flask, cap flask tightly, and lay flask horizontally until agar is completely cooled, approximately 30 minutes. Store at 4°C until use.

Preparation of Brucella broth: Dissolve 28 g of Brucella Broth powder per liter deionized water while stirring on a heated hot plate. Alternatively, dissolve the Brucella Broth powder in deionized H20 directly in the fermentor. Cool and adjust pH to 7.2. Steam sterilize at 121°C for 30-45 minutes. Store at 4°C until use if not used within 24 hours.

Preparation of Hemin Stock Solution (5 mg/ml in 10 mM NaOH, pH 7.2) : Add 2.5 g hemin per 0.5 liter of 10 mM NaOH while stirring (using magnetic stir bar) on a heated hot plate (do not boil). Stir for one hour. Cool and adjust pH to 7.2 using IN HCL. Autoclave for 30 minutes at 121°C. Store at 4°C until use. Stir on a magnetic stir plate for approximately 10 minutes prior to use to resuspend hemin.

Preparation of Bartonella Broth: Add Hemin Stock Solution to Brucella Broth at 1: 20 for a final hemin concentration of 250 Fg/ml.

Measuring Broth Culture Growth by Spectrophotometric Determination Growth is monitored by determining the optical density using a BioRad SmartSpec 3000 spectrophotometer or equivalent. The samples were prepared as follows: 432 ßl of 5M NaOH was added to a 12 ml culture sample and mixed to solubilize most of the hemin stock solution. The resulting mixture was centrifuged at 1,329 x g for 10 minutes at 20°C, the liquid was then poured off, and the pellet resuspended in 12 ml of phosphate buffered saline (PBS). The sample was again centrifuged at 1, 329 x g at 20°C for 10 minutes, the liquid poured off, and the pellet resuspended in 3 ml PBS. The absorbance was then read at 500 nM after recalibrating the instrument with the same PBS used for resuspending the pellet.

Seed Passage and Scale-up X+1 agar culture: The X+1 culture was prepared as follows: One vial of B. henselae seed (X+0 culture) was thawed and diluted 1: 20 in Brucella Broth. Two 175-cm2 flasks of Columbia Blood Agar (5% rabbit blood) were inoculated each with 1.5 ml of the diluted seed. The flasks were loosely capped and incubated at 37°C with 5% C02 for approximately 4 days. After 4 days, the bacterial growth was collected from the agar surface using sterile 7 mm glass beads and 8.5 ml Brucella Broth per flask, to dislodge growth. Then, the flask was shaken to dislodge the bacteria. The broth containing the dislodged Bartonella from the 1st agar flask was then transferred to the 2nd agar flask containing glass beads using a pipette. The 2"''agar flask was then shaken and the broth containing the dislodged Bartonella growth pipetted from both flasks to a sterile 50 ml tube. This process was repeated with an additional 8.5 ml of broth to harvest the remaining Bartonella. The harvest resulted in approximately 17 ml of collected broth containing Bartonella cultures. The 50-ml tube of collected Bartonella material was then vortexed immediately prior to use for seeding the X+2 broth culture.

X+2 broth culture: The X+2 culture was prepared as follows: 500 ml of Bartonella Broth was inoculated in a 1,000 ml shake flask with 1,250 u, l of the collected X+1 culture. A sterile cotton stopper (covered with aluminum foil) was used to cover the flask. As precaution, to assist in preventing contamination, the stopper was flamed before replacing on culture flask. The X+2 culture was heated at 37°C +/- 2°C in air on a shaking platform at approximately 100 rpm for approximately 72 hours.

X+3 broth culture: The X+3 culture was prepared as follows: The X+2 culture is passed 1: 40 to an X+3 culture. The X+3 culture will need to be of sufficient volume to inoculate the fermentor with a 1: 10 (v/v) inoculum (this X+3 example provides for 10 liters of X+3, which is 10 times the volume necessary for inoculating a 10 liter X+4 fermentation culture as described below). Larger vessels may also be used. Inoculating the fermentor at 1: 20 will also work but will typically delay the culture reaching peak growth by 12-24 hours. The X+2 culture was then swirled to adequately resuspend the Bartonella and the hemin stock solution. 250 ml of X+2 culture solution was then added to 9,750 ml Bartonella Broth in a 20 liter vented plastic carboy, by pipette. The resulting culture was heated at 37°C +/-2°C in air on a shaking platform at approximately 100 rpm for 42-66 hours to make the X+3 culture.

Fermentor Preparation and Settings Prior to running a fermentation, the Fermentor should be Set-up, by at least calibrating the pH probe. Brucella broth was prepared directly in the fermentor by adding 280 g of dehydrated Brucella Broth to 10 liters of deionized water. 1 ml per 10 liters of Sigma Antifoam-A was added to the media in the fermentor to arrive at a final volume concentration of 0.01%, which corresponds to a ratio of lml per 53.46 inches 2 of the media surface area. The culture media was then steam sterilized at 121°C for 30-45 minutes.

The dissolved oxygen (DO) probe was calibrated at 100% in the Brucella Broth in the fermentor, using air to increase the DO to 100%. Next, the DO probe was calibrated at 0% in the Brucella Broth using N2 or C02 to decrease the DO level to 0%. The addition of C02 caused the pH to drop dramatically.

The Hemin Stock Solution was then added to the Brucella Broth in the fermentor vessel (to create Bartonella Broth) at 1: 20 ratio (0.5 liter hemin stock per 10 liters of Brucella Broth) by addition through a sterile port. This is expected to increase the DO to approximately 2.5-3. 5%. The DO level is left near 0% after calibrating the DO probe at 0% by leaving the DO control in the off position until after addition of seed.

The parameters of the fermentor were set as such (for a ten liter fermentation culture in a twenty liter vessel): Temperature= 37. 5°C DO= 5% with air pH2 control= STIR + VALVE Gas Flow ratio control = 1. 5-8 lpm (start with 1.5 and gradually increase to 1. 5-8 lpm as needed); Control= TOTAL; Ratio= 100%; Proportional band setting= MINIMUM Auto/Valve pH 7.2 with 5N NaOH and 4N HCL RPM: minimum= 75; maximum= 200 Next, the acid/base lines were connected and primed. Base was added to manually adjust the pH to approximately 7.2.

Fermentation The DO was ensured to be between 0 and 5% before beginning fermentation. If the DO is >5%, the DO is lowered with N2 or CO2. Each vessel was inoculated with X+3 broth seed at approximately 1: 10 v/v (1.0 liter seed for every 10 liters Bartonella Broth).

A sample was taken to measure the optical density immediately after seeding the fermentor.

The DO controls were then turned on and the fermentation process initiated.

Samples were taken to measure the OD at time intervals after initiation of log growth phase. The culture was in log growth phase when the AIR valve is either cycling regularly or is open continuously, and when the STIR speed is either cycling regularly between the minimum and maximum speeds or is increased to the maximum speed continuously. The bacteria was harvested when the OD at 500 nm became greater than 0.9 but before it reached 1.0.

Example 2: Growth Comparison of bacteria in shake flasks using broth medium Table 1-Representative experiment comparing the growth of B. henselae for 2,3, 5,7, 8, 9, and 10 days in shake flasks using broth medium. The peak titer occurred at 2-3 days post-inoculation. Day Titer 2 7.80E+07 7. 80E+07 5 3. 60E+07 7 2. 12E+07 8 1. 44E+07 9 2. 26E+06 10 5.80E+05 Example 3: Growth Comparison of bacteria in shake flasks using broth medium Table 2-Representative experiment comparing the growth of B. henselae for 3,4, 5,6, 7, 8, and 10 days in shake flasks using broth medium. The peak titer occurred at 4 days post-inoculation.

Day Titer CFU/ml 3 1.06E+07 4 2. 10E+07 5 1.86E+06 6 1.40E+06 7 7.60E+04 8 4.80E+04 10 5.60E+04 Example 4: Growth Comparison of bacteria in shake flasks using broth medium Table 3-Representative experiment comparing the growth of B. henselae in shake flasks using broth medium for various time points. The peak titer occurred at 63 hours post-inoculation (HPI).

HPI Titer CFU/mi 2 1. OOE+06 8 1.02E+06 14 1.62E+06 24 4.40E+06 29 8.40E+06 32 1. 48E+07 39 2.46E+07 48 8.14E+07 53 1.20E+08 56 1.58E+08 63 2.22E+08 72 5.56E+06 78 3. 00E+05 Example 5: Growth Comparison of bacteria in scale-up cultures Table 4-Representative experiment comparing successive broth scale-up cultures and the time points of peak growth titer. For the first broth culture (X+3), peak growth was at 74 HPI. For later cultures (X+4 and X+5) peak growth occurred from 42-57 HPI. X+3 X+4 X+5 HPI Titer CFU/ml HPI Titer CFU/ml HPI Titer CFU/ml 0 1.20E+05 0 1.04E+06 0 6.02E+06 9 1.60E+05 9 2.24E+06 9 1.94E+07 18 4.60E+05 18 7.54E+06 18 4.64E+07 26 8.20E+05 26 1.68E+07 26 6.82E+07 31 1.60E+06 33 2.72E+07 33 9.80E+07 42 6.14E+06 42 7.22E+07 42 1.52E+08 48 1.08E+07 47 1.12E+08 47 1.26E+08 57 3.44E+07 50 1.52E+08 50 9.72E+07 zizi 8. 76E+07 57 2.50E+08 57 6.50E+07 74 1.38E+08 66 7.06E+07 66 3.94E+07 74 3.12E+07 74 1.42E+07 Example 6: Growth Comparison of bacteria in scale-up cultures Table 5-Representative experiment comparing successive broth scale-up cultures and the time points of peak growth titer. For the first broth culture (X+3), peak growth was at 74 HPI. For X+4 and X+5 cultures, peak growth occurred at 57-66 HPI. X+3 X+4 X+5 HPI Titer CFU/ml HPI Titer CFU/ml HPI Titer CFU/ml 0 2.08E+04 0 8.00E+04 0 9.80E+05 9 2.74E+04 9 2.20E+05 9 3.84E+06 18 3.68E+04 18 7.20E+05 18 1.06E+07 26 1.00E+05 26 1.72E+06 26 4.30E+07 33 2.52E+05 31 3.00E+06 33 4.22E+07 42 7.40E+05 42 9.60E+06 42 1.02E+08 47 1 32E+06 48 2.12E+07 47 1.70E+08 47 1.32E+06 48 2.12E+07 47 1.70E+08 50 1.86E+06 57 5.52E+07 50 2.06E+08 57 5.84E+06 66 1.32E+08 57 2.34E+08 66 1.60E+07 74 1.24E+08 66 5.54E+07 74 3.52E+07 74 4.62E+07 Example 7: Optimization of hemin concentration The optimal concentration of hemin in broth medium is 50-250 Fg/ml.

Table 6-An experiment comparing broth growth at three different concentrations of hemin. The highest titer occurred in broth media with a final hemin concentration of 250 µg/ml.

Day Hemin conc. Titer CFU/ml D3 50 2.24E+07 D3 150 2.96E+06 D3 250 2.96E+07 Example 7: Development and determination of fermentation conditions Numerous fermentation experiments were performed. The objective was to determine and optimize growth conditions and also to determine time of peak growth.

Graphs of four representative fermentation runs are shown in Figures 2-5. Each graph shows a typical successful Bartonella fermentation run. Characteristics of each fermentation phase are described as the following: 1) Bartonella Lag Phase: The cultures typically do not require regular input of air. Also, the stirring mechanism is controlled at the minimum stir speed. These parameters will gradually increase (the culture requiring air and stir speed increasing) as the culture transitions to log growth phase.

2) Bartonella Log Phase: The input of air and stir speed are either increasingly cycling and/or continually active.

3) Bartonella Stationary Phase: Air input and stir speed (from minimum setting to maximum) may still be continuously cycling on and off, but do so at a decreasing frequency/amplitude.

4) Bartonella Death Phase: There is a greatly decreased frequency/amplitude of air input and stir speed cycling.

Eventually, air input ceases and the stir speed will be running constantly at the minimum setting. There is an exponential death rate of Bartonella during this phase.

*Note : Although Figures 2-5 detail the fermentation process through stationary or death phase, the fermentation is usually interrupted at the end of the log phase for harvest of antigen for vaccine production.

5) Evaluation of anti-foam agents: Antifoam agents reduce the oxygen transfer rate but may be necessary for reducing excess bubbling and foaming of the culture during the stirring and addition of air. Addition of an antifoam agent interrupts renewal of the bubble surface by bubble movement, which in the case of fermentation is caused by the input of air and the mechanical action of the stirrer paddles. a. An experiment was performed to evaluate the effect of different concentrations (1%, 0.1%, and 0. 01% final concentrations) of Sigma corn oil and Sigma anti-foam A on the titers of Bartonella henselae broth cultures. Corn oil reduced growth 100% at all concentrations tested. Anti- foam A used at 1% concentration reduced growth 100%; at 0.1%, growth was reduced 95.5% ; and at 0.01%, growth was only reduced 26.6% The reduction in titer from use of Sigma anti-foam A at 0.01% concentration was acceptable.

6) Evaluation of scale-up growth in carboys: Using plastic carboys as the last growth vessel prior to inoculating the fermentor has several advantages, namely the ease of use and safety. We optimized scale-up in plastic carboy vessels and determined that the optimal conditions are as follows: a. 10 liters of culture in a 20 liter carboy b. Use of a vented cap and culture maintenance in an air environment c. Shaken on a rotating shake table at 100 rpm 7) Development of methods to determine optimal fermentor harvest time. a. Two experiments compared the measured number of viable Bartonella throughout the fermentation culture and compared them with the OD of the culture. The OD measurement was performed according to the method described in the following section, "Measuring Culture growth by spectrophotometric determination".

While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and the appended Claims are intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth whether now existing or after arising. Further, while embodiments of the invention have been described with specific dimensional characteristics and/or measurements, it will be understood that the embodiments are capable of different dimensional characteristics and/or measurements without departing from the principles of the invention and the appended Claims are intended to cover such differences. Furthermore, all patents, patent applications, articles, and other publications mentioned herein are herby incorporated by reference, including those patents, patent applications, articles, and other publications referenced in the patents, patent applications, articles, and other publications mentioned herein.

For a further understanding of various embodiments of the present invention, reference should be had to the following examples: