RU1149467C - Vaccine against colibacteriosis of young cattle and sheep - Google Patents

Description

 The invention relates to veterinary microbiology, in particular to methods for producing vaccines used for the prevention of colibacteriosis (Escherichiosis) of farm animals.

 Colibacteriosis (Escherichiosis) is an infectious disease caused by enteropathogenic Escherichia, is widespread in many countries of the world and causes significant economic damage to livestock due to the high mortality of young animals (calves in the first days of life, and lambs in the post-weaning period), lower weight gain, loss of productivity in sick animals .

 The main directions in the fight against colibacteriosis of farm animals are chemotherapy and specific prevention. However, the emergence of resistant forms of enteropathogenic Escherichia noticeably reduces the therapeutic efficacy of drugs. In this regard, vaccinoprophylaxis of colibacillosis is becoming increasingly important.

Currently, according to biological characteristics, enteropathogenic Escherichia, causing diarrhea in young farm animals and humans, are conventionally divided into three groups:
a) enteropathogenic Escherichia producing thermolabile and thermostable enterotoxins;
b) enteropathogenic Escherichia belonging to certain serogroups;
c) enteropathogenic Escherichia, with pronounced adhesive properties due to the presence of K-88 and K-99 antigens.

In this regard, to obtain an effective means of specific prophylaxis of colibacteriosis, the vaccine should include:
a set of strains of certain serogroups of enteropathogenic Escherichia, most often causing colibacteriosis in this animal species;
enterotoxin-producing strains or purified enterotoxins;
strains having adhesive factors (adhesion factors) K-99 K-88 or purified K-antigens.

 A known vaccine for native immunization against enteric form of colibacteriosis, containing a mixture of 4 strains of serovars 0101, 09, 09, 08, formalin inactivator, adjuvant aluminum hydroxide.

 However, due to the insufficiently complete set of 0-antigen lipopolysaccharides and the absence of exotoxins in the vaccine, it has a limited immunogenic spectrum and will not protect animals from enterotoxemic and partially septic forms of colibacteriosis.

 A vaccine against diarrhea caused by E. coli is also known, containing an aqueous suspension of semi-purified K99 antigen, K-antigen polysaccharides, 0-antigen lipopolysaccharides and thermostable enterotoxin from Escherichia serovarov strains 09: K35: K99; 0101: K30: K99; 08: K85: K99, cell fragments and culture fluid with a molecular weight of more than 1000 daltons.

 However, the immunogenic spectrum of action of the vaccine is limited for the prevention of enterotoxemic form of colibacteriosis due to the absence of thermolabile enterotoxin. In addition, the technology for manufacturing the vaccine is complex, time-consuming and requires sophisticated equipment.

 A known vaccine against colibacteriosis of calves, lambs, taken as a prototype, containing a mixture of 13 strains of E. coli serovars 08, 09, 015, 020, 026, 041, 055, 078, 086, 0101, 0115, 0117 and 0119 at a concentration of 24 billion microbial bodies, inactivator 03-0.4% formalin, adjuvant 30% by volume to the back mass of 6% aluminum hydroxide.

 However, the vaccine has weak immunogenic activity and has an increased reactogenicity.

 The purpose of the invention is to increase the immunogenicity of the vaccine against colibacteriosis (Escherichiosis) of calves and lambs and the expansion of its spectrum of action.

This goal is achieved by the fact that the vaccine against colibacteriosis of young cattle and sheep, containing Escherichia antigens, a preservative based on formalin and thiomersal, additionally contains Hottingen broth and physiological saline, and as antigens a mixture of Escherichia coli strains VGNKI N 305-37 / 10 , E. coli VGNKI N 355-37 / 12, E. coli VGNKI N 286-37 / 9, E. coli VGNKI N 296-37 / 10, E. coli VGNKI N 301-37 / 10, E. coli VGNKI N 301 328-37 / 11, E. coli VGNKI N 289-37 / 10, E. coli VGNKI N 288 / -37 / 9, E. coli VGNKI N 288 / -37 / 9, E. coli VGNKI N 58-37 / 2 , E. coli VGNKI N 67-37 / 2, E. coli VGNKI N 59-37 / 2, E. coli VGNKI N 293-37 / 10, taken in equal amounts Wearing at the following content of components per 1 liter of vaccine:
Escherichia antigens
(cells) 7.7˙10 ¹² 9.2˙ 10 ¹²
Aluminum hydroxide (cm ³ ) 280-330
Formalin (cm ³ ) 2.8-3.5
Thiomersal (g) 0.13-0.18
Hottinger Broth (cm ³ ) 100-160
Saline solution Up to 1 L
The vaccine contains Escherichia strains of 13 serogroups (08, 09, 015, 020, 026, 041, 055, 078, 086, 0101, 0115, 0117, 0119), which are responsible for the enzootia of colibacteriosis in calves and lambs. The strains possess capsular antigens K-99, K-43, K-14, K-30, K-60, K-61, K-62 K-69 and K-80, are capable of producing toxins and hemolysins.

 The Escherichia strains used for the manufacture of the vaccine are selected for immunogenic and antigenic activity. Strains of Escherichia coli VGNKI N 305-37 / 10, E. coli VGNKI N 288-37 / 9, E. coli VGNKI N 296-37 / 10, E. coli VGNKI N 3016 -37/10, E. coli VGNKI N 328-37 / 10, E. coli VGNKI N 289-37 / 10, E. coli VGNKI N 297-37 / 10, E. coli VGNKI N 288 / 1-37 / 9 , E. coli VGNKI N 58-37 / 2, E. coli VGNKI N 67-37 / 2, E. coli VGNKI N 59-37 / 2, E. coli VGNKI N 299-37 / 10 deposited in the collection of microorganisms of the All-Union State Scientific and Control Institute of Veterinary Medicines.

 The strains of Escherichia coli included in the vaccine are characterized by the following properties.

 Morphological properties: strains are small gram-negative rods without spores and capsules. Size 1.5-2 microns, width 0.3-0.5 microns. Cells of strain N 305-37 / 10, N 289-37 / 10, N 297-37 / 10, N 288-37 / 9, N 288 / 1-37 / 9, N 58-37 / 2, N 301-37 / 10 are motile due to peretrichial flagella.

Cultural properties: meat-peptone broth for all strains produce blurred after 6-8 hours of growth at 37-38 ° ^C. On the meat-peptone agar form a matte, round, smooth, fine, slightly convex colonies ( "S" shape) having a diameter of 2-4 mm.

 Enzymatic properties: all strains ferment lactose, glucose, mannitol, arabinose, xylose, sorbitol, sucrose (with the exception of strain N 328-37 / 11, N 58-37 / 2, N 59-37 / 2, N 297-37 / 10, N 305-37 / 10, N 289-37 / 10); raffinose (with the exception of strains N 288-37 / 9, N 289-37 / 10, N 296-37 / 10, N 288 / 1-37 / 9); dulcite (with the exception of strains N 297-37 / 10, N 355-37 / 12).

 All strains form indole and coagulate milk.

 Strains N 305-37 / 10, N 288-37 / 9, N 296-37 / 10, N 67-37 / 2 have pronounced hemolytic properties.

Antigenic structure: strains have the following antigenic structure:
E. coli strain N 305-37 / 10 is agglutinated with 0-coli with serum 078 in a titer of 1: 6400 and serum K-80 on four crosses, strain N 355-37 / 12 with serum 09 in a titer of 1: 3200 and K-30, N 288-37 / 9 with serum 015 to 1: 6400 and serum K-14, N 296-37 / 10 with serum 026 in a titer of 1: 12800 and serum K-60, N 301-37 / 10 is agglutinated with serum 086 in a titer of 1: 6400 and serum K-61, N 328-37 / 11 serum 0101 to 1: 6400 and serum K-99 to a titer of 1: 640, N 289-37 / 10 serum 0117 in a titer of 1: 12800 and serum K-62, N 297-37 / 10 with serum 0119 to 1: 6400 and serum K-69, N 288 / 1-37 / 9 is agglutinated with serum 020 in the titer to 1: 3200, N 58-37 / 2 with serum 041 in the titer 1: 6400, N 67-37 / 2 series swelling 0115 in a titer of 1: 12800, N 59-37 / 2 with a serum of 055 in a titer of 1: 6400 and N 299-37 / 10 with a serum of 08 in a titer of 1: 3200 and K-43 serum

Virulent properties: LD ₅₀ for mice, strain N 305-37 / 10 is 57 million, strain N 355-37 / 12 500 million, N 288-37 / 9 18 million, N 296-37 / 10 56 million, N 301-37 / 10 98 million, N 328-37 / 11 380 million, N 289-37 / 10 92 million, N 297-37 / 10 32 million, N 288 / 1-37 / 9 94 million, N 58-37 / 2 75 million, N 67-37 / 2 63 million, N 59-37 / 2 38 million, N 299-37 / 10 79 million microbial cells of the daily agar culture.

 Toxic and toxigenic properties: strains N 328-37 / 11, N 67-37 / 2, N 297-37 / 10, N 288-37 / 9, N 305-37 / 10, produce thermolabile (TL) toxin, strain N 355-37 / 12 thermolabile and thermostable enterotoxins.

 All strains possess endotoxins.

PRI me R 1. Escherichia strains N 305-37 / 10, 355-37 / 12, 288-37 / 9, 296-37 / 10, 301-37 / 10, 328-37 / 4, 289-37 / 10, 297-37 / 10, 288 / 1-37 / 9, 58-37 / 2, 67-37 / 2, 59-37 / 2 and 299-37 / 1 are sown individually 0.5 cm ³ in 200 ml vials containing 80-100 cm ^{3 of} Hottinger broth having amine nitrogen 150-250 mg% tryptophan 50-100 mg /% and pH 7.8-8.0. At the same time, to control the strains for purity, they are sown in plates with MPA and Endo medium.

Strains were grown for 8-10 hours at 37-38 ° ^C. The grown culture is checked for purity by known methods.

Pure cultures inoculated separately from the half-filled vials in broth Hottinger 10 liter bottles and cultured for 8-10 hours at 37-38 ^{° C} shaking periodically. The resulting brood is checked for cleanliness before inoculation into the reactor.

The strains were inoculated into the reactor groups based on their kolitsinogennosti and grown for 10-14 hours at 37-38 ° ^C under continuous aeration and stirring.

At the end of the cultivation, the pH, the concentration of microbial bodies and the purity of the culture are determined by microscopy and culture on culture media. The bacterial mass obtained in each reactor is diluted to a concentration of 10 billion / cm ^{3 of} microbial bodies and mixed in one reactor.

Formalin diluted with physiological saline 1: 1 is added to the mixture of cultures in the reactor with the stirrer switched on, so that its concentration in the culture is 0.3% and thiomersal in the amount of 0.01%. Inactivation of the culture is carried out for 15-16 days at 37-38 ^about C. In the process of inactivation, the culture is mixed.

 A sterile 6% solution of aluminum hydroxide is added to an inactivated culture to a concentration of 30% by volume and 0.05 g of thiomersal per liter of vaccine and mixed thoroughly.

Then the pH is adjusted with 10% NaOH to 7.2-7.4 leave vaccine at 18-20 ^{° C} and are monitored for sterility.

 The vaccine tested for sterility is packaged with the stirrer in bottles, which are closed with rubber stoppers, wrapped in metal caps and labeled.

 The packaged vaccine is checked for sterility, safety and activity.

The content of the components in the resulting vaccine is as follows: Component of the vaccine Content in 1 l
minimum maximum Escherichia (cell) antigens 7.7˙10 ¹² 9.2˙ 10 ¹² E. coli VGNKI N 305-37 / 10 (Serovar 078: K-80) 0.6˙10 ¹² 0.7˙ 10 ¹² E coli VGNKI N 355-37 / 12 (serovar 09: K-30) 0.6˙10 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 288-37 / 9 (serovar 015: K-14: H30) 0 , 5˙10 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 296-37 / 10 (Serovar 026: K-60) 0.6 1210 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 301-37 / 10 (serovar 086: K-61: H32) 0.6˙10 ¹² 0.7˙10 ¹² E. coli VGNKI N 328-37 / 11 (serovar 0101: K-99) 0.6˙10 ¹² 0.7 ˙ 10 ¹² E. coli VGNKI N 289-37 / 10 (serovar 0117: K-62: H4) 0.6˙10 ¹² 07˙ 10 ¹² E. coli VGNKI N 297-37 / 10 (serovar 0119: K-69 : H4) 0.6˙10 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 288 / 1-37 / 9 (Serovar 015: K-14: H30) 0.6˙10 ¹² 0.7˙ 10 ¹² E coli VGNKI N 58-37 / 2 (serovar 041) 0.6˙10 ¹² 0.8˙ 10 ¹² E. coli VGNKI N 67-37 / 2 (serovar 0115) 0.6˙10 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 59-37 / 2 (serovar 055) 0.6˙10 ¹² 0.7˙ 10 ¹² E. coli VGNKI N 299-37 / 10 (serovar 08: K-43) 0.6˙10 ¹² 0, 7˙ 10 ¹² Adjuvant:
aluminum hydroxide
6% solution, cm ³ 280 330 Preservative:
formalin, cm ³ 2.8 3.5
thiomersal, g 0.13 0.18
Hottinger broth, cm ³ 100 160
saline up to 1 l
PRI me R 2.

The resulting vaccine is administered intramuscularly twice to cows and ewes 1.5-2 months before calving or lambing, respectively, and to lambs before weaning in doses:
to stylish cows, cm ³ 10.15 and 15-20
to sheep sheep, cm ³ 3-4 and 4-5
to the lambs before
weaning, cm ³ 1-1.5 and 1.5-2
The vaccine is used in colibacteriosis-poor households for vaccination, pregnant cows and pregnant sheep, and 1.5-2 months before calving (lambing), as well as lambs before weaning.

The vaccine is administered intramuscularly in the neck, following the rules of asepsis twice with an interval of 10-15 days in the following doses:
Type of animals Doses of the vaccine, cm ³
first second
Cows 10-15 15-20
Sheep 3-4 4-5
Lambs (before weaning) 1-1.5 1.5-2
Young animals are vaccinated before weaning no earlier than at 21 days of age. Immunity occurs 18-20 days after the first dose of the vaccine and persists in adult animals for 5-6, in young animals 3-4 months.

 The immunogenic properties of the vaccine were tested in 6 households on an adequate number of animals. Control (unvaccinated) groups of animals (deep-shelled cows and ewes), as well as young animals born, were kept under the same conditions of keeping, feeding and caring in the same premises and at the same time with experimental animals born from vaccinated cows and ewes .

 The animals were vaccinated 1.5 months before calving and lambing twice with an interval of 10-15 days. Health monitoring of calves was carried out up to 10-15 days of age, lambs up to 30-40 days of age.

 The test was conducted on 1510 cows (of which 800 control), 1495 calves (including 89 control) and 404 sheep.

 The test results showed (Table 1) that calves from vaccinated cows get colibacillosis in 63% versus 100% in the control group, their disease was mainly mild and calves in this group were 6.2 times less than in calves born to unvaccinated cows.

 PRI me R 3. The resulting vaccine was compared by immunogenic activity with the vaccine of the prototype on 24 pregnant cows (12 cows for each vaccine) and 4 cows served as a control.

Vaccination was carried out 1.5-2 months before calving twice with doses of 10 + 15 cm ³ with an interval between doses of 10 days. 24 hours after birth and 3 times colostrum, calves were infected with control strain 078: K80 at a dose of 4-10 ¹¹ microbial cells. The results are presented in table. 2.

 As can be seen from the table, the prototype vaccine had increased reactogenicity (caused lameness and abortion in vaccinated cows), as well as a low immunogenicity. The waste of calves obtained from cows vaccinated with the prototype vaccine and infected with control strain 078 was 16.6% with 100% survival of young animals obtained from mothers vaccinated with the proposed (new) vaccine.

 The proposed vaccine has well-defined antigenic properties, as well as a wide range of immunogenicity, which ensures the prevention of colibacillosis in calves and lambs in various regions of the world.

Claims (1)

Vaccine against colibacteriosis of young cattle and sheep, containing Escherichia antigens, a preservative based on formalin and thiomersal and an adjuvant, characterized in that, in order to increase immunogenicity and expand the spectrum of action, it additionally contains Hottinger broth and physiological solution, and as strains of Escherichia coli VGNKI N 305-37 / 10, E. coli VGNKI N 288-37 / 9, E. coli VGNKI N 296-37 / 10, E. coli VGNKI N 296-37 / 10, E. coli VGNKI N 301 -37/10, E. coli VGNKI N 328-37 / 10, E. coli VGNKI N 289-37 / 10, E. coli VGNKI N 297-37 / 10, E. coli VGNKI N 288 / 1-37 / 9 , E. coli VGNKI N 58-37, E. coli VGNKI N 67- 37/2, VGNKI N 59-37 / 2, E. coli VGNKI N 299-37 / 10, taken in equal proportions with the following content of components per 1 liter of vaccine:
Escherichia antigens (cells) 7.7 · 10 ^{1 2} 9.2 · 10 ^{1 2}
Aluminum hydroxide (cm ³ ) 280 330
Formalin (cm ³ ) 2.8 3.5
Thiomersal (g) 0.13 0.18
Hottinger broth (cm ³ ) 100 160
Saline solution Up to 1 L

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