WO2001032205A1

WO2001032205A1 - Mucosal preventives for mastitis

Info

Publication number: WO2001032205A1
Application number: PCT/JP2000/007445
Authority: WO
Inventors: Takuya Koji; Takuji Seo; Toshikatsu Hayashi
Original assignee: Takeda Schering-Plough Animal Health K.K.
Priority date: 1999-10-29
Filing date: 2000-10-24
Publication date: 2001-05-10
Also published as: JP2003171291A; AU7956400A

Abstract

Preparations to be orally or nasally administered for preventing mastitis of mammalians which contain a microbial cell lysate containing inactivated microbial cells of microbial outer wall proteins or polysaccharides; and cattle feeds containing a microbial cell lysate containing inactivated microbial cells or microbial outer wall proteins or polysaccharides. Thus, mucosal vaccines for mastitis of mammalians, which can be orally or nasally administered, being much superior in efficacy and practical availibility to the existing ones can be provided.

Description

Membrane Mastitis mucosal prophylactic agent Technical field

The present invention relates to an orally or nasally-administered mucosal preventive agent for mastitis, which is useful for preventing mastitis diseases such as cattle caused by bacterial infection, particularly to mammals such as livestock, and livestock containing the same. For feed. Background art

Livestock such as cows, horses, sheep, goats, and pigs are fed by lactating pups after delivery. During the feeding period or milking by humans, mastitis develops due to bacterial infection of the breast. Mastitis has also been found to occur during the dry period before cows give birth. The incidence of mastitis in cattle is particularly high, with 50% of dairy cows reported in Japan and the United States of America (Asato, Veterinary Society, 136, 27-36 (1993), Cul lor, Clinical Veterinary Medicine, Vol. 12, No. 1, pp. 17-27 (1994)) Mastitis in cattle is a clinical mastitis with hot sensation, pain, redness, and swelling, and these symptoms. Although there is no indication, there is latent mastitis that causes abnormalities in milk, such as an increase in the number of somatic cells, etc. The damage caused by mastitis is a significant decrease in milk yield. It is estimated to be about 30 billion yen, and the price of milk will be reduced due to quality deterioration, and the economic loss due to cattle death and culling will be significant. The rate of abolition was second only to surgical diseases, with 129 deaths per 10,000 Accounted for 19% of the incidence (Hamaoka addition, clinical veterinarian, Vol. 17 (No. 5), 198- 217 pp., (1998)).

For prevention and treatment of bovine mastitis, penicillins (eg, cloxacillin), cephalosporins (eg, cefazolin), streptomycin, oki Antibiotic ointments such as citetracycline and erythromycin have been used. For prevention, an intramammary injection of an antibiotic ointment is performed on the day of drying two months before delivery. For treatment, intramammary injection of antibiotic ointment and intramuscular administration of antibiotic injection are performed for several days. During the period of administration of the antibiotic and the suspension of the drug, milk cannot be sold due to legal regulations, so the economic loss is significant. Significant expenditures are also made on veterinarian rewards and medications needed for treatment.

Well, if measures other than antibiotics are used for infections of pathogenic microorganisms such as bacteria, prophylactic treatment with vaccines is common. However, no mastitis vaccine has been sold in Japan yet. In the United States of America, only one vaccine for subcutaneous administration using Escherichia coli J-5 strain is marketed by AppJon (Morin et al., J. Am. Vet. Med. Assoc. 212, 1423-1431 (1998)), vaccines such as Suphylococcus aureus and Streptococcus uberis are injected subcutaneously or intramuscularly. It has only been studied experimentally as an agent (Giraudo et al., J. Dairy Sci. 80, 845-853 (1997), Finch et al., Vaccine 15, 1138-1143 (1997)). Disclosure of the invention

The present invention provides a more practical preventive agent for mucosal mastitis in mammals by oral or nasal administration, that is, a far more effective preventive agent than previously known. That is, according to one aspect of the present invention, there is provided a mucosal vaccine for mammalian mastitis which is more practical and useful by the oral or nasal administration method.

Under these circumstances, the present inventors have conducted intensive studies and found that S. filococcus aureus (hereinafter referred to as "S. aureus"), which is the causative agent of mammalian mastitis. Oral or nasal administration of bacterial cells obtained by inactivating cultured bacterial cells such as "Reus") or lysed bacterial cells with an adjuvant is caused by S. aureus, etc. The present inventors have found that the present invention has a preventive effect on mastitis, and have further studied based on these findings. As a result, the present invention has been completed.

That is, according to the present invention, a breast of a mammal containing inactivated bacterial cells or a bacterial cell elution-treated product containing a protein or a polysaccharide (such as a mucopolysaccharide) such as an inactivated bacterial cell (such as aoi membrane and cell wall) An oral or nasal formulation for preventing inflammation is provided.

In addition, according to the present invention, livestock containing an inactivated microbial cell or a bacterial cell elution-treated product containing a protein or a polysaccharide (mucopolysaccharide or the like) of an outer wall (aoi membrane, cell wall, etc.) of the microbial cell is used. Feed is provided.

Immunization by oral or nasal administration of the vaccine relies on the induction of secretory IgA (slgA) in the secretions. That is, the mucous membranes of the whole body, such as the mammary gland, gastrointestinal tract, and respiratory tract, are in direct contact with the outside world. The defense mechanisms here include (1) non-specific defense mechanisms by lysozyme and lactoferrin, and (2) A protective mechanism by an immune reaction specifically corresponding to a specific foreign antigen can be mentioned. The latter defense mechanism by the immune response is further divided into two groups: (2-1) local immune system (local immune system) and (2-2) systemic immune system (sys temic immune system). is there. The local immune system is an immune system that involves immunoglobulin, an IgA that is produced and secreted from lymphocytes widely distributed in mucous membranes. It is often referred to as the mucosal immune system because it plays a major role in the immune response in the mucosal system of the whole body. The systemic immunity mechanism is the immunity that is mainly carried out by IgG, which responds to foreign antigens that have invaded the living body by breaking specific and non-specific defense mechanisms on the mucosal surface, and lymphocytes associated therewith. System. In the case of mastitis, what is needed to prevent new infections is the nonspecific immune mechanisms described above and the slgA It is a membrane immune mechanism. When bacteria pass through the mucous membranes and invade the mammary gland tissue to establish infection, the IgG-mediated systemic immune system operates.

IgA is induced by antigen stimulation through the mucosal surface, and the process has been analyzed in the intestine of mammals. In other words, well-developed Peyer's patches (P.P.) are scattered in the mammalian intestinal tract, and this site is thought to be the IgA-inducing tissue. For example, orally administered antigens that reach the intestinal tract are taken up by M cells, special epithelial cells that cover P.P. M cells have no antigen-presenting ability, and macrophage II dendritic cells located in the dome region below treat antigens as antigen-presenting cells and activate T lymphocytes. IgA-producing progenitor B cells affected by antigen stimulation and activated T lymphocytes enter the circulatory system via mesenteric lymph nodes and thoracic ducts, and the mucous membrane includes not only the intestinal tract but also the respiratory system and mammary gland It is distributed in the eigenlayer. From the above, it is preferable to sensitize the mucosa directly to the mucous membrane via the oral or nasal route in order to activate the mucosal immune mechanism by IgA.

The bacterium that can be used for preparing the preparation for oral or nasal administration of the present invention is not particularly limited as long as it is a bacterium that is involved in mastitis. Sexually active Sirophycoccus (Coagulase negative Staphylococci), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus' ice galactia (Streptococcus dysgalact iae), Streptococcus-uberis (Streptococcus uberisept) bovis), Enterococcus faecal is, Corynebacterium bovis, Actinomyces pyogenes, Escherichia coli and Klebsiella lemonella pneumoniae) And the like. These bacterial species can be used alone or in combination. Bacteria for vaccine production can be obtained by culture. The culture may be performed using either a known liquid medium or solid medium, and the medium and culture conditions may be appropriately selected so that the cells can be obtained efficiently. For example, a broth culture medium, a normal agar medium and the like can be mentioned. Further, the culture medium may be improved by adding milk whey or the like to increase the yield of cells or to increase the disease-causing property. The cultivation may be carried out by culturing the target bacterium in a medium obtained by sterilizing the above-mentioned medium according to the usage thereof, and shaking or standing at 37T: for about 8 hours to 1 week, preferably for 20 to 48 hours.

The cells after culturing may be in a state of suspension in a culture solution.However, in order to perform the subsequent processing more efficiently, a known method such as centrifugal sedimentation, filtration or coagulation sedimentation from the culture after culturing is required. Preferably, the cells are recovered as a precipitate or a concentrate by the method described in (1).

The “inactivation” treatment in the “inactivated cell body” in the present invention refers to a treatment for losing the pathogenicity of bacteria, that is, infectivity. Specifically, it suffices to lose the proliferative ability, such as sterilization.

The inactivation of the cells can be achieved by a method of treating the cells with a weak acid such as formalin or acetic acid, a black form, a method of irradiating the cells with ultraviolet rays, a method of heating the cells, and the like. In particular, it is preferable to inactivate the cells by formalin treatment. In this case, it is appropriate that formalin is allowed to act on the cells at a concentration of usually 0.2 to 1.0%.

In the present invention, the term "a cell elution-treated product containing a cell outer wall protein or a polysaccharide" refers to a treated product obtained by subjecting a cell to "a treatment for eluting at least a cell outer wall protein or a polysaccharide". means.

The bacterial cell elution product in the present invention is prepared so as to contain at least the outer wall protein or polysaccharide of the bacterial cells, but may contain other bacterial cell components such as intracellular components in addition thereto. The cells may be contained as they are. In the preparation of such a bacterial cell elution product, "at least the cell outer wall protein or Is a treatment to elute polysaccharides ”(hereinafter sometimes simply referred to as“ elution treatment ”) is a treatment to release or elute at least all or part of the bacterial cell outer wall protein or polysaccharide from the cells. Say. Typical examples of this treatment include a heat treatment or a lysis treatment of the cells. It can also be achieved by the above-mentioned inactivation treatment.

The heat treatment is carried out by treating the cultured cells or a suspension thereof at 60 to 100, preferably 70 to 80, for 10 to 60 minutes, preferably for 20 to 30 minutes. The processing conditions are appropriately selected according to the amount of the cells and the material and shape of the processing container in which the cells are placed and heated.

The lysis treatment is a treatment for dissolving or releasing at least a part of a protein or a polysaccharide (for example, mucopolysaccharide) of a cell wall, an outer membrane of a cell, and, for example, a lytic enzyme treatment or a physical crushing method. Physical treatments such as (eg, ultrasonic crushing method) or a method of repeating freezing and thawing (freezing and thawing method) or a combination of these physical treatments. In addition, autolysis of bacteria, which can occur depending on the culture conditions, is also included in the lysis treatment.

The lytic enzyme may be any hydrolyzing enzyme that does not impair the antigenicity of the cell walls, outer membrane proteins or polysaccharides, and may be any of lysozyme, cellulase, achromopeptidase, and serratiopeptidase. (Trade name: DAISEN, manufactured by Takeda Pharmaceutical Co., Ltd.), and particularly, lysozyme is preferably used. Lysozyme should be made of readily available, commercially available egg white. The lytic enzyme treatment may be performed according to a known method depending on the hydrolase to be used.If the cells are difficult to lyse, an anionic surfactant is particularly added after or simultaneously with the lytic enzyme treatment. Good.

As the anionic surfactant, the hydrophobic group is preferably an aliphatic hydrocarbon group or an aromatic hydrocarbon group, the hydrophilic group is a carboxylate (general formula: R'COONa), and the hydrophilic group is a sulfate. (general formula: R ² 0S0 ₃ Na) or hydrophilic group scan A surfactant which is a sulfonate (general formula: R ² S 0 ₃ Na or R ² C ₆ H ₄ S 0 ₃ Na) [In the above general formula, R ¹ is C¾ (CH ₂ ) _m On is 2 or more R ² represents a group represented by C (CH ₂ ) „(n represents an integer of 4 or more and 15 or less, preferably 7 or more and 11 or less) Among the above-mentioned anionic surfactants, those having excellent water-solubility and strong bacteriolysis in a temperature range in which the lytic enzyme can act, particularly in the vicinity of room temperature, are preferable, and they are easily available. Specifically, for example, the following compounds may be mentioned: As a compound represented by R'COONa, R 'is decanoic acid of CH ₃ (CH ₂ ) ₈ or R ¹ is CH ₃ (CH ₂ ) such as lauric acid _1Q are preferably used. Examples of the compounds represented by R ² OS0 ₃ Na, sodium lauryl sulfate R ^z is CH ₃ (CH ₂₎ " And the like are preferably used. As the compound represented by R ² S0 ₃ Na, R ² is preferably sodium decanesulfonate of CH ₃ (CH ₂ ) ₉ , and as the compound represented by R ² C ₆ H ₄ S0 ₃ Na, Sodium laurylbenzenesulfonate wherein R ² is CH ₃ (CH ₂ ) „is preferably used.

Ultrasonic treatment in physical crushing method The crushing method may be performed by a known method using a commercially available ultrasonic generator.For example, a cell suspension is oscillated at 20 KHz and output at 200 W for 5 to 30 minutes. It is good to process to the extent.

The freeze-thawing method may be performed by a known method.For example, a method of freezing the cells at about 20 to 30 and then thawing at 30 to 40 to repeat the operation of thawing the cells is to destroy a part of the cells. No. Dehydration drying using an organic solvent such as acetone, heating vacuum drying, freeze drying, etc. may be performed, and a mechanical treatment used for crushing solids or powders may be added.

In the present invention, an inactivated cell, a cell elution treatment containing the outer wall protein or polysaccharide of the cell, and a cell elution treatment containing the inactivated cell outer wall protein or the polysaccharide are included. And any one of them may be contained, and two or three of these may be contained. In such a case, by inactivating the cells as described above, some of the cells lose their growth ability or are sterilized. As a result, a part of the cells can be used as the product of the present invention as a cell-eluted product in which the cell outer wall protein or polysaccharide is eluted.

The solution containing the inactivated cells, the cell-eluted product containing the outer wall protein of the cells, or the polysaccharide prepared as described above can be directly orally or nasally administered to mammals, but is frozen or refrigerated. It is also possible to preserve it. Thaw it when necessary, and, if necessary, dilute it appropriately with water or the like, and then orally or nasally administer it to mammals such as livestock. Alternatively, the powder may be dried and powdered by freeze-drying, dehydration-drying with acetone, or the like, and further appropriately diluted and prepared with starch or saccharide before administration.

When preserving the processed cells, methods such as centrifugal sedimentation and filtration, and methods of precipitating protein substances with organic solvents such as acetone and ethanol, are used to prevent contamination of various bacteria and the stability of the active ingredient. Alternatively, it is preferable to concentrate by a salting-out method using a saturated aqueous solution of sodium sulfate, ammonium sulfate, or the like, collect as a precipitate, and then dry dry and store. When dry powdering is carried out, it is adsorbed on an appropriate medium, for example, starches such as potato starch and corn starch, soybean flour, rice bran cereal (cake) powder, dried by vacuum, ventilation, freeze-drying, etc. to be powdered. You may.

In order to effectively incorporate the inactivated cells or the cell-eluted product containing the outer wall protein or polysaccharide of the cells, an adjuvant is usually added. Adjuvants include cholera toxin (cholera toxin, CT), Escherichia coli heat-labile exotoxin (heat-lab lile enterotoxin, LT) or non-toxic CT and LT derived from mutants excluding these virulences, or Examples include phosphoryl lipid A (monophosphoryl lipid A) and sahonin (Qui 1 laj a saponin).

The inactivated cell or the cell elution treated product containing the outer wall protein or polysaccharide of the cell described above has low toxicity and excellent safety in mammals. Rat, mouse, guinea pig, hidge, goat, bush, ゥ It can be used as a preventive agent for mastitis in mammals such as horses, horses, cats, dogs, monkeys, and humans (preferably, livestock such as horses, horses, sheep, goats, and bush).

For example, in the case of oral administration to mammals such as livestock, usually, a lysate or powder containing inactivated cells or a cell elution-treated product containing the outer wall protein or polysaccharide of the cells is used. It may be added to the feed and given. The feed may be a compound feed, a compound feed for private use, or a cereal (corn, grain sorghum, barley, barley, oats, rye, brown rice, flour, kinako, etc.) which are generally commercially available as feeds for the above mammals. (Rice bran, rice bran oil cake, wheat bran, bran, etc.), vegetable oil cake (soybean cake, amaji oil cake, cottonseed oil cake, peanut oil cake, safflower oil cake, palm oil cake, palm oil cake, sesame oil cake, sunflower oil cake, tanane oil cake , Kapok oil cake, etc.), animal feed ingredients (fish meal, fish soluble, meat meal, meat-and-bone meal, blood meal, power meal, shrimp meal, silkworm, skim milk powder, dried whey, etc.), and other ingredients (chlorella, wakame, And the like, such as konbu, etc. alone or as a mixture.

In the case of intranasal administration, a lysate containing inactivated cells or a cell lysate containing a cell surface protein or a polysaccharide containing a cell lysate containing a polysaccharide is manually or electrically sprayed (nebulizer 1) or the like. It can be sprayed into the nasal cavity.

In addition, during oral administration, by minimizing degradation by enzymes and microorganisms in the digestive tract of mammals, the availability of absorption from the intestinal tract is increased, and in order to exert a strong infection-protecting ability with a smaller amount, Preparation of dried bacterial cell components coated with a coating agent such as a fatty acid such as decanoic acid or a physiologically acceptable fat-soluble substance such as hardened oil or fat, or an enteric polymer compound used in the production of enteric-coated preparations, A lysate using an oil or fat and a surfactant may be used as a water-in-oil emulsion that is not dispersed in water, or may be prepared by applying a known formulation technique such as a liposome formulation. Of course, those preparations are only for oral administration Alternatively, it may be used for nasal administration.

Examples of the fat-soluble substance used as the coating agent include fatty acids such as decanoic acid (capric acid), stearic acid, lauric acid, and palmitic acid, and fats and oils such as hardened tallow or hardened soybean oil. The coating with a fatty acid fat is performed by a known coating preparation technique, for example, by adding a solution of a fat-soluble substance to a bacterial cell powder, mixing the mixture, and then cooling and solidifying the mixture to pulverize and granulate. In addition, coating with an enteric polymer compound involves dispersing the bacterial cell powder in a solution of a cellulose derivative such as hydroxypropylmethylcellulose phthalate and carboxymethylethylcellulose in a solvent such as alcohol, and then removing the solvent. A known method such as pulverization may be used.

Fats and oils used in the water-in-oil type emulsion include soybean oil, palm oil, corn oil and the like, and surfactants include glycerin fatty acid ester, sorbin fatty acid ester, and propylene dalycol fatty acid ester. No. The water-in-oil emulsification is performed by a known method such as emulsifying a water-soluble bacterial cell solution and a fat or oil to which a surfactant has been added by stirring or ultrasonic treatment.

Phospholipids used in ribosome preparations include saturated or unsaturated phosphodiesters such as egg yolk lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and dioleoylphosphatidylcholine. And a phospholipid ゃ lysolecithin whose hydrophilic group is choline, ethanolamine, serine, inositol, glycerol or the like. Of course, a plurality of the above phospholipids may be used in combination. Formulation is performed by a method known per se such as a reverse phase evaporation method and a sonication method.

The dose, duration, and interval of oral or nasal administration of the preparation (formulation or feed) described above to mammals (eg, cattle, horses, sheep, goats, pigs, etc.) are described below. Adjusted appropriately depending on the type of mammal to be administered can do. For example, the dosage may be 0.01 to 1000 mg (wet weight) of bacterial cells per 1 kg of the mammal's body weight, administered orally or nasally 1 to 10 times at intervals of 1 to 21 days.

According to another aspect of the present invention, there is provided a method for preventing mastitis by orally or nasally administering the agent for preventing mucositis for mastitis of the present invention to a mammal. Further, a method for preventing mastitis by orally administering to a mammal by mixing the feed with the mucosal prophylactic agent for mastitis of the present invention and feeding the feed to the mammal is provided. Embodiment of the Invention

Hereinafter, the present invention will be described specifically with reference to Test Examples, but it goes without saying that this does not limit the scope of the present invention.

Test example 1

A 20-hour culture of S. aureus was inactivated with formalin and killed cells or lysozyme-treated cells were orally administered with cholera toxin as an adjuvant. Examination of the ability to protect against infection showed that it had an effect of protecting against infection.

That is, No. 46 strain of S. aureus (S taphys) suspended in 20% glycerol-added Trypticase Soy Broth (manufactured by BBL, hereinafter abbreviated as TSB) at 180. After thawing L. ococcus aureus No. 46), 1 Ese was spread on a Mannitol salt medium (manufactured by Nissui), and one colony after culturing at 37 for 24-36 hours was inoculated into TSB. After culturing for 20 hours at 37, centrifuge at 10, OOOr. Pm, 4 for 10 minutes to collect the cells, and phosphate buffer containing 0.5% formalin (ν / ν). (Dulbecco Ca, Mg-free, manufactured by Dainippon Pharmaceutical Co., Ltd., PBS) was added, and the killed cells obtained after the inactivation treatment at 25 to 24 hours were used as immunizing antigens. This antigen was stored at 4 until tested for immunization.

For immunization, the above immunizing antigen was centrifuged at 10,000 rpm at 4 for 10 minutes. After separation, the 1, 10 or 50 mg (wet weight) was suspended in 0.5 ral PBS, and 10 / zg cholera toxin (manufactured by Wako Pure Chemical Industries, Ltd.) was added as an adjuvant. BALB / c female mice, aged 8 to 10 weeks, weighing about 20 g, were orally administered twice at 14-day intervals. Study of infectious defenses against intramammary attack bacterial solution 0. lml containing 10 ⁴ CFU of No. 46 strain of S. § © Reus cultured 1. 5% skim milk broth 21 days after the second immunization Was inoculated into the left and right fourth mammary glands of mice 10 to 15 days after delivery. At this time, mice that were not immunized as a control group were also challenged under the same conditions. Necropsy was performed 48 hours after the challenge, and S. aureus was isolated from the mammary gland. Individuals who died by 48 hours after the challenge were necropsied as soon as possible and bacteria were collected from the mammary gland. Table 1 shows the mortality and the number of bacteria recovered from the mammary gland 48 hours after the challenge.

Average soil standard deviation (number of cases)

^2> Significantly different from non-immune group p <0.05, Dunne tt's test) Test Example 2

A PBS suspension of the immunizing antigen treated in the same manner as in Test Example 1 was prepared, and egg white lysozyme (Wako Pure Chemical Industries, Ltd.) at 1/10 (w / w) of the antigen (wet weight) was added. Thereafter, the cells were left standing at 37 for 30 minutes to perform lysis treatment. Thereafter, cholera toxin (manufactured by Wako Pure Chemical Industries, Ltd.) was added as an adjuvant, and immunization and challenge were performed in the same manner as in Test Example 1. Table 2 shows the mortality and the number of bacteria recovered from the mammary gland 48 hours after the challenge. Table 2

u Average soil standard deviation (number of cases)

² ) There is a significant difference from the non-immunized group (* 'p <0. OK Dunne tt's test) The number of deaths in the non-immunized group was 1/5 and 3/9 in Test Examples 1 and 2, respectively. Met. No mortality was observed in the immunized group except for 1/7 of the lmg immunized group in Test Example 2. The average bacterial count of S. aureus recovered from the mammary gland tended to decrease with immunization. In particular, the mean number of bacteria recovered from the mammary gland in the 50 mg immunized group was significantly higher at 4.1 and 4.5 l ogCFU / g in Test Examples 1 and 2, respectively than in the non-immunized group (Test Example 1: p <0.05, Test example 2: p <0.01) decreased.

As a result, by immunizing mice by orally administering the formalin-inactivated antigen of No. 46 strain of S. aureus or its lysate-treated lysozyme together with cholera toxin to mice, it was possible to prevent infection of the strain against intramammary gland attack. It became clear that an effect was obtained.

S. aureus No. 46, which was used in Test Examples 1 and 2 above, was isolated from the milk of a pest with clinical mastitis and showed a high level of mastitis. 11 Deposited with the Yeast Research Institute (IF0) as IF016333 from October 28, 2011. Test Example 3: Infection protection and antibody response to intramammary gland attack by S. aureus in mice intranasally administered with S. aureus inactivated cells

Female BALB / c mice aged 8 to 10 weeks were tested and immunized intranasally or subcutaneously with inactivated S. aureus cells twice at 14 day intervals. Test mouse is the first exemption Immediately after the epidemic, they started living with the male, and then lived together for 7 days. The No.46 strain of S. Aureusu cultured skim milk broth were inoculated into 10 ⁵ to 10 ⁶ CFU includes fourth mammary gland of the left and right bacterial dilutions 0. lml to 21 days after the second immunization. 48 hours after the intramammary gland challenge, S. aureus was collected from the mammary gland, and at the same time, serum was separated from the collected blood and the anti-S. Aureus-specific IgA and IgG antibody titers were measured by ELISA. The inactivated S. aureus cells used for intranasal administration were subjected to ultrasonic treatment at an oscillation frequency of 20 kHz, an output of 200 W, and for 30 minutes.

Tables 3 and 4 show the results.

Table 3: Protective effect of infection against intramammary gland attack by S. aureus

n 0: No symptoms, 1: Mild weakness, 2: Moderate to severe weakness, 3: Death

²⁾ Average of clinical score (total score of individual animals Z number of test animals)

³⁾ Me an earth SD

⁴ ) Significantly different from non-immunized group (* p <0.05, "p <0.01, Student's t-test)

Table 4: Mouse antibody response

"ELISA antibody titer (Mean soil S D, n = 9 to 12)

²⁾ There is a significant difference between alphabetic characters (p <0.05, one-way analysis of variance and

LSD test) According to Tables 3 and 4, the clinical score of the intranasally immunized group (hereinafter referred to as “intranasally immunized group”) of the inactivated S. aureus inactivated bacterial cells treated with sonication was as follows:

(Hereinafter referred to as “subcutaneous immunized group”), which was lower than that of the non-immunized group. In addition, the number of bacteria recovered from the mammary gland of the intranasal immunization group was almost the same as that of the subcutaneous immunization group, and was significantly (P 0.01) lower than that of the non-immunization group. Serum IgG antibody in the intranasal immunization group was significantly higher than that in the subcutaneous immunization group.

(p <0.05) Although the value was lower, a significant (P <0.05) increase in anti-S. aureus serum IgG antibody was observed in the non-immunized group. Test example 4: Antibody response of immunized mice immunized intranasally with antigens (ovalbumin, ovalbumin) and adjuvant encapsulated in liposomal preparations

The antigens (ovalbumin, OVA) and adjuvants (cholera toxin, CT, and saponin) shown in Table 5 were encapsulated in ribosome preparations in Holstein breeding female females (weight around 150-200 kg). After intranasal administration and immunization, the antibody response of the mouse on day 21 was examined.

Table 5 shows the formulation of the test preparation, and Tables 6 to 9 show the results.

Unformulated (immunized)

Table 6: Antibody response (anti-OVAIgG antibody) and effect of ribosome treatment

D administration: Twice every 14 days

²⁾ Unformulated (immunized)

³⁾ Ribosome preparation

⁴ ) ELIS Α antibody titer (absorbance: 492nm, Mean ± SD, n = 3)

⁵⁾ There is a significant difference compared to the value on day 0 (before the first immunization) ('ρ <0.05) Intranasal immunization of V245120 and V245121 with cholera toxin as adjuvant did not show an increase in anti-OVA and anti-CT serum IgG and IgA antibody titers. Intranasal immunization of V245122 and V245123 with saponin as adjuvant showed a significant (p <0.05) increase in anti-OVA serum IgA antibody. Industrial applicability

The preparation of the present invention is extremely practical since it has an excellent protective effect on mammalian mastitis infection and can be used by oral or nasal administration.

Claims

Scope of Claim 1. For oral or nasal administration to prevent mastitis in mammals containing inactivated cells or cell lysates containing polysaccharides or outer wall proteins of the cells. Formulation.

2. The inactivated microbial cells or the cell lysate containing the outer wall protein or polysaccharide of the microbial cells are obtained by formalin treatment of the microbial cells or the microbial cell lysate containing the outer wall proteins or polysaccharides of the microbial cells. The preparation according to claim 1, which is prepared.

3. The preparation according to claim 1, wherein the cell-eluted product containing the inactivated cell outer wall protein or polysaccharide is obtained by heat-treating the cells.

4. The method according to claim 1, wherein the cell-eluted product containing the outer wall protein or the polysaccharide of the inactivated cells is obtained by lysing the inactivated cells.

5. The preparation according to any one of claims 1 to 4, further comprising an adjuvant.

6. Adjuvants are non-toxic CT and LT derived from cholera toxin (CT), heat-lablile enterotoxin (LT), or mutants that have been deprived of these toxins; Oryl lipid A

6. The preparation according to claim 5, wherein the preparation is at least one member selected from the group consisting of (monophosphoryl lipid A) and saponin (Quillaja saponin;).

7. The preparation according to any one of claims 1 to 6, wherein the cells are cells of bacteria involved in mastitis.

8. The cells are Staphylococcus aureus, Coagulase negative Staphylococci, Streptococcus agalactiae, Streptococcus dyspactae, Streptococcus dystrophy Coccus Peveris (Streptococcus uberis), Streptococcus bovis, Enterococcus faecalis, Corynebacteriuin bovis, Actinomyces pyogenes; Escherichia cole 8. The preparation according to claim 7, wherein the preparation is at least one member selected from the group consisting of Klebsiella pneumoniae).

9. The preparation according to any one of claims 1 to 5, which is a (1) fat-soluble preparation, (2) a water-in-oil emulsion or (3) a ribosome preparation.

10. Livestock feed containing the inactivated cells or a cell-eluted material containing the outer wall protein or polysaccharide of the cells.

1 1. A method for preventing mastitis by orally or nasally administering the preparation of any one of claims 1 to 9 to a mammal.

12. A method for preventing mastitis by providing the animal feed of claim 10 to a mammal.