Vaccine aimed against actinobacillous pleuropneumonia and method of obtaining thereof

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to field of veterinary. Claimed are: vaccine, aimed against actinobacillous pleuropneumonia, including lipopolysaccharide in complex with one or more repeats of ApxI, ApxII and ApxIII toxins, separated from bacterial culture and polymyxin to reduce symptoms of endotoxic shock, caused by lipopolysaccharide, method of obtaining such vaccine, application of polymyxin to reduce endotoxic shock symptoms when vaccine is introduced, in which polymixyn is added into vaccine in dose from 2.6 to 60 mcg/ml.

EFFECT: claimed group of inventions provides effective means and methods to reduce endotoxic shock symptoms, caused by lipopolysaccharide, when vaccine against actinobacillous pleuropneumonia is introduced to animal.

14 cl, 4 tbl, 4 ex

 

The present invention relates to a vaccine directed against actinobacillus pleuropneumonia, widespread diseases in the world causing significant economic losses in industrial pig farming. Etiologic agent actinobacillus pleuropneumonia is aActinobacillus pleuropneumoniae (APP), gram-negative bacterium belonging to the familyPasteurellaceae. The disease is transmitted by aerosol or by direct contact with an infected pig and is characterized by hemorrhagic, fibrinous and necrotic lesions of the lungs. The clinical picture can vary from very acute to chronic, and asymptomatic carriers pigs can transmit the disease when they get into uninfected herds. On the basis of capsular polysaccharides and lipopolysaccharides (LPS, LPS) components Of the circuits described 15 serovars. Serotyping and other genetic typing methods forActinobacillus pleuropneumoniaemade an enormous contribution to the study and epidemiological studies. These tools provide important information for decision control aimed at eradication of virulent types of bacteria. In North America the serovars 1, 5 and 7 are the most common, while the serovars 2 and 9 are the most common in Heb�pyo, and serovar 15 predominantly among Australian pigs.

Virulent factors described for theA. pleuropneumoniaeinclude LPS, capsular polysaccharides, Apx toxins I-IV (the so-called repetitions of RTX toxins or toxins), outer membrane proteins (OMP) and different systems of iron uptake. However, the overall contribution of each component in the infection process remains unclear, and the mechanisms of pathogenesis of this bacterium. Almost all existing vaccine againstA. pleuropneumoniaeare either inactivated whole cell bacterin, or combinations of subunits Apx toxins and proteins, optionally in combination with the outer membrane fractions. In any case, it became clear that to obtain the necessary protection, preferably acting against multiple serovars in the vaccine must contain a lipopolysaccharide (LPS) (Julien Gouré et al. in: BMC Genomics 2009,10:88, published February 24, 2009; Dubreuil et al. in: Animal Health Research Reviews 1(2): 73-93, December 2000). Moreover, since the 1980s, it was known that the vaccine, based only on the lipopolysaccharide, is already having a positive effect on reducing the level of infection or clinical signs of the disease and induces cross-protection (Fenwick et al. in: American Journal of Veterinary Research, Volume 47, No. 9, September 1986; Rapp et al. in: Canadian Veterinary Journal, 1988, Volume 29, Number 7, pp 585-587; Lembras et al. in: Comparative Immunology, Mirobiology & Infectious Diseases 20(1):63-74, January 1997).

A serious drawback of the lipopolysaccharide is endotoksicski nature, thus, is associated with many undesirable side effects, such as lethargy, diarrhea, vomiting, loss of appetite and even death. At first glance, there is the temptation to reduce the number of LPS in the vaccine or detoxify LPS. However, adequate APP the vaccine depends on the presence of LPS, and thus, as is well known, reducing the number of LPS, respectively, reduces the effectiveness of the vaccine. Also, various attempts have been made reduce endotoksicski nature of LPS and also get effective APP vaccine. Lembras et al. (mentioned here and above) investigated the effect detektirovanie LPS on the efficacy of the vaccine against APP and came to the conclusion that mice immunized with (vegetarierinnen) LPS were protected, while the vaccine is based on detektirovanie LPS, failed to protect mice (see page 71 of the publication Lembras, third paragraph). This study was re-conducted on pigs (Research in Veterinary Science, 1998, 65, 165-167). It has been shown that commercially available bacteriana vaccine (containing nudetoxicology LPS) provides good protection against stimulation with wild type APP. The vaccine, based on detektirovanie LPS were significantly less effective (60% Swee�she still had damage to the lungs). Actually, this corresponds to the widespread understanding that after LPS detoxification is much less immunogenic.

The aim of the present invention to provide a vaccine directed against actinobacillus pleuropneumonia, with efficiency comparable to commercially available vaccine, but with greatly reduced symptoms endotoksicski shock, especially when no or almost imperceptible signs of diarrhea, vomiting and increased respiratory in pigs after the administration of an effective dose of the vaccine.

In this regard, developed a vaccine based on the inclusion of lipopolysaccharide, where the vaccine further comprises a polymyxin to reduce the symptoms of endotoksicski shock caused by lipopolysaccharide. Unexpectedly it was discovered that the detoxification of LPS by polymyxin does not have a significant negative effect on the immunogenicity of LPS and thus on the effectiveness of the vaccine. This implies that through the use of polymyxin symptoms endotoksicski shock, such as diarrhea, vomiting and increased respiration, can be significantly reduced, at the same time, while maintaining the efficacy of the vaccine at a sufficient level. In particular, this implies that the vaccine may even have the same efficacy as a vaccine without added polymyxin.

The vaccine � sense of the present invention is a structure to prevent the development or decrease of infections caused by microorganism, or clinical symptoms of the disease or disorder caused by infection by inducing antibodies that cause harm to the organism or its metabolic processes (either inside or caused by a microorganism) in the body of the host. The vaccine may contain a subunit of a microorganism (such as, for example, LPS) as the primary antigen, but may also contain attenuated or killed whole microorganisms, or perhaps a metabolite of an organism (such as a secreted toxin). The vaccine contains an immunologically effective amount of antigen (i.e., able to stimulate the immune system of the animal target, enough at least to reduce the negative effects of wild-type microorganisms, usually in combination with a pharmaceutically acceptable carrier, such as liquid-containing water (often phosphate-saline buffer solution), and optionally contains immune-boosting means (adjuvants), stabilizers, substances that alter the viscosity or other components depending on the purpose of use or desired properties of the vaccine. There also may be additional antigens of other microorganisms that the vaccine provided protection against m�of egesta of wild-type organisms.

The polymyxins are compounds with antibacterial properties that can be extracted fromBacillus polymyxa(B. aerospora) or artificially produced, and compounds with antibacterial properties, are directly derived from them. Polymyxins produced byB. polymixaa common structure consisting of a cyclic peptide with a long hydrophobic tail. Typical polymyxins such as polymyxin A (also called aeroporia), polymyxin B (often called polymyxin), polymyxin E (also called colistin), polymyxin M (also known as matachin (mattacin)) and the polymyxin nonapeptide.

Noted that has been previously described that the polymyxins can be used for a successful detox lipopolysaccharides in immunogenic compositions containing gram-negative bacteria. For example, Cooperstock et al. revealed in the journal “Infection and Immunity”, July 1981, pp 315-318 that the use of polymyxin b endotoksicski activity in suspensions of several gram-negative bacteria (namely,Bordetella pertussis, Escherichia coli, Haemophilus influenzaeandPseudomonas aeruginosa) significantly decreased. However, Cooperstock also clearly notes that "it remains doubtful whether the immunogenic properties of bacteria, polymyxin-treated, intact" and that "the ability of several components...Nar�l membrane to bind preformed antibody in immunoelectrophoretic system was partially lost after treatment or a bacteria, or isolated membranes with high concentrations of polymyxin b". In other words: immunogenic properties detektirovanie LPS were questionable, and the loss of at least part of these properties had predicted.

Actually, also Bannatyne et al. reveal in the Journal of Hygiene (Cambridge), 1981, 87, pp 377-381, polymyxin b at a concentration of 5000 µg (polymyxin B sulfate) per ml (equivalent to approximately 42000 International Units per dose of vaccine) was effective for suppression of the toxic effect of LPS. However, he also States: "Remain, however, reservations about such polymyxin-containing vaccines. The most important is whether the reduction endotoksicski component by reducing the amount of protective antigen and is it related to the drop in defensive efficiency". A few years later Larter et al. (in American Journal of Diseases of Children, Vol 138, March 1984) and in fact finds that the pertussis vaccine antibody titers in animals receiving pertussis vaccine and polymyxin simultaneously (more than 150,000 IU per dose) was lower and grew more slowly or not at all was found.

These discoveries in the early 1980-ies can explain why for effective RDAs vaccine in which LPS plays a dominant role, detoxification with the use of polymyxin has never been considered possible to successfully obtain secure, but still� effective vaccine. This is confirmed by the fact that over 25 years have passed since the observations noted here and above, up to the present the use of polymyxin b RDAs vaccine, including LPS, as a Central component.

In a variant implementation of the vaccine includes the lipopolysaccharide isolated from bacterial culture (i.e. concentrated relative to other cellular components such as cell organelles, nucleus, components of the cell wall, etc.), in combination with one or more repetitions of toxins. It is known that the RDA vaccine based on bacteria, provides less protection than a vaccine based on the combination of subunits LPS and Apx in the form of the complex. Thus, such a combination of subunit vaccines, including LPS, isolated from the bacterial culture, in combination with one or more of Apx toxins is preferred. It is known that the specific links between LPS and these toxins play a major role in improving the immunogenicity of toxins (see including Ramjeet et al. inMolecular Biology,2008, 70(1), pp 221-235), which may explain enhanced protection for such combination subunit vaccine. It was unexpectedly found that polymyxin, despite its strong binding properties of LPS, apparently, has no negative impact on the complex LPS-toxin, as the combination of subunit vaccines, including�I polymyxin, provides protection equivalent to the same combinations of subunit vaccines, including polymyxin. Replays of toxins, for example, can be those known from EP 453024. Also can attend any of these replays ApxII toxins or ApxIV. It may depend on the RDA serovar(s) from which you want to receive protection. We know that along withActinobacillus pleuropneumoniaeApx toxins (I and II) also producedActinobacillus suis.Last toxins can also be used in the vaccine in accordance with this variant implementation. Preferably one or more toxins Apx I, Apx II, Apx III included in the vaccine, more preferably, each one of them.

In a variant implementation of the vaccine includes a minimum of 2,000 IU of polymyxin b per dose. It was unexpectedly discovered that an effective reduction endotoksicski of symptoms can be achieved by using only 2000 IU/dose (which can be achieved by, for example, adding approximately 60 μg of polymyxin B sulfate per ml of the vaccine dose 4 ml). In the links given here and above, an effective amount of at vaccination are in the range 42000-160000 IU of polymyxin b per dose, which represents an approximate range for use of polymyxin b as a preservative (see also the 9thedition of the Merck Veterinary Manual, ed. 2005, which recommends a dose of at least 50000 IU n� dose for pet up to 10 kg). The applicant unexpectedly found that only a fraction (less than 5%) is sufficient to reduce the symptoms of endotoksicski shock. As the polymyxins, as a rule, are not infinitely stable, the amount that is present in the vaccine, will be less than the added amount, typically from 50 to 80% when measured one year after receipt. It should be noted that to reduce the clinical symptoms can be used a small amount of polymyxin. May be that in the breed of pigs that are less affected by LPS in the vaccine, the number of 15-30 IU per dose is effective adequate to mitigate symptoms endotoksicski shock. The positive effect of such a low number was confirmed experimentally. It should be noted that the minimum effective amount can not only depend on the breed of pig, but also from the required reduction of clinical symptoms, the number of LPS in the vaccine, like polymyxin b and D. In the embodiment of the vaccine includes less than 1000 and even less than 500 IU per dose.

In a variant implementation of lipopolysaccharides derived fromActinobacillus pleuropneumoniae. Actually, as it is well known that LPS in the vaccine does not require the allocation of the RDA of bacteria to be effective vaccine against actinobacillus pleuropneumonia. It can be obtained, for example�EP, fromEscherichia coli, recombinante expressed on bacteria-host cell is an animal or other type of host. However, the authors found that LPS derived from the RDA, gives very good results vaccination. Such LPS can be isolated directly from the ARD bacteria, but can also be expressed in a suitable host.

In a variant implementation of the vaccine includes a 42 kDa protein of the outer membrane ofActinobacillus pleuropneumoniaeas is known from EP 453024. The present invention also relates to a method of producing vaccines directed against actinobacillus pleuropneumonia, including the production of lipopolysaccharide, the mixing of lipopolysaccharide with a pharmaceutically acceptable carrier and detektirovanie of lipopolysaccharide by the addition of polymyxin. Detektirovanie in this sense means that the amount of added polymyxin is appropriate at least to reduce the symptoms of endotoksicski shock, in particular, diarrhoea, vomiting and increased respiration. Preferably less than 2000, or even less than 1000 or 500 IU of polymyxin is used to provide the desired dose of the vaccine. In a variant implementation of the polymyxin is added when lipopolysaccharide is mixed with a carrier.

The invention also relates to a method of reducing the symptoms endotoksicski shock with the introduction of a vaccine aimed �contrast actinobacillus pleuropneumonia, containing lipopolysaccharide, including the introduction of a vaccine in accordance with the present invention.

The invention will be further illustrated by the following examples.

Scheme of the experiment

In the first study was used 150 specific free from pathogens (SPF) pigs at the age of 6 weeks, and was formed three groups of 50 animals each. Each group was vaccinated intramuscularly 4 ml dose in 6 weeks. Group 1 received standard RDA vaccine (Porcilis APP, available from Intervet International BV, Boxmeer, the Netherlands) without polymyxin. The vaccine contains (1 ml) 25 units of repeats toxins ApxI, II and III, as well as a 42 kDa protein of the outer membrane. LPS is present in the amount of approximately 5-6×105IU/ml Group 2 received the same vaccine, but with the addition of polymyxin b (as sulfate salt) in an amount of 30 μg/ml (approximately 250 IU per ml). Group 3 received the standard vaccine, but with the addition of polymyxin E (as the sulfate salt), also in an amount of 30 μg/ml (approximately 250 IU per ml). The polymyxins were added at the final stage of receiving the vaccine, i.e. when all other antigens were mixed with the carrier Diluvac Forte (available from Intervet International BV, Boxmeer, the Netherlands).

Rectal temperature was measured 20 hours before vaccination, just before the vaccination and at 2, 4, 7 and 24 �Asa after vaccination. All groups of animals controlled about the appearance of clinical symptoms (indicative endotoksicski shock) after 30 minutes, 1, 2, 4, 7 and 24 hours after vaccination. In particular, animals were checked for the following symptoms: decreased activity, tremors, cough, diarrhea, vomiting, increased frequency of breathing, abdominal breathing, shortness of breath and death. From 2 to 6 days after vaccination was only made daily inspection. Compared temperature values and clinical symptoms in group 2 and 3 with the values obtained in group 1.

In the second study tested two different concentrations of polymyxin. This study was conducted on 42 pigs that were randomly divided into 3 groups of treatment in 14 pigs each. Group 1 received Porcillis APP (without polymyxin), group 2 received the same vaccine with 30 μg of polymyxin b per ml of vaccine, and group 3 received the same vaccine with 60 μg of polymyxin b per ml of vaccine. Further, the experiment consisted of the same as in the first study.

In the third study tested the effectiveness of RDAs vaccines based on FSC and containing polymyxin B. Polymyxin b commonly known as polymyxin best LPS binding properties, and thus, theoretically, polymyxin b has the strongest negative effect on EF�objectivity of the vaccine. Therefore, if the vaccine is obtained with this polymyxin is effective, it is proof that with another polymyxin can be obtained equally effective vaccine. The vaccine for this study was obtained by adding to the standard Porcillis APP vaccine 30 μg of polymyxin b per ml of vaccine. Fourteen pigs were vaccinated intramuscularly at the age of 6 to 10 weeks to 2 ml of the vaccine, thus, get a dose of approximately 500 IU of polymyxin. Seven pigs received a placebo vaccine (40 mm Tris-HCl buffer). Antibody titers against the RTX toxins ApxI, ApxII and ApxIII, and OMP were identified at 13 week. Three weeks after the secondary vaccination, pigs were infected withActinobacillus pleuropneumoniae, sarovaram 2.

In the fourth study, the inventors tested the safety of the vaccine, where polymyxin was added during the course of batch fermentation ofActinobacillus pleuropneumoniaeultimately, achieving a far smaller amount of polymyxin b vaccine (approximately 2-4 μg/ml). In this study the vaccine was obtained by adding 30 μg of polymyxin b or polymyxin E sulfate per ml of supernatant enzymatic broth respective cultures, then the toxins ApxI, II, III, together with LPS and 42 kDa OMP, were isolated from the supernatant. These selected subunit were�learned in Diluvac forte to achieve per ml of the vaccine is about 25 subunits of toxins and OMP and approximately 1×10 6IU LPS. It was found that in these vaccines, the concentration of polymyxin b is approximately 22 IU (2.6 µg/ml) of polymyxin E and about 35 IU (4,3 µg/ml) of polymyxin b per ml. To test the safety of two groups of ten 6-week-old SPF pigs received 4 ml of the appropriate vaccine. Clinical symptoms were evaluated by the same way as in the first study.

Results

Polymyxins had no significant effect on rectal temperature. However, there were significant effects in terms of symptoms endotoksicski shock. In table 1, located here and below are a summary of the symptoms in each group. In group 1 the typical symptoms were observed in more than half of the animals, such as vomiting (60%) and increased respiration (66%). In this group, two pigs (4%) died as a result endotoksicski shock. In contrast, only very mild symptoms were observed in groups 2 and 3. In both groups, 2% of pigs (one of 50 animals) was vomiting. In addition, 2% of pigs in group 3 were increased respiration. Thus, it is concluded that the polymyxins are appropriate to reduce the severity of symptoms endotoksicski shock caused by LPS in the RDA vaccine.

Table 1
Symptoms endotoksicski shock after VA�Canazei different RDAs vaccines
Vaccinediarrheavomitingthe increase in respiratory ratedeath
Porcillis APP10%60%66%4%
Porcillis APP + 30 μg/ml of polymyxin b0%2%0%0%
Porcillis APP + 30 μg/ml of polymyxin E0%2%2%0%

Table 2 shows the results of the second study. In group 1 (standard vaccine) showed typical clinical symptoms, such as vomiting (54%) and increased respiration rate (15%). In groups 2 and 3 showed the symptoms from mild to nonexistent. Based on these results it is concluded that the addition of 60 μg of polymyxin per ml of vaccine or even just 30 μg/ml is effective to almost completely block LPS in inducing severe symptoms endotoksicski shock.

Table 2
Symptoms endotoksicski shock after vaccination with different RDAs vaccines
Vaccinediarrheavomitingthe increase in respiratory ratedeath
Porcillis APP15%54%15%0%
Porcillis APP + 30 μg/ml of polymyxin b0%0%8%0%
Porcillis APP + 60 µg/ml of polymyxin b0%0%0%0%

In the third study were obtained good results on efficiency (equal to the results obtained with the standard Porcillis APP vaccine, having the same composition, except polymyxin) with vaccine containing polymyxin B. the Results are summarized in table 3. In the control group, almost 60% of the animals died, while animals in the vaccinated group were protected from infection. Titles in the latter group were comparable to the titers obtained with the standard Porcillis RDAs vaccine that does not contain at�of mixin ("n.d." indicates not determined used in the test). Obtained extremely good results with RDAs vaccine containing polymyxin b, and the fact that this polymyxin has the strongest binding properties toward LPS, make understandable what with other polymyxins can be derived vaccine, which is at least equal in effectiveness.

Table 3
Average results of the efficacy of a vaccine containing polymyxin b
VaccineCaption Apx ICaption Apx IICaption Apx IIICaption OMPDeath
Porcillic APP + 30 μg/ml of polymyxin b9,310,18,59,10/14
controln.d.n.d.n.d.n.d.4/7

In the fourth study it was shown that even with a vaccine comprising only 20 IU of polymyxin per ml, can be obtained good results on safety. Average data are shown in the table . The resulting effect is that even with a significantly higher content of polymyxin (see study 3) can still be obtained good results for security, makes it obvious that vaccine fourth research can also be obtained good results in efficiency.

Table 4
Symptoms endotoksicski shock after vaccination with different RDAs vaccines
Vaccinediarrheavomitingthe increase in respiratory ratedeath
Porcillis APP50%90%70%0%
Porcillis APP ctg 4.2 µ g/ml of polymyxin b0%10%10%0%
Porcillis APP ctg 2.6 µg/ml of polymyxin E0%20%30%0%

1. A vaccine directed against actinobacillus pleuropneumonia, including lipopolysaccharide in combination with od�them or more repetitions of toxins ApxI, ApxII and ApxIII, isolated from a bacterial culture, wherein the vaccine includes polymyxin to reduce symptoms endotoksicski shock caused by lipopolysaccharide.

2. The vaccine according to claim 1, characterized in that the vaccine comprises less than 2000, ME, polymyxin b per dose.

3. The vaccine according to claim 2, characterized in that the vaccine comprises less than 1000 IU of polymyxin b per dose.

4. The vaccine according to claim 3, characterized in that the vaccine comprises less than 500 ME of polymyxin b per dose.

5. The vaccine according to claim 1, characterized in that a is a polymyxin polymyxin B.

6. The vaccine according to claim 1, characterized in that the lipopolysaccharide is obtained from Actinobacillus pleuropneumoniae.

7. The vaccine according to claim 1, characterized in that the vaccine comprises a 42 kDa outer membrane protein of Actinobacillus pleuropneumoniae.

8. A method of producing vaccines directed against actinobacillus pleuropneumonia, including isolation and purification of lipopolysaccharide in combination with one or more repetitions of toxins ApxI, ApxII and ApxIII of bacterial culture, the mixing of purified lipopolysaccharide in combination with one or more repetitions of toxins with a pharmaceutically acceptable carrier, and detektirovanie of lipopolysaccharide by the addition of polymyxin.

9. A method according to claim 8, characterized in that the polymyxin was added to obtain maximum 2000 ME per dose of vaccine.

10. A method according to claim 9, wherein �the polymyxin was added to obtain a maximum of 1000 IU per dose of vaccine.

11. A method according to claim 10, characterized in that the polymyxin was added to obtain maximum 500 ME on a dose of the vaccine.

12. A method according to any one of claims.8-11, wherein the polymyxin is added when lipopolysaccharide is mixed with a carrier.

13. The use of polymyxin to reduce the symptoms of endotoksicski shock with the introduction of a vaccine directed against actinobacillus pleuropneumonia containing lipopolysaccharide in combination with one or more repetitions of toxins ApxI, ApxII and ApxIII, isolated from a bacterial culture, which polymyxin is added to the vaccine in a dose of from 2.6 to 60 μg/ml prior to administration of the experimental animal.

14. A method for reducing symptoms endotoksicski shock with the introduction of a vaccine directed against actinobacillus pleuropneumonia containing lipopolysaccharide in combination with one or more repetitions of toxins ApxI, ApxII and ApxIII, isolated from a bacterial culture, where to reduce symptoms endotoksicski shock caused by lipopolysaccharide in the vaccine add polymyxin b dose from 2.6 to 60 μg/ml.



 

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EFFECT: method can be used for preparing the microbiological nutrient media for erysipelas strain upstream.

FIELD: biotechnologies.

SUBSTANCE: method of obtaining of brucellous L-antigen is performed as follows. The strain Brucella abortus 19 is cultivated in the L-form in the dense nutrient medium prepared on the basis of meat-peptonic hepatic glucose-glyceric agar (1.3-1.5%) with adding of 10-15% normal horse Serum at the temperature 20-22°C within 2-3 days. The culture, obtained in the L-form, is emulsified with 0.5% phenolised normal saline solution, inactivated at the temperature 85-90°C within 60 minutes. The suspension is centrifuged at 3000-5000 rpm within 15-20 minutes and the concentration of brucellas is established at the level 50-60 billion m.k. The obtained antigen is titrated, standardised by specificity and activity. It is used for agglutination test in serum diagnostics of brucellosis.

EFFECT: invention allows to minimise the time of antigen preparation and to increase the yield of bacterial mass.

4 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes a composition for induction of immune response against P. gingivalis, which contains an effective amount of one of the above chimeric or hybrid proteins, a prophylaxis method of a state or a disease related to P. Gingivalis, and a method for reduction of incidence or severity of the state or disease related to P. gingivalis with their application. Besides, the invention describes use of the above chimeric or hybrid proteins for determination of antibodies to P. Gingivalis in a biological specimen.

EFFECT: invention allows effective induction of immune response against the specified etiologic agent.

16 cl, 7 dwg, 4 tbl, 22 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to a recovered polypeptide which is a biological target for methane-producing cell inhibition, as well as to a recovered polynucleotide which codes this polypeptide. There are disclosed expression vector and cloning vector containing this polynucleotide, and host cells containing the above expression vector. There are described conjugated molecules or fused molecule for methane-producing cell inhibition, as well as antibody or its functional fragment which binds to the above polypeptide. The invention also covers a pharmaceutical composition and methods for inhibiting and identifying the methane-producing cell with the use of the above conjugated molecule or fused molecule and the antibody or its fragment.

EFFECT: invention enables inhibiting the methane-producing cell effectively.

19 cl, 9 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: wound-healing agent is a concentrate of the culture liquid of strain Trichoderma harzianum Rifai deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM: F-180, as a producer of L-lysine of alpha-oxidase, and can be applied as a wound-healing agent for skin lesions.

EFFECT: invention enables to expand the range of means that provide wound healing of skin lesions.

1 tbl, 2 ex

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

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: medicine.

SUBSTANCE: invention refers to a method of treating tuberculosis with multiple drug resistance characterised by prescribing a combination of six anti-tuberculosis preparations in the intensive phase of chemotherapy and five preparations - in the phase of the 20-month therapy continuation, wherein the intensive phase duration makes at least 8 months until obtaining four negative culture results every month in tuberculosis with multiple drug resistance and until obtaining two negative culture results in all other cases of tuberculosis with multiple drug resistance, the phase of the therapy continuation makes 12 months.

EFFECT: higher clinical effectiveness.

1 tbl, 9 ex

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