FR3074912A1 - METHOD FOR DISTINGUISHING AN IMMUNIZATION STATE BY THE ABSTRACT BRUCELLA VACCINE FROM A NATURAL INFECTION STATE - Google Patents

Abstract

The present invention relates to a method for distinguishing a state of immunization by the vaccine against Brucella abortus from a state of natural infection. The method consists in constructing a sensitive animal model based on a method for detecting Brucella IgG and IgM subtype antibodies, in order to obtain the growth and decline regimes of the immunized antibodies by attenuated anti-brucella vaccines ( strain S2 of Brucella suis and Brucella abortus strain A19) commonly used in China and natural infection with a standard virulent strain (Brucella abortus strain 2308). Based on the characteristics of the natural infection groups and the vaccinated groups, 5 states are determined and a method for distinguishing natural infection from vaccine immunization is established. The method is verified and its reliability is confirmed. The method distinguishes natural infection from vaccine immunization by detecting IgG and IgM from only one sample at a given time. The method is simple, convenient and very accurate, it is fast and cheap, suitable for various types of laboratory and meets the needs of large scale detection. The method addresses technical gaps in the field, at home and abroad, and provides technical assistance for the prevention and control of brucellosis.

Description

The present invention relates to a method for distinguishing a state of immunization with the Brucella abortus vaccine from a state of natural infection; it belongs to the technical field of detection of biological products.

Prior art

Brucellosis being an important zoonosis it seriously threatens public health and the healthy development of the livestock industry. For farm animals, the main symptoms of brucellosis are undulating fever, weight loss, wasting and decreased milk production and, for pregnant females, it causes symptoms such as abortion, stillbirth and long-term infertility. Brucellosis poses a serious threat to international trade and also affects the export of animal products; it is considered by the Office international des épizooties (OIE) as a class B infectious disease, and has been placed in China on the B list of diseases. With a few developed countries being the exception, brucellosis has occurred in more than 170 countries around the world and is particularly widespread in Central Asia and the Middle East.

Brucellosis was well controlled in China from the 1960s to the 1980s of the last century, but in recent years the number of epidemics of brucellosis and the incidence on the animal population have increased from year to year. Since brucellosis is a zoonosis that seriously affects public health and the healthy development of the livestock industry, its prevention and control have become urgent. Brucellosis was included in China in the "National plan for the medium and long term against animal diseases (2012-2020)". In countries or regions where brucellosis is eliminated, measures such as vaccination, quarantine and slaughter are commonly combined to achieve the goal of purifying the population.

Vaccination is one of the most effective measures to control and prevent brucellosis in animals. Brucella abortus strain S19 and Brucella suis strain S2 play an important role in the prevention and control of brucellosis in China. In particular, the strain S 2, developed in China in the 1960s, has good protective effects on animals susceptible to brucellosis and is characterized by low virulence and a relatively low threat to public health posed by the secondary reactions of the vaccination. However, the above two vaccines are smooth strains and the antibodies they produce are not distinguishable from those produced during natural infections, making the use of vaccines very difficult.

Summary of the invention

The aim of the present invention is to establish a technical method capable of distinguishing between immunization with the vaccine against Brucella abortus and natural infection. The present invention is based on the indirect ELIS A method previously established for antibodies to IgG and IgM subtypes of Brucella abortus (CN105067812A and CN106771182A), comprising: continuous monitoring of the growth and decline regime of the IgG subtypes and IgM in the blood of adult cattle vaccinated subcutaneously with a live vaccine against Brucella abortus (strain A19), vaccinated orally with a live vaccine against Brucella abortus (strain S2), and who have received an injection of live barium from the virulent strain 2308 of Brucella abortus to obtain an artificial infection, respectively, and the determination of a critical value for different states (immunization / infection), in order to establish a model of the growth and decline regimes of the d subtypes antibody in cases of natural immunization / infection with Brucella abortus vaccine, and to provide a technical method for identifying nti-Brucella and natural infection using antibody subtypes, thus meeting the requirements of the epidemiological investigation of brucellosis in China.

Technical solutions of the present invention

1. A method for distinguishing a state of immunization by the vaccine against Brucella abortus from a state of natural infection according to the present invention, characterized by determining the state of infection / immunization of a cattle tested by analysis the level of antibodies to IgG and IgM subtypes specifically against Brucella in cattle, so as to differentiate between immunization with the vaccine against Brucella abortus and natural infection and thus meet the requirement of investigation epidemiological of brucellosis, knowing that this is not a method of diagnosing the disease.

2. The method for distinguishing a state of immunization by the vaccine against Brucella abortus from a state of natural infection according to the present invention, further characterized in that the detection of the level of antibodies of IgG subtypes and IgM specifically antiBrucella is based on a specific kit for detection of IgG antibodies to Brucella abortus and a specific kit for detection of IgM antibodies to Brucella abortus.

3. The method for distinguishing a state of immunization by the vaccine against Brucella abortus from a state of natural infection according to the present invention, further characterized in that the determination of the state of infection / immunization of cattle tested is carried out according to the following stages: establishment of growth and decline regimes of IgG and IgM in cattle immunized with a Brucella vaccine and growth and decline regimes of IgG and IgM in cattle where natural infection of Brucella is simulated, and delimitation of ranges for immunization with the Brucella vaccine and natural infection, that is to say that the indicators for judging the state of infection / immunization of the animals tested are the following:

when the P% value of a serum sample is greater than or equal to 20%, this indicates that the serum sample is positive for the antibody of the IgG subtype of Brucella abortus, and when the P% value of l the serum sample is less than 20%, this indicates that the serum sample is negative for the IgG antibody of Brucella abortus; and when the P% value of a serum sample is greater than or equal to 85%, this indicates that the serum sample is positive for the antibody of the IgM subtype of Brucella abortus, and when the P% value of the serum sample is less than 85%, this indicates that the serum sample is negative for the Brucella abortus IgM antibody.

4. The method for distinguishing a state of immunization with the Brucella abortus vaccine from a state of natural infection according to the present invention, further characterized by the fact that the indicators making it possible to judge the state of infection / immunization of the tested animal are established based on the following test results:

when the test results are: IgG; S / P> 60%; and IgM, S / P <85%, they are marked as state 1, i.e. natural infection;

when the test results are: IgG; S / P> 60%; and IgM, S / P> 85%, they are marked as state 2, i.e. natural infection or early stage of immunization A19;

when the test results are: IgG, S / P between 20% and 60%; and IgM, S / P> 85%, they are marked as state 3, i.e. immunization A19;

when the test results are: IgG, S / P between 20% and 60%; and IgM, S / P <85%, they are marked as state 4, i.e. advanced stage of immunization A19 or advanced stage of infection; and when the test results are: IgG, S / P <20%; and IgM, S / P> 85%, they are marked as state 5, i.e. S2 immunization or acute infection.

5. The method for distinguishing a state of immunization with the Brucella abortus vaccine from a state of natural infection according to the present invention, in which the processing of the data can be carried out by computer means.

Specific embodiments of the present invention

The present invention determines the antibody titers using, respectively, an indirect ELISA kit for detection of IgG antibodies against Brucella abortus and an indirect ELISA kit for detection of IgM antibodies against Brucella abortus (CN 105067812A and CN106771182A) and the specific method refers to note 1.

1. The present application has established the antibody evolution regimes in cattle vaccinated with the live brucellosis vaccine (strain A19) (hereinafter called the A19 vaccine).

More than 50 brucellosis-negative cattle have been selected and vaccinated by injection according to the directions for use of the A19 vaccine. Blood samples were taken at different times and the serum was separated. Antibody titers were determined using, respectively, an indirect ELISA kit for detection of IgG antibodies against Brucella abortus and an indirect ELISA kit for detection of IgM antibodies against Brucella abortus (CN105067812A and CN106771182A ) and the P values measured at each step were recorded.

In the present invention, 92 adult cattle negative for anti-Brucella antibodies were vaccinated by injection of the A19 vaccine, and serum samples were taken respectively on days 7, 15, 30, 60, 90, 120 and 150 after vaccination. for the purpose of detecting antibodies, this so as to define the regimes for the development of IgG and IgM in cattle after vaccination with the A19 vaccine. The results of the study (see Figure 1) showed that IgG and IgM antibodies in cattle began to appear on day 7 after vaccination with the A19 vaccine, peaked on day 15, and then gradually decreased. With regard to IgG, after the 15th day, the level of antibodies in the group vaccinated with the A19 vaccine decreased significantly, and the average value of IgG was close to the critical value on the 90th day; and the positive rates in the population at different times were 62.64% (7th day), 84.78% (15th day), 78.26% (30th day), 75% (60th day), 36.95% (90th day), 27.17% (120th day) and 14.22% (150th day). There were statistical differences in antibody levels at different times, with the most significant difference between day 60 and day 90 (p <0.01) indicating that the IgG content in the animal's body significantly reduced after the 60th day. Compared with IgG, the IgM of the group vaccinated with the A19 vaccine changed more significantly. 15 days after vaccination the IgM antibody level peaked, then decreased rapidly. On the 60th day, the mean value was close to the critical value, and the IgM antibodies had completely disappeared on the 120th day after vaccination. The IgM content varied significantly between the different periods. Antibody titers were significantly different between the 15th and the 30th day, between the 30th and the 60th day, and between the 90th and the 120th day (p <0.01). The profile of changes in antibody titers was plotted on the basis of the arithmetic means of the antibody levels of the population at different times after vaccination (see Figure 2). Figure 2 shows that the IgG level was above the critical value in the 15 days following the A19 vaccination, then that it gradually decreased and was significantly below the critical value on the 90th day. The IgM peaked on the 15th day, then decreased continuously and was below the critical value after 60 days.

2. The present application has established the antibody evolution regimes in cattle vaccinated with the live brucellosis vaccine (strain S2) (hereinafter called the S2 vaccine).

The S2 vaccine is the most widely used live Brucella vaccine in China. It is of great importance to study the evolution profile of the different antibody subtypes and to find important indicators to distinguish the different immune states.

More than 50 brucellosis-negative cattle have been selected and vaccinated orally according to the instructions for use of the S2 vaccine. Blood samples were taken at different times and the serum was separated. Antibody titers were determined using, respectively, an indirect ELISA kit for detection of IgG antibodies against Brucella abortus and an indirect ELISA kit for detection of IgM antibodies against Brucella abortus (CN105067812A and CN106771182A ) and the P values measured at each step were recorded.

Unlike A19, the recommended route for S2 vaccination in cattle was the oral route, and the antibody titer in vaccinated animals exhibited the characteristics of a low titer and rapid decay (see Figure 3). Specifically, in most cattle in the experiment, antibodies of the IgM subtype began to be generated on the 7th day after vaccination and peaked on the 15th day. At the time of the peak, the level of antibodies (P% value) was approximately 3 times higher than the critical value, and the P% values of most samples were between 100 and 200. After the 15th day, their level of antibody was rapidly decreasing and by day 60 the mean value in the population was below the critical value. By the 90th day, most of the population samples (55/56) were below the critical level. Unlike the A19 vaccine, the population vaccinated with the S2 vaccine had very low levels of IgG subtype antibodies, except in some samples (4/56) whose antibody levels were slightly above the critical value on the 30th day after vaccination, and in most samples the IgG subtype antibody levels were below the critical value during the entire surveillance period, which was one of the most important characteristics of cattle vaccinated with S2 vaccine . Compared with the A19 vaccine group, the IgM subtype antibody levels in the S2 vaccine group evolved more radically over time. After vaccination with the S2 vaccine, the levels of IgM subtype antibodies changed significantly between the 7th and the 15th day, between the 15th and the 30th day, between the 30th and the 60th day, between the 60th and the 90th day , and between the 90th and 120th day (p <0.05). For IgG, although antibody levels in most samples were below the critical value during the entire monitoring period, there was also a significant difference between the 30th and the 60th day and between the 60th and 90th day (p <0.05). The antibody evolution profile was plotted on the basis of the arithmetic means of the antibody levels of the population at different times after vaccination (see FIG. 4). Comparing FIG. 2 and FIG. 4, it can be seen that the vaccinated cattle of group S2 have an average level of IgG antibodies significantly lower than that of group A19, and as regards IgM, although their peak value was lower than that of the A19 vaccine group, their level and the extent of their decrease were closer to those of the A19 group after 15 days.

3. The present application established the regimes for the evolution of the antibodies detected by the regimes for growth and decline of IgG and IgM in cattle infected with a reference virulent brucellar strain (strain 2308).

Antibodies from cattle infected with strain 2308. 4 cattle negative for brucellosis were selected to be artificially infected in a P3 laboratory. Blood samples were taken at different times and the serum was separated. Antibody titers were determined using, respectively, indirect ELISA kits for detection of IgG antibodies against Brucella abortus and indirect ELISA kits for detection of IgM antibodies against Brucella abortus (CN 105067812A and CN106771182A) and P values measured at each stage were recorded.

In the present application, the natural infection has been simulated in experimental cattle in a test with a virulent reference strain of brucella abortus (strain 2308), and the growth and decline regimes of the antibodies have been followed ( see Figure 5). The result was that, compared to the vaccinated group (including the A19 vaccine group and the S2 vaccine group), the infected group exhibited the characteristics of late and sustained production of antibodies. Antibody of the IgM subtype was detected for two weeks after infection and peaked on day 30, then decreased rapidly and was below the critical value on day 90. Furthermore, the highest titer of IgM antibodies was only 2 times higher than the critical value and the differences in the antibody levels between the different infected subjects were relatively small. Compared to IgM, although IgG antibodies were produced later, IgG continued to increase thereafter and remained stable throughout the surveillance period.

4. The present application has established an evolution profile of the different antibody subtypes and has determined the ranges for different states of infection.

In order to better understand intuitively the differences between the different groups, we have combined the evolution profile of IgG and IgM antibodies in accordance with the change regime of the antibody subtype in the infected group (strain 2308) and the vaccinated groups ( including Al9 and S2), and we obtained an analytical curve making it possible to distinguish vaccination with the brucella vaccine and natural infection (see Figure 6). In this figure, lines 1 and 2 represent the antibody evolution profiles after an artificial infection with strain 2308, lines 3 and 4 represent the antibody evolution profiles after vaccination with the A19 vaccine, lines 4 and 5 represent the profiles after vaccination with the S2 vaccine; lines 1, 3 and 5 with the arrowhead represent IgM, and solid lines 2, 4 and 6 without arrowhead represent IgG. The combined regime curve of the antibody titer was divided into 3 regions on the vertical axis (Y axis, S / P value, indicating the antibody level) and the horizontal axis (X axis, days, indicating the time since infection / vaccination) respectively according to the level of antibodies in each model. On the vertical axis, the negative region of IgG antibodies corresponds to an S / P of less than 20%, and the positive region of IgG antibodies corresponds to an S / P of more than 20%; and the negative region of IgM antibodies corresponds to an S / P less than 85%, and the positive region of IgM antibodies corresponds to an S / P greater than 85%. The evolution profile shows that the IgGs of group S2 were still in the negative region, while in group A19 the average value of IgG had remained around 35% from the 15th day, then decreased after the 60th day and started to drop below critical value on day 90. In the group infected with strain 2308, its mean value, after having been greater than the critical value on day 30, had increased and remained at a relatively high level. The time of the X axis can be artificially divided into three phases according to the differences in the regimes of antibody titers between the different groups. The first phase is that of the 15 days following infection / vaccination, which can be called the antibody raising step, during which the IgG and IgM of the A19 vaccine group and the antibodies of the IgM subtype of the S2 vaccine group both increased to their peak on the 15th day; however, in the group infected with strain 2308 the IgM reached its peak during the next phase and during this phase of antibody increase, although the IgM increased, their level was still below the critical value. The second phase, from the 15th to the 90th day after infection / vaccination, has been known as the antibody decay phase. During this phase, the antibody titers of the A19 and S2 vaccine groups showed a downward trend, and the mean values of IgM subtype antibodies of the A19 and S2 vaccine groups were both close to the critical value on the 60th day and lower than the critical value on the 90th day. In the A19 vaccine group, the downward trends in IgG and IgM antibodies were basically the same, both relatively slow. The infected group showed a completely different trend during this phase, their IgM showed a peak profile for 30 days, the antibody rapidly increasing in 15 days then decreasing rapidly, and their average value was greater than the critical value during this phase; IgG started to appear after the 30th day and remained at a high level. The third phase, i.e. 90 days after infection / vaccination, has been designated as the phase of disappearance of the antibodies. At this point, with the exception that the IgG antibody of the group infected with strain 2308 was still above the critical value, not all antibody subtypes of all other groups had been tested. detected. The analysis in FIG. 6 shows that during the second phase, in particular from the 60th to the 90th day, the group infected with the 2308 strain was crossed with the group vaccinated with the A19 vaccine, which makes it difficult to assess precisely the actual situation of infection with the virulent bacterial strain or immunization by vaccine. In this case, error in judgment at a specific stage can be avoided by taking samples again (an interval of more than 2 weeks from the first sample taken) for detection.

The levels of IgG and IgM antibodies were divided into 5 infection / vaccination states according to the infection / vaccination model established in combination with the detection results of a large number of clinical samples (the results are presented in Table 1): (1) natural infection (IgG; S / P> 60%; IgM, S / P <85%; called state 1), (2) natural infection or initial stage of immunization with the Al9 vaccine ( IgG; S / P> 60%; IgM, S / P> 85%;

called state 2), (3) immunization with the A19 vaccine (IgG, S / P 20% - 60%; IgM, S / P> 85%;

called state 3), (4) advanced stage after vaccination with the A19 vaccine or advanced stage after infection with strain 2308 (IgG, S / P 20% - 60%; IgM, S / P <85%; called state 4) , and (5) S2 vaccine immunization or acute infection (IgG, S / P <20%; IgM, S / P> 85%;

called state 5). Regarding states 2 and 4, in order to guarantee the authenticity of the results, the samples must be taken again after 15 days to carry out the detection. The results of the additional test can be used to accurately judge the infection / immunization status of the test animals, and the indicators to judge the infection / immunization status of the test animal are defined as follows:

when the P% value of the serum sample is greater than or equal to 20%, this indicates that the serum sample is positive for the antibody of the IgG subtype of Brucella abortus, and when the P% value of l the serum sample is less than 20%, this indicates that the serum sample is negative for the IgG antibody of Brucella abortus; and when the P% value of the serum sample is greater than or equal to 85%, this indicates that the serum sample is positive for the antibody of the IgM subtype of Brucella abortus, and when the P% value of the serum sample is less than 85%, this indicates that the serum sample is negative for the Brucella abortus IgM antibody.

Table 1. Intervals of the different states

States IgG (S / P * States 100%) IgM (S / P * 100%) 1 > 60% <85% Natural infection 2 > 60% > 85% Natural infection or initial stage of immunization with the A19 vaccine 3 20% - 60% > 85% Immunization with the A19 vaccine Advanced stage of immunization A19 or stage 4 20% - 60% <85% advanced infection (a new sample must be taken within 15 to 30 days to carry out the detection) 5 <20% > 85% Immunization with the A19 vaccine or acute infection

5. The detection carried out on clinical samples with a known past confirms the reliability of the present invention.

The condition of 50 clinical samples with a known past was evaluated using the method established in the present invention, and 92% of the assessments carried out (46/50) were in accordance with real situations (for more details, see Table 2 from example 1). There were two possible situations for states 2 and 4, and the results of the verification showed that a total of 6 samples had been classified in state 2, knowing that 5 of these samples were natural infections and 1 a immunization with the A19 vaccine, which indicates that at this stage natural infection was the majority. With regard to state 4, a total of 6 samples were tested, of which 2 samples, representing 33% of the total, were immunizations with the A19 vaccine, in this borderline condition it is difficult to distinguish between natural infection and immunization by the A19 vaccine and you have to combine the results of the second detection to be able to make a judgment. The above results have shown that the method established by the present invention makes it possible to more reliably identify the state of immunization with a Brucella vaccine and the state of natural infection.

Brief description of the illustrations

Figure 1 is a graph relating the antibody titer of cattle vaccinated with the A19 vaccine and the time since vaccination (day). Graph A shows the relationship between the IgG antibody titer (P%) and the time since vaccination (day); and graph B shows the relationship between the IgM antibody titer (P%) and the time since vaccination (day) (*** indicates a very significant difference p <0.001, ** indicates p <0.01 and * indicates p <0.05).

Figure 2 shows the growth and decline regimes of IgG and IgM antibodies in cattle after immunization with the A19 vaccine.

Figure 3 is a graph relating the antibody titer of cattle vaccinated with S2 vaccine and the time since vaccination (day). Graph A shows the relationship between the IgG antibody titer (P%) and the time since vaccination (day); and graph B shows the relationship between the IgM antibody titer (P%) and the time since vaccination (day) (*** indicates a very significant difference p <0.001, ** indicates p <0.01 and * indicates p <0.05).

Figure 4 shows the growth and decline regimes of IgG and IgM antibodies in cattle after immunization with the S2 vaccine.

FIG. 5 is a graph relating the antibody titer and the time since immunization (day) in 5 cattle after an artificial infection with the virulent strain 2308. Graph A shows the relationship between the antibody titer IgG (P %) and the time since immunization (day) in cattle after infection; and Graph B shows the relationship between the IgM antibody titer (P%) and the time since immunization (day) after infection.

Figure 6 shows the growth and decline regimes of IgG and IgM antibodies in the case of vaccination and natural infection. Lines 1 and 2 in the figure are, respectively, the growth and decline regimes of the IgG and IgM antibodies after infection with strain 2308; lines 3 and 4 are the growth and decline regimes of the IgG and IgM antibodies after vaccination with the A19 vaccine; and lines 5 and 6 are the growth and decline regimes of IgG and IgM antibodies after vaccination with the S2 vaccine.

Information on microbial resources of the present invention

The microorganisms involved in the present invention are: The S2 strain of Brucella suis (CVCC70502), which is a Brucella suis biovar 2 and constitutes a reference strain for a live attenuated vaccine against brucellosis; the A19 strain of Brucella abortus (also called strain S19, CVCC 70202), which is a Brucella abortus biovar 1 and constitutes a reference strain for a live attenuated vaccine against brucellosis; and Brucella abortus 2308 (CVCC 788) which is a virulent strain. The three strains above have all been identified, preserved and supplied by the China Veterinary Culture Collection Center of China Institute of Veterinary Drug Control (No. 8 Zhongguancun South Street, Haidian District, Beijing) (see "China Veterinary Strains Catalog", 2 ^nd edition, China Agricultural Science and Technology Press, published by the China Institute of Veterinary Drug Control and China Veterinary Culture Collection Center, 2002, p35, p25, p24).

Advantages of the present invention

The present invention relates to a method for distinguishing a state of immunization with the Brucella abortus vaccine from a state of natural infection. The method is based on the detection methods of Brucella antibodies of IgG and IgM subtypes to construct a sensitive animal model in order to obtain the antibody regimes of the attenuated anti-brucella vaccines (S2 strain of Brucella suis and A19 strain of Brucella abortus) and a standard virulent strain (strain 2308 of Brucella abortus). Then, based on the characteristics of naturally infected and vaccinated groups, 5 states are determined and a method for distinguishing natural infection from vaccine immunization is thus established. The method is verified and its reliability is confirmed. The method distinguishes between natural infection and immunization by detecting IgG and IgM from only one sample at a given time. The method is simple, practical and very precise, it is fast and inexpensive, suitable for various types of laboratory and meets the needs of large-scale detection. The method fills technical gaps in the field, at home and abroad, and provides technical assistance for the prevention and control of brucellosis.

Specific embodiments of the invention

The following examples are provided for the purpose of further illustrating the technical solutions of the present invention and are not intended to limit the present invention.

Example 1

- The detection carried out on clinical samples with a known past confirms the reliability of the present invention.

The state of 50 clinical samples with a known past was evaluated using the method established in the present invention, and 92% of the assessments carried out (46/50) were in accordance with real situations (the results are presented in Table 2) . There are two 5 possible situations for states 2 and 4, and the results of the verification showed that a total of 6 samples had been classified in state 2, knowing that 5 of these samples were natural infections and 1 sample was immunization with the A19 vaccine, indicating that at this stage natural infection was predominant. With regard to state 4, a total of 6 samples were tested, of which 2 samples, representing 33% of the total, were 10 immunizations with the A19 vaccine. In this borderline condition it is difficult to distinguish between natural infection and immunization by the A19 vaccine and you have to combine the results of the second detection to be able to make a judgment. The above results prove that the method established by the present invention makes it possible to more reliably identify the state of immunization with a Brucella vaccine and the state of natural infection.

Table 2 Validation results of clinical samples

Serum number IgG (S / P * 100%) IgM (S / P * 100%) Assessment results Actual situation 1 35.695 132.12 State 3 A19 vaccination 2 97.7803 88.37395 State 2 Natural infection 3 15.2121 172.3887 State 5 Vaccination S2 4 27.7576 191.2862 State 3 A19 vaccination 5 47.803 145.5244 State 3 A19 vaccination 6 23.3939 111.609 State 3 A19 vaccination 7 45.4621 59.52626 State 4 Natural infection 8 35.7349 132.2636 State 3 A19 vaccination 9 40.3485 119.3555 State 3 A19 vaccination 10 18.1818 182.2514 State 5 Vaccination S2 11 21.2803 152.8153 State 3 A19 vaccination 12 63.6894 45.2027 State 1 A19 vaccination 13 60.9924 149.9164 State 2 Natural infection 14 34.3636 156.5207 State 3 A19 vaccination 15 29.9621 136.1519 State 3 A19 vaccination 16 53.3258 121.1886 State 3 A19 vaccination 17 70.5758 184.5183 State 3 Natural infection 18 52.7576 232.0784 State 3 A19 vaccination

19 40.2857 160.3134 State 3 A19 vaccination 20 59.0214 80.22535 State 3 A19 vaccination 21 27 97.30986 State 3 A19 vaccination 22 35.3143 109.8592 State 3 A19 vaccination 23 16.0143 105.507 State 5 Vaccination S2 24 47.7571 135.6197 State 3 A19 vaccination 25 61.9357 91.90141 State 2 A19 vaccination 26 41.4429 83.59155 State 4 Natural infection 27 43.0929 130.9437 State 3 A19 vaccination 28 44.0143 15.37576 State 4 Natural infection 29 41.7357 17.55758 State 4 A19 vaccination 30 98.3654 22.14545 State 1 Natural infection 31 105.369 18.81818 State 1 Natural infection 32 78.9625 41.58788 State 1 Natural infection 33 80.3335 29.99394 State 1 Natural infection 34 64.3382 11.84848 State 1 A19 vaccination 35 3.3365 116.6016 State 5 Vaccination S2 36 4.3987 121.7383 State 5 Natural infection 37 11.6985 105.8959 State 5 Natural infection 38 4.3697 94.2578 State 5 Vaccination S2 39 88.625 143.5142 State 2 Natural infection 40 120.669 180.5054 State 2 Natural infection 41 68.331 124.6882 State 2 Natural infection 42 77.125 33.641 State 1 Natural infection 43 16.336 163.221 State 5 A19 vaccination 44 8.369 36.952 Negative Negative 45 12,345 148.672 State 5 Vaccination S2 46 77.366 33.664 State 1 Natural infection 47 100.111 20.741 State 1 Natural infection 48 12,987 62.341 Negative Negative 49 21.336 55.227 State 4 Natural infection 50 30.125 34.6521 State 4 A19 vaccination

Note 1

Instructions for the iELISA method

1. Use (1) Treatment of the sample: the whole blood of the animal was taken then, after coagulation, the blood was centrifuged at 4000 r / min for 10 minutes and the supernatant was taken to obtain a clear serum without hemolysis.

(2) Preparation of the washing solution: before use, a solution for washing 20x was heated to room temperature (20 to 25 ° C) and stirred to dissolve the crystals (which can be heated in a water bath at 37 ° C for 5-10 minutes), then diluted with deionized water (1:20 dilution) and mixed well.

(3) Dilution of the test serum and the control serum: the test serum, the negative control serum and the positive control serum were diluted with the sample diluent (dilution 1:50) in a serum dilution plate.

(4) Operation steps

1) Addition of sample: the plate coated with antigen has been taken (it can be disassembled and used in batches according to the number of samples), the plate was washed once with 300 μl of washing solution per well, and the washing solution was discarded. The diluted test serum, the diluted positive control serum and the diluted negative control serum were added respectively to the ELIS A plate (100 μΐ / well), the positive control serum and the negative control serum being each added to 2 wells (well 1, well 2). After adding the sample, an incubation at 37 ° C was carried out for 30 minutes. The reaction plate was taken, the reaction solution was discarded, and 300 μΐ of lx washing solution were added to each well to perform 3 washes, and finally the reaction plate was dried by centrifugation or by tapping after the last wash.

2) Addition of an antibody labeled with the enzyme: The IgG HRP-rabbit anti-bovine antibody labeled with the enzyme or the IgM HRP-goat anti-bovine antibody labeled with the enzyme was diluted with diluents ( 1: 200 dilution), then added to each well (100 μΐ / well) and incubated at 37 ° C for 30 minutes. The reaction plate was taken, the reaction solution was discarded, and 300 μΐ of lx washing solution were added to each well to perform 3 washes, and finally the reaction plate was dried by centrifugation or by tapping after the last wash.

3) Staining and termination: the solutions of substrate A and B were mixed at 1: 1 (V / V), and immediately added to the ELIS A reaction plate, 100 μΐ / well. After staining at room temperature in the dark for 15 minutes, a stop solution was added (50 μΐ / well) to terminate the reaction.

2. Determination of the results: the OD45011111 value was measured with a microplate reader within 15 minutes after the end of the reaction.

(1) Calculation method:

(well 1+ well 2) OD _450nm

The mean OD ^ oiim value of the positive control = (well 1+ well 2) OD _450nm

OD ₄₅₀ nm mean value of negative control = OD ^ nm value of sample x

Positive percentage P% = --------------------------------------------- Average OD ^ nm value of the positive control 100% (2) Conditions for establishing the experiment:

The experiment was established when lOD450n _m mean of the positive control is greater than or equal to 0.8, rOD45o _nm mean of the negative control is less than 0.2 and P% is less than 20% (IgG) or 85% (IgM ).

(3) Determination of results:

when the P% value of the serum sample is greater than or equal to 20%, this indicates that the serum sample is positive for the antibody of the IgG subtype of Brucella abortus, and when the P% value of l the serum sample is less than 20%, this indicates that the serum sample is negative for the IgG antibody of Brucella abortus; and when the P% value of the serum sample is greater than or equal to 85%, this indicates that the serum sample is positive for the antibody of the IgM subtype of Brucella abortus, and when the P% value of the serum sample is less than 85%, this indicates that the serum sample is negative for the Brucella abortus IgM antibody.

Claims (4)

1. An in vitro method for distinguishing in cattle an immunization state provided by the Brucella abortus vaccine from a state of natural infection, characterized in that the growth and decline regimes of the antibodies are determined. IgG and IgM subtype specifically directed against Brucella in a sample of cattle serum and in that ranges for different states of infection or vaccination are determined. 2. Method according to claim 1, characterized in that the indicators making it possible to judge the state of infection / immunization of the animal whose serum is tested are the following: when the stock of IgG specifically directed against Brucella is S / P> 60%; and the titer of IgM specifically directed against Brucella is S / P <85%, the animal whose serum has been tested is marked as state 1, ie. natural infection; when the ratio of IgG specifically directed against Brucella is S / P> 60%; and the titer of IgM specifically directed against Brucella is S / P> 85%, the animal whose serum has been tested is marked as state 2, ie. natural infection or early stage of immunization with the vaccine using strain A19; when the ratio of IgG specifically directed against Brucella is S / P between 20% and 60%; and the titer of IgM specifically directed against Brucella is S / P> 85%, said animal whose serum has been tested is marked in state 3, ie. immunization with the vaccine using strain A19; when the ratio of IgG specifically directed against Brucella is S / P between 20% and 60%; and the titer of IgM specifically directed against Brucella is S / P <85%, said animal whose serum has been tested is marked in state 4, ie. advanced stage of immunization with the A19 strain vaccine or advanced stage of infection; and when the ratio of IgG specifically directed against Brucella is S / P <20%; and the titer of IgM specifically directed against Brucella is S / P> 85%, said animal whose serum has been tested is marked state 5, that is to say. immunization with the vaccine using the S2 strain or acute infection. 3. Method according to claim 2, characterized in that a second sample is taken within 15 to 30 days after the first detection when the animal whose 5 the serum has been tested is classified in category 4 and in that the titer of the antibodies IgG and IgM specifically directed against Brucella is again determined. 4. The method for distinguishing a state of immunization by the vaccine against Brucella abortus from a state of natural infection according to claim 1, characterized in that the processing of the data can be carried out by computer means.