JP2008074787A - Mastitis therapeutic agent and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for treating mastitis capable of attaining excellent effect to enable the shake-up, disposal and milking-stop of cows. <P>SOLUTION: The agent for treating mastitis comprises a combination of lactoferrin and antibiotics. A method for treating mastitis is also provided, comprising administering a combination of lactoferrin and the antibiotics to bovine mammary gland under dry period. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  It has been conventionally known that bovine mastitis is a disease located at the top of the cause of injury and illness in dairy cows (see, for example, Non-Patent Document 1). The economic loss is said to be 12 billion yen per year, which is a serious management impediment to dairy farming (see Non-Patent Document 2). The cause of mastitis is mainly bacterial infection in the mammary gland, and the causative bacteria range from E. coli, Staphylococcus aureus, Streptococcus. Various control measures are taken against this, but the current situation is that it is finally dealt with by treating the affected cows such as drought, disuse and blind milk. Moreover, it is known that the onset time is often in the dry period in which milking is artificially stopped to prepare for delivery and immediately after delivery.

  As the ointment used for the treatment of bovine mastitis, the ointment for the milking period needs immediate effect, so the viscosity of the ointment base is adjusted to be low so that antibiotics compatible with the causative bacteria are quickly dispersed in the milk tank. . In addition, since the ointment for the dry period maintains the effectiveness of the antibiotic for a long time, the viscosity of the ointment base is adjusted to be high so that the dispersion occurs slowly.

  The dry-season ointment used in the dry season, where mastitis is common, is due to the fact that milk protein dries and solidifies at the mouth of the teat after the milking is stopped. Infusion and no subsequent re-administration. For this reason, it is highly durable but lacks immediate effect, so it is actually used prophylactically. Therefore, in the treatment of acute dry-stage mastitis, an immediate-acting lactating ointment may be used, but the therapeutic effect of antibiotic ointment in the dry period is approximately 50%, and the lactating period In the dry period, when the clinical symptoms of mastitis are more severe, many cattle are culled and discarded.

  On the other hand, lactoferrin is a typical defense substance in milk, and its physiological action is diverse. Typical physiological actions include antibacterial action, that is, action that suppresses the generation, growth, and proliferation of microorganisms. In particular, the peptide lactoferricin contained in the hydrolyzate of lactoferrin shows strong antibacterial action. It is known (see Non-Patent Documents 3 and 4). It is known that lactoferrin itself or lactoferrin hydrolyzate alone is injected into the breast using this property to treat bovine mastitis. However, the effect on mastitis caused by some causative bacteria with weak pathogenicity (see Non-Patent Document 5) and latent mastitis (see Non-Patent Document 6) in which clinical symptoms do not appear clearly. It has only been obtained.

In addition, lactoferrin is known to exhibit an anti-inflammatory effect when bound to lipopolysaccharide (LPS) of Escherichia coli. However, since Staphylococcus aureus and streptococci do not have LPS, it has been considered that acute mastitis caused by these causes does not exhibit an anti-inflammatory effect.
Nakamoto “Actual Results of the Livestock Mutual Aid Project in FY2000” Livestock Practice May 49, 2002 p.335-340 Eguchi “Current Status of Bovine Mastitis Vaccine” Livestock Practice January 47, 2000 Issue 1 Pages 15-23 Bellamy et al. “Identification of the bactericidal domain of lactoferrin” Biochimica et Biophysica Acta. 1992 1121 p.130-136 Tanida et al. “Lactoferrin Peptide Increases the Survival of Candida albicans-Inoculated Mice by Upregulating Neutrophil and Macrophage Functions, Especially in Combination with Amphotericin B and Granulocyte-Macrophage Colony-Stimulating Factor” Infection and Immunity June 2001 Vol. 69 No. 6 p. .3883-3890 Kai et al. “Effects of Bovine Lactoferrin by the Intramammary Infusion in Cows with Staphylococcal Mastitis during the Early Non-Lactating Period” J. Vet. Med. Sci. 2002 64 (10) p.873-878 Kawai et al. “Effect of Infusing Lactoferrin Hydrolysate into Bovine Mammary Glands with Subclinical Mastits” Veterinary Research Communications 2003 27 P.539-548

  One object of the present invention is to provide a therapeutic agent for mastitis that achieves an excellent therapeutic effect. Another object of the present invention is to reduce cow straw, waste, and blind milk.

  As a result of intensive studies to achieve the above object, the present inventors have found that a remarkably excellent mastitis therapeutic effect can be obtained by administering lactoferrin and antibiotics in combination.

  That is, one embodiment of the present invention relates to a therapeutic agent for mastitis that combines lactoferrin and antibiotics. One embodiment of the present invention also relates to a method for treating mastitis, wherein lactoferrin and antibiotics are administered in combination to bovine breasts in the dry period. Furthermore, one embodiment of the present invention relates to a method for suppressing the production of inflammatory cytokines through the effect of suppressing the activity of NFκB by the combination therapy of lactoferrin and antibiotics in the mammary gland of clinical mastitis during the dry period. Furthermore, one embodiment of the present invention relates to a method for suppressing the production of inflammatory mediators and inflammatory lactoferrin molecules by combination therapy of lactoferrin and antibiotics in the dry mammary gland of clinical mastitis. Furthermore, one embodiment of the present invention relates to a method for enhancing the sterilizing action of antibiotics by injecting lactoferrin into the mammary gland of clinical mastitis during the dry period. Furthermore, one embodiment of the present invention relates to a method for alleviating clinical symptoms in the breast by a combined therapy of lactoferrin and antibiotics in the mammary gland of dry-type clinical mastitis. Furthermore, one embodiment of the present invention relates to a method for preventing the onset of postpartum mastitis by a combination therapy of lactoferrin and antibiotics in the mammary gland of clinical mastitis during the dry period. Furthermore, one embodiment of the present invention relates to a method for inducing normal lactation after delivery by a combination therapy of lactoferrin and antibiotics into the dry mammary gland of clinical mastitis.

  According to one embodiment of the present invention, it is possible to achieve a markedly superior mastitis therapeutic effect. In addition, according to another embodiment of the present invention, it is possible to reduce cow straw, waste, and blind milk. Furthermore, according to one embodiment of the present invention, clinical symptoms of mastitis can be improved. Furthermore, according to one embodiment of the present invention, clinical mastitis caused by Staphylococcus aureus in the dry period can be treated.

  The mastitis treatment method of the present invention comprises a step of administering lactoferrin and a dry or lactating antibiotic in combination to a dairy cow.

  Bovine mastitis can be classified into subclinical mastitis and clinical mastitis. Latent mastitis is determined to be mastitis by a known mastitis determination test, for example, somatic cell count (SCC), modified California Masterytis test (CMT modified method) or visual observation of breasts in milk. This refers to cases where clinical symptoms have not appeared. In addition, clinical mastitis refers to clinical symptoms such as breast swelling, induration, and atrophy, and general symptoms such as fever and inability to stand, regardless of the results of the mastitis test described above. . Clinical mastitis is often more difficult to repair.

  Treatment of clinical mastitis consists of eradication of the causative bacteria and relief of clinical symptoms. Here, sterilization refers to the action of suppressing the generation, growth and proliferation of microorganisms. Normally, the eradication of the causative bacteria is performed only with antibiotics, but when inflammation occurs in the tissue, the causative bacteria enter the tissue easily due to the damage of the epithelial tissue, and the effect of the antibiotics is inhibited. Therefore, it is necessary to quickly improve inflammation that inhibits the sterilization effect of antibiotics. According to the study by the present inventors, it has been found that lactoferrin exerts an anti-inflammatory action against causative bacteria not having LPS by using antibiotics and lactoferrin together. In the treatment of mastitis, lactoferrin, which is an in-vivo substance contained in body fluids such as milk and blood, is an optimal substance without harm to the body.

  As used herein, lactoferrin includes lactoferrin, its related peptides and salts thereof. Examples of the related peptide of lactoferrin include lactoferricin. Examples of the lactoferrin salt include sodium salt of lactoferrin. Lactoferrin is preferably derived from bovine milk.

  Antibiotics that match the antibacterial spectrum of the causative bacteria of mastitis are used. For example, erythromycin is mentioned when the causative bacteria are staphylococci and streptococci.

  Lactoferrin and antibiotics may be administered simultaneously as the same drug. Moreover, you may administer as a separate chemical | medical agent simultaneously or at different time, in other words, you may administer in combination, ie, you may use together.

  Examples of the dosage form include tablets, granules, powders, ointments, and liquids. These can be manufactured using a conventionally known technique. Preferably, it is an ointment or a liquid. Ointments and liquids can be directly injected into the affected area, that is, the drug can be injected into the mammary gland from the nipple using a cannula, and the healing effect is high.

  In the case of an ointment, an ointment base is used. Examples of the ointment base include a lipophilic ointment base and a hydrophilic ointment base. Examples of the lipophilic ointment base include white petrolatum, yellow petrolatum, liquid paraffin, olive oil, peanut oil, and soybean oil. Examples of the hydrophilic ointment base include polyethylene glycol, sodium polyacrylate, stearic acid, aluminum stearate, glycerin, and carboxymethyl cellulose.

  In the case of a liquid agent, a solvent is used. Examples of the solvent include propylene glycol and polyethylene glycol in addition to the above-described base material.

  In addition to the therapeutic agent, buffers, isotonic agents, stabilizers, preservatives and the like can be added as necessary.

  The dose of lactoferrin is preferably administered so that lactoferrin is 100 to 200 mg per quarter at a time, and is appropriately changed depending on the condition of the cow. The lactoferrin concentration at the time of administration is preferably 100 to 2,000 μg / ml in milk.

  The dose of antibiotic is the effective therapeutic amount specified for each antibiotic. For example, in the case of erythromycin, 300 mg is administered per minute per day.

  When lactoferrin and antibiotics are administered at the same time, they are administered simultaneously according to the dosage and administration specified for each antibiotic. Moreover, when administering separately, it is preferable to administer antibiotics according to the usage and dosage designated for each antibiotic, and to administer lactoferrin within 4 days after antibiotic administration.

  The administration method is not particularly limited, and includes oral, injection, cannula injection, and the like, but it is preferable to directly inject the drug from the nipple into the mammary gland using the cannula.

  Hereinafter, it demonstrates more concretely using a test example.

  Antibiotics and lactoferrin were administered to the quarters that developed clinical mastitis caused by Staphylococcus aureus between 7 and 14 days after introduction of the dry period (that is, after milking was artificially stopped). The following items (1) to (3) were observed over time. For comparison with this, antibiotics alone or lactoferrin alone was administered and observed similarly. Administration was directly injected as an ointment from the nipple into the mammary gland using a cannula.

(1) Clinical Symptoms Before administration of antibiotics and / or lactoferrin, and on the 3rd and 7th days after the start of treatment, the quarters were examined by palpation and visual inspection.

(2) Number of causative bacteria Staphylococci were measured as causative bacteria. The method of measurement is to dilute the milk collected from the mammary gland on the 7th day after the start of treatment immediately before the administration of antibiotics and / or Lf, respectively, with diluted physiological saline to 10, 100, 1000 times. Was applied to an agar medium (No. 110 medium) 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.

(3) Milk secretion state after delivery Specifically, the milk secretion state was examined based on the somatic cell count (SCC), the modified California Masterytis test (CMT modified method), and the presence or absence of lumps.

  The number of SCC was measured with a flow cytometer after staining nucleic acid of cells in milk with propidium iodide. Somatic cells are composed of white blood cells and epithelial cells, and white blood cells occupying most of the somatic cells appear in milk as a response to inflammation. Therefore, if the SCC number is low, it is normal milk, and if it is high, it is abnormal milk.

  Regarding the modified CMT method, the presence or absence of a gelation reaction and the change in color tone were examined by adding a CMT reagent to milk. If the mixture of milk and reagent remains watery, it is normal milk with a small number of cells, and if it is in a gel or aggregated state, it is abnormal milk with a large number of cells. Further, if the color mixture does not change in the mixture of milk and reagent, it is normal milk, but if it changes even slightly, it is abnormal milk mixed with body fluid components such as blood.

  As for the presence or absence of irregularities, it is normal milk if a small mass coagulated in the milk is not visually observed, and abnormal milk having a large number of cells if even one mass is visible.

(Test Example 1)
Of the 25 quarters of clinical mastitis in the dry phase caused by Staphylococcus aureus, lactoferrin and antibiotics were administered in combination for 9 quarters (hereinafter referred to as combination therapy). As an antibiotic, a lactating antibiotic (cephazoline sodium) was used. The dosage of lactoferrin is 200 mg per quarter, and the dosage of antibiotics is 150 mg. Moreover, about 10 quarters, lactoferrin was administered alone so that it might become 200 mg per quarter (henceforth lactoferrin therapy). Further, for the 6 quarters, the above antibiotics were administered alone so as to be 150 mg per quarter (hereinafter referred to as antibiotic therapy).

  In combination therapy, lactoferrin was administered simultaneously with antibiotic administration, or lactoferrin was administered on the third day after antibiotic administration.

  The lactoferrin and / or antibiotic administration date is the treatment start date (day 0), but when lactoferrin is administered on the third day after antibiotic administration, the lactoferrin administration date is the treatment start date (day 0). ).

(1) Clinical symptoms On the third day after the start of treatment, clinical symptoms such as swelling, induration, and fever disappeared from 44.4% of the combination therapy quarters. On the other hand, the disappearance of clinical symptoms was not seen from the antibiotic therapy quarter and the lactoferrin therapy quarter. On the 7th day after the start of treatment, clinical symptoms disappeared from 80% of the combination therapy quarters. On the other hand, the therapeutic effect was recognized in about half of the quarters in the antibiotic therapy quarter and the lactoferrin therapy quarter. Table 1 shows the disappearance rate of clinical symptoms. That is, according to the present invention, there is a remarkable effect of relieving clinical symptoms.

（２）起因菌数
治療開始後７日目に、併用療法分房の全９分房から得られた乳汁については、黄色ブドウ球菌数が大きく低減した。これに対し、抗生物質療法分房及びラクトフェリン療法分房から得られた乳汁については、約半分の乳汁において菌数が低下したが、残り半分の乳汁は菌数の変化がないか菌数が増加した。図１に黄色ブドウ球菌数の変化を示す。つまり、抗生物質単独療法では除菌効果が認められたのは約半分だったが、抗生物質とラクトフェリンとの併用療法では全分房に顕著な除菌効果が認められた。つまり、本発明によると、ラクトフェリンにより抗生物質の除菌効果が増強した。 Moreover, even when the antibiotic and lactoferrin were administered at the same time, the same effect was exhibited when lactoferrin was administered on the third day after the antibiotic administration.

(2) Number of causative bacteria On the 7th day after the start of treatment, the number of Staphylococcus aureus was greatly reduced for the milk obtained from all 9 quarters of the combination therapy quarter. In contrast, for milk obtained from antibiotic therapy and lactoferrin therapy, the number of bacteria decreased in about half of the milk, but the number of bacteria in the other half of milk did not change or the number of bacteria increased. did. FIG. 1 shows changes in the number of Staphylococcus aureus. In other words, about half of the antibiotic monotherapy showed a sterilization effect, but the combination therapy with antibiotics and lactoferrin showed a significant sterilization effect in all quarters. That is, according to the present invention, lactoferrin enhanced the sterilizing effect of antibiotics.

(3) Postpartum lactation status The postpartum lactation status was examined in order to know whether normal mastitis that can be shipped after delivery can be secreted by treatment of clinical mastitis during the dry period.

Regarding the number of SCC, in the milk obtained from the combination therapy quarters, the number of somatic cells on the 7th day after delivery was 44 ± 140,000 cells / ml, which was a value that could be shipped. On the other hand, the milk obtained from the antibiotic therapy quarter and the lactoferrin therapy quarter was more than five times higher than the combination therapy quarter, although it could be shipped. In particular, in the antibiotic therapy quarter, the number of SCCs increased rather than before the start of treatment. The number of SCC in each milk is shown in Table 2. That is, according to the present invention, there is an excellent effect of inducing the secretion of normal milk after parturition.

With regard to the presence or absence of modified CMT and punctures, 77.8% of milk obtained from the combination therapy quarter was normal on the 7th day after delivery, as judged by the presence or absence of modified CMT and puncture. On the other hand, normal milk was 20% in milk obtained from the antibiotic-treated quarters and 33.3% in milk obtained from lactoferrin-treated quarters. Table 3 shows the percentage of normal milk judged from the CMT modified method and the presence or absence of lumps. That is, according to the present invention, there is an excellent effect of inducing the secretion of normal milk after parturition.

  Conventionally, clinical mastitis has been easily refractory due to the pathogenesis of mastitis, for example, biofilms formed by Staphylococcus aureus, but it is likely to be refractory. However, the combination of antibiotics and lactoferrin is high. A therapeutic effect is obtained.

(Test Example 2)
Next, the effect of the therapeutic effect of lactoferrin was examined using the mammary epithelial cell line (BMEC) (Sakamoto, K., et al. 2005. J. Dairy Res. 72: 264-270.). .

  First, a small-sized lactoferrin molecule that appears in mastitis milk and exhibits inflammatory effects (inflammatory lactoferrin; Komine, K., et al. 2005. J. Vet. Med. Sci. 67: 667-677.) And normal lactoferrin were subjected to stimulation culture using BMEC. And, tumor necrosis factor (TNFα) and interleukin (IL-6) which are inflammatory cytokines, and IL-8 of chemokine which is a leukocyte chemotactic factor which infiltrates into tissues during inflammation and damages surrounding tissues The expression ability of mRNA in milk cells of Monocyte Chemotactic Protein (MCP-1) was measured by RT-PCR.

  In normal lactoferrin medium (Lf), mRNA expression was increased in IL-6, TNFα and MCP-1 as compared to the unstimulated group (medium), but no significant difference was observed. Further, almost no change was observed in the expression of IL-8.

  In inflammatory lactoferrin-stimulated medium (inflammatory Lf), both cytokines and chemokines were significantly (P <0.01) higher than the unstimulated group (medium) and normal lactoferrin-stimulated medium (Lf) mRNA expression was confirmed.

  In the mixed stimulation medium of inflammatory lactoferrin and normal lactoferrin, the mRNA expression level of stimulation culture with inflammatory lactoferrin alone was significantly lower (P <0.01), and decreased to the same level as that of stimulation culture with normal lactoferrin alone Was. FIG. 2 shows the mRNA expression level.

From the above results, it was found that lactoferrin has an anti-inflammatory action against the inflammatory action of inflammatory lactoferrin.

  Furthermore, in order to investigate the specific mechanism of this anti-inflammatory action, Nuclear Factor-κB (NFκB), an intracellular transcription factor involved in the induction of production of various inflammatory mediators such as inflammatory cytokines, chemokines, and superoxide. After BMEC stimulation culture with normal and inflammatory lactoferrin, intracellular NFκBp65 activity was measured by ELISA.

  As a result, an increase in the activity of NFκB was confirmed to be about 5-fold higher for inflammatory lactoferrin than to stimulation with normal lactoferrin. On the other hand, in the mixed stimulation culture of normal lactoferrin and inflammatory lactoferrin, the activity was suppressed to half or less as compared with single stimulation of inflammatory lactoferrin. See Figure 3.

  That is, it was revealed that lactoferrin has an action of suppressing an inflammatory reaction caused by inflammatory lactoferrin. And it was strongly suggested that this anti-inflammatory action is induced by a mechanism different from the anti-inflammatory action caused by the binding of lactoferrin and LPS in the previous report.

  According to the present invention, there is an excellent effect of suppressing the production of inflammatory cytokines through the effect of suppressing NFκB activity. Moreover, according to this invention, there exists an outstanding effect which suppresses production of an inflammatory mediator and an inflammatory lactoferrin molecule.

It is a graph which shows the change of staphylococcal count. It is a graph which shows the production inhibitory effect of the inflammatory cytokine and chemokine by lactoferrin. It is a graph which shows the activity suppression effect of the intracellular transcription factor NFκB by lactoferrin.

Claims (8)

  A therapeutic agent for mastitis in which lactoferrin and antibiotics are combined.   A method for treating mastitis comprising administering lactoferrin and antibiotics in combination to a bovine breast in the dry period.   A method for suppressing the production of inflammatory cytokines through the inhibitory effect of NFκB activity by the combination therapy of lactoferrin and antibiotics in the mammary gland of clinical mastitis during the dry period.   A method for inhibiting the production of inflammatory mediators and inflammatory lactoferrin molecules by a combination therapy of lactoferrin and antibiotics into the mammary gland of clinical mastitis during the dry period.   A method for enhancing the sterilization effect of antibiotics by injecting lactoferrin into the mammary gland of clinical mastitis during the dry period   Clinical symptom alleviation of lactation by combination therapy with lactoferrin and antibiotics in the mammary gland of clinical mastitis during the dry period.   A method to prevent postpartum mastitis by combining lactoferrin and antibiotics into the mammary gland of clinical mastitis during the dry period.   A method for inducing normal lactation after parturition by the combination therapy of lactoferrin and antibiotics into the mammary gland of dry-type clinical mastitis.

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