Leucocyte function inhibitor, sampling kit, method for pretreating body fluid, and method for identifying bacterium

Abstract

The present invention provides a technique used to identify a bacterium present in a leucocyte-containing body fluid collected from a subject, the technique making it possible to retain the bacterium in the body fluid without killing the bacterium. A leukocyte function inhibitor 3 according to the present invention contains a chloride as an active ingredient, the chloride being formed from at least one metal selected from the group consisting of alkali metals and alkaline earth metals, and can suppress the bactericidal activity of leucocytes in the leucocyte-containing body fluid. A sampling kit 1 for collecting the leucocyte-containing body fluid includes: a container 2; and a leucocyte function inhibitor 3 that contains NaCl in an amount of 4-40 mg / mL on the basis of the volume of the container 2.

Description

Leukocyte function inhibitor, sampling kit, method for pre-treating body fluid, and method for identifying bacteria

[0001] The present invention relates to a pretreatment for identifying bacteria contained in a body fluid.

[0002] In our daily lives, we consume milk from livestock such as cows and goats, as well as dairy products made from milk such as butter, yogurt, and cheese, every day, and the development of dairy farming remains important today.

[0003] On the other hand, dairy farming is also known to be a heavy economic burden. For example, if livestock become infected with bacteria and are unable to ship milk, or if livestock die due to the worsening of the infection, the economic burden on dairy farmers is heavy and the dairy farm may become unsustainable. Therefore, appropriate treatment for bacterial infections in livestock has become an unavoidable issue for dairy farmers.

[0004] An example of an infectious disease is mastitis. Mastitis is a type of infectious disease that occurs when pathogenic microorganisms such as bacteria invade the udder and multiply. Milk collected from livestock suffering from mastitis is often of low quality, making it unsuitable for market and making it difficult to make a profit. Mastitis can also recur, requiring appropriate treatment each time. For this reason, mastitis is known to be a major problem troubling dairy farmers.

[0005] Mastitis is usually treated by culturing bacteria contained in milk to identify the type of bacteria and then administering antibacterial drugs to livestock. If the type of bacteria cannot be accurately identified, the symptoms of the livestock will not improve even if the drug is administered, and there is also a risk that the bacteria will acquire drug resistance.

[0006] Non-Patent Document 1 discloses the change in the number of viable pathogenic bacteria during storage of milk collected from cows with subclinical mastitis, the correlation between the reduction rate of viable bacteria during storage and the number of somatic cells, and the correlation between the reduction rate of viable bacteria during storage and the values ​​of antibacterial peptides, lactoferrin, and lactoperoxidase.Non-Patent Document 1 suggests that the viable count of pathogenic bacteria in milk decreases during storage at room temperature after collection, and it is speculated that this reduction is due to leukocytes or antibacterial components present in the milk.

[0007] Furthermore, Non-Patent Document 2 discloses further investigation into factors that reduce the number of viable pathogenic bacteria during storage of milk collected from cows with subclinical mastitis. Non-Patent Document 2 suggests that most pathogenic bacteria contained in milk with a high somatic cell count are reduced during storage at 15 to 25°C by both cellular components (including leukocytes) and antibacterial components contained in the milk, and discloses that the cellular components in particular significantly reduced the number of bacteria.

[0008] Hisaeda K, Koshiishi T, Watanabe M, Miyake H, Yoshimura Y, Isobe N (2016) Change in viable bacterial count during Preservation of milk derived from dairy cows with subclinical mastitis and its relationship with antimicrobial components in milk. The Journal of Veterinary Medical Science 78(8):1245-1250. Koshiishi T, Watanabe M, Miyake H, Hisaeda K, Isobe N (2017) Cellular and soluble components decrease the viable Pathogen counts in milk from dairy cows with subclinical mastitis. The Journal of Veterinary Medical Science 79(8):1389-1393.

[0009] As mentioned above, due to the antibacterial activity of white blood cells in milk, bacteria may be reduced or killed between the time of milk collection and the start of culture, which may make it difficult to accurately identify the type of bacteria.

[0010] The present invention has been made in consideration of the above-mentioned problems, and its purpose is to provide a technology that can preserve bacteria in leukocyte-containing body fluids collected from a subject without killing them, in order to identify the type of bacteria in the body fluids.

[0011] To achieve the above-mentioned object, the present inventors, through extensive research, have discovered that chlorides of alkali metals or alkaline earth metals inhibit the bactericidal effect of leukocytes, thereby enabling bacteria in body fluids to be preserved without being killed. The present invention provides the following technologies: [1] A leukocyte function inhibitor for identifying the type of bacteria contained in a leukocyte-containing body fluid, comprising, as an active ingredient, a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals. [2] The leukocyte function inhibitor described in [1] above, wherein the metal is selected from the group consisting of potassium, sodium, and calcium. [3] A sampling kit for sampling a leukocyte-containing body fluid, comprising: a container capable of holding a predetermined volume of the leukocyte-containing body fluid; and a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals, the chloride having a mass relative to the volume of 4 to 40 mg / mL. [4] The sampling kit described in [3] above, wherein the mass of the chloride relative to the volume is 4 to 20 mg / mL. [5] A pretreatment method for identifying the type of bacteria contained in a leukocyte-containing body fluid, comprising the step of adding 4 to 40 mg / mL of a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals relative to the volume of the leukocyte-containing body fluid. [6] The treatment method according to [5] above, wherein the leukocyte-containing body fluid is selected from the group consisting of saliva, milk, cerebrospinal fluid, and sputum. [7] The pretreatment method according to [5] above, wherein the leukocyte-containing body fluid is collected from a subject selected from the group consisting of humans, cows, goats, pigs, dogs, cats, and chickens. [8] A method for identifying bacteria in a leukocyte-containing body fluid, comprising the steps of: (a) pretreating the leukocyte-containing body fluid with the pretreatment method according to claim 4; (b) culturing bacteria in the leukocyte-containing body fluid after step (a); and (c) identifying the bacteria cultured in step (b).

[0012] NaCl can inhibit the bactericidal action of white blood cells, and therefore can prevent bacteria from dying out between the time of collection of body fluid and the time of bacterial culture.

[0013] Fig. 1 is a perspective view showing the configuration of a sampling kit according to one embodiment of the present invention. Fig. 2 is a graph showing the relationship between the NaCl concentration and the viable count ratio of E. coli (0.5h / 0h) in milk collected from a mastitis-affected goat (milk with a high cell count (high SCC)). Fig. 3 is a graph showing the relationship between the NaCl concentration and the viable count ratio of Staphylococcus aureus (0.5h / 0h) in milk collected from a mastitis-affected goat (milk with a high SCC). Fig. 4 is a graph showing the relationship between the NaCl concentration and the viable count ratio of Klebsiella pneumoniae (0.5h / 0h) in milk collected from a mastitis-affected goat (milk with a high SCC). Fig. 5 is a graph showing the relationship between the NaCl concentration and the viable count ratio of E. coli (0.5h / 0h) in milk collected from a healthy goat (milk with a normal SCC). Figure 6 is a graph showing the relationship between the NaCl concentration in milk collected from a healthy goat (milk with normal SCC) and the phagocytic rate of leukocytes against Staphylococcus aureus. Figure 7 is a graph showing the relationship between the NaCl concentration in milk collected from a healthy goat (milk with normal SCC) and the growth rate ratio of E. coli (3h / 0h). Figure 8 is a graph showing the relationship between the NaCl concentration in milk collected from a healthy goat (milk with normal SCC) and the growth rate ratio of Staphylococcus aureus (3h / 0h). Figure 9 is a graph showing the viable cell count of Klebsiella pneumoniae in milk in the presence of NaCl. Figure 10 is a graph showing the viable count of Staphylococcus aureus in milk in the presence of NaCl, where Figure 10(a) shows the results for NaCl concentrations of 0 to 40 mg / mL and incubation times of 0 to 24 hours, and Figure 10(b) shows the results for NaCl concentrations of 0 to 10 mg / mL and incubation times of 0 to 4 hours. Figure 11 is a graph showing the relationship between KCl concentration and E. coli growth ratio (3h / 0h) in milk collected from mastitis-affected goats (milk with high SCC). Figure 12 is a graph showing the relationship between CaCl concentration and E. coli growth ratio (3h / 0h) in milk collected from mastitis-affected goats (milk with high SCC). ２13(a) is a graph showing the viable cell counts in milk (0 h) immediately after collection from a cow suffering from mastitis, milk left for 5 hours without the addition of NaCl (control (5 h)), and milk left for 5 hours with the addition of NaCl (NaCl (5 h)), and Fig. 13(b) is a graph showing the viable cell count ratio (5 h / 0 h) obtained by dividing the values ​​for control (5 h) and NaCl (5 h) in Fig. 13(a) by the value for 0 h.

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

[0015] <Leukocyte Function Inhibitor> Leukocyte function inhibitors are used to identify the types of bacteria contained in leukocyte-containing body fluids (hereinafter sometimes simply referred to as "body fluids").

[0016] White blood cells are blood cells that do not contain hemoglobin and are responsible for the immune system's defenses against bacterial and viral infections. White blood cells are classified into several types based on their function and morphology. The main function of white blood cells is bactericidal activity, which includes phagocytosis.

[0017] Leukocyte function inhibitors can suppress the bactericidal action of leukocytes, but do not themselves have bactericidal action, i.e., they do not kill bacteria, and therefore can keep bacteria alive in body fluids. This allows leukocyte function inhibitors to be used to identify the type of bacteria. The "bactericidal action by leukocytes" is, for example, phagocytosis, but it may also be bactericidal action by other mechanisms.

[0018] The leukocyte function inhibitor contains, as an active ingredient, a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals. "A chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals" is sometimes abbreviated to "metal chloride." "Containing a metal chloride as an active ingredient" means that, in the leukocyte function inhibitor, the metal chloride has the effect of inhibiting the function of leukocytes. Furthermore, the metal chloride does not exhibit bactericidal activity within the preferred concentration range described below.

[0019] In the metal chloride, the metal is preferably selected from the group consisting of potassium, sodium, and calcium. Chlorides of these metals are generally highly safe for humans and other animals, inexpensive, and can be stored at room temperature for a long period of time.

[0020] The leukocyte function inhibitor is preferably used so that the amount of metal chloride added to the volume of body fluid (sometimes referred to as "metal chloride concentration") is 4 to 40 mg / mL.

[0021] Adding a metal chloride amount of 4 mg / mL or more can suppress the bactericidal activity of white blood cells. High concentrations of metal chlorides can inhibit bacterial growth and even kill bacteria. Although sensitivity to metal chlorides varies depending on the type of bacteria, a metal chloride concentration of 40 mg / mL or less can preserve typical infectious disease-causing bacteria. Furthermore, a metal chloride concentration of 20 mg / mL or less allows many bacteria to survive, making it applicable to a wider variety of infectious diseases. Furthermore, if the type of causative bacteria can be narrowed down to a certain extent based on symptoms, the metal chloride concentration can be set based on the metal chloride sensitivity of those causative bacteria.

[0022] The target bacteria are not limited to a specific type, and examples include bacteria that cause various infectious diseases, such as E. coli, Staphylococcus aureus, and Klebsiella pneumoniae, which are causative bacteria of mastitis.

[0023] The leukocyte function inhibitor may contain a component other than a metal chloride. The component other than a metal chloride may be a component capable of suppressing the bactericidal action of leukocytes, or may be a component used for another purpose.

[0024] "Leukocyte-containing body fluid" refers to a body fluid that can be collected from a subject and contains leukocytes. The metal chloride concentration of the body fluid to which the leukocyte function inhibitor is applied is lower than the concentration of metal chloride added by the leukocyte function inhibitor; for example, the metal chloride concentration contained in the body fluid itself is preferably less than 4 mg / mL or 2 mg / mL or less. Such body fluids are, for example, selected from the group consisting of saliva, milk, cerebrospinal fluid, and sputum.

[0025] Although leukocytes generally refer to leukocytes contained in a subject, they may also be leukocytes that have been mixed in from outside (i.e., leukocytes that do not originate from the subject). That is, a leukocyte-containing body fluid may contain at least one of leukocytes derived from the subject and leukocytes that have been mixed in from outside.

[0026] The "subject" may be any animal, preferably a mammal or bird, since mammals and birds have white blood cells. The subject may be selected from the group consisting of, for example, humans, cows, goats, pigs, dogs, cats, and chickens. Non-human subjects may be either domestic or wild animals. The subject may also be a healthy animal or an animal suffering from a disease. Preferably, the disease is a bacterial infection.

[0027] For example, the subject may be a cow suffering from a bacterial infection, and the leukocyte-containing body fluid may be milk, including, but not limited to, all known bovine bacterial infections, such as mastitis, respiratory disease syndrome, and bovine colibacillosis.

[0028] In this embodiment, the subject is preferably a goat suffering from a bacterial infection, and the leukocyte-containing bodily fluid is goat's milk, including, but not limited to, bacterial infections such as mastitis, including all known bacterial infections in goats.

[0029] <Sampling Kit> One embodiment of the present invention is a sampling kit. Note that the terms and configurations already explained may not be explained again.

[0030] The sampling kit is a kit for sampling a leukocyte-containing body fluid, and comprises a container capable of holding a predetermined volume of the leukocyte-containing body fluid, and 4 to 40 mg / mL of a metal chloride relative to the volume.

[0031] An example of the sampling kit is shown in Figure 1. As shown in Figure 1, the sampling kit 1 comprises a container 2 and the leukocyte function inhibitor 3 described above.

[0032] The container 2 has a transparent or translucent container body and a lid that can liquid-tightly seal the container body. The side of the container body is marked with a scale that indicates the amount of body fluid to be poured. In this embodiment, the inner surface of the bottom of the container 2 is convex and rounded to facilitate mixing of the leukocyte function inhibitor 3 with the body fluid. However, the shape of the container 2 is not limited to this.

[0033] The "predetermined volume" that the container 2 can hold may be, for example, a volume that is greater than the graduation and less than the upper limit of the capacity of the container body. By allowing a certain degree of flexibility in the applicable volume, the user can easily measure bodily fluids. In this embodiment, the container 2 is used with the lid closed, so the upper limit of the capacity is the volume when the lid is closed.

[0034] The amount of leukocyte function inhibitor 3 is set so that the amount of metal chloride for the above-mentioned predetermined volume of body fluid is 4 to 40 mg / mL. In this embodiment, leukocyte function inhibitor 3 is pre-placed in container 2, but it may also be attached separately from container 2. If attached separately, the amount of leukocyte function inhibitor 3 that fits the volume of one container 2 may be individually packaged, or a device for measuring this amount may be attached. Only metal chloride may be used as leukocyte function inhibitor 3.

[0035] By adding metal chlorides to leukocyte-containing body fluids in the above-mentioned concentration range, the bactericidal activity of leukocytes can be effectively suppressed without killing bacteria, allowing accurate identification of bacteria contained in body fluids collected using this sampling kit.

[0036] The metal chloride concentration is the ratio of the mass of the metal chloride to the volume of the body fluid, but since the volume of the metal chloride is very small even when the metal chloride concentration is at its maximum, the volume of the leukocyte function inhibitor 3 to be added can be ignored even when the leukocyte function inhibitor 3 is placed in the container 2 beforehand, as in the example of Figure 1. However, in some cases, such as when the volume of components other than the metal chloride in the leukocyte function inhibitor 3 is large, the scale may be set taking into account the volume of the leukocyte function inhibitor 3.

[0037] <Pretreatment Method> Metal chlorides are used in a pretreatment method for body fluids, which is carried out to identify the type of bacteria contained in leukocyte-containing body fluids. This pretreatment method includes adding metal chlorides to collected body fluids so that the metal chloride concentration per volume is 4 to 40 mg / mL. The environment in which the metal chlorides are added may be room temperature. After addition, it is preferable to mix the body fluids so that the metal chloride concentration in the body fluids is uniform. When using the sampling kit 1, mixing can be achieved by turning the container 2 upside down.

[0038] The "leukocyte-containing body fluid" and "subject" are as described above. The metal chloride to be added may also be contained in the leukocyte function inhibitor. The sampling kit described above is preferably used for carrying out this pretreatment method. That is, the matters described for the leukocyte function inhibitor and the sampling kit also apply to the pretreatment method. For example, the metal chloride concentration is preferably 20 mg / mL or less.

[0039] <Method for Identifying Bacteria> The above-described pretreatment method may be included as one step in a method for identifying bacteria, which comprises: (a) a step of pretreating a leukocyte-containing body fluid; (b) a step of culturing bacteria in the leukocyte-containing body fluid after the step (a); and (c) a step of identifying the bacteria cultured in the step (b).

[0040] Step (a) is the pretreatment method described above. In step (b), the body fluid (bacteria contained therein) is cultured under appropriate conditions. In step (c), a staining test and a biochemical property test are performed, and the type of bacteria is identified based on the bacterial morphology, the staining test results, and the biochemical property test results. Known methods can be applied to steps (b) and (c).

[0041] To identify the causative bacteria of bovine mastitis using the sampling kit 1, the following specific procedures may be performed. First, milk from a cow showing symptoms of mastitis is collected and mixed with a leukocyte function inhibitor 3 containing a metal chloride in the container 2 of the sampling kit 1. By culturing the bacteria in the milk stored in this container 2, the type of bacteria causing the infection can be identified. By identifying the bacteria in this way, appropriate treatment can be administered to the subject. The bacteria causing the infection is not limited to one type, but may be two or more types.

[0042] In this specification, mass, parts by mass, and mass % are used synonymously with weight, parts by weight, and % by weight, respectively.

[0043] [Experimental Example 1] Effect of NaCl on the number of viable bacteria in the milk of mastitis-affected goats Test tubes containing different masses of NaCl were placed in a clean bench and sterilized by irradiating them with an ultraviolet lamp for 10 minutes, and the mouths of the test tubes were then sealed with lids.

[0044] The day after lipopolysaccharide (LPS) administration to goats, milk was collected and confirmed to have significantly higher than normal cell counts (SCC). Ten milliliters of milk collected on the same day was poured into each of the test tubes mentioned above, and various bacteria were added. The milk was then left at room temperature for 0, 0.5, or 3 hours. After the milk was left at room temperature, it was plated on an agar medium and the bacteria were cultured at 37°C for 24 hours.

[0045] After culturing the bacteria, the viable cell count was calculated from the number of colonies formed by the bacteria. For each NaCl concentration, the ratio of the viable cell count obtained from the sample left at room temperature for 0.5 or 3 hours was calculated relative to the viable cell count obtained from the sample left at room temperature for 0 hours. This ratio is shown as the "viable cell count ratio" on the vertical axis of Figures 2 to 4. Figure 2 shows the viable cell count ratio (0.5 h / 0 h) of E. coli at NaCl concentrations of 0, 1.8, 3.6, 7, and 14 mg / mL. Figure 3 shows the viable cell count ratio (3 h / 0 h) of Staphylococcus aureus at NaCl concentrations of 0, 5, 10, 20, and 40 mg / mL. Figure 4 shows the viable cell count ratio (3 h / 0 h) of Klebsiella pneumoniae at NaCl concentrations of 0, 5, 10, 20, 40, and 80 mg / mL.

[0046] As shown in Figures 2 to 4, the addition of NaCl increased the viable cell count ratio. In particular, as shown in Figure 2, the viable cell count ratio increased for E. coli at NaCl concentrations of 3.6 mg / mL or higher, up to at least 14 mg / mL. Furthermore, as shown in Figures 3 and 4, the viable cell count increased for S. aureus and K. pneumoniae at NaCl concentrations of 5 mg / mL or higher, up to at least 40 mg / mL. These results demonstrate that NaCl inhibits leukocyte function.

[0047] On the other hand, as shown in Figure 4, for K. pneumoniae, the viable cell count ratio at a NaCl concentration of 80 mg / mL was lower than that at 40 mg / mL and was equivalent to that without NaCl, which is thought to be because at least a portion of the K. pneumoniae was killed by the high NaCl concentration.

[0048] Experimental Example 2: Effect of NaCl on the Phagocytic Activity of Leukocytes Milk was collected from a healthy goat and poured into a test tube containing NaCl. Killed bacteria were added and the mixture was left at room temperature for 30 minutes. After the milk was left, the sample was smeared on a glass slide, stained with Giemsa, and observed under a microscope to count the number of phagocytic cells. The ratio of the number of phagocytic cells to the total number of observed cells (number of phagocytic cells / total number of observed cells) is shown in Figures 5 and 6 as the phagocytic rate. Since the phagocytic rate decreased with the addition of NaCl, it was thought that NaCl reduces the phagocytic activity of leukocytes. Furthermore, this effect of reducing phagocytic activity was observed at least in the range of 2.5 to 20 mg / mL.

[0049] Experimental Example 3: Effect of NaCl on the Viable Count of E. coli and S. aureus in Milk The effect of NaCl on bacterial survival was examined. Milk was collected from a healthy goat and poured into a test tube containing NaCl. After adding bacteria, the mixture was left at room temperature for 0 or 3 hours. The mixture was then plated on an agar medium and cultured for 24 hours, after which the number of colonies was counted to obtain the viable count. For each NaCl concentration, the ratio of the viable count obtained from the sample left at room temperature for 3 hours to the viable count obtained from the sample left at room temperature for 0 hours was calculated. This ratio is shown on the vertical axis of Figures 7 and 8 as the "viable count ratio."

[0050] As shown in Figure 7, the viable cell count ratio of E. coli increased particularly at NaCl concentrations of 5 mg / mL to 20 mg / mL. The viable cell count ratio of E. coli at NaCl concentrations of 40 mg / mL to 160 mg / mL was lower than the viable cell count ratio at a NaCl concentration of 20 mg / mL, and the average value was close to 1, but exceeded the viable cell count ratio at a NaCl concentration of 0 mg / mL, which was less than 1. As shown in Figure 8, the viable cell count ratio of Staphylococcus aureus was not killed even when the NaCl concentration was increased to 160 mg / mL, because Staphylococcus aureus is salt-tolerant.

[0051] The reason why the number of bacteria did not decrease when no NaCl was added is that no operation to increase the number of white blood cells was performed in order to observe only the effect of salt.

[0052] [Experimental Example 4] Examination of the durability of the effect of NaCl Different concentrations of NaCl and live bacteria (K. pneumoniae and S. aureus) were added to the milk of healthy goats and cultured for 0 to 24 hours. After a certain time had passed from the start of culture, the milk was inoculated onto an agar medium and cultured for 24 hours, and the number of colonies was counted.

[0053] As a result, the viable cell count continued to decrease at a NaCl concentration of 0 mg / mL, but the decrease was suppressed at 10 mg / mL or higher. Specifically, as shown in Figure 9, NaCl concentrations of 5 mg / mL or higher were effective against K. pneumoniae for at least 6 hours. Similarly, NaCl concentrations of 5 mg / mL or higher were effective against S. aureus for at least 4 hours (Figure 10(b)), and for up to 24 hours at 10 mg / mL (Figure 10(a)). Thus, it was revealed that the effect of NaCl persists even during long-term storage of body fluids.

[0054] Experimental Example 5: Effect of KCl and CaCl on the viable cell count ratio The viable cell count ratio (3 h / 0 h) was measured in the same manner as in Experimental Example 1, except that KCl and CaCl were used instead of NaCl.

[0055] As shown in FIGS. 11 and 12, whether KCl or CaCl was used, a higher viable cell count ratio than 0 mg / mL was obtained at least at chloride concentrations of 5 to 40 mg / mL.

[0056] Experimental Example 6: Effect of NaCl on viable bacterial count in milk from cows with mastitis. NaCl was added to milk from cows with mastitis to a concentration of 10 mg / mL and allowed to stand at room temperature for 5 hours. After standing, the milk was plated on an agar medium, and the bacteria in the milk were cultured at 37°C for 24 hours. After culture, the viable bacterial count was calculated from the number of colonies formed by the bacteria.

[0057] Figure 13(a) shows the results of inoculating agar media with milk immediately after collection (0 h), milk left for 5 hours without the addition of NaCl (control (5 h)), and milk left for 5 hours with NaCl added (NaCl (5 h)). The viable cell count ratio (5 h / 0 h) obtained by dividing the values ​​for control (5 h) and NaCl (5 h) in Figure 13(a) by the value for 0 h is also shown.

[0058] As shown in Figures 13(a) and (b), even when the bacterial species in the sample had not been identified, the effect of NaCl on inhibiting leukocyte function was observed at about 10 mg / mL.

[0059] The above experimental examples confirmed that NaCl, KCl, and CaCl all inhibit the function of leukocytes (particularly phagocytosis), thereby increasing the survival rate of bacteria in milk.

Claims

1. A leukocyte function inhibitor for identifying the type of bacteria contained in a leukocyte-containing body fluid, which contains, as an active ingredient, a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals.

2. The leukocyte function inhibitor of claim 1, wherein the metal is selected from the group consisting of potassium, sodium, and calcium.

3. A kit for sampling a leukocyte-containing body fluid, comprising: a container capable of holding a predetermined volume of leukocyte-containing body fluid; and a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals, the chloride having a mass relative to the volume of 4 to 40 mg / mL.

4. The sampling kit according to claim 3, wherein the mass ratio of the chloride to the volume is 4 to 20 mg / mL.

5. A pretreatment method for identifying the type of bacteria contained in a leukocyte-containing body fluid, comprising the step of adding 4 to 40 mg / mL of a chloride of at least one metal selected from the group consisting of alkali metals and alkaline earth metals relative to the volume of the leukocyte-containing body fluid.

6. The pretreatment method according to claim 5, wherein the leukocyte-containing body fluid is selected from the group consisting of saliva, milk, cerebrospinal fluid, and sputum.

7. The pretreatment method according to claim 5, wherein the leukocyte-containing body fluid is collected from a subject selected from the group consisting of humans, cows, goats, pigs, dogs, cats, and chickens.

8. A method for identifying bacteria in a leukocyte-containing body fluid, comprising: (a) a step of pretreating a leukocyte-containing body fluid using the pretreatment method described in claim 5; (b) a step of culturing bacteria in the leukocyte-containing body fluid after step (a); and (c) a step of identifying the bacteria cultured by step (b).

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