RU1149466C - Colibacillosis vaccine for pigs - 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), 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 piglets both in the first days of life and in the post-weaning period, a decrease in weight gain and loss of productivity in sick animals.

 The main methods of combating colibacteriosis are chemotherapy and antibiotic therapy. However, the emergence of resistant forms of enteropathogenic Escherichia significantly reduces the therapeutic effectiveness of the drugs. In this regard, the development of specific prophylaxis of colibacteriosis, in particular vaccines, is of current importance.

 Enteropathogenic Escherichia is known to have a complex of virulence factors, of which the production of thermolabile and thermostable toxins and hemolysin, the presence of adhesion antigens K-88 and K-99 can be considered the main ones. The presence and severity of these factors in Escherichia also determine the manifestation of colibacillosis in piglets: enterotoxemic, septic and edematous. The first two are observed in piglets in the first days of life, the last during weaning.

 In this regard, a highly effective vaccine against colibacteriosis of piglets should neutralize the main virulence factors of enteropathogenic Escherichia.

 Known inactivated alum-oil vaccine against colibacteriosis of piglets, containing a mixture of Escherichia strains of serovars 08, 020, 054, 078, 0101, 0138, 0139, 0141, 0147 and 0149, formalin, mineral oil, alum, saponin, lanolin and toluene. However, the vaccine has a limited spectrum of action due to the absence of adhesive antigens and enterotoxins.

 Known vaccine against colibacteriosis of piglets containing thermostable toxin and adjuvant. However, the vaccine is not highly effective in various forms of colibacteriosis due to the lack of a complete set of factors responsible for the breadth of the immunogenic spectrum of the drug.

 A known vaccine against colibacteriosis (Escherichiosis) of piglets containing a mixture of 10 strains of Escherichia coli serovars 08, 09, 078, 0137, 0138, 0139, 0141, 0142, 0147, 0149 with a concentration of 24 billion microbial bodies, formalin inactivator and aluminum hydroxide adjuvant.

 The vaccine does not provide sufficiently intense immunity in vaccinated animals with enterotoxemic forms of colibacteriosis. In addition, due to the high concentration of antigen, it has a high reactogenicity and causes abscesses at the injection site.

 The purpose of the invention is to increase the immunogenicity of the vaccine against colibacteriosis of piglets and the expansion of its spectrum of action.

This goal is achieved in that the vaccine against colibacteriosis of piglets, containing Escherichia antigens, a preservative based on formalin and thiomersal and an adjuvant, additionally contains Hottinger broth and 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 357-37 / 12, E.coli VGNKI N 331-37 / 12, E.coli VGNKI N 359-37 / 12, E. coli VGNKI N 360-37 / 12, E.coli VGNKI N 299-37 / 10, E.coli VGNKI N 330-37 / 11 with the following content of components per 1 liter of vaccine: Escherichia antigens, cells 7.7x10 ¹² -9.2x10 ¹⁰ Alumina hydroxide - minia, 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, l Up to 1
The vaccine contains Escherichia strains of 8 serogroups (08, 09, 078, 0138, 0139, 0141, 0147 and 0149), most often affecting piglets. The strains contain capsular antigens K30, K80, K43, K81, K85, K88, K91, are capable of producing hemolysin, thermostable and thermolabile toxins.

 The Escherichia strains used for the manufacture of the vaccine are selected for immunogenic and antigenic activity.

 Strains of E. coli VGNKI N 305-37 / 10, E. coli VGNKI N 299-37 / 10, E. coli VGNKI N 330-37 / 11, E. coli VGNKI N 330-37 / 11, E. coli VGNKI N 330 357-37 / 12, E.coli VGNKI N 331-37 / 12, ET.coli VGNKI N 359-37 / 12, E.coli VGNKI N 360-37 / 12 deposited in the collection of microorganisms of the All-Union State Scientific and Control Institute of Veterinary Preparations Ministry of Agriculture of the USSR.

 Strains have the following properties.

 Morphological properties. Strains E. coli N 299-37 / 10, N 355-37 / 12, N 330-37 / 11, N 305-37 / 10, N 357-37 / 12, N 331-37 / 12, N 359-37 / 12, N 360-37 / 12 are small gram-negative rods without spores and capsules. Size 1.5-2 microns, width 0.3-0.5 microns. Cells of strains N 305-37 / 10, N 360-37 / 12, N 330-37 / 11, N 331-37 / 12, N 359-37 / 12 are mobile due to peretrichnally located flagella.

Cultural properties. On meat-peptone broth all strains produce intense turbidity after 6-8 hours of growth at 37-38 ° ^C. On MPA grow as round matt, smooth fine, slightly convex colonies (S-form) with a diameter of 2-4 mm.

 Enzymatic properties. All strains ferment lactose with the exception of strain N 331-37 / 12, glucose, mannitol, arabinose, sorbitol and sucrose with the exception of strain N 305-37 / 10, N 360-37 / 12, N 299-37 / 10, raffinose strain N 360-37 / 12, dulcite with the exception of strain N 360-37 / 12. All strains coagulate milk and form indole.

 Strains N 305-37 / 10, N 357-37 / 12, N 330-37 / 11, N 355-37 / 12, N 360-37 / 12, N 331-37 / 12 have pronounced hemolytic properties.

 Antigenic structure. The Escherichia strains have the following antigenic structure: strain N 305-37 / 12 is agglutinated with O-cola with serum 078 to a titer of 1: 6400 and serum K80. strain N 299-37 / 10 is agglutinated by O-coli with serum 08 to a titer of 1: 3200 and serum K-43; strain N 355-37 / 12 is agglutinated with O-coli with serum 09 to a titer of 1: 3200 and serum K-30; strain N 330-37 / 11 is agglutinated with O-coli with serum 0138 to a titer of 1: 6400 and serum K-81; strain N 357-37 / 12 with serum 0139 to titer 1: 12800, strain N 331-37 / 12 with serum 0141 to titer 1: 12800 and serum K-88 to titer 1: 320 and K-85; strain N 359-37 / 12 is agglutinated with O-coli serum 0147 to a titer of 1: 6400; strain N 360-37 / 12 with serum 0149 in a titer of 1: 6400, serum K-88 to a titer of 1: 640 and serum K-91.

Virulence of strains. Strain N 305-37 / 12 has an LD ₅₀ value, for mice equal to 57 million strain N 299-37 / 10 79, N 355-37 / 12 500, N 330-37 / 11 125, N 357-37 / 12 45 , N 331-37 / 12 280, N 359-37 / 12 275, N 360-37 / 12 94 million microbial cells of the daily agar culture.

 Toxic and toxigenic properties. Strain N 305-37 / 12 produces thermolabile (TL) enterotoxin, strain N 355-37 / 121 TL and thermostable (TS) toxin, strains N 359-37 / 12, N 360-37 / 12, 331-37 / 12 form TL and TS toxins, strain N 330-37 / 11 TL toxin.

 All strains possess endotoxin.

PRI me R 1. A method of manufacturing a vaccine against colibacteriosis (Escherichiosis) of piglets is as follows. The Escherichia strains are each inoculated separately in 0.5 cm ³ into 200 cm ³ bottles containing 80-100 cm ³ Hottinger broth having amine nitrogen 150-200 mg% tryptophan 50-100 mg and pH 7.8-8.0.

Strains were grown for 8-10 hours at 37-38 ° ^C. The grown culture is checked for purity. Pure cultures of vials inoculated separately in the half-filled broth Hottinger 10-liter bottles and cultured for 8-10 hours at a temperature of 37-38 ^{° C,} shaking occasionally. Strains are seeded in groups in the reactor, taking into account their colicinogenicity.

In each reactor is inoculated with 5-7% by volume of the medium matrovyh rasplodok grown for 10-14 hours at a temperature of 37-38 ^{° C,} continuous aeration and stirring.

At the end of the cultivation, the pH, the concentration of microbial bodies, the purity of the culture by microscopy and culture on culture media are determined. The resulting bacterial mass in each reactor is diluted with sterile saline to a concentration of 10 billion microbial bodies in 1 cm ³ , pumped 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 temperature 37-38 ^about C.

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 1 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 resulting vaccine has a component composition shown in table 1.

PRI me R 2. The resulting vaccine is administered intramuscularly twice to pregnant sows 1.5-2 months before farrowing, as well as to piglets before weaning in doses: pregnant sows, 4-5 and 5-6 cm ³ , piglets before weaning 1 -1.5 and 1.5-2 cm ³ .

 The vaccine is used in colibacteriosis-poor households to vaccinate pregnant sows 1.5-2 months before farrowing, as well as piglets before weaning.

 The vaccine is administered intramuscularly in the neck region in compliance with aseptic rules, twice with an interval of 10-15 days in the doses shown in table 2.

 Young animals are vaccinated before weaning no earlier than at 21 days of age. Weak young animals are vaccinated in half doses. Immunity occurs 18-20 days after the first dose of the vaccine and persists in adult animals for 5-6 months, in young animals 3-5 months.

 The immunogenic properties of the vaccine were tested in 3 households. The control (unvaccinated) groups of animals (pregnant sows), as well as the young calves born, were in similar conditions of keeping, feeding and caring in the same premises and at the same time together with experimental animals born from vaccinated sows.

 The test was conducted on 464 pregnant sows. The test results showed (see Table 3) that piglets from vaccinated mothers contract colibacteriosis in an average of 8.2% of cases versus 32.6% in the control group, their disease is mild and the piglets left in this group 6.6 times less than in piglets born from unvaccinated sows.

 PRI me R 3. The experimental series of vaccines was compared by immunogenic activity with the prototype vaccine in 10 pregnant sows (5 animals per vaccine). Two sows served as control.

Vaccination was carried out 1.5-2 months before farrowing twice in doses of 4 + 5 s ³ with an interval between doses of 10 days. 24 hours after birth, piglets were infected intraperitoneally with a control strain of E. coli 078 at a dose of 2 × ^{10 10} cells of daily agar culture in physiological saline. The results are presented in table 4.

 Thus, the results of experimental infection of piglets from mothers vaccinated with two vaccines indicate the advantage of the new vaccine, where the safety of piglets was 100% versus 39% for those vaccinated with the prototype vaccine. In addition, the prototype vaccine had increased reactogenicity and caused abscesses and sows at the injection site.

 The proposed vaccine has well-defined antigenic properties, as well as a wide range of immunogenicity, which helps prevent colibacillosis of piglets in various regions of the world.

Claims (1)

Piglet vaccine containing colibacteriosis, 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 saline, and Escherichia coli strain VGNKI N as antigens 305-37 / 10, E.coli VGNKI N 355-37 / 12, E.coli VGNKI N 357-37 / 12, E.coli VGNKI N 331-37 / 12, E.coli VGNKI N 359-37 / 12, E. coli VGNKI N 360-37 / 12, E. coli VGNKI N 299-37 / 10, E. coli VGNKI N 330-37 / 11, taken in equal proportions with the following 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, l To 1

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