JPH07501334A - Pasteurella maltocida toxoid vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Pasteurella maltocida toxoid vaccine Field of the Invention Generally, the present invention is in the field of livestock vaccines, vaccine compositions and methods of making the same. More particularly, the present invention provides a method for combating diseases associated with infection by toxin-producing strains of Pasteurella multocida. The present invention relates to vaccine compositions and protective methods for protecting against diseases. BACKGROUND OF THE INVENTION Pasteurella multocida is associated with disease in a variety of animals, including humans, cattle, sheep, and pigs. Typically affects the animal's nasopharynx and lungs. Boss. For example, toxigenic strains of Bordetella bronchiseptica and B. multocida (capsular type A) or D) causes atrophic rhinitis in pigs. Atrophic rhinitis (AR) causes atrophy of the nasal turbinates and deformity of the nose and face in pigs. The pathogenicity of B. multocida is due in large part to the production of strong pronecrosis (also called predermal necrosis (DNT)). Hereafter, this will be referred to as a "toxin." The toxin is a molecule It is characterized as a heat-labile protein with an amount of about 140,000 to 160,000. B. multocida stands out from other species of Pasteurella due to its growth characteristics: can be distinguished from Hemolytic/negative (90%); growth on MacConkey agar, negative, indole production: positive, urease production: negative: mannitol metabolism: positive. Zinsset, Microbiology (edited by Joklik et al.) (=!-See Appleton-Century-Crofts, York, 1980). This is incorporated herein by reference. Currently available solutions to protect animals from B. multocida infection-related diseases. Cutin is derived from inactivated toxin-producing B. maltocida cells, partially purified B. maltocida cells, and partially purified B. maltocida cells. cida toxin inactivated products, and mixtures of P. multocida cell-free preparations with other inactivated P. multocida strains or B. bronchiseptica strains [e.g., M. Kobisch] et al., Vet.Record, Vol. 124, pp. 57-61 (1989); and N.T. See N. T. Foged et al., Veterinary Records, Vol. 125, pp. 7-11 (1989)]. However, these vaccine preparations are not fully protective against disease because they fail to induce sufficient amounts of antibodies (known as ``antitoxins'') to neutralize the toxin. In the field of veterinary medicine, effective methods against infection of animals by B. multocida have been identified. Kuching is necessary. SUMMARY OF THE INVENTION The present invention provides novel vaccine compositions and components that protect animals from diseases associated with infection by toxigenic B. multocida. These vaccine compositions are characterized by their ability to induce significant amounts of circulating antitoxin. In a first aspect, the present invention provides novel vaccine compositions containing whole Pasteurella multocida killed bacteria (bacterins) with cell-binding toxoids. Compared to free soluble toxoid, this composition induces superior antitoxin responses in previously unvaccinated animals. This composition is associated with a carrier suitable for internal use, preferably an aluminum hydroxide gel. In another embodiment, the present invention provides: (1) a whole package with a cell-binding toxoid; Sturella multocida bacterin (which induces an antitoxin response when used internally in animals) and (2) a free soluble toxoid of P. maltocida. This vaccine composition produces an unexpected synergistic antitoxin response that is much greater than the sum of the effects of the two components. Preferably, a carrier is associated with the composition. A soluble cell-free toxoid of B. multocida is prepared by a method that exposes the toxin to various pHs and temperatures. This method is also a novel aspect of the invention. "Tokisoi The term "toxoid" refers to a toxin preparation that has been inactivated ("toxoidized") by a method that removes its toxicity without losing its ability to induce the production of a specific neutralizing antitoxin. In a further embodiment, in combination with an immunogenic amount of one or more additional antigens. The above vaccine composition may be varied by changing the vaccine composition. Preferred antigens are Bronchisebtica bacterin, Erysipelothrix rhusiopathiae bacterin, Mycoplasma hyopneum6niae extract vaccine. However, other conventional vaccines Other ingredients may be added to the vaccine composition of the present invention. The vaccine composition of the present invention is administered internally as a dosage unit. In the specific example of 1, the vaccine The dose of Pasteurella multocytoid with an immunogenic dose of cell-bound toxoid is It consists of 0.5 to 3 ml of a sterile suspension of Dabacterin (which induces an antitoxin response when used internally in animals). In another embodiment, the dosage unit is - 0.5 to 3 ml of a sterile mixture of cell-bound and free toxoids of M. multocida. In yet another embodiment, the dosage unit is It consists of 0.5 to 3 ml of a sterile mixture of the toxoid and one or more additional antigenic components. In yet another aspect, the invention provides serum-free and substantially bacterial cell-free Provided is a method for producing Lysiperosrix vaccine components. A further aspect of the invention is a method of administering a vaccine to an animal against atrophic rhinitis. The method comprises administering to the animal an effective amount of one or more B. maltocida toxoid vaccine components. Other aspects and advantages of the invention are further described in the detailed description of the preferred embodiments below. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to toxigenic P. multocida and The present invention provides vaccine compositions useful for preventing diseases caused by infections caused by other pathogenic organisms, including B. siae, B. bronchisebtica, and M. hyopneumoniae. Such diseases include atrophic rhinitis (AR), pleurisy and pastures of the lungs. This includes erythrosis, erysipelas, and pneumonia. One embodiment of the invention is a method containing a stable toxoid entrapped within bacterial cells. The whole batataline toxoid of B. maltocida is provided. This cell-binding toxoid can be prepared by the known method described in Example 1 or preferably by the following vaccine group. Prepare by any of the methods for preparing a mu-negative bacterial solution. The inactivating agent is added to P. multocida cultures that are still in logarithmic growth phase and have not yet released toxins into the growth medium. This causes the toxoid to become entrapped within the bacterial cell. Tokisoi The dead bacteria (i.e. bacterins) that have isolated the bacteria within them allow the immune process to take place. It is an ideal antigen particle to present to host cells. This is especially true for animals that have not been previously exposed to toxins or toxoids and generally lack antivenom. It is important. The B. maltocype cell-binding toxoid of the present invention is extremely stable. No decrease in antigenicity was observed even after storage at 4°C for more than 2 years. For the purposes of the present invention, the above cell-binding toxoids are combined with toxigenic pea malt. It may be derived from any strain of cider. Several such strains are available, for example, from the American Type Culture Collection, Rockville, Maryland, or from various veterinary universities or laboratories. Examples of the latter are P. multocida type D.8 and 4677 strains. Currently, P. maltocida type D 4677 strain or cell-associated toxovirus preferred for the preparation of This strain is particularly advantageous because when using equal numbers of individual cells, strain 4677 produces twice as much toxin as strain 8 (previously known as the best toxin producing strain). This ability of the 4677 strain to produce twice the amount of toxin is because it requires half as many cultures of the 4677 strain, or half as many cells, as it would if eight strains produced the same amount of toxin. This ability of strain 4677 to produce is advantageous. Furthermore, the volume of the combined vaccine is limited to, for example, a 2 ml dose. The fact that the 4677 strain fluid accounts for only half of the strain fluid is a significant advantage over prior art vaccines. Therefore, B. Maltosa Ida 4677 strain provides prescription benefits not achievable with previously used strains. I can do it. Suitable media for culturing P. multocida can be selected by those skilled in the art. However, Herriott et al. J. Bact, Volume 101: 513-516, [Defined Medicine The medium described in Defined Medium for Growth of Hemaphilus Influenzae J (1970) is preferred, but not limited thereto. To induce an antitoxin response that prevents the pathological changes characteristic of atrophic rhinitis. The novel whole cell-binding toxoids described above can be used separately in vaccine compositions to You can stay there. Preferably, the vaccine composition is suitable for injection into mammals, especially pigs. In the formulation, an immunogenic amount of cell-binding toxoid is mixed with a suitable conventional vaccine adjuvant and pharmaceutical carrier. B. multocida free toxoids (below) and cell-bound toxoids (above) A more preferred vaccine composition can be obtained by synergistic combination of the following. Immune amount play Prepare isolated toxoids and such combination vaccines. Immune dose of cell-bound toxo by mixing the compound with a suitable adjuvant and a pharmaceutical carrier for mammalian injection. Then, such a combination vaccine is prepared. A preferred adjuvant is aluminum hydroxide Includes Umgel. As mentioned above, free soluble bee maltoside toxoid is It is useful in synergistic combinations with cell-binding toxoids. The free soluble toxoid is the subject of co-pending US Patent Application No. 071,537.457. This is incorporated herein by reference. Toxin-producing pi - maltocida strains are also used for the preparation of soluble toxoids, but are not used in the examples below. The present strain is P. maltocida type D.8. This stock is available from the University of Illinois upon request. Generally, soluble toxoids are added to bacterial cells. The toxin is extracted from the cells, and the cell-free toxin is purified at pH 9 or higher and at about 12°C to 19°C. prepared by partial denaturation by incubation for 24 hours. make More specifically, free toxoids are prepared as follows. selected toxins A viable P. multocida strain is grown in a suitable medium. At the end of the breeding cycle At this time, toxins are released from the cells by conventional physical and chemical means such as, for example, French press or ultrasonic disruption, and cell debris is removed by centrifugation or filtration. For large-scale production, it is recommended to apply the following pH cycling method for incubation. and then the pH is less than about 10.5 for a period of at least 21 hours. Incubate the toxin in the cell-free extract by cycling three times between Turn on. Laboratory production is carried out using similar methods described in the patent applications cited above. This method can completely detoxify toxins and yield toxoids that are soluble in aqueous solutions (eg, phosphate-buffered saline, Tris-buffered saline). Soluble B. maltocida toxoid preparations have both antigenic and immunogenic properties. In particular, the soluble toxoid induces antibodies that can bind to the toxin and neutralize its toxicity. Furthermore, the soluble toxoid of the present invention has the characteristic of being stable for 24 months at 4°C, making it possible to store this vaccine for later use. I can do it. In the vaccine administration experiment using animals described in Example 11 below, it was found that free and cell-bound toxoids act synergistically in a single vaccine preparation. It was issued. That is, the vaccine has a positive effect on each vaccinated animal. This produces a much greater effect than when the two components are administered separately. This combination vaccine significantly stimulates antitoxin production in test animals. This mixture The efficacy of this treatment was determined by sequential administration of cell-bound toxoid vaccine followed by soluble vaccine. It can also be caused by injection. Without wishing to be limited by theory, it is currently believed that cell-conjugated vaccines prime animals, particularly immunologically native animals that are unable to respond to soluble toxoids. A second administration of cell-bound toxoid induces a mild secondary immune response. Once primed with toxoid-rich cells, the animal becomes highly responsive to free toxoid. It is as if cell-bound toxoids are good priming agents and soluble toxoids are good boosters. Still other preferred vaccine compositions of the present invention include cell-binding toxoids of the present invention (which may be mixed with free toxoids or mixed with other vaccine agents). It is obtained by A typical example is obtained by mixing whole B. bronchisebtica bacterin with B. multocida cell-binding toxoid. Alternatively, the composition may further contain free toxoid of B. maltocida. This free toxoid was absorbed into the capsular type B. maltocida (fine (different from the type used for the preparation of cell-binding toxoids). difference Furthermore, the vaccine composition of the present invention may contain an E. rhusiopathiae component. stomach. Thus, in another aspect, the invention provides Provides a method for preparing the ingredients. Other effective vaccine agents that can be mixed with the vaccine composition of the present invention are Escherichia coli, B. maltocida, Stre. Types C and D Xoid, Pseudorabjes virus vaccine (modified live cow) virus and/or dead virus), Rotavirus vaccine (modified live virus), Coronavirus vaccine (modified live virus), but are not limited to these. In preferred example 1 Mycoplasma hypopneumoniae is also present in at least B. maltosa. The vaccine composition of the present invention is mixed with a cell-binding toxoid and included in the vaccine composition of the present invention. The M. hypopneumoniae strain useful for the preparation of the vaccine of the present invention was tested in pigs. It can be isolated from pigs infected with wild type or other known strains that cause Icoplasma pneumonia. Other known strains of M. hibopneumoniae (virulent and (both non-virulent and non-virulent) are also useful in the compositions of the present invention. City Useful Strains May be obtained through marketing channels or universities. A particularly preferred strain of M. hypopneumoniae for embodiments of the present invention is strain P-5722-3 (strain ATCC #55052, deposited on May 30, 1990 and made available upon request from the United States Patent and Trademark Office). be. The method for preparing the M. hypopneumoniae vaccine component is described in Example 10 below. I will post it. More detailed information regarding these vaccines can be found in co-pending US patent application Ser. No. 07/634.237. It is incorporated herein by reference. The vaccines of the present invention may be prepared as pharmaceutical compositions containing an effective immunogenic amount of a cell-binding toxoid as an active ingredient in a non-toxic, sterile, pharmaceutically acceptable carrier. A preferred embodiment of the vaccine of the invention comprises an aqueous suspension or solution containing a cell-binding toxoid, preferably buffered to a pH of about 6.5. and in a form that can be easily injected. In addition, cell-bound toxoids (which may be administered alone or in combination with free toxoids) The inventive product can be prepared by mixing or adsorbing it with a conventional adjuvant. It may also be added to cutin compositions. Adjuvants enhance specific antitoxin responses Used as a non-specific reagent for Such adjuvants include amphigen (manufactured by Hydronics, Inc.) or other desirable oils, aluminum hydroxide, muramyl dipeptide, and saponins such as QuilA. In yet another embodiment, the cell-bound toxoid (which may be administered alone or with free toxoid) is administered to B. bronchisebuteica bacterin, E. luciopanae bacterin, or M. hypopneumoniae. It can be administered in conjunction with another antigenic preparation such as A. nigra (prepared by known methods, preferably by the methods described herein). One of the preferred methods for preparing the bacterial components of the present invention is a novel method for preparing Gram-negative bacteria for vaccine formulation. This method can be applied and used to prepare any type of antigen concentrate, including, but not limited to, whole cell suspensions and cell-free extracts of Gram-negative bacteria. This new method allows joint applications to be accepted simultaneously. This is the subject of a patent application filed in the United States and is described below. According to the invention, selected Gram-negative bacteria (e.g., the Gram-negative bacteria described herein such as Pasteurella) are cultured under conventional conditions (in any conventional suitable medium or medium). may be grown in other media). Appropriate media are within the ordinary knowledge of those skilled in the art. Vine selected by knowledge. Preferably, the bacterial culture temperature is in the range of 35 to 38°C. When whole bacteria are used in the resulting vaccine component, growth may be in a transition phase or logarithmic increase. During the propagation phase, an inactivating agent is added. Preferably, the inactivating agent is an immobilizing agent capable of binding cellular structures and preventing degradation of the cell wall. The first fixative is formalin (formaldehyde solution, US Pharmacopoeia) and is generally used at a concentration of about 0.3% (V/V) to about 1.0% (V/V). Another fixative that is useful when using synthetic media is glutaraldehyde. For example, a 25% glutaraldehyde solution can be used at about 0.5 to 1% (V/V). Ru. Other suitable inactivating agents, such as beta-propiolactone, may also prove useful, and appropriate concentrations are readily determined by those skilled in the art. When adding a deactivator, turn off the ventilation and automatic pH control. If desired, the pH may be adjusted to the optimum value for inactivation. Reduce agitation to minimum and then increase speed to allow effective mixing. Inactivation conditions will depend on the particular bacterial species and are readily determined by those skilled in the art, but typically inactivation is carried out at between about 28 and 38°C. Alternatively, when preparing cell extracts for the resulting vaccine composition, inactivated Does not require sexualization. In such cases, the culture is cooled (usually below 20°C) to prevent growth. The extraction can be performed before or after the concentration step described below.The bacteria can be concentrated by centrifuging or filtering the G1° cells to obtain a concentrated aqueous suspension. or extract. Preferably, centrifugation is carried out at a force of about 10,000 g. When adjusting the flow rate at which the culture medium passes through the centrifuge to obtain approximately 100% cell recovery, use an appropriate strong or weak force, depending on the type of bacteria. Exists. Appropriate flow rates can also be determined by those skilled in the art by measuring the absorbance of the supernatant (discarded). Instead of centrifugation, other concentration methods such as ultrafiltration may be used. The methods as well as the conditions and equipment necessary to permit recovery of almost all cells are within the skill in the art. Depending on the concentration method selected, the cells in one batch after another are collected in as small a volume as possible. Preferably, the cell concentration is about 109 to about 10'1 cells/ml culture solution. This same concentration of cell concentrate may be used prior to extraction to prepare a concentrated cell extract. The resulting cell concentrate is diluted with a small amount of water, saline, or buffer to the selected standard concentration (depending on the concentration method). Standard concentrations will depend on the particular bacterium and will be selected by one of ordinary skill in the art given the above teachings. The concentrated aqueous suspension or extract is then adsorbed onto a suitable inorganic carrier. Supports useful in the present invention include aluminum hydroxide, aluminum phosphate, alum (usually polymer). tatshu alam (potassium aluminum sulfate) or calcium phosphate. may be included. These compounds bind tightly to endotoxin. At present A preferred carrier is aluminum hydroxide gel, which has high binding strength. Such aluminum hydroxide gels include, for example, Rehydra gel wax viscocity gel. Rehydagel 101 viscosity gel or Rehydagel 101 viscosity gel LaGel HPA (High Power) Gel is commercially available from Reheis Chemical Co., Berkeley Heights, New Chassis. Preferably, the carrier is passed through the diluted bacterial cell suspension or cell extract. The concentration is usually between 15 and 60% (V/V). This concentration is determined by titration to ensure optimal binding strength of the carrier and to reduce endotoxin and immune Optimal binding of both epidemiogenic bacterial antigens. The titration end point is the point at which the free endotoxin concentration is between about 2 and about 50 ng/ml. Titration methods are well known to those skilled in the art. If a lehydracel support is used, the adsorption is carried out at room temperature (20-25°C), preferably at a pH of about 6.5, with stirring for about 1 hour. Those skilled in the art will understand when using the method of the present invention. These conditions may be changed if necessary. For example, adsorption can be carried out at lower temperatures (eg 4° C.) but with longer mixing times. Similarly, adsorption may be carried out at higher temperatures (eg 37°C), but the stability of the antigen at this temperature allows for shorter mixing times. After the adsorption is complete, the resulting adsorption suspension or extract is diluted for use in vaccine compositions. Depending on the standard concentration of suspension or extract chosen, dilution ranges from about 5 to about 50 times. A suitable vaccine composition is obtained. This method uses the strong binding of endotoxins present in Gram-negative bacterial concentrates to a selective carrier. concentrated fine The binding of endotoxin in the cells or extracts, after dilution, makes it difficult to use the vaccine in animals. make the resulting liquid safe. This binding allows the vaccinated animal to Systemic reactivity of the vaccine in the body is eliminated. At the same time, The low concentration of inorganic carrier in the formulation avoids the disadvantageous injection site reactions associated with the use of conventional carrier concentrations (eg, 10% or more). Although not intending to be limited by theory regarding the method of the present invention, the inventors have determined that the injection site The Gram-negative cells of the present invention We believe that products containing bacterial cell suspensions or extracts are systemically safe. Rapid uptake of endotoxin into the system causes shock. When modifying or optimizing the binding strength of a carrier gel by the method of the invention, some antigens (eg bacterial cells) will not bind. Others (e.g. important protein antigens such as endotoxin) may be more loosely bound (less rigid) than in a fully binding gel. (b) binding, allowing for uptake by macrophages. Therefore, good immunogenicity is maintained. Then, only the bacterial solution obtained by processing and diluting as described above is used as a vaccine specimen. It may be used as a vaccine or mixed with other vaccine components. Preferably, The resulting vaccine composition containing lum-negative cells or cell extracts contains about 0.1 to 10% (v/v), more preferably about 0.5 to 5% (v/v) of It may also contain a aluminum carrier. Vaccine compositions prepared according to the invention may significantly contain endotoxin after adsorption and before dilution at a concentration of about 2 to 250 nanograms/ml, preferably 2 to 50 ng/ml. The pH of the vaccine composition prepared by the method of the present invention is about 6.5±0.5, preferably or adjust to ±0.2. The concentration of free endotoxin in the vaccine solution is preferably determined by the LAL method [Levin et al. Bull, Johns napkins Tlosp, Vol. 115, p. 265 (1964) and Levin et al., Thromb, Diath, Haem orrh, Vol. 19: p. 186 (1968). year)]. Other methods may be used to assay endotoxin levels. According to the present invention, the active ingredient may be only a cell-bound toxoid, a mixture of a cell-bound toxoid and a free toxoid, or these two formulations may further contain an active ingredient, preferably. is a pharmaceutical composition (comprised of an immune dose of active ingredients). (sterile preparation) is provided in unit doses of 0.5 to 3 ml, more preferably 2 ml. For purposes of the present invention, the immunological amount of B. maltocype cell-binding toxoid is measured in units called opacity or absorbance (0, U, or A, U,). These units are the optical density (0, D, ) per ml measured on a Spectronic 20 spectrophotometer at a wavelength of 625 nm. The immunogenic amount of cell-binding toxoid is preferably between 1 and 5 A, U. More preferably, the free toxoid is administered at about 2 A,U. For purposes of the present invention, an immunological dose of free toxoid is defined as When administered in combination with a cell-binding toxoid, approximately 65 to 8.1 my in clograms/dose. In contrast, only free toxoids are immunologically native. It does not induce antivenom when administered to healthy animals. This indicates an interaction between free toxin and cell-associated toxoid. These immunogenic amounts of free toxoid were determined in relative toxoid units (rRUJ). may be justified. Preferably, a free toxoid/cell-associated toxoid vaccine combination The composition shall contain from 80 to 1000 RU/dose, more preferably from 80 to 150 RU/dose. The RU value is LD. Mapping purified toxins with approximately 30 It is determined experimentally based on the estimated amount of toxoid that induces an antitoxin response that protects half of the mice from lethal effects when administered parenterally to mice. Therefore, the R U value is the PD of the mouse. approximately equal to the value. The cell-bound toxoid of B. maltocida of the present invention (which may or may not be administered together with the free toxoid of B. multocida) It may contain other active ingredients. At present, the most preferred further activity The sexual components include B. bronchisebuteica component, E. lunopanae component, and E. It is a component of M. hypopneumoniae. For the purpose of the present invention, E. luciobasiae was prepared as described in Example 7 below. When manufacturing, the minimum immunogenic amount of this component is 3.40. It is U. The degree units of opacity are the same as defined above, except that the O, D readings are taken at a wavelength of 650 nm. For the purposes of the present invention, B. bronchise prepared as described in Example 8 below is used. The immunogenic amount of Butica is measured in units called turbidimetric units. inactivated The required immunogenic amount of this component in immunogenic form depends on the method used to obtain it. It is right. For example, when B. bronchisbutica is inactivated with beta-propiolactone (BPL), at least 1500 nephelometric units are required to exhibit immunogenicity. When inactivated by the glutaraldehyde method, less Both require 3000 turbidimetric units. When inactivating with formaldehyde, at least 4500 nephelometric units are required. If other inactivation methods are used, one skilled in the art will be able to determine other appropriate immunogen amounts. This ingredient is the active ingredient of the present invention. Contained in Chin, it protects against atrophic rhinitis. For the purpose of the present invention, M hyponucleus prepared as described in Example 10 below is used. The immune dose of M. monniae was determined in color change units (CCU) [Rodwell and Whitecomb, Meth. MycoplasmologY J, Vol. 1, p. 188 et seq.・Press (^ academic Press) (1983)]. Preferably, the vaccine composition of the present invention will contain about 5X10'CCU. However, by optimizing the conditions this amount can be reduced to approximately 5X10'. It is also possible to reduce it. A desirable dosing regimen involves administering the desired vaccine composition twice. The amount of antigen in each fraction is as described above. The method of administering the vaccine of the invention can be any suitable method that can deliver the vaccine to the host. However However, preferably the vaccine is administered by subcutaneous or intramuscular injection. desired Alternatively, other methods of administration such as intradermal or intravenous injection may be used. Current research in pigs involves two intramuscular injections of the vaccine two weeks apart. Ru. These studies have shown that for each of the above vaccine compositions, secondary immunization of newborn animals is successfully initiated during the first week of life. Then leave Booster at milk time. For primary immunization of pregnant sows, two doses are recommended. A second vaccination is recommended approximately two weeks before delivery. Following birth immunization is recommended. Revaccinating male pigs every six months is recommended. As shown by the in vitro animal test described below, the vaccine composition of the present invention The specific mechanism of protection against P. multocida induced by induction of toxin-neutralizing antibodies (antitoxins) in treated animals. Preparation of the cell-binding toxoid of the present invention and various vaccines containing the novel component of the present invention Preferred methods for the preparation and testing of tins are illustrated below by way of example. These examples are merely illustrative and do not limit the scope of the invention. Specific examples of the compositions include pea malts in which the toxoid is stabilized within bacterial cells. Tocyda's cell-bound toxoids are included. P. multocida type D 4677 strain was obtained from the Illinois Animal Diseases Laboratory. (Dr. Douglas Hoefling). The strain was subcultured twice on agar. Cells from the second passage were incubated with 10% NZA. suspension in a sterile solution of 1% gelatin and 10% glycerol. An ampoule of the suspension was frozen in liquid nitrogen. This master node is maintained at SmithKline Beecham Animal Health (White Hall, IL). Seed and production cultures of P. multocida strain D type 4677 are grown in the following media. 30 g without dextrose (Dirco) Ribsin soy broth and deionized water (now adjusted to 1 liter), pH 7.5 for seed culture, pH 7.0 for production culture. High pressure for at least 20 minutes at 121°C. Sterilize the medium by sterilizing it. After autoclaving, add 5 Qml of filter-sterilized yeast extract solution (10% w/v) and 4ml of autoclaved dextrose solution (50% w/v). During the incubation, additional dextrose solution may be added if necessary. Thaw the viable seeds (subcultured from the master seeds obtained as above) and transfer their contents to the seed medium container described above. The seed culture was incubated at 37°C for 12 to 24 hours. Incubate with stirring for a while. If the culture is titrated as determined by Gram staining, use it to inoculate the production culture. Alternatively, this may be used to inoculate a secondary seed culture. The inoculum is in 2-10 culture volumes. Incubate the production culture at 37°C for 2-6 hours. Maintain dissolved oxygen at approximately 35% of saturation. The temperature is 37°C and the pH is maintained at 7 by adding 1ON NaOH as needed. Proliferation is monitored by periodic optical density (OD) reading at 625 nm. Near the end of exponential growth (when OD, is approximately 2), aeration is discontinued and the form is removed. Add the aldehyde solution to a final concentration of 0.5% V/V. Stir gently or intermittently. Inactivation is continued for 4 days at 37° C. with stirring. 2 players 15 OD and less A pure culture (determined by Gram staining) with a tensile value of 6 units/ml is considered sufficient for use in production. For toxin-antitoxin titration in mice As shown, L 1,000 units of toxin is equivalent to 1 unit of standard antitoxin. Seven days after subcutaneous injection of a 4 ml volume of the culture, guinea pigs must be alive and healthy. At this point, the intracellular toxin is This) xoid is safe and very stable. and has the ability to induce the production of neutralizing antitoxin when injected into animals. At the end of the inactivation phase, the culture is cooled and then aseptically transferred to a recipient. The culture is aseptically separated into cellular and liquid fractions by sterile continuous flow centrifugation. Collect 100 ryo into a sterile container for further processing. Depending on the OD at the time of inactivation, dilute the cell fraction to a calculated OD of 12.5. This includes a portion of the liquid fraction and a constant volume. of sterile saline (0,85% NaC1) and the final liquid fraction contains bacterial cells suspended in 40% by volume culture liquid, or in a liquid containing 1% w/v peptone and yeast extract. Do it like this. After inactivation and concentration of the culture is complete, sterile aluminum hydroxide gel is added to a final concentration of 25% v/v. Free formaldehyde content of the liquid Quantify the amount and reduce this to 0.2% or less with sodium bisulfite. Ru. Add thimerosal-EDTA solution (or other suitable preservative) as a preservative to give a final thimerosal concentration of 0.01% w/v. Check the pH and adjust to 6.5 ± 0.2 if necessary. The product is standardized to contain 1,875 absorbance units calculated from the OD upon inactivation. This standardized volume is for 0.2ml administered internally alone. It may be obtained in amounts or in combination with other vaccine components. Such a combination Cutin usually has a total dose of 2 ml. Example 2 Preparation of P. maltocida cell-binding toxoid Also, the cell-binding toxoid of the present invention The combined toxoid may be prepared from P. maltocida toxin-producing strains other than the 4677 strain described in Example 1 above. For example, using P. maltocida type 8 strain D, cell-bound toxoid was detected by the above method. can be prepared. However, compared to the 4677 strains, 8 strains had a lower degree of toxicity. Several minor changes must be made to accommodate xoid production capabilities. stomach. P. multocida, type D, 8 strains are grown in the following medium: Dext 30 g tryptic soy broth without loin (Difco), 5 g yeast extract (Difco): 4 g dextrose; deionized water (this makes 1 liter), pH approximately 7; autoclave at 121°C. The culture was then grown, inactivated, concentrated, and enough supernatant was taken to make a suspension of OD 4.2 (optical density at 625 nm, measured in a Spectronic 20 spectrometer). , adjuvanted essentially as described above. This product was adjusted to contain 3.75 absorbance units calculated from the OD at the time of inactivation. Standardize sea urchins. This standardized amount can be administered alone or with other vaccines. It can also be administered in combination with the chin component. Typically, such combination vaccines usually have a dose of about 2 ml. P. multocida type D (type 8) [Dr. Ross Covart, subcultured for 1 day in defined medium. The pH of the assembly medium is adjusted to 7.3±12 with sterile NaOH. Cells from this culture are transferred to fresh synthetic medium and, once expanded, the culture is combined with cryopreservation and stored at -70°C. After inoculation, live A production culture is grown and harvested during a 3-24 hour incubation at approximately 36"±1°C. The dissolved oxygen content of the culture is removed by aeration of sterile air and agitation. Maintain quantity. Control foam using sterile antifoam solution. The pH of the culture is maintained at 73±0.2. At the end of the growth cycle, the P. multocida culture was examined and the cells were measured at absorbance at 650 nm. Measure cell density. The agitation is then reduced and aeration and pH control are discontinued. The toxin content of the lysate is determined by mouse lethality (LD) and by enzyme-linked immunosorbent assay (ELIS^) as described in Example 6 below. B. Pre-detoxification treatment After the growth of the organism, a sterile melamine in an amount less than or equal to 0.01% weight/volume Add olate to the culture. The culture may be transferred aseptically through a closed connection into a sterile closed container. Devices used to physically lyse cells and release cell contents, e.g. For example, connect the container to a GAULIN J Model 15M Laboratory Homogenizer using a closure device. Bacterial cells in the culture solution are lysed by continuous passage through the pressure chamber of the homogenizer. , the cells are immediately depressurized from an initial pressure of about 2000 to about 5000 psi to an ambient pressure of 15 psi. The lysed cells are aseptically placed into another closed container. Centrifugation and/or pore filtration The lysate is clarified by one successive culm of sterilization. The clarified solution may be concentrated before or after sterilization by filtration. Before concentration and sterilization by filtration, an amount of ethylenediaminetetraacetic acid (EDTA) to a final concentration of 5 mM or less is added. and an amount of glycerol up to a final concentration of 1.0% (vol/vol) to prevent aggregation of the concentrated protein. C1 Detoxification Slowly and aseptically add sterile 5N NaOH to the sterile toxin to bring the pH to about 10. 5 5 ± 0.10 pH units. The mixture is heated at about 15 ± 1 °C for at least 7 hours. Detoxification occurs at this pH with slow stirring for a while. Sterile 5N HCl is then slowly and aseptically added to adjust the pH to 6.80±0.20 pH units. The pH is maintained at this lower value long enough to take an aliquot. each alico The residual toxicity of mice was determined and expressed as mouse L Dso per ml. The mixture was then readjusted to a pH of approximately 10.55 ± 0.10 pH units as described above. Condition and maintain for 7 hours. At the end of this time, the mixture is again adjusted to pH approximately 6.80±0.20 pH units for sufficient time to take an aliquot. Then, the mixture Cycle the material through these processes again. Typically, 21 hours after the start of this pH cycling step, a preparation with an initial value of about 10.000 LDso/mI is detoxified without any apparent reduction in quantifiable antigen content. The toxovirus is then combined with other ingredients to form a vaccine composition. Store the id at 26-7°C. Example 4 Formulation of a Vaccine Containing Free Toxoid An exemplary free toxoid vaccine was formulated by preparing the soluble free toxoid described in Example 3 above. To prepare the vaccine, the toxoid is diluted in sterile buffered saline at neutral pH. Adsorb the toxoid using sterile aluminum hydroxide gel as an adjuvant. Add sufficient amount to coat, typically 12% ± 1% (volume/volume). completely mixed Then, add the indicated amount of toxoid and aluminum hydroxide gel to 500 ml of the gel. The vaccine composition is prepared by taking the vaccine into a maker. Then add sterile saline Ru. The mixture is stirred and stored at 4°C. A dosage of 2 ml/dose is preferred, giving about 450 relative dosage units/dose. Table ■ shows the composition of two types of free toxoid vaccines. Table 1 Experimental Lot Ingredients Total Volume A Toxoid Concentrate 1.50.0ml Aluminum Hydroxide Gel 36. O ml Sterile saline 114.0 ml Total 300.0 ml B Toxoid concentrate 235.0 ml Aluminum hydroxide gel 41. Oml Sterile Saline 304.0ml Total 580.0ml These free toxoid vaccine formulations are useful as an aid in the prevention of atrophic rhinitis in pigs caused by P. multocida infection. Code of free toxoid vaccines Typical tests are conducted by injecting the formulation into pigs (piglets and mother pigs) as described below. Example 5 Experiments Using Free Toxoid Vaccine Using the formulation of Example 4, the vaccine is intramuscularly administered to randomly selected piglets and parent pigs according to the following method. In each study, after vaccination, the animals were challenged with purified toquinone at a dose known to consistently induce clinical signs of atrophic rhinitis in pigs. Ru. Toxicity of DNT was evaluated in mice before and after challenge. The total dose of 1-xin administered to each pig was 8.4 μg 1 or 50 mice LD. And It was. In three equal doses, poisoning was achieved over a period of 3 days starting approximately 2 weeks after administration of the vaccine. The substance was administered. The outcome of the challenge was evaluated approximately 28 days after the first toxin administration. Gain 28 Days Post-Challenge Weight percent gain was calculated by dividing the number of pounds by the weight in pounds at the time of challenge. Attenuation - Nasal turbinate atrophy was evaluated by nasal cross-section at premolars as follows: score 0, normal: score 1, minimal atrophy: score 2, moderate to low atrophy, score 3, substantial atrophy: score 4. , complete atrophy; and score 5, complete atrophy. Protocol: Four gilts were vaccinated with a 2 ml dose of P. maltocida free toxoid (A) as described in Example 4 above. Two pains The poor pigs did not farrow because they were infected with porcine parvovirus. disease is evident As soon as this occurred, they were removed from the facility. The piglets born from the remaining two pigs were tested at 13 days old (637.7 piglets) and 9 days old (638.4 piglets). Pea maltocida play described in 4. A 2 ml dose of toxoid (B) was administered as the vaccine. After 2 weeks all the piglets A secondary vaccine was administered to the animals. Two weeks after the secondary vaccine administration, a fixed dose of poison He attacked the piglet with his bare hands. Pigs with similar calving dates to vaccinated distressed pigs. Suffering pigs from one herd received contemporaneous unvaccinated control piglets. After challenge, vaccinated control piglets and pigs were killed until the final score was given. A control piglet not treated with cutin was mixed. Table II shows pigs born to parent pigs that received vaccine A twice and were themselves vaccinated twice with free toxoid vaccine B. administration (vx) 1. Figure 2 shows the effectiveness of challenge on piglets compared to non-vaccinated (NonVX) pigs. These results show significantly lower nasal scores and significantly better weight gain in the vaccinated group. Table II At attack At slaughter Weight increase Weight increase Average nose group Number Weight Weight (pounds) % Score VX 10 26.20 39.60 13.40 54.27],,0ONon-VX 8 22.88 31.56 8. 69 35.30 2゜34 Protocol II: Vaccinating 4 suffering pigs with 2 ml dose of Vaccine 8 Chin was administered. One distressed pig did not farrow due to infection with porcine balhovirus. The patient was removed from the facility as soon as the disease became apparent. Piglets from the remaining distressed pigs were challenged with the toxin as follows: 9 piglets (10 days old) from 1 distressed pig; 2 piglets from the second distressed pig ( 12 days of age); and third tribulation block 6 piglets (4 days old) from Ta. Suffering pigs from the same group with a similar farrowing date as vaccinated distressed pigs were fed control piglets that were not vaccinated at the same time. Vaccinated and unvaccinated control piglets were challenged prior to weaning and then stratified and mixed until final scoring. Table III summarizes the effects of challenge on piglets born to piglets that received two doses of vaccine A. Data are presented (a) independently from a litter of pigs, and (b) between pigs across a litter. Give by average. These results indicate significantly lower nasal scores and significantly lower nasal scores in the vaccinated group. Shows better weight gain. These observations indicate that two doses of vaccine A to the parent pigs induced the production of antitoxin, which was otherwise passively transmitted to the infected piglets. Furthermore, the duration of passive defense is at least It was also 10 to 12 days. Table III (a) At attack At slaughter Weight increase Weight increase Average nose group No. Weight Weight (pounds) % Score VX 15 6.87 21.00 14.13205.83 3. 02NonJX 5 8.20 16.40 g, 20 100.00 3. 70(b) Vx Suffering Pig 629 7 8.71 21.50 12.79147.09 3.6g Young Suffering Ta 639 2 g, 00 29.25 21.25268.65 2J8 Sow 633 6 4.33 17. 67 13.33310.28 2.46 young sow Ta Mean 67.02 22.81 15.79 242.01 2.84NonJ Samples were tested for antibodies against the toxin by kinetic ELISA. Briefly, purified toxin (250 ng/well) in 0.1 M sodium borate (pH 9.1) was adsorbed to flat-bottomed 96-well Nunc microtiter plates overnight at 4°C. Then, it was blocked with 10% dry skim milk in PBS containing 0.05% Tween-20 (blocking buffer) for 30 minutes at 37°C. Blocking buffer was added to the plate glass twice with PBSlo, 05% Tween-20 (PBS/Tween). After that, I rinsed it with PBS. Serum was diluted 1:100 in blot buffer and 50 μl of sample was added to each of 4 wells. Plates were incubated for 60 minutes at 37°C and then rinsed as above. Goat anti-pig IgG (heavy and light chain specific) - Horseradish peroxidase (key chain specific) Kirkegaard and Perry Laboratories (Gaithersburg, MD) was diluted 1:500 in blocking buffer and added to each well (50 μl). After incubating for 60 minutes at 37°C, plate and rinsed as above. ABTS substrate (2,2'-axinodi-3-ethyl Rubenzothiazoline sulfonate) (Kirkegaard and Perry) was added to the V max ELISA Reader [Molecular Devices Corp.] at 405 nm. Molecular Devices Corporation, Palo Alto, Calif. read the text. Each well was read eight times at 1 minute intervals and the rate of enzyme reaction was calculated. Rates were calculated as millimeter change in optical density (mOD) per minute. did Therefore, a reading of 100 m0D per minute is equal to 1.0 OD for 10 minutes. Values were then corrected for the amount of serum used per well and expressed as moDZ min/m+serum. For example, if a 50 μm serum exhibits 100 mOD/min, the value shown would be 2.000 mOD units/min/ml. Include the following controls on each ELISA plate. (1) Serum Control: Each diluted piglet serum was placed in a well containing no antigen and then exposed to all subsequent reagents to check for non-specific adsorption to the plate. Pig blood used Upon dilution of serum serum (1:100), no color stronger than that obtained with the negative serum control was observed. I couldn't. (2) Negative piglet serum. Each plate contained three wells of known negative piglet serum diluted 1100 in blocking buffer. (3) Positive piglet serum control Serum containing toxin-specific antibodies was diluted in negative piglet serum to obtain serum containing high, medium and low concentrations of specific antibodies. These three sera were diluted 1:100 in blocking buffer and added to each plate. I put it in three layers. In wells containing all reagents except piglet serum, remove the buffer. Kugland, or non-specific reactivity, was measured. The ELISA titers of parent pigs and piglets vaccinated with toxoid vaccines A and B, respectively, according to Protocol II (Example 6) are summarized in Table IV below. This table shows the geometric mean titers of serum taken before the next and second brood vaccinations, and the geometric mean titers of colostrum, compared to non-vaccinated controls (Non-Vx). , and - subsequent and secondary piglet vaccination, challenge and Give the geometric mean titer of the serum taken before sacrifice. - Second parent group Second parent group - Second offspring Second offspring group Ta VX Ta VX Colostrum Evening VX Evening VX Attack Slaughter 1'X 21.71 0 173.00 0.99 1.73 109.03 139. 07Non25.35 12.34 83. 38 1.45 1.72. 38 8.64X These results indicate that two doses of vaccine to parent pigs, Two doses of Vaccine B were given to the piglets in the later stages, which would otherwise be susceptible to infection. Induced immunity against the toxin in piglets. From the same study (Protocol II, Example 6), parent pigs vaccinated (vaccine A), parent pigs vaccinated with the tJ vaccine and those not vaccinated. The ELISA titers of these piglets are summarized in Table ■. Table V Geometric mean ELISA titer Primary parent Secondary parent Sow 629 21.80 5.80 70.60 1.66 29.15 young Sow 639 29.20 - 18.60 26.0?25.39 Not suffering Pig 636 23.40 10.80 66.80 - 12.40 sow 631 30.60 11.90 135.90 - 0.20 Sow 62 6 19.80 17.60 76.70 - 9.40 Sow 635 40. 60 20.40 Immunochallenge studies on parent pigs and piglets vaccinated from free toxoid formulations 2 at various doses (expressed in relative toxoid units, RU) are summarized in Table VII. Table VI Dose RU Significant nasal turbinate atrophy Parent pig Piglet No. Weight loss Protection against weight loss876th32 307±70 10 Yes Yes876±32 0 15 Yes Yes 391Sat52 0 1.0 No No0391±52 9 No No The data are twice with a vaccine containing 876 ± 32 RU of free toxoid. Figure 2 shows significant protection in piglets born to cutin-treated parents. piglet or pregnancy In pregnant distressed pigs, two administrations of experimental lots containing 300-400 RU/dose showed no induction of protection. Example 7 - Preparation of E. RHUSIOPATHIAE Vaccine Ingredients Erysipelothrix rhusipathiae, alone or preferably in combination with the P. multocida component described above. Prepared for use in vaccine compositions. The E. rhusiopathiae component is preferably derived from serotype 2, which has the highest immunogenic yield common to all E. rhusiopathiae serotypes [R. , Wood (R, L, Wood), Journal of American Veterinary Medical Association (J, AmervetJ4ed, As5oc,), Eng. 84: pp. 944-948 (1984)]. Currently, the preferred strain is CN3342 [Sumi S++1th K1ine Beecham Co. Ru. A. Cell Culture The following culture media are free of crude organic substances, such as serum and bile, two components commonly used in Erysiberothrix culture. This has the effect of reducing the frequency of adverse reactions and the severity of symptoms. A centrifugation step to remove bacterial cells also helps reduce the number and intensity of harmful reactions. Conveniently, the usual immunogen is present in the supernatant fluid. E. rhusiopathiae was grown in deionized water (12.0-3.0% of Difco or equivalent). Tease Vep [manufactured by Difco or equivalent]. 25-0.75% yeast extract, 0.1-3% Tween-80 [polyoxyethylene-sorbitan monooleate-1], 1.0-2.0% The cells are cultured in a seed medium consisting of 2HPO4. Adjust the pH to approximately 7.0 + 0.21 m with 1ON NaOH. The medium is sterilized by autoclaving at 121°C for 30 minutes. After autoclaving, no Add a 50% dextrose solution of the bacteria to a final concentration of 0% v/v. Those skilled in the art will be able to vary the components of the culture media described above and the amounts of each used. For example, protein digests other than protease peptone may be used in the medium described above, Tween 80 may be replaced by other soluble compounds, such as Tween 85; or growth factors may be supplemented. Alternatively, the yeast extract may be replaced with, for example, Hermit. Thaw the ampoule of vor kingseed and transfer its contents to a container of seed medium. Incubate the seed culture at 37°C for 12 to 24 hours with agitation. Ru. If microscopic examination of Gram-stained slides shows a satisfactory culture, the culture is used to inoculate the production culture. Alternatively, it may be used to inoculate a second seed culture that is then used to inoculate the production culture. The inoculum is equal to or less than 2 of the culture volume. and 10%. The production culture is incubated at 30-39°C with stirring for an average of 5 to 8 hours. cuveate. Sterile ION sodium hydroxide solution was added throughout the incubation period. Add to the culture and maintain pH at 7.0-7.6. Sterile 50% dextrose solution may be added if desired. Once maximum ○D is reached (stationary phase), inactivating agents are added to the culture as described below. Alternatively, inactivation may occur during the earlier logarithmic or transitional phase of growth. B. Inactivation of bacteria Inactivation is performed as follows. Samples of E. rhusiopa thiae are taken from the culture and Gram stained. If this is pure Once a good culture is found, add enough formaldehyde solution to give a final concentration of 0.5% by volume. Transfer the culture to a sterile bath and place in a constant temperature bath at 37°C for 24 hours with constant agitation (the formaldehyde concentration may be decreased or increased; however, the incubation time may be adjusted to satisfy Inactivation is determined by the bulk terility test I: according to 9 CFR 113.26. The product is stored at 4-10°C until ready for further processing. C. Preparation of Vaccine Fluid After inactivation, the culture is sterilized and aseptically transferred to a container.The culture is then passed through a continuous flow centrifuge. After clarification, the fluid fraction is subjected to ultrafiltration. Concentrate to a calculated OD value of 67. The result is a sterile immunogenic fluid. This degree of concentration, i.e. the degree obtained by concentrating to 0D16.67, was 3.5% in 3ml of the mixture volume (after adding the aluminum hydroxide gel to a final concentration of 25%). It will contain an antigen dose of 75 opacity units (1 opacity unit contains ODl in 1 ml). This 0.3 ml is the E. rhus iopathiae volume used for a combination vaccine with a total dose of 2 ml. Example 8 - Preparation of B. BRONCI'1ISEPTIC^ vaccine component. Prepared for use in tin compositions. B. bronchiseptica inoculum (preferably one derived from pigs suffering from atrophic rhinitis) Yes. Currently, the preferred strain is the 2-9 NADC strain [National Animal Deposit National Animal Disease Center, Ames, Iowa]. death 144 strain SmithKline Beecham) may be used. A. Cell Culture and Manufacturing The culture medium used for the propagation of Bordetella bronchiseptica was modified Steinerskolt synthetic medium. It is a modified stainer-8choLte defined 5 synthetic minimal medium. 1 liter of synthetic base medium 2.4 g L-proline, 67 g L-glutamic acid, 2.5 g NaCl. The following procedure is used to prepare 0.5 g KH2PO4, 0.2 g KCI, 6.075 g Triz+obase (Sigma) #T 1503]. These components are sequentially dissolved in 10100 O distilled or deionized water with stirring. The pH is adjusted to 7.0+0.2 units with HCI and the medium is sterilized by autoclaving. Prepare 1.00 mL of each of the three IOX stock solutions as follows. The L-7 stain solution is prepared by dissolving 0.4 g of L-7 stain in 4 mL of 4N HCl and then making up to a volume of 100 mL with distilled or deionized water. The iron sulfate (I), calcium chloride, magnesium chloride solution contains 0.125 g iron (II) sulfate heptahydrate, 0.3 g calcium chloride dihydrate, and 1.0 g magnesium chloride. Prepare ium hexahydrate by dissolving it in 100 mL of distilled or deionized water. child These ingredients are each dissolved in sequence with stirring before adding the next ingredient. As The corbic acid, nicotinamide, and sodium acetate solution was added to 1.00 mL of distilled water or or deionized by dissolving 0.2 g ascorbic acid, 0.10 g nicotinamide, and 2.0 g sodium acetate per bottle. These three solutions are then filter sterilized and the L-cysteine and vitamin solutions are stored at 26-7°C. A culture medium is prepared by adding 10 mL of each of these stock solutions to the above-mentioned minimal salt solution. A 1 to 5% suspension of the reconstituted freeze-dried master inoculum (master 5eed) or thawed inoculum is inoculated into flasks containing the culture medium described above. Ru. The culture was incubated for 16 to 30 hours at 36°C ± 1°C with air supply. Bate. After sufficient growth, this culture medium is transferred to seedlings containing fresh medium. Transfer to a seeding flask using a 1 to 5% inoculum. This second subculture is incubated as described above. Manufacturing capacity The production culture is inoculated with a 1 to 5% inoculum in a second actively growing subculture. Production culture of B. bronchiseptica The fermenters are grown in fermenters. The culture was incubated at 36°C ± 1°C for 16 days after inoculation. and incubate for 40 hours. The dissolved oxygen setpoint is set at 80% of maximum equilibrium (saturation). The set value of dissolved oxygen is preferably set to 40%. This is rapid and therefore requires a short incubation period of between 6 and 16 hours. I can do it. Dissolved oxygen content is maintained by sterile air supply and agitation. Inoculate the medium Add a sterile antifoam solution before washing. The pH of the culture was adjusted by adding propionic acid. Maintain at 7.2-7.4 by adding B. Fluid Inactivation and Preparation Any aldehyde can be used to inactivate the culture. Using BPL If necessary, add it to the culture at the end of the growth phase. A second additional BPL addition is made after 2 to 18 hours. The final concentration of BPL should not exceed 1:500 (0.2%). The culture is incubated at less than 20° C. for at least 12 hours with constant agitation. Alternatively, at the end of the growth phase First, formalin (formaldehyde solution USP) is added to the culture at a final concentration of 0.4% of the total volume. The culture was incubated at 35° with constant stirring. Incubate at 37°C for at least 24 hours. After inactivation, representative samples were taken and directly plate counted on tryptiase say agar plates using 0.5 mL of inoculum on each of the three plates. Test for completion of inactivation by: Bulk samples are tested for inability to grow in thioglycolic acid at 37°C and tryptic say broth at 22°C. Inactivated cultures are kept sterile. It may be transferred to a storage container and stored at 2°C to 7°C until collected. Sterile aluminum hydroxide gel (equivalent to 2% Alto3) was added to a 5% volume/volume concentration. to the inactivated culture to control free endotoxin. Total adsorbed Cal Chi + - (whole culture) is obtained. Alternative Methods of Fluid Preparation As a preferred method, rather than inactivating the culture with BPI, Gluta as described in U.S. Pat. No. 4,888,169 to Brovn et al. The culture used in vaccines can be prepared by the Ruraldehyde method. Ru. This method involves inactivating the culture by adding glutaralchthon to the culture medium. B. bronchiseptica was prepared at a glutaraldehyde concentration of 0.2 to 0. Adjust by increasing the volume to 25% V/V and incubating at 37° C. for about 24 hours. Gluculaldehyde acts to bind endotoxin, obviating the need for the culture to adsorb to the aluminum hydroxide. D Standardization of antigen concentration When inactivating by BPL, 1,500 turbidimetric units (nepheLo + aetricυn1t) or more per dose; if inactivating by the nigeltaldehyde method, 3,000 turbidimetric units or more; inactivating with formaldehyde. If The B. bronchiseptica force is standardized to contain more than 4000 turbidimetric units per hour. The turbidimetric unit is based on measurements at the time of harvest. When this component is used in a vaccine composition (having a 2 mL dose) containing several other antigenic components, it is approximately 0.2 to 1. Configure OmL to be accomplished. Example 9 - Combination Vaccine The combination vaccine was prepared using other inactivated microorganisms such as B. bronchjseptica, other strains of B. multocida (P. multocida), M. h.pneumoniae (M. hyopneumoniae) and/or the soluble toxoid of Example 3. One particular effective vaccine composition contains free and cell-bound toxoids of the B. maltocida CP, mυ1tocida) type D, described above, together with the B. bronchseptica B, bronchjseptica) component described in Example 8. do. One specific formulation of a combination vaccine consists of the following ingredients: Ingredients Volume/Dose (ml) Volume (ml) P, m Cell-bound toxoid 0.2 00 25.00 Free toxoid (650U/ml)' 0.242 30. 25B, bronchiseptica O, 30037, 50 oil runtine 0.100 12.50 Tween 80 0. 0567,00 Span 80 0.024 3.00 Loss of Stars'' Jiang-1-Saririkage-------- Two L Yul total amount 2.000 250.00 In emulsification, these components are simply Combined as a batch and emulsified for 2 minutes. Manufacturing regulations It is expected that in-line metering would be preferable to batch compounding. Other ingredients may be added to, or substituted for, the ingredients present in the special formulations described above. In addition to oils/lectins, hydroxylated amines have adsorbed cell-bound toxoids. Luminium gel also serves as an adjuvant. one or more of each fraction Whole or partial bulk lots can be adjuvanted. and diluted saline to achieve standard antigen concentrations. Example 10 - M, flYOPNEtlliONI AE vaccine components Mycoplasma hyopneumoniae vaccine components are incorporated into the combination vaccine of the present invention. It is useful in preventing mycoplasmal pneumonia in pigs. Currently inactivated The desired immunogenic amount of the product is about 109 color changing units (XCCU). However, it is anticipated that this dose may be reduced to about 5X10'' to 5X10'CCU under best conditions. Approximately 0.1 to 0.3 mL is required to obtain the volume. Typically, in the preparation of combination vaccines containing the M. hyopneumonae component, this culture is simply added to the liquid bulk solution. Add to Chin formulation. A. Growth of M. hyopneumoniae and CultureM hyopne+monia e) strain P-5722-3 was kindly provided by Dr. Charles Armstrong of Purdue University and was donated by American Co., Ltd. under accession number 55052. It has been deposited in the Type Culture Collection. This strain has immunological and biochemical properties: mannose-positive, arginine-negative, and urease-negative. The strain was positive for growth inhibition by anti-M. hyopneumoniae (M. hyopneumoniae) antiserum and anti-K. pneumoniae fluorescein binding. A direct fluorescent antibody test using a combined antibody is positive. This strain grows as follows. I let it grow. A culture medium was prepared by the following procedure. Add the broth to the anion exchange resin. - Amberlite, Sigma IRA 400 - Chlorine Type] for 1 to 4 hours at a ratio of 500 g of resin to every 104 parts of broth. 83% PPL○ broth without crystal violet (Difco Laboratories, Detroit, Mich.) was prepared by IJRv treatment. Yeast extract was prepared by adding 500 g of active yeast granules to 31 g of deionized water and stirring at room temperature. completely mixed After that, 16.2 ml of ION NaOH was added dropwise to the suspension, and then further Stir for 15-45 minutes. The slurry was then placed in the oven at 121°C for 1.5-45 minutes. autoclaved. The supernatant was decanted into a container and clarified either by centrifugation or microfiltration. 1NHCl was added to the clarified supernatant at a ratio of 2 ml per 100 ml of extract. The extract was stirred at room temperature for at least 15 minutes and then clarified as above. The clarified extract was sterilized by autoclaving or microfiltration as described above. The following medium components were added to the pretreated broth: o, oi% thallium acetate; 0.005% ampinlin + 0.0125% cysteine hydrochloride; 6.25% yeast extract, 1% dextrose, 10% pig. Serum (Gibco) heat inactivated and optionally 0.0026% phenol red. mosquito The pH of the Lucher medium was adjusted to pH 7.5±02 and filter sterilized. To begin serial production, a frozen M. hyopneumoniae master inoculum was thawed and a 5-20% suspension was inoculated into 1.0000-3000 of the above culture medium. The cultures were incubated at 30°C to 39°C for 36 to 168 hours. After sufficient growth, the culture The char was transferred to a nurturing container containing fresh medium using 5-20% inoculum. This culture was incubated at 37°C±1°C for 36 to 96 hours. Production culture of M. hyopneumoniae The ink was grown in a fermenter and inoculated for 36 to 96 hours at 37°C ± 1°C after inoculation. cuveate. The dissolved oxygen content of the culture was determined by supplying sterile air and stirring. Maintain 20-40%. Sterile antifoam agents may be used to control foam. At the end of the growth phase, the pH of the culture rises to 76±0.2 and the pH is maintained in this range for about 1 hour. To inactivate the organism, 2-bromoethyl A filter-sterilized aqueous solution of amine hydrobromide (BEA) was added to a final concentration of approximately 4.0 mM. BEA is converted to the inactivating agent binary ethyleneimine (XBEI) in the culture at elevated pH. The culture was incubated at 37°C ± 1°C with constant stirring. Both were incubated for 24 hours. After incubation for 24 hours, a filter-sterilized aqueous solution of sodium thiosulfate, a standard neutralizing agent, was added to a final concentration of approximately 4 mM to neutralize excess BEI. The culture was incubated for an additional 24 hours at 37°C ± 1°C until completely inactivated. turned into B. Vaccine Preparation After inactivation of the vaccine components of the previous example, inactivated M. hyopneumoniae was combined with P. multocida (P. multocida) cell-bound toxoid and/or other vaccine components of the invention. can be added to It was formulated according to the following. A sufficient amount of inactivated M, hyo pneumoniae was combined with the bulk liquid vaccine loft to obtain a minimum antigen concentration of approximately 10'CCU per 2 ml dose. Sterile 10% merthiolate and 10% ethylene Diaminetetraacetic acid (EDTA, disodium or tetrasodium salt) solution was added as a preservative. Sterile mineral oil (Drakeol) containing 5% to 40% by weight of lentin (Central 5oya) was emulsified in phosphate buffered saline and added to the bulk vaccine stream. Add to the body at a final concentration of 5% v/v. This oil/cintin mixture is used as an adjuvant. Serve as a gift. Tween 80 with a final concentration of 0.7% to 3.2% and Span 03 to 18% were added as emulsifiers. Selected parabens (p-hydro Methyl xybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate) may be added as further preservatives for oils and emulsifiers. Example 11 - Vaccine studies in animals - Synergy between soluble and cell-bound toxoids of P. multocida (P, MLI LTOCID) During testing, a mixture of cell-bound and free toxoids from P. multocida was used to evaluate antibody responses in pigs. antitoxin induction compared to the use of cell-bound toxoid alone or free toxoid alone. The surprising fact has been discovered that cell-bound toxoids have a greater than additive effect. A herd of pigs was vaccinated in Kuching. Table ■ shows the detailed results for free toxoid alone. Figure 3 shows antibody responses to vaccination with cell-bound toxoid alone and with a combination of these two vaccine compositions. The ELTSA titer is It shows the synergistic effect of cutin. This combination vaccine composition is believed to induce the best immunity in pigs. Post-vaccination serum was collected using Roberts (R (+berts)) and Swearinge. The method of wearingin [American Journal of Betari Antitoxin neutralizing, actual protective antibodies were assayed by Nalley Research (AOI, J. Vet, Res.), 49:216 8 (1988)]. Antitoxin values indicate strong synergy between free and cell-bound toxoids. (Table ■). (Aluminum hydroxide content is 12% v/v; Amphigen content is 5% v/v). A vaccine containing whole cells (Bb) was used in guinea pigs to determine serum antibody levels using an EBL tissue culture assay [J.M., Rutter, et al., Veterinary Record, 114. : pp. 393-396 (1984) are doing. In this experiment, the dosage unit of the combination vaccine is 2 ml/dose. In this experiment, 600 RU of free toxoid had no observable anti-toxoid content. No elementary reaction was induced. In contrast, free ibis in combination with 600 toxoids) multocida) induces an antitoxin response level of 128. Provides a further example of immunological synergy of char. Table ■ Number of pigs Bound 1 Free 1 Adjuvant Serum antibody level Neutralizing antibody Toxin (ml) (RU) Vaccination Vaccination Before/after Unit/m1 Before vaccination 8 0ml 20OA40H3〈10 13 <18 0ml 200 7mph ijix7-AbOHs <l 0 16 <18 2ml OAIto)13 <io 93 208 2ml Oambuidiene-AitOHs<10 46 208 2ml 12OA40Hs <10 252 40 bags-! Bound Toi 74F Free) + 74F Dose Fraction Adjuvant Serum Antibody Level (RU) Before Vaccination After Vaccination Bb+PmD 600 1/25 A Amphisien-Al*OHs <2 128Bb+PmD 300 1/25 Amphisien-AbOHs <2 4Bb+PmD 0 1/25 Amphisien Zy-Al, OH, <2 <2Bb 600 1/25 Amphisien-A lxOHs <2 4Bb 300 1/25 Amphisien-Aj', OH, <'2 (2 Numerous modifications and variations of the present invention are described above. For example, B. bronchiseptica, E. rhus iopathiae or M. Suitable inactivated pathogens other than P. hyopneu moniae may be used in the combination vaccines of the present invention. Similarly, other conventional adjuvants and inactivated vaccine components may be used in the formulations and will be readily understood by those skilled in the art. The dosage and administration protocols for these vaccine compositions may also be adjusted by those skilled in the art based on the animal to be vaccinated, the disease desired to be prevented, and other relevant factors. All such modifications and changes to operation are intended to be encompassed within the scope of the claims set forth herein.Continued from Front Page (72) Inventor Mouth Parts, Dipid Niss Nebulus, United States 68 510, 1020 Rockburst Drive, Lincoln (72) Inventor Wellingin, Leroy A. 68510, Nebraska, United States, Linker No. 9, South 33rd

Claims (1)

[Claims] 1. Cell-bound toxoids that induce neutralizing antibodies against the toxin when used internally in animals Pasteurella multocida (Pasteurella multocida) da) Type D 4677 strain bacterin and a carrier suitable for internal use Vaccine composition. 2. P. multocida organisms in logarithmic growth phase. Produced by treatment with formaldehyde for a sufficient period of time to inactivate intracellular toxins. The vaccine composition according to claim 1, wherein the vaccine composition is prepared as follows. 3. 4. Further comprising an immunogenic amount of one or more additional antigens. Vaccine composition. 4. The additional antigen is Bordetella pronchiseptica. 4. The vaccine according to claim 3, comprising a bacterin. Composition. 5. The additional antigen is Erysipelozurix rhusiopathiae. thrix rhusiopathiae) bacterin according to claim 1. vaccine composition. 6. the additional antigen consists of a soluble free toxoid of Pasteurella multocida The vaccine composition according to claim 3. 7. Pasteurella multocida type with immunogenic amounts of cell-bound toxoids A vaccine consisting of 0.5 to 3 ml of a sterile suspension of bacterin D.4677. Dosage unit. 8. A cell-binding toxoid that induces the production of neutralizing antivenom when used internally in animals. Bacterin associated with Pasteurella multocida type D strain 4677. 9. Pasteurella multocida type with immunogenic amounts of cell-bound toxoids P. maltocytosis consists of internally administering D.4677 strain bacterin to animals. Methods for administering vaccines to animals against Da. 10. It consists of internally administering the vaccine composition according to claims 1 to 6 to an animal. , a method for administering vaccines to animals against P. multocida. 11. (1) When used internally in animals, cell condensation induces the production of antitoxin against toxins. Pasteurella multocida bacterin with synthetic toxoids and (2) P. ・Maltocida・Vacuum consisting of soluble free toxoid and a carrier suitable for internal use Chin composition. 12. Contains at least 150 relative toxoid units per mL of said free toxoid The vaccine composition according to claim 11. 13. Claim 11 further comprising an immunogenic amount of one or more additional antigens. Vaccine compositions. 14. The additional antigen consists of Bordetella bronchisebtica bacterin. 14. The vaccine composition according to claim 13. 15. The additional antigen is from Erysiperoshrix luciobasiae bacterin. The vaccine composition according to claim 13. 16. Immunogenic doses of P. maltocida free toxoids and cell-bound toxoids A sterile solution consisting of P. maltocida bacterin with id 0.5 to 3 Vaccine dosage unit consisting of ml. 17. (1) Pasteurella multocida bacteria with cell-binding toxoids and (2) Bee Maltocida soluble free toxoid and suitable for internal use. P. maltosa consists of internally administering to animals a vaccine composition consisting of a carrier. Methods for administering vaccines to animals against Ida. 18. When used internally, an antitoxin response is induced in the animal, and the antitoxin response is Antitoxin responses to cell-bound toxoids and soluble free toxoids 18. The method of claim 17, wherein the antitoxin response is greater than or equal to the sum of the antitoxin responses. 19. P. multocida cell-binding toxoid, P. multocida release Toxoid, B. bronchiseptica - Bacterins and E. rhusiopathiae - Vaccine composition consisting of bacterin. 20. 20. The vaccine composition of claim 19, further comprising an adjuvant. 21. The adjuvant is aluminum hydroxide, saponin, magnesium hydroxide. , the group consisting of aluminum phosphate, magnesium phosphate and calcium compounds The vaccine composition according to claim 19, selected from: 22. Atrophy, comprising administering the vaccine composition according to claim 19 to an animal. Methods for administering vaccines to animals against rhinitis and erysipelas.