RU2488117C2 - Immunoenzymometric test system for serologic diagnosis of reovirus infection in cattle and monitoring of post-vaccination immunity level - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to immunoenzymometric test system for serologic diagnosis of reovirus infection in cattle and the monitoring of post-vaccination immunity level. The presented immunoenzymometric test system comprises a specific antigen of the strain "Reo 1 Lang-DEP" of reovirus of type I, inactivated by 0.08% solution of 1,2-aminoethylaziridine representing a specific protein in a concentration of 5-10 mkg/cm³ adsorbed on the surface of polystyrene cavities in carbonate-bicarbonate buffer with merthiolate in a final concentration of 0.1-0.2 mg/cm³, a control positive serum obtained to the antigen of reovirus of type I with the activity in IFA 1:3200-1:6400, control negative serum, anti-species conjugate, potassium dihydrogen phosphate, potassium phosphate dibasic, sodium chloride, a chromogen (ortho-phenylenediamine), hydrogen peroxide, stop reagent and panels for carrying out reaction of the immunoenzymatic assay.

EFFECT: test system enables to diagnose reovirus infection of cattle with high sensitivity.

2 dwg, 3 tbl, 7 ex

Description

The invention relates to veterinary virology and biotechnology.

For modern livestock breeding, the most common viral respiratory and intestinal infections of calves and the pathology of the reproductive organs of adult livestock associated with infertility, early embryonic mortality and abortion, decreased milk production, decreased reproductive functions, growth retardation, and increased risk of secondary bacterial infections are of great economic importance. infections and increased waste, especially among young animals.

In the development of these forms of cattle pathologies, the causative agents of a viral nature, belonging to various taxonomic groups, are dominant. Among this vast group of viruses, reoviruses are little known and little studied.

Cattle reoviruses belong to the genus Orthoreovirus of the Reoviridae family. Virions are spherical particles with a diameter of 75-80 nm, with two capsids: external and internal. The genome of reoviruses consists of 10 double-stranded RNA segments. The name reoviruses (reo - the initial letters of the words respiratory - enteric - orphans) comes from the fact that they have a pronounced tropism for the respiratory and digestive organs (Sabin A.B., 1959).

Reoviruses are quite resistant to physicochemical effects: they do not lose their infectivity when heated to 500 ° C in the presence of MgCl2. In the cold, samples can be stored at -200C for 8-10 months. up to 2 years. At 40C, the virus retains its infectious properties for 2 months. Defrosting and thawing five times higher does not affect the infectivity of reoviruses (Ritova, V.V., Trofimov N.M., 1977). They tolerate a wide range of pH (from 2.2 to 8.0), but are completely inactivated by 70% ethanol or 3% formalin at 56 ° C for 30 minutes (Stanley N.F., Dorman D.C., Pansford I., 1953).

For the first time, reoviruses as pathogenic agents were reported by Sabin in 1959, isolating type I reovirus from a child with a symptom complex of diarrhea and nasal discharge. According to the results of RVN and RTGA in humans and mammals, three related serotypes have so far been detected. ECHO-10 virus (Lang strain) became the prototype type I reovirus. Reoviruses of types I, II and III are isolated from cattle, horses, dogs, cats, and mice. According to RNA hybridization, viruses of serotypes I and II are 70% homologous, and the degree of homology between them and serotype II viruses is only 10% (Gaillard R.K., Ioklik W.K., 1982). Reovirus infection affects many species of animals and humans, regardless of age. Most often, the infection is inapparent, latent in nature and is accompanied by prolonged virus carriage and virus isolation. Human and animal reoviruses of the same type are serologically indistinguishable and closely related in biological properties. Strains of human and animal reoviruses cause the same changes, multiply in the same tissue cultures and cause similar reactions in natural hosts. Possible diseases of people with infection with bovine reovirus type 1 and calves with infection with any of the serotypes of the human virus.

Cattle reovirus infection was first recorded in the USA, Belgium, Germany, France and other countries. In these countries, a high percentage of seropositive animals was detected. So, for example, in Germany, the participation of reoviruses in the defeat of the respiratory tract of calves has been proven, and their role in the pathology of the fetus and newborns has been established. When examining the blood serum of 155 calves and paired sera from 62 adult animals in RTHA, it was shown that re-virus of cattle of type I reacted with 70% of the studied animals with type II reovirus - 16% and type III - 5%. In three of the 24 herds of cattle, a serological study of paired sera confirmed the complicity of reoviruses in the acute course of infection (Syurin V.N., Samuilenko A.Ya. et al. 1998; Wizigmann G., Reinhardt G., 1971).

Serological studies in the RTGA of 194 calf blood serum samples during the acute course of respiratory enzootia conducted at the Institute of Virology of the University of Vienna using type I reovirus (Lang strain) as an antigen in the RTGA showed that antibodies were detected in 54% of the examined animal population . Moreover, 39 sera reacted at a dilution of 1:32, 20 at a dilution of 1:64, 12 at a dilution of 1: 128, and 5 at a dilution of 1: 256 and higher. These data indicated that the dominant serotype is type I reovirus (Böckmann I., 1976).

Enough convincing evidence has accumulated indicating the etiological role of reoviruses in the pathology of respiratory and intestinal infections of young cattle. So, Rosen L., Abinanti F.R., Hovis I.F. (1963), in a study in the USA of 154 cases of an infection process in calves the first 2 years of life, reovirus infections caused by all three types of reoviruses were diagnosed in 109 animals from three farms. Of these, type I also prevailed.

According to Rosen L. (1962), Böckmann I., (1976), reoviruses are able to cause pneumoenteritis in calves in the first 3 months of life, which are manifested clinically by diarrhea, rhinitis, fever and pneumonia, while in adult animals, reovirus infection occurs latently and is accompanied by a decrease in milk yield in dairy cows. When examining livestock in Maryland (USA), almost every calf by the age of one was infected with one of the serotypes of bovine reovirus (Rosen L. (1964). Based on the studies Dawson PS (1966) concluded that a clinically pronounced form of the course was rarely observed However, complication of reoviruses was found in 29 cases out of 111 recorded enzootia of calf respiratory infections.

It should be noted that during the experimental infection of calves-gnitobionts with all three serotypes, fever, cough, discharge from the nasal cavity were observed, and interstitial pneumonia was histologically detected (Lamont P.H. et al., 1968).

Despite the poor knowledge of reovirus infection, there are reports of the introduction of a reovirus antigen as a component in a multivalent inactivated vaccine consisting of PG-3 virus, adeno- and reoviruses (Sulbo J., Petermani X. et al., 1973). The vaccine is proposed to be used for calves from 6-8 weeks of age, and in the absence of colostrum antibodies, animals are immunized at a weekly age. The duration of immunity is about a year (Perreau P., 1973).

It should also be noted that the authors of the claimed invention developed the “Associated vaccine against parvovirus, reovirus, herpesvirus type I infections and viral diarrhea-disease of cattle mucous membranes” in the conditions of the virology laboratory of the Federal State Budget Institution FTSTR-VNIVI. The vaccine is intended for specific prophylaxis of respiratory and intestinal infections of young animals and pathologies of the reproductive organs of adult livestock.

Summarizing the cited material, it should be noted that under natural conditions in cattle, reovirus infection with a bright clinical sign does not always occur and reoviruses are mainly involved in the formation of acute mixed viral respiratory and intestinal infections of calves, especially in the presence of stress factors.

It is known that pronounced antigenic activity is characteristic of reoviruses: neutralizing antibodies and antibodies that suppress the hemagglutination reaction appear in response to reovirus infection. All mammalian reoviruses have hemagglutinating properties in relation to human red blood cells. The temperature conditions during the formulation of the RGA do not affect the hemagglutinin titer, as well as the pH in the range from 6.0 to 8.0.

According to foreign researchers, a retrospective diagnosis of reovirus infection is established by examining paired sera taken at intervals of 3-4 weeks, by examining them in rtga with type I reovirus. An increase in titers of antihemagglutinins in the second samples is 4 or more times considered diagnostic. With latent reovirus infection in the herd, the percentage of animals having antibodies ranges from 40 to 80% (Brudaker M.M., 1964; Moreno-Lopez I. 1979; Wizigmann G., Reinhardt G., 1971).

From literature it is known that laboratory diagnosis of reovirus infection in cattle is carried out by isolating the virus in cell culture and the results should be confirmed by detecting an increase in antihemagglutinins or virus-neutralizing antibodies during convalescence compared with blood serums taken in the acute stage of the disease.

To isolate the causative agent, mainly feces, nasal and conjunctival swabs, blood, lungs and lymph nodes are used. The material is processed according to standard methods, preparing for infection of cell cultures. An isolated cytopathogenic agent is checked for hemagglutinating activity against human erythrocytes and, if any, is identified in rtga using standard serums of three serotypes of reovirus. Mixed infections are often serologically diagnosed: PG-3, VD-BS, reovirus infection and chlamydia (Thomas L., Collins A., 1974).

However, to date, no studies have been conducted in the Russian Federation to study the role of reoviruses in cattle pathology; many aspects of this disease, in particular, the sources and transmission routes, virus carrier and virus isolation in naturally infected cattle, have not been studied, and the breadth and extent of reovirus infection remained outside the field of view of the country's veterinary service. Obviously, the solution of such issues as features of the epizootology of the disease and post-infectious immunity, the tasks associated with the development of methods and means of laboratory diagnostics and the specific prevention of reovirus infection in cattle, requires serious attention.

In view of this situation, in order to identify the degree of spread of reovirus infection among cattle in the Middle Volga region, we carried out seromonitoring for this infection by examining 1031 blood serum obtained from 16 households in 6 regions of the Republic in the hemagglutination inhibition test (RTGA). Tatarstan, in which there were respiratory and intestinal diseases of calves and impaired reproductive organs of the uterine livestock. The inactivated reovirus type I strain “Reo 1 Lang” obtained on a Vero cell culture with hemagglutinating activity 1: 512-1: 1024 was used as antigen. From the results of the studies presented in table 1, it follows that the highest antibody titers in the range of 1: 160-1: 320 are found both in deep-skinned cows and heifers of a random age, and in calves. These data indicate the activity of reovirus infection in the examined livestock. Therefore, a generalization and analysis of the results showed that a fairly high percentage of positive reactions in diagnostic titers (1:40 and above) in an adult population (58.9%) and in calves up to three months of age (31.5%) indicates the facts type I reovirus circulation among the examined livestock in 16 households in 6 regions of Tatarstan and the prospects of further large-scale studies of this viral infection unexplored in the Russian Federation.

At present, the veterinary service of the Russian Federation does not have modern methods and laboratory diagnostic tools, including any serological monitoring kits for this little-known reovirus infection in cattle, which can infect many species of animals and humans, regardless of age and participating in the formation of most often mixed infections. Therefore, the problem of creating an enzyme immunoassay test system has become very urgent.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, allowed to establish that the applicant did not find sources characterized by features that are identical (identical) to all the essential features of the invention. From the identified prototype analogues, the hemagglutination inhibition response was determined according to Rosen L. 1962, as the closest in combination of features with respect to the technical result perceived by the applicant as the distinctive features of the proposed ELISA test system, set forth in the independent claim.

In all foreign countries, serological diagnosis of rheovirus infection in cattle and identification of isolated strains of rheoviruses is carried out by the inhibition of hemagglutination, using mainly the antigen of the Reo 1 Lang strain of reovirus type I. Using the inhibition of hemagglutination in almost all cases of natural and experimental animal reovirus infection and a person manages to detect a 4-fold or higher increase in the titer of homologous antibodies in paired blood serum

A well-known solution adopted for the prototype of the inventive method for the serological diagnosis of rheovirus infection in cattle and for monitoring the intensity of post-vaccination immunity by the enzyme immunoassay system is the hemagglutination inhibition test (Rosen L., 1969; - Diagnostic procedures for viral and Rickettsial infections, p. 291: Moscow “Medicine”, 1974 translation from English.).

The essence of rtga is to neutralize the hemagglutinating properties of the virus with specific antibodies contained in the blood serum, as a result of which the red blood cells do not glue (agglutinate) and settle to the bottom, forming a dense precipitate in the form of a button

The components of the reaction:

- reovirus antigen;

- 0.5% suspension of red blood cells About the human group;

- 25% suspension of kaolin;

- control positive serum;

- control negative serum.

RTGA is carried out by a micromethod according to the method described by Rosen L. (1972) against 4 hemagglutinating units of the reovirus antigen. Before setting rtga, serum is diluted 1: 5, heated in a water bath (to remove thermolabile hemagglutinins) at + 58 ° C for 30 minutes, treated with an equal volume of 25% suspension of kaolin for 20 minutes. The supernatant, after centrifugation at 1500 rpm for 10 minutes, is used in the hemagglutination inhibition reaction.

The disadvantages of the prototype is a well-known method of serological diagnosis of reovirus infection, are:

- the effect of non-specific hemagglutination inhibitors, the elimination of which requires preliminary treatment of sera;

- a relatively low sensitivity of the reaction, which makes it difficult to detect low concentrations of antibodies;

- instability of the reaction ingredients, mainly native red blood cells, whose shelf life is short (3-4 days);

- the complexity and duration of this method, requiring one working day, which limits the possibility of simultaneous study of a large number of blood serum samples;

- frequent discrepancies in the results of rtga when examining the same sera in different laboratories due to differences in the procedure for setting and accounting for the reaction.

These disadvantages of the prototype are eliminated by the invention.

It is absolutely obvious that the modern diagnostic service must rely on such highly specific and sensitive diagnostic methods that would make it possible to obtain reproducible, reliable and maximum quality results within the method that do not require complex instrumental equipment, provided with standard reagents and recognized as suitable for conducting large-scale studies in the conditions practical veterinary laboratories. The enzyme-linked immunosorbent assay largely meets these requirements.

The aim of the invention is the development of an enzyme-linked immunosorbent assay system as the most sensitive and reliable method for the serological diagnosis of reovirus infection in cattle and for monitoring post-vaccination immunity.

This goal is achieved by the fact that the inventive test system contains a specific antigen of reovirus type I strain "Reo 1 Lang-DEPT" inactivated with a 0.08% solution of 1,2-aminoethylaziridine, which is a specific protein at a concentration of 5-10 μg / cm ³ adsorbed on the surface of polystyrene wells in carbonate-bicarbonate buffer with merthiolate in a final concentration of 0.1-0.2 mg / cm ³ , control positive serum obtained for type I reovirus antigen with an activity in ELISA of 1: 3200-1: 6400 control negative serum conjugate anti-species, potassium phosphate monosubstituted, potassium phosphate disubstituted, sodium chloride, chromogen (ortho-phenylenediamine), hydrogen peroxide, stop reagent and panels for setting the enzyme immunoassay. The main advantage of the invention is that the strain “Reo 1 Lang-DEPT” is used as an antigen, which refers to the first serotype that is actively circulating among livestock in all countries of the world, including the territory of the Russian Federation.

According to the “Instructions for the use of the ELISA test system for the serological diagnosis of cattle reovirus infection and determination of the level of post-vaccination antibodies”, one test system kit is designed for simultaneous analysis of 46 blood serum samples in one dilution (in duplicate) on one tablet and 2 control sera (in duplicate) or 20 test serum samples (in four dilutions) and 2 control sera (in four dilutions). One test system is designed to study 230 samples of blood serum in one dilution and 100 samples in four dilutions.

Therefore, the claimed invention has significant differences and is characterized by a new set of features, which allows to obtain a higher expected effect, in contrast to the result that was obtained using the method of serological diagnosis of rheovirus infection of cattle - prototype.

Feasibility study:

- safety (inactivated antigen is used);

- convenience of work (automation of intermediate stages of reactions);

- the use of a Bio-Rad Model 680 spectrophotometer device, with which it is possible to measure light absorption in 96 wells of a tablet in a few seconds and obtain objective optical density indicators of the studied blood serum samples, which can significantly increase the number of analyzes and simplify the methodological procedure IFA.

Diagnostic parameters and quality indicators of the developed ELISA test system for serological diagnosis of cattle reovirus infection and control of post-vaccination immunity tension meet the requirements of the OIE standards (Manual for the Diagnostics and Production of Vaccines, OIE publication, 2004, p.21-29).

The composition of the proposed ELISA test system includes:

1. Polystyrene 96-well microplate for enzyme-linked immunosorbent assay adsorbed with purified Reovirus type I antigen, strain Reo 1 Lang-DEPT, at a concentration of 5-10 μg / cm ³ - 5 pcs.

2. Control positive serum, lyophilized (K ⁺ ), volume 0.5 cm ³ , working dilution 1: 400, titer in ELISA 1: 3200-1: 6400-2 amp. No. 1

3. Control negative serum, lyophilized (K ^- ), volume 1 cm ³ , working dilution 1: 400-2 fl. Number 2

4. Adsorption 0.01 M carbonate-bicarbonate buffer solution (0.01 M KBB pH 9.6) containing merthiolate in a final concentration of 0.1-0.2 mg / cm ³ .

5. 10-fold concentrate of phosphate-buffered solution with Tween-20 for washing tablets (FBI-T), 100 cm ³ - 2 fl. Number 3

6. The buffer for dilution of the conjugate (DBK), the volume of 10 cm ³ - 1 fl. Number 4

7. Diagnostic antibodies against immunoglobulins G (IgG) of cattle labeled with horseradish peroxidase (conjugate), volume 0.2 cm ³ - 1 amp. Number 5

8. Fostfatno-citrate buffer (FCB), volume 10 cm ³ - 5 fl. Number 6

9. Chromogen, ortho-phenylenediamine (OFD), 4 mg - 5 fl. Number 7

10. Hydrogen peroxide (H ₂ O ₂ ) 3% solution, a volume of 10 cm ³ - 1 fl. Number 8

11. 1M Sulfuric acid (Stop reagent), volume 50 cm ³ - 1fl. Number 9

Cooking kit components.

1. Preparation of working buffers

1.1. Adsorption 0.01 M carbonate-bicarbonate buffer solution (KBB pH 9.6) containing merthiolate in a final concentration of 0.1-0.2 mg / cm ³ is intended for dissolution and sorption of antigen in the well of tablets. 5 cm ³ are taken from the mother liquor, another 95 cm ³ dist. water and 10-20 mg of merthiolate. This solution is used in the reaction (0.01 M KBB pH 9.6).

1.2. Phosphate-buffer solution with Tween-20 is intended for dilution of control, test sera and panel washing. The contents of the vial with a 10-fold concentrate of phosphate-buffered saline with Tween-20 was adjusted with distilled water to 1000 cm ³ . The working solution of the buffer is a clear solution with a pH of 7.2-7.4.

1.3. The conjugate dilution buffer is a clear phosphate-free buffer solution without Tween with a pH of 7.2-7.4

1.4. Working chromogen-substrate solution. The solution is prepared immediately before use. For this, 4 mg of OPD is dissolved in 10 cm ^{3 of} phosphate-citrate buffer and 0.14 cm ^{3 of a} 3% hydrogen peroxide solution is added. The solution should be transparent and have a light yellow color.

2. Antigen Preparation

In the inventive ELISA test system, designed to detect and quantify antibodies to cattle reovirus, a specific antigen is prepared using reovirus strain "Reo 1 Lang".

The strain "Reo 1 Lang" reovirus, was obtained in 1979 by Professor Gaffarov H.Z. from the CVL of the UK Ministry of Agriculture and accepted by the applicant as a production one, has high biological, antigenic and immunogenic activity, retains its native immunobiological properties after inactivation, in its native form and after inactivation, are highly productive in sensitive biological cultivation systems, recognized as suitable for the manufacture of highly sensitive and specific diagnostics.

Reovirus - RNA-containing virus. Virions are spherical particles with a diameter of 75-80 nm, with two capsids: external and internal. The genome of reoviruses consists of 10 double-stranded RNA segments. The strain "Reo 1 Lang-DEP" reovirus, resistant to ether, 1% hydrogen peroxide, 1% phenol, 3% formaldehyde, stable in a wide range of pH values from 2.2 to 8.0, inactivated 70 % ethyl alcohol or 3% formalin at 56 ° C for 30 minutes. The virus agglutinates human erythrocytes of the 0-group. In vivo, cattle are susceptible to the virus.

The strain “Reo 1 Lang-DEP” of type I cattle reovirus was deposited in the laboratory of the Museum of Microorganism Stresses of Highly Dangerous Diseases (SAR) of the Federal State Budgetary Institution “FTsTRB-VNIVI” on December 9, 2009, registration number 2. The laboratory of the strain of microorganisms was designated the storage location for the strain. OOB FSBI FTsTRB-VNIVI, 420075, Kazan, Scientific Town-2.

Reovirus is reproduced in a transplanted culture of green monkey kidney cells (Vero) and / or in a transplanted culture of piglet kidney cells (PK-15), the reovirus-containing material is subjected to three freezing and thawing, followed by clarification by low-speed centrifugation (3000 rpm) for 30 minutes , inactivate a 0.08% solution of 1,2-aminoethylaziridine. In order to concentrate the antigen, the supernatant is collected and further centrifuged at 30,000 rpm for 1 hour. The concentrated antigen is purified by ultracentrifugation at 30,000 rpm for 2 hours on a 10-20-30% sucrose density gradient.

Example 1. In the inventive test system, the reovirus antigen is prepared as follows: the reovirus is reproduced in the transplanted culture of green monkey kidney cells (Vero) and / or in the transplanted culture of piglet kidney cells (PK-15) until the virus accumulates in titer 1: 32-1 : 128 GAE. For infection with reovirus, mattresses with a complete monolayer of Vero and / or PK-15 cells are selected, growth medium is removed, 5 cm of viral material is introduced into each mattress. Incubate in an incubator for an hour. After that, without removing the viral material, a supporting medium is introduced in a volume of 150-200 cm and the culture is placed in a thermostat. The characteristic cytopathogenic effect of reovirus on Vero and / or PK-15 cell culture (granularity, rounding and cell destruction) is controlled by microscopy of mats within 72-96 hours after infection, frozen three times at -20 ° C, then samples are taken from each mattress using 5 cm ³ for inoculation on sterility and determination of the titer in the RGA.

Then reovirus-containing material is subjected to three times freezing and thawing, followed by clarification by low-speed centrifugation (3000 rpm) for 30 minutes. Inactivate the virus with a 0.08% solution of 1,2-aminoethylaziridine. In order to concentrate the antigen, the supernatant is collected and centrifuged at 30,000 rpm for 2 hours. The concentrated antigen is purified by ultracentrifugation at 30,000 rpm for 2 hours on a 10-20-30% sucrose density gradient.

The precipitate is resuspended in carbonate-bicarbonate buffer (pH 9.6-9.8) containing merthiolate in a final concentration of 0.1-0.2 mg / cm ³ and is used as a reovirus antigen to sensitize the wells of polystyrene plates. The degree of purification and concentration of the antigen is estimated by the hemagglutinating activity determined in the DGA, and by the optical density of the viral antigen protein on a LOMO SF-46 spectrophotometer at wavelengths of 235 and 280 nm, adjusted for dilution in a cuvette according to the following formula:

According to the formula:

$$\frac{\mathrm{ABOUT}{\mathrm{<figure-callout\; id="2"\; label="P"\; filenames="00000004.png,00000005.png"\; state="\{\{state\}\}">P</figure-callout>}}_{235}-\mathrm{<figure-callout\; id="2"\; label="ABOUT\; P"\; filenames="00000004.png,00000005.png"\; state="\{\{state\}\}">ABOUT\; </figure-callout>}{P}_{}{}_{280}}{2,5\mathrm{one}}\times 30$$

Where

-OP ₂₃₅ ; OP ₂₈₀ - optical density at a wavelength of 235 and 280 nm, respectively;

- 30 - dilution of the test sample in the cuvette (1:30).

According to the results of chess titration of antigen and control sera, the optimal working concentration of reovirus antigen is established in ELISA, which in this case is 5-10 μg / cm ³ .

3. Preparation of control sera

The production of a positive positive type-specific serum against the Reo 1 Lang-DEP strain of type I reovirus is carried out by hyperimmunization of clinical healthy young cattle of 6-8 months of age, concentrated, purified and inactivated virus suspension with hemagglutinating activity of at least 1: 256-1 : 512.

14-16 days after the last virus administration, blood samples were taken from the jugular vein in animals, serum was obtained and a hemagglutination inhibition test (RTGA) and ELISA were performed.

Serum with an antihemagglutinating titer in rtga not lower than 1: 1280 and in ELISA - 1: 3200-1: 6400 are packaged in 0.5 ml ampoules and lyophilized.

Example 2. The manufacture of a control positive serum-specific serum against the Reo 1 Lang-DEP strain of type I reovirus is carried out by hyperimmunization of clinical healthy young cattle of 6-8 months of age, concentrated, purified and inactivated virus suspension with hemagglutinating activity of at least 1: 256-1: 512.

Prior to the start of immunization, animals from the jugular vein are sampled and the serum is checked for the presence of antihemagglutinins for cattle reovirus. In the absence of specific antibodies in them, animals are considered suitable for hyperimmunization.

To obtain immune sera, a viral antigen is deposited in Freund's incomplete adjuvant, which is prepared by thoroughly mixing 15 cm ^{3 of} liquid paraffin and 5 cm ^{3 of} dehydrated lanolin. The resulting mixture is subjected to fractional sterilization in a water bath for 3 consecutive days for 60 minutes. A mixture of antigen with adjuvant (in equal volume) is heated in a water bath to + 37 ° C, mix thoroughly and injected subcutaneously in a dose of 2 cm ³ . Subsequently, hyperimmunization of animals is carried out by three intravenous administration of inactivated culture virus suspension, purified from cells, in increasing doses of 10 cm ³ , 20 cm and 30 cm ^3, respectively, with an interval between administrations of 14-16 days.

In order to prevent an anaphylactic reaction, starting from the second cycle of immunization, the animal is injected with 2 cm ^{3 of the} virus subcutaneously 40 minutes before the main dose of the virus.

14-16 days after the last virus introduction, blood samples are taken from the jugular vein in animals, serum is obtained and a hemagglutination inhibition test is performed.

Production bleeding is allowed in the presence of antibodies in the blood serum of a producer to cattle reovirus not lower than 1: 1280-1: 2560 in RTGA. The first blood collection from the producers in volume should not exceed 0.6 liters of blood per 100 kg of animal weight, and in the subsequent - 1.6 liters per 100 kg of weight.

Blood from the jugular vein into sterile cylinders moistened with saline is placed in a thermal room for 30-40 minutes at a temperature of + 37-38 ° C, after which the serum is circled and the cylinders are left for 18 hours at a temperature of + 4-6 ° C for settling serum. Then the serum is taken, merthiolate is added in a ratio of 1: 10000, the titer of antibodies in rtga and ELISA with a homologous antigen is determined.

Serum with an antihemagglutinating titer in rtga not lower than 1: 1280 and in ELISA - 1: 3200-1: 6400 are packaged in 0.5 cm ³ ampoules and lyophilized.

4. Preparation of negative control serum

Cattle are kept in quarantine for 2 months, and blood serum is examined in ELISA. In clinically healthy non-immunized animals that do not contain specific antibodies to the first serotype reovirus during ELISA, sterile blood sampling from the jugular vein is performed at a rate of 600 cm ^{3 of} blood per 100 kg of animal weight. The blood is kept for 30 minutes in an thermostat at + 37 ° C, then circled and defended at 4 ° C. The settled serum is poured into vials under sterile conditions, packaged in vials of 1 cm ³ and lyophilized.

Example 3. Standardization of the basic conditions for research, the claimed test system ELISA

The effect of the concentration of cattle reovirus antigen on the results of ELISA is investigated at a content of 30, 15, 5, 10, 3, μg / cm ³ . The research results showed that the maximum value of the coefficient of specificity is observed during adsorption of cattle reovirus antigen at a concentration of 5-10 μg / cm ³ . The use of its other concentrations leads to a decrease in the specificity of the reaction.

When developing enzyme immunoassays in which a single dilution of the test serum is used for the study, it is important to select the conditions for sorption of the immune serum on a sensitized antigen plate. For this, control sera in dilutions from 1: 100 to 1: 12800 are introduced into the wells of the plate immobilized by antigen. The conjugate is used in a working dilution of 1: 5000, which is determined in preliminary experiments by the method of "chess" titration.

The optimal antigen concentration and working dilutions of the components of the enzyme immunoassay system are considered to be their final values, which ensure that in wells with a positive control serum OD490 was at least 0.7, and in wells with a negative serum, not more than 0.2. The following reaction parameters meet these conditions: antigen concentration - 5-10 μg / cm ³ , dilutions of control sera - 1: 400 and working dilution of the conjugate 1: 5000. The optimal time for antigen adsorption was its 18-hour incubation at + 4 ± 1 ° С, for serums this time is 2 hours at + 37 ± 1 ° С.

Example 4. Determination of the positive-negative threshold of the ELISA test system.

To take into account and interpret the results obtained by the ELISA test system, a positive-negative threshold of the test system was determined, which is in the range of <16% - ≥25%. In the future, all tests, the value of Ksv. for which there was less PNP, were considered negative, and samples with a value of Ksv. positive or equal to this indicator.

Example 5

The sensitivity and specificity of the test system was determined based on the correlation of the results of studies of blood serum in rtga and ELISA (table 2).

The results are shown in table 2, indicate that the diagnostic sensitivity of the developed test system in relation to RTGA was 111: 115 × 100% = 96.5%; diagnostic specificity 68: 73 × 100% = 93.1%; the coincidence of the results of the two methods 179: 188 × 100% = 95.2%.

At the same time, in the study of blood serum in ELISA and RTHA obtained from obviously negative (clinically healthy control animals from a well-off economy for this disease, n = 90) and obviously positive (experimental animals vaccinated with “Associated vaccine against parvo-, reovirus, herpesvirus type I infections and viral diarrhea-disease of the mucous membranes of cattle inactivated emulsion ”, n = 90) cows, 100% coincidence of results was observed. In the first case, all research results were negative, in the second - positive.

The reproducibility of the ELISA test system was determined in 5 replicates both within the same tablet (ELISA kit), and between different ELISA kits of the same series and sets of different series. The value of the coefficient of variation of the Ksv values. in the study of positive samples (n = 15) by the ELISA test system of different series did not exceed 6.3%, and in the study of negative (n = 13) - 7.4%, which corresponds to the recommended parameters and does not exceed 10%. In this case, the correlation coefficient of the results in the study of positive samples was 0.9, and in the study of negative samples - 0.95.

Example 6. Illustration of the application of the ELISA test system

6.1. Preparation of biological material for research. For analysis, blood serum of cattle is used. If the analysis is carried out within 24 hours after selection, samples of the biomaterial are stored at a temperature of + 4 ° C. During longer storage, the samples are frozen at a temperature of -20 ° C. Before the study, frozen samples are quickly (within 5-10 minutes) heated in a water bath at a temperature of + 37 ° C. In the event of a precipitation of red blood cells or protein, the samples must be clarified by centrifugation for 10 min at 2000 g. Repeated freezing and thawing of samples is not allowed. Do not use pooled sera from different animals. Hemolyzed and sprouted serums are not subject to research.

6.2. Preparation of biological components of the ELISA test system.

Control negative and positive serum (amp. Or fl.) No. 1 and No. 2 are dissolved in FBI-T and diluted 1: 400.

The contents of the ampoule No. 5 with anti-species immunoperoxidase conjugate before use, dilute the required amount of 5000 times, for example, to 0.01 cm ³ conjugate add 50 cm ³ DBK.

6.3. All initial kit components are stable until the expiration date.

6.4. Enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) is carried out in an indirect solid-phase version using the components included in the ELISA test system for serological diagnosis of reovirus infection in cattle and for monitoring post-vaccination immunity.

Before starting work, the tablet is washed twice with a working solution of FBI-T in a volume of 0.15 cm ³ per well.

In a single dilution study, the test and control sera are used in a dilution of 1: 400 phosphate-buffered solution with tween (FBI-T) No. 4. For example, 0.01 cm ^{3 of} test serum is added to 3.99 cm ^{3 of} FBI-T.

0.1 cm ³ K + is added to wells E12-F12 at a working dilution of 1: 400.

In wells G12-H12 contribute 0.1 cm ^{3 To -} in a working dilution of 1: 400.

0.1 cm ^{3 of the} test sample (PI) of the serum in a dilution of 1: 400 (two wells for each serum sample) is added to the remaining wells of the tablet according to Scheme 1.

When tested in 4 dilutions, the test and control sera were used at a dilution of 1: 800.

For example, 0.01 cm ^{3 of} test serum is added to 7.99 cm ^{3 of} FBI-T.

Previously, 0.1 cm ^{3 of} FBI-T is added to all wells of the plate. 0.1 cm ³ K + is added to well A11 at a working dilution of 1: 800.

0.1 cm ³ K is added to well A12 in a working dilution of 1: 800.

The remaining wells of the vertical row of the tablet (A1-A10) contribute 0.1 cm ^{3 of the} test serum at a dilution of 1: 800 and sequential double dilutions in four wells are carried out according to Scheme 2.

- Cover the plate with a lid and incubate for 2 hours at a temperature of 37 ° C.

- The tablet is washed 3 times with FBI-T buffer (0.15 cm ³ per well), the solution is removed by shaking, followed by tapping on filter paper until all residual solution is removed from the walls and bottom of the wells.

- 0.1 cm ³ of the conjugate working solution is added to each well.

- The tablet is closed with a lid and incubated for 1 h at 37 ° C.

- The tablet is washed 3 times with FBI-T buffer.

- Contribute 0.1 cm ^{3 of} working chromogen-substrate solution to each well.

- The tablet is incubated in the dark for 5-15 minutes at room temperature.

- Stop the reaction by adding 0.1 cm ³ stop reagent (1M H ₂ SO ₄ ).

- After stopping the reaction, the optical density of the substrate mixture is measured on a spectrophotometer with a vertical beam at a wavelength of 490 nm (OD ₄₉₀ ).

Example 7. Accounting and interpretation of the results.

When spectrophotometric accounting of the results of studies of blood serum in one dilution produce the following calculations:

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for samples K + (OD ₄₉₀ K + _sr );

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for samples K- (OD ₄₉₀ K- _cf) ;

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for each test sample SP (OP ₄₉₀ SP _avg );

- Calculate the binding coefficient (Ksv) of the conjugate with serum antibodies according to the formula:

, где $$\mathrm{TO}\mathrm{from}\mathrm{at}=\frac{\left(\mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{AND}{P}_{\mathrm{from}R}-\mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{TO}{-}_{\mathrm{from}R}\right)}{\left(\mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{TO}{+}_{\mathrm{from}R}-\mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{TO}{-}_{\mathrm{from}R}\right)}\times \mathrm{one}00$$

where

$$\begin{array}{c}\mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{AND}{P}_{\mathrm{from}R},\\ \mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{TO}{-}_{\mathrm{from}R}\\ \mathrm{ABOUT}{P}_{\mathrm{four}90}\mathrm{TO}{+}_{\mathrm{from}R}\end{array}\}\begin{array}{l}\mathrm{from}Redn\mathrm{and}e\mathrm{but}R\mathrm{and}fmet\mathrm{and}he\mathrm{from}\mathrm{to}\mathrm{and}esn\mathrm{but}hen\mathrm{and}\mathrm{I\; am}\mathrm{about}Pt\mathrm{and}he\mathrm{from}\mathrm{to}\mathrm{about}\mathrm{th}Pl\mathrm{about}tn\mathrm{about}\mathrm{from}t\mathrm{and}\hfill \\ \mathrm{at}l\mathrm{at}n\mathrm{to}\mathrm{but}x\mathrm{from}\mathrm{and}\mathrm{from}Pst\mathrm{at}em\mathrm{about}\mathrm{th}PR\mathrm{about}b\mathrm{about}\mathrm{th},\mathrm{about}tR\mathrm{and}c\mathrm{but}telbnsm\mathrm{and}\hfill \\ P\mathrm{about}l\mathrm{about}\mathrm{well}\mathrm{and}telbnsm\mathrm{to}\mathrm{about}ntR\mathrm{about}l\mathrm{I\; am}m\mathrm{and}\mathrm{from}\mathrm{about}\mathrm{about}t\mathrm{at}et\mathrm{from}t\mathrm{at}enn\mathrm{about}.\hfill \end{array}$$

The sample was considered negative if the value of Ksv less than 16%.

If the value Ksv more than 25% of the sample is considered positive.

The revealed results of serum studies in this range (from ≥16% to <25%) are considered doubtful, and the analysis should be repeated.

When taking into account the results of blood serum studies in four dilutions, the specificity coefficient is calculated, which is equal to the ratio of the optical density of the reaction product in wells with a control positive (OD ₄₉₀ K +) or test serum (OD ₄₉₀ PI) to the optical density of the control negative serum (OD ₄₉₀ K -). The reaction is considered positive if the specificity coefficient is not lower than 2.1 and negative if lower than 2.1.

The detection of positive samples of blood serum in one dilution during an epizootological examination of animals that have not previously been vaccinated against reovirus infection in cattle indicates circulation in the herd of the causative agent of the disease.

The identification of the growth dynamics of specific antibodies in paired serum samples, in the study of samples in four dilutions, allows you to diagnose reovirus infection in the studied livestock.

The results of titration of sera in four dilutions determine the intensity of immunity in vaccinated animals 2-3 weeks after the second vaccination by dividing the number of samples with an antibody titer of 1: 400 and higher in vaccinated calves of 10-20 days of age, and in deep-bred cows and heifers of random age with an antibody titer of 1: 800 and higher to the total number of tested sera and expressed as a percentage.

Our seromonitoring using the claimed ELISA test system for reovirus infection in 35 households of the Republic of Tatarstan, the Republic of Bashkortostan and the Saratov Region clearly demonstrates that out of 1378 samples of cattle blood serum specific antibodies to type I reovirus in ELISA in 638 ( 46.2%) of animals (table 3). The results obtained indicate the fact of the circulation of this pathogen among the examined livestock.

Thus, the proposed test system allows us to conduct large-scale studies of the little studied and little known in the Russian Federation reovirus infection of cattle and to evaluate the effectiveness of vaccine prevention.

Information sources

1. Ritova, VV, Trofimov N.M. Reoviruses and their role in infectious diseases of humans and animals // Issues of Virology. - 1977. - 2. - P.134-141.

2. Syurin VN, Samuylenko A.Ya., Soloviev BV, Fomina NV, - Viral diseases of animals. / Moscow, VNITIBP. - 1998. - S.18-19

3. Sulbo J., Petermani X., Terr J., Brun A., Shapui G., Tivro M. // In coll. “Summary Report. 2nd Int. symp VAVMI and special. dawn bol. " Varna - 1973. - 23 p.

4. Abinanti, F.R.:Vet.Bull32., - 1962. - 150 p.

5. Böckmann, J. Serologische erhebungen über die verbreitung von reovirusinfektionen im österreichischen mastkälberstrapel. / Wien. tierärztl. mschr. - 1976. - 63. - P. 358-360.

6. Brubaker, M.M., West, B., Ellis Human blood group influence on reovirus hemagglutinatination liters. / Proc. soc. exp. boil. (N.Y.). - 1964. - 115. - P.1118-1120.

7. Lamont, P. H., Darbyshire, J. H., u. Dawson, P.S.:J. Comp. Path 78. - 1968. - 23 p.

8. Moreno-Lopez, J. A serosurvey of viruses during outbreaks of acute respiratory and / or enteritic disease in Swedish cattle. / Zbl.vet.med Reihe B. - 1979. - 26. - 8. - P.634-640.

9. Perreau P. “Res. Ved. Veter ", 1973. - 149. - No. 9. - P.1147-1162

10. Rosen, L. Reoviruses in animals other than man. / Ann. N.Y. acad. sci. - 1962. - 101. - P. 461-465.

11. Rosen L. Diagnostic procedures for viral and Rickettsial infections, Moscow: “Medicine”. - 1974 translation from English. - S.285-291.

12. Rosen, L., Abinanti, F.R, u. Hovis, J.F. // Amer. J. Hyg. 77. - 1963. - P.38.

13. Sabin, A.B. Reoviruses. / Science. - 1959. - 130. - P.1387.

14. Wizigmann, G., Reinhardt G. Untersuchungen über Reovirus-Infektionen beim Rind in der Bundesrepublik Deutschlan // Zbl. vet, med. - 1971. - 18 - P.564.

15. Gaillard R.K., loklik W.K. Quantitation of the relatedness of the genjmes of reoviruses serotype 1, 2 and 3 at the gene level. // Virology., 1982. - P.123. - 152.

16. Stanley N.F., Dorman D.C., Pansford I., 1953 - Aust.I. exp. "Biol. med Sci "1953. - V.31. - P.149-159.

17. Sharpe A.H., Fields B.N., Pathogenesis of reovirus infection. In: The Reoviridae, ed by W.K. loklik, Plenum, New. York. - 1983 .-- P.229-285.

18. Thomas L., Collins A., - Veter. Rec., 1974. - 94. - No. 22. - R.506-509.

Table 1 Serological screening of cattle for type I reovirus antigen Region Name Age group n Antibody titers in rtga Total positive 1:40 1:80 1: 160 1: 320 and higher number percent Arsky rt RT cows 60 8 9 2 0 19 31 calves thirty one 0 one 0 2 6 Atninsky district of the RT cows 120 eighteen 12 6 22 58 48 calves 60 5 6 5 13 29th 48 Apastovsky rt RT cows 385 93 31 6 5 135 35 calves - - - - - - - Baltasinsky district of the Republic of Tatarstan cows 135 24 23 12 5 64 47 calves 45 8 9 5 one 23 51 Kamskoustinsky district of the Republic of Tatarstan cows 12 2 0 5 one 8 67 calves 44 7 four 2 one fourteen 31 Kaybitsky district of the Republic of Tatarstan cows 110 5 10 9 3 27 25 calves thirty 7 one 0 0 8 27 Rep. Bashkortostan cows 40 2 12 0 12 26 65 calves 10 one four 2 one 8 80

table 2 Comparative evaluation of the results in ELISA and RTGA. ELISA results, number of samples: RTGA result, number of samples: positive (n = 115) negative (n = 73) positive 111 5 negative four 68

Table 3 Indicators of the level of specific antibodies to type I reovirus antigen - in blood serum of the examined livestock of cattle No. p / p Farm Name Number of samples Of them seropositive in ELISA Qty % one 2 3 6 7 Arsky district of the Republic of Tatarstan one Ak Bars-Agro LLC 90 43 47.7 Atninsky district of the Republic of Tatarstan 2 SKHPK PZ "them. Lenin 90 36 40,0 3 LLC Tukaevsky 90 41 45.5 Baltasinsky district of the Republic of Tatarstan four Smail LLC 5 2 40,0 5 LLC Igenche 95 35 33.3 6 LLC "Trud" fifteen 5 33.3 7 LLC "Pine" 45 twenty 44,4 8 LLC Mayak 55 fifteen 27.3 Kaybitsky district of the Republic of Tatarstan 9 LLC Kubnya 140 54 38.6 Nurlat region of the Republic of Tatarstan 10 Peasant farm "Suleymanova" 13 3 23.0 Novosheshminsky district of the Republic of Tatarstan eleven A / F "Tatarstan" Dep. "Tubylgy Tau" 21 5 23.8 12 A / F "Pendant" twenty 5 25.0 13 A / F "Tatarstan" Dep. "Azeevo" twenty fifteen 75.0 Kamskoustinsky district of the Republic of Tatarstan fourteen LLC "Syukeyevo" 56 35 62.5 Rybnoslobodsky district of the Republic of Tatarstan fifteen LLC "Slobodskoye" 26 four 15.4 Apastovsky district of the Republic of Tatarstan 16 LLC SHP "them. Rakhimov thirty fifteen 50,0 17 LLC SHP "Gran" thirty 13 43.3 eighteen LLC SHP "Ibragimov and K" thirty eleven 36.6 19 LLC SHP "Alga" d. Tumaevo thirty eighteen 60.0 twenty MTF "Utyamyshevo" 28 21 75.0 21 LLC SHP “Enali d. St. Yumraly 32 twenty 62.5 22 MTF "Azbaba" 25 13 52.0 23 LLC SHP "Enali" d.Sr. Baltai 25 fifteen 60.0 24 LLC SHP "Enali" d.Devlekeevo thirty 16 53.3 25 LLC SHP Tabar 45 thirty 66.6 26 MTF "B.Kokuzi" thirty 22 73.3 27 000 SHP "Alga" d.Shugay fifty 38 76.0 Verkhneuslonsky district of the Republic of Tatarstan 28 "Red East - Agro" 52 fourteen 26.9 Tetyushsky district of the Republic of Tatarstan 29th LLC Mayak fifteen 7 46.6 Mamadyshsky district of the Republic of Tatarstan thirty A / F "Takanysh" twenty 3 15.0 Saratov region of the Russian Federation 31 CJSC Plemzavod “Tudovoy” 26 10 38,4 32 LLC "Sergeevskoe" 45 5 11.1 Republic of Bashkortostan 33 them. Lenin fifteen fifteen 100.0 34 them. Marcel Khuzin thirty 25 83.3 35 Aktay 9 9 100.0 Total samples 1378 638 46.2

Claims (1)

An enzyme-linked immunosorbent assay system for the serological diagnosis of cattle reovirus infection and for monitoring post-vaccination immunity, characterized in that it contains a specific antigen of the Reo 1 Lang-DEP strain of type I reovirus inactivated with a 0.08% solution of 1.2- aminoethylaziridine, which is a specific protein at a concentration of 5-10 μg / cm ³ adsorbed on the surface of polystyrene wells in carbonate-bicarbonate buffer with merthiolate in a final concentration of 0.1-0.2 mg / cm ³ , control position total serum obtained for type I reovirus antigen with ELISA activity of 1: 3200-1: 6400, control negative serum, antispecific conjugate, monosubstituted potassium phosphate, disubstituted potassium phosphate, disubstituted potassium chloride, chromogen (ortho-phenylenediamine, stop ortho-phenylenediamine) -reagent and panels for staging the reaction of enzyme immunoassay.

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