RU2211705C1 - Vaccine against canine coronaviral enteritis - Google Patents

Abstract

FIELD: veterinary virology. SUBSTANCE: the suggested vaccine contains inactivated and viral suspension of "Karat" canine coronaviral strain deposited at aluminum hydroxide at titer of infectious activity being TCD⁵⁰/cu.cm. Formalin is applied to inactivate the virus. Additionally, vaccine contains lactalbumin hydrolyzate upon Hanks' solution at the following ratio of components, weight%: viral suspension of "Karat"'s strain 50-70, lactalbumin hydrolyzate upon Hanks' solution 30- 50. Vaccine in question is safe and enables to develop immunity of high degree in dogs and minimize sick rate of the present infection. EFFECT: higher efficiency of canine vaccine. 4 tbl

Description

 The invention relates to the field of veterinary virology, in particular to an inactivated vaccine against coronavirus enteritis in dogs.

 Known inactivated vaccine containing inactivated pig gastroenteritis virus (TGEV) (1).

 A vaccine against coronavirus enteritis of dogs is also known, containing, in basis, an inactivated and precipitated on alumina hydrate virus suspension of the strain Canine coronavirus 1-743 (2).

 Known vaccines in the Russian Federation have not been used, and the information provided in the indicated patent sources does not allow us to make a judgment about their advantages compared to the proposed one. The vaccine, which basically contains the pig transmissible gastroenteritis virus (HES), is less effective due to the fact that the used HES strain is not closely related to dog coronavirus.

 The aim of the invention is a vaccine for the prevention of coronavirus enteritis in dogs, which is harmless and highly immunogenic.

и дополнительно гидролизата лактальбумина в растворе Хенкса при следующем соотношении компонентов (мас.%):
Инактивированная и осажденная на гидрате окиси алюминия вирусная суспензия штамма Coronavirus canine "Карат" ВГНКИ 29-ДЕП с титром инфекционной активности 10^5,5-10^6,5

- 50-70
Гидролизат лактальбумина в растворе Хенкса - 30-50
Штамм Coronavirus canine "Карат" ВГНКИ 29-ДЕП является известным и используется для контроля иммуногенной активности вакцин и получения сывороток для лечения и диагностики коронавирусной инфекции собак (3).The goal is achieved by using as a vaccine the coronavirus enteritis of the dog as an antigen of the inactivated and precipitated on aluminum oxide hydrate virus suspension of the Coronavirus canine strain "Carat" VGNKI 29-DEPT with an infectious activity titer of 10 ^5.5 -10 ^6.5

and additionally lactalbumin hydrolyzate in Hanks solution in the following ratio of components (wt.%):
Inactivated and precipitated on alumina hydrate virus suspension of the Coronavirus canine strain "Carat" VGNKI 29-DEPT with a titer of infectious activity 10 ^5.5 -10 ^6.5

- 50-70
Lactalbumin hydrolyzate in Hanks solution - 30-50
The strain Coronavirus canine "Carat" VGNKI 29-DEPT is known and is used to control the immunogenic activity of vaccines and to obtain sera for the treatment and diagnosis of coronavirus infection in dogs (3).

 The invention is illustrated by the following examples.

 Example 1

A vaccine is prepared for the prevention of dogs coronavirus enteritis. To obtain the viral material of the Karat strain of dog coronavirus enteritis, a primary trypsinized culture of dog kidney cells of 1-3 months of age is used. As a growth medium, a medium containing lactalbumin hydrolyzate is used - 50%, Igla medium with glutamine - 20%, medium 199 - 20%, bovine serum - 10% and antibiotics (gentamicin 0.1 mg per 1 cm ^{3 of} medium) .

в дозе 20 см³ на полуторалитровые матрасы. Зараженную культуру клеток выдерживают для адсорбции вируса в течение 1 ч при температуре 37^oС, после чего добавляют поддерживающую среду в объеме 200 см³ и инкубируют в условиях термостата в течение 5-7 сут. В качестве поддерживающей используют среду, содержащую те же компоненты, что и ростовая, но без добавления сыворотки. Размножение вируса оценивают по цитопатическому действию. Поражение 70% клеточного монослоя свидетельствует о высоком уровне накопления вируса и необходимости прекращения его культивирования. Цитопатическое действие в чувствительных культурах клеток характеризуется округлением и скоплением эпителиоподобных клеток, а фибробластоподобные клетки удлиняются, образовывая своеобразные тяжи. Монослой принимает вид узорчатого кружева. На 4-5 сут зараженные клетки начинают отделяться от стекла. Для более полного выхода вируса из зараженных клеток матрасы с вирусной суспензией трехкратно замораживают при -40^oС и оттаивают при 20^oС. Полученную вируссодержащую культуральную суспензию штамма "Карат" с титром инфекционной активности 10^5,5-10^6,5

сливают в одну стерильную емкость. Для удаления клеточного детрита полученную вирусную суспензию подвергают фильтрации на системе "Миллипор" с диаметром фильтра 293 мм и размером пор 0,22 мкм при давлении 15 Пси. Вирус инактивируют формалином сначала в условиях термостата, а затем при комнатной температуре.To obtain the virus, growth medium is removed from mattresses with a formed cell monolayer, the cell monolayer is carefully rinsed twice twice with warm serum-free nutrient medium in a volume of 50 cm ³ and infected with the Karat strain with a virus activity of 10 ^5.5 -10 ^6.5

in a dose of 20 cm ³ on one and a half liter mattresses. The infected cell culture is kept for adsorption of the virus for 1 h at a temperature of 37 ^o C, after which add support medium in a volume of 200 cm ³ and incubate in a thermostat for 5-7 days. As a support, a medium is used that contains the same components as the growth, but without the addition of serum. Reproduction of the virus is evaluated by cytopathic effect. The defeat of 70% of the cell monolayer indicates a high level of virus accumulation and the need to stop its cultivation. The cytopathic effect in sensitive cell cultures is characterized by rounding and accumulation of epithelial-like cells, and fibroblast-like cells lengthen, forming peculiar cords. The monolayer takes the form of patterned lace. At 4-5 days, infected cells begin to separate from the glass. For a more complete exit of the virus from infected cells, mattresses with a viral suspension are frozen three times at -40 ^° C and thawed at 20 ^° C. The resulting virus-containing culture suspension of the Karat strain with an infectious activity titer of 10 ^5.5 -10 ^6.5

poured into one sterile container. To remove cell detritus, the resulting viral suspension is filtered on a Millipor system with a filter diameter of 293 mm and a pore size of 0.22 μm at a pressure of 15 psi. The virus is inactivated with formalin, first in a thermostat, and then at room temperature.

 A sterile aluminum hydroxide (GOA) solution is added to the formalin inactivated virus, thoroughly mixing, and left to stand at room temperature, after which 30-50% of the supernatant is removed and the lactalbumin hydralizate is added to the initial volume on the Hanks solution.

 Get 3 series of vaccines of the following composition: (see table.A).

 The finished vaccine is a pink or reddish-pink liquid with a loose precipitate that breaks easily when shaken into a homogeneous suspension.

 Example 2

 The safety of the vaccine series 3 against coronavirus enteritis in dogs is tested in laboratory animals (guinea pigs weighing 250-300 g and mice weighing 14-16 g) and naturally susceptible animals.

 The vaccine is administered to experimental animals and observations are made for 10 days.

 The verification results are presented in table. 1.

 From the table. 1 shows that the vaccine is harmless. During the period of observation of the inflammatory reaction at the injection site, no cases of animal disease are noted.

 Example 3

 Check the harmlessness of the vaccine against coronavirus enteritis in dogs of series 1 and 2 in the same way as in example 2, on 10 guinea pigs and 20 white mice.

 Set the safety of these series in laboratory animals.

 Example 4

The study of the optimal timing of the use of vaccines against coronavirus enteritis in dogs of series 3 is carried out on 3 groups of puppies aged 1,5, 3 and 4 months. Animals are vaccinated intramuscularly twice with an interval of 14 sut in a dose of 0.5 cm ³ . The dynamics of the accumulation of antibodies to dog coronavirus in the reaction - indirect hemagglutination is shown in table. 2.

 From the table. 2 it follows that the proposed vaccine allows you to create intense immunity in puppies not only 3 and 4 months of age, but also in puppies of 1.5 months of age - in puppies of an early age, especially susceptible to coronavirus enteritis.

 Example 5

 Evaluation of the immunological effectiveness of the vaccine against coronavirus enteritis of dogs is carried out on 12 experimental and 4 control puppies of dogs of 3 months of age by experimental infection with the control strain "Carat".

Experimental animals (4 goals. In the group) are pre-vaccinated with a vaccine against coronavirus enteritis of dogs of series 1, 2, 3 intramuscularly at a dose of 1 cm ³ .

On the 21st day from the start of the experiment, all animals are infected with a control strain at a dose of 10 ^6.0 TCD ₅₀ / cm ³ .

 All series of the proposed vaccines in experimental puppies do not cause post-vaccination complications and create a high protective effect during infection control.

 On the 3rd day after infection in puppies of the control group, an oppressed state, frequent defecation, feces of a liquid consistency are observed. On day 7, 3 puppies in the control group fell with signs of severe diarrhea. At autopsy, dead animals noted dehydration, an increase in intestinal lymph nodes. The intestinal mucosa is thickened, hyperemic, with spot hemorrhages and areas of necrosis.

 Example 6

The antigenic activity of the vaccine is determined on puppies of a dog of 3 months of age and rabbits. Animals are vaccinated intramuscularly or subcutaneously with a vaccine against coronavirus enteritis of dogs of series 1 at a dose of 1 cm ³ and blood is taken on days 1, 14 and 21 to determine the level of vaccine-induced humoral antibodies.

 Antibodies to dog coronavirus are determined in an indirect hemagglutination reaction. The results are presented in table. 3.

 According to the table. Figure 3 shows that an increase in the level of antibodies to the antigen included in the vaccine is most pronounced 2 weeks after the 2nd vaccination.

 This series of vaccines has antigenic activity, since the titer of antibodies in the blood serum of vaccinated puppies of dogs and rabbits to dog coronavirus increases 2-4 times or more.

 The use of rabbits instead of puppy dogs to control the antigenic activity of this vaccine will simplify and reduce the cost of research.

 The use of a vaccine based on the Coronavirus canine Karat strain VGNKI 29 DEP allows for the formation of high tension immunity in dogs in a short time and to reduce the incidence of dogs with this infection to a minimum.

Sources of information
1. US 5911999, 06/15/99.

 2. US 5464621, 11/7/95.

 3. RU 2100433, 11.27.97.

Claims (1)

A dog coronavirus enteritis vaccine containing a dog coronavirus enteritis antigen inactivated and precipitated on alumina hydrate, characterized in that as a dog coronavirus enteritis antigen, it contains an inactivated and precipitated on alumina hydrate suspension of the Karat DE titan Coronavirus canine titan infectious activity 10 ^5.5 -10 ^6.5 TCD ⁵⁰ / cm ³ and additionally lactalbumin hydrolyzate in Hanks solution in the following ratio of components, wt. %:
Inactivated and precipitated on an alumina hydrate suspension of the Coronavirus canine strain "Carat" VGNKI 29 DEPT with a titer of infectious activity 10 ^5.5 -10 ^6.5 TCD ⁵⁰ / cm ³ - 50 - 70
Lactalbumin hydrolyzate on Hanks solution - 30-50

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