Vaccine against lawsonia intracellularis

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to veterinary science, and can be applicable for using a composition for protection against an infection caused by Lawsonia intracellularis. That is ensured by using a non-living composition containing carbohydrate which is also found in living cells of Lawsonia intracellularis in association with an external cell membrane of the above cells. The vaccine is presented in the form applicable for intramuscular introduction, and contains an oil-in-water adjuvant containing oil drops with an average size of 400 nm.

EFFECT: using the given non-living composition leads to effective immunisation in intramuscular introduction with using small drops of the oil-in-water adjuvant which provides protection of animals against Lawsonia intracellularis.

7 cl, 9 tbl, 4 ex

 

This invention relates to a vaccine for protection against infections caused byLawsonia intracellularisthe vaccine, in this sense, represents a composition that, at least, reduces the negative impact of infections caused byLawsonia intracellulariswhere specified a negative impact represents, for example, tissue damage and/or clinical signs such as reduced weight gain, diarrhea, etc.

Proliferative enteropathy (also called enteritis or eleita) in many animals, especially in pigs, is a clinical symptom or pathological syndrome with hyperplasia of immature epithelial cells of the crypts of the mucosa, mainly in the terminal ileum. Other parts of the intestine that can be affected include the small intestine, blind intestine and the colon. Affects mainly the piglets weaning and young adult pigs with typical clinical manifestation of rapid weight loss and dehydration. Natural clinical disease of pigs occurs everywhere. The disease is invariably associated with the presence of intracellular bacteria curved shape, now known asLawsonia intracellularis.

In General, it is shown that oral vaccination againstLawsonia intracellularisis cost the ski effective measure for the control of ileitis and ensure the best use of the genetic growth potential of the pig (Porcine Proliferative Enteropathy Technical manual 3.0, July 2006; available at Boehringer Ingelheim). In addition, oral vaccination rather than parenteral, reduces the transmission of bloodborne infections such as PRRS, through reusable needles and reduces adverse reactions from injections and the number of needles left in the body of the animal. Oral vaccination reduces stress in the animal and the person, time, cost, labor and effort, compared to individual vaccination (McOrist: "Ileitis - One Pathogen, Several Diseases" at the IPVS Ileitis Symposium in Hamburg, June 28th, 2004).

In General, it goes without saying that the advantage of the approach using attenuated live vaccine is that the effectiveness of the immune system is usually relatively good, because the host's immune system is exposed to all antigenic properties of the microorganism more "natural" way. Consider, in particular, that for intracellular bacterial agents, such asLawsonia intracellularisthe approach using a live attenuated vaccine provides the best protection available vaccinated animals with a complete and adequate T-cell immune response. Specified immune response differs with variations from a weak immune system, associated with the types of subunit or killed vaccines against intracellular bacteria. This statement is also true, in castnet is, for obligate intracellular bacteria, such asLawsonia intracellularisorChlamydia spthat cause pathogenic infections of the mucosa. Studies show that full live attenuated forms discussed intracellular bacteria are best delivered to the target mucosa that they are needed in the form of whole live bacterial forms to obtain full protective immune response targets, mucous membranes, and also that they are better immunological vaccines prepared using individual bacterial components.

It became common practice that vaccineLawsonia intracellularisit should be administered orally (see Technical Manual 3.0, as described herein above). This way of introducing the fact that the basis for the stability of the organism to eleito is the local immunity of the intestine, which is a product of cell-mediated immunity and local defense by antibodies, especially IgA. According to current data of serum antibodies (IgG) do not provide protection, simply because they do not reach the intestinal lumen. Studies have shown that oral vaccination induces cell-mediated immunity, as well as local production of IgA in the gut (Murtaugh, in Agrar - und Veterinar-Akademie, Nutztierpraxis Aktuell, Ausgabe 9, Juni 2004; and Hyland et al. in Veterinry Immunology and lmmunopathology 102 (2004) 329-338). In contrast, intramuscular injection does not lead to the formation of the protection. In addition, along with the conventional view that a successful vaccine against intracellular bacteria should induce cellular immunity, as well as the products of local antibodies skilled in the art it is known that only a very small percentage received oral antigens really absorbed by enterocytes, and that the introduction ofLawsonia intracellularisinside cells is an active process initiated by the bacterium. Thus, inactivated vaccine would be incomplete bowel immunogenic antigen (Haesebrouck et al. in Veterinary Microbiology 100 (2004) 255-268). Therefore, I believe the only attenuated live vaccines induce sufficient cell-mediated protection of cells of the intestine (see Technical Manual 3.0, as noted herein above). Currently on the market presents only one vaccine to protect againstLawsonia intracellularisnamely Enterisol® Ileitis offered by Boehringer Ingelheim. This vaccine is a live vaccine for oral administration.

The present invention is the provision of an alternative vaccine to protect against infections caused byLawsonia intracellularis. To this end developed for use inorganic composition containing carbohydrate specified uglev the d is also found in living cells Lawsonia intracellularisin Association with the outer cell membrane of these cells, the production of vaccines to protect against infections caused byLawsonia intracellularisthe vaccine is in a form suitable for systemic administration. Unexpectedly, in spite of sustainable common idea about how to deal withLawsonia intracellularisfound that when using inanimate kompoziia containing carbohydrate, for example, extracted from the outer cell membraneLawsonia intracellularisas the antigen in the vaccine, the carbohydrate can induce protection againstLawsonia intracellularisthat is similar to or exceeds the protection provided by live vaccine Enterisol® Ileitis (input in accordance with the relevant instructions)if the antigen is administered systemically, i.e. the way in which he reaches the circulatory system of the body (including cardiovascular and lymphatic system), acting thus on the body as a whole and not on a specific area, such as the gastrointestinal tract. System introduction can be carried out, for example, by introducing antigens into the muscle (intramuscularly), into the skin (intradermally), under the skin (subcutaneously), under the mucosa (submucosa), vein (intravenously), etc. in Addition to receiving a good defense, an important advantage of the present nonliving vaccine is that it has pressey DNAs is Rennie security compared to the live vaccine.

In General, a composition comprising a carbohydrate, can be applied for the production of vaccines using known in the art methods, which mainly include mixing a composition containing antigenic carbohydrate (or compositions derived from it, such as dilution or concentrate original composition or extract one or more purified components and so on) with a pharmaceutically acceptable carrier, for example with a liquid carrier, such as (optionally buffered) water or solid carrier, such as a medium customarily used to obtain freeze-dried vaccines. Essentially vaccine production can occur in an industrial environment, but also antigens can be mixed with other components of the vaccinein situ(i.e. veterinarians, farm and so on), for example, (immediately) before the actual introduction of the animal. In the vaccine antigens must be presented in immunologically effective amounts, i.e. in amounts capable of stimulating the immune system of the target animal at least to reduce the negative effects after vaccination in the control of infection by the wild-type microorganisms. Optionally, other substances such as adjuvants, stabilizers, viscosity modifiers or other components added to C is depending on the intended use or the desired properties of the vaccine. For systemic vaccination fit many forms, in particular liquid preparations (dissolved, emulsified or suspended antigens), as well as solid dosage forms, such as implants, or an intermediate form, such as a solid carrier for antigen suspended in the liquid. Systemic vaccination, in particular parenteral vaccination (i.e., not through the digestive tract), and appropriate (physical) forms of vaccines for systemic vaccination has been known for over 200 years.

Note that the subunit cellLawsonia intracellularisdescribed as antigens in the vaccine for protection against specified bacteria. However, these antigens are predominantly recombinant proteins to the present time none of them was able to provide good protection. Killed bacteria (which by their nature contain carbohydrate, which is also found in living cellsLawsonia intracellularisin Association with the outer cell membrane) is also proposed as antigens in vaccines againstLawsonia intracellularis,but in fact, vaccines based on killed whole cells were not tested and described to provide good protection. In addition, systemic injection is not used in combination with these dead cells on the overall view that there are sufficient grounds for a successful system the roadways to the introduction of antigens for local (i.e. in the intestine) anti - Lawsonia intracellularis.

In this regard, it is noted that WO 97/20050 (Daratech PTY Ltd) is the use of killed bacteriaLawsonia intracellularisfor immunization of pigs. However, the system introduction is not mentioned. Based on current knowledge about the effectiveness of vaccination only after oral administration, usually understand that oral path was by introducing selected for the experiments described in the patent application, Daratech. Another patent application that mentions killed bacteria, is WO 2005/011731 (Boehringer Ingelheim). However, in fact, disclosed is the use of live vaccines administered orally. Not shown that killed vaccine can be effective, especially as the killed vaccine can be introduced systemically. EP 843818 (Boehringer Ingelheim) describes intramuscular injection of a killed vaccine (paragraph [01 15] in combination with paragraph [01 19]). In paragraph [01 15] argues that the bacteria were killed by their store at 4°C under normal atmospheric conditions. However, it is well known that under these conditions, the bacteriumLawsonia intracellularisremain alive. Thus, this document does not report the subject matter of the present invention. It is also noted that a composition comprising a carbohydrate, where carbohydrates are also found in living cellsLawsonia intracellularisin Association with the outer to emochnoy membrane of these cells, known from Kroll et al. (Clinical and Diagnostic Laboratory Immunology, June 2005, 693-699). However, this composition is used for diagnostics. The above composition was not tested as a protective antigen for the reasons outlined above.

In one of the embodiments containing the carbohydrate composition is a material obtained by killing bacteriaLawsonia intracellularis. Found that a very convenient way to obtain carbohydrate for use in the present invention is the elimination of cellsLawsonia intracellularisand application of the resulting material as a source of carbohydrate. Theoretically it is also possible the extraction of carbon from living cells (similar to obtaining a "shadow" of living cells by removing the cell wall), but it requires more complex and therefore more expensive technologies. The material can be used in General form, for example in the form of a suspension of whole cells, or lysates of cellsLawsonia intracellularisor carbohydrate can be cleaned or even isolated from the material. The method may be implemented by using relatively simple technologies known in the art.

In the preferred embodiment a composition comprising a carbohydrate, contains whole cells of killed bacteriaLawsonia intracellularis. It is proved that the composition of t which provides the most convenient way of providing carbohydrate as antigen in the vaccine. In addition, vaccine effectiveness is further increased, possibly due to the fact that the presentation of antigen to the immune system of the animal target best mimics the natural environment of carbohydrate.

In the embodiment, the vaccine contains an adjuvant type oil-in-water containing oil droplets of submicron size. In General, the adjuvant is a non-specific immunostimulating agent. In principle, any substance that is able to support or reinforce a specific process in the cascade of immunological events leading ultimately to increased immunological response (i.e. the integral system response to an antigen, in particular the response mediated by lymphocytes and usually includes the recognition of antigens by specific antibodies or previously sensitized lymphocytes)may be indicated as adjuvant. It is shown that the use of adjuvant type oil-in-water containing oil droplets of submicron size, provides very good protection fromLawsonia intracellularis. Indeed, the inclusion of oil in water adjuvants essentially usually applied to non-living antigens. However, in General, it is known that the best immune-boosting properties is obtained if oil droplets have a large diameter. In particular, oil droplets with a diameter less than 1 micrometer is used is, if you think that the important issue is security. In this case you could use a small drop, since it is known that they cause less damage to tissues, clinical symptoms, etc. However, in the case of obtaining protection from diseases associated with the bowels, using a system of vaccination (as in the case of the present invention), you could select the large drops, because it was assumed that the immune response is significantly increased. On the contrary, the inventors have found that the use of small drops of oil in the composition provided very good results in terms of protection fromLawsonia intracellularis.

In another preferred embodiment the adjuvant includes drops of biodegradable oil and drops of mineral oil, drops of biodegradable oils have an average size that is different from the average size of the droplets of mineral oil. It is shown that the application of a mixture of biodegradable oils and mineral oil gives very good results in terms of efficacy and safety. In addition, the stability of the composition is very high, which is an important economic advantage. It is proved that the stability is very good, especially if the average size (weighted by volume) drops of biodegradable oils or mineral is the SLA is less than 500 nm (preferably about 400 nm).

In the embodiment, the vaccine further includes antigensMycoplasma hyopneumoniaeand circovirus pigs. Previously combined vaccineLawsonia intracellularishave been proposed in the prior art. However, few of these combinations were actually tested for effectiveness. The reason for this is that, in General, understand that the combination of antigens by antigenLawsonia intracellulariscan lead to successful protection only if the antigensLawsoniapresented in the form of live (attenuated) cells. In this regard, the inventors refer to WO 2005/011731, which also offers all kinds of combination vaccines based onLawsonia intracellularis. However, taking into account the structure of the descriptions and claims of the patent application, the patent owner (Boehringer Ingelheim), apparently confident that combination vaccines are only suspected to have a reasonable chance of success if the antigensLawsoniapresents them in the form of living cells. This statement is true for WO 2006/099561, also fixed for Boehringer Ingelheim. Indeed, on the basis of publicly available information, the above opinion is an obvious consideration.

Hereinafter the invention will be explained using the following Examples.

Example 1 describes a method of obtaining a mainly protein-free composition containing a carbohydrate, and the vaccine, which is created when using the years of the specified composition. Example 2 describes an experiment in which the vaccine of the present invention is compared with the vaccine currently on the market, and with an experimental vaccine, including subunit proteinsLawsonia intracellularis. Example 3 describes an experiment in which two different vaccines of the present invention is compared with the vaccine currently on the market. Example 4 describes an experiment in which set the exposure dose of the vaccine of the present invention.

EXAMPLE 1

In the present example describes a method of obtaining a composition mainly free from protein carbohydrate associated with the outer membrane of cellsLawsonia intracellularisand vaccines, which can be obtained using this composition. In General, the carbohydrate is an organic compound that includes carbon, hydrogen and oxygen, usually in the ratio 1:2:1. Examples of carbohydrates are sugars (sugars, starches, cellulose and resin. They usually serve as the main energy sources in animal nutrition.Lawsonia intracellularisis a gram-negative bacterium, which, thus, has an outer membrane, which built not only of phospholipid and protein, but also includes carbohydrates, in particular a polysaccharide (usually polysaccharides, such as lipopolysaccharide, lipooligosaccharide, or even the polysaccharides in non-lipid form).

The FRACTION of CARBOHYDRATE FOR the preparation of VACCINES

Took twenty milliliters of buffered water (0,04 M PBS, phosphate buffered saline)containing cellsLawsonia intracellularisat concentrations of 3,7E8 (=3.7 x 108) cells/ml Cells were literally by keeping them at 100°C for 10 minutes. Added protease K (10 mg/ml) 0.04 M PBS at a final concentration of 1.7 mg/ml of This mixture were incubated at 60°C for 60 minutes to destroy all the protein and keep the carbs intact. The mixture is then incubated at 100°C for 10 minutes to inactivate the protease K. the resulting material, which was a composition comprising a carbohydrate, in particular containing carbohydrates, which are presented in living bacteriaLawsonia intracellularisin Association with their outer membrane (see following paragraph), was stored at 2-8°C until further use. The composition was given in Freund Diluvac Forte. This adjuvant (see also EP 0 382 271) includes a 7.5 percent by weight of drops of vitamin E acetate with an average volume-weighted size of approximately 400 nm, suspended in water and stabilized with 0.5% by weight of Tween 80 (polyoxyethylenesorbitan monooleate). Each milliliter of vaccine contained material that was extracted from 1,2E8 cells<> Lawsonia intracellularis.

IMMUNOPRECIPITATE CARBOHYDRATE ANTIGENS LAWSONIA

Two batches of monoclonal antibodies (MoAb's)raised against whole cells ofLawsonia intracellularis, precipitiously saturated Na2SO4at room temperature in accordance with standard methods. The precipitate was besieged by centrifugation (10000 g for 10 minutes). The precipitate was washed with 20% Na2SO4and resuspendable 0.04 M PBS. Activated talasila Dynal beads (DynaBeads, DK) pre-washed with 0.1 M NaPO4(pH 7,4), according to manufacturer's instructions. From each batch of MoAb took 140 mcg and added to 2E8 pre-washed beads and incubated overnight at 37°C. Beads were besieged by centrifugation and unrelated MoAb was removed by aspiration of the supernatant. Spectrophotometric measurements showed that 20 to 35% added MoAb was associated with the beads.

Two batches of cellsLawsonia intracellularisa volume of 1 ml (3,7E8/ml) 0.04 M PBS was treated with ultrasound for 1 minute. The obtained cell lysate was added to tylosin-activated beads - monoclonal complexes and incubated over night at 4°C. Tylosin-activated beads - monoclonal complexes were washed three times with 0.1 M NaPO4(pH of 7.4). Related compounds were suirable by washing the beads in 0.5 ml of 8 M urea, 0.04 M PBS (E1); 0.5 ml of 10 mm glycine pH of 2.5 (E2); and 0.5 ml of 50 mm HCl (E3),consistent manner. After elution of E2 and E3 neutralized with 100 ál and 200 ál of 1 M Tris/HCl (pH 8.0).

Samples were taken at each stage and was placed on the gels LTO-PAG. Gels were stained with Kumasi brilliant blue (CBB) or silver or blokirovala. The blots showed similar MoAb, which was mentioned above. Checking gels and blots showed that strips identified by MoAb, with an apparent molecular mass of 21 and 24 kDa, which is invisible on the gels CBB, were visible on the gels, stained with silver. In addition, it was found that fraction of cells, which was associated with MoAb, was resistant to protease K. Thus, from the results it can be concluded that this fraction comprises carbohydrates (as protein literoitca, and processed by the ultrasound faction DNA does not appear as clear bands when stained with silver), and that the carbohydrates are in the Association (i.e. form part of or associated with the outer cell membraneLawsonia intracellularis(i.e. MoAb, induced against the specified faction, also recognized by whole cellsLawsonia intracellularis). Given thatLawsonia intracellularisis a gram-negative bacterium, believe that the carbohydrate composition includes a polysaccharide(s).

EXAMPLE 2

This experiment was conducted to test a convenient way to obtain carbohydrate antigen in the vaccine, namely using killed whole cells (known what Noah as bacteria). As controls were used commercially available vaccine Enterisol® ileitis and experimental subunit vaccine containing protein subunit. Along with this used unvaccinated animals as control.

EXPERIMENTAL DESIGN of EXAMPLE 2

Inactivated whole cell vaccine produced as follows. Collected live cellsLawsonia intracellularis,derived from pig intestines with PPE. Cells iactiveaware 0,01% BPL (beta propiolactone). The resulting material, which is essentially an inanimate composition comprising a carbohydrate, in the present invention (in particular, since it includes carbohydrates, which are presented in living bacteriaLawsonia intracellularisin Association with the outer cell membrane), received in Freund Diluvac Forte (see Example 1) at a concentration of approximately 2.8×108cells per ml of vaccine.

Subunit vaccines contain recombinants P1/2 and P4, which are known from EP 1219711 (proteins 19/21 and 37 kDa, respectively), and the recombinant proteins expressed genes 5074, 4320 and 5464, as described in WO2005/070958. Proteins were received in the Freund Diwali Forte. The vaccine contained approximately 50 Khramov each protein per milliliter.

Used forty pigs breed SPF at the age of 6 weeks. Pigs were divided into 4 groups of ten pigs in each is. Group 1 were vaccinated orally once (T=O), 2 ml of a live vaccine "Enterisol® ileitis" (Boehringer Ingelheim) according to the manufacturer's instructions. Groups 2 and 3 were vaccinated twice intramuscularly (at T=O and T=4w), 2 ml of inactivated vaccine from whole cellsLawsonia and recombinant subunit combination vaccine, as described above, respectively. Group 4 was left as unvaccinated controls. At T=6w all control pigs were infected orally by using a homogenized mucosa infectedLawsonia intracellularis. Then there was the daily observation of all pigs on the appearance of clinical signs of porcine proliferative enteropathy (PPE). Periodically before and after control of infection in pigs took samples of blood serum (for serology) and faeces (PCR). At T=9w to all pigs used to be euthanized and dissected them. Took histological samples of ileum and perform microscopic examination.

The inoculum for infection control were prepared from infected mucosa: 500 grams infected mucosa (erased from infected intestines) was mixed with 500 ml of physiological solution. The mixture is homogenized in a mixer omnipixel for one minute at maximum speed on ice. All control pigs were infected orally by 20 ml of the inoculum is for the control of infection in T=6w.

At time T=0, 4, 6, 7, 8 and 9 took a sample of faeces (g) and blood serum sample of each pig and kept frozen until testing. Samples of faeces were tested using analysis of quantitative PCR (Q-PCR) and data were expressed as the logarithm of the detected number in pilgrammage (GHG)emissions. Samples of serum were tested using commonly used test IFT (reaction immunofluorescence assay for detection of antibodies against whole cells ofLawsonia intracellularisin the serum). For histological evaluation took suitable sample of the ileum were fixed in 4% buffered formalin, poured in the usual way and prepared for microscopic preparations. These preparations were stained with hematoxylin-eosin (dye HE) and immunohistochemical dye with the use of monoclonal antibodies againstLawsonia intracellularis(dye IHC). The preparations were examined under a microscope. Histological evaluation of the submitted as specified below.

The dye HE
pathology was not detectedscore = 0
questionable damagescore = 1/2
light damage score = 1
moderate damagescore = 2
heavy damagescore = 3

Dye IHC
there is no explicit bacteriaL. intracelluarisscore = 0
the questionable presence of bacteriascore = 1/2
the presence of a single/small number of bacteria in the productscore = 1
the presence of moderate numbers of bacteria in the productscore = 2
the presence of large numbers of bacteria in the product, heavy damagescore = 3

All data was recorded for each pig individually. The evaluation group was calculated as the average number of positive animals for various parameters after control of infection. Non-parametric U-test Mann-Whitney was used to test statistical significance (two-sided test, the significance level was set to 0.05).

The RESULTS of EXAMPLE 2

Serology

Before PE the howling vaccination, all pigs were seronegative when tested IFT antibody titers. After vaccination, the whole cell bacteria (group 2) in pigs showed high titers of antibodies IFT, whereas the controls and the pigs vaccinated with the subunit vaccine remained negative to the control of infection (table 1). Two vaccinated with Enterisol® pigs (group 1) had average titers IFT, whereas the rest of the pigs in this group remained seronegative. After control of infection in all pigs showed high titers of antibodies IFT. The results as average values described in table 1 (used by breeding the detection limit on the underside was 1.0).

3
Table 1
Average values of IFT antibody titers (2log) in the serum of pigs after vaccination and infection control
GroupT=0 weeksT=4 weeksT=6 weeksT=9 weeks
1<1,01,11,7>11,4
2<1,03,7>11,8>to 12.0
<1,0<1,0<1,0>the 11.6
4<1,0<1,0<1,0>to 12.0

PCR real-time samples of faeces

To control contamination of all samples of faeces were negative. After control of infection positive reaction was found in all groups. Group 1 (p=0.02), group 2 (p=0.01) and group 3 (p=0.03) had significantly lower weight compared to control. An overview of the results after control of infection are presented in table 2.

Table 2
The results in the form of average values of PCR analysis of faecal samples (log PG) after vaccination and infection control
GroupT=6 weeksT=7 weeksT=8 weeksT=9 weeksThe result, after controlling infection
101,33,61, 6,3
200,82,81,95,5
300,5the 3.82,05,9
400,8a 4.9a 4.910,0

Histological evaluation

Group 2 had the lowest histological evaluation of HE (p=0.05), assessment of IHC (p=0,08) and total histological score (p=0,08). Other groups had higher grades and did not differ significantly from the control group. Cm. table 3.

Table 3
The average histological evaluation of ileum
GroupHE scoreIHC scoreThe overall score
11,81,53,3
2 1,31,52,7
31,81,63,4
42,42,3the 4.7

CONCLUSION of EXAMPLE 2

From the results it can be concluded that systemic injection of inanimate whole cell vaccineLawsonia intracellularis,which essentially comprises a carbohydrate, which is also found in Association with the outer membrane of living cellsLawsonia intracellularis, induced at least partial protection. All investigated parameters and histological scores were significantly or nearly significantly better compared to the controls.

EXAMPLE 3

This experiment was conducted to test the vaccine comprising a composition comprising a carbohydrate as antigen. The second vaccine, which was tested that included the addition of killed whole cells ofLawsonia intracellularisthe antigensMycoplasma hyopneumoniaeand swine circovirus vaccine combination ("Combi"). As control was used a commercially available vaccine Enterisol® ileitis. Along with this, as a second control used unvaccinated animals.

EXPERIMENTAL DESIGN of EXAMPLE 3

VA is the CIN on the basis of mainly free of protein composition, containing carbohydrate, obtained as described in example 1.

Experimental combinatin contained the antigen of inactivated whole cells ofLawsonia intracellularis(method of obtaining of inactivated bacteria, see example 2) at a concentration of 1.7×108cells/ml. in Addition, the vaccine contains an inactivated antigen PCV-2 (20 Khramov/ml protein encoded ORF 2, PCV-2; a protein that is expressed in the baculovirus expression system, which is well known in the art, for example as described in WO 2007/028823) and an inactivated antigenMycoplasma hyopneumoniae(the same antigen in the same dose, as is known from commercially available vaccine Porcilis Mhyo®, obtained from Intervet, Boxmeer, The Netherlands). The antigens were obtained in the double emulsion adjuvant "X". This adjuvant is a mixture of 5 parts by volume adjuvant "A" and 1 volume part adjuvant "B". Adjuvant "A" consists of drops of mineral oil with an average size (volume weighted) of approximately 1 μm, stabilized by Tween 80 in water. Adjuvant "A" comprises 25 wt.% mineral oil and 1 wt.% The twin. The remainder represents water. Adjuvant "B" consists of drops of biodegradable vitamin E acetate with an average size (measured in volume) of approximately 400 nm, stabilized also by Tween 80. Adjuvant "B" comprises 15 wt.% vitamin E acetate and 6 the AC.%. Tween 80, the remainder represents the water.

Used sixty-four pig breeds SPF at the age of 3 days. Pigs were divided into four groups of 14 piglets and one group of 8 piglets (group 4). Group 1 was vaccinated intramuscularly at the age of 3 days 2 ml combinatin, and then re-vaccination at the age of 25 days. Group 2 was vaccinated intramuscularly once with 2 ml of combinatin at the age of 25 days. Group 3 were vaccinated orally with 2 ml of Enterisol® ileitis (Boehringer Ingelheim) at the age of 25 days according to the recommendations. Group 4 were vaccinated intramuscularly at the age of 3 and 25 days, 2 ml of protein-free carbohydrate vaccines. Group 5 was left unvaccinated as a group for the control of infection. At the age of 46 days all control pigs were infected orally by a homogenized infected mucous. Then there was the daily observation of all pigs on the appearance of clinical signs of porcine proliferative enteropathy (PPE). Periodically before and after control of infection in pigs took samples of blood serum for serology and PCR analysis, respectively. At the age of 68 days for all pigs used to be euthanized and dissected them. Conducted histological examination of the ileum.

Other issues on the design of the experiment were similar to those described in example 2, if not specified and the ache.

The RESULTS of EXAMPLE 3

Serology

Before the first vaccination, all pigs were seronegative when tested IFT antibody titers. After vaccination combinatinos (groups 1 and 2) and non-protein carbohydrate vaccine (group 4) most of the pigs discovered antibody titers IFT, whereas the controls and the pigs vaccinated with Enterisol remained seronegative to control infection. After control of infection of pigs with the exception of two in the group Enterisol) found antibody titers IFT. Overview of the obtained average values are presented in table 4 (due to higher cultivation in comparison with Example 2, the detection limit was equal to 4.0).

Table 4
Average values of IFT antibody titersLawsonia(2log) in the serum of pigs after vaccination and infection control
GroupT=3 daysT=25 daysT=46 daysT=67 days
1<4,0<4,07,910,3
2<4,0<4,0 4,89,8
3<4,0<4,0<4,08,5
4<4,0<4,06,910,6
5<4,0<4,0<4,09,0

In respect of Mhyo, at the beginning of the experiment, and also on the day of vaccination (age 25 days) all pigs were seronegative for Mhyo. After revaccination in group 1 showed high titers of antibodies Mhyo comparable to titers obtained using commercially available vaccines. In relation to PCV, at the age of 3 days, the piglets had titers of maternal antibodies against PCV. On the day of revaccination (age 25 days) vaccinated animals (group 1) had a titer similar to the titer of antibody 2 and antibody titer of the control group. Titers of antibodies against PCV at the age of 25 days was slightly lower compared with the titer determined at the age of 3 days. After vaccination at the age of 25 days titers of group 1 (2 of vaccination on days 3 and 25) and group 2 (one vaccination at day 25) remained at a high level, while the pigs of the control group on what has monsterously normal reduction of maternal antibodies. Received PCV-titres comparable to titers obtained with commercially available vaccines.

PCR real-time samples of faeces

Three weeks after control of infection in pigs of groups 1, 2 and 4 contained fewerLawsonia(DNA) in the faeces compared with groups 3 and 5. Statistically significant were the only differences between groups 1 and 3 (Enterisol) and between groups 4 and 3 (p<0,05, U-test Mann-Whitney). The results as average values presented in table 5.

Table 5
The average values of the results of PCR analysis of faecal samples
(log, PG) after vaccination and infection control
GroupAverage
11,0
21,2
32,0
40,6
51,8

Histological evaluation

Histological evaluation of groups 1 and 4 were significantly lower compared with estimates of groups 3 and 5 (p<0,05, two-sided U-test Mann-Whitney (see table 6). The number of pigs under the approved diagnosis PPE was 2/13 in group 1, 6/12 in group 2, 12/14 in group 3, 2/7 in group 4 and 12/14 in the control group 5. Groups 1 and 4 had a significantly lower incidence of PPE, compared with groups 3 and 5 (p<0,05, two-sided Fisher's exact test).

Table 6
The average histological evaluation of ileum
GroupHE scoreIHC scoreThe overall score
10,40,61,0
20,70,71,4

31,61,43,0
40,40,40,8
51,91,53,4

CONCLUSION of EXAMPLE 3

From the results it can be concluded that systemic injection of the whole cell bacterinLawsoniathe standard deviation is binyavanga with PCV antigen and Mhyo, as well as vaccines containing (mainly protein-free) carbohydrate, applied at the age of 3 days and at the age of 25 days, induces partial protection against experimental infectionLawsonia intracellularis. Especially unexpected that the vaccine is effective, if the initial introduction is to weaning (age 21-25 days). Indicated that in examples 2 and 3 vaccines, when talking about the antigensLawsoniacontain ml antigenic material derived from more than 1E8 cellsLawsonia intracellucaris. Taking into account that these vaccines, despite the fact that they used the soft adjuvants (namely adjuvants containing small drops, not containing or containing a small amount of mineral oil), give good protection against ileitis, especially in comparison with the commercially available vaccine Enterisol® Ileitis, the dose of antigens could be reduced. The reduction can be performed by introducing a smaller amount of vaccine (for example, to 0.2 ml, which is suitable, for example, for intradermal use) or by reducing the antigen content of the vaccine. Based on the unique technology of vaccines, I believe that with the help of antigenic dose (vaccine) derived from or contained in 1E7 cells, in particular 2,5E7 cells or more, can be obtained comparable or even better results than using existing commercial and affordable vaccines. Given that the combination vaccine provides antibody titers against Mhyo and PCV in concentrations comparable to the concentrations obtained using commercially available individual vaccines, with the combination vaccine also provides protection againstMycoplasma hyopneumoniaeand circovirus pigs.

EXAMPLE 4

This experiment was conducted to determine the impact of dose of vaccine according to the invention. In this experiment also used unvaccinated animals as control.

EXPERIMENTAL DESIGN of EXAMPLE 4

Inactivated whole cell vaccine was received as shown in example 2. Antigenic material was prepared in Freund Diluvac Forte at a concentration of approximately 2.0×108cells per ml of vaccine, respectively, of 5.0×1071.25×107cells per ml of vaccine. Used sixty pigs breed SPF at the age of 3 days. Pigs were divided into four groups of 15 pigs in each. Pigs of groups 1, 2 and 3 were vaccinated intramuscularly (in the neck) at the age of 3 days and 25 days, each time with 2 ml of vaccine. Group 4 was left as unvaccinated controls. At the age of 46 days all control pigs were infected by oral bacteriaLawsoniaas shown in example 2. All pigs at the age of 67 days was used euthanasia and opened them. The tests were performed as shown in the example is 2. Along with this was performed by PCR analysis of samples of mucous. For this purpose, samples were taken ileum from each animal, as appropriate, of the plot, which demonstrates thickening.

The RESULTS of EXAMPLE 4

Weight gain

In the time period from 14 days and then between groups were there significant differences in the total weight gain. Group 1 showed the average total weight gain of approximately 5350 grams. In group 2, this figure amounted 5150 grams. Group 3 showed a weight gain 4250 grams, whereas group 4 showed a weight gain 4550 grams.

PCR real-time samples of faeces

Three weeks after control of infection positive reaction was found in all groups. Group 1 and group 2 had a significantly lower level of weight reduction in comparison with control. An overview of the number of infected animals (which were determined using analysis PCR) after control of infection are presented in table 7.

Table 7
The result of PCR analysis of samples of faeces after vaccination and infection control
GroupThe number of infected animals after infection control
11/15
22/15
37/15
48/15

PCR real-time samples of the mucous membrane

Three weeks after control of infection positive reaction was found in all groups. Group 1 and group 2 had a significantly lower level of weight reduction in comparison with control. Overview after control of infection the number of infected animals (which were determined using PCR analysis) are presented table 8.

Table 8
The result of PCR analysis of samples of the mucosa after vaccination and infection control
GroupThe number of infected animals after infection control
10/15
22/15
35/15
46/14 (no sample from pig No. 8)

Histological evaluation

General histological assessment of the ka and the number of animals with confirmed diagnosis of PPE, described in table 9.

Table 9
The average histological evaluation of ileum
GroupThe overall scoreThe number of animals with PPE
10,30/15
20,82/14
31,14/15
41,97/15

CONCLUSION of EXAMPLE 4

Contrary to the expectations, the results show that is very unexpected decrease in the protective effect when used in experiments, the lowest dose.

Although the dose of antigenic material derived from a 2.5×107cells, still provides a protective effect comparable to the effect of commercially available vaccines, the fact that the reduction effect compared with the dose in excess of specified only on 0,6log is so significant (almost not see the effect on weight gain, the number of infected animals and the result of the PCR analysis of the mucous membrane; but still on logout reducing the number of animals diagnosed with PPE), suggests that in practice the minimum effective dose of antigen, as a rule, can be set in accordance with the following condition: the amount of antigen that is less than the amount derived from or contained in 1x107cells, in practice, in modern market conditions, does not lead to economically meaningful results. The reason for the existence of the specified explicit thresholds is not clear at 100%. Usually expect a more gradual reduction of protection at lower doses. It is possible that to counter local infection of the intestinal mucosa by systematically produced by the immune response need some minimum number of antigens.

Along with the above, an unexpected effect observed in example 3, namely that a vaccine based on the carbohydrate antigen is introduced systematically, is effective if the initial introduction is before weaning (age 21-25 days), confirmed in the specified experiment with a different adjuvant. Therefore, with a sufficient basis to believe that the specified feature is a genetic feature of nonliving vaccines containing carbohydrate antigen.

1. The use of inanimate compositions containing carbohydrate, which is also found in living cells Lawsonia intracellularis in Association with N. the outer cell membrane of these cells, in the manufacture of a vaccine for protection against infection caused by Lawsonia intracellularis, where the vaccine is in a form suitable for intramuscular injection, and includes adjuvant-type oil-in-water containing oil droplets with an average size of 400 nm.

2. The use according to claim 1, characterized in that a composition comprising a carbohydrate, is a material derived from dead bacteria Lawsonia intracellularis.

3. The use according to claim 2, characterized in that the composition containing the carbohydrate comprises whole cells of killed bacteria Lawsonia intracellularis.

4. The use according to claims 1 to 3, characterized in that the adjuvant includes drops of biodegradable oil and a drop of mineral oil where the drops of biodegradable oils have an average volume-weighted size that differs from the average volume-weighted size drops of mineral oil.

5. The use according to any one of paragraphs. 1-3, characterized in that the vaccine further comprises antigens of Mycoplasma hyopneumoniae and swine circovirus.

6. The use according to claim 4, characterized in that the vaccine further comprises antigens of Mycoplasma hyopneumoniae and swine circovirus.

7. Inanimate composition comprising a carbohydrate, which is also found in living cells Lawsonia intracellularis in Association with the outer cell membrane of these cells, for the production of the vaccine is to protect against infection, caused by Lawsonia intracellularis, where the vaccine is in the form intended for intramuscular injection, and includes adjuvant-type oil-in-water containing oil droplets with an average size of 400 nm.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: invention relates to compositions and polymeric materials for biomedical use, comprising silver nanoparticles (0.0005-0.02 wt %) stabilised by amphiphilic copolymers of maleic acid (0.0008-0.05 wt %), low molecular weight organic amines (0.0002-0.04 wt %) and water. In addition, the said composition may additionally comprise the polymeric structure-forming agent.

EFFECT: introduction to the composition of the polymer structure-forming agent enables to obtain the macroporous structured hydrogel materials having prolonged bactericidal and antifungal action.

3 cl, 2 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: inorganic clay, represented by sodium-calcium, and/or calcium and/or ferrous forms of montmorillonite, is modified with a water solution of silver nitrate with a concentration 0.16-9.9 wt % in a weight ratio clay:water solution of silver nitrate 1:5. Modification is carried out with mixing from 3 to 7 hours at a temperature in the interval from 10°C to the temperature of boiling. The obtained material is washed with distilled water to pH ≈6-5, until excess of silver nitrate is removed, stood at room temperature and decanted. The material is dried at a temperature of 20-160°C.

EFFECT: obtaining an efficient antibacterial material for traditional and veterinary medicine.

2 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: given invention refers to a compound of formula its stereoisomers, including R and S isomers, wherein: 'A' represents N; Y and Y' represent O; '---' is absent; R1 and R2 are identical or different, and independently represent hydrogen or C1-12 alkyl; R3 represents hydrogen; R4 represents heteroaryl which can be optionally substituted in any acceptable position by one or more substitutes Ra; Z represents -(CH2)n-heteroaryl which can be optionally substituted in any acceptable position by one or more substitutes Ra; T, U, V and W are identical or different, and independently represent hydrogen or halogen; Ra is independently specified in hydrogen, halogen, C1-12 alkyl, C1-12 haloalkyl, -C(=Y)OR7, -(CH2)nYR7, each of which can be optionally substituted in any acceptable position by halogen; R7 represents hydrogen or C1-12 alkyl; m represents 1; m′ represents 0; n represents 1; wherein: the above heteroaryl is specified in 1,2,3-triazolyl, pyridinyl, 1-oxypyridinyl (pyridinyl-N-oxide), pyrazinyl, isoxazolyl, imidazo[1,2-α]pyrimidinyl, imidazo[1,2-α]pyrazinyl. The compounds of the given invention are applicable to prevent, relieve and/or treat bacterial infections in an individual. The bacterial infection is caused by the drug-resistant species Staphylococcus, Streptococcus, Enterococcus, Bacterioides, Clostridia, H. influenza, Moraxella, acid-resistant species like Mycobacterium tuberculosis, as well as linezolid-resistant species Staphylococcus and Enterococcus.

EFFECT: phenyloxazolidinone compounds as antimicrobial agents.

12 cl, 8 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: claimed is a cocrystalline form of fenbufen with pyrazinamide, where molar ratio of fenbufen with pyrazinamide constitutes 1:1, which has an endothermal peak from 148 to 152°C by the data of measurements by means of differential scanning calorimetry and peaks at 2θ(°) 7.38, 10.43, 11.04, 21.67 by the data of measurement of polycrystal X-ray radiation diffraction.

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2 ex, 7 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology, molecular biology and genetic engineering. There are presented an immunogenic composition containing a mixture of staphylococcal proteins, and comprising a staphylococcal protein binding an extracellular component, and a staphylococcal transport protein, or the staphylococcal protein binding the extracellular component, and a staphylococcal virulence regulator or a toxin, or the staphylococcal transport protein and the staphylococcal virulence regulator or the toxin. There are also presented vaccines, methods of treating, using and methods for preparing a staphylococcus vaccine.

EFFECT: invention may be used in medicine for treating and preventing a staphylococcal infection.

23 cl, 8 tbl, 7 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical formulations and is applicable for providing bactericidal efficacy. A pharmaceutical formulation contains two various antibiotics as active ingredients in the form of a synergetic combination of a fixed dose in a parenteral dosage form. The first antibiotic represents carbapenem or its pharmaceutically acceptable salts, while the second antibiotic represents aminoglycoside which represents etimycin or its pharmaceutically acceptable salts. The above first antibiotic and the above second antibiotic are found in weight ratio of 6:1 to 13:1. Besides, the pharmaceutical formulation contains one or more additives specified from a group of synthetic/natural amino acids/vitamins/stabilisers/polymers/antioxidants/micronutrient elements.

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8 cl, 3 tbl, 6 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: composition includes a bactericidal substance - catapol - in amount of 2.1-2.5 wt %, zosterin in amount of 1.1-5.0 wt % and distilled water.

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1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry and medicine and deals with novel 4-(pyrrolidine-1-yl)quinoline compounds, a method of their obtaining and application for treatment of bacterial or fungal infection.

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17 cl, 3 dwg, 1 tbl, 47 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of pharmaceutics, namely represents compositions for treating nail and nail bed diseases and methods of treating nail diseases.

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30 cl, 2 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: in a complex preparation containing a carrier representing an enterosorbent; the enterosorbent is modified by immobilising high-disperse silver - nanosilver in a concentration of 0.01 - 1.0 wt % on its surface. The enterosorbent represents activated carbon, kaolin, bentonit, or enterodesum, or monocrystalline cellulose. A modifying silver-containing solution - a nanosilver source - is silver clusters in an aqueous solution.

EFFECT: higher specific antimicrobial activity.

2 cl, 3 tbl, 2 ex

FIELD: veterinary medicine.

SUBSTANCE: method of prevention of infectious conjunctivitis-keratitis of cattle comprises vaccination with vaccine associated against infectious conjunctivitis-keratitis of cattle based on antigens of bacteria Moraxella bovis and herpesvirus type I, while 29-31 days prior to vaccination the immunostimulatory agent "Kerokonvitin" is injected to the animals subcutaneously in the upper third part of the neck at a dose of 0.045-0.055 ml/kg body weight, obtained on the basis of cytotoxic serum from the blood of donor horses by their hyperimmunisation with antigen prepared from tissues of eyes - the conjunctiva and cornea of cattle which had an infectious disease of conjunctivitis-keratitis. Eye tissues of cattle are obtained during slaughtering animals.

EFFECT: improving the efficiency of prevention of infectious conjunctivitis-keratitis of cattle, higher immune status of body of animals.

3 tbl, 1 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to produce thermolabile enterotoxin (LT-enterotoxin) and Hafnia alvei anatoxin when producing a vaccine. The strain is deposited in the State Collection of Pathogenic Microorganisms of FBSI Scientific Centre for Evaluation of Medical Products of the Ministry of Public Health and Social Development of Russia under number 294.

EFFECT: strain has high capacity for producing thermolabile LT-enterotoxin.

1 tbl, 2 ex

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.

SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.

EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.

3 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to medicine. There are presented a composition containing an immunologically effective amount of the live avirulent strain Mycoplasma hyopneumoniae, and a method of inducing the immune response on Mycoplasma hyopneumoniae, involving the stage of administering the above composition to an animal. What is presented is a method of preventing or relieving an attack of Mycoplasma hyopneumoniae. There are also presented methods of enhancing the immune response on Mycoplasma hyopneumoniae, involving the stages of administering single or double doses of the above composition to the animal.

EFFECT: group of inventions is effective to provide the immunity in the animal and to protect from the infection with the virulent strain Mycoplasma hyopneumoniae, thereby reducing a severity and/or preventing the diseases caused by one or more virulent strains Mycoplasma hyopneumoniae.

18 cl, 8 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary microbiology, immunology, biotechnology, namely to technology of an antigen for diagnosing brucellosis. The method for producing brucellous L-antigen is implemented as follows. The strain Brucella abortus 19 is cultured in the L-form on a solid nutrient medium prepared of meat-peptone liver glucose-glycerol agar (1.3-1.5% agar) with added 10-15% normal horse serum and streptomycin in a dose of 2.5-5.0 Units/ml at 37-38°C for 4-5 days. Then the prepared L-form culture is emulsified by 0.5% phenolised physiological saline; the suspension is inactivated at temperature 85-90°C for 60 minutes, centrifuged at 3000-5000 rpm for 15-20 minutes; the brucella concentration is specified at 50-60 bln microbial cells; the prepared antigen is titred, standartised by specificity and activity. The latter is used for the purpose of an agglutination test in serological diagnosis of brucellosis. The invention enables higher effectiveness and reliability of diagnosing brucellosis by 20-25%.

EFFECT: invention may be used for diagnosing brucellosis in carrier animals with persistent changed L-forms of the agent.

4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: method comprises immunising the animals with the use of surface melioidosis antigens in the liposomal form, and for the purpose of the additional stimulation of the cell immunity mechanisms, the immunomodulator Bestim is added to cytokines. The stimulant action of the preparations on the immune system is assessed in dynamics of the immune response as shown by the TDTH level and a degree of chemiluminescence response to zymosan of peritoneal macrophages in the immunised mice. The protective properties of the antigens are stated by mortality rates (the percentage of surviving animals and the average life expectancy) after the control infection with the virulent culture of B. pseudomallei. The immunogenic and protective properties of the antigens are substantially increased through the use of the antigen in the liposomal form and the addition of the cell immunity activator Bestim into the immunisation schedule.

EFFECT: method shall provide higher immunogenicity of the melioidosis antigens while developing the preparations for specific prevention of melioidosis infection.

6 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: invention may be used for production of antigens, diagnostic and preventive preparations. The method includes preliminary water and salt treatment of bacterial cells with their subsequent lysis by a buffer solution, containing 0.1 M Teis-HCI pH 8.0, 10 mM EDTA, 1% Triton X-100. Destruction with ultrasound is carried out. The untreated extract of LPS is treated with a ferment complex of proteovibrin until the final concentration of 160 mcg/ml and incubated at 37°C, pH 7.8-8.0 for 18 h. Treatment from nucleic acids is carried out by acidification of a sample with an ice acetic acid to pH 3.2-3.4 and their deposition at 5000 g for 30 min.

EFFECT: invention makes it possible to improve quality of LPS preparations, to simplify and cheapen methodology, to reduce time of LPS extraction.

2 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to medical microbiology, particularly to molecular-genetic typing of the strains Helicobacter pylori. What is presented is a method for differentiation of the strains H.pylori by multilocal VNTR-typing with the PCR involving oligonucleotide primers on VNTR-comprising loci of H.pylori - HpA, HpD, HpE and HpF; the strains H.pylori are differentiated by a number of repetitions in the amplified fragments in each VNTR-comprising locus of H.pylori - HpA, HpD, HpE and HpF that enables genetic typing of the studied strains.

EFFECT: what is presented is the method for differentiation of the strains Hpylori by multilocal VNTR-typing.

1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: new strain of bacteria Pasteurella trehalosi is proposed, where the specified bacteria are positive in respect to beta-haemolysis, positive in respect to oxidase, positive in respect to catalase, negative in respect to urease, positive in respect to nitrates, negative in respect to indole, MacConkey-positive, positive in respect to glucose, positive in respect to saccharose, positive in respect to mannitol, negative in respect to arabinose, negative in respect to cellobiose, positive in respect to xylose, negative in respect to salicin, negative in respect to ornithine, negative in respect to esculin, negative in respect to alpha-fucosidase, positive in respect to beta-galactosidase. Also the strain of bacteria Mannheimia haemolytica is proposed. These bacteria are deposited under registration numbers ATCC No. PTA-3667, ATCC No. PTA-3668, ATCC No. PTA-3669.

EFFECT: immunisation of chickens with the purpose to prevent disease caused by above bacteria.

7 cl, 22 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and aims at intraoperative and early postoperative infusion therapy. A method involves administering a crystalloid balanced solution of sterofundin iso and a balanced colloidal solution of tetraspan. In the intraoperative period, sterofundin is administered with tetraspan in the volume ratio 3:1. In the early postoperative period, for the first day, sterofundin and tetraspan are administered in the volume ratio 6:1.

EFFECT: method is simple, safe, enables effectively correcting aqueous electrolyte disorders, stabilising systemic hemodynamic parameters, causing no negative effect on haemostasis and electrolyte composition of the blood serum.

1 ex, 1 tbl

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