EA009170B1 - The use of fermented wheat-germ in the feeding and veterinary practice - Google Patents

Abstract

This invention relates to new uses of fermented wheat-germ extract, in paticular, to the use of fermented wheat-germ extract for manufacture of preparation for preventing and/or treating animal's Mycoplasma infections, to the use said extract for manufacture of preparation for preventing and/or treating coccidiosis infection of poultry, and also to the use said extract for manufacture of preparation for increasing antibody titer of vaccinated poultry, specific to such infection of poultry as Mycoplasma synoviae infection or coccidiosis.

Description

The invention relates to new uses of the extract of fermented wheat germ, in particular for veterinary purposes.

The extract of fermented wheat germ (hereinafter referred to as UET-HBM) and its preparation are described in UO 99/08694, where its immunostimulatory and metastasis-inhibiting effect is disclosed. (The above document is included in this description by reference.) This extract is obtained by fermenting wheat germ Sassiaceae segeusae. then drying the filtered fermented liquid. The resulting material is characterized by a content of 2,6-dimethoxy parabenzoquinone, amounting to about 0.4 mg / g of dry material.

In the course of research, the inventors unexpectedly discovered. that UET-HBM can be used with high efficiency in livestock farming for feeding animals. WEH-HBM provides faster weight gain and increases the resistance of animals to diseases. primarily to infectious diseases. Is he. in particular. serves to increase meat production and to improve the quality of meat of farm animals. bred commercially. primarily poultry and pigs. and at the same time provides better feed utilization (better feed absorption ratio).

Additionally, the inventors investigated the effect of UET-HBM on infections. usually occurring in poultry farms. and it was unexpectedly discovered. that WEH-HBM is able to protect animals from mycoplasmic infections. called in particular. Garbage dasherszerit and Garbage 5 Utop. as well as from coccidiosis. caused by E1tepa 1ene11a. and is able to increase resistance to other infectious diseases of poultry (for example, Gumboro disease).

Infection of poultry M. daNyrisit and M. zupoue still leads to large economic losses in the poultry industry. Due to this infection, weight gain and egg production are reduced. and mortality due to the loss of non-hatching chicks. rejection at slaughter, etc. are increasing. Damage to the epithelium of the respiratory tract contributes to the secondary. bacterial infection. thereby further increasing losses.

To reduce economic losses, the use of various antibiotics is suggested (for example, tylosin, tiamulin, norfloxacin, enrofloxacin, etc.). However, in recent years, various states have decided to reduce the use of antibiotics (for example, the use of certain antibiotics, including tylosin, is prohibited as a means of increasing productivity). and banned the use of antibiotics. used to treat people. For this reason, the inventors investigated the effect of UET-HBM. showed very good results in feed studies against common mycoplasmic infections.

The inventors unexpectedly discovered. that mycoplasmic infection can be prevented by administering UET-HBM in a manner similar to that of thiamutine. the best known antibiotic against mycoplasma. Due to the significant incidence of mycoplasmic infections around the world. This fact is of great economic importance. Economic loss. caused by these infections. can be reduced by using WET-HBM. This may be particularly preferable at present. when seeking to eliminate the use of antibiotics in veterinary practice and to increase productivity.

Besides. The inventors have conducted studies on pigs in connection with the Garbage collection of yuorpeitoshae. which is present in the normal population of pigs and leads to large economic losses; inventors unexpectedly discovered. that WEH-HBM was able to protect pigs from pneumonia. caused by M. yuorpeitoshae.

Besides. The inventors investigated the effect of UET-HBM against another parasitic disease, coccidiosis. caused by E1tepa! epe11a. which is a very common parasitic poultry disease. The number of cases of coccidiosis has increased everywhere in the world in recent decades. since mass dilution maximized the likelihood of coccidial infection. Very likely. 7 Enpep strains play a role in the development of coccidiosis; among them are pathogenic and less pathogenic strains. Among the strains. causing hemorrhagic enteritis. Essential! causing coccidiosis of the appendix. Therefore, studies of the authors of the invention were carried out on chickens. infected with the pure strain E1tepa! pe11.

The inventors unexpectedly discovered. that oocyst excretion by artificially infected chickens. receiving supplement WET-HBM. significantly decreased compared with the control; it means. that the intermediate forms and parasites of E.! nep11a were destroyed. causing a lot of damage to the appendix. were not able to develop. Consequently. WEH-HBM may be able to protect chickens from serious illness. caused by E.! pepe11a.

In addition to the above. The inventors investigated the effect of UET-HBM on the change in the level of antibodies in chickens. vaccinated against Gumboro disease (CEUAS vaccine). The inventors have discovered. what is WED-HBM. introduced into the feed. significantly increased the production of antibodies in chickens and, together with an increase in the content of antibodies in the blood, it increased the effect of the CEUAS vaccine during the fattening period of chickens. providing animals with a higher degree of protection.

Besides. The inventors have observed. that the introduction of UET-HBM significantly reduced economic losses. caused by stress (eg heat and transport stress).

The additive HET-HBM according to the invention is used. mixing accordingly with the initial feed. middle and final fattening stage. partially or during the whole feeding. Wets-NVM can also

- 1 009170 to apply by introducing animals into drinking water.

The present invention also relates to the use of an extract of fermented wheat germ to prevent and / or reduce mycoplasmic infections and coccidiosis in poultry and to increase antibody titers in vaccinated poultry. The extract of fermented wheat germ can be mainly used for the prevention of infections of Garlica daSchrekisit or Garbagetape, for the prevention and / or reduction of coccidiosis of poultry, as well as for the prevention of pig pneumonia caused by M. yuoriyitosae. The invention relates to the use of an extract of fermented wheat germ in the manufacture of preparations for the above purposes.

Veterinary preparations according to the invention, containing an extract of fermented wheat germ, can be obtained in the usual way by mixing the active ingredient with one or more veterinarily acceptable auxiliary materials to form drugs that increase the resistance of animals to diseases, drugs that prevent and / or treat mycoplasmic infections and infectious inflammations , drugs that prevent and / or treat coccidiosis of poultry, and drugs that increase the value of the titer antibodies in vaccinated poultry.

Preparations using assistive materials commonly used in veterinary practice may be in the form of tablets, pills, capsules, gels or pastes. These auxiliary materials include gelatin, natural sugars from a range of sugars such as lactose, maltose and dextrose, lecithin, pectin, cyclodextrin, dextran, polyvinylpyrrolidone, polyvinyl acetate, acacia gum, xanthan gum, tragacanth, agar-agar, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid, alginic acid. sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or similar cellulose derivatives, emulsifiers, oils, fats, in particular glycerol esters and polyglycerol esters derived from saturated fatty acids.

The number of components in the preparation may vary, and it depends on various factors, such as the individual characteristics of the animals to be treated. The doses administered may depend, among other factors, on the size of the animal to be treated and the type of disease to be prevented or treated. The daily dose can be administered in a single dose or divided into a larger number of partial doses per day.

The invention is further illustrated by the following examples, which, however, are not intended to limit the invention.

Examples

UET-HBM, used in the following examples, was obtained in accordance with the following technology, which essentially corresponds to Example 2 from PP # 99/08694.

300 kg of wheat germ milled into flour (in accordance with the Hungarian standard) and 100 kg of yeast (Bassagotusek segeuil) were placed in a 5 m ³ fermenter and drinking water was added until the total volume reached 4000 l. The fermentation period was 18 hours, during which constant aeration was used (0.5 l of air / l of fermented liquid / min) and slow mixing (30 rpm). To inhibit foaming, 1 l / m ^{3 of} sunflower oil was added to the mixture. After cessation of aeration and mixing of the fermentable mass, the fermented liquid was separated first in a screw decanter, then in a separator and, finally, in a sterilizing separator.

Obtaining fraction 1.

The fermented liquid was quickly filtered and its sterility was checked under a microscope. The filtered, fermented liquid contained almost no cells, which meant that a maximum of 1 yeast cell was detected on 10 fields (of the microscope). The resulting fermented liquid, which contained about 1.5% by weight of dry material, was evaporated in a vacuum condenser at a temperature of 40-50 ° C and, after stopping the evacuation, the contents were boiled at atmospheric pressure for about 15 minutes. After that, the content of dry material in the solution was determined and so much maltodextrin (first dissolved in hot and then cold water) was added so that the dry matter content in the solution was about 30% by weight. After that, the solution was subjected to spray drying in a rotating spray dryer with a mixing nozzle, and the temperature of the outgoing air was about 90 ° C. The resulting final product in powder form contained 60% by weight of the fermented plant material according to the invention and 40% by weight of maltodextrin. The content of dimethoxyparabenzoquinone, which was determined by HPLC, was 0.15 mg / g ± 20% dry material.

Obtaining fraction 2.

Biomass from 25-27 wt.% Dry material separated on a screw decanter was dried at a ratio of 1: 1 on a finely divided carrier of corn flakes in the equipment for drying the fluidization and its particle size was adjusted to a range from 0.2 to 0.8 mm by granulation.

Getting the final product.

Fraction 1 and fraction 2 were combined in a homogenizer of the Bbuscht device and thoroughly homogenized. The content of 2,6-dimethoxyparabenzoquinone in the preparation thus obtained was 0.11 mg / g ± 20% dry material.

Example 1. The study of the action against Garbage dakshicit.

Investigations were carried out on Agyog Asgekk chickens of the meat type, not infected with M. cupouiae. From

- 2 009170 Presence of animals Kupou1ae was checked by systematic serological screening in the agglutination test with the help of antigens M. daShkerysit and M. kupou1ae (1 July 1 st 1 jupe 1 VW; Voteg, TN No 1 1 1 ibe). In addition, animals were tested in an enzyme-linked immunosorbent assay (ЕЫ8А) on the basis of monoclonal antibodies and using the MUSA test kit (O | adocx1 | Kit Ky, Vibarek !, Nipdagu) and the MU8A kit (8uaoua, Irrka1, 8 tebepep) | C / | Gga. Soo. e! a1 .: au1ap Όίκ. 37, 680-688 (1993)].

Serological tests gave negative results. In addition, the same brood, from which experimental animals originated, tried to isolate mycoplasmas from the nasal cavity, trachea and air pocket of 20 one-day-old chicks using the medium B [Eto, N., Appy 8yrkoyk, L .: As! A Ue !. 8sapb. 14, 436-449 (1973)] and Rteu’s media [Regue, MS e! A1 .: At. 1. Ye1. Century. 29, 21642171 (1068)]. This study also gave a negative result.

The 120 animals included in the experiment were divided into 4 equal groups with the same number (30-30 animals) so that the average weight of 4 groups did not deviate from each other when used! Student criterion.

For the infection of experimental animals, M. dasherkitsit No. 1226, previously attenuated in medium B for 24 hours, was used. The content of microorganisms was 9.5x10 ⁸ pGi / ml (pGi = colony forming units).

Experimental groups were treated and infected as follows.

Group 1 was placed in a 200-liter box that could be sealed; 10 ml of sterile medium B were sprayed in the box, and then the animals were kept in the box for 20 minutes. Then this group was placed in a separate room, and the animals did not receive any treatment; this group was considered as negative control.

Group 2 was placed in an identical box in which 10 ml of M. daShkerysit broth culture was sprayed, then the animals were kept in this box for 20 minutes. This group was then placed in a second separate room, and the animals received no treatment; this group was considered as a control for monitoring infection.

Group 3 was infected in the same way as group 2, then after being placed in the third room, the animals were asked feed for breeding chickens containing UET-HBM at a concentration of 3 g / kg during the experiment.

Group 4 was infected in the same way as group 2, then after being placed in the fourth room, the animals were asked to feed containing 200 mg / kg of tiamutine (Vyuseyne Strn, Ki1b, AikDga) during the experiment.

To determine the effectiveness of treatment, the following indicators were investigated: clinical symptoms, weight change, unit feed costs; in addition to them, pathological, histological and serological studies were performed, as well as re-secretion of mycoplasma. In the course of their evaluation, the following results were obtained.

Results.

1. Clinical study.

Clinical symptoms and possible deaths were investigated every day. The treated animals did not have any clinical symptoms (groups 3 and 4), whereas the infected, not receiving treatment group had respiratory symptoms on the 6th day, moreover, one death occurred on the 7th and 9th day .

2. Gains.

The gains were statistically significantly lower in the infected, non-treated group than in the control, non-treated group, and in two groups treated with UET-HBM. At the same time, in the two treated groups, the increase was the same degree as in the negative control group.

3. Unit feed costs.

The specific feed costs increased in the infected, non-treated group, reaching 0.45 kg / kg, although they remained at the same level in both the treated groups as in the negative control group.

4. Pathomorphological study.

At the end of the experiment, all birds conducted a pathological study of the air pocket and peritoneum to detect the inflammation characteristic of M. daShkraytsit infection.

In group 1, all animals had negative results, whereas in group 2, all animals showed inflammation of the air pocket and peritoneum of varying severity. In group 3 and group 4 treated with UET-HBM, subtle pathological changes developed and their severity was significantly less than in animals of group 2. The results obtained for the groups treated with UET-HBM and thiamutin, respectively, did not differ from each other.

5. Histological examination.

In group 2, due to infection, the number of cases of lymphohistiocytic bronchitis and lobar interstitial pneumonia increased significantly compared with the uninfected group 1. At the same time, the indicators in group 3 and group 4, treated respectively with UET-HBM and tiamutine, remained at the same level as in group 1, with the exception of lobar interstitial pneumonia, the incidence of which was significantly higher in group 3 than in the control group. Group 3 and

- 3 009170 group 4 did not differ from each other statistically with respect to the changes studied.

6. Serological examination.

The blood plasma of all the chickens was examined on a glass slide in the agglutination test for the presence of M. daSchreishitis. The degree of reaction was determined by scoring and the number of animals that responded and the sum of scoring was compared by the CHI system. Group 1 remained negative until the end of the experiment. In the treated group 3 and group 4, a significantly smaller number of animals showed a serological reaction (respectively, 6 and 8 versus 25), and the scores were significantly lower (respectively, 6 and 11) than in the untreated group 2 (75 points).

7. Re-excretion of Garbage.

Repeated isolation of the infectious strain of Garbage was performed as follows. After infection, after 1 h, 5 animals were killed. Tracheal pieces of 1 cm in length were placed in 2 ml of liquid medium B and after 3 minutes of shaking, the microorganisms were counted in the medium. At the end of the experiment, an attempt was made to re-isolate the strain used to infect birds from the respiratory organs (trachea, lungs, air pocket) and other organs (brain, liver, spleen, kidneys, heart) of all chickens so that samples of each of these organs transferred to solid medium B using a tampon. Oblique agar media were cultured for 10 days and then evaluated. A portion of the isolates was identified by an epifluorescent method using a specific immune serum.

Immediately after infection, Garbage was not isolated from the trachea of animals of group 1. At the same time, it was possible to isolate 1x10 ² -2.7x10 ³ pGi / ml M. daShkursit from the trachea of infected animals of group 2.

At the end of the experiment, it was impossible to recultivate the strains used for the infection in group 1, whereas in group 2 they could be reclaimed in 64 cases, primarily from the trachea, lungs and air pockets. On the other hand, re-isolation was significantly less successful in groups 3 and 4 (respectively, in 10 and 3 cases) and only from some lungs and trachea, but not from other internal organs. There was no significant difference between the groups treated with UET-HBM and Tiamutin respectively.

Example 2. Investigation of the effect against E. lepe11a.

day-old chicks, divided into 4 groups (12 animals), were included in the experiment. Chickens were kept in groups in cages; during the experiment, the room temperature was 28 ° C. During the fattening, the animals consumed the usual initial feed and drinking water without restriction until the age of 14 days (group 1 and group 2). 0.3 g OET-HBM per 1 kg of feed was added to the feed of group 3 and group 4.

Animals of group 2 and group 4 were orally infected with a suspension containing 2x10 ³ sporulated oocysts of Eppep 1ep11a.

Starting from the 7th day of infection, feces were examined for the presence of oocysts. The daily amount of faeces of animals belonging to the same group was weighed and homogenized separately. The same amount was weighed from the faeces of each animal and homogenized with a 2.5% K ₂ Cr ₂ O ₇ solution. The daily isolation of oocysts in this group was determined in a McMa ^ em cell with three repetitions. The results are presented in the table below.

Determination of daily excretion of oocysts in the chamber MsMayet

Control group Processed group Group 1. Uninfected Group 2. Infected, mean + standard deviation Group 3. Uninfected Group 4. Infected, mean ± standard deviation 1. day 0 21,500 ± 0,02 1, day 0 23800 ± 0.03 2. day 0 120,500 ± 0,31 2. day 0 27,250 ± 0,07 p <0.0001 3. day 0 84500 + 0.11 3. day 0 20300 ± 0.08 p <0.0001 4. day 0 75800 + 0.22 4. day 0 15900 ± 0.14 p <0.0001 5. day 0 5700 ± 0.13 5. day 0 1800 ± 0.02 p <0.0001 6. day 0 950 + 0.03 6. day 0 200 ± 0.01 p <0,001 7. day 0 350 ± 0.02 7. day 0 15 ± 0.01 p <0,001

- 4 009170

The table above shows that the development of oocysts in an infected group 4 receiving νΕΤ-ΗΒΜ was significantly less (p <0.0001 and p <0.001) than in animals of an infected group 2 receiving traditional food. In this group, the increase in oocysts was significantly higher than that of the treated group on all days, and this higher level remained until the end of the experiment.

Weight measurements on the 0th, 7th and 14th days confirmed that the weight of infected animals fed traditional food gradually decreased until the last day of the experiment, whereas in groups of infected and uninfected chickens consuming νΕΤ-ΗΒΜ, significant (p <0.001) weight gain.

Example 3. Measuring the level of antibodies in vaccinated chickens.

7 one-day-old chickens of type K.O55-308 (source of supply: Baloia, Niidatu) were included in the experiment, and the chickens received a single treatment while the vaccine against Gumboro's disease was in the egg (vaccine ΤΈνΑί. 'From Ryu1ah1a, Vibarei, Niidatu) . In the main feed, half of the chickens were given νΗΒΜ-0,3 in the amount of 0.3 g / kg feed. The animals consumed food and drinking water without restriction. The control and treated chickens took blood on the first day and every week. Their blood was collected separately, the serum was separated by centrifugation, then stored at -18 ° C before processing.

The amount of antibodies was determined by test Е1818А. During the test, the antigen is usually adsorbed to the wall of polystyrene 96-well plates. Specific serum antibodies to be studied are bound to the antigen, however, unbound antibodies are removed by washing, then the system is completed with such a species-specific antiglobulin serum that has been conjugated to horseradish peroxidase or another enzyme. Molecules of the antiglobulin conjugate that did not react were removed by washing. The sandwich of the antigen-antiglobulin antibody conjugate becomes visible in the form of a color reaction after the addition of the enzyme substrate. In this experiment, a kit testing antibodies of infectious bursitis was used for the measurement (the Proglok® 1BIE11AA kit, manufactured by Kpkedaatb & Repu LaBotaFpeck, SibGog. IR; catalog number 54-81-01).

The measurement was carried out in accordance with the methodology described above. For this measurement, 50 μl of antigen-sensitized serum was added to the wells of the plates. Positive and negative control sera were placed in the wells on the front part (-1, +2, -3) and the end part (-94, +95, -96) of Е8А tablets. Serum plates were incubated for 30 min at room temperature, then they were washed with a washing solution (300 μl), the solution remained in the wells for 3 min, then it was drained. This washing step was repeated after 2 minutes. After that, 100 μl of the conjugate from the kit was added to each well of the samples, then they were incubated for 30 min at room temperature and, finally, they were washed twice according to the procedure described above. Then 100 μl of the substrate was injected into the system. And it was incubated at room temperature for 15 minutes. Reactions were stopped with 100 μl of stopping solution. The developed green-blue color was read in a device for ЕЫ8А at a wavelength of 405-410 nm. Antibody titers were calculated from the obtained absorbance data, which were evaluated by weekly decomposition.

According to these measurements, it can be established that the titer values of the sera of animals subjected to bloodletting on day 1 were the same as the normal values of the control group (on average, 13.5). The magnitude of the titers of serum samples taken on the 1st week increased compared with the values of the control group (on average, 17.4). At week 2, this increase still increased compared with the control (on average, 21.2). On the 3rd week, a powerful increase in titer values was observed under the influence of the treatment νΕΤ0ΗΒΜ compared with the control (on average, 30.3). On the 4th week, the titer values of the treated groups tripled compared with the control (on average, 42.1). On week 5, titer values were 4 times higher than control values (on average, 55.6). On the 6th week, the titer values were almost 5 times higher than in the control group, measured on the 6th week (on average, 69.4).

Based on the statistical evaluation, it was proved that the obtained values were significant (p <0.001) and they showed a faster growth compared with the control.

Claims (8)

CLAIM 1. The use of an extract of fermented wheat germ for the manufacture of a preparation for the prevention and / or treatment of mycoplasmic infections of animals. 2. The use according to claim 1, in which the infection is caused by at least one mycoplasma from the group consisting of Rubbish da Skerbsit, Rubbish 8ujou and Rubbie yuorieitoshae. 3. The use according to claim 1 or 2, in which the animal is a farm animal selected from the group consisting of cattle, horses, rabbits, piglets, pigs, broiler chickens, hens, turkeys, geese and ducks. 4. The use of an extract of fermented wheat germ for the manufacture of a preparation for - 5 009170 treatment of bird coccidiosis caused by E1tepa 1sps11a. 5. The use of an extract of fermented wheat germ for the manufacture of a preparation for increasing the titer of antibodies in vaccinated poultry specific for poultry infections such as mycoplasmic infections or coccidiosis. 6. The use according to any one of claims 1 to 5, in which the extract of the fermented wheat germ is obtained from a fermented liquid and biomass, said fermented liquid and biomass being obtained by fermenting a wheat germ. 7. The use according to any one of claims 1 to 6, in which the preparation is a feed in which the extract of the fermented wheat germ is in an amount of from 0.1 to 6 g per kg of feed. 8. The use according to any one of claims 1 to 6, in which the specified extract of fermented wheat germ is used by mixing it with food in an amount of from 0.1 to 6 g per kg of feed.