Method for manufacturing of polyvalent serum against psevdomenosis of agricultural animals

FIELD: veterinary science.

SUBSTANCE: method includes hyperimmunisation of stud bulls with polyvalent antigen, which is produced by cultivation of epizootic strains grown separately in Hottinger broth, having pH 7.2 -7.4, and mixed in equal proportions with further inactivation with formalin, blood draw to prepare serum, separation of serum with further preservation and control for activity. At the same time as epizootic strains they use Pseudomonas aeruginosa of serotypes 01, 02, 03, 04, 06, 010, 011, 013, 018, 019, and immunisation is carried out in cycles with growing doses of antigen. Antigen is injected subcutaneously not more than 14 times in 3-4 days for 51 days, starting from 5 ml, besides two subsequent doses each time are increased twice, and the remaining ones - 1.3 times more compared to each previous one, and the last four doses of not more than 150 ml. Blood draw in servicing animals for control definition of titres is done in 8, 22, 32 days from the beginning of antigen injection. In order to produce serum, blood is drawn at 52-53 day from the start of antigen injection.

EFFECT: serum has high efficiency for treatment of pigs and calves, as well as for sows.

2 cl, 1 tbl, 1 ex

 

The invention relates to veterinary medicine, in particular to methods of manufacture of sera, and can be used in institutions for the creation of whey products.

A method of obtaining polyvalent O-serum (ed. St. No. 789116, CL A61K 39/40, 1980), namely that animal producers are subjected to immunization increasing doses politicheskogo preparation obtained by mixing in equal proportions of O-antigens isolated from different serological groups of one species of bacteria pre-treated by boiling for 20-25 h, while immunization are cycles of four injections with an interval between cycles 12-14 days, and between injections - 5-6 days, and the first injection are subcutaneously, followed by intravenous.

The closest in technical essence is a method of obtaining a hyperimmune serum against colibacillosis, salmonellosis of Klebsiella calves, piglets, lambs (see RF patent № 2306954, CL A61K 39/40, 2007 - prototype), including hyperimmunization bulls-producers polyvalent antigen, obtained by cultivation of epizootic strains were grown separately in broth of Hottinger having a pH of 7.2 to 7.4, and mixed in equal proportions with subsequent inactivation by formalin, drawing blood to obtain serum, Department of serum posleduyushim canning.

However, the known serum to prevent Pseudomonas farm animals is not applied.

Technical solution to the problem is to increase the efficiency of preventive measures against diseases caused by the pathogen Pseudomonas aeruginosa, and expansion of the spectrum.

This object is achieved in that in a method of producing polyvalent serum against Pseudomonas farm animals, including hyperimmunization bulls-producers polyvalent antigen, obtained by cultivation of epizootic strains were grown separately in broth of Hottinger having a pH of 7.2 to 7.4, and mixed in equal proportions with subsequent inactivation by formalin, drawing blood to obtain serum, the separation of serum, followed by canning, according to the invention, as epizootic strains using Pseudomonas aeruginosa serotypes O1, O2, O3, O4, O6, O, O11, A13, 18O, O and conduct immunization in cycles of increasing doses of antigen injected antigen subcutaneously not more than 14 times in 3-4 days for 51 days, starting with 5 ml, and two subsequent doses every fold in half, and the rest in 1, 3 times each previous and the last four doses of not more than 150 ml, and the taking of the blood in animals-producers for the control definition titles carry through by 8.22, 32 days from start of injection of the antigen, and serum blood take 52-53 day from the start of injection of the antigen. In the method of obtaining a polyvalent serum against Pseudomonas agricultural animal control serum on the activity carried out on white mice.

The novelty of the claimed proposal is that the use of epizootic strains of Pseudomonas aeruginosa serotypes O1, O2, O3, O4, O6, O, O, A13, 18O, O provides the possibility of increasing the effectiveness of preventive measures against diseases caused by the pathogen Pseudomonas aeruginosa, and the application of the proposed scheme hyperimmunization animal-producers and control serum on the activity by white mice simplify the process and reduce costs for preparation of serum.

According to scientific-technical and patent literature is not detected is similar to the claimed combination of features, allowing us to obtain a technical result, which has not previously been achieved by known means, which allows to judge about the inventive step of the claimed proposal.

The proposed method of obtaining serum against Pseudomonas farm animals meets the criterion of "industrial applicability"because reproduce, and possibly widespread use in cultivation of agricultural animals.

An example of the spiral is tion of the method of obtaining the polyvalent serum against Pseudomonas farm animals.

The process of making whey can be divided into several stages.

The 1st stage. Pre-prepared nutrient medium for cultivation of epizootic strains, which are used Pseudomonas aeruginosa serotypes O1, O2, O3, O4, O6, O, O, A13, 18O, O. To do this, use the broth of Hottinger prepared from primary parivara of Hottinger, this prepared minced meat, freed from bones, fat, ligaments and tendons. Per I kg of meat take 1.5 l of distilled water (pH of 6.8 to 7.0), heated to a temperature of 40-42°C, and alkalinized chemically pure sodium bicarbonate (GOST 2156.76) or 10%solution of sodium hydroxide (GOST 4328-77) to a pH of 7.8 to 8.0. In I liter of the mixture add 150-200 g purified from membranes and minced in a meat grinder pancreas of bovine or 20-30 g of Pancreatin and 20 ml of chemically pure chloroform. After adding the ingredients, the mixture is thoroughly stirred and left to digest at a temperature of 40-42°C for 3-4 days. The first 6 hours, the mixture is stirred every hour, and then 3-4 times a day. In the process of digesting daily determine the pH of the medium. In the case of reducing the pH prewar alkalinized to 7.8 to 8.0 by adding 10%aqueous solution of sodium hydroxide. After these deadlines, the meat becomes friable greyish precipitate, which when right Pereverzeva the AI of the upper liquid layer has a straw-yellow color. The decrease in the percentage of tryptophan indicates the readiness of parivara. By this time the pH is stabilized in the range of 7.6 to 8.0. Chemical characteristics of parivara: total nitrogen 800-1200, amino nitrogen 700-900 and tryptophan 100-200 mg, %.

Then prepare a broth of Hottinger used for this transparent supernatant main parivara, which is diluted with distilled water to a content in the broth 200-300 mg % amino nitrogen. Medium heat and add 0.5% peptone (GOST 13805-76), 0.5 % sodium chloride (GOST 4233-77), 0,3% chemically pure Doonbeg phosphate sodium (GOST 11773-76) and 10% water to boiling. Wednesday boiled for 30 minutes. During boiling establish a pH of 6-8,0 by adding a 10%solution of sodium hydroxide. Wednesday again boiled for hours, leave in the same digester to lag on 1-1,5 hours and filtered to full transparency through a thick layer of cotton wool and gauze. The medium is pumped into another pre-prepared sterile reactor, which fill no more than 2/3 of the volume and sterilized at a temperature of 118-120°C for 45-50min. Prepared nutrient medium should have the following characteristics: pH 7,2-7,4; 200-250 amino nitrogen and tryptophan 50-100 mg, %.

The 2nd stage. After preparation of the nutrient medium, proceed to the preparation of inoculum. For the manufacture of series of each antigen is the first time they use a separate ampoule of freeze-dried culture of strain. The culture of each strain in the ampoule resuspended to the original volume of sterile meat-peptone bouillon or broth of Hottinger sow from MPA 3-4 bacteriological Cup according to the following procedure: 0.1 to 0.2 ml of the suspension culture of strain contribute to the surface of the agar and a glass spatula spread it over the surface of the agar. This spatula is carried out on the surface of agar the second, then the third Cup, incubated for 18-24 hours at 37-37,5°C. In the third Cup should be the growth of isolated colonies. At the same time doing the sowing in tubes with BCH, MPA MPB under paraffin oil and the environment Saburo, incubated at the same temperature (environment Saburo 20-24°C) crops on MPA and MPB three days, and on MPB under paraffin oil and the environment Saburo 5 days. After a period of incubation the culture of each strain individually checked for purity and the nature of the growth of the visual view and the view of smears stained by gram stain. From cultures grown on agar in cups, take 2 - 3 pigmentproducing colony and sown in the tubes of broth, Hottinger each strain separately and incubated at 37-37 .5°C 18-24 hours. Check the purity of the growth browsing smears, gram stained, and sow each strain separately on the broth of Hottinger vials or bottles in the ratio of 1:100 and grown.

To obtain the matrix is asplode of vials or bottles clean daily broth culture of each strain individually subcultured in warmed up to 36-37°C broth of Hottinger cylinders based 80-100 ml per 10 liters of medium. Crops cultivated at 37-37 .5°C for 18-24 hours, check the cleanliness of growth. The optical concentration of the culture should be not less than 0.8-1.0 billion microbial cells in I ml

The 3rd stage. Further, the growing culture is carried out in reactors to produce the bacterial mass.

After checking matrix of the seed purity by microscopy of stained smears for the visual detection of gram and visual definition character growth in the reactor or tank broth of Hottinger, warmed up to 36-37°C, make a matrix seed is obtained in the amount of 5-8% by volume of the nutrient medium. To reduce foaming to a nutrient medium in the reactor add 0,03-0,05% sterile antifoam (sunflower, plum, apricot or peach oil). The culture of each strain inoculated and grown in a separate reactor or tank.

After making the matrix of the seed in the reactor the reactor is stirred with a mechanical stirrer for 1-2 minutes with the following speed and leave for the cultivation of 1.5-2 hours. After 1.5-2 hours cultivation continue with continuous stirring and simultaneous aeration with sterile air. Speed mechanical mixer should be in the range of 150-240 revolutions per minute, the amount of blown air 1-1,5 volume per minute of the total volume of aerated culture. Cultivated the e carried out at a temperature 37-37 .5°C and stopped after 24-36 hours under the condition of a slight increase or preventing the accumulation of bacterial mass. By this time the concentration of the bacterial mass should be no higher than 20 billion M.K. in I ml of optical turbidity standard of gisk named after. Tarasevich. 6-8 hours after the beginning of cultivation and subsequently every 2-3 hours to carry out sampling. In the samples tested the concentration of microbial cells on the optical turbidity standard of gisk named after. Tarasevich. To maintain within the pH to 7.2 to 7.4 using 10% solution of sodium hydroxide. With the increase of pH from the original in the alkaline side add sterile 40%glucose solution to a concentration of 0.1%. After a period of incubation, the heating of the reactor was stopped and cooled to a temperature of 6-8°C. the Grown culture of each strain separately check for indicators: clean - microscopy of smears stained by gram stain; culture on the MPA, MPB, Wednesday, Saburo, MPB under vaseline oil; pH - electrometric method and concentration of microbial cells on the optical turbidity standard of gisk named after. Tarasevich. Culture must be clean, to contain not less than 20 billion microbial cells in I ml, pH 7,2-7,4.

4-th stage. Preparation of antigen for hyperimmunization oxen-producers carried out after the establishment of purity, the concentration of bacterial mass and pH of the culture O1, O2, O3, O4, O6, O, O, A13, 18O, O serotypes in reactors bred appropriate number of sterile physical and the logical solution to a concentration of 10 billion/ml of microbial cells, then the biomass of each serotype is pumped and mixed in the same container in equal volumes. When the surplus obtained bacterial mass estimated number of received culture is pumped into another (other) reactors or tanks, providing mixing and maintaining the required temperature of 6-8°C. After mixing, the suspension cultures subjected to inactivation by formalin.

The amount of polyvalent culture, intended for production of antigen for subcutaneous injection, when the stirrer is added formalin (GOST 1625-75) to a concentration of 0.3% in total volume and incubated at a temperature of 37-38°C for 20 days. Formalin should contain not less than 36% formaldehyde. The prepared antigen test for sterility, pH, residual formalin and emissions introduction subcutaneously five white mice in a dose of 0.5 ml Mouse must remain alive for five days, and the crops of sterile within 10 days of observation, the pH should be between 7.0 to 7.2, the content of formalin should be no more than 0,09%, control serum on the activity performed on white mice. Tested for sterility, potency and safety of the antigen used for hyperimmunization animals. The shelf life of polyvalent antigen for up to six months if kept in a dark place at a temperature of 2-15°C. 5-th stage. Then avodat immunization in cycles of animals-producers of increasing doses of antigen, but pre-training animals-producers, which use oxen.

Brought to biopreparation oxen are subjected to quarantine, then examination and appropriate treatment against infectious diseases in accordance with the "Basic veterinary harvesting regulations, animals and procurement of eggs used in the biological industry, approved the BS of the Ministry of agriculture of the USSR 17.03.81,

After quarantine, examination and processing of healthy oxen transferred to serum shop for hyperimmunization. Admitted to the operation of the animals aged 2.5-5 years old, weighing not less than 400 kg

Hyperimmunization oxen-producers carried out according to the following scheme (see table 1).

As can be seen from the scheme, administered antigen subcutaneously not more than 14 times in 3-4 days for 51 days, starting with 5 ml, and two subsequent doses every fold in half, and the rest in 1, 3 times each previous and the last four doses of not more than 150 ml, and the taking of the blood in animals-producers for the control definition titles carry through 8, 22, 32 days from start of injection of the antigen, and serum blood take 52-53 day from the start of injection of the antigen.

Table I
The proposed scheme hyperimmunization p is duzentos
No.The days of injections of antigenNumber of days through which the injected antigenThe dose of antigen in mlNumber of days engaged in drawing blood after injection of antigen
Grandemente
I105,0
24310,0
38420,08
411330,0
515440,0
618350,0
722 470,022
825390,0
9294110,0
10323150,032
Hyperimmunization
11353150
12394150
13434150
1451815051

After subcutaneous administration of antigen in large doses it is administered in different places of the neck, upper body, but not of the former is 10-15 cm from the rear edge of the blade. Through 8, 22, and 32 days after the start of injection of the antigen from the producers of the jugular veins take blood in tubes for the control definition titles. Antibody titers to each serotype is determined in vitro agglutination reaction generally accepted method. As the antigen used daily suspension culture of each serotype grown on MPA in cups, washed with a saline solution (pH of 7.2 to 7.4, containing 0.5 billion tons of microbial cells in I ml of optical standard turbidity), inactivated by formalin. Antibody titers in RA after the 10th injection of antigen must be at least 1:800 estimated at three to four cross to each serotype. In operation, after each blood collection is carried out, depending on the mass producer, two-time introduction of antigen: the first time in the half-dose (75-100 ml) subcutaneously in 5-6 days after blood collection, the second time in full dose (150-200 ml) after 5-6 days after the first injection of antigen. After 5-6 days after the second injection of antigen produce another blood sample, etc. Exploited ox-producers annually provide a month's vacation from blood collection (better in the summer). In the second half of rest (15-20 days) producers subcutaneously three times with an interval of 5-6 days taking into account the weight of the animals injected polyvalent antigen, inactivated by formalin: first time at a dose of 80-100 ml, p the reappointment 110-150 ml and third time 150-200 ml and begin the next cycle of operation of the producers.

The 6th stage. The blood sampling and obtaining serum. A test blood sample from the producers produce at a rate of not more than 0.8 liters per 100 kg of live weight, and further to 1.6 liters. Blood is collected from producers with a normal body temperature after a preliminary 12-hour endurance on the starvation diet with unlimited watering. Blood will be drawn into a sterile graduated bottles with a capacity of 15-20 liters. Then the blood sephirot, and the resulting plasma defibrilator. To prevent blood from clotting used anticoagulant - 1%solution of sodium citrate, which is prepared with distilled water or saline solution and sterilized in an autoclave at a temperature of 120°C for 30 minutes

7-th stage. Control serum. Check serum emissions, sterility known methods, and the activity on white mice.

The effectiveness of the proposed sera confirmed by comparative data (see table 2), which were obtained by conducting experiments with 9 groups of farm animals (pigs, calves and sows), 3 of which each species was used, we offer whey, 3 other used traditional methods of treatment by chemotherapy, antibiotics, and group 3 animal control, which was not treated.

td align="right" namest="c0" nameend="c5"> Table 2
Comparative data confirming the effectiveness of the proposed whey.
No.AnimalThan treated doseAll animals in the groupCuredThe efficiency percentage, %
1.Pigsserum, 0.5 ml/kg of body weight22918982,5
2.Pigsstreptomycin, polymyxin925660,9
3.Pigscontrol241145,8
4.Calvesserum, 0.5 ml/kg of body weight282485,7
5.Calvesstreptomycin, polymyxin2462,5
6.Calvescontrol7342,8
7.Sowsserum, 0.5 ml/kg of body weight544888,9
8.Sowsstreptomycin, polymyxin483777,1
9.Sowscontrol362158,3

Table 2 shows that compared with the traditional method of treatment offered serum against Pseudomonas farm animals is effective for the treatment of young pigs and calves and sows, in all cases, the percentage of treatment effectiveness is quite high, with an average of 85.7 %.

1. A method of obtaining a polyvalent serum against Pseudomonas farm animals, including hyperimmunization bulls-producers watering entnum antigen, obtained by cultivation of epizootic strains were grown separately in broth of Hottinger having a pH of 7.2 to 7.4, and mixed in equal proportions with subsequent inactivation by formalin, drawing blood to obtain serum, Department of serum with subsequent preservation and control of the activity, characterized in that as the epizootic strains using Pseudomonas aeruginosa serotypes 01, 02, 03, 04, 06, 010, 011, 013, 018, 019 and conduct immunization in cycles of increasing doses of antigen, when administered antigen subcutaneously not more than 14 times in 3-4 days for 51 days starting with 5 ml, and two subsequent doses every fold in half, and the rest in 1, 3 times each previous, and the last four doses of not more than 150 ml, and the taking of the blood in animals-producers for the control definition titles carry through 8, 22, 32 days from start of injection of the antigen, and serum blood take 52-53 day from the start of injection of the antigen.

2. A method of obtaining a polyvalent serum against Pseudomonas farm animals according to claim 1, characterized in that the control serum on the activity carried out on white mice.



 

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4 tbl

FIELD: medicine, veterinary and food processing industry.

SUBSTANCE: claimed method includes manufacturing of corn-shell-type latex, wherein corn represents polystyrene latex and shell represents styrene-zinc methacrylate copolymer latex in repeating unit mass ratio of 1:0.8-1:0.4. Then latex is sensitized with monoclonal antibodies of lgG2a and lgG2b fractions in 0.01 M glycine buffer at pH 7.2-8.8 without preliminary conditioning followed by incubation primarily at 37°C for 130-150 min and further at 8°C for 18 hours. Obtained diagnosticum has specificity of 97±0.6, sensibility up to 10 pg/ml and makes it possible to obtain investigation results for 10 min or less.

EFFECT: accelerated method for diagnosticum production.

3 tbl

FIELD: medicine, veterinary and food processing industry.

SUBSTANCE: claimed method includes manufacturing of corn-shell-type latex, wherein corn represents polystyrene latex and shell represents styrene-zinc methacrylate copolymer latex in repeating unit mass ratio of 1:0.8-1:0.4. Then latex is sensitized with monoclonal antibodies of lgG2a and lgG2b fractions in 0.01 M glycine buffer at pH 8.2-8.6 without preliminary conditioning followed by incubation primarily at 36-38°C for 120-180 min and further at 2-8°C for 1-20 hours. Obtained diagnosticum has specificity of 99±0.89, sensibility up to 10 pg/ml and makes it possible to obtain investigation results for 10 min or less.

EFFECT: accelerated method for diagnosticum production.

6 tbl

FIELD: veterinary microbiology.

SUBSTANCE: claimed method includes providing of stable culture from B.abortus 19 strain in L-form followed by rabbit immunization. Culture is obtained in dense broth containing additionally 10-15 % of normal hoarse serum wherein broth is singly exposed to streptomycin action in dose of 2.5-5.0 U/ml of medium. Then slurry is prepared from obtained culture, inactivated at 85-90°C for 160 min and triply intravenously administrated to rabbits in increasing doses with 72 h intervals.

EFFECT: method for more exact estimation of brucelliasis epizootic situation on basis of typical, dissociated and deep-altered brucella forms.

6 tbl, 4 ex

FIELD: molecular biology, veterinary.

SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.

EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.

19 cl, 17 dwg, 8 ex

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