KR102368216B1 - Process for detecting latent infected cow with bovine tuberculosis using ELISA boosting method - Google Patents

Abstract

The present invention provides a method for detecting tuberculosis-positive cattle by applying the ELISA boosting test to cattle infected with tuberculosis latent tuberculosis that are not detected by the tubecurin intradermal injection reaction method and the gamma interferon method, which are cellular immune methods that are currently approved and used for screening for bovine tuberculosis disease. would like to provide The above method utilizes the fact that the ELISA test results (OD, SP values) are boosted before and after intradermal inoculation of the PPD diagnostic solution used in the tubercurin intradermal injection reaction method. The ELISA boosting test method of the present invention is a method of detecting whether the tuberculosis bacillus is infected based on the S/P (%) value of the ELISA antibody before and after the tubercurin intradermal injection reaction method (PPD inoculation). It can be usefully used as a detection method that can eradicate latent Mycobacterium tuberculosis in ".

Description

{Process for detecting latent infected cow with bovine tuberculosis using ELISA boosting method}

The present invention relates to a method for detecting latent infection with bovine tuberculosis disease, and more particularly, to a tuberculin intradermal injection reaction method for tuberculosis-positive livestock. Infection with tuberculosis disease undetectable by cellular immunity method using the fact that intradermal inoculation with tuberculosis diagnostic solution (PPD-T) boosts the measured value of the ELISA antibody test result (absorbance OD, and sample-measured value S/P value compared to positive) It relates to a detection method that can detect latent or energized "latent tuberculosis-positive cattle with small tuberculosis disease".

Bovine tuberculosis disease is a zoonotic disease transmitted by Mycobacterium bovis , and is designated as a second kind of livestock infectious disease in Korea. In the literature, bovine tuberculosis disease shows clinical symptoms such as fever, weakness, thinness, enlarged lymph nodes, anemia, and decreased milk flow in severe cases. is known as It is a chronic, debilitating disease that has not been eradicated for a long time and causes severe economic loss. However, despite huge budgets for tuberculosis testing, it is not easily eradicated worldwide.

In Korea, the method for detecting tuberculosis disease in cattle in slaughterhouses is that the slaughter inspector visually collects samples for tuberculosis-suspicious lesions during the 'shipment live/disassembly test', and then confirms the 'positive tuberculosis disease livestock' through a detailed PCR test of the sample. there is.

On the other hand, the method for testing bovine tuberculosis disease on farms uses two cellular immunoassays worldwide. Since the 1970s, the 'Tubercurin Intradermal Injection Reaction Method', which inoculates a purified protein derivative (PPD) diagnostic solution for tubecurin intradermal testing, has been used since the 1970s. In addition, since 2013, the 'gamma-interferon (γ-INF) method', which is used in advanced livestock countries such as the UK and Australia, and has excellent sensitivity and specificity, has been introduced and has been used as a test method until now.

The tubercurin intradermal injection reaction method is a cellular immunodiagnostic method using delayed-type hypersensitivity reaction, by intradermally inoculating PPD (purified protein derivative) tuberculosis diagnostic solution (PPD-T), a cultured product of Mycobacterium tuberculosis, into the coccygeal cortex wall of cattle with high sensitivity. , If the individual is already infected with Mycobacterium tuberculosis, immune cells gather to remove the antigen, PPD, and as a result, a nodule is formed at the inoculation site near the root of the tail to confirm the infection axis. Disadvantages are that the cellular immune response shows a high detection rate in the early and middle stages of infection, and the detection rate drops in the late stages of tuberculosis infection (see Fig. 1), and false positives due to non-specific reactions such as tuberculosis-like bacteria (Johne's disease, paratuberculosis, Johne's disease) It is known that a false-negative reaction can occur when co-infection with a reaction and immunosuppressive disease.

In the gamma interferon test, when a PPD antigen is added to whole blood collected from an animal, antigen-specific memory cells (T cells) are rapidly restimulated to secrete interferon gamma, a type of cytokine, which interferon gamma is induced by the antigen. It is used as a specific marker of a cellular response and is a method of measuring it. As a disadvantage, the gamma-interferon method also has a lower detection rate in the late stages of tuberculosis infection, and may cause false-negative reactions when co-infected with immunosuppressive disease. In addition, after collecting blood from cattle on site, at least 5 ml of whole blood is collected in a tube treated with heparin anticoagulant for each individual, and transported to the laboratory at room temperature (22±3℃) for 16 to 24 hours. Incubation time is required.

In order to compensate for the above shortcomings in Korea, from 2009 to 2013, in order to increase the positive detection rate, the humoral immunization method known to have a high detection rate in the late stages of tuberculosis infection, the 'ELIZA test (Bionot's BTB 2.0, Central Vaccine Research Institute) ProCheck) was used. After blood collection, ELISA test was performed. If a positive individual was confirmed, the 'Tubercurin intradermal injection reaction method' was used a second time to confirm. However, the ELISA test method was abolished and replaced with the current gamma interferon method after being abolished because the sensitivity was too high when applied alone and the coincidence rate was low when compared with the tubecurin intradermal injection reaction method currently in use.

When the tubercurin intradermal injection method or gamma interferon method, which are cellular immune methods currently used worldwide for screening bovine tuberculosis, was applied, negative results were obtained in both diagnostic methods. As a 'latent infection cow', there is a risk of developing tuberculosis infection in the farm. It is worth paying more attention to the diagnostic method using cellular immunity, such as the use of anti-inflammatory drugs in cattle farms, calving, leucosis, bovine viral diarrhea (BVD), immunosuppressive diseases, etc. Simultaneous infection with tuberculosis may cause false negatives due to cellular immunity, which may limit detection.

As in the present case, if no tuberculosis disease-positive livestock with reduced cellular immunity or late infection is detected during a diagnostic test using only cellular immunoassay in a tuberculosis-infected farm, at an appropriate time, 'cows with latent infection with bovine tuberculosis disease' remain undetected at the tuberculosis-infected farm. It can be a factor in the transmission of tuberculosis and limit the eradication of the disease.

Korean Patent Laid-Open Publication No. 10-2017-0115211

The inventors of the present invention conducted the tuberculin test, gamma-interferon method (γ-INF), ELISA for about 3 years from December 2013 to December 2016 targeting cattle tuberculosis-infected farm households and tuberculosis-infected cattle. By studying the correlation and concordance rate of the law, it was possible to confirm the positive axis of bovine tuberculosis (PPD, γ-INF positive) through the close examination of the autopsy sample from the individuals confirmed to be positive only by the ELISA method as well as the correlation between each other. axis), it was found that the ELISA method result value (OD, S/P(%) value) value increased depending on the individual 7 days after PPD inoculation.

Therefore, the present invention confirmed the reason that the agreement rate with the conventional tubecurin intradermal injection reaction method is low in order to detect latent tuberculosis-positive cattle that cannot be detected by existing cellular immunity methods such as the tubercurin intradermal injection reaction method and the gamma interferon method. The purpose of this study is to clarify the improvement plan of the ELISA Act, which was abolished in the inspection items, and to provide a plan for eradicating bovine tuberculosis disease, a zoonotic infectious disease, by detecting the currently undetectable tuberculosis latent infected cattle by proposing the 'ELISA boosting test method'.

In order to achieve the object of the present invention, the present invention (a) obtains a primary sample by collecting primary blood before inoculation with purified protein derivative (PPD) from cattle suspected of having a latent infection with Mycobacterium tuberculosis, 7 to 14 days after PPD inoculation Thereafter, a second blood sampling step to obtain a second sample;

(b) calculating the S/P (%) value according to Equation 1 below by measuring the OD values of the antibodies of the primary and secondary samples collected by the ELISA test; and

<Formula 1>

(c) when the measured S/P (%) value of the first sample is 80% or more, and the S/P (%) value of the second sample is also 80% or more; When the S/P(%) value of the first sample is 50% or more and less than 80%, and the S/P(%) value of the second sample is 50% or more and less than 80% or 80% or more; When the S/P (%) value of the first sample is 30% or more and less than 50%, and the S/P (%) value of the second sample is 50% or more and less than 80% or 80% or more; Or, if the S/P(%) value of the first sample is 0% or more and less than 30%, and the S/P(%) value of the second sample is 50% or more but less than 80% or 80% or more, the step of judging tuberculosis It provides a method for detecting whether or not tuberculosis infection in cattle, including.

In one embodiment of the present invention, when the cattle suspected of being infected with Mycobacterium tuberculosis in step (a) are cattle of a farmhouse where the tuberculosis group occurs, the S/P (%) value of the primary sample in step (c) is 30% or more and 50% If the S/P (%) value of the secondary sample is 30% or more and less than 50%, or 50% or more but less than 80%, or increases to 80% or more, it can be determined as tuberculosis cow.

In one embodiment of the present invention, the cow suspected of having the Mycobacterium tuberculosis latent infection in step (a) may be a cow that is negative in the tuberculin intradermal injection reaction method and the gamma interferon test method.

In one embodiment of the present invention, the cattle suspected of having the Mycobacterium tuberculosis latent infection in step (a) may be Korean cattle or dairy cattle.

In one embodiment of the present invention, the secondary sample of step (a) may be blood collected 7 to 10 days after PPD inoculation.

In one embodiment of the present invention, the antibody in step (b) may be retested when the OD value is 0.15 or more in the negative control group and 1.5 or less in the positive control group.

According to the present invention, in the detection method for Mycobacterium tuberculosis infection, the humoral immune method ELISA boosting test method is simultaneously applied to false-negative individuals that are not detected by these two cellular immunoassay methods, such as the tubecurin intradermal injection reaction method and the gamma interferon method. It has been found that tuberculosis-positive cattle can be detected.

Therefore, in the ELISA boosting test method of the present invention, the tuberculin intradermal test (PPD) diagnostic solution is inoculated into the body of a cow, and the ELISA antibody before and after inoculation is S/P (%) to detect the presence of Mycobacterium tuberculosis infection. This method can be usefully used as a detection method that can eradicate latent Mycobacterium tuberculosis that remains undetected and causes farm spread in "bovine tuberculosis-inducing farms" infected with Mycobacterium tuberculosis.

1 is a cellular immune response vs. It is a graph schematically showing the humoral immune response.
Figure 2 shows autopsy lesions such as lungs and lymph nodes at the time of autopsy of a positive individual only in the ELISA test method.

Hereinafter, the present invention will be described in detail.

The present invention relates to (a) cattle suspected of having latent tuberculosis infection by collecting primary blood before inoculation with purified protein derivative (PPD), obtaining a primary sample, and collecting secondary blood 7 to 14 days after PPD inoculation to obtain a secondary sample. to obtain;

(b) calculating the S/P (%) value according to Equation 1 below by measuring the OD values of the antibodies of the primary and secondary samples collected by the ELISA test; and

<Formula 1>

(c) when the measured S/P (%) value of the first sample is 80% or more, and the S/P (%) value of the second sample is also 80% or more; When the S/P(%) value of the first sample is 50% or more and less than 80%, and the S/P(%) value of the second sample is 50% or more and less than 80% or 80% or more; When the S/P (%) value of the first sample is 30% or more and less than 50%, and the S/P (%) value of the second sample is 50% or more and less than 80% or 80% or more; Or, if the S/P(%) value of the first sample is 0% or more and less than 30%, and the S/P(%) value of the second sample is 50% or more but less than 80% or 80% or more, the step of judging tuberculosis It provides a method for detecting whether or not tuberculosis infection in cattle, including.

Bovine tuberculosis is a zoonotic disease transmitted by Mycobacterium bovis , and is designated as a type 2 livestock infectious disease in Korea. In farms, tuberculosis intradermal injection reaction method and gamma-interferon (γ-INF) test are used as methods for testing bovine tuberculosis disease. If it is determined to be a tuberculosis livestock (positive by the PPD method and/or γ-INF method), it is transferred to the slaughterhouse. At the slaughterhouse, during the dismantling examination after bleeding, the inspector visually identified tuberculosis nodules, pearlescent nodules, and cheesy granulomatous stools in the lungs, mediastinum lymph nodes, larynx lymph nodes, chest wall, and abdominal organs, and sampled suspected lesions from the slaughterhouse. The institution will confirm the positive axis through a PCR test. Because gross lesions are specific and typical compared to other diseases, if gross lesions are confirmed, most of the PCR tests for tuberculosis lesions are positive. For testing, tubecurin intradermal injection reaction method and gamma interferon test are performed at the same time.

In addition, tuberculosis testing will be performed on nearby farms that are epidemiologically related to recent purchases and sales from this farm. In other words, if tuberculosis infection is found in the slaughterhouse, follow-up testing for tuberculosis with a livestock confirmation test or epidemiological-related farmhouse test is a common route to detect tuberculosis.

However, although it is possible to detect tuberculosis-positive livestock through close examination after live decomposition at the slaughterhouse, it is difficult to prevent disease in a timely manner because many tuberculosis transmissions have already occurred.

In the case of slaughterhouse tuberculosis-infected farms, confirmation of livestock living together is performed three times at 2-3 month intervals. However, it is not confirmed by the tubercurin intradermal injection reaction method or the gamma interferon method, and it is sometimes found as tuberculosis-positive cattle during shipment from the slaughterhouse or during tuberculosis diagnosis. In this case, the tuberculosis-positive cattle are progressed to 'latent infection cows' without being confirmed by the tubercurin intradermal injection reaction method and the gamma interferon test method. have a risk

In the method for detecting Mycobacterium tuberculosis infection of the present invention, a primary sample is obtained by collecting primary blood before inoculation of purified protein derivative (PPD) into cattle suspected of having a latent Mycobacterium tuberculosis infection, and secondary blood is collected 7 to 14 days after PPD inoculation. obtaining a secondary sample (ie, step (a)). In step (a), the cattle suspected of having a latent infection with Mycobacterium tuberculosis may be cattle that are negative in the tubercurin intradermal injection reaction method and/or gamma interferon test method, and may be Korean cattle or dairy cattle. In general, Korean cattle often live 2-3 years, and dairy cows often live 7-8 years because they produce milk. Therefore, in the case of cows infected with Mycobacterium tuberculosis and having a latent infection, it can affect the milk produced, so it is particularly important to find out the latent infected cows.

In the present invention, the secondary sample of step (a) may be inoculated with PPD and blood may be collected from 7 to 10 days. In the case of a humoral immune response, since the reaction proceeds later than the cellular immunity after antigen sensitization (see Fig. 1), the ELISA method for detecting the humoral immune response can be usefully used in the late detection after antigen sensitization, When the ELISA test is additionally performed by supplementing the tubecurin intradermal injection reaction method and the gamma interferon test method, which are cellular immune responses, it is possible to additionally detect tuberculosis-infected cattle that were not detected in the cellular immune response, and to detect latent tuberculosis-infected cattle. And because it can contribute to the eradication of Mycobacterium tuberculosis.

The Mycobacterium tuberculosis infection detection method of the present invention measures the antibody titer (OD)S/P(%) value of the primary sample and the secondary sample collected by the ELISA boosting test method, and the S/P(%) value according to the following Equation 1 calculating [i.e., step (b)].

<Formula 1>

In step (b), the ELISA assay may be performed using a commercially available ELISA test kit.

In addition, the present invention is when the S/P (%) value of the measured primary sample is 80% or more, and the S/P (%) value of the secondary sample is also 80% or more; When the S/P(%) value of the first sample is 50% or more and less than 80%, and the S/P(%) value of the second sample is 50% or more and less than 80% or 80% or more; When the S/P (%) value of the first sample is 30% or more and less than 50%, and the S/P (%) value of the second sample is 50% or more and less than 80% or 80% or more; Or, if the S/P(%) value of the first sample is 0% or more and less than 30%, and the S/P(%) value of the second sample is 50% or more but less than 80% or 80% or more, the stage of determining tuberculosis step (c).

In the present invention, if the cattle suspected of being infected with Mycobacterium tuberculosis in step (a) in the present invention are cattle from a farmhouse with a tuberculosis group, the S/P (%) value of the primary sample in step (c) is 30% or more and less than 50%, and the secondary If the S/P(%) value of the sample is 30% or more and less than 50%, or 50% or more but less than 80%, or increases to 80% or more, it can be judged as tuberculosis cow.

In the present invention, cattle in the tuberculosis cluster outbreak farmhouse refers to cattle where tuberculosis-positive individuals were found in more than 1/3 of the total number of cattle in a single farm, and lived together in the same barn for tuberculosis outbreaks for more than 3 to 4 months. there is.

In the step (b) of the present invention, the retest can be performed when the OD value of the antibody is 0.15 or more in the negative control group and 1.5 or less in the positive control group.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Tuberculosis Cow Test

(1) Materials and methods

From December 2013 to December 2016, the tubercurin intradermal injection reaction method, gamma interferon test method, The incidence of tuberculosis by three test methods, including the ELISA test, was investigated, and the correlation between each test method was compared and analyzed. In addition, lesion sites and blood samples from cattle determined to be positive tuberculosis in the slaughterhouse dismantling test and tuberculosis diagnostic test were collected and PCR and biopsy were performed to confirm Mycobacterium tuberculosis.

The test kits and test subjects for each diagnostic method are as follows. The tuberculin intradermal injection reaction method was performed on tuberculosis-infected farm cattle, epidemiological-related cattle, and dairy cattle (over 1 year old). Gamma interferon test method (TB-Feron, Bionote Co., Ltd., centered on Korean beef) is a tuberculosis farm cattle, epidemiological-related cattle, Korean cattle monitoring inspection and various tests request axis (Breeder Periodical Inspection Center, Breeding Screening Breeder Improvement Association request axis, livestock Quarantine support headquarters monitoring). For the ELISA test method (BTB 2.0, Bionote Co., Ltd.), cattle from a farm with chronic tuberculosis were screened using the PPD-T method and the gamma interferon test method. PCR test (PCR-RT PCR-Digital PCR) and histopathologic examination include lesion tests such as lungs, chest wall, and mediastinum lymph nodes of cows suspected of being positive for tuberculosis during slaughterhouse dismantling examination, and larynx and larynx of cattle determined to be positive in various tuberculosis diagnoses. Neck lymph nodes such as the following lymph nodes and blood samples were collected and tested.

(2) Tubercurin intradermal injection reaction method (PPD-T diagnostic solution, Central Vaccine Research Center)

In accordance with the "Practical Guidelines for Prevention of Tuberculosis and Brucellosis", inoculate the PPD diagnostic solution for tubecurin intradermal test into the skin of the coccyx of the target animal to be screened for right tuberculosis, and then measure the skin thickness of the inoculated area before and after intradermal inoculation. did The skin thickness at the inoculation site is determined between 48 and 72 hours after inoculation. By measuring the skin thickness at the inoculation site, positive when the difference in swelling is 5 mm or more, pseudo-positive when it is 3 mm or more and less than 5 mm, and negative when it is less than 3 mm. was judged as

(3) Gamma Interferon (TB-Feron, Bionote Co., Ltd.)

1) blood sample collection

At least 5 ml of blood was collected from the jugular vein and tail veins of cattle. The collected blood was placed in a heparin tube and mixed with an anticoagulant. Shake gently so that hemolysis does not occur, and the collected blood samples were kept at room temperature (18-25 ° C) and transported to the laboratory within 24 hours after blood collection.

2) dispensing of blood

The blood sample contained in heparin was mixed well by gently shaking the sample tube up and down about 10 times (because it is an experiment that requires live lymphocytes, it is mixed in a range that does not cause damage to cells). Whole blood prepared above was dispensed to each well of a 24-well cell culture tray by 1.5 ml each in 3 wells. 100 μl of each of sterile PBS, PPD A, and PPD B were aseptically added to three wells in which whole blood was dispensed. It was rotated 10 times clockwise and counterclockwise on a flat surface (care was taken not to generate bubbles or contamination with other wells at this time). Since whole blood with hemolysis may be judged to be false-negative, care was taken to avoid hemolysis during blood collection and dispensing.

3) culture

A 24-well cell culture tray containing the sensitizing antigen and whole blood was incubated at 37° C. in a CO ₂ incubator for 16 to 24 hours.

4) Collection of plasma samples

After incubation, centrifuged at 500 x g for 10 minutes (room temperature), and the plasma of the supernatant except for the precipitated red blood cells was transferred to a microtube for a clean serological test using a pipette. At this time, red blood cells were not mixed, and when mixed, the supernatant plasma was recovered by centrifugation again.

5) Storage of plasma specimens

If plasma is not used on the day of collection, it can be stored at 2~8℃ for 7 days. It can be stored at -20 ℃ for 2-3 months, but before the ELISA test, it was left at room temperature (18-25 ℃) for about 15-30 minutes and used after vortexing.

6) γ-INF ELISA test (TB-Feron, Bionote Co., Ltd.)

The γ-INF level of each sample was measured according to the manufacturer's instructions.

7) Interpretation

① Check (Test validation)

- Average absorbance of negative control (NC) < 0.2, average absorbance of positive control (PC) ≥1.0

② Interpretation

- Positive: PPD Bovine absorbance - PBS absorbance ≥ 0.1 and PPD Bovine absorbance - PPD Avian absorbance ≥ 0.1

- Negative: PPD Bovine Absorbance - PBS Absorbance < 0.1 or PPD Bovine Absorbance - PPD Avian Absorbance < 0.1

Cows whose absorbance value of plasma to which PPD Bovine is added is 0.1 or more higher than that of plasma added with sterile PBS and 0.1 or more higher than that of plasma to which PPD Avian has been added is B. tuberculosis bacillus. It means there is an infection.

(4) ELISA test method (BTB 2.0, Bionote Co., Ltd.)

Purely isolated BTB antigen by enzyme immunoassay was adsorbed to a plate, and MPB70 enzyme was conjugated to attach to and react with the bovine tuberculosis antibody in the sample, and the bovine tuberculosis antibody was detected in bovine serum/plasma. After reacting the sample on the plate at 37 °C for 1 hour and washing, the substrate solution was added and reacted at room temperature for 15 minutes, and then the absorbance OD (optical density), which is the degree of color development at a measurement wavelength of 450 nm and a reference wavelength of 620 nm, was measured. Thus, negative and positive antibodies to bovine tuberculosis were distinguished.

1) Inspection procedure

The absorbance of each sample was measured according to the manufacturer's instructions.

2) Result Judgment

The S/P value was calculated by Equation 1 below, and the result was determined based on the calculated S/P value.

<Formula 1>

At this time, the average OD value of the positive control group was about 2.5 to 3.0, and the average OD value of the negative control group was about 0.05 to 0.1.

The criteria for determining the S/P (%) value are as follows.

- Negative: [S/P (%) value less than 30%] A sample showing an S/P (%) value of less than 30% was judged to be negative.

- Pseudo-positive: [30% ≤ S/P(%) value < 50%] Samples with an S/P(%) value of 30% or more and less than 50% were judged as pseudo-positive, and a pseudo-positive sample was tuberculosis outbreak status. Accordingly, it was included in the secondary PPD confirmation test.

- Positive: [50 ≤ S/P(%) value] A sample showing an S/P(%) value of 50% or more was judged as positive. Samples that were positive in the first test were retested for 2 wells or more, and if more than 1 well was judged positive in the retest result, the final positive was judged. The final diagnosis was performed by a professional veterinarian by using other clinical or experimental results (PPD test or r-IFN test, etc.) for the final positive specimen.

2. Correlation comparison of each test method

For about 3 years from December 2013 to December 2016, the correlation and concordance rate between the tuberculin intradermal injection reaction method, the γ-INF method, and the ELISA method were studied for cattle tuberculosis-infected farms and livestock with tuberculosis. correlation was found. In addition, an autopsy sample was collected from an individual that was positive only in the ELISA method, and it was confirmed that it was a bovine tuberculosis positive axis through a close examination. In addition, in the case of tuberculosis-inducing animals (positive tubecurin intradermal injection method and/or γ-INF method), it was found that there was a change in test results around 7 days after PPD inoculation in the ELISA method.

Specifically, the response results for the three screening methods from 2013 to 2016 are shown in Table 1 below (the tubecurin intradermal injection reaction method, γ-INF method and ELISA method concordance survey).

As shown in Table 1 above, the ratio of cattle identified as positive in the ELISA method among cattle identified as benign according to the current policy of judging individuals positive for PPD method and/or gamma interferon method as tuberculosis positive cattle was investigated. , 6/10 head (60%) in 2013, 51/66 head (77.3%) in 2014, 6/37 (16.1%) in 2015, and 13/57 head (22.8%) in 2016, In 2013 and 2014, the concordance rate was clearly high, indicating that there was a difference in the concordance rate by year.

As a result of finding the cause of this difference, in 2013 and 2014, the tubercurin intradermal injection reaction method was used in the ELISA test method. After inoculation of the PPD diagnostic solution, blood was collected during the slaughter of positive individuals and proceeded with a time interval (around 1 week) after the tubercurin intradermal inoculation. Accordingly, it was determined that the timing of blood sampling after PPD inoculation affects the accuracy of the ELISA test, and additional experiments were conducted to confirm this.

That is, in 2015 and 2016 (at the time of screening for tuberculosis farms in 2015 and 2016, 1 week after PPD inoculation and before PPD inoculation for cattle infected with tuberculosis (PPD method and/or γ-INF method positive) and ③ individuals showing positive only in ELISA test method The ELISA test results were compared by collecting blood before and after each, and as a result, the ELISA antibody titer significantly increased in about 80% of the cases of tuberculosis-infected cattle, and those who tested positive only in the ELISA test. In some of them, it was confirmed that the ELISA antibody titer increased in the same way as in tuberculosis-infected cattle.These individuals are judged to be tuberculosis-infected cattle under the current policy of judging those positive for tubercurin intradermal injection and/or positive for gamma interferon as tuberculosis-positive cattle. However, it was determined that the possibility of an individual infected with Mycobacterium tuberculosis was high.

3. Confirmation of tuberculosis in a positive individual only by the existing accredited test method negative or ELISA test method

As a result of an autopsy on an individual (③) that showed positive only in the ELISA test even though the tuberculin intradermal injection reaction method or the γ-INF method was negative in a farmhouse where tuberculosis was clustered, 17 out of 17 heads were determined to be positive for tuberculosis in total head count. (necropsy and PCR). Therefore, it was confirmed that the ELISA test method is useful for detecting latent tuberculosis cows (see Table 2 below, the tuberculosis-positive data in the autopsy and PCR detailed test of positive individuals only in the ELISA test method). In addition, when a positive ELISA test method is found in a farmhouse with a cluster outbreak of tuberculosis, if the ELISA S/P (%) value is 40% or higher, shipment to the slaughterhouse (culling) is recommended. there was no In other words, in the case of tuberculosis-infected farms after 2013, livestock with tuberculosis (positive for tubecurin intradermal injection and/or γ-INF method) were culled, followed by ELISA testing to recommend culling. After the conversion from 20-30 tuberculosis-infected farms to negative farms, the rate of tuberculosis recurrence decreased significantly.

4. Classification according to change in ELISA S/P (%) value before and after PPD inoculation of tuberculosis-positive animals

Among 170 tuberculosis-positive animals that were confirmed positive by domestic approved test methods (Tubercurin intradermal injection method and gamma interferon method), 112 cows with ELIZA test antibody levels before and after inoculation of PPD diagnostic solution during Tubercurin intradermal injection reaction method Table 3 (change in ELISA S/P (%) value before and after PPD inoculation of tuberculosis-positive animals) was classified as follows.

As shown in Table 3 above, a significant number of tuberculosis-positive cattle had an S/P (%) of 12% or less (D value of 0.3 or less) before PPD inoculation and increased after PPD inoculation (78 out of 112). (Group 4 in Table 3), it was confirmed that the probability of latent infection in this case is high. In addition, most of the tuberculosis-positive animals (104 out of 112) had increased ELISA antibody titers after PPD inoculation.

5. Positive axis determination according to the change of ELISA S/P (%) value before and after PPD inoculation

Intradermal Inoculation Injection Method Pre and post PPD inoculation values were measured in bovine serum/plasma and antibodies to bovine tuberculosis were measured by the ELISA test (BTB 2.0, Bionote Co., Ltd.), and the S/P (%) value was calculated by Equation 1 below. did

<Formula 1>

* PC: 2.5 ~ 3.0, NC 0.05 ~ 0.1

Tuberculosis positivity was classified according to Table 4 below (examples of positive axis detection according to ELISA S/P (%) change before and after PPD inoculation).

* In a farm with three or more repeated cases of tuberculosis, positive individuals occurred in more than one-third of the total number of cattle, and if they lived together for three to four months or more in the same barn with tuberculosis-infected cattle (BTB group 5), S after boosting Positive if /P value is included in BTB 1 or BTB 2 or BTB 3

As described above, tuberculosis disease did not recur in cattle breeding farms (Wonju, Hoengseong, Hongcheon) of 21 farms that used BTB 2.0 (humoral immunity) together with cellular immunity in tuberculosis-infected farms as described above. In tuberculosis-infected farms, especially in cluster farms, most of the PPD and γ-INF test methods were negative in some animals, and the boosting conjugated BTB 2.0 method was confirmed as positive, and autopsy and PCR confirmed positive.

In addition, as a result of comparing before and after PPD inoculation in the tuberculosis final positive axis (PPD, γ-positive axis), it was confirmed that strong boosting occurred.

※ The expected section of the ELISA boosting method among the tuberculosis diagnostic methods (blue section)

※ Tubercurin intradermal injection reaction method (T), gamma interferon method (γ), ELISA method (E), intersection (∩)

※ Group 1 (E only)/ Group 0- ELISA BOOST- True positive object confirmation (currently not detected)

Claims (6)

(a) Obtaining a primary sample by collecting primary blood before inoculation with purified protein derivative (PPD) from cattle suspected of having a latent infection with Mycobacterium tuberculosis, and obtaining a secondary sample by collecting secondary blood 7 to 14 days after PPD inoculation ;
(b) calculating the S/P (%) value by Equation 1 below by measuring the OD values of the antibodies of the primary and secondary samples collected by the ELISA test; and
<Formula 1>

(c) when the measured S/P(%) value of the first sample is 80% or more, and the S/P(%) value of the second sample is also 80% or more; When the S/P(%) value of the first sample is 50% or more and less than 80%, and the S/P(%) value of the second sample is 50% or more and less than 80% or 80% or more; When the S/P (%) value of the first sample is 30% or more and less than 50%, and the S/P (%) value of the second sample is 50% or more and less than 80% or 80% or more; Or, if the S/P(%) value of the first sample is 0% or more and less than 30%, and the S/P(%) value of the second sample is 50% or more but less than 80% or 80% or more, the step of judging tuberculosis Including, a method for detecting whether or not the tuberculosis infection in cattle.
The method according to claim 1, wherein when the cattle suspected of being infected with Mycobacterium tuberculosis in step (a) are cattle of a farmhouse with a tuberculosis cluster, the S/P (%) value of the primary sample in step (c) is 30% or more and less than 50%, and , A detection method, characterized in that if the S/P (%) value of the secondary sample is 30% or more and less than 50%, or 50% or more but less than 80%, or increases to 80% or more, it is determined as tuberculosis cow.
The detection method according to claim 1, wherein the cattle suspected of having the Mycobacterium tuberculosis latent infection in step (a) are negative in the tuberculin intradermal injection reaction method and the gamma interferon test method.
The method according to claim 1, wherein the cattle suspected of being infected with Mycobacterium tuberculosis in step (a) are Korean cattle or dairy cattle.
According to claim 1, wherein the secondary sample of step (a) is characterized in that the blood is collected 7 to 10 days after the PPD inoculation, the detection method.
The method of claim 1, wherein the retest is performed when the OD value of the antibody in step (b) is 0.15 or more in the negative control group and 1.5 or less in the positive control group.

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