RU2030916C1 - Vaccine against chicken salmonellosis and a method of prophylaxis of chicken salmonellosis - Google Patents

Abstract

FIELD: veterinary biotechnology. SUBSTANCE: vaccine has suspension of strain Sal. enteritidis ВГНКИ 204 and gelatin-sucrose stabilizing agent taken at effective concentration. Attenuated strain Sal. enteritidis DUYRB 204 is prepared by insertion of plasmid

Description

 The invention relates to veterinary biotechnology, in particular to means for the specific prevention of chicken salmonellosis.

 Salmonellosis of chickens caused by Salmonella enteritidis is recorded in many countries of the world and regions of the USSR. Over the past decade, it has grown into a serious economic and social problem.

 Sal. enteritidis, adapted to the body of chickens, causes significant waste of chickens (up to 10-12%) and salmonella in adult chickens. Salmonella-infected poultry slaughter products are one of the main sources of salmonella toxicoinfection in humans.

 The widespread prevalence of Sal.enteritidis among chickens, significant material damage caused to poultry farming, danger to humans, and the lack of effective medications make it necessary to find means of specific prevention of this salmonellosis in chickens.

 A vaccine against typhoid typhoid chickens is known, containing a suspension of Sal.gallinarum strain pullorum N 9 cells and a stabilizer (Gordon et al., 1959). The vaccine was used on a limited number of chickens. The vaccine was administered subcutaneously to an adult bird in order to obtain transovarial immunity in chickens. Strain 9R does not have genetic markers for differentiation from epizootic strains and, like any strain in the R form, has a defective antigenic structure due to the loss of side chains in the polysaccharide molecules of the cell wall. In addition, the vaccine from strain 9R causes pathological and morphological changes in the ovaries characteristic of pullorosis in the vaccinated bird, which made this vaccine unsuitable for practical widespread use.

 The aim of the invention is a vaccine against salmonellosis of chickens with harmlessness, high antigenic and immunogenic activity.

This goal is achieved by obtaining a vaccine containing a suspension of cells of strain Sal. enteritidis 204, sucrose, gelatin and distilled water in the following ratio of components wt./vol.%:
Cell suspension
strain Sal.enteritidis
204 with concentration
175-250 billion

microbial cells in 1 cm ³ 42.0-55.0 Sucrose 5.0-7.5 Gelatin 1.5-2.0
Distilled water
A method for the prevention of chicken salmonellosis involves administering to the body of chickens 2-3 days old with water a vaccine containing a suspension of cells of the strain Sal.enteritidis 204 with a concentration of 175-250 billion / cm ^{3 of} microbial cells in an amount of 42.0-55.0 cm ³ and sucrose-gelatin stabilizer in an amount of 45-48 cm ³ at the rate of 0.5-1.0 billion / cm ^{3 of} microbial cells per chicken, twice with an interval of 2 days after 1-24-hour fasting.

 The strain Sal.enteritidis 204 used for the manufacture of the vaccine is obtained by targeted selection from the epizootic strain Sal.enteritidis 126. The strain is deposited in the collection of bacterial cultures of the All-Union State Scientific and Control Institute of Veterinary Medicines at number 204.

PRI me R 1. Get resistant to rifampicin mutant (Rif ^r ) virulent strain Sal.enteritidis 126, sowing a suspension of bacteria (1 billion / cm ³ ) on MPA containing 100 μg / cm ³ rifampicin. The rif ^r mutant retains the original virulence for mice (LD ₅₀ = 36 microbial cells).

Through conjugation to Rif ^r plasmid mutants administered ^VD ₃ R :: Tn7 not capable of replicating in Sal.enteritidis cells at 43 ^° C Grow transkonyuganty Sal.enteritidis 126 Rif ^r (P ^VD ₃ :: Tn7) in IPA with streptomycin (20 g / cm ³⁾ at 43 ^{° C,} selected insertional mutants Sal.enteritidis 126 Rif ^r :: Tn7.

In experiments on white mice, clones of mutants were detected, upon infection of which (intraperitoneal dose of ⁵ × 10 ⁴ microbial cells) 100% of the animals would survive.

 Strain Sal.enteritidis 204 was detected in the analysis of 54 insertion mutants.

 Strain Sal. enteritidis 204 is characterized by the following properties and symptoms.

 Morphology - small gram-negative rods without spores and capsules, have active mobility, peretrich. The size of the sticks is 2-3 x 0.6-0.7 microns.

Cultural properties - on meat-peptone broth forming uniform turbidity after 5-7 hours of incubation at ³⁷ ° C; then a delicate film forms and a gray-white, easily-breaking sediment falls out. On the meat-peptone agar forms after 18-20 hours of growth at ³⁷ ° C colonies of 0.8-1.5 mm diameter in the S form, gray-white, translucent, round, smooth, fine-grained, convex. After 2-3 days, the size of the colonies increases to 2-3 mm. Optional aerob.

 Enzymatic properties - ferments with the formation of acid and gas glucose, maltose, mannitol, sorbitol, xylose, levulose, galactose, mannose, trihalose, dextrin, glycyrin, dulcite inositol, forms hydrogen sulfide, does not ferment sucrose, lactose, arabinose, raffinose, inulin, , adonite, does not clot milk, does not form indole, nitrites reduces to nitrates.

 Antigenic structure - it is agglutinated by salmonella monoreceptor “0” serums: 1, 9, 12 and “H” serums: g, 1, 7. The non-specific phase “H” of antigen (1 and 7) is not constantly detected.

Genetic markers - the strain is resistant to streptomycin (150 μg / cm ³ ), rifampicin (200 μg / cm ³ ), trimethoprim (200 μg / cm ³ ). Resistance markers are stably maintained after 10-fold passages on meat and peptone agar, 5-fold passage through white mice and 5-fold passage through chickens.

Virulence for white mice. The initial strain Sal.enteritidis 126 has an LD ₅₀ for Balb / s-18 mice, and 5 microbial cells for outbred white mice. The LD _{50 of} the Rif ^r strain is 36 and 14 microbial cells, respectively, and for the attenuated Sal.enteritidis 204 strain is 1.6 × 10 ⁶ and 5.6 × 10 ⁶ microbial cells.

Immunogenicity — the survival rate of white mice and chickens immunized with an attenuated strain of 204 is 80–95% of the number of animals infected with the death of 90–100% of the control in the control after infection with 5–7 LD _{50 of the} virulent initial Salmonella enteritidis strain.

EXAMPLE Example 2. The reactor tank is filled with 200-500 l 1/3 Hottinger broth and sterilized at ¹²⁰ ° C for 1 hour. Matrovuyu rasplodku Sal.eteritidis 204 attenuated strain is introduced into the reactor in an amount of 4,5- 5.5% of the volume of the medium. Cultivation was carried out for 10-12 hours at a temperature of 37 ± 1 ^{° C} and continuously working mixer at a speed of 120 ± 5 revolutions / min and feeding sterile air in an amount of 2 ± 0.2 volume / min. The accumulation of microbial cells is 35-50 billion / cm ³ environment.

 The grown culture is besieged on a rotary centrifuge type SGO-100 at 12-15 thousand rpm. The bacterial mass is removed from the rotor in a laminar box and mixed in a 1: 2 ratio with a stabilizer containing sucrose, gelatin and distilled water.

The vaccine is packaged in 10 cm ³ vials and freeze-dried.

The vaccine against salmonellosis of chickens of series 1 has the following component composition, wt. /about. %: Suspension of cells of the strain Sal.enteritidis 204 with a concentration of 175 billion / cm ³ 55.0 Sucrose 5.0 Gelatin 1.5 Distilled water
The vaccine against chicken salmonellosis series 2 has the following component composition, wt./vol.%: Suspension of cells of strain Sal.enteritidis 204 with a concentration of 212.5 billion / cm ³ 48.5 Sucrose 6.25 Gelatin 1.75 Distilled water The rest
The vaccine against salmanolles of chickens of series 3 has the following component composition, wt./vol.%: Suspension of cells of strain Sal.enteritidis 204 with a concentration of 250 billion / cm ³ 42.0 Sucrose 7.5 Gelatin 2.0 Distilled water The rest
PRI me R 3. Determine the optimal immunizing dose of the proposed vaccine series 2 for 2-3-day-old chickens. The chickens are incubated for 18-24 hours without water and food, and then immunized by feeding the vaccine with water twice with an interval of 2 days at a dose of 0.5 billion live microbial cells per chicken. The chickens are infected 10-12 days after drinking the second dose of the vaccine intramuscularly at a dose of 10 LD _{50 with the} virulent strain Sal.enteritidis N 126.

The experiment is performed in three repetitions. In each experiment, 20 vaccinated and 20 unvaccinated chickens are used. The results are shown in the table.
Among vaccinated chickens, the survival rate in three groups was 90% with the death of 83.3% of chickens in the control.

 The use of the proposed vaccine in veterinary practice will improve the effectiveness of antiepizootic measures in chicken salmonellosis.

Claims (1)

 VACCINE AGAINST CHICKEN SALMONELLOSIS AND METHOD FOR PREVENTION OF CHICKEN SALMONELLOSIS. Chicken salmonellosis vaccine containing a suspension of vaccine strain cells and a sucrose-kelatin stabilizer, characterized in that it contains a strain of Salmonella enteritidis VGNKI 204 with a concentration of living microbial cells (175-250) · 10 ⁹ cells / cm ³ as follows the ratio of components, vol.%:
Suspension of Salmonella enteritidis strain VGNKI
N 204 with a concentration of (175-250) · 10 ³ cells / cm * 9
93 - 42.0 - 55.0
Sucrose - 5.0 - 7.5
Gelatin - 1.5 - 2.0
Distilled Water - Else
2. A method for the prevention of chicken salmonellosis, comprising administering a vaccine preparation to the chickens, characterized in that a vaccine containing the following components is used as a vaccine preparation, vol.%:
Suspension of Salmonella enteritidis strain VGNKI
N 204 with a concentration of (175-250) · 10 ⁹ cells / cm ³ - 42.0 - 55.0
Sucrose - 5.0 - 7.5
Gelatin - 1.5 - 2.0
Distilled Water - Else
which is administered to chickens of 2-3 days of age with water by drinking from a calculation of (0.5-1.0) · 10 ⁹ microbial cells per chicken, twice with an interval of 48 hours after an 18-24 hour fast exposure.

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