RU2463073C1 - Swine transmissible gastroenteritis vaccine and method for preparing it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns preparing and using the vaccine for specific prevention of swine transmissible gastroenteritis. The described vaccine contains a virus material of the VN-96 strain of swine transmissible gastroenteritis virus in titre 7.0-7.2 lg TCD₅₀/ml, while the excipient is physiologic saline. A method for using the presented vaccine consists in immunisation of pregnant sows by the intramuscular single introduction in dose 4.5-5 ml per one head 40-45 days before farrow; it is added with immunisation of 23-27-day suction piglets by the intramuscular single introduction in dose 3.0-3.5 ml.

EFFECT: presented inventions enable the combined activation of cell immunity in pregnant sows to provide colostral protection of newborn piglets and humoral immunity providing protection of pregnant sows against infection.

2 cl, 6 tbl, 9 ex

Description

The invention relates to biotechnology and relates to the manufacture and use of a vaccine for the specific prevention of transmissible gastroenteritis in pigs.

Transmissible gastroenteritis of pigs (TGS) is an infectious highly contagious disease, mainly of piglets up to 3 months of age, characterized by catarrhal hemorrhagic gastroenteritis, vomiting, dehydration of the body and accompanied by atrophy of villi of the upper small intestine and high mortality (often up to 100%) among piglets up to 2 weeks of age.

A known vaccine against transmissible gastroenteritis and a method for its use (see RF patent No. 2306951, IPC A61K 39/225, publ. 09/27/2007) containing the inactivated virulent strain “TB” of the TGS virus in the titer of 7.0-8.5 lg TCD _{50 / ml} and adjuvant. Vaccines are administered to the main and repair pigs intramuscularly twice for 75-80 and 90-100 days of gestation.

However, inactivated vaccines, along with a positive effect, in some cases can cause allergic reactions due to the addition of adjuvants to them. Their use is laborious, involves multiple immunization of animals. In addition, they do not provide effective protection due to the lack of antigenic identity to the virulent pathogen.

Known live vaccine against transmissible gastroenteritis of pigs based on the strain "Gorsky-95" and the method of its use (see RF patent No. 2140982, IPC C12N 7/00, publ. 10.11.1999). This strain is designated as Gorsky-95 strain and deposited in the collection of microorganism strains of the State Scientific Institution All-Russian Research Institute of Veterinary Virology and Microbiology (GNU VNIIVViM) inv. No. 1941.

The vaccine is administered twice to pregnant sows orally, respectively 6-8 and 2-3 weeks before farrowing.

However, the effectiveness of the vaccine is low due to the low activity of the strain, which is able to accumulate only up to 10 ⁵ TCD 50 / cm ³ , has not very high stability (up to 50 passages), is not suitable for the prevention of transmissible gastroenteritis in piglets of all age groups, including weaners.

Closest to the claimed is a live vaccine against transmissible gastroenteritis and the method of its use (see RF patent No. 2347585, IPC A61K 39/225, publ. 02.27.2008), containing viral material from the Transmissible gastroenteritis strain VGNKI No. 5, sucrose, peptone, enzyme peptone, casein, thiourea, aerosil AM-1-300 and resin KB-4P-2. The biological activity of the drug is not less than 6.0 lg TCD _{50 / ml} . The vaccine is administered to pregnant sows by feeding with dry food before morning feeding at a dose of 10 g per head for 3 consecutive days 10-14 days before farrowing. 10-14 days after farrowing, the vaccine is given to piglets also before morning feeding at a dose of 5 g per head.

However, this vaccine is intended for oral use. In addition, it requires considerable labor in the manufacture and use, as it involves the use of dry food and three times the introduction of animals. The mechanism of action is aimed at activating only cellular immunity, which provides only colostral protection for newborn piglets and does not prevent the development of an infectious process in vaccinated sows.

The invention is aimed at solving the problem of creating an avirulent effective live vaccine against transmissible gastroenteritis, an easy-to-use method of its use, aimed at simultaneously activating cellular immunity in sows, providing colostral protection of newborn piglets, and activating humoral immunity in sows, which protect them from infection.

To solve the problem, a vaccine against porcine transmissible gastroenteritis containing viral material and an excipient according to the invention contains viral material as a viral material from strain VN-96 of porcine transmissible gastroenteritis virus in a titer of 7.0-7.2 lg TCD _{50 / ml} , and as a filler, physiological saline in the following ratio of components (wt.%):

Viral material from the strain "VN-96" - 0.09-0.1%

Saline solution - the rest.

A method of using a live vaccine against transmissible gastroenteritis of pigs is intramuscular single immunization of pregnant sows 40-45 days before farrowing at a dose of 4.5-5 ml per head, while a vaccine containing viral material from the strain “VN-96” is used as a vaccine "Pig transmissible gastroenteritis virus in titer of 7.0-7.2 lg TCD _{50 / ml} and physiological saline in the following ratio of components (wt.%): Viral material from strain" VN-96 "- 0.09-0.1% , saline - the rest.

To provide additional protection for piglets in the post-weaning period, immunization of suckling piglets at the age of 23-27 days is carried out once intramuscularly at a dose of 3.0-3.5 ml.

The sources of patent and scientific and technical information known to the authors do not describe a live vaccine against transmissible gastroenteritis of pigs and an easy-to-use method of its application, which provide effective prevention of the disease due to the simultaneous activation of cellular and humoral immunity in sows, which contributes to colostral protection of newborn piglets and sows themselves from infection.

A single injection of a vaccine based on a mixture of viral material from the strain "VN-96" of the virus of transmissible gastroenteritis of pigs in the titer of 7.0-7.2 lg TCD _{50 / ml} with saline in the ratio (1-0.9) :( 999-999 ,one).

Currently known vaccines against transmissible gastroenteritis in pigs are aimed mainly at enhancing cellular immunity. None of the known vaccines allows the simultaneous activation of cellular (to a greater extent) and humoral (to a lesser extent) immunities.

The foregoing allows us to conclude that there is an inventive step in this invention.

The strain intended for the manufacture of the vaccine was deposited at the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and Feed (VGNKI) (certificate of deposit No. 1103/19 of May 27, 2009).

Strain VN-96 was isolated in 1988 from the Rims vaccine (GDR) by passaging 30 times in a transplanted culture of pig embryonic kidney cells and using the dilution method, followed by 5-fold cloning of micro-plaques of the initial porcine transmissible gastroenteritis virus, 96 were performed passages.

The storage location of the production strain “VN-96” -DEP of the transmissible gastroenteritis virus of pigs is a collection of microorganisms of the State Scientific Institution of the Saratov Scientific Research Veterinary Institute of the Russian Academy of Agricultural Sciences (GNU Saratov Scientific Research Institute of Agricultural Sciences).

The strain is characterized by the following properties.

Morphological features

An electron microscopic examination of the virus-containing material revealed that the virus of the “VN-96” strain is enveloped, pleomorphic, with a diameter of 80-150 nm, has one layer of club-shaped processes (peplomeres) in the form of a crown 12-20 nm long, i.e. the strain is morphologically identical to representatives of the genus Coronavirus of the family Coronaviridae.

Cultural signs

The strain "VN-96" of the virus of transmissible gastroenteritis of pigs propagates in the transplanted line of kidney cells of the pig embryo - SPEV (GNU VNIIVViM). The permissible number of passages is 10.

The infectious activity of the strain in the SPEV culture is 5.25 ± 0.25 lg TCC _{50 / cm} ³ . After the 7th passage, the titer of the TGES virus of the strain “VN-96” is 7.0-7.2 lg TCC _{50 / cm} ³ , further passage does not increase the titer of the virus.

Antigenic properties

The strain "VN-96" of the virus of transmissible gastroenteritis of pigs causes the formation of virus-neutralizing and precipitating antibodies to the THES virus in the body of pigs and rabbits in titers 4-6 log ₂ .

Virulent properties

Not virulent.

Pathogenicity for naturally susceptible and laboratory animals

The strain "VN-96" is apatogenous for piglets of all age groups with oral and subcutaneous methods of administration in doses of 5.0-6.5 lg TCC _{50 / cm} ³ .

Contamination with bacteria, fungi, mycoplasmas and extraneous viruses

Not contaminated.

Molecular genetic feature of the strain

Strain "BH-96" is an RNA-containing virus. The RT-PCR method amplified a portion of the THES virus genome, including 800 n.a. at the 5'-end of the S gene.

The virus contains 3 main structural proteins: a large surface glycoprotein (S) with a molecular weight of 170-200 kDa, a nucleocapsid protein (N) with a molecular weight of 50-62 kDa, and a small enveloped glycoprotein (M) with a molecular weight of 25-35 kDa.

PCR analysis showed that the only characteristic fraction of a specific PCR product (cDNA of 886 bp) was detected in viral nucleic acid tracks, indicating a rather large amount of the THES virus genome.

Storage conditions

Strain "VN-96" is stored in lyophilized form in hermetically sealed ampoules at a temperature of minus 20-40 ° C for 5 years. During the storage period, a decrease in activity of not more than 1.5 lg of the SCC _{50 / cm} ^{3 is} allowed.

The stability of the basic properties of the strain

Strain "VN-96" is stable for 10 serial passages in the cell culture SPEV.

The invention is illustrated by the following examples.

Example 1

To obtain viral material from the strain "VN-96" use culture SPEV grown for 3 days at a temperature of 37 ° C with a formed monolayer of cells with a seeding dose of 150 thousand / ml. The growth medium is replaced with a support medium of the same composition, but without serum, and the virus is added in a ratio of 1:10. The infected culture is maintained for 3 days at a temperature of 37 ° C, then frozen and thawed three times. The resulting suspension is centrifuged at 3000 rpm for 30 minutes. The supernatant is collected and titrated. The viral material is selected whose titer is 7.0-7.2 lg TCD _{50 / ml} .

Viral material is stored at a temperature of minus 18-20 ° C.

Example 2

The vaccine is prepared as follows.

Take 1 ml of viral material with a titer of 7.0-7.2 lg TCD _{50 / ml} .

Add it to 999 ml of physiological saline. The mixture is mixed and used for the prevention of TSH.

The finished vaccine is a clear liquid, without color and odor.

Example 3

Justification of the titer of the virus of transmissible gastroenteritis and the percentage of components of the vaccine against TSV.

The experiments were carried out in an unsuccessful TSE farm. The vaccine was administered to sows 40-45 days before farrowing in different titers of the virus. The first group (174 sows) - in the titer 6.0-6.2 lg TCD _{50 / ml} , the second (195 sows) - in the titer 7.0-7.2 lg TCD _{50 / ml} , the third (270 sows) titer 8.0-8.2 lg TCD _{50 / ml} . Controls were 311 sows.

Vaccination efficacy was assessed by the number of business pigs received per farrowing sow.

The results are presented in table 1.

From table 1 it follows that the highest yield of business piglets per farrowing sow (6.5) was obtained in the group that was subjected to a single immunization 40-45 days prior to farrowing with a vaccine with a virus titer of 7.0-7.2 lg TCD _{50 / ml} and with a ratio of components of 1: 999. Reducing or increasing the titer of the virus and the ratio of the components of the vaccine reduced its effectiveness.

Table 1 The results of the effectiveness of vaccination of sows with different titers of the TSE virus and the ratio of the components of the vaccine Characterization of sow groups The number of sows in the group Virus titer lg TCD _{50 / ml} The ratio of the components of the vaccine (wt. Parts) Received business piglets Total per pig. 1 experienced 174 6.0-6.2 1:99 820 4.7 2 experienced 195 7.0-7.2 1: 999 1274 6.5 3 experienced 270 8.0-8.2 1: 9999 1526 5.7 Control 311 - - 1451 4.7

Example 4

The rationale for the frequency of administration of the vaccine was carried out on three groups of sows (80 animals each), one of which was the control, and the other two were experimental. The first experimental group of sows was immunized once, the second two times with an interval of 14 days.

A vaccine with a virus titer of 7.0-7.2 lg TCD _{50 / ml} was administered to sows intramuscularly 40-45 days before farrowing at a dose of 5 ml per head. Vaccination efficacy was evaluated by the number of piglets received. Clinical observations were made of the pigs, incidence and mortality were recorded until weaned age. The result is presented in table 2.

table 2 The effectiveness of vaccination of sows depending on the frequency of administration of the vaccine. Characterization of sow groups The number of pigs in the group Got sick Fell goals % goals % 1 Experienced (vaccinated twice) 528 528 100.0 327 61.7 2 Experienced (vaccinated once) 542 125 23.1 72 13.3 Control 532 532 100.0 394 74.1

From table 2 it follows that all piglets from unvaccinated sows fell ill with TSE, of which 394 heads fell (74.1%).

In the experimental group of sows vaccinated twice, all pigs also fell ill, 327 of them fell (61.9%). In the experimental group of sows vaccinated once, out of 542 piglets only 125 (23.1%) fell ill, 73 (13.3%) fell.

Thus, a single injection of the vaccine provides the greatest protection of suckling pigs from the disease.

Example 5

The dose of vaccine was justified on two groups of sows (50 animals each), while one group of sows was given a vaccine at a dose of 4.5-5 ml per head, and the second group - 6-8 ml per head.

Vaccination efficacy was evaluated by the number of piglets received. Clinical observations were made of the pigs, incidence and mortality were recorded until weaned age. The result is presented in table 3.

Table 3 The effectiveness of vaccination of sows depending on the dose of vaccine Vaccine dose The number of pigs in the group Got sick Fell goals % goals % 4.5-5 ml 357 105 29.4 57 16,0 6-8 ml 363 295 81.3 151 41.6

From table 3 it follows that from the first group of sows that were vaccinated with a dose of 4.5-5 ml, 357 piglets were obtained, of which 105 (29.4%) fell ill and 57 (16.0%) fell. From the second group of sows, to which the vaccine was administered at a dose of 6-8 ml, 363 pigs were obtained, of which 295 (81.3%) fell ill and 151 (41.6%) fell.

Thus, an increase in the vaccination dose (more than 5 ml) reduces the protection of suckling pigs from transmissible gastroenteritis in dysfunctional farms. Similar results were obtained at a dose of less than 4.5 ml.

Example 6

Justification of the timing of vaccination of sows

For this, 5 groups of sows were formed, of which 4 were experimental and one was a control. All sows of the first four groups were immunized with a vaccine with a virus titer of 7.0-7.2 lg TCD _{50 / ml} . The vaccine was administered intramuscularly at a dose of 5 ml per head. The first experimental group (48 sows) was immunized 9-10 days before farrowing. The second (46 goals) - 22-25 days before farrowing, the third (41 heads) - 30-32 days before farrowing, the fourth (43 heads) - 40-45 days before farrowing.

Vaccination efficacy was evaluated by the number of piglets received. Clinical observations were made of the pigs, incidence and mortality were recorded until weaned age. The result is presented in table 4.

Table 4 The effectiveness of vaccination of sows depending on the timing of the vaccine before farrowing Characterization of sow groups Sows The number of pigs in the group Got sick Fell goals % goals % 1 Experienced (vacc. 9-10 days before farrowing) 48 432 303 70.1 230 53,2 2 Experienced (vacc. 22-25 days before farrowing) 46 414 81 19.6 27 6.5 3 Experienced (vacc. 30-32 days before farrowing) 41 369 twenty 5,4 8 2.2 5 Experienced (vacc. 40-45 days before farrowing) 43 387 10 2.6 2 0.5 Control 25 225 135 60.0 103 45.8

From table 4 it follows that the smallest number of sick (2.6%) and dead (0.5%) piglets was observed when vaccinating sows 40-45 days before farrowing.

Example 7

Justification of the timing and dose of vaccination of piglets

It has been experimentally proved that in order to provide additional protection for piglets in the post-weaning period, they are immunized at the age of 23-27 days. These dates are justified, because in the first 3 weeks of life in piglets the natural - colostral defense prevails, transmitted to them through sows milk. By 21-30 days of age, a significant decrease in colostral protection occurs, and at the age of 35-40 days, piglets become unprotected from infection.

The vaccination dose of 3-3.5 ml piglets was also experimentally selected taking into account the weight of the animals.

Example 8

The study of the antigenic activity of the vaccine was carried out on first-aid and sows, farrowing two or more times. The vaccine was administered intramuscularly once 40 days before farrowing at a dose of 5 ml per head. The antibody titer was determined: in serum 1-2 days before farrowing, colostrum in the first 12 hours after birth, milk - 5-6 days after the start of lactation.

Table 5 Antibody titer in blood serum, colostrum and milk of vaccinated and unvaccinated sows Group number Characterization of sows Number of researches samples Antibody titer (Ig ₂ ) a blood serum colostrum milk First sprinklers one - vaccinated 12 4.4 ± 0.40 7.5 ± 0.56 5.8 ± 0.74 2 - control 17 0.9 ± 0.29 4.3 ± 0.47 1.3 ± 0.24 Farrowed two or more times 3 - vaccinated 12 4.6 ± 0.47 7.6 ± 0.48 6.6 ± 0.40 four - control 23 2.8 ± 0.33 4.5 ± 0.29 2.4 ± 0.15

As can be seen from table 5, the average titer of antibodies in vaccinated sows in blood serum, colostrum, milk is significantly higher than similar parameters of unvaccinated animals.

Namely, in vaccinated animals, an increase in humoral and cellular immunities occurs. An increase in humoral immunity is evidenced by a change in titer in blood serum from 0.9 ± 0.29 to 4.4 ± 0.40 in primiparous and from 2.8 ± 0.33 to 4.6 ± 0.47 in sows, farrowing two and more times. This protects the vaccinated sows against TSV infection.

An increase in cellular immunity is evidenced by a change in titer in colostrum and milk also in all sows. For example, in primoriformes, the titer rises from 4.3 ± 0.47 to 7.5 ± 0.56. This protects newborn piglets from TSV infection.

Example 9

The study of the immunogenic activity of the vaccine was carried out on sows who were injected with the vaccine intramuscularly once 45 days before farrowing at a dose of 5 ml per head.

The titers of specific immunoglobulins of various classes were determined in the colostrum of all sows on the first day after farrowing. Immunoglobulin titers were determined by setting up an indirect immunofluorescence reaction using specific erythrocyte dagnosticum against transmissible porcine gastroenteritis and specific antibodies to specific classes of immunoglobulins. The results are presented in table 6.

Table 6 Titles of specific immunoglobulins of various classes in colostrum of sows to TSE virus Characterization of sows The titer of immunoglobulins (Ig ₂ ) in the immunofluorescence reaction IgA SIgA IgG IgM Experienced 1.44 ± 0.60 5.33 ± 0.76 6.11 ± 0.65 3.78 ± 0.30 Control 1.56 ± 0.58 2.60 ± 0.76 7.89 ± 0.48 0.78 ± 0.40

From table 6 it follows that after vaccination of sows on the first day after farrowing, activation of secretory immunoglobulins SIgA (from 2.60 ± 0.76 to 5.33 ± 0.76) and immunoglobulins IgM (from 0.78 ± 0.40 to 3.78 ± 0.30). A significant increase in these indicators indicates an increase in colostral protection of newborn piglets.

Thus, the claimed live vaccine against transmissible gastroenteritis of pigs is effective, simple to operate, and at the same time it activates cellular and humoral immunity in sows, while cellular immunity provides colostral protection for newborn piglets, while humoral immunity provides protection for sows themselves against transmissible gastroenteritis.

Claims (3)

1. The vaccine against transmissible gastroenteritis of pigs, containing viral material and an excipient, characterized in that as the viral material it contains material from strain "BH-96" of the virus of transmissible gastroenteritis of pigs in the titer of 7.0-7.2 lg TCD _{50 / ml} , and as a filler - saline in the following ratio of components, wt.%:
Viral material from the strain "VN-96" 0.09-0.1 Saline Rest 2. The method of using the vaccine against transmissible gastroenteritis of pigs according to claim 1, which consists in immunizing pregnant sows, intramuscularly once 40-45 days before farrowing at a dose of 4.5-5 ml per head. 3. The method of using the vaccine according to claim 2, characterized in that it additionally immunize the suckling piglets at the age of 23-27 days once intramuscularly at a dose of 3.0-3.5 ml.