BG3644U1 - Biologically active polybacterial product for immunostimulation in animals - Google Patents

Abstract

The utility model refers to a biologically active polybacterial product, in the form of oral powder or tablets, applicable as a veterinary immunostimulant in acute chronic infections, which are of particular importance for the health and nutrition of animals. The product according to the present utility model contains a polybacterial complex of a substance of killed bacterial bodies and their lysates of strain Escherichia coli 29 expressing type 1 and P files, Escherichia coli Rc mutant and Staphylococcus aureus with a stabilizer in an amount by weight % respectively: polybacterial substance from 30 to 50, stabilizer from 50 to 70, as well as suitable one or more excipients, where each of the two strains of E. coli of the polybacterial substance is in an amount of from 20 to 45 wt. %, and strain St. aureus is in an amount of 20 to 60 wt. %. The amount of agents involved in the dry powder form is in the following weight %, respectively for: the polybacterial complex from 96.9 to 99.9 and for excipient (s) from 0.1 to 3.1, and for the tablet form respectively for the polybacterial complex 35 to 41, and excipient (s) from 59 to 65. The biologically active polybacterial complex, together with the whole bacterial cells used and their lysate in the product according to the present utility model, provide full stimulation of all defense mechanisms of the organism of mammals and birds. The selected excipients with their quantities ensure the stability of the product and improve its taste and efficiency. It is designed for all age groups of animals, incl. newborns - sucklers and weaned, pregnant, lactating mothers, as well as newly hatched birds and laying birds.

Description

Field of technology

The utility model relates to a biologically active polybacterial product, in the form of oral powder or tablets, useful as a means of immunostimulation in veterinary medicine in acute chronic infections, which are of particular importance for the health and proper nutrition of animals.

BACKGROUND OF THE INVENTION

Infections in animals are a serious problem, especially in animals kept in groups for the purpose of their economic use in the food chain. Immunoprophylaxis is a key factor in the fight against infectious diseases, thanks to immunoprophylactic activities, a large proportion of life-threatening and fatal infections can be prevented and avoided.

Of particular importance for the limitation, eradication and termination of an infectious disease is the application of permanent preventive measures, as well as the possibility of immunoprophylaxis and immunotherapy with drugs. Bacterial preparations and combined ones that stimulate the immune response occupy an important place in the group of immunoprophylactic and immunotherapeutic agents and successfully replace the use of antibiotics prohibited in veterinary practice. Moreover, there is a constantly growing antibiotic resistance of different types of bacteria.

It is known from BG 1141 U1 polybacterial complex for immunoprophylaxis and immunotherapy in mammals and dosage form containing a polybacterial substance of the complex of killed bacterial bodies and lysates of two strains of Escherichia coli 29, expressing type 1 and P files and Escherichia together with Staphylococcus aureus and stabilizer dextran 40 in an amount in% by weight, respectively for polybacterial substance from 30 to 50 and dextran from 50 to 70. The tool is used in the form of ordinary tablets obtained by standard methods, intended for the prevention and treatment of acute chronic infections, especially in cases of choleenteritis and bronchopneumonia.

The task of the utility model is to create a biologically active polybacterial preparation for immunostimulation, suitable for both mammals and birds with suitable excipients in the form of oral powder or tablets intended for use in acute and chronic infections to help the full absorption of food and increase animal growth.

Technical essence of the utility model

The utility model relates to a biologically active bacterial product for immunostimulation in mammals and birds, containing a polybacterial complex, a stabilizer and one or more excipients.

The biologically active polybacterial complex is a polybacterial substance of killed bacterial bodies and lysates of two strains of Escherichia coli 29 expressing type 1 and P files and Escherichia coli Rc mutant, together with Staphylococcus aureus, for the three strains regardless of the deposit number by weight. % from 30 to 50 and a stabilizer in an amount by weight. % from 50 to 70.

Like each of the two strains of Escherichia coli, as part of the polybacterial substance is in the range of 20% to 45%, and the strain Staphylococcus aursus - from 20% to 60%. The bacterial substance was in an amount of 10 x 10 ⁹ cfu (bacterial bodies) in mg of active substance for each of the three strains and with a density of 7.5 x 10 ⁹ / ml before lyophilization using the stabilizer.

The excipients are selected from: colloidal silicon dioxide, macrogol 6000, crospovidone, silicified microcrystalline cellulose or a combination thereof in amounts by weight. %, respectively for: colloidal silicon dioxide from 0.1 to 3.1, macrogol 6000 from 2.88 to 3.12, crospovidone from 10.66 to 11.34, silicified microcrystalline cellulose from 45.46 to 50.54. The amount of the polybacterial complex in the tablet dosage form is from 35 to 41% by weight, and for the powder form is from 96.9 to 99.9.

3980 Descriptions to utility registration certificates № 05.2 / 29.05.2020

The polybacterial product is suitable for tableting or packaging in sachets or banks. It is administered orally to mammals and birds at a dose of 0.1 to 10 mg / kg body weight with food or water.

The advantages of the biologically active polybacterial complex and the product containing it according to the utility model are determined by its content and quantity, where the used whole bacterial cells and their lysate provide full stimulation of all defense mechanisms of mammals and birds. Thus selected and used strains induce an effective specific and non-specific immune response at different levels of the immune system and create broad-spectrum immunity to the most common causes of infections, ensuring a lasting effect in the absence of any toxicity and side effects.

The selected excipients with their quantities ensure the stability of the product and improve its taste and efficiency. The polybacterial complex and its product are suitable for immunostimulation in mammals and birds, especially in acute and chronic infections, especially in cases of cholenteritis and bronchopneumonia, as well as for complete digestion and increased growth of animals, which is especially valuable in animals kept in groups for commercial use, where it exists in veterinary practice and a ban on the use of antibiotics. The product is non-toxic, suitable for long-term use. The administration of the polybacterial product according to the present utility model can be discontinued at any time of administration without the risk of side effects.

They are designed for all age groups of animals, incl. Newborns - sucklers and weaned, pregnant, lactating mother animals, as well as newly hatched birds and laying birds.

Clinical studies of the biologically active polybacterial product according to the present utility model demonstrate its use as an immunostimulant in both mammals and birds.

Examples of implementation of the utility model

Example 1.

The agent in one dose contains:

A / Composition of the active bacterial mixture

The active polybacterial mixture contains lyophilized killed cultures of the following microbial species: E. coli 29, E. coli Rc and S. aureus in an amount corresponding to 10 x 10 ⁹ cells of each species per 1 mg of the bacterial mixture, and a mass of density 7 , 5x10 ⁹ / ml before lyophilization using dextran stabilizer 40.

Feed data / Example 1.A.1. 1A2. 1A3. 1Λ.4. 1 A.5 . 1 A6. 1.A7 1A9 Escfientiia coil 29 25 20 35 33.3 45 35 30 40 37 Escherichia aoit J 5 25 20 35 33.3 35 40 30 35 30 Staphylococcus autous 50 60 30 33.3 20 25 40 25 25

27/53

B / Composition of the polybacterial complex

The active biological polybacterial complex contains as a stabilizer dextran 40 included in the polybacterial substance, in a ratio by weight. %, as in the following embodiments:

Ingoadients / Poimeo 1 B.1. 1.B.2. 1.B.Z. 1.B.4. 1 B.5, 1.B.6. 1.B.7. 1.B. & Polybac. substance 30 33 37 40 42 44 46 50 Dextran 40 70 67 63 60 58 56 54 50

3981 Descriptions to utility registration certificates № 05.2 / 29.05.2020

Example 2.

A / Composition of dry form - oral powder product of the polybacterial complex and PV in wt. %:

I ngr (schie * gti.1 P r i mor 2.Аь1. 2.A.2. 1L.4. IAS. 2.A.a. Α.Α.Τ. 2JLS 2 .AS Ι.Λ.1Ό, 2 A.11 ΪΛ12 2.A15 PCln & Dk. КЙМПЛЙкС 97.16 B7.B5 B7.9 35.15 ¢ 5.2 BS. & 5 Ίΐ, ί pimochkn uQutfrciea (colloidal silicon J.1 2fi 2.35 2.1 1, F5 1.5 1.35 1.1 Oh, SS 0.5 Ϊ3 » 0.1

dioxpd]

B / Composition of tablet form - from the polybacterial complex and PV in wt. %:

Iga radmsn1M1rim £ r 2.B.1. 2.B.2. 2.B.Z. 2.B.4. 2AB5. 2.B.S. 2.E.7 35 3S 37 for for 40 41 M2 з.оа 3.04 3.0 2.96 2.90 2.SS К ^; КгХ1в> <д <и 11.34 11.22 11.13 11.0 10.88 10.70 10.66 Spimifim karana mmrochryastapna cellulose 50.54 49.70 40.63 4ΐ ·, ϋ 47,? 0 46.4П 45 45 PV 65 04 03 62 01.05 W 59

Example 3.

Method of preparation:

The bioactive polybacterial substance with the described lyophilized bacterial strains was obtained by standard classical methods. The resulting individual bacterial suspensions were pre-killed and treated (killed with a chemical agent and mixed in ratios to ensure the presence of 10 x 10 ⁹ of the three strains - Escherichia coli 29, Escherichia coli J5 and Staphylococcus aureus 27/58 in 1 mg). The bacterial mixture thus obtained is lyophilized and stored in vacuum-sealed plastic containers for no more than 36 months from the date of lyophilization.

The preparation of the product includes the screening and screening phases of the active complex, to which the pre-screened excipients are successively added. The resulting mixture in the tablet form is homogenized and tableted according to conventional methods, and in the case of oral powder is dosed in sachets or jars. The finished form is stored at room temperature in a dry place.

Example 4.

Product application and storage.

The polybacterial product is administered orally at a dose of 0.1 to 10 mg / kg body weight. It is included in a small part of the food or dissolved in a limited amount of the daily ration of drinking water, and 30 minutes later the rest of the animal's feeding is continued, incl. newborns - sucklers and weaned, pregnant, lactating mother animals, as well as newly hatched birds and laying birds.

4.A. In pigs - pregnant sows and sows

The sow is given 1.5 mg of polybacterial complex per kg / weight per day for 30 days before term. Admission is stopped on the 10th day after birth. In another embodiment, the administration is continued throughout the lactation period. In a further embodiment, the administration is stopped on the 20th day.

4.B. In newborn piglets

1. From 5-20 days of age

Newborn piglets from 5 to 20 days after birth are given 1 mg / kg body weight per day polybacterial complex in the morning on an empty stomach.

3982 Descriptions to utility registration certificates № 05.2 / 29.05.2020

1.1. Through drinking water - the daily dose is dissolved in a small amount of water and given through a dispenser through the mouth.

1.2. By spray method - the daily dose is dissolved in a small amount of clean water and the resulting solution is sprayed evenly on the prestarter mixture. After half an hour, the rest of the prestarter is fed and allowed to suckle from the mother.

2. Newborn piglets from 21 to 45 days of age. Newborn piglets from 21 to 45 days after birth are given 2 mg / kg body weight polybacterial complex for each animal.

2.1. By spray method - the daily dose is dissolved in a small amount of clean water and the resulting solution is sprayed evenly on the prestarter mixture. Half an hour later, the rest of the prestarter is given and allowed to suckle.

2.2. Through the feed - the daily dose is mixed with about 70 g of feed mixture from the morning ration. After about half an hour, the remaining certain amount of feed is given, and during the mammalian period they are allowed to the mothers.

3. Through the drinking water - the piglets are left in the morning for 1-2 hours, after which the daily dose is dissolved in a small amount of water and the ready solution is given through drinking bowls. After consuming the solution, the feed mixture is set with free access to the drinking water, and the suckers are allowed to the mothers.

4. C. In newborns and adult dogs

The product is given at a dose of 1 mg / kg body weight with water or the first sips of food.

4.G. In broiler chickens

The product is administered at 4 mg / kg live weight for 28 days and the intended daily amount is dissolved in drinking water.

4.D. In laying hens

The treatment is performed in 3 stages - after hatching (1-2 weeks), before transfer (20-21 weeks) and at 50-51 weeks of age. The bacterial product according to the utility model is given at 4 mg / kg live weight. The treatment time at each stage is 14 days. It is applied dissolved in drinking water.

4. F. In newborn and adult cats

The product is given at a dose of 1 mg / kg live weight, by water or with the first sips of food.

5. Testing of the preparation in scientific and economic experiments

5.A. The experiment was conducted in the experimental base for poultry at the Institute of Agriculture, Stara Zagora.

In birds - a total of 100 broiler chickens Ross 500 were observed, divided into two groups of experimental and control of 50 pieces. Feeding is done with compound feed with and without a product obtained according to Example 2.A. Feeding is carried out according to a schedule providing all the necessary nutrients for the respective stage of fattening - newly hatched (1-10 days), adolescents (11-35 days), adults (36-49 days). All birds were weighed at the beginning of the 10th, as well as on the 21st, 35th and at the end of the period - 49th day - table. 1. The experimental chickens are treated with the product from 2 to 11 days at a dose of 300 mg / kg of feed per day. The product is dissolved in 50 ml of water and sprayed in 3 kg of feed. The first blood samples were collected at 20 days of age from 25 chickens and the dose was increased to 400 mg / l in drinking water for 10 days to achieve a better immune response. The second blood sample is at 40 days of age. Serum lysozyme concentrations and complement activity (defined as an alternative route of complement activation) were studied.

Table 1 Live weight of broiler pipette (e) _________________

Groups Day 10 Day 21 Day 35 Day 49 Control 262.02111.82 963.02 ± 47.37 1976.66 ± 36.66 3066.66133.33 Experimental 265.6219.74 1034.34 ± 19.98 2180120 *** 3250150 *

* p <0.001

3983 Descriptions to utility registration certificates № 05.2 / 29.05.2020

On day 21, the experimental group was 71.32 g or 6.9% heavier. The tendency for more intensive growth of broilers treated with Natstim® is maintained until the 35th day, when the chickens in the experimental group are 203.34 g (9.3%) heavier.

Animal mortality, average live weight of chickens and their health status were monitored daily.

Table 2 Feed consumption (kg / kg)

Groups Day 1-10 Day 11-21 Day 22-35 day 36-49 Control 1,387 th most common 1,583 th most common 1,762 th most common 2,055 th most common Experimental group 1,340 th most common 1,561 th most common 1,765 th most common 1 988

Table 3 Productivity index (kg / kg) (IP)

Groups Live weight at 49 l * day i'kg Premium% Corresponding of fodder consumption (cd / cd) Full IP Sinsipenem IP Control 3,066 th most common 98 2,055 th most common 293 09.3 Experimental 3.25 100 1.9ВЙ 333.7 100

group

The results of the present study show a tendency towards higher live weight, higher productivity index, better breast and thigh proportions and a significant effect on serum lysozyme in broiler chickens treated with the product.

5.B. In newborns - suckling and weaned and pregnant, lactating mothers

The test was performed in newborns - suckling and weaned pigs and pregnant and lactating sows. Pregnant women are given 1.5 mg / product per kg / live weight for 30 days before the due date. The intake is stopped 10 days after birth, and for the second batch the intake continues throughout the lactation period of the sows.

In newborn piglets from 5 to 20 days of age after birth, 1 mg / kg live weight per day is given a polybacterial complex with water or part of the first dose of food. From 21 to 45 days after birth, 2 mg / kg live weight per day is given for each animal in the morning on an empty stomach. Live weight of pigs in kg, average daily gain in g and feed utilization kg / kg growth, morbidity and mortality of experimental animals were observed. A control group of 77 pigs and an experimental group of 74 pigs were observed.

The test was conducted in the period February - August in two pig farms, and in the experiments were included 12 sows in a group of experimental and control, and their offspring, which are an average of 10 piglets per mother. The attached table presents the data on the number of animals and the variants for research (Table 4).

3984 Descriptions to utility registration certificates № 05.2 / 29.05.2020

Table 4

“EXPERIMENTAL GROUP CONTROL GROUP FARM <o ¥ £ + 3 I 4 1 i 4- 1 I I s & 1 S £ h- . Ή 3. = 1 !! ^!! s I 3 1 1 & I s L. AGROSL AVYANI M “aod Stara Zagara is - 5 1- 30 30 * for - G s 14 and 11 зй зй for 'АК & Ц- СЮД гр. Соепиосч? 1- 6 - Θ + 70 7P - 6 - ^s . and ¹

Note:, + * - With Deliana «product

- <nz doba ™ per product, GT pdztugbo

The sows from both experiments were matched in the order of farrowing. The experimental period for mothers included the last month of pregnancy. The offspring were placed under the conditions of the corresponding experimental variant with respect to the test preparation. Their cultivation corresponded to the accepted order on the farms. The duration of the experiment in pigs included the mammalian period and after weaning - up to 5 5 days of age. The animals were further monitored after the end of the experiment, ie. during adolescence and fattening. The preparation is given according to Example 4.

Experimental animals (sows), incl. controls are obtained from the polybacterial complex 32 days during the last phase of pregnancy. The lactation period was 28.7 for the control and 28.5 days for the experimental group. The experimental period for offspring is 53.7 days. The tests showed a positive effect of the product in terms of morbidity and mortality in newborn piglets receiving the polybacterial complex compared to the control group of animals. These results are presented in Table. 5.

3985 Descriptions to utility registration certificates № 05.2 / 29.05.2020

Table. 5. Morbidity and mortality of the offspring of the control and elite sows (Agro-Slavs)

Indicator boaaen g * eriod Pschchod after weaning Kyitro LmZ> grute Experimental group Nontrodpa group Experimental group 1- f product 1. -without product Total 1. t oraduct 7. would product Total P [»SGTYA, number 77 ZV * DV * 74 ' 73 35 * 36 * 71 Ill «t nolmbyitsridoa; - number W B 7.8 - . 2.6 1 1.35 - - 1 2 73 1 1.41 Duration of illness 2 - 2 2 - - 2 ---— Weight yaa the clinical picture ++ 4 + - * 4- Died; - number -K 4 5.19 1 3-7V 2 5.26 3 4L5 1 1.37 ' > 3? 1-1 f-d 1 L41

ЗвВапежга.

Ch Rpagaptspa V · brel № prodenivta OmZyynii period i poddasteagets □ result Na sma * », j OT deyugta

Example 6.

Immunological activity of the product

The test was conducted in the experimental base for poultry at the Institute of Agriculture, Stara Zagora and two pig farms with control and experimental animals.

In the case of pigs, the experiment was performed with 12 sows and 10 piglets per mother, reared according to the accepted order on the farms.

The pigs received a product obtained according to Example 2.B.5 for 30 consecutive days before farrowing and in piglets from 5 days to 45 in an amount according to Example 6.B.

Tests were performed on mothers at 0 and 54 days and at 0 and 45 days in piglets.

Serum IgA, IgM and IgG antibodies determined by ELISA were monitored (Fig. 1).

The results show a statistically significant increase in antibodies after 45 days of treatment of the drug in newborns compared to baseline - self-monitoring at day 0. This increase varies from 21 to 60% in the animals tested, depending on the type of antibody.

In clinical trials in all animals, the polybacterial complex and its dry powder formulation showed convincing activity in stimulating specific antibodies to IgG, IgM and IgA against the bacterial strains contained therein. Convincingly, the preparation most actively stimulates the production of specific IgM antibodies. The product stimulates a systemic humoral immune response, which is largely responsible for overcoming bacterial infections, which is demonstrated by the smaller number of sick and dead animals.

In birds, serum lysozyme concentrations and alternative complement activation pathway (APCA) activity were observed at different ages and for a product administered by different pathways.

3986 Descriptions to utility registration certificates № 05.2 / 29.05.2020

Table. 6 Serum lysozyme concentration and alternative complement activation pathway (APCA) activity in chickens treated with the product according to the present utility model

Group parameters __________ Lysozyme Controls, 20 days of age 0.4810.04 T reted, 20 days of age Q.40 ± 0 049 ARSA ________________ 36b./9 ± 14.91 37В.621В.74 Controls, 40 day age 31.1117.34 ** ' Tretprani, 40 days of age 14.12 ± 2.52 526.33121.ВЯ 42 ± 13.66 ‘**

Table. 7, Serum lysozyme concentrations and alternative complement activation pathway (APCA) activity in immunomodulator-treated chickens According to utility model ____

Follower and ost of tests LYSOCYME ARSA controls treated controls treated Before treatment 0.391D.O6S D.74 ± fl 25 596.25i20.2la 5e6.B4i1D.17 Product in feed fl 8510.29 9.7910.1 572.16116.70 595.18124.51 Product in water 0.610.06 D.S3 ± O.D0 *** a 583.2 ± 14.23 631.92117.48 ^ 5 Blood test 0.5110.04 о.е ± о.о4 | _463.1219.05 455.56112.00

- P <0.Q5; *** ’P <0.001 -group differences a - P <0.001; B - P <D.O1 - intrastructure differences

The material and method were according to Example 5 A. Serum lysozyme was observed by the method of Lie et. al., 1985) and ARSA - by the method of Sotirov (1986). The data are summarized and presented in Table. 6 and 7. The product stimulates humoral immunity in birds as the serum lysozyme is reduced by a total of 50% compared to the control (P <0.001) and significantly activates APCA (P <0.01).

The product is non-toxic and suitable for long-term use.

Claims (5)

A bioactive polybacterial product for immunostimulation in animals, comprising a polybacterial complex of a substance of killed bacterial bodies and their lysates of a strain of Escherichia coli 29 expressing type 1 and P files, Escherichia coli Rc mutant and Staphylococcus aureus with a stabilizer characterized by that contained in wt. %: polybacterial substance from 30 to 50, stabilizer from 50 to 70, and suitably one or more and excipients, where each of the two strains of E. coli of the polybacterial substance is in an amount of from 20 to 45 wt. %, and strain St. aureus is in an amount of 20 to 60 wt. %. Bioactive polybacterial product according to claim 1, characterized in that the polybacterial substance is in an amount of 10 x 10 ⁹ bacterial bodies in rag active substance from each of the three strains and a mass with a density of 7.5 x 10 ⁹ / ml before lyophilization with use of the stabilizer. Bioactive polybacterial product according to claim 1, characterized in that it is dry, intended for use in dry powder or tablet form. Bioactive polybacterial product according to claim 3, characterized in that the amount of agents involved in the dry powder form is in the following weights. %, respectively for: the polybacterial complex from 96.9 to 99.9 and excipient (s) from 0.1 to 3.1. Bioactive polybacterial product according to claim 3, characterized in that the amount of agents involved in the tablet form is in the following weights. % respectively for: the polybacterial complex from 35 to 41, and excipient (s) from 59 to 65.