RU2396068C1 - Remote vaccine and veterinary preparation delivery system - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to systems for remote delivery of vaccines, and medicinal, therapeutic, immobilisation and other veterinary preparations to animals. A system represents a lead bullet of calibre 5.6 mm wherein a cavity used as a container and closed from above with a ballistic head is created. The cavity has volume 0.03-0.09 cm³ and is created without changing the external size of the bullet. On its internal surface, the cavity has a thin layer of a non-toxic protective compound presented with edible varnish or polymericfluorine varnish, or (0.5%-1%) agar.

EFFECT: use of the invention allows for safe-distance delivery of a required immunising dose of the vaccine to animals with ensuring minimum damage of an animal, and fast and complete release of the preparation in its tissue.

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Description

FIELD OF THE INVENTION

The invention relates to devices for the remote delivery of vaccines to animals, as well as pharmaceutical, prophylactic, immobilizing and other veterinary drugs.

The problem of vaccination of wild animals in order to prevent zoonoses and prevent the transmission of these infections to farm animals and humans has always been of great importance and relevance. The increasing intrusion of man into the world of wildlife aggravates the significance of this problem.

For the effective prevention of infectious diseases in the wild, two components are necessary: 1) an effective and safe vaccine for animals, and 2) an effective and safe for humans method of delivering vaccines to wild animals.

Currently, there are several methods for the remote delivery of drugs and prophylactic drugs to both an individual animal and animal populations. These include top dressing, aerosols, darts, implants, biopools, etc. Each of them has its own advantages and disadvantages.

The use of methods such as top dressing and aerosol delivery does not allow you to control the output of the active substance, its exact dosage and delivery to a specific target animal. In addition, the use of these methods, as well as the use of darts and devices for trapping and fixing animals (for the introduction of implants), does not meet the requirement of minimizing the impact on the environment and on the behavior of natural herds of wild animals.

In accordance with this, the use of such methods of remote delivery of medicines and preventive drugs to animals is prohibited in many areas, especially in nature reserves and other nature-protected areas.

State of the art

Given the above, the most appropriate method for the remote delivery of vaccines and other veterinary drugs may be the method using bullets.

The human safety distance required for guaranteed vaccine delivery depends on the type of animal. For most wild animals, it is at least 60-80 meters, and for shy species of animals - deer, elk, roe deer, etc. - even more.

Currently, resorbable bioplugs of 0.25 (6.43 mm diameter) and 0.20 (5.08 mm) caliber are used for the delivery of biological and veterinary drugs to domestic and wild animals [1-4].

Such bio-bullets consist of an external absorbable container containing biological material in the form of a solid, semi-solid or liquid form. The main components of the container, which, when pressed under high pressure, becomes hard, like plastic, are hydroxypropyl cellulose and calcium carbonate.

Upon entry into the animal and in contact with tissue fluids, the container dissolves and completely melts within 24 hours. Depending on the composition of the formulation, the vaccines or drugs placed in the cavity of the bullet have a different dissolution rate. Currently produced bullets capable of delivering 125-300 mg of payload.

Biopools are usually delivered using a gun, which requires compressed air to fire. A significant drawback of such biopools is that the maximum range of their flight is an average of 25 m, which is completely insufficient for the safety of the personnel conducting the vaccination. In addition, a special container is required for storing compressed air, which is connected to the gun by a hose and which must be transported with it, which is extremely inconvenient. The disadvantages should also include possible difficulties when filling the tank with gas.

Known bullet (prototype) for immobilization (immobilization) of animals [5], which is a hollow body, one end of which is open and the other is closed. On the body of the above-mentioned body are guides extending in mutually perpendicular directions along which the above-mentioned body deforms as a result of an impact on the animal.

A solid immobilizing substance and a means for compaction are placed inside the hollow body, after which the open end is closed by a ballistic head.

The prototype bullet is made of lead, has a caliber of 5.6 mm and allows you to deliver a solid preparation at a distance of 60-80 m, without causing significant injury to the animal.

The disadvantages of the bullet prototype are:

1) a small volume of the cavity for embedding biological material, which allows the use of only solid preparations and does not allow the use of preparations in liquid form, since they occupy a larger volume, and it is not possible to place the necessary dose of liquid preparation for the animal in a limited volume of the cavity (for example, dose of vaccine);

2) the inability to use the bullet for the delivery of live vaccines, since the bullet material (lead) is toxic to live bacterial cells or viral material.

Disclosure of invention

The task of creating the proposed technical solution was to create a ballistic device (bullet) that allows, from a safe distance (at least 80 m), to deliver the necessary immunizing dose of the vaccine (at least 10 ¹¹ cells), while maintaining its viability, while ensuring a minimum damage to the animal, as well as the rapid and complete release of the drug in the tissue of the animal.

The problem is solved in that for remote delivery of the vaccine, a device is proposed, which is a lead bullet of 5.6 mm caliber, inside of which without changing the external dimensions of the bullet a cavity of 0.03-0.09 cm ³ volume is created, which serves as a container for live vaccine or veterinary the drug. Below the cavity is closed by a partition, which ensures the preservation of the contents of the bullet and protects it from the effects of powder gases during firing.

After filling, the cavity from above is closed with a head made of non-toxic plastic material (for example, plasticine or paraffin), which does not allow the contents of the cavity to leak out and ensures the straightness of the bullet’s flight.

If the device is used to deliver a live vaccine, in order to maintain its viability, a non-toxic protective composition is applied on the inner surface of the cavity with a thin layer (0.01-0.02 mm), for example, food varnish, fluoropolymer varnish, (0.5% -1%) agar and others

For the manufacture of the claimed device can be used bullet caliber 5.6 mm factory-made (hereinafter: standard bullet caliber 5.6 mm).

Brief Description of the Drawings

The drawing shows the inventive bullet in longitudinal section.

The implementation of the invention

The presented bullet consists of a housing 1, a cavity 2 for drug insertion, which has a partition at the bottom 3. The open end of the cavity 2 is hermetically closed by a convex-shaped head 4, and the inner surface of the cavity is covered with a protective layer 5. The partition 3 has a concave shape (may also have flat and cone-shaped).

A live vaccine or veterinary drug is placed in cavity 2, which can occupy the entire cavity or part of it, then the cavity is hermetically closed by the head 4.

The bullet is placed in the barrel of the cartridge case and is used to charge the gun under the 5.6 mm small caliber cartridge. To fix and fix the bullet in the barrel barrel on the case of a standard 5.6 mm bullet inside which a cavity is created, there is a transverse ring groove 6 (flute) with a smooth or corrugated surface. Fixation prevents the displacement of the bullet, pushing the bullet into the sleeve under the action of recoil.

To carry out ballistic immunization, a single shot is fired at a target animal from a distance of 80-100 m.

When shooting with such bullets, it is necessary to aim at the large muscles of the limbs of the animal (thigh or croup). When the lead body of a bullet having thin walls hits the target in the muscle tissue of the animal, it begins to deform (flatten or tear into small fragments), releasing the contents of the bullet into the surrounding muscle tissue. The usual depth of penetration of a bullet into the muscle tissue of an animal when fired from a distance of 80-100 m is 3-5.5 cm. The bullet itself or its fragments remain in the tissues of the animal and do not disturb it. The bullet wounds are small and heal within a few days.

Creating inside a standard bullet of a caliber of 5.6 mm a cavity of a specified volume without changing its external dimensions allows you to:

- deliver the necessary immunizing dose of the vaccine or drug;

- not to violate the ballistic characteristics of the bullet from a distance of 80-100 meters, to achieve accurate penetration of the bullet into the target area of the animal tissue to a depth of 3-5.5 cm (i.e. to achieve minimal damage to the animal when the vaccine is introduced into the intended area);

- to achieve rapid and complete release of the vaccine or veterinary drug in the tissue of the animal due to flattening or disintegration into small fragments of the refined walls of the bullet.

The inventive bullet allows you to maintain the viability of the vaccine and deliver it in the optimal immunizing dose, which is achieved both by applying a non-toxic protective layer to the inner surface of the cavity, and by optimizing the wall thickness of the modified bullet, which is sufficient to protect against the effect of increased temperature on cell survival the vaccine strain in the pool when fired from a gun from different distances, while the size of the resulting cavity makes it possible to deliver the animal dose of vaccine.

The invention and its practical applicability is illustrated by examples.

Example 1. Preparing a bullet for use

A cavity is made along the longitudinal axis of a standard 5.6 mm bullet using a 3.5 mm drill and an Einhell SB 1020 / 1W 42.507.85 drilling machine with a low number of revolutions per minute (about 120 rpm). To fix the bullet in the machine, clamps are used that prevent the expansion of the bullet walls during the drilling of the hole, i.e. increase the caliber of the bullet. When drilling, the drill is lowered into the bullet to a depth of 8.5 mm, not reaching the base of the bullet, thus forming a cavity of volume 0.082 cm ³ , closed from the bottom by a partition. The latter ensures the preservation and protection of the contents of the cavity from the effects of powder gases during firing.

The inner surface of the cavity formed is covered with food varnish EP-547 (composition (%): ethyl cellosolve - 95, ethyl alcohol - 5). To this end, the bullet cavity is filled to the top with varnish, then the bullet is turned over and left for 1 hour in air to remove the varnish and form a thin film. The coating thickness is ~ 0.01 ± 0.003 mm.

Example 2. Determination of cell viability of B. abortus 82 in modified bullets

This vaccine strain is most often used in Russia and the CIS countries.

The initial museum culture of the vaccine strain B. abortus 82 was grown on peptone-hepatic-glucose-glycerol agar for 48 hours at a temperature of 37 ° C. Cells were washed with gelatin-sucrose medium (sucrose 10%, gelatin 1.5%) and the concentration was adjusted to ~ 1 × 10 ¹² CFU / ml.

0.08 ml of the resulting suspension (containing a dose of 10 ¹¹ CFU cells) is placed in the bullets prepared according to Example 1 without coating with a protective compound (n = 30, where n is the number of prepared samples) and coated with a protective compound (n = 30). On top of the cavity is closed with a paraffin head.

As a control, the same volume of the cell suspension is placed in plastic microtubes (n = 30). Bullets and tubes are stored at 4 ° C.

At certain time intervals, a suspension of cells of the vaccine strain B. abortus 82 is removed from the bullets, diluted in physiological saline, tenfold dilutions are made and plated on erythritol agar plates. Crops are incubated at 37 ° C for 5 days, after which the number of colonies grown is counted.

The experimental results are presented in table 1.

Table 1 Duration of observation (day) The number of viable cells (CFU) Microtubes (control) (n = 30) Bullet without a protective coating (n = 30) Protective Coated Bullets (n = 30) 0 (11.2 ± 2.2) × 10 ¹⁰ (9.5 ± 2.6) × 10 ¹⁰ (9.4 ± 2.6) × 10 ¹⁰ 2 (1,1 ± 1) × 10 ⁹ (0.66 ± 0.04) × 10 ⁹ (1.1 ± 0.1) × 10 ⁹ four (5.6 ± 0.4) × 10 ⁸ (0.26 ± 0.04) × 10 ⁸ (3.7 ± 0.2) × 10 ⁸ 6 (5.0 ± 0.4) × 10 ⁸ (0.15 ± 0.08) × 10 ⁸ (3.0 ± 0.3) × 10 ⁸ 8 (4.8 ± 0.3) × 10 ⁸ (3.2 ± 0.3) × 10 ⁶ (3.2 ± 0.2) × 10 ⁸ 10 (4.1 ± 0.2) × 10 ⁸ (1.6 ± 0.2) × 10 ⁵ (3.3 ± 0.1) × 10 ⁸ 12 (4.2 ± 0.2) × 10 ⁸ (1.3 ± 0.1) × 10 ⁵ (3.0 ± 0.2) × 10 ⁸

The results show that the lead from which the bullet is made has a toxic effect on brucella cells of the vaccine strain B. abortus 82. The survival dynamics of brucella cells in bullets coated with a protective composition practically does not differ from the control.

Example 3. Determination of cell viability of B. abortus 19 in modified bullets

This vaccine strain is most commonly used in the United States, North America, Latin America, and Europe.

The experimental conditions are similar to example 2. The results for a suspension of cells of B. abortus 19 are presented in table 2.

Comparing the survival rates of B. abortus 19 cells, it can be noted that in modified bullets coated with a protective composition, the number of living cells during 48 days of storage at 4-6 ° С practically did not change and is comparable to the control (microtubes). In bullets without a protective coating, the number of living cells is significantly lower.

table 2 Duration of observation (day) The number of viable cells (CFU) Microtubes (control) (n = 30) Bullet without a protective coating (n = 30) Protective Coated Bullets (n = 30) 0 (109.8 ± 3.2) × 10 ⁹ (98 ± 2.2) × 10 ⁹ (110.8 ± 2.5) × 10 ⁹ 5 (98.5 ± 2.2) × 10 ⁹ (0.71 ± 0.02) × 10 ⁹ (104.0 ± 2.2) × 10 ⁹ 8 (105.3 ± 1.6) × 10 ⁹ (2.9 ± 0.2) × 10 ⁷ (108.0 ± 1.1) × 10 ⁹ eleven (103.2 ± 2.1) × 10 ⁹ (5.5 ± 0.8) × 10 ⁵ (106.5 ± 2.6) × 10 ⁹ eighteen (103.0 ± 1.8) × 10 ⁹ (3.2 ± 0.3) × 10 ⁵ (104.3 ± 1.5) × 10 ⁹ 26 (108.4 ± 1.9) × 10 ⁹ (2.0 ± 0.2) × 10 ⁵ (100.4 ± 1.8) × 10 ⁹ 33 (106.6 ± 3.0) × 10 ⁹ (1.6 ± 0.1) × 10 ⁵ (98.20 ± 3.7) × 10 ⁹ 48 (90.5 ± 2.3) × 10 ⁹ (6.8 ± 0.3) × 10 ⁴ (91.5 ± 1.5) × 10 ⁹ 68 (82.05 ± 1.9) × 10 ⁹ (5.2 ± 0.2) × 10 ⁴ (80.5 ± 2.4) × 10 ⁹

Thus, coating the inner surface of the bullet cavity with a protective composition allows preserving the viability of the cells of vaccine strains of various origin inside the modified bullet.

Example 4. Determination of cell viability in bullets when fired from different distances

It is known that in the process of a shot a powder charge explodes and almost simultaneously releases powder gases, the temperature and high pressure of which affect a bullet. The action of the powder gases continues even after the bullet leaves the barrel, until the pressure of the powder gases at the bottom of the bullet is balanced by air resistance. In addition, the heating of a bullet can occur not only during its passage through the barrel of the gun, but also when flying through the air as a result of friction. In this regard, it was important to clarify the possible effect of elevated temperature on the survival of the vaccine strain cells in the pool when firing a gun from a different distance.

In the experiments, use the bullets of example 1 (n = 40), coated inside with a protective composition and containing cells of the vaccine strain B. abortus 82. Shots are carried out from a distance of 0.5 and 100 m into a polypropylene tube filled with sterile physical. solution. The number of living cells in the pool after the shot is determined by the bacteriological method by seeding the appropriate dilutions of the culture of strain B. abortus 82. The content of the unshooted bullet is used as a control (n = 20). The experimental results are presented in table 3.

Table 3 Experiment Distance 0.5 m (n = 20) Distance 100 m (n = 20) CFU / ml concentration (log) CFU / bullet concentration CFU / ml concentration (log) CFU / bullet concentration The control 10.03 ± 0.06 (8.98 ± 1.35) × 10 ⁸ 9.01 ± 0.03 (7.01 ± 0.57) × 10 ⁷ Experience 9.95 ± 0.06 (6.64 ± 0.78) × 10 ⁸ 9.03 ± 0.04 (7.22 ± 0.63) × 10 ⁷

The data obtained showed that a statistically significant difference between the number of living cells of B. abortus 82 in bullets after a shot and in bullets that did not undergo a shot was not found. Therefore, the shot itself, the movement of the bullet along the barrel of the gun and its flight through the air have a minimal effect on the survival of the cells of strain B. abortus 82 inside the inventive bullet.

Example 5. Determining the suitability of a modified bullet for the delivery of dry preparations

The experiment uses the bullets of example 1, but without coating the inner surface of the cavity of the bullet with a protective composition (n = 10).

20 mg of dry preparation (ditilin) are poured into the cavity to immobilize the animal. The cavity is covered with a plasticine head from above. Shots are fired from a distance of 100 m in cattle.

15 minutes after the bullet hit the animal paralyzed. Thus, the possibility of using a bullet to deliver dry preparations to animals at a distance of 100 m was demonstrated.

Example 6. Determining the accuracy of the hit of modified bullets in the target

For the experiment, use the bullets of example 1 with an empty cavity (n = 10) and containing 0.08 ml of a suspension of cells of the vaccine strain B. abortus 82 (n = 10). As a control, a standard 5.6 mm bullet is used. Shots are carried out from a 5.6 mm rifled rifle in the open air from a distance of 100 m into a paper target. The results are presented in table 4.

Table 4 No. Bullet design Deviation of the bullet from the center of the target (cm) one Modified, containing the vaccine (n = 10) 7.7 ± 0.4 2 Modified, vaccine-free (n = 10) 8.0 ± 0.5 3 Unmodified (control) (n = 10) 7.5 ± 0.3

The results show that when firing from a distance of 100 m, the accuracy rating of modified bullets (both containing and not containing vaccine) is close to the accuracy rating for a standard bullet, which indicates the stability of the ballistic characteristics of the modified bullet and their immutability when laying vaccine material in them.

The bullet holes are rounded, which indicates the correct aerodynamic properties of the bullets.

Example 7. Determination of the depth of penetration of a bullet into animal tissue

The bullets of example 1, containing 0.08 ml of a suspension of cells of the vaccine strain B. abortus 82, from a distance of 100 m carry out shots in cattle (n = 7) and bison (n = 1).

After 56 days, all animals were euthanized and had complete necropsy with internal organs taken for bacteriological examination.

With necropsy, no serious damage to the organs and tissues of animals, as well as the presence of inflammatory infiltrates along the bullet channels were found. Bullet channels have smooth edges and are clearly visible in the section.

Bullets were found in the muscle tissue of all animals at a depth of 3-5.5 cm (table 5). Detected bullets are deformed (flattened or broken into small fragments).

Table 5 No. Kind of animal Bullet penetration depth (cm) The condition of the bullets in the muscle tissue of the animal one. Cattle heifers (weight 150 kg) 4.0 ± 1.5 Flattened or broken up into small fragments 2. Bison (weight 600 kg) 3.0 Flattened

Example 8. Analysis of the immune response of cattle to the delivery of live brucellosis vaccines

Heifers of cattle 13-15 months of age (weighing 160-180 kg) in an amount of 7 goals before immunization are divided by random selection into two groups. The experimental group (4 animals) is vaccinated with a ballistic method using a modified bullet, the control group (3 animals) is injected. As vaccines, B. abortus 82 and B. abortus 19 strains are used (control). 5 weeks after immunization, all animals are re-immunized with similar Brucella cultures, which are administered in the same doses and in the same ways, but in the opposite limbs. 9 weeks after vaccination, the animals were euthanized and completely necropsy with blood and internal organs taken for examination.

Humoral immunity in vaccinated animals was evaluated by determining the level of anti-brucellosis antibodies in the blood serum of vaccinated animals in serological reactions and by ELISA.

The presence of vaccine strain cells in the internal organs of animals was detected by PCR.

1-2 weeks after immunization, high titers of specific R-antibodies, low titers of S-antibodies (for vaccine from strain B. abortus 82) and high titers of S-antibodies (for vaccine from strain B. abortus 19) were detected in blood serum of heifers. . The dynamics of antibody formation in both methods of vaccination was of the same type.

The induction of an immune response in animals was also evidenced by the high proliferative response of blood lymphocytes and an increase in the phagocytic activity of blood cells. The nature of changes in the indicators of cellular immunity and phagocytosis in animals vaccinated with both strains of B. abortus using ballistic and injection methods was practically the same.

Example 9. Analysis of the immune response of bison to the delivery of live brucellosis vaccine

A bison weighing 600 kg is immunized with a vaccine from strain B. abortus 82 directly in the herd by the ballistic method using the modified bullet of Example 2 twice from a distance of ~ 90 m to the left and right croup. After vaccination, the bison is placed in a separate paddock. The animal was carried out daily veterinary supervision.

36 days after vaccination with necropsy, fragments of two bullets were found in bison muscle tissue at a depth of 3 cm. Using PCR, the presence of cells of the vaccine strain B. abortus 82 was detected in the lymph nodes and spleen. A high level of cellular and humoral immunity was detected in bison blood serum (antibody titer in ELISA = 1: 1280000), and S-antibodies were detected using agglutination reaction (in titer 1:50), i.e. induction of a tense and persistent immunological response in the bison organism is shown.

Thus, the described results confirm the possibility of using the claimed bullet for delivery to cattle and wild animals (bison) from a distance of 80-100 m of the vaccine in the required dose and with preserved cell viability. Atraumaticity of the claimed bullet for animals and a small depth of its penetration into animal tissue is shown.

Due to the aerodynamic, ballistic and structural characteristics characteristic of the developed bullet, it with high accuracy falls into the target place of the vaccinated animal, penetrates the muscle tissue of the animal to a depth of no more than 3-5.5 cm and, due to flattening or disintegration into small fragments, the vaccine quickly and completely leaves and effectively colonizes the surrounding muscle tissue of the animal, followed by cell multiplication.

Ballistic vaccination of animals from a distance of 80-100 m does not cause the formation of extensive injuries, has a minimal effect on anxiety and behavior of animals, which allows vaccination of almost all animals in one herd.

As a result of ballistic vaccination using the claimed bullet in the animal’s body, a tense and persistent immunological response is formed that protects the animal from infection.

The remains of a bullet in the muscle tissue of an animal do not cause serious clinical manifestations and may remain in the body of the animal for a long time.

Bibliography

1. Keith A, Kreeger TJ, Roffe TJ. Overview of delivery systems for the administration of vaccines to elk and bison of the Greater Yellowstone Area. In Kreeger TJ, editor. Brucellosis in Elk and Bison in the Greater Yellowstone Area. Cheyenne: Wyoming Game and Fish Department; 2002, p. 66-79.

2. Jessup D., DeForge J.R., Sandberg S. Biobullet vaccination of captive and free-ranging bighorn sheep. Proceedings of the 2 International Game Ranching Symposium, 1992, p. 429-34.

3. Roffer TJ, Jones LC, Coffin K, Sweeney SJ, and Hansen R. Parenteral delivery of vaccines to free-ranging bison in Yellowstone National Park. Proceedings U.S. Animal Health Association 2001; 105: 135-41.

4. Drake, Jr., James F .; Paul, Jr., Fred R. Ballistic animal implant. US Patent No. 3948263.

5. Komarov V.A. Shell for the immobilization of animals. US Patent No. 3616758.

Claims (3)

1. A device for the remote delivery of vaccines and veterinary preparations to an animal, which is a lead bullet of 5.6 mm caliber, in which a cavity is created that serves as a container and closes on top with a ballistic head, characterized in that the cavity has a volume of 0.03-0.09 cm ³ and is created without changing the external dimensions of the bullet. 2. The device according to claim 1, characterized in that a non-toxic protective composition is applied in a thin layer on the inner surface of the cavity. 3. The device according to claim 2, characterized in that as a protective composition use food varnish, or fluoropolymer varnish, or (0.5% -1%) agar.