Method for preparing hyperimmune serum against porcine circovirus, reproductive-respiratory syndrome and hemophilosis

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science, namely veterinary epizootiology and immunology. The method implies as follows. The producing pigs are hyperimmunised intramuscularly in various points using monovalent inactivated porcine circovirus infection (PCVI), reproductive and respiratory syndrome (PRRS) and hemophilosis vaccines in graded doses every 7 days; that is combined with administering the preparation Gamavit for additional immunogenesis stimulation.

EFFECT: higher preventive effect and reducing length of treating of associated viral-bacterial diseases in weaned pigs due to maintaining high serum protective antibody titers to circovirus infection min 1:25600, to reproductive and respiratory syndrome min 1:12800 (in ELISA), to hemophilosis min 1:512 (in agglutination reaction).

3 tbl, 4 ex

 

The invention relates to veterinary medicine, namely to epizootiology and veterinary immunology and AIDS for specific passive prophylaxis and treatment of associated diseases viral and bacterial etiology.

In the structure of infectious respiratory diseases of piglets at weaning, circovirus infection is high specific vespolina positive results in serological study in ELISA among different age and sex groups: growing 50%, pullets - 81,0%, sows - 62,7%, the grunts of 30%, an average - 55,9% (Scaeva M.A., Verkhovsky with O.A. al., 2005). The disease is characterized by the fact that on the background of immunodeficiency layered co-infections of viral and bacterial etiology, in particular the virus reproductive-respiratory syndrome, the causative agent of hemophilia and others (Shahi A.G. and others, 2003, 2008, 2009; Fedorov, YU.N., 2006).

Associated forms of circoviruses infection cause pig-breeding complexes considerable economic damage resulting from the high mortality of the patients, reduction in weight, high cost of treatment and holding of the General veterinary-sanitary and specific preventive measures.

The peculiarity of the infectious process is the multiplication of circovirus in actively dividing cells, in which lastnosti in macrophages. Experimental or natural immunoactivity can lead to high levels of macrophages and, therefore, increases the replication of circovirus (Krakowka S.A. et al., 2001; Segales J., 2002; Allan J.M. et al., 2000).

In vivo clinical signs of circoviruses infection of pigs reported in 5-12 weeks piglets in the form of the syndrome paleochannel Multisystem depletion with the further development of respiratory pathology in the layers of concomitant viral and bacterial pathogens. In different households, the incidence of youngsters is usually 5...20% (sometimes 50...70%), the mortality rate reaches 80...100%. The frequency of the disease and the severity of its course are directly dependent on the concentration of animals on the farm.

At the present stage of development of veterinary science in the field of industrial pig and effective means of specific prophylaxis and treatment of pigs with associated form of circoviruses infection does not exist. Preventive measures are reduced to a thorough disinfection of premises, to prevent the introduction of infected animals from disadvantaged households and other quarantine action. The implementation of such strategies may reduce the incidence of pigs and sanitation measures as early weaning of piglets from sows, fattening groups matched on age, turned out to be really successful in the prevention of viral diseases paleochannel period piglets (orlyankin astray freight, Aliper So, Nepoklonov E.A., 2002, 2003, 2007, 2009; drew T., 2005).

Syndrome Multisystem paleochannel depletion piglets link several respiratory pathogens, both viral and bacterial origin. Most often in this regard are circovirus type II, the virus reproductive and respiratory syndrome, the causative agent of hemophilia.

Now for the specific prevention of circoviruses infection of pigs in our country is inactivated tissue vaccine and undergoes extensive production testing of the vaccine "Verres - Circo" production LLC Velikim", Moscow. However, it is not designed multivalent vaccine and hyperimmune serum against CVIS and secondary infections (Grechukhin A.N., 2008).

Proposed biological product containing polyvalent hyperimmune serum of pigs with antibodies to the causative agents of salmonellosis, colibacillosis, pasteurellosis, faces and transmissible gastroenteritis, and transfer factor obtained from leukocyte mass of blood hyperimmunizing pigs after separation of the serum (Shcherbakov P.N., Yurov C.P., Karavaev UD, Shcherbakova T.B., 2002).

Its disadvantage is the lack of specific antibodies against the most common is osmotically respiratory infections and requires additional introduction of appropriate serum.

Known hyperimmune anti-adhesive and anti-toxic serum against escherichiosis farm animals, containing a complex of antibodies against somatic 09, 078, 0141, protein adhesive K, K, R, 741 and polisaharidnykh capsule C, K, K antigens (Malakhov Y.A., Tugarinov O.A., Pies, M.K., Solodkov B.C., 1993).

The disadvantage of this drug is that it does not have immunospecificity activity when associated forms of infection processes.

The closest analogue (prototype) is hyperimmune serum against hemophilia, streptococcosis and pasteurellosis pigs received from producers oxen (Students EE, Colomina GF, Skorodumov DI 2004; grin S.A., 2008). Known hyperimmune serum get 7...10 multiples of intramuscular injections of antigens oxen-producers with an interval of 2 to 3 days. The proposed method obtain hyperimmune serum is time-consuming and costly, as it is based on the prior receipt of the required number of antigens by plating on a nutrient medium with the subsequent stage of their inactivation. Not provided by the use of immunostimulating drug to increase the level of the antibodies produced after injection of antigens animal producers.

The efficiency of known serum insufficient the a, because it contains only antibodies neutralizing agents of bacterial origin, but it is not effective against the causative agents of viral respiratory infections in pigs. Piglets in group rearing, as a rule, the main trigger in the development of respiratory disease agents are viral nature or a combination of several viruses such as circovirus and the causative agent of reproductive-respiratory syndrome, which strongly suppress the immune system and against this background, multiply secondary bacterial infection. Increasing the number of antigens and the number of injections is impractical because puts extra strain on the immune system of producers and not profitable from an economic point of view.

The objective was to develop the best technology for the production of polyvalent serum against circoviruses infections, reproductive-respiratory syndrome and hemophilia pigs, providing effective passive immunity in the shortest time possible and the reduction treatment in the event of illness.

The objective of the invention is to obtain immunospecificity polyvalent serum for the prevention and treatment of associated viral-bacterial infections of pigs.

The technical result of the invention is to improve prophylactic effect and the reduction of treatment time AC is oneiromancy viral and bacterial diseases in piglets at weaning due to the content in serum high titers of protective antibodies to circoviruses infection is not less than 1:25600 and reproductive-respiratory syndrome not less 1:12800 (ELISA), Hemophilus not less than 1:512 (RA).

The essence of the invention. Polyvalent hyperimmune serum for the prevention and treatment of associated respiratory disease of pigs contains antibodies to pathogens, circoviruses infection in the titer of at least 1:25600 (ELISA), reproductive-respiratory syndrome in titer of at least 1:12800 (ELISA), pathogen hemophilia in titre not less than 1:512 (RA).

The product is a clear, slightly opalescense, straw-yellow color with a reddish tint liquid. Release packaged in glass bottles with a capacity of 100 cm3. Store the drug in a dry, dark place at +4...+10°C. shelf Life 12 months from date of manufacture.

The serum is injected subcutaneously pigs prophylactic doses for 7...10 cm3on the head twice with an interval of 10 days. For therapeutic purposes, the drug is administered in a double dose of 15...20 ml cm3on the head in combination with antibiotics.

In recommended doses, the drug causes no complications and no side effects.

A method of manufacturing a polyvalent hyperimmune serum as follows.

Example 1. Serum is obtained by 6 fold hyperimmunization pigs monovalent inactivated vaccines against circoviruses infections, reproductive-respiratory syndrome and hemo is ilesa intramuscular injections in increasing doses with an interval of 7 days with the parallel introduction of the immunostimulant amavita. Pig producers, live weight of 150...160 kg, pre-quarantinious, subjected to investigation and appropriate treatment against infectious diseases according to the current "Instructions on the procedure of preparation and sanitary treatment of animals used for production of biological products". Hyperimmunization pigs is carried out according to the scheme given in table 1.

Table 1
Scheme hyperimmunization pigs-producers
The number of injectionsIIIIVVVI
The total dose of drug (cm3)Verrès-Circo (Wetbike)405080115
Conc. inact. against PRRS (Wetbike)405075105
Inact. Sobirov. against hemophilia (ARRIAH)45 6090125
Gamavit
(immunostimulant)
30405060
The activity of serumCVIS in ELISA1:64001:128001:256001:25600
PRRS ELISA1:32001:64001:128001:12800
Gamefiles in RA1:1281:2561:5121:512

From table 1 it follows that the maximum activity of the serum obtained after five multiple injections of vaccines to circoviruses infection at 1:25600 and reproductive-respiratory syndrome - 1:12800 (ELISA), pathogen hemophilia 1:512 (RA). A further increase in the number of injections of vaccine does not improve the level of antibody titers

The blood sample must be conducted in the presence of a level of antibodies to circoviruses infection 1:25600, reproductive-respiratory syndrome in the credits of at least 1:12800 (ELISA) and schemafilename polyserositis in titre not less than 1:512 (RA).

Blood sampling is carried out by a total of bleeding during slaughter after 3...4 weeks after the final hyperimmunization in sterile 20...30 litre bottles. The serum of the blood is obtained citrate method. To prevent blood from clotting used anticoagulant 10%solution of citric acid sodium, prepared with saline solution, sterilized in an autoclave at a temperature of 120°C for 30 min In sterile bottles and pouring the solution citric acid sodium based 34 ml per 1 liter of blood. During and after blood collection the solution of anticoagulant and blood mixed, by shaking the bottle gentle circular movements.

To separate the formed elements obtained citrate blood sephirot separator brand g-as-2ZH with subsequent defibrinating plasma in a sterile defibrilator with a stirrer. To the plasma, while the mixer, through the fitting add 30%solution of calcium chloride based 1.3 ml per liter of plasma and fresh serum containing the enzyme thrombin, at the rate of 10 ml per 1 l the Process of defibrinate plasma usually lasts 25...30 minutes after this time take samples to check for completeness of the translation of fibrinogen into fibrin, which in the test tube pour 7...8 ml of the test serum and 5...6 ml of a saturated solution of calcium chloride, the mixture carefully what about the shake and leave to rest for 10 to 15 minutes... The transparency of the mixture indicates the completeness of the transition of fibrinogen to fibrin. The resulting serum test menganalisis method for calcium, the presence of which in the serum should not exceed 35 mg%. Next, the serum can 5%phenol solution until the concentration in the total volume of serum to 0.5%. After that, the whey is pumped into the sump and incubated for 2...2.5 months at 2...15°C. defending the serum can be replaced by separation. Then the whey is subjected successively advanced and sterilizing filtration through plate marks "F2" and "SF", and then tested for sterility as described in example 2.

Example 2. Prepared a series of serum tested for appearance, sterility, safety and potency.

To determine the appearance, color, presence of impurities, mold, presbyacusis flakes, integrity capping all serum vials shaken, viewing in transmitted light and turn down the tubes. Sterility serum checked by plating on a meat-peptone agar, meat-peptone broth, Saburo, regenerated environment Kitt-Tarozzi. Crops are cultivated at a temperature of 37...38°C. two days from liquids do subcultures on meat-peptone agar, meat-peptone broth, meat-peptone-liver agar under adelineven oil. Primary crops was incubated for 10, and the secondary is 8 days. Sterility is confirmed by the lack of growth in normal and selective nutrient media. The harmlessness of the serum was determined on 10 white mice weighing 14...18 g and 10 Guinea pigs weighing 350 to 400 g in a subcutaneous respectively 0.5 and 2.0 cm3drug. The serum is considered harmless if all animals remain clinically healthy for 10 days.

Example 3. Obtained in example 1 hyperimmune serum is used for the prevention of respiratory disease of pigs. The experiment was conducted at 80 piglets at the age of 28 days before the transfer in group rearing, with an average live body weight at 7,1-7,5 kg Piglets divided into 4 groups according to the principle analogues. Animals 1, 2 and 3 groups (experienced) on 20 goals) was subcutaneously injected obtained in example 1 serum respectively 5, 7 and 10 cm3at the head. Piglets 4th group of 20 heads were injected serum (prototype) in the dose of 10 see the Serum was injected three times, with an interval of 10 days. The observation period of 60 days.

Prototype (control)
Table 2
The incidence of respiratory diseases of piglets at weaning
IndicatorsThe experimental group
IIIIII
The number of animals, (goal.)20202020
Dose serum (cm3)571010
Multiplicity of3 injections with an interval of 7 days
Ill respiratory diseases (%)25101020

From table 2 it follows that the optimal dose of the serum is 7 cm3 on the head, which received a high preventive effect with increasing dose up to 10 cm3 efficiency remained at the same level.

Example 4. Obtained in example 1 serum used for the treatment of associated respiratory disease in piglets at growing 60...65 days age and live weight 20...22 kg In patients piglets are lagging behind in growth, exhaustion, cough, about the cap, raising the temperature to 41°C. In the experiment 60 piglets, which were divided into four groups according to the principle of the steam-analogues. The piglets of the first, second and third groups were administered obtained in example 1 serum doses of 10, 15, 20 cm3at the head. Pigs fourth group was injected serum (prototype) at a dose of 20 cm3at the head. Pigs of all groups were injected intramuscularly gentamicin in recommended doses. The observation period of 60 days. The results of the experiments are presented in table 3.

Table 3
The serum's effectiveness for respiratory diseases of pigs
IndicatorsThe experimental groupPrototype (control)
IIIIII
Dose serum (cm3)10152020
The number of animals (animals)15151515
Recovered a goal.12141412
Palo goal.3113
Safety, %8093,393,380

From the above data table 3 shows that the proposed hyperimmune serum in a dose of 15 cm3on the head with the re-introduction of the serum after 48 hours, up to 3-5 injections, has a more pronounced therapeutic effect in comparison with the hyperimmune serum - prototype. In severe cases, the clinical course of the disease daily dose whey you must enter fractional 2-3 times at intervals of 3-4 hours, which provides a reliable therapeutic effect.

References

1. Grechukhin, A.N. Features displays circoviruses infection of pigs and its specific prevention/ A.N. Grechukhin // Pig. - 2010. No. 2. - P.48-50.

2. Grin, S.A. Modern biotechnological processes and immunological methods for the industrial production of veterinary drugs: dis...Prof. Biol. Sciences / S.A. grin. - Shchyolkovo,2008. - P.á305.

3. Drew, T. Syndrome paleochannel multi-systemic wasting: the role of swine circovirus in its etiology / Industrial and breeding pigs, 2005. No. 4. - P.51-52.

4. Malakhov, Y.A. Specific prevention escherichiosis animals / Waallahu, Oisteanu., Mckergow // Veterinary Medicine. - 1993. No. 3. - P.5-7.

5. Orlyankin, astray freight is Circovirus infection / astray freight orlyankin, So Aliper, E.A. Nepoklonov // veterinary medicine. - 2002. No. 8. - P.24-25.

6. Orlyankin, astray freight is Circovirus infection / astray freight orlyankin, So Aliper, E.A. Nepoklonov // Livestock Russia, 2003. No. 11. - P.48-51.

7. Orlyankin, astray freight is Circovirus infection / astray freight orlyankin // Problems of infectious pathology of pigs: proceedings of the XVI international. Moscow. Congress, 2007. - Moscow. - 2007. - P.19-24.

8. Orlyankin, astray freight is an Infectious respiratory disease of pigs / astray freight orlyankin // Livestock Russia. - 2009. No. 5. - P.35-36.

9. Reference Vidal veterinary, "Medicinal product for veterinary use", Moscow, Astra Pharm Service, 2003-2004. - S.

10. Fedorov, YU.N.. The immune status of pigs in farms industrial type / N. Fedorov [and other] // veterinary medicine. - 2006.-№6. - P.18-21.

11. Shcherbakov, PN. Prevention of gastrointestinal and respiratory disease of pigs / Shcherbakov PN. [and other] // Pig. - 2002. No. 5. - S-26.

12. Patent 2180239 Russian Federation, AK 39/395. Biological drug for prevention and cured what I gastrointestinal and respiratory disease of pigs / Shcherbakov P.N., Yurov C.P., Karavaev UD, Shcherbakova T.B.; applicant and patentee of the Urals. States. Academy of veterinary medicine.- No. 2000122079/13; Appl. 22.08.2000; publ. 10.03.2002.

13. Patent 2357756 C2 of the Russian Federation, AT 39/40. The way to obtain hyperimmune serum against hemophilia, streptococcosis and pasteurellosis pigs / Students EE; applicant and patentee educat. nauch.-the experiments. And technologist, Institute of biology, prom.-Ty, No. 200713692/13; Appl. 26.06.2007; publ. 10.06.2009.

14. Patent 2043772 C1 of the Russian Federation, AK 39/108, AK 39/40. A method of obtaining a serum anti-adhesive and anti-toxic against escherichiosis farm animals / Malakhov Y.A., Tugarinov O.A., Pies, M.K., Solodkov B.C.; applicant and patentee Malakhov Y.A., Tugarinov O.A., Pies, M.K., Solodkov B.C. - No. 93009805/13; Appl. 25.02.1993; publ. 20.09.1995.

15. Shakhov, A.G. Application of immunomodulators with vaccination of animals against salmonellosis / AG Shahi [and other] // veterinary medicine.- 2006. No. 6. - P.21-26.

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17. Shakhov, A.G. the Influence of immune status on Vozniknovenie and the development of respiratory disease of pigs / AG Shahi, M.I. Turkish, YU.N.. Masanov etc. // Reports of the RAAS. - 2009. No. 4. - P.55-58.

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19. Students, EA Test serum against hemophilia and pasteurellosis pigs / Anachronical, Gracilaria, Thismaradona // Veterinary biotechnology: present and future: proceedings of the Intern. nauch.-the practical. conference, 2004. - Shchyolkovo. - 2004. - P.56-57.

20. Allan, G.M. Immunostimulation, PCV-2 and PMWS / G.M. Alan [et al.] // Vet. Rec, 2000. - V. 147. No. 6. - P.170-171.

21. Krakowka, S. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2) / S. Krakowka, J.A. Ellis, F. McNeilly [et al.] // Vet. Pathol, 2001. - V. 38. - P. 31-42.

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The way to obtain hyperimmune serum against circoviruses infections, reproductive-respiratory syndrome and hemophilia pigs, including hyperimmunization pig producers with subsequent kremovatam and selection of the target product, characterized in that hyperimmunization conduct monovalent vaccines against circoviruses infections, reproductive-respiratory syndrome and hemophilia pigs increasing doses, 6-multiples of intramuscular injections with an interval of 7 days, the total dose of each vaccine 105-125 cm3with the addition of the immunostimulant amavita.



 

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13 cl, 112 ex

FIELD: biotechnology, in particular production of vaccines and diagnosis objects.

SUBSTANCE: bacterium Lawsonia intracellularis is cultivated in Hep-2, Me Coys or IEC-18 cells in suspension at oxygen concentration from more than 0 to about 18 %. Vaccines, containing bacterium L. intracellularis obtained by claimed method, is useful in inducing of immune response to said bacteria in animals.

EFFECT: improved method for large-scale cultivation of bacterium L. intracellularis.

21 cl, 5 tbl, 6 ex

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 37-37.5°C for 60-90 min and further at 2-8°C for 22 hours. Obtained diagnosticum has specificity of 98±0.2, 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.

5 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 3.5-37°C for 100-120 min and further at 6-8°C for 20 hours. Obtained diagnosticum has specificity of 98±1.0, 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.

5 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 37-39°C for 90-159 min and further at 10°C for 10 hours. Obtained diagnosticum has specificity of 99±0.3, 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.

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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