KR20200013511A - Method of Screening Odor Causing Substance that Reduces or Increases Immunity - Google Patents

Abstract

The present invention relates to a method and an apparatus for screening an odor-generating substance that reduces or increases immunity, wherein the method and the apparatus can minimize stress due to breeding environment of an animal model, so that it is possible to objectively analyze an effect of odor components on immunity. Therefore, the method and the apparatus can be usefully utilized for screening the odor-generating substance that can affect immunity of humans or animals.

Description

Method of Screening Odor Causing Substance that Reduces or Increases Immunity

The present invention relates to methods and apparatus for screening odor generating materials that reduce or increase immunity.

Large and concentrated livestock raising causes serious problems such as complaints from manure odors, deterioration of the natural environment, livestock managers and livestock productivity.

Odors from the barn have a serious impact on the living environment of the residents living around the farm. These odor components can also affect the health of the people working in the barn, including problems with lung function, bronchitis, nasal mucosa, esophagitis, and headaches. It is known to increase.

In addition, the odor of the livestock manure is also related to the health of the livestock, the odor generated from the excrement causes respiratory diseases and infectious diseases, and also has a great impact on the health and productivity of the livestock. To solve this problem, antibiotics are usually used all the time. However, antibiotics used indiscriminately during livestock raising not only cause the emergence of resistant bacteria to antibiotics, the intake of human antibiotics through meat, and environmental pollution, but also the economic burden on livestock farms due to expensive antibiotics. This is a problem.

However, little has been studied about how odor from livestock manure causes such problems to humans and livestock, and there is no standard of which patents of odor generating materials should be avoided, so there is insufficient awareness of odor. . Therefore, the barn workers and livestock are still exposed to such a bad smell.

Accordingly, the present inventors completed the present invention by preparing a model for analyzing the correlation between odor and immunity, and screening for an odor generating substance that reduces or increases immunity using the same.

Republic of Korea Patent Publication No. 10-2018-0044611

It is an object of the present invention to provide a method and apparatus for screening odor generating materials.

One aspect of the present invention comprises the steps of exposing the odor generating material to animals other than humans; And it provides a method for screening the odor generating material to reduce or increase the immunity including the step of evaluating the immunity of the animal.

The screening method of an odor generating substance that reduces or increases the immunity of the present invention inhales and exposes the odor component to the animal to evaluate the immunity, and because the stress other than the smell to the animal can be minimized in the process of exposing the odor component, The effect on the immunity of the trigger can be objectively assessed. In addition, various odor-generating substances can be evaluated for immunity, as well as screening at low cost. Therefore, by evaluating immunity against various odor-producing substances, it can be utilized to screen for substances that increase immunity or to avoid substances that reduce immunity, and thus can be usefully used to maintain the health of humans and livestock. .

Odor generating materials that can be used in the screening method of the present invention may be materials that generate components that stimulate the sense of smell around the material in a general air environment. The odor generating material may be, for example, a material generating propionic acid, ammonia, 3-methylindole and / or dimethyl disulfide, but is not limited thereto. On the other hand, since the screening method of the present invention is a method of evaluating immunity by inhalation exposure to animals, the substance that may cause animal death, such as acute toxicity by inhalation exposure, is not suitable for the screening method of the present invention. .

Exposing the odor generating material to an animal other than a human according to one embodiment of the present invention preferably allows the odor component to be inhaled and exposed to the respirator of the animal without the odor generating material contacting the skin of the animal.

According to one embodiment of the invention, the step of exposing the odor generating material is to place the odor generating material at the bottom of the cage with a hole formed in the bottom, the animal is odor component diffused into the cage through the hole It may be to inhale.

Cage according to an embodiment of the present invention may be a cage of a size suitable for breeding animals other than humans, when using the cage to minimize the stress of the animal according to the environment of the cage while changing the animal's immunity due to the odor component You can evaluate it more objectively.

Holes formed in the bottom of the cage according to an embodiment of the present invention may be formed so that the odor component as a whole in the cage can be diffused without inconvenience to the animal.

For example, the pore size can be formed from 1 to 5 mm. If the pore size is less than 1mm, the pore can be easily blocked due to animal breeding, so that the diffusion of odor components is not easy. If the pore size is greater than 5 mm, the animal's body can be exposed out of the pore, not only can the animal come into direct contact with the odor-producing substance, but it can also cause walking problems and cause stress, so an objective assessment of immunity Is difficult to achieve. In addition, the spacing between the holes may be formed of 2 to 4 cm. If the gap between the holes is less than 2cm, the odor component may be excessively diffused in the cage, and it may cause stress in the animal's life, and if it is more than 4cm, it is difficult to diffuse the odor component sufficiently, making it difficult to accurately assess immunity. .

According to one embodiment of the present invention, the odor generating material may be disposed at the bottom of the cage for 5 to 20 minutes in a day.

If the odor generating material is disposed in less than 5 minutes, the animal is not sufficiently exposed to the odor component, and if it is placed over 20 minutes, the odor generating substance may be excessively exposed to the odor component, and thus it is difficult to accurately reflect the effect on the immunity. Therefore, in consideration of the difference in physical properties such as volatility of the odor component may be placed on the lower end of the cage perforated for 5 minutes to 20 minutes to inhale exposure to the odor to the animal.

When the odor generating material is disposed at the bottom of the cage, the odor generating material is less likely to be directly exposed to the surface of the animal even if the cage is impacted by the animal's abrupt action or the environment outside the cage.

According to one embodiment of the invention, the exposure may be made for 3 to 5 weeks.

Exposure less than 3 weeks does not fully reflect the effects on immunity, and exposure beyond 4 weeks may increase the effects of conditions other than immunity, so an accurate assessment of immunity may not be made.

According to one embodiment of the invention, the step of assessing immunity is by evaluation of one or more functional changes selected from the group consisting of hematologic function change, cytoimmune function change and humoral immunological function change of the animal. Can be.

Evaluating the immunity according to an embodiment of the present invention may be performed by evaluating the results of analyzing changes in the hematologic function of the mouse.

The evaluation of hematological function changes may be performed by an evaluation method of hematological function changes conventionally made in the art, for example, evaluation of total leukocyte count, lymphocyte count or granulocyte count, or evaluation of red blood cell and platelet count. .

Evaluating the immunity according to an embodiment of the present invention may be made by evaluating the results of analysis of changes in the cell's immunological function. Evaluation of cytoimmune functional changes can be, for example, analysis of interferon-γ (IFNγ), interleukin (IL) -12, tumor necrosis factor-α (TNFα) or IL-4, IL-13 levels present in cell culture fluids, Or CD4 T helper T cells, CD8 cytotoxic T cells, and B cell composition ratio analysis, such as those performed in the art can be performed by the method for evaluating the changes in cytoimmune function commonly made in the art.

Evaluating the immunity according to an embodiment of the present invention may be made by evaluating the results of analysis of changes in humoral immunological function of the mouse.

The assessment of humoral immunological function changes may be performed by an evaluation method of hematological function changes conventionally made in the art such as, for example, evaluation of the levels of IgG1, IgG2a, IgG2b, IgG3, IgE and / or IgA.

According to one embodiment of the invention, the odor causing substance may be manure of livestock.

In order to analyze the effect of the major harmful gas generated from pig manure on the immune system, the immunity of the mouse was evaluated using the screening method according to an embodiment of the present invention. Specifically, we analyzed the changes in immunity of mice according to high-dose / low-dose subacute exposure of four major pig odor-producing substances, such as ammonia, methyl disulfide, propionic acid, and skatol, and evaluated hematological, cytoimmune, and humoral immunological immunity. The overall effect of this study was to analyze the effects on the immune function of each substance. As a result of comprehensively reflecting the evaluation items, the immunity of the mouse was decreased in the order of ammonia> scatol> propionic acid> methyl disulfide, and the smell of the present invention was changed in that the change in immune function was induced differently for each substance. It was confirmed that the material screening method worked properly.

According to another aspect of the present invention, in an animal breeding cage for screening an odor generating substance that reduces or increases immunity, a plurality of holes are formed in the bottom of the cage, and the bottom is the common surface below the cage. A space separate from the cage is formed, and the cage and the space are connected to a hole formed in the bottom, so that the odor component generated from the odor generating material disposed in the space can diffuse into the cage through the hole. Provided are animal breeding cages for screening odor generating materials that reduce or increase immunity.

In the animal breeding cage of the present invention, a separate space is formed under the bottom of the cage so that the smell generating material can be disposed, and a plurality of holes are formed in the bottom of the cage, so The generated odor component can diffuse into the cage through the hole. Therefore, the animals raised in the cage and the odor-producing substance may not be in direct contact with each other, and thus may maintain an environment similar to the existing breeding environment, thereby minimizing the induction of stress caused by factors other than the odor component. This can be useful for objectively assessing impact.

Animal breeding cage according to an embodiment of the present invention can be prepared as follows.

By modifying a conventional animal breeding cage can be made of a cage suitable for odor exposure, the cage can be manufactured using acrylic, but is not limited thereto.

The size of the cage is typically the size of the cage used for animal breeding, for example, in the case of a mouse may be a size of 20.9cm (width) × 25.9cm (length) × 12cm (height), but It is not limited.

Holes formed in the bottom of the cage may be formed to facilitate the diffusion of odor components while minimizing stress in the life of the animal living in the cage.

Specifically, the hole size may be formed of 1 to 5mm. If the pore size is less than 1mm, the pore can be easily blocked due to animal breeding, so that the diffusion of odor components is not easy. If the pore size is greater than 5 mm, the animal's body can be exposed out of the pore, not only can the animal come into direct contact with the odor-producing substance, but it can also cause walking problems and cause stress, so an objective assessment of immunity Is difficult to achieve. In addition, the spacing between the holes may be formed of 2 to 4 cm. If the gap between the holes is less than 2cm, the odor component may be excessively diffused in the cage, and it may cause stress in the animal's life, and if it is more than 4cm, it is difficult to diffuse the odor component sufficiently, making it difficult to accurately assess immunity. .

The animal breeding cage of the present invention has a separate space formed under the bottom of the cage, and the odor component generated from the odor generating material disposed in the space is diffused through the hole in the bottom of the cage and exposed to the animal in the cage.

In the space formed under the bottom of the cage can be opened and closed doors, through which the odor generating material can be placed or removed.

According to the method and apparatus for screening an odor generating substance that reduces or increases immunity according to the present invention, stress can be minimized according to a breeding environment of an animal model, so that the effect of an odor component on immunity can be objectively analyzed. It can be useful for the screening of odor generating substances that may affect the immunity of humans or livestock.

1 is a diagram showing the specific size and shape of the mouse cage for identifying the odor-causing substances affecting the immune system.
Figure 2 is a photograph showing a plan view (A), side view (B and C), rear view (D) and front view (E) of the mouse cage produced to identify the odor-causing substances affecting the immune system.
Figure 3 is a photograph showing a state of breeding mice in a mouse cage to identify the odor-causing substances affecting the immune system. The feed is fed from the top of the cage, and masking tape is used to close the gaps in the doors to feed the odor-producing substances to prevent them from spreading out of the cage.
Figure 4 is a graph showing the body weight change of mice inhaled exposure to ammonia (A), propionic acid (B), methyl disulfide (C) and skatol (D) for 4 weeks.
FIG. 5 shows the major indices related to anti-cancer and antiviral immunity versus atopic immunity (IFNγ / ( IL-4 × 10)).
FIG. 6 is a graph showing TNFα levels of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks.
FIG. 7 is a graph showing IgE levels of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks.
8 is a graph showing the IgG2a / IgG1 ratio of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks.
FIG. 9 is a graph showing and IgA levels of mice aspirated with ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks.
10 is a graph showing the composition ratio of immune cells in the spleen of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks. .
FIG. 11 is a graph showing changes in the total white blood cell count of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C), and skatole (D) for 4 weeks.
12 is a graph showing the change in the neutrophil number of mice inhaled and exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatol (D) for 4 weeks.
FIG. 13 is a graph showing target cell death rate by natural killer cells in mice exposed to ammonia (A), propionic acid (B), methyl disulfide (C) and skatole (D) for 4 weeks.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Selection of odor component for analysis

Swine manure is stored in the form of slurry and undergoes anaerobic fermentation or decomposition to produce various malodorous substances. Based on the chemical characteristics of the odor-causing substances, representative odor-causing substances were selected as the odor components to be analyzed.

Specifically, propionic acid as a volatile fatty acid material, ammonia as ammonia / volatile amines, 3-methylindole as a phenol / indole based material, and methyl disulfide as a sulfur compound based material were selected.

Then, in order to observe the changes in the hematological, cytoimmune and humoral immunological functions of the mice according to the exposure of the odor component, the selected compound was inhaled and exposed to the mice.

Example 2 Analysis of Changes in Immune Function in Animal Models Following Exposure to Odor Components

2-1. Animal model preparation and odor exposure

Mouse models used locally to analyze physiological and behavioral effects of odor (Matsukawa et al., 2016, Brain Research 1631: 46-52; Janitzky et al., 2015, Brain Research 1599: 1-8; Hacquemand et al., 2013, Behavioral Brain Research 238: 227-231; Capone et al., 2005, Acta Neurobiol.Exp. 65: 29-38) exposed the odor components selected in Example 1 and all experimental procedures. The program was approved with the Daegu Catholic University Animal Ethics Committee (IACUC-2017-001).

Specifically, a conventional mouse cage was modified and used as a cage suitable for odor exposure (FIGS. 1 and 2). The cage is made of acrylic, 20.9 cm (width) × 25.9 cm (length) × 12 cm (height), and 50 holes (hole size: 3 mm, spacing between holes: 2.65 to 2.97 cm) in the bottom of the cage Pierced. An acrylic box of 16.5 cm (width) x 18.4 cm (length) x 5.43 cm (height) with a door opening and closing 11.8 cm (width) x 3.9 cm (length) was formed under the cage.

The odorous substance selected in Example 1 was divided into low-dose group (10 µl) and high-dose group (200 µl), respectively, and the petri dish was dropped, and then put through the door under the cage, and the mouse was able to smell the smell moved to the cage through the hole. To make it possible.

Specifically, as an odorous substance, ammonia, propionic acid and methyl disulfide were prepared as a stock solution, and statol was prepared by dissolving about 0.02 g of powder in 200 μl of olive oil. In the case of the low dose group, 10 μl of the odorous substance prepared in Petri dishes was instilled, and in the case of the high dose group, 200 μl of the prepared odorous substance was instilled.

For 10 minutes daily, mice were exposed to odors for 4 weeks, and in the case of the control group, drinking water was added dropwise (FIG. 3). Each cage was tested with one mouse, and the total number of mice for each substance was 8 in consideration of statistical significance test. The mice were exposed to the odor component for 4 weeks and sacrificed for hematological, Cytoimmune and humoral immune function changes were analyzed.

2-2. Check your mouse's weight

Changes in body weight of mice exposed to odor components were confirmed.

Specifically, the body weight was measured every week while exposing the mouse to the odor component of Example 1 according to Example 2-1 over 4 weeks to analyze the weight according to the odor component exposure.

As a result, no significant body weight change was observed in the ammonia, propionic acid, methyl disulfide and skatol between the control, low dose and high dose exposure groups (FIG. 4). Through this, it was confirmed that the concentration of the odor component exposed to the mouse in this experiment is not a degree that fatally cause acute toxicity.

2-3. Identification of weakened odor components based on analysis of major indicators related to immunity

High levels of IL-4 indicate a strong atopic appearance. High levels of IFN-γ indicate strong anticancer and antiviral immunity. In other words, higher levels of IFN-γ compared to IL-4 indicate high immune function as a whole ( Korean Journal of Occupational Health Care 23.4 (2013): 393-401).

In order to identify which of the odor components of Example 1 weakens immunity, mice are exposed to the odor components of Example 1 for 4 weeks according to Example 2-1, and then sacrificed for anticancer and antiviral immunity vs. atopy. Major indicators related to immunological capacity were analyzed.

Specifically, the index value was confirmed by analyzing the relative production rate of IFNγ, a key cytokine in the defense against viral infection or cancer, and IL-4, which contributes to atopic changes, by the following equation.

[IFNγ / (IL-4 × 10)]

IFNγ: IFNγ concentration

IL-4: IL-4 concentration

After the end of the odor exposure, the spleen of the mouse was taken and cultured into single cells by the following method. The collected spleen is pulverized using a sterile slide, lysed red blood cells using RBC lysis buffer, and then single cells are formed to complete RPMI (RPMI 1640, 1 mM sodium pyruvate) and non-essential amino acid. ), 1% sodium bicarbonate, 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin solution, 50 mM 2-mercaptoethanol Incubated in 24-well plates. T lymphocytes were activated using immobilized anti-CD3 mAb (5 μg / well), 5 ng PMA (phorbol12-myristate 13-acetate), 500 ng ionomycin, and 10 U IL-2. After incubation for 48 hours in a 5% CO₂ incubator, the supernatant was recovered by centrifugation at 4 ℃, 10000rpm for 15 minutes, and stored at -80 ℃ until analysis.

After monocellularization, IFN-γ and IL-4 in the cell culture obtained by activating T cells for 48 hours were quantified by a sandwich ELISA method as follows. Immunlon IV plates were aliquoted with 100 μl / well of capture antibody for each and left overnight at 4 ° C. The next day, the plates were washed using an autowasher, and then 10 μl FBS-PBS was dispensed at 200 μl / well in order to prevent nonspecific binding and left at room temperature for 2 hours. After washing the plate with an automatic washer, 100 μl / well of the standard calibration reagent subdivided according to each indicator value and a reference sample of known concentration were dispensed to adjust the difference between the sample and the plate and overnight at 4 ° C. It was left. Passage dilution standards for IFN- [gamma] are 2000 pg / ml, 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 0 pg / ml, and 1/50 using 10% FBS-PBS. The culture was diluted and used. IL-4 passage dilution standards were 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 15.625 pg / ml, and 0 pg / ml. The next day, the plates were washed using an autowasher, 100 μl / well of the detection antibody was dispensed, and then left at room temperature for 2 hours in the dark. The plates were then washed using an auto washer and 100 μl / well of streptavidin-horseradishperoxidase conjugate was added and allowed to stand at room temperature for 2 hours in the dark. Finally, the plate was washed using an automatic washer and 100 μl / well of 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate solution was added to induce color development. Absorbance was measured at 450 nm (Reference 570 nm) using a Gen5 reader.

As a result, in the case of ammonia, the indicator value decreased depending on the concentration of ammonia, whereas propionic acid, methyl disulfide, and skatol were found to increase depending on the concentration of each component (FIG. 5).

2-4. Confirmation of Proinflammatory Cytokine Production

In order to identify the components that increase the production of proinflammatory cytokines in the odor component of Example 1, the cytokine TNFα, which is cited as an proinflammatory indicator early in the development of allergic diseases such as allergic contact dermatitis and asthma. The production was analyzed.

Specifically, using capture and detection antibodies against TNFα, and passage dilution standards were 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 0 pg / ml ( The culture solution was not diluted), and the concentration of TNFα was measured from the cell culture solution by measuring in the same manner as in Example 2-3 using the send position ELISA method.

As a result, it was confirmed that the concentration of TNFα increased depending on the concentration of propionic acid and skatol, but there was no significant difference in ammonia and methyl disulfide (FIG. 6).

2-5. Confirmation of changes in body immune system

IgE, IgA, and IgG isotype levels were analyzed to identify the components that undermine immunity among the odor components of Example 1.

Specifically, the level of IgE present in serum was quantified by sandwich ELISA method. Rat anti-mouse IgE capture antibody was immunized with 2 μg / ml and 100 μl of each well on an Immulon II plate and allowed to stand for 24 hours in a cold state. To prevent nonspecific binding, 1% BSA-PBS was added, allowed to stand at room temperature for 2 hours, 1/5 diluted serum sample and purified mouse IgE mAb isotype standard (IgE), and then refrigerated 24 Let time stand. The next day, 1 μg / ml and 100 μl of biotinylated rat anti-mouse IgE antibody were dispensed and allowed to stand at room temperature for 2 hours, followed by addition of avidin-peroxidase and standing at room temperature for 1 hour. . Since color development was induced with TMB substrate solution, absorbance was measured at 405 nm (reference: 570 nm) using Biotek Gen5. Passage-diluted standard IgE is 250.0 ng / ml, 125.0 ng / ml, 62.5 ng / ml, 31.2 ng / ml, 15.6 ng / ml, 7.8 ng / ml, 0 ng / ml.

IgA was also measured using rat anti-mouse IgA capture and detection antibodies in the same manner as IgE. Serum was diluted to 1 / 10,000 and 5% BSA-PBS was added to prevent nonspecific binding. Passage diluted standard IgA is 62.5ng / ml, 31.25ng / ml, 15.625ng / ml, 7.8125ng / ml, 3.90625ng / ml, 1.953125ng / ml, 0ng / ml.

In addition, to assess the levels of IgG1 and IgG2a, the IgG isotypes present in serum, IgE using a peroxidase conjugated anti-mouse IgG detection antibody with goat-anti mouse IgG1, gG2a capture antibody The same method was followed. Serum was all diluted to 1 / 100,000 and subdivided standard IgG1 and IgG2a were 250.0ng / ml, 125.0ng / ml, 62.5ng / ml, 31.2ng / ml, 15.6ng / ml, 7.8ng / ml, 0ng / Ml. Color development was induced by TMB substrate solution and measured by absorbance at 405 nm (reference: 630 nm) with ELISA reader.

As a result, it was confirmed that the IgE level, which is a representative indicator of atopy propensity, increased in a concentration-dependent manner in the case of ammonia, but decreased in a concentration-dependent manner in the case of skatol (FIG. 7).

In addition, as a result of analyzing the ratio of IgG2a isotype switched by IFNγ and IgG1 isotype converted by IL-4, the concentration was reduced in ammonia, while in the methyl disulfide exposed group. The ratio was increased, and it was confirmed that there was no significant difference between the control group and the exposed group in propionic acid and scatol (FIG. 8).

On the other hand, when the mucosa is stimulated by exposure to pathogens, chemicals, etc., it was confirmed that the IgA level increased significantly except for propionic acid (FIG. 9).

2-6. Confirmation of changes in spleen immune cell composition

In order to identify among the odor components of Example 1 that weaken the immunity, the distribution of spleen constituent lymphocytes was analyzed.

Specifically, the composition ratio of immune cells in major immune organs such as spleen, axillary lymph nodes, and thymus was analyzed using a Fluorescence-Activated Cell Sorting (FACS) instrument.

Specifically, the spleen, thymus and mesenteric lymph nodes were subjected to single cellization in the same manner as in Example 2-3, and then supplemented T lymphocytes using anti-CD3 PE antibody and anti-CD4 FITC antibody for T lymphocyte activation. Cell disruption T lymphocytes were analyzed with anti-CD3 FITC antibody and anti-CD8 PE, and anti-B220 FITC antibody was used for B lymphocyte activation. Single cells of the spleen, thymus and mesenteric lymph nodes were dispensed into 1 × 10 ⁶ cells in a FACS tube, then centrifuged at 1500 rpm for 4 minutes at 4 ° C. with 3 ml staining buffer, followed by nonspecific antigen-antibodies. Fc block (1 μg / 10 ⁶ cells) was added to prevent binding and left at 4 ° C. for 30 minutes. Then, CD3, CD4, CD8, B220 antibody was added, and allowed to stand at 4 ° C for 30 minutes to fluoresce. Stained cells were washed three times with staining buffer before FACS analysis.

As a result, in the case of ammonia, the composition ratio of helper T cells and cytotoxic T cells was decreased in the exposed group compared to the control group, while the composition ratio of B cells was confirmed to increase. Scatol also showed a similar distribution pattern to ammonia, but propionic acid did not confirm this trend. On the other hand, in the methyl disulfide exposed group, the composition ratio of helper T cells was confirmed to increase compared to the control (Fig. 10).

2-7. Confirmation of hematological changes

Peripheral blood immune cell composition was analyzed to identify the components that weaken immunity among the odor components of Example 1.

Specifically, after exposing the mouse to the odor component of Example 1 for 4 weeks according to Example 2-1, by mixing the anticoagulant with the blood obtained through heart blood collection leukocytes and neutrophils (automatic hematology analyzer, Hemavet 950 FS, Drew Scientific Inc, TX, USA) and compared with the values measured prior to exposure to odor components.

As a result, it was confirmed that the total leukocyte count decreased with exposure to all substances (Fig. 11).

Meanwhile, in the case of ammonia, the composition ratio of neutrophils (main granules coping with pathogens such as viruses) decreased in concentration-dependent manner, while in the methyl disulfide and skatol-exposed group, it was increased compared to the control group (FIG. 12).

2-8. Identify changes in natural killer cell function

In order to identify components that weaken immunity among the odor components of Example 1, the function of natural killer cells, which are representative cells of natural immunity, was analyzed.

Specifically, target cell attack ability of NK cells in the spleen of the mouse was analyzed by staining with MitoTracker Green FM, using K562 human leukemia cells as target cells. Activity analysis of spontaneous killer cells in splenocytes was performed by staining mitochondria in K562 human leukemia cells with Mitotracker Green FM dye, followed by incubating splenocytes from experimental animals. After attacking, the dead K562 cancer cells were stained with propidium iodide, differentiated, and the percentage was calculated.

A stock solution of Mitotracker Green FM dye 1 mM was prepared with dimethyl sulfoxide (DMSO). The target cells, K562 human leukemia cancer cells 2 × 10 ⁶ cells and 1 mM stock solution of 300 nM Mitotracker Green FM dye diluted with medium were mixed and stained for 3 hours in a 5% CO ₂ incubator. The medium was RPMI 1640, Penicillin-Streptomycin solution (PSN), 1M sodium bicarbonate, 1M HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), heating fetal bovine serum) and the medium was preheated in a CO ₂ incubator 30 minutes before use. Stained K562 cells and single-celled splenocytes were mixed at a ratio of 1:10 and 1:50, incubated for 3 hours, and then propidium iodide (2 μl from 1 mg / ml stock solution, Sigma) immediately before analysis. Dead cells were stained and FACS analysis was performed.

As a result, in the propionic acid exposed group, the target cell attacking ability of the natural killer cells was significantly reduced compared to the control group, and the activity of the natural killer cells was also reduced in scatol concentration (FIG. 13).

Taken together, it was confirmed that ammonia and scatol had a negative effect on overall cellular immunity, but methyl disulfide and propionic acid were not found to have a positive or negative effect on immune function.

So far, the present invention has been described with reference to the embodiments. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown not in the above description but in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (7)

Exposing the odor generating substance to animals other than humans; And
Evaluating the immunity of the animal
Screening method of odor generating material to reduce or increase the immunity comprising a.
The method of claim 1, wherein exposing the odor generating material
Placing the odor generating material at the bottom of the cage with a hole formed at the bottom to allow the animal to inhale the odor diffused into the cage through the hole.
The method of claim 2, wherein the odor generating material is disposed at the bottom of the cage for 5 to 20 minutes in a day.
3. The method of claim 2, wherein said exposing is for three to five weeks.
The method of claim 1, wherein evaluating the immunity is
A method for screening an odor generating substance that reduces or increases immunity by evaluating at least one functional change selected from the group consisting of a change in hematologic function, a cytoimmune function, and a humoral immunological function of the animal.
The method of claim 1, wherein the odor generating material is manure of livestock.
In the animal breeding cage for screening odor generating substances to reduce or increase immunity,
A plurality of holes are formed in the bottom of the cage,
A space separated from the cage having the bottom as a common surface is formed under the bottom of the cage,
The cage and the space are connected to a hole formed in the bottom, so that the odor component generated from the odor generating material disposed in the space can be diffused into the cage through the hole.
Animal breeding cages for screening odor generating substances that reduce or increase immunity.

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