CS252037B1 - Bivalent inactivated vaccine against red murrain and infection atrophic rhinitis of pigs - Google Patents

Abstract

The solution relates to inactivated preparation a seedling agent that can be used for specific prophylaxis of piglets and to produce a high titer specific agglutinating serum and colostral antibodies in sows against bordetel antigens, by way of a colostral transfer they create a state of passive protection in piglets in the early postnatal period. bivalent the vaccine contains 40 vol. % bacterin Erysipelothrix insidiosa and 60 vol. % Bordetelia bronchiseptica. mixed the bacterin is adsorbed onto the hydroxide gel of aluminum into the resulting aluminum content 6 to 10 g / l. The bivalent vaccine replaces fully in the immunoprophylaxis of breeding pigs so far produced and used two monovalent vaccines. It finds use in livestock production, where it contributes to better economic efficiency in the production of pork by that it minimizes the number of vaccination interventions against these infectious diseases, decreases the application rate and the amount of stress the burden of vaccinating animals. Implementation of production manufacturers will reduce production costs.

Description

(54) Bivalent inactivated vaccine against robin and infectious airophic rhinitis in pigs

The present invention relates to the preparation of an inactivated vaccine which can be used for the specific prophylaxis of swine roughy and for the production of high titer specific agglutinating serum and colostral antibodies in sows against brothetel antigens which, through colostral transfer, create a passive post-protection status in pigs. The bivalent vaccine contains 40 vol. % of Erysipelothrix insidiosa and 60 vol. % of Bordetelia bronchiseptica bacterin. The mixed bacterin is adsorbed onto an aluminum hydroxide gel to a final aluminum content of 6 to 10 g / l. The bivalent vaccine fully replaces two monovalent vaccines produced and used in immunoprophylaxis of breeding pigs.

It is used in large-scale livestock production, where it contributes to better economic efficiency in pork production by minimizing the number of vaccination interventions against these infectious diseases, reducing the application rate and the number of stress burdens for vaccinating animals. Manufacturing will reduce production costs for manufacturers.

232037

The present invention relates to a bivalent inactivated vaccine from bacterial germs of the species Erysipelothrix insidiosa and Bordetella bronchiseptica, which can be used for the specific prophylaxis of robin rouge and for the production of high titer specific agglutinating serum and colostral antibodies in sows against bordetella colostral transfer antigens piglets in the early postnatal period passive immune status against infectious atrophic rhinitis.

Currently, a purified robin adsorbate vaccine, technologically prepared according to the Czechoslovak patent purely 148,091, is used for immunoprophylaxis of swine robins, which produces a high degree of specific protection without the side effects of allergic effects in vaccinees. The vaccine is given at a dose of 2 ml twice a year, but must be given at least 14 days before the expected birth in the case of high-gravidity pests.

For the immunoprophylaxis of infectious atrophic rhinitis in pigs, an inactivated adsorbate vaccine is currently used. The vaccine is administered to sows in the second half of pregnancy at intervals of 14 days with doses of 10 ml. The second dose must be given at least 14 days before the expected birth. Following this immunization procedure, sows produce, in addition to the production of serum antibodies, specific secretory antibodies that accumulate in the colostrum. The postnatal transfer of immune colostrum from sow to piglet provides for the passive creation of a specific protection status in neonates in the most critical period in terms of etiopathogenesis of this infectious disease.

The current immunization scheme against these infectious diseases is very demanding in terms of operation and technology. It represents a heavy burden for veterinary and animal care staff, especially in large-scale farms, with a high degree of mechanization and a small number of workers. By repeated vaccination procedures, animals are subjected to stress stresses, resulting in a reduction in their performance. Possible simplification of the immunization schedule means reduction of vaccination interventions, technological savings of bioproducts, time and effort involved in their implementation. This can be achieved by technological preparation and inclusion in the immunization schedule of a bivalent vaccine.

Our experiments have shown that individual inactivated adsorbate antigens prepared from Erysipelothrix insidiosis and Bordetella bronchiseptica microorganisms in a predetermined ratio will enhance immunostimulation with each other after parenteral administration of the pigs. This ratio is strictly specific since the mutual immunostimulatory effect was not achieved by experimental verification of other ratios.

The inactivated vaccine, which contains 40 vol. % of Erysipelothrix insidiosa at a final inactivated microorganism concentration of 1 to 4 x 10 ^{1 (J} bacterial bodies in 1 ml and 60 vol% Bordetella bronchiseptica at a final inactivated microorganism concentration of 1 to 4 x 10 ¹⁰ bacterial bodies in 1 ml.

Furthermore, we have shown that by using the experimentally proven immunostimulatory effect of the two types of microorganisms, it is possible to prepare a bivalent vaccine having qualitatively higher parameters than the individual mono-vaccines. The practical consequence of this is that a substantially lower dose of 5 milliliters of bivalent vaccine is capable of inducing a higher degree of specific receptivity in vaccinated pigs than individual monovaccines are capable of. The doses of these mono-vaccines are listed on page 1, paragraphs 2 and 3. A higher degree of specific protection against infectious atrophic rhinitis is documented by the fact that the average titer of agglutinating antibodies is 1: 640 for mono-vaccine. Using a bivalent vaccine prepared by us, titers of serum agglutination antibodies of 1: 640 to 1: 1280 and colostral antibodies of 1: 1280 to 1: 2560 were achieved.

The benefit of the present invention is to replace two monovalent vaccines with the need for a double application, a total of four administrations with one bivalent vaccine with a two-phase application. This will significantly reduce immunoprophylactic interventions in the breeding pig category and reduce the application dose of the bivalent vaccine to a total of 5 ml, achieving greater specific protection for the pigs against these infectious diseases. In addition to the above advantages, the use of the present invention will result in a reduction in manufacturing cost in the field of packaging and adjusting material by 60-70%.

Example

Bacterial mass of single species of microorganisms is technologically prepared separately.

First

Submerged cultivation of Erysipelothrix insidiosa

To a sterile soy nutrient medium (representing an amino acid solution obtained by enzymatic hydrolysis of delipidized soy flour containing 350 mg% total nitrogen, 150 mg% amine nitrogen and 0.4 g / 100 ml sodium chloride) which is tempered to 37 ° C with Aseptic conditions add 1 vol% albumin fraction (blood serum or swine plasma albumin fraction), 1.2 vol% 40% glucose solution and 0.2 vol% aqueous polyoxyethylene sorbitan monooleate (Tween 80). prepared production medium are inoculated with a sub-culture in a quantity by volume. ° / a. subculture should contain 1 ml to 7 χ 10 ⁸ viable microorganisms. Cultivation is carried out at 36 ° C with stirring at 170-360 rev / min and aeration with sterile air 3-4 The pH is maintained at 7.8 and cultured until the permeability has dropped to 12 to 8% (measured per second). a spectrophotometer against a reference sample at 575 nm), but no longer than 15 hours. After completion of the culture, the bacterial culture is inactivated with a 5% aqueous solution of formalin at a ratio of 100 ml per liter of culture. After inactivation, the bacterial mass is concentrated by flow centrifugation so that the final concentration of microorganisms is 1 to 4 x 10 x 10 ¹⁰ in 1 ml.

Second

Submerged culture of Bordetella bronchiseptica

To a sterile soy nutrient medium (representing an amino acid solution obtained by enzymatic hydrolysis of delipidized soy flour containing 350 mg% total nitrogen, 150 mg% amine nitrogen, and 0.4 g / 100 ml sodium chloride), a. Bacto CasamiiiO 'acids (trade name for a mixture of amino acids obtained by acid hydrolysis of casein containing 10% by volume of total nitrogen, 14% by volume of sodium chloride, 15% by volume of ash and 2% by volume of phosphorus pentoxide), 0.08% by weight . Yeast extract (company name for aqueous solution of fresh autolysed yeast) and 0.01 vol. % albumin fraction (albumin active fraction of swine serum or plasma). Subculture in an amount of 1.0 vol. %.

The subculture must contain 2 to χ 10 ⁷ live micro-organisms per ml. The cultivation is carried out at 36-37 ° C with constant stirring at 170 rpm and the supply of sterile air at 7 l / min is not sustained throughout the cultivation. The pH is maintained at 7.6. Cultivation is terminated when the transmittance decreases to 50% (measured on the specimen against the reference sample at 575 nm). The length of the culture is 20 to 36 hours. After completion of the culture, the bacterial culture is inactivated with a 5% aqueous formalin solution in a ratio of 4% to the culture volume. Subsequently, the bacterial mass is concentrated by flow centrifugation so that the final concentration of microorganisms is 1 to 4 χ 10 ^{l ()} in 1 ml.

The inactivated concentrated bacterial masses are then leveled at a rate of 40 percent by volume of Erysipelothrix insidiosa bacterin and 60 percent by volume of Bordetella bronchiseptica bacterin. The mixed bacterin thus prepared is adsorbed onto an aluminum hydroxide gel to a final aluminum content of max. 8 g / l ^-1 .

A bivalent inactivated vaccine is satisfactory if it is free from bacterial and fungal contamination. It is harmless if, after subcutaneous administration of 0.5 ml of bivalent vaccine, none of the vaccinated mice die within an observation period of 7 days after administration.

The immunogenic value of the vaccine is tested on pigs by administering a subcutaneous dose of 2 x 5 ml bivalent vaccine to a group of pigs 14 days apart. 14 days to 21 days after revaccination, the immunized group is tested for immune status against swine robin in the form of an intra-cutaneous infection with skin-ultrulent Erysipelothrix insidiosa strains. Against the antigenic component of Bordetella bronchiseptica, immunized pigs are tested serologically by determining the titer of specific agglutinating antibodies.

The vaccine is of good immunogenic value if, after an intra-cutaneous infection, 48 to 72 hours neither one of the immunized pigs is infected with the cutaneous skin form, nor does it produce an overall thermal response above 40 ° C and elicits a titer of specific agglutinating antibodies against Bordetella bronchiseptica antigens at 1: 640 up to 1: 1280 in serum and 1: 1280 to 1: 2560 in colostrum.

Claims (2)

252037, tempered to 37 ° C, 1 vol% albumin fraction (albumin fraction of blood serum, but also porcine plasma), 1.2 vol% 40% non-glucose solution and 0.2 vol% aqueous solution were added under aseptic conditions. flow of polyoxyethylene sorbitan monooleate (Tween 80). The production medium thus prepared is inoculated with a subculture of 2 vol. The subculture must contain 3 to 7 x 108 live microorganisms per ml. The cultivation is carried out at 36 ° C with constant agitation at 170 to 360 rpm, and aeration with sterile air of 3-4 l / min fed through the surface of the medium does not persist for the entire time of cultivation. The pH is maintained at 7.8. It is cultured until the permeability drops to 12 to 8% (measured on a spectrophotometer for a reference sample at 575 nm), but for a maximum of 15 hours. Upon completion of the culture, the sabacterial culture was inactivated with a 5% aqueous solution of formalin at a ratio of 100 ml per liter of culture. After inactivation of the bacterial mass, it is concentrated by flow centrifugation so that the final concentration of microorganisms is 1 to 4 x 10 10 10 in 1 ml. 2. Submerged cultivation of Bor-detella bronchiseptica To sterile soybean nutrient medium (represents an amino acid solution obtained by enzymatic hydrolysis of delipidized soy meal containing 350 mg% total nitrogen, 150 mg amine nitrogen and 0.4 g / 100 ml sodium chloride) under asep % by weight, 0% by wt. Bacto Casamino, acids (company name for a mixture of amino acids obtained by acid hydrolysis of casein containing 10 vol% total nitrogen, 14 vol% sodium chloride, 15 vol% ash and 2 vol% oxide) % phosphorous), 0.08 wt. Yeast extract (company name for fresh autolysed yeast water solution) and 0.01 vol% albumin fraction (albunar fraction of blood serum or plasma of nude). Subculture is inoculated into the volume of this production medium in a volume of 1.0 vol%. The subculture must contain 2 to 5 x 10 7 live microorganisms per ml. The cultivation takes place at 36-37 ° C with continuous stirring at 170 rpm and the sterile air intake 7 rpm does not persist throughout the culture. The pH is maintained at 7.6. The 50% permeation rate (measured on a Spike versus 575nm reference sample) is discontinued. The length of the culture is 20 to 36 hours. Upon completion of the culture, the bacterial culture is inactivated with a 5% aqueous formalin solution at a ratio of 4% to the culture volume. Subsequently, the bacterial amase is concentrated by flow centrifugation such that the final concentration of microorganisms is 1 to 4 x 101 () in 1 ml. The inactivated concentrated bacterial masses are subsequently leveled at a ratio of 40 percent by volume of the bacterium of Erysipelothrix insidiosa microorganism and 60 percent by volume of Borde-tella bronchiseptica bacterin. The mixed bacterin thus prepared is adsorbed onto the aluminum hydroxide gel to a final aluminum content of max. 8 g / l. The bivalent inactivated vaccine is useful if it is free of bacterial and fungal contamination. It is harmless if, after subcutaneous administration of 0.5 ml of the bivalent vaccine, none of the vaccinated mice is present at the observation time of 7 days after administration. The immunogenic value of the vaccine is tested in pigs by administering to the pig group a subcutaneous dose of 2 x 5 ml of a bivalent vaccine at 14 days apart. 14 days to 21 days after revaccination, the immune group is tested for immune status in swine pigs by intra-ocular infections with Ery-sipelothrix insidiosa skin-fluffy strains. Immunized pigs tested serologically against the antigenic component of Bordetella bronchisepticase with the determination of the titer of specific agglutinating antibodies. The vaccine has a good immunogenic value when, after an intradermal infection in 48-72 hours, none of the immunized pigs in the red skin form nor does it undergo a total thermal response above 40 ° C and induce a titer of specific agglutinating antibodies against the Bordetella bronchiseptica antigens in 1: 641) to 1: 1,280 and 1: 1,280 to 1: 2560 in colostrum. OBJECT What is claimed is: 1. A bivalent inactivated red robin and infectious atrophic rhinitis vaccine comprising 40% by volume of Erysipelothrixinsidiosa bacterin at a final concentration of inactivated microorganisms of 1 to 4 x 10 < 10 > the volume of Bordetella bronchiseptica bacterium in the resultant concentration of inactivated microorganisms 1 to 4 x 10 10 bacterial bodies per ml. 2. A bivalent inactivated vaccine according to claim 1, wherein the particulate bacterin is adsorbed onto the aluminum hydroxide gel to a final aluminum content of 6 to 10 g / l, preferably 8 g / l.