Method of obtaining toxin actinobacillus pleuropneumoniae apxi, applying culture medium, containing calcium-borogluconate complex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

5 cl, 4 tbl

 

The invention relates to a method for RTX-toxin ApxI by culturing bacteria Actinobacillus pleuropneumoniae in the cultural environment that ensures the growth of bacteria, to this medium was added calcium salt for education in the environment of calcium ions.

Pleuropneumonia of swine, the main respiratory disease of pigs, is distributed worldwide and causes severe economic losses in pig production due to lightning deaths, ill pigs and delays in sales because of chronically infected animals. The aetiological agent is Actinobacillus pleuropneumoniae. It is transmitted primarily through direct contact between animals, and the infection leads to disease from lightning to chronic. The disease mainly is an infection of the respiratory tract, with clinical signs of high fever, severe respiratory distress, cough and anorexia. Onset rapid, morbidity and mortality are high. One way to control infection by bacteria Actinobacillus pleuropneumoniae (hereinafter also referred to as "ADR") is a program of vaccination. These programs were used passivated bacteria, but aware of their severe side effects. Currently widely used in subunit vaccines on the again of toxins ARR.

The RDA produces the so-called RTX-toxins (RTX indicates the repeat in toxin). The presence of these RTX-toxins contribute to the pathogenic nature of this bacteria. RTX-toxins have been discussed in detail previously and described in the literature. As is well known, not all serotypes of APP produce all RTX-toxins. For example, serotypes 1, 5, 9 and 11 produce ApxI and ApxII. Serotypes 2, 3, 4, 6 and 8 produce ApxII and ApxIII. Serotype 10 produces only ApxI, and serotypes 7 and 12 produce only ApxII. Current commercially available vaccines against the RDA is based on the toxins ApxI, ApxII and ApxIII. Recently it was discovered that all serotypes of APP produce the fourth RTX-toxin, currently called ApxIV (see EP 0875574).

It is widely known how to produce RTX-toxin ApxI through cultivation of Actinobacillus pleuropneumoniae in the culture medium in which the salt of calcium (i.e. a chemical compound based on the acid formed by replacing all or part of the hydrogen ions of the acid to one or more of calcium ions). In particular, earlier in the EP 0453024 described this way (see "example 2", paragraph 2 "Purification and characterization of hemolysin", subsection "Methods"). You should take into account that the used ApxI should be marked "HLY" (see Frey et al. in the journal “J Gen Environ.”, August, 1993; 139(8): 1723-8). This EP patent is known about how to add in with the food calcium (CaCl 2). In fact, the article Environ Pathogenesis 37 (2004) 29-33 indicated that the transcriptional activity of the ApxI operon is amplified when you add in the growth environment of calcium. Thus, it can be ensured of the highest levels of toxin ApxI. The environment must support the growth of bacteria ARR. It is well known how to construct an environment that ensures the growth of bacteria. Classical culture medium was originally developed by Eagle, ham and other in 1950-60, They found that an environment that meets the needs of growth, must contain inorganic salts, a nitrogen source (for example, in the form of nitrogen-containing compounds, such as peptides or proteins), the source of carbon and vitamins. Environment mainly sautereau to prevent them from either acidification or alkalization. This basic recipe is available a large number of different compositions. For example, to provide the amino acids you can choose components of animal origin, but you can also choose chemically defined amino acids. Other connections are also possible large number of variants. Actually, to make an environment that ensures the growth of bacteria, is relatively easy. However, optimization of growth and/or receiving metabolites may take some time to develop, in particular, if preferred with the food, which does not contain serum or other components of animal origin. Strategies to improve fermentation environment, however, well known in this field and are described in detail in the literature (see, for example, a review article Kennedy and Krouse in journal of Industrial Microbiology & Biotechnology (1999) 23, 456-475). This optimization is part of routine experiments in the laboratory of fermentation. In the case of the cultivation of the RDA, NAD (nicotinamide adenine dinucleotide) essentially is part of the environment because the bacterium RDAs is a NAD-dependent. In the absence of NAD environment will not support the growth of bacteria Actinobacillus pleuropneumoniae and therefore can not be considered as a liquid medium to support the growth of the RDA from the point of view of this application and the attached claims. Environment to support the growth of bacteria or components for making such media are commercially available from numerous companies such as Sigma Aldrich, Quest International, Oxoid, Becton Dickinson, Pharmacia, VGD Inc, Mediatech, Invitrogen, Marcor, Irvin Scientific, etc.

Although the methods of the prior art sufficient to obtain economically meaningful output toxin ApxI, the applicant has realized that there is a possibility of improvement. It is in the process of fermentation medium becomes turbid. The merit of the applicants was the realization that this may be due to precipitation of one (or bore the channels at) calcium salts. It RDAs produces carbon dioxide, which in the environment is transformed into carbonate ions. Calcium carbonate is a salt with low solubility. Consequently, you may experience some problems. First, suppose that the precipitation takes involved calcium ions, making them unavailable to bacteria ARR. Secondly, precipitiously calcium salts cause problems associated with processing. In particular, the filters tend to clog. Therefore, the applicant added on Wednesday, a large number of complexing agents to see whether they can prevent the precipitation of salt. Actually, for example, adding EDTA environment can be more or less transparent. However, the use of these complexing agents have a negative effect on obtaining ApxI. Thus the assumption, apparently, incorrectly or incompletely. However, there is still a need to improve production ApxI.

Unexpectedly, it was found that when using borogluconate (for example, in the form of 2,3-dihydroxy-3-[2-hydroxy-5-(hydroxymethyl)-1,3,2-dioxaborolan-4-yl]propanoate; see also Herbert Taylor MacPherson and James Stewart of the Institute Moredun in Biochemical Journal: “Investigations on the nature of calcium borogluconate,” published November 16, 1937) for the formation of a complex with calcium ions can be obtained ApxI at a high level on sravneniya methods of the prior art, not applicable (not added) complexing agents or based on other complexing agents. Obviously, when using this particular complexing agent so that the environment contains a complex of calcium-borogluconate (for example, available in the form of D-gluconic acid, cyclic 4,5-ester with boric acid, calcium salt of 2:1), can be prevented significant precipitation of calcium ions with other negative ions, at the same time, calcium ions remain able to enhance the transcriptional activity of the ApxI operon of the bacterium Actinobacillus pleuropneumoniae. Apparently, calcium ions remain "trapped" in the complex salt, where communication "capture" on the one hand strong enough to prevent the formation of calcium ions precipitate, for example, carbonate or other negative ions, but on the other hand allow the bacteria to use calcium ions, if they are in free solution (i.e. form a complex only with water molecules). Apparently, borogluconate fully satisfies the critical balance that is necessary for the production of ApxI bacteria ARR.

In one embodiment, the implementation of the concentration of borogluconate is less than 60 mmol/l Over this concentration is found that the products ApxI falls on the low levels. Even if possible, it is preferable that the concentration remained below this figure. More preferably, the concentration was in the range of from 25 to 45 mmol/l, in particular 40 mmol/l, which seems to be optimal for some environments.

Although it is not essential to the present invention, the medium may not contain components of animal origin. The lack of many methods of the prior art is that they are based on the use of media containing components of animal origin, such as Colombian culture medium. Other components of animal origin referred to in the prior art, are, for example, a modified Colombian culture medium or cardio-cerebral infusion environment. As is well known, the use of components of animal origin has several significant drawbacks. First, the chemical composition can vary significantly between batches. In addition, supplements of animal origin may be contaminated by infectious agents. The most dangerous is the presence of prions that cause TSE in humans or animals. You can simply choose the environment that does not contain animal ingredients (often referred to as "ACF"). Component of animal origin" is this sense means any component, which is present as such in the animal (e.g., blood or protein) or is of such a component (for example, a modified serum obtained from the blood, or amino acids derived from protein). The applicant, however, found that the production efficiency of the ApxI significantly lower when using these environments ACF compared with media containing components of animal origin, even if the concentration of calcium is adequate. Not referring to theory, it is possible that when using serum, the problem of precipitation of the calcium salt will be not so heavy due to the presence of agents that form soluble complexes with calcium ions. In any case, when using borogluconate for the formation of a complex with calcium ions in these environments can also be obtained a significant increase in the yield of ApxI.

In another embodiment, the calcium salt is borogluconate calcium. Although it is possible even, for example, the use of calcium chloride as a source of calcium and adding borogluconate salt for the formation of a complex with calcium ions, preferably calcium was added in the form of borogluconate salt. Thus, there is no need to wait for equilibrium between a large number of physical reactions (precipitation, dissolution, destruction of the complex, formation of the complex)that occur in the environment. This saves time and therefore cost-efficient.

In another embodiment, in the process of cultivation through fluid leak air, and the air contains carbon dioxide gas above atmospheric level. Unexpectedly, it was shown that carbon dioxide increases the volume of production ApxI even more. Noted that most is known about the use of elevated levels of carbon dioxide during the culturing of bacterial colonies on plates (see, for example, U.S. patent 6019984: EXAMPLES of Bacterial strains and growth conditions"). However, this applies to the cultivation of colonies of bacteria, which are then used for inoculation of the fermenter. At this stage, the products RTX-toxins insignificant. More precisely, in General, it is clear (see, for example, Microbial Pathogenesis 37 (2004) 29-33)that the maximum production of Apx occurs when a high density of cells in fermenters, i.e. at the end of the exponential phase of growth. It is clear that at this stage, carbon dioxide is not suitable as a stimulating factor. Therefore, no attempt was made to increase the production of Apx through the use of increased levels of carbon dioxide. In particular, the applicant has found that when the content in the air to 5% vol. carbon dioxide (net carbon dioxide to the volume of normal air) products ApxI is away at a very high level. It is noted that in this embodiment, for the transmission of air through the environment can be used many techniques, usually with the help of a device that allows air bubbles to leak anywhere in the environment (i.e. under the surface of the medium). Under the "air" in the context of the present invention realize a gaseous environment containing one or more gaseous components that are normally present in atmospheric air, such as oxygen, nitrogen, carbon dioxide, helium, neon, argon, xenon, radon, etc.

The invention further optionally explained using the following non-limiting examples.

MATERIALS AND METHODS

Bacterial strain and environment

The studies were carried out using a strain of Actinobacillus pleuropneumoniae, produce ApxI, serotype 10, later in this document called the RDA 10. In all cases, work seed of this strain was restored using a Cup with a base Columbia blood agar (BAB) (production company Becton, Dickinson, USA). Used liquid medium consisted of either Colombian culture medium (production company Becton, Dickinson USA)maintained at pH of 7.3 using NaOH and acetic acid, or the environment, does not contain components of animal origin (called "environment ACF"). Last Wednesday contains MgSO4(0.75 g/l), the cyst is n·HCl (0.1 g/l), FeCl3(0.1 g/l), NaNO3(0.1 g/l), KCl (0.1 g/l), trace elements (for example, 2.5 ml of a solution SL-10 specified in the guidance Handbook of Microbiological Media, 3rdrdition, Ronald Atlas, CRC press, 2004), 50% glucose solution (10 ml) and 10 mm solution of amino acids (containing all 20 amino acids, except tryptophan), HEPES buffer (6 g/l; for example, available from the company Sigma Aldrich) and yeast extract (10 g/l; for example, available from Becton, Dickinson).

These have been used in rekultivirovanie and fermentation. Nicotinamide adenine dinucleotide (0,01%) and calcium (in various concentrations) was used preculture and fermentation. All the media were sterilized by filtration with a pore diameter of 0.22 μm. Before using fermentation environment was heated at 85°C for one minute.

Cultivation

Work seed strain ARR 10 were sown in a Cup with Columbia agar VAV, and incubated for approximately 24 hours at 37°C. Several colonies were selected for inoculation vessel with a volume of 500 ml containing 75 ml Colombian medium. The vessel was incubated for approximately 6 hours at 37°C with shaking for education preculture. Through this preculture carried out several fermentati. Some of them carried in containers with a volume of 500 ml In this case 75 ml of medium was inoculable 1 ml preculture. The vessels were incubated at 3°C with shaking. Alternative cultivation was carried out in fermenters SIXFORS (production company Infors AG, Switzerland), containing approximately 400 ml of culture medium, to which was added 20 ml of preculture as inoculum. The cultivation temperature is 37°C.

Tests

Cell growth was determined by measuring optical density (OD) at 660 nm. The concentration of antigen ApxI was measured using the enacted ELISA analysis.

RESULTS

The first experiment was conducted to determine whether more calcium, despite the formation of the complex with borogluconate for bacteria ARR. This experiment was carried out in vessels, as described herein above. The results are presented below in table 1.

Table 1
EnvironmentAntigen ApxI (IU/ml)
Colombian environment without adding Sa0
Colombian medium with addition of 25 mm Sa borogluconate7

As indicated by the data of table 1, in the formation of complex ions of calcium borogluconate can be obtained in good yield ApxI. An important advantage of the formation of this complex SOS is the RTO is the precipitation of the calcium salt is no longer a significant impact on the course of the process.

The second experiment was carried out to understand the effects of borogluconate in an environment that does not contain animal ingredients. To do this, the authors compared the addition of 20 mm solution of CaCl2with the addition of 20 mm solution of Sa borogluconate. The results are presented in table 2.

Table 2
EnvironmentAntigen ApxI (IU/ml)
ACF added 20 mm CaCl21
ACF added 20 mm Sa-borogluconate24

Obtained two results. First, it is clear that the use of calcium chloride obtaining sufficient quantities of ApxI environment ACF difficult even with the creation of normal levels of calcium. In the formation of a complex of calcium with borogluconate can be obtained a high yield of ApxI. Comparable results can be obtained in other operating environments. The authors have carried out such an experiment in an environment that did not contain any iron chloride or magnesium sulfate ("ACF-alt"), but otherwise was the same environment as the environment ACF described in the word document above. And this time the formation of a complex of calcium with borogluconate received increased significantly the levels.

The third experiment was carried out to study the effect of concentration of borogluconate. The authors used three different concentrations, namely 20, 40 and 60 mm borogluconate calcium. The results are presented in table 3.

Table 3
EnvironmentAntigen ApxI (IU/ml)
ACF added 20 mm Sa-borogluconate4
ACF added 40 mm Sa-borogluconate31
ACF, added 60 mm Sa-borogluconate1

As is clear from table 3, the optimal concentration of about 40 mm.

In the fourth experiment, the authors examined the effect of elevated levels of carbon dioxide to obtain ApxI. For this purpose, the authors used the environment ACF-alt, described herein above, and increased the level of sodium nitrate to 0.5 g/L. the Concentration of borogluconate varied between 40, 50 and 70 mm. The increased concentration of CO2received, maintaining a constant flow of air into the fermenter 1 vvm (volume of gas to volume of medium in a mine is) for a mixture of air/CO 295/5.about. The experiments were carried out in a fermenter SIXFORS, as described herein above. The results are presented in table 4.

Table 4
EnvironmentAntigen ApxI (IU/ml) ELISA
ACF-alt, added 40 mm Sa-borogluconate, 5% CO2520
ACF-alt, added 50 mm CA-borogluconate, 5% CO2357
ACF-alt, added 70 mm Sa-borogluconate, 5% CO2222

Based on these results, we can conclude that carbon dioxide has a positive effect on the production of ApxI: even at a concentration of 70 mm borogluconate calcium can be obtained with acceptable levels of ApxI. And in this case, the optimal concentration of 40 mm.

CONCLUSION

The applicant has found that in a liquid culture medium that supports the growth of bacteria RDAs, borogluconate can provide the ultimate balance between preventing precipitation of calcium ions with negatively charged ions, on the one hand, and maintaining the calcium ions available for stimulation of Actinobacillus pleuropneumoniae to products ApxI, on the other hand. This can be the t can be preferably used in any environment for the cultivation of Actinobacillus pleuropneumoniae, containing negative ions, which form a precipitate with calcium ions. In fact, depending on the environment and optimize its components bacteria RDAs will produce higher or lower levels of ApxI. But because of the shielding effect of borogluconate will work regardless of the actual speed of production of the bacteria, this solution can be successfully used for all environments, in particular because essentially all environments contain carbonate ions, which are ions that can form a precipitate with calcium ions.

1. The method of obtaining the RTX-toxin ApxI by culturing bacteria Actinobacillus pleuropneumoniae in the cultural environment that ensures the growth of bacteria, and to this medium was added calcium salt for education in the environment of calcium ions, wherein the culture medium contains borogluconate in concentrations of less than 60 mmol/l for education in an environment of complex calcium-borogluconate.

2. The method according to claim 1, characterized in that the concentration of borogluconate is in the range from 25 to 45 mmol/l, preferably 40 mmol/L.

3. The method according to claim 1 or 2, characterized in that the calcium salt is borogluconate calcium.

4. The method according to claim 1, characterized in that in the process of cultivation through the liquid medium is passed the air, the rich air contains carbon dioxide gas above atmospheric level.

5. The method according to claim 4, characterized in that the air contains 5 vol.% of carbon dioxide.



 

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FIELD: biotechnology.

SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.

EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.

2 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.

EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of obtaining preparation based on vaccine strain of plague microbe. Claimed invention includes preparing inoculation native culture of plague microbe, concentration of microbe suspension, preparing vaccine suspension and obtaining dry form of preparation, with process of preparing inoculation culture including cultivation of microbes in liquid nutritional medium in flasks for 48 h at temperature 26…28°C and contibuous aeration with not less than 10 l min-1. with passaged stabilised starting culture, obtained as a result of three successive passages through organism of guinea pigs and mixed with glycerol-lactose-polyglucinum liquid in ratio 2:1; for preparation of vaccine suspension used is optimised in component composition protective drying medium, lyophilisation being carried out with observance of the specified regimen.

EFFECT: claimed solution makes it possible to obtain product with higher activity with reduced duration of process of its manufacturing.

3 dwg, 6 tbl

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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