RU2376034C1 - Method for manufacturing of polyvalent serum against psevdomenosis of agricultural animals - Google Patents

Abstract

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

Description

The invention relates to veterinary medicine, in particular to methods for the manufacture of serums, and can be used in institutions involved in the creation of serum preparations.

A known method for producing polyvalent O-serum (ed. St. No. 789116, class A61K 39/40, 1980), which consists in the fact that animal producers are immunized with increasing doses of a polyantigenic preparation obtained by mixing in equal proportions of O-antigens isolated from different serological groups of one bacterial species, previously treated by boiling for 20-25 hours, while immunization is carried out in cycles of four injections with an interval between cycles of 12-14 days, and between injections - 5-6 days, moreover, the first injection spend subcutaneously, and p following - intravenously.

The closest in technical essence is a method for producing hyperimmune serum against colibacteriosis, salmonellosis of klebsiellosis of calves, piglets, lambs (see RF patent No. 2306954, CL A61K 39/40, 2007 - prototype), including hyperimmunization of bulls-producers with a polyvalent antigen, obtained by culturing epizootic strains grown separately on Hottinger broth, having a pH of 7.2-7.4, and mixed in equal proportions followed by formalin inactivation, taking blood to obtain serum, separating serum from leduyuschim canning.

However, the known serum for the prevention of pseudomonosis of farm animals is not used.

The technical solution to the problem is to increase the effectiveness of preventive measures against diseases caused by the pathogen Pseudomonas aeruginosa, and to expand the spectrum of action.

The problem is achieved in that in a method for producing polyvalent serum against pseudomonosis of farm animals, comprising hyperimmunizing bulls with a polyvalent antigen obtained by culturing epizootic strains grown separately on Hottinger broth, having a pH of 7.2-7.4, and mixed in equal ratios followed by inactivation with formalin, blood sampling to obtain serum, separation of serum, followed by conservation, according to the invention, as an epizootic of their strains use Pseudomonas aeruginosa serotypes O1, O2, O3, O4, O6, O10, O11, O13, O18, O19 and carry out immunization in cycles of increasing doses of antigen, inject the antigen subcutaneously no more than 14 times after 3-4 days for 51 days starting with 5 ml, and the two subsequent doses are doubled each time, and the rest are 1, 3 times more than each previous and the last four doses are not more than 150 ml, while blood is taken from animal producers for the control determination of titers after 8, 22, 32 days from the start of antigen administration, and blood is taken to obtain serum ie 52-53 days after the onset of administration of the antigen. In a method for producing polyvalent serum against pseudomonosis of farm animals, the control of serum activity is carried out on white mice.

The novelty of the proposed proposal is that the use of serotypes O1, O2, O3, O4, O6, O10, O11, O13, O18, O19 as epizootic strains - Pseudomonas aeruginosa, provides the opportunity to increase the effectiveness of preventive measures against diseases caused by the pathogen Pseudomonas aeruginosa, and the application of the proposed scheme for hyperimmunization of animal producers and control of serum activity through white mice simplify the process and reduce the cost of preparing serum.

According to the scientific, technical and patent literature, a similar set of features has not been found that allows obtaining a technical result that was not previously achieved by known means, which allows one to judge the inventive step of the claimed proposal.

The proposed method for producing serum against pseudomonosis of farm animals meets the criterion of "industrial applicability", since it is reproducible, and widespread use is possible when growing agricultural animals.

An example of a specific implementation of the method for producing polyvalent serum against pseudomonosis of farm animals.

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

1st stage. Nutrient medium is preliminarily prepared for culturing epizootic strains, for which Pseudomonas aeruginosa serotypes O1, O2, O3, O4, O6, O10, O11, O13, O18, O19 are used. To do this, use the Hottinger broth, prepared from the main Hottinger pass, for this mincemeat is prepared from meat freed from bones, fatty tissue, ligaments and tendons. 1.5 l of distilled water (pH 6.8-7.0), heated to a temperature of 40-42 ° С, is taken for I kg of minced meat and made alkaline with chemically pure sodium bicarbonate (GOST 2156.76) or 10% sodium hydroxide solution ( GOST 4328-77) to a pH of 7.8-8.0. To I liter of the mixture add 150-200 g of cattle, cleaned from shells and crushed in a meat grinder, or 20-30 g of pancreatin and 20 ml of chemically pure chloroform. After adding the ingredients, the mixture is thoroughly mixed and left for digestion 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 digestion daily determine the pH of the medium. In the case of a decrease in pH, the digest is alkalinized to 7.8-8.0 by the addition of a 10% sodium hydroxide solution. After the indicated dates, the minced meat turns into a loose grayish precipitate, over which, with proper digestion, the upper liquid layer has a straw-yellow color. A decrease in the percentage of tryptophan indicates the readiness of the digest. By this time, the pH is stabilized in the range of 7.6-8.0. Chemical parameters of the digest: total nitrogen 800-1200, amine nitrogen 700-900 and tryptophan 100-200 mg,%.

Then, Hottinger broth is prepared; for this, a transparent supernatant of the main digest is used, which is diluted with distilled water to contain 200-300 mg% of amine nitrogen in the broth. The medium is heated and 0.5% peptone (GOST 13805-76), 0.5% sodium chloride (GOST 4233-77), 0.3% chemically pure dibasic sodium phosphate (GOST 11773-76) and 10% boiling water are added . The medium is boiled for 30 minutes. During boiling, a pH of 6-8.0 is established by adding a 10% sodium hydroxide solution. The medium is again boiled for an hour, left in the same digester to lag for 1-1.5 hours and filtered until completely transparent through a dense layer of cotton wool and gauze. The medium is pumped into another pre-prepared sterile reactor, which is filled with no more than 2/3 of the volume, and sterilized at a temperature of 118-120 ° C for 45-50 min. Ready nutrient medium should have the following indicators: pH 7.2-7.4; amine nitrogen 200-250 and tryptophan 50-100 mg,%.

2nd stage. After preparing the nutrient medium begin to prepare the seed. For the manufacture of a series of antigen each time use a separate ampoule with a lyophilized culture of the strain. The culture of each strain in the ampoule is resuspended to the initial volume with sterile meat-peptone broth or Hottinger broth, seeded from MPA into 3-4 bacteriological plates according to the following procedure: 0.1-0.2 ml of the suspension of the strain culture is applied to the surface of the agar and spread with a glass spatula her on the surface of the agar. The same spatula is carried out on the surface of the agar of the second, and 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, inoculations are made in test tubes with MPB, MPA MPPB under vaseline oil and Saburo medium, incubated at the same temperature regime (for Saburo medium 20-24 ° С), crops on MPA and MPB for three days, and on MPPB under vaseline oil and Saburo medium 5 days. At the end of the incubation period, the culture of each strain is individually checked for purity and growth by visual inspection, as well as viewing smears stained by Gram. From cultures grown on agar in cups, 2–3 pigment-producing colonies were selected and each strain was plated in tubes with Hotinger broth separately and incubated at 37–37.5 ° С for 18-24 hours. The purity of growth is checked by viewing smears stained according to Gram, and each strain is plated individually on Hottinger broth in bottles or bottles in a ratio of 1: 100 and cultivated.

To obtain a matrix seedling from vials or bottles, the pure diurnal broth culture of each strain is individually subcultured into Hottinger broth heated to 36-37 ° C in containers at the rate of 80-100 ml per 10 liters of medium. Crops are cultivated at 37-37.5 ° C for 18-24 hours, check the purity of growth. The optical concentration of the culture should be at least 0.8-1.0 billion microbial cells in I ml.

3rd stage. Next, the culture is carried out in reactors to obtain a bacterial mass.

After checking the matrix seedling for purity by microscopy of stained smears of Gram visual determination and visual determination of the nature of growth in the reactor or cylinder with Hottinger broth, heated to 36-37 ° C, the matrix seed is introduced in the amount of 5-8% of the volume of the nutrient medium. To reduce foaming, 0.03-0.05% of a sterile antifoam (sunflower, plum, apricot or peach oil) is added to the reactor medium. The culture of each strain is seeded and grown in a separate reactor or balloon.

After introducing the matrix seedling into the reactor, the contents of the reactor are stirred with a mechanical stirrer for 1-2 minutes with the speed indicated below and left for cultivation for 1.5-2 hours. After 1.5-2 hours, cultivation is continued with continuous stirring and simultaneous aeration with sterile air. The frequency of rotation of the mechanical mixer should be in the range of 150-240 revolutions per minute, the amount of purged air is 1-1.5 volumes per minute to the total volume of the aerated culture. Cultivation is carried out at a temperature of 37-37.5 ° C and stopped after 24-36 hours, provided that the accumulation of bacterial mass is slightly increased or stopped. By this time, the concentration of the bacterial mass should be no higher than 20 billion m. in I ml according to the optical turbidity standard GISK im. Tarasevich. 6-8 hours after the start of cultivation and subsequently every 2-3 hours sampling is carried out. In the selected samples, the concentration of microbial cells is checked according to the optical turbidity standard of GISK named after Tarasevich. To maintain a pH of 7.2-7.4, a 10% sodium hydroxide solution is used. With increasing pH from the initial alkaline side, a sterile 40% glucose solution is added to a concentration of 0.1%. After the incubation period, the heating of the reactor is stopped and cooled to a temperature of 6-8 ° C. The grown culture of each strain individually is checked for indicators: purity by microscopy of smears stained by Gram; sowing on MPA, MPB, Saburo medium, MPPB under liquid paraffin; pH - electrometric method and the concentration of microbial cells according to the optical turbidity standard GISK them. Tarasevich. The culture should be clean, contain at least 20 billion microbial cells in I ml, pH 7.2-7.4.

4th stage. Antigen preparation for hyperimmunization of producer oxides is carried out after establishing purity, concentration of bacterial mass and pH, cultures of O1, O2, O3, O4, O6, O10, O11, O13, O18, O19 serotypes in reactors diluted with an appropriate amount of sterile physiological saline to a concentration of 10 billion / ml of microbial cells, then the biomass of each serotype is pumped and mixed in one tank in equal volumes. With an excess of the obtained bacterial mass, the calculated amount of the obtained culture is pumped into other (other) reactors or tanks, which provide mixing and maintaining the required temperature regime of 6-8 ° C. After mixing, the suspension of cultures is subjected to formalin inactivation.

Formalin (GOST 1625-75) is added to the volume of the polyvalent culture intended for the manufacture of antigen for subcutaneous administration with the stirrer switched on to a concentration of 0.3% in the total volume and kept at a temperature of 37-38 ° С for 20 days. Formalin must contain at least 36% formaldehyde. The prepared antigen is checked for sterility, pH, residual formalin and for harmless administration by subcutaneous injection to five white mice at a dose of 0.5 ml. Mice should remain alive for five days, and the cultures were sterile for 10 days of observation, the pH should be 7.0-7.2, the formalin content should be no more than 0.09%, and serum activity was also tested on white mice. Tested for sterility, activity and safety, the antigen is used to hyperimmunize animals. The shelf life of the polyvalent antigen is up to six months, provided that it is stored in a dark place at a temperature of 2-15 ° C. 5th stage. Then immunization is carried out in cycles of animal producers with increasing doses of antigen, but preliminary preparation of animal producers is carried out, in which oxen are used.

The oxen brought to the biological enterprise are quarantined, then examined and necessary treated against infectious diseases in accordance with the Basic Veterinary Rules for the Harvesting, Keeping of Animals and the Purchase of Eggs Used in the Biological Industry, approved by the GUV Ministry of Agriculture of the USSR on March 17, 81.

After quarantine, inspection and processing of healthy oxen are transferred to the serum workshop for hypermunization. Animals aged 2.5-5 years and weighing at least 400 kg are allowed for use.

Hyperimmunization of producing oxen is carried out according to the following scheme (see table 1).

As can be seen from the scheme, the antigen is injected subcutaneously no more than 14 times after 3-4 days for 51 days, starting with 5 ml, with the next two doses being doubled each time, and the rest - 1, 3 times more than each previous and the last four doses are not more than 150 ml, while blood sampling from animal producers for the control determination of titers is carried out after 8, 22, 32 days from the start of antigen administration, and to obtain serum, blood is taken at 52-53 days from the start of antigen administration.

Table I
The proposed scheme of hyperimmunization of producers No. Days of antigen injection The number of days through which the antigen was administered Doses of antigen in ml The number of days through which blood was taken after administration of antigen Grundimmunization I one 0 5,0 2 four 3 10.0 3 8 four 20,0 8 four eleven 3 30,0 5 fifteen four 40,0 6 eighteen 3 50,0 7 22 four 70.0 22 8 25 3 90.0 9 29th four 110.0 10 32 3 150.0 32 Hyperimmunization eleven 35 3 150 12 39 four 150 13 43 four 150 fourteen 51 8 150 51

With subcutaneous administration of antigen in large doses, it is administered in different places of the neck, upper body, but not closer than 10-15 cm from the posterior edge of the scapula. 8, 22, and 32 days after the start of antigen administration, jugular vein producers draw blood into tubes for the control determination of titers. Antibody titers for each serotype are determined in an agglutination test tube by a conventional method. The daily suspension of each serotype culture grown on MPA in plates washed with physiological saline (pH 7.2-7.4, containing 0.5 billion tons of microbial cells in I ml according to the optical turbidity standard) inactivated with formalin is used as antigen. Antibody titers in RA after the 10th administration of antigen should not be lower than 1: 800 with an estimate of three to four crosses for each serotype. In the process of operation, after each blood sampling, depending on the weight of the producer, two-fold administration of antigen is carried out: the first time in half the dose (75-100 ml) subcutaneously 5-6 days after the blood draw, the second time in the full dose (150-200 ml ) 5-6 days after the first injection of antigen. 5-6 days after the second injection of antigen, another blood sample is taken, etc. Produced oxen-producers are annually provided with a monthly rest from taking blood (preferably in the summer). In the second half of the rest (after 15-20 days), the producers are injected three times subcutaneously with an interval of 5-6 days, taking into account the weight of the animals, the polyvalent antigen inactivated by formalin: the first time at a dose of 80-100 ml, the second time 110-150 ml and the third time 150- 200 ml and begin the next cycle of operation of the producers.

6th stage. Taking blood and getting serum. Trial blood sampling from the producers is carried out at the rate of not more than 0.8 liters per 100 kg of live weight, and the subsequent 1.6 liters. Blood is taken from producers with normal body temperature after a preliminary 12-hour exposure to a hungry diet with unlimited drinking. Blood is taken in sterile graduated bottles with a capacity of 15-20 liters. Then the blood is separated, and the resulting plasma is defibrinated. To protect blood from clotting, an anticoagulant is used - a 1% solution of sodium citrate, which is prepared in distilled water or physiological saline and sterilized in an autoclave at a temperature of 120 ° C for 30 minutes.

7th stage. Serum control. Check the serum for harmlessness, sterility by known methods, and activity on white mice.

The effectiveness of the proposed serum is confirmed by comparative data (see table 2), which were obtained as a result of experiments with 9 groups of farm animals (pigs, calves and sows), in 3 of which each type of animal was used, the proposed serum 3 others used traditional methods of treatment by means of chemotherapy, antibiotics and 3 groups of animals - control, which were not treated.

table 2
Comparative data confirming the effectiveness of the proposed serum. №№ Animal species What was treated, dose Number of animals in the group Cured Percentage of efficiency,% one. Piglets serum, 0.5 ml / kg of body weight 229 189 82.5 2. Piglets streptomycin, polymyxin 92 56 60.9 3. Piglets the control 24 eleven 45.8 four. Calves serum, 0.5 ml / kg of body weight 28 24 85.7 5. Calves streptomycin, polymyxin 24 fifteen 62.5 6. Calves the control 7 3 42.8 7. Sows serum, 0.5 ml / kg of body weight 54 48 88.9 8. Sows streptomycin, polymyxin 48 37 77.1 9. Sows the control 36 21 58.3

Table 2 shows that, compared with the traditional method of treatment, the proposed serum against pseudomonosis of farm animals is effective both for the treatment of young piglets and calves and sows, in all cases the percentage of treatment efficiency is quite high and averages 85.7%.

Claims (2)

1. A method of producing polyvalent serum against pseudomonosis of farm animals, comprising hyperimmunization of bulls with a polyvalent antigen obtained by culturing epizootic strains grown separately on Hottinger broth, having a pH of 7.2-7.4, and mixed in equal proportions followed by formalin inactivation taking blood to obtain serum, separation of serum, followed by preservation and control for activity, characterized in that as epizootic strains used Pseudomonas aeruginosa serotypes 01, 02, 03, 04, 06, 010, 011, 013, 018, 019 are used and immunization is carried out in cycles with increasing doses of antigen, while the antigen is administered subcutaneously no more than 14 times after 3-4 days for 51 days starting with 5 ml, and the next two doses are doubled each time, and the rest are 1, 3 times more than each previous one, and the last four doses are not more than 150 ml, while taking blood from animal producers for control determination titers are carried out after 8, 22, 32 days from the start of antigen administration, and to obtain serum, blood is taken at 52-53 d Hb from the start of administration of the antigen. 2. The method of obtaining polyvalent serum against pseudomonosis of farm animals according to claim 1, characterized in that the control of serum activity is carried out on white mice.