JP2004292462A - Prophylactic agent and prophylactic method for bovine mastitis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a prophylactic agent and to provide a prophylactic method for preventing from being suffered with bovine mastitis in the transition from bovine lactation period to bovine dry period, and in the dry period. <P>SOLUTION: The prophylactic agent and the prophylactic method for preventing from being suffered with the bovine mastitis are provided, wherein the prophylactic agent for bovine mastitis contains lactoferrin as an active ingredient and the prophylactic method for bovine mastitis comprises injecting the prophylactic agent containing the lactoferrin as an active ingredient into bovine mamma. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an immune system activator and immunostimulation method in the mammary gland of lactating cows. More specifically, the present invention relates to an immunostimulator and an immunostimulation method for smoothly transitioning the mammary gland of a lactating cow from the lactation period to the dry period.

  Dairy cows are inseminated during lactation and become pregnant. When five months have passed since insemination, the placenta of the pregnant mother actively secretes hormones such as estrogen, the milk yield decreases, and some tissues and cells of the mammary gland begin to regress.

  Furthermore, as the fetus grows, the mammary gland regresses in the mother's womb and the milk production function declines. Dairy farmers introduce the mammary glands during the dry season by stopping milking these lactating cows at an appropriate time before delivery. This encourages dairy cows to deliver smoothly and resume smooth lactation after delivery.

During the dry period, new mammary gland tissue is being made into components for feeding calves after calving. In the component formation, the old mammary epithelium in the mammary mucosal tissue is regressed by apoptosis, and many kinds of blood cells are transferred instead. When component formation begins, the cellular tissue in the breast is composed of cells of extremely diverse elements such as granulocytes, monocytes, macrophages, dendritic cells, NK cells, T cells, B cells, and mast cells. Therefore, phagocytic cells such as granulocytes and macrophages are distributed in mammary gland tissue and milk, and lymphocytes are derived from helper-type CD4 ⁺ T cells derived from blood circulation and B cells producing antibodies. It becomes distributed (for example, refer nonpatent literature 1). For this reason, during the dry period, the mammary gland becomes highly resistant to infection.

  Conventionally, in order to introduce lactating cows in the dry period, milking of lactating cows was stopped at once, and the method of suppressing lactation by increasing the intramammary pressure was mainly used. However, even if dairymen introduced lactating cows in the dry season by this method, the lactating cow's mammary gland actually did not move smoothly into the dry season. In such a case, there has been a problem that foreign bacteria easily infect the mammary gland and increase the incidence of mastitis.

In addition, if this transition is not smoothly performed and delayed, the development of the mammary gland of the mother cow is also delayed.
When calves are reared by mother cows whose mammary gland has been delayed, calves suffer from growth failure and suffer from infectious diarrhea caused by various bacteria and viruses.

These problems are serious problems for dairy farmers, and it is desired to solve them as soon as possible. However, no effective means for solving these problems has been found.
Vet.Immunol. & Immunopathol., 65, 51-61, 1998; J. Dairy Sci., 82, 1459-1464, 1999

  The object of the present invention is to rapidly transfer the mammary glands of lactating cows to the dry period. Another object of the present invention is to smoothly advance mammary gland development and colostrum formation.

  As a result of extensive research, the present inventors activated a mother cow's immune system by administering a small amount of lactoferrin to the mother cow, and smoothly transferred the mother's mammary gland from the lactation period to the dry period. I found what I could do.

That is, in order to solve the above-mentioned object, the present invention provides an immunostimulating agent for mammary glands in lactating cows containing lactoferrin or a pharmaceutically acceptable salt thereof as an active ingredient.
Furthermore, the present invention provides a method for immunostimulating mammary glands in dairy cows, comprising administering to lactating cows a pharmaceutical composition containing lactoferrin or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, it is possible to activate the immune system of the mammary gland of lactating cows.
In addition, according to the present invention, the mammary gland of a lactating cow can be smoothly and quickly transferred from the lactation period to the dry period.
Furthermore, according to the present invention, it becomes possible to form clean colostrum that contains abundant antibodies for transfer to calves and is less contaminated with bacteria, and further prevents the growth failure of calves. Make it possible.
Furthermore, according to the present invention, it is possible to smoothly advance the transition of the perinatal period such as parturition, formation of colostrum, and start of adult milk secretion.
Furthermore, the present invention makes it possible to prevent the mammary gland of the mother cow from being directly infected with bacteria during the transition from the lactation period to the dry period or during the dry period.
Furthermore, the present invention makes it possible to prevent the development of mastitis during the transition from the lactation period to the dry period or during the dry period.

  The present invention relates to an immunostimulating agent and an immunostimulating method for mammary glands in dairy cows containing lactoferrin or a pharmaceutically acceptable salt thereof as an active ingredient.

  Lactoferrin (hereinafter abbreviated as Lf) used in the present invention is a huge glycoprotein having a molecular weight of about 80,000 contained in mammalian milk, saliva, tears, bronchial mucus, bile, and intestinal fluid.

  The pharmaceutically acceptable salt of Lf is not particularly limited as long as it is highly safe for bovine mothers and fetuses, and may be, for example, ammonium salt, alkali metal salt, hydrochloride, acetate, or carbonate.

  In the present invention, these Lf or Lf salts may be used alone or in admixture of two or more.

  Lf and Lf salts used in the present invention are those isolated and purified from human breast milk, those isolated and purified from the breast milk of mammals such as cows, goats and sheep, and those obtained by genetic recombination of transgenic animals and microorganisms. It may be.

  Examples of the dosage form of the present invention include ointments and liquids. Examples of the base used to make an ointment include lipophilic ointment bases such as white petrolatum, yellow petrolatum, liquid paraffin, olive oil, peanut oil, soybean oil, lanolin, polyethylene glycol, sodium polyacrylate, stearyl alcohol, Examples thereof include hydrophilic ointment base materials such as stearic acid, aluminum stearate, glycerin, sodium alginate, and carboxymethylcellulose. In addition to the above, examples of the solvent used for preparing the liquid include propylene glycol, polyethylene glycol, and glycerin.

  An additive may optionally be added to the pharmaceutical composition of the present invention. Examples of additives include buffering agents, isotonic agents, stabilizers, and preservatives.

  The dose of the present invention is preferably 100 to 250 mg per quarter, more preferably 100 to 200 mg in terms of Lf, considering the effects and economics obtained by administration. The Lf concentration in the milk is preferably administered such that the measured value of Lf when measuring milk milked immediately after administration is 1000 to 2500 μg / ml, more preferably 1000 to 2000 μg / ml.

  The number of administrations may be such that the above-mentioned amount of the drug may be administered once or several times during this period from the date of introduction of dry milk to 3 weeks after the introduction of dry milk. good.

  Examples of the dosage form of the present invention include injection, preferably direct injection, and more preferably injection into the breast.

  Examples 1 and 2 below show formulation examples when the pharmaceutical composition of the present invention is used as a liquid and as an ointment.

(Prescription Example 1)
Bovine lactoferrin 100mg
Sodium chloride 80mg Potassium chloride 2mg
Potassium dihydrogen phosphate 2mg
Sodium dihydrogen phosphate 2mg
The above compounds were mixed and made up to 10 ml with distilled water to obtain the solution of the present invention.

(Prescription example 2)
Bovine lactoferrin 100mg
Cocoa butter 4g
Ascorbic acid 25mg
Sodium chloride 80mg
The above compound was mixed and made up to 10 ml with distilled water to obtain an ointment of the present invention.

(Test Example 1)
Lf was administered to the breast of a lactating cow on the day of introduction of dry milk, and the number of somatic cells in the milk (Somatic Cell Count, hereinafter abbreviated as SCC) was quantified. For comparison with this, SCC in milk was also measured for lactating cows that did not receive Lf.

  Somatic cells are mostly composed of white blood cells and appear in milk as a response to inflammation. Furthermore, it is known that when it is introduced in the dry period, the SCC once increases, returns to the original value, and then takes a stable value.

  This SCC quantification was specifically performed by the following method. On the day of introduction of dry milk (day 0), the liquid prepared in Formulation Example 1 was injected into 29 breasts of healthy lactating cows. The dose of the solution was injected so as to be 100 to 250 mg per breast in terms of lactoferrin contained in the solution.

  In addition, the liquid prepared in Formulation Example 1 was not injected into 11 breasts of healthy lactating cows.

  0, 1, 3, 7 for milk obtained from the breast group infused with the liquid (hereinafter abbreviated as Lf administration quarter) and the breast group that was not infused (hereinafter abbreviated as non-administration quarter) On the 14th and 14th days, the SCC was measured and the average values were calculated. SCC was measured by washing a milk sample with 20 mM ethylenebistetraacetic acid sodium phosphate physiological buffer (EDTA-PBS), staining with propidium iodide, and then using a flow cytometer (trade name: Fax Caliber, Japan). Becton Dickinson). These results are shown in Table 1 and FIG.

表１および図１に示されるように、Lf投与分房では、投与直後からSCCが増加
して３日目には最高値に達し、７日過ぎにはその増加は止み、Lf投与前の低値にもどる。これに対して、非投与分房では、１日経過後からSCC数が徐々に上昇し始め、７日目付近に最高値を示した後減少し、１４日目の時点でも減少が続きSCC値は安定しなかった。また、Lf投与分房のSCC最高値は、非投与分房のSCC最高値と比較して、より高かった。

As shown in Table 1 and FIG. 1, in the Lf-administered quarter, the SCC increased immediately after the administration, reached the maximum value on the third day, stopped increasing after the seventh day, and decreased before the Lf administration. Return to value. In contrast, in non-administered quarters, the number of SCCs began to gradually increase after the first day, decreased after reaching the highest value around the seventh day, and continued to decrease even at the 14th day. It was not stable. Moreover, the SCC maximum value of the Lf-administered quarter was higher than the SCC maximum value of the non-administered quarter.

  From the above results, it can be seen that the Lf-administered quarter rapidly accelerates the transfer of blood-derived cells into the mammary gland tissue as compared to the non-administered quarter.

  Therefore, by administering Lf, it becomes possible to obtain a significant sterilization effect at an early stage.

(Test Example 2)
The number of CD11b antigen-positive cells, which are representative cells of the phagocytic function, was quantified for the milk obtained from the Lf-administered and non-administered quarter groups used in Test Example 1.

  When introduced in the dry period, the number of CD11b antigen-positive cells is known to increase once and then stabilize. Thus, by measuring the number of CD11b antigen-positive cells, the effect of Lf administration on phagocytic function was examined.

  Specifically, milk cells were collected by centrifugation, stained with bovine CD11b monoclonal antibody and FITC-labeled mouse IgG2b monoclonal antibody, and positive for CD11b antigen on days 0, 1, 3, 7 and 14 using a flow cytometer. Cell number was measured. The number of CD11b antigen-positive cells obtained is shown in Table 2 and FIG.

表２および図２に示されるように、Lf投与分房では、投与直後からCD11b抗原
陽性細胞数が増加し、３日目に最高値を示した後、７日目には既に安定した値となった。これに対して、非投与分房では、１日経過後から上昇し、３日目に高値を示した後減少し、１４日目の時点ではまだ減少が続き安定しなかった。

As shown in Table 2 and FIG. 2, in the Lf-administered quarter, the number of CD11b antigen-positive cells increased immediately after administration, showed the highest value on the third day, and was already stable on the seventh day. became. On the other hand, in the non-administered quarters, it increased after 1 day, decreased after showing a high value on the 3rd day, and continued to decrease at the 14th day and was not stable.

  In addition, the maximum value in the Lf-administered quarter was extremely high compared to the maximum value in the non-administered quarter.

  From the above results, it can be seen that the number of blood-derived cells increases rapidly and significantly in the Lf-administered quarters compared to the non-administered quarters.

  Therefore, it is possible to obtain a significant sterilization effect at an early stage by administering Lf.

(Test Example 3)
In order to examine the effect of Lf on T lymphocyte cells, which are immune response cells, in milk obtained from the Lf-administered and non-administered breast groups used in Test Example 1, CD4 ⁺ T / CD8 ⁺ T Was quantified.

Among the T lymphocyte subtypes, CD4 ⁺ T is known to be contained as a main component in milk and mammary gland during the dry period, and CD8 ⁺ T is known as a main component contained in milk and mammary gland during the lactation period. CD4 ⁺ T cells are known to function as helper T cells in the antibody production system, and CD8 ⁺ T functions as suppressor cells.

Specifically, the experiment was performed as follows. Concentrations of CD4 ⁺ T cells and CD8 ⁺ T cells were measured for the milk obtained from the Lf-administered and non-administered quarter groups used in Test Example 1. The CD4 ⁺ T cells were stained with bovine CD4 monoclonal antibody and FITC-labeled mouse IgG1 monoclonal antibody, and CD8 ⁺ T cells were stained with bovine CD8 monoclonal antibody and FITC-labeled mouse IgM monoclonal antibody. And measured on days 0, 1, 3, 7 and 14. The average values of the obtained CD4 ⁺ T cells / CD8 ⁺ T cells are shown in Table 3 and FIG.

表３および図３に示されるように、Lf投与分房では２日目からCD4^＋T／CD8^＋T値は急激に上昇し、その効果は７日目まで継続した。これに対して、非投与分房では、CD4^＋T／CD8^＋T値は特に大きな変化は見られないまま緩やかに上昇した。

As shown in Table 3 and FIG. 3, in the Lf administration quarter, the CD4 ⁺ T / CD8 ⁺ T value increased rapidly from the second day, and the effect continued until the seventh day. On the other hand, in the non-administered quarter, the CD4 ⁺ T / CD8 ⁺ T value gradually increased without any significant change.

  From the above results, it was found that the Lf-administered quarter greatly changes the ratio of helper type T cells and killer type T cells compared with the non-administered quarter.

  Therefore, it was found that administration of Lf enables the mammary gland to be transferred to the dry stage early.

(Test Example 4)
Further, in order to examine the influence of Lf addition of CD4 ⁺ T / CD8 ⁺ T following Test Example 4, each of the CD4 ⁺ T / CD8 ^{+ of} the medium added with Lf and the medium added with Lf and Lf antibodies was used. T concentration ratio was measured.

  Specifically, first, the medium used here is RPMI 1640 medium (manufactured by Nissui Pharmaceutical) containing 5% fetal bovine serum. Lymphocytes were collected from peripheral blood of healthy lactating cows by specific gravity centrifugation, and (1) medium, (2) medium supplemented with 100 μg of Lf, and (3) bound to 100 μg of Lf and Lf of 100 μg of Lf. Each of these was cultured for 3 days using a medium supplemented with a bovine Lf rabbit affinity antibody having the titer to be used, and T lymphocyte subsets before and after 3 days of culture were analyzed by a flow cytometer. The results are shown in Table 4 and FIG.

表４及び図４に示されるように、Lfが添加された培地のCD4^＋T/CD8^＋T値は、無添加の培地と比較して大きく増加した。これに対して、Lf及びLf抗体を添加した培地のCD4^＋T/CD8^＋T値は、無添加の培地と比較して減少した。このことから、CD4^＋T/CD8^＋T値はLfの添加に大きく依存することが理解される。

As shown in Table 4 and FIG. 4, the CD4 ⁺ T / CD8 ⁺ T value of the medium to which Lf was added was greatly increased compared to the medium to which no additive was added. In contrast, the CD4 ⁺ T / CD8 ⁺ T value of the medium supplemented with Lf and Lf antibody was decreased compared to the medium without addition. From this, it is understood that the CD4 ⁺ T / CD8 ⁺ T value greatly depends on the addition of Lf.

(Test Example 5)
The increase of immune protein component by Lf input was investigated. As the immunity protein, immunoglobulin IgG, particularly IgG1, which is most abundant in the subclass, was quantified.

  Specifically, it was performed as follows. On the day of introduction of dry milk (day 0), the liquid prepared in Formulation Example 1 was injected into 5 breasts of healthy lactating cows. The liquid dosage was 100 mg per breast (concentration in milk 1000 μg / ml) in terms of lactoferrin contained in the liquid.

  In addition, the liquid prepared in Formulation Example 1 was not injected into 3 breasts of healthy lactating cows.

For milk obtained from the breast group injected with the liquid (hereinafter abbreviated as Lf-administered quarter) and the breast group not injected (hereinafter abbreviated as the non-administered quarter), IgG1 was administered on days 0 and 7 14 days, 30 days before parturition, 14 days before parturition and each day when colostrum was obtained.
Measurement of the concentration of IgG1 was carried out using the one-way radioimmunodiffusion (SRID) method, and the average value of the obtained IgG1 concentrations was calculated. The results are shown in Table 5 and FIG.

表５及び図５に示されるように、Lf投与分房では、非投与分房後と比較して、いずれの日においてもIgG1濃度の増加が顕著であった。

As shown in Table 5 and FIG. 5, in the Lf-administered quarter, the increase in the IgG1 concentration was remarkable on any day as compared with that after the non-administered quarter.

  Thus, Lf administration was found to activate the mammary gland immune system during the dry period.

(Test Example 6)
In order to examine the Lf dependency of IgG1, the IgG1 concentration was measured in the same manner as in Test Example 5 with the doses of Lf per quarter being 10, 100, 200 and 500 mg. The results are shown in Table 6 and FIG.

表６及び図６に示されるように、Lfの投与量が10mgの場合は、IgG1濃度の増加が小さい。これに対し、Lfの投与量100mg以上では、IgG1濃度が大きく増加する。従って、１分房あたりLfを100mg以上投与すると、IgG1の分泌を強く刺激することが分かる。

As shown in Table 6 and FIG. 6, when the dose of Lf is 10 mg, the increase in IgG1 concentration is small. On the other hand, the IgG1 concentration greatly increases when the dose of Lf is 100 mg or more. Therefore, it can be seen that administration of 100 mg or more of Lf per quarter strongly stimulates the secretion of IgG1.

(Test Example 7)
In order to examine the sterilization effect and mastitis treatment effect of Lf, Lf was administered to lactating cows in the dry breast affected by mastitis, and the state of mastitis and the number of infectious bacteria were examined.

  Specifically, the liquid prepared in Formulation Example 1 was injected into 12 quarters (8 cases of overt mastitis and 4 cases of overt mastitis) in the dry period affected by mastitis. The dosage of the liquid was 100 mg per quarter of breast in terms of Lf contained in the liquid.

  For comparison, dry ointment used as a mastitis therapeutic agent in the dry period was administered by injection into the mammary gland for 29 cases in the dry period suffering from mastitis. As the dry milk ointment, product name Cefamedin DC manufactured by Nippon Zenyaku Co., Ltd. was used.

  About the Lf administration quarter and the ointment administration quarter, the mastitis symptom was investigated until one week after delivery. In addition, the number of staphylococci, which are the main cause of mastitis, was measured.

  The method for measuring staphylococci was as follows. The milk collected from the mammary gland immediately before administration of Lf and 6 days after administration and colostrum after delivery are diluted 10, 100, and 1000 times with sterilized physiological saline, respectively, and this is diluted with an agar medium ( No. 110 medium) was applied by a plate smearing method, cultured at 37 ° C. for 48 hours, and then the number of staphylococci was counted from the number of colonies formed on the agar medium.

  In addition, mammary inflammation was examined by palpation and visual observation at the time of Lf or ointment administration and one week after delivery.

  These results are shown in Table 7 for the Lf administration quarters and in Table 8 for the ointment administration quarters.

Here, regarding the evaluation of the mastitis symptoms in the figure,-: a state not affected by mastitis, +: mild mastitis symptoms, ++: moderate mastitis, and +++: acute mastitis. The number of staphylococci is expressed in colony forming units (CFU) per ml, and the evaluation is −: <2.0 × 10 ² CFU / ml, ±: 2.0 × 10 ^{2 to} 4.0 × 10 ² CFU / ml, + : 4.0 × 10 ^{2 to} 1.0 × 10 ³ CFU / ml, ++: 1.0 × 10 ^{3 to} 5.0 × 10 ³ CFU / ml, +++:> 5.0 × 10 ³ CFU / ml

表７及び８に示されるとおり、Lf投与分房の初乳におけるブドウ球菌の陽性率（（＋、＋＋及び＋＋＋例）/測定例、2例/8例）は25%であった。これに対して、軟膏投与分房の初乳中の陽性率は55.2%（16例/29例）であった。

As shown in Tables 7 and 8, the positive rate of staphylococci ((+, ++ and ++ cases) / measurement example, 2 cases / 8 cases) in the colostrum of the Lf-administered quarter was 25%. In contrast, the positive rate of colostrum in the ointment-administered quarter was 55.2% (16/29 cases).

  The incidence of mastitis was 8.3% in Lf-administered quarters ((+, ++ or +++ cases) / measured cases, 1 case / 12 cases), compared with 50% in ointment-administered quarters. (15/29 cases).

  From this, it is understood that the therapeutic effect of mastitis can be obtained by administering Lf to lactating cows in the dry period.

(Test Example 8)
In order to examine Lf's mastitis-preventing effect, Lf was administered to lactating cows in the dry period where no mammary inflammation symptoms were observed, and then the number of infectious bacteria and the presence of mammary inflammation symptoms were examined.

  Specifically, the liquid prepared in Formulation Example 1 was injected into 17 cases of dry quarters in which no symptoms of mastitis were observed. The amount of liquid injected was 100 mg per quarter in terms of Lf.

  In addition, for comparison, ointment was injected into 17 breasts in the same manner as in Test Example 7 using lactating cows in the dry period in which no symptoms of mastitis were observed.

About the above-mentioned Lf administration quarter and ointment administration quarter, the mammary inflammation state which was affected by one week after delivery was investigated. In addition, the number of staphylococci, which are the main cause of mastitis, was measured.
The measurement method is the same as in Test Example 7.

  These results are shown in Tables 9 and 10. Each evaluation is the same as in Test Example 7.

表９及び１０に示されるとおり、乳房炎発症率（（＋、＋＋及び＋＋＋例）/
全例）は、Lf投与分房では0%であったが、軟膏投与分房では47.1%（8例/17例）であった。

As shown in Tables 9 and 10, the incidence of mastitis ((+, ++ and ++++ examples) /
All cases) were 0% in Lf-administered quarters, but 47.1% (8/17 cases) in ointment-administered quarters.

  Regarding the number of staphylococci, the positive rate in the colostrum of the Lf-administered quarter was 11.8% ((+, ++ and +++ cases) / measurement example, 2 cases / 17 cases). In contrast, the positive rate in the colostrum of the ointment-administered quarter was 55.8% (10 cases / 17 cases).

  Thus, it can be understood that mastitis can be prevented by administering Lf to lactating cows in the dry period.

It is a graph which shows the fluctuation | variation of SCC in a Lf administration quarter and a non-administration quarter. It is a graph which shows the fluctuation | variation of the CD11b antigen positive somatic cell count in a Lf administration quarter and a non-administration quarter. It is a graph which shows the fluctuation | variation of the CD4 ⁺ T cell / CD8 ⁺ T cell concentration ratio in a Lf administration quarter and a non-administration quarter. It is a graph which shows the fluctuation | variation of the CD4 ⁺ T cell / CD8 ⁺ T cell concentration ratio in each culture medium. It is a graph which shows the fluctuation | variation of IgG1 density | concentration in a Lf administration quarter and a non-administration quarter. It is a graph which shows the fluctuation | variation of IgG1 density | concentration with respect to Lf administration of each density | concentration.

Claims (4)

 A preventive agent for bovine mastitis containing lactoferrin as an active ingredient.  A method for preventing bovine mastitis, which comprises injecting a prophylactic agent containing lactoferrin as an active ingredient into bovine breast.   The method for preventing bovine mastitis according to claim 2, wherein the dose of the prophylactic agent is 100 to 250 mg per breast in terms of lactoferrin.  The method for preventing bovine mastitis according to claim 2, wherein the prophylactic agent is administered from the day of introduction of dry milk to 3 weeks after the introduction of dry milk.

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