JPH06172213A - Vaccine for mycoplasma pneumonia - Google Patents

Abstract

PURPOSE: To provide vaccine for the diseases induced by mycoplasma- hyopneumoniae and more particularly the vaccine which protects swine against a mycoplasma pneumonia. CONSTITUTION: This vaccine contains the surface suspension of the cultured matter of the mucoplasma-hyopneumoniae having antigen effectiveness of at lest about 1024 in a metabolism inhibition test and an approved medicine base and protects the swine against the mycoplasma pneumonia. The surface suspension is preferably obtd. by culturing mycoplasma-hyopneumoniae-strain-PRIT-5 having high antigenicity and high proliferation rate to obtain the cultured matter having density of a color change unit of 10<9> to 10<10> per 1 ml and subjecting the cultured matter to a centrifugal sepn. at a rate between 1,000 × g and 1,600 × g. The vaccine contains bacterial at a density of 1.5×10<9> to 10<10> ccu/ml and water-soluble antigens at a concentration of 65 to 80 μg/min and is preferably used by oral administration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a vaccine against the plague caused by Mycoplasma hyopneumoniae, and more particularly to a vaccine for protecting pigs against Mycoplasma pneumonia.

[0002]

BACKGROUND OF THE INVENTION Mycoplasma suinoimoniae (My
Mycoplasma hyonoimoniae, also known as coplasma suipneumoniae), is a carrier of mycoplasma pneumonia in pigs, also known as swine endemic pneumonia, viral pneumonia, infectious pneumonia, and anterior lobe pneumonia. is there. Pigs are the only one known as the owner of Mycoplasma. Since pig populations can be well controlled, it is possible to establish and maintain pig breeding completely free of Mycoplasma pneumonia.

Mycoplasma pneumoniae, one of the most prevalent epidemics of the respiratory tract of pigs among pig-rearing countries in the world,
Mortality is low but has high morbidity (30-80%). The disease generally results in reduced animal growth, incompetence, and considerable economic loss as it causes the disease. The disease is transmitted from pig to pig through the nasal cavity by airborne microorganisms excreted by infected pigs. Mycoplasma penetrates deep into the pneumonia of the lungs and lobes near the heart, where pigmented plums or gray lesions are seen, leading to dyspnea and weight loss. Primary infections with Mycoplasma hyonoimoniae are secondary to other mycoplasma species (eg, Mycoplasma hyonoimoniae and Mycoplasma hyonoimoniae) as well as bacterial etiology (species of the genus Pasteurella and Bordetella). Accompanied by infection.

These respiratory illnesses caused by Mycoplasma hyonoimoniae result in weight loss when the animals are bred for market. Therefore, animals infected with these microorganisms will have less value at slaughter than uninfected animals.

There are many attempts to provide a vaccine that protects pigs against Mycoplasma pneumonia. However, such vaccines have generally not been successful. Pigs recovered from Mycoplasma hyonoemoniae infection are resistant to the following provocative exposures and give some protection against pneumonia when given a sufficient amount of immune serum. Therefore, vaccines that protect against mycoplasma epidemics have been proposed. For example, active vaccines using attenuated microorganisms (eg, US Pat. No. 3,534,136), or surface suspensions of whole cell cultures, thermosonication, thin film strips or extractions of mycoplasma sterilized by chemical agents. All vaccines containing min have been tested experimentally. A vaccine containing non-viable Mycoplasma hyonoimoniae with incomplete or complete Freund's adjuvant experimentally reduced pneumonia in pigs, but immunized pigs with Mycoplasma hyonoimoniae without a non-viable adjuvant Attempts to try have been largely unsuccessful in field experiments.

"Goodwin (1973) Br. Ve
t. J. 129 : 465-470 "report the preparation of a formalin-treated vaccine against endemic pneumonia tested in two field experiments. In the first experiment, pigs were single subcutaneously at 3 weeks of age. Received administration.

In the second experiment, subcutaneous administration was performed twice at the age of 3 weeks and 5 weeks. Based on age and weight similarities of slaughtered pigs, and no significant differences in lung lesion assessment values between vaccinated and control animals, the vaccines showed growth rate or at slaughter time. It is concluded that it has no effect on the incidence and extent of pneumonia.

"Christensen et al. (1981) Am
J. Vet. Res. 42 : 784-788 "vaccinated pigs with the heat-inactivated Mycoplasma hyonoimoniae organisms in complete Freund's adjuvant. However, the vaccinated animals were exposed to natural infection. At times, no protective effect on the immune response was detected.

"Research of Ethelide and Lloyd (1982) Res. Vet. Sci. 33 : 188" is effective for lung infection and transplantation of Mycoplasma hyonoimoniae when a live vaccine is administered intravenously, subcutaneously or intraperitoneally. In contrast, it showed incomplete (60%) protection. However, there were two drawbacks associated with the approach used in this study. (1) Many injected pigs develop arthritis,
(2) The injected antigen did not reach the immunologically reactive site in all animals.

[Ross et al. (1984) Am. J. Ve
t. Res. 45 : 1899-1905 "reported altered protective activity of whole cells in a porcine model of experimentally induced Mycoplasma pneumoniae or Mycoplasma hyonoimoniae vaccine prepared from crude extraction. Whole cells, ice-salting Vaccines prepared from solution extracts, surface suspensions of cultures or saline washes of cells were treated with 0.15% formalin combined with Freund's incomplete adjuvant and were administered intramuscularly in two doses. 25 to 42 days after the first administration of the vaccine, it was induced in the respiratory tract with a surface suspension of lung homogenate containing only Mycoplasma hyonoimoniae. Salt-washing of suspensions and cells produced comparable protection: the extracts prepared by the thaw procedure were in protective activity. It was variable and in some cases enhanced lesion development.

"Kisima et al. (1985) Am. J. Ve
t. Res. 46 : 456-462 "evaluated the effect of dextran sulphate on the protective effect of incapable Mycoplasma hyonoimoniae administered dextran sulphate intramuscularly. Pigs vaccinated with Mycoplasma hyonoimoniae and dextran sulphate Although experimentally induced pneumonia was less severe than unvaccinated pigs, protection against spontaneous pneumonia was not tested in field experiments.

The concept of the normal mucosal immune system is supported for the oral immunity of herds in animal farms (Bergmann et al. (1986) Int. Arch. Allergy).
Appl. Immunol. 17 : 357-365).
Oral administration of the antigen can effectively induce antibodies through the terminal small intestinal lymphoid population known as Peyer's patches (Bergmann et al. (1986) supra). Wen (1985) Ph.D. Diss
publication, Wotson College, C
Cambridge, U.S.A. K. , (1985), 95-
109 "was inoculated intramuscularly with Mycoplasma hyonoimoniae inactivated with incomplete Freund's adjuvant formalin, and it was possible to give an auxiliary injection in the intestinal tract. The absorption of Mycoplasma hyonoimoniae through the patch of Paiya is GI system. Not only in the respiratory system but also in the respiratory system, the normal mucosal immune system is generated to obtain the effect of immune function.

However, the practical use of such an immune route is prevented by the deterioration of the antigen by the acidity of the GI stomach and the proteolytic enzyme. "Tuan et al. (198
9) J. Chinese Soc. Vet. Sci. 1
5 : 199-206 "reported conditions of culture temperature and pH (pH) at which the viability of Mycoplasma hyonoimoniae antigen was retained." Tuan et al. (1989) B.
iotech. Tech. 5 : 139-144 "found that sululose acetate phthalate was used to prepare enteric coated microspheres of the Mycoplasma hyonoimoniae vaccine mixed with porcine serum. These microspheres were administered orally. For mixed administration with pig feed.

"Covish et al. (1987) Ann. I.
nst. Pasteur Immunol. 138 : 6
93-705; World Patent Publi
Cation WO 86/000019 "indicates that pregnant pigs inoculated with Mycoplasma hyonoimoniae cells (combined with aluminum hydroxide gel) show mycoplasma pneumonia and mycoplasma hyonoimoniae lung after passive transfer of colostrum. Reported that it stimulated the development of immunoglobulins that significantly protect the piglets from transplantation into the piglets.As the vaccine was evaluated for only 10 weeks, it has long-term immunity until it is post-development and matures. Not shown.

"Lloyd et al. (1989) Aust. Ve
t. J. 66 : 9-12 "reported that intraperitoneal injection of a strain of live LKR of Mycoplasma hyonoimoniae provided some protection against infection from artificially infected pigs. Was significantly lower in the injected population than in the control population (P <0.05),
Moreover, the proportion of severely infected pigs was significantly lower in the injected population than in the control population (P <0.01). This study suggests that living microorganisms raise questions about whether they should be used to protect pigs in a commercial environment.

Currently, there is no commercially available vaccine against Mycoplasma epidemics (Fitztelstone (1990) Abl. Suppel. 20, Gust.
avFischer Verlag, Stuttga
rt, New York, pp 254-259).
For a variety of reasons, none of the experimentally prepared and tested vaccines performed satisfactorily in field experiments and, as a result, none are currently in common use.

[0017]

SUMMARY OF THE INVENTION The present invention relates to Mycoplasma hyonoimoniae exhibiting unique physical and immunological biological properties. In an example, the invention relates to Mycoplasma hyonoimoniae strain (strain) PRIT-5. The PRIT-5 strain (strain) is characterized by strong antigenicity and high growth rate. PR measured by metabolism test
The antigenicity of the IT-5 strain is a standard J strain (American culture population (ATCC) accession number 2
5934), which is about 4 to 16 times more effective than the other strains of Mycoplasma hyonoimoniae. Mycoplasma hyonoimoniae antigens cultivated in the culture medium of PRIT-5 are heat-labile,
It is acid-sensitive and its antigenicity is reduced at 50 ° C or higher, at PH3 or lower, and by exposure to trypsin. Furthermore, Mycoplasma hyonoimoniae strain PRIT-5 grows rapidly, and after about 48-72 hours at 37 ° C., 10 ⁹ -10 ¹⁰ color changing units (c) per milliliter.
cu / ml) is reached.

The present invention provides useful vaccines to protect pigs from mycoplasma pneumonia. The vaccine of the present invention has a high antigenicity when administered to pigs and comprises surface suspension from a culture of Mycoplasma hyonoimoniae capable of eliciting an immune response. Vaccine prepared from Mycoplasma hyono imoniae PRIT-5 (adjusted)
Preferably. The vaccine of the present invention is about 1.5 ×
It preferably contains bacteria at a density of 10 ⁹ -10 ¹⁰ ccu / ml and water-soluble antigen at a concentration of about 65-80 μg / ml, and is preferably used by administration (dosing).

The present invention also provides a method of vaccinating pigs against Mycoplasma pneumonia, wherein the vaccinated pigs show a statistically significant reduction in lung lesions, and the vaccinated pigs result in weight loss. No decrease is shown. In the examples of the invention, the vaccinated pigs maintained normal (control or non-vaccinated) body weight at slaughter, and in a more preferred embodiment, the vaccinated pigs showed weight gain at slaughter.

[0020]

EXAMPLES The following definitions are provided to provide clarity as to the intent or scope of use in the specification and claims. As used herein, the term "antigenic active substance (anti
genically active substance) "is" Taylor Robinson other (1969) J.Gen.Micrbiol. 5
5 : 127-137 ", and 1 from a culture of Mycoplasma hyonoimoniae that exhibits an antigenicity of at least about 1024 in an antimetabolite experiment.
Reference is made to surface suspensions of 20,000 xg and 16,000 xg. The term includes antigens and (a) epitopes that give rise to antibodies recognized by the antigen,
(B) It contains a fragment of an antigen that immunoreacts with the serum of an animal (especially, a pig) infected with Mycoplasma hyonoemoniae.

Antigenic efficacy or antigenicity (antigenic po
tency or antigenicity) is the degree of antigen concentration, and as is well known in the art, refers to the degree of inhibition obtained by a standardized Metabolic Inhibition Test, and refers to the degree of inhibition by a specific antibody. Based on the characteristics of Mycoplasma regarding the prohibition of. This test bans metabolic activity with mycoplasma, especially
It is based on determining the amount of growth inhibition by the antibody as measured by glucose fermentation, arginine hydrolysis or tetrazolium reduction. As for the present invention, the metabolism experiment is described in “Taylor Robinson et al.
9) J. Gen. Microbiol. 55 : 127
-137 "method based on the microtiter plate
er plate). A culture of Mycoplasma hyonoimoniae 24-hour broth diluted to 10 ⁻³ was used as the antigen. The term "potent antigenicity or enhanced antigenicity"
genicity) "as defined herein is a mycoplasma showing a value of at least about 1024 in an antimetabolite test.
References to Hyonoimoniae.

The term “culture surface or suspension from a culture of Mycoplasma hyonoimoniae”.
e of Mycoplasma hyopneumonia e) "is 10,000 × g
And the surface suspension obtained by centrifugation of the culture of Mycoplasma hyonoimoniae at a rate of between 16,000 × g.

The term “protecti” when used with respect to the vaccine of Mycoplasma pneumonia described herein.
"On or protecting)" means that the vaccine prevents mycoplasma pneumonia and / or reduces the severity of mycoplasma pneumonia as statistically judged from the aggregation of lung lesions.

The term "positive-con
”) refers to pigs that have not been vaccinated, but have been subjected to the challenge test, as further defined in Table 1.

The term "negative-con
”) refers to pigs that have not been vaccinated and have not been challenged, as further defined in Table 1. "Color changing units per milliliter or ccu / ml" is the highest of mycoplasma suspensions that produces a change of half a PH unit of a standard broth base medium with additives at a specific time. Reference to dilution.

Effective dose of vaccine
vaccine) refers to an amount of vaccine sufficient to protect pigs against Mycoplasma pneumonia. This administration elicits an immune response in the subject animal. In an embodiment of the present invention, an effective dose of vaccine is 2 ml containing organisms at a level of 10 ⁹ -10 ¹⁰ ccu / ml and protein at a concentration of 65-80 μg / ml.
Was determined to be an aliquot of.

[0027] A drug-accepting carrier (base) (pharmacologic
“Ally acceptable carrier” refers to a carrier (base) adopted for an antigen, and may be any one of a wide variety of carriers. Representative examples of suitable carriers are, for example,
Mineral oil, alum, synthetic polymers, etc. Vaccine carriers are well known in the art and selection of an appropriate carrier is considered to be within the skill of one in the art.
The choice of suitable carrier also depends on the way the vaccine is administered. The vaccine may be in the form of injectable, for example, intramuscular, subcutaneous, intraperitoneal, or intravenous. In addition, it is also possible to administer the vaccine orally by mixing the active ingredient with feed or water by presenting a tablet form or the like.

Adjuvant refers to a substance whose admixture with an injected immunogen increases the response. Widely used procedures are inorganic gels such as alum,
Involve administration of aluminum hydroxide or aluminum phosphate. Some of the most effective adjuvants are emulsions of water in oil,
In particular, a suspension of live or dead mycobacteria, such as complete Freund's adjuvant (c
Omplete Freund's adjuvant). However, mycobacteria-free emulsions (incomplete Freund's adjuvant) are less irritating and are often used.

Evaluation value of lung or evaluation value of lesion of lung (lun
g score or lung lesion score) refers to the degree of lesion detected in lung cells. The evaluation system is "Goodwin and Whittleston (1973) Br. Vet.
J. 129 : 456-462 ". 10 points are assigned to each pneumonia and lobes near the heart, 5 points to the middle lobe, and 5 points.
A point is assigned to the beginning of each phrenic lobe. Therefore,
If all these areas are fully strengthened, the evaluation value is 55.
become.

Market weight refers to a weight of about 90 kg, at which time the pig is handed over to the slaughterhouse.

The present invention arises from the need to control the onset and spread of Mycoplasma pneumonia in pigs in many countries of the world.
There is a long-standing interest in the development of vaccines against Mycoplasma pneumonia because the economic impact of the plague has not been resolved by other means.

Mycoplasma hyonoimoniae (Myc
oplasma hyopneumoniae) is a vector that has been known to cause mycoplasma pneumonia in pigs, and only pigs are known to be hosts (hosts) of Mycoplasma hyonoimoniae. Therefore, live, attenuated, or dead Mycoplasma hyonoimoniae, or a specific antigen thereof, is considered to be the best candidate to protect pigs from Mycoplasma pneumoniae.

Applicant used mycoplasma hyonoimonia estrain PRIT from lung tissue of 83 infected pigs.
-5 was separated. This PRIT-5 strain is characterized by enhanced antigenicity when compared to the strains of Mycoplasma hyonoimoniae et al. The efficacy of the antigen measured in the anti-metabolism test of Mycoplasma hyonoemoniae is about 1024, and the standard J
Other strains, including strain (ATCC 25934), have efficacy values between 64 and 256. In addition, the Applicant has found that Mycoplasma hyonoimoniae strain PRIT-5 has a temperature of between 35 and 38 ° C.
It was observed after 72 hours that a culture density of approximately 10 ⁹ to 10 ¹⁰ ccu / ml was reached. Other strains of Mycoplasma hyonoimoniae achieved densities of about 10 ⁸ to 10 ⁹ ccu / ml under the same growth conditions.

In addition, Applicants have discovered that the culture media used to culture Mycoplasma hyonoimoniae affected the tissue and antigenicity of Mycoplasma. Applicants used essentially Friis culture medium as follows. Hank's solution 600-800 ml Distilled water 900-1100 ml Bacte brain heart infusion 10-13 gm Bacterial PPLO fluid medium 11-13 gm yeast extract 80-100 ml HEPES buffer 5-8 gm Porcine serum 400-500 ml The pH of the medium was adjusted to a value between 7.4 and 7.6 and the medium was filtered to remove the precipitate. Prior to the addition of porcine serum, the serum was quiesced for 30 minutes at 56 ° C., then for 48 hours at 37 ° C. before centrifugation at 10,000 × g for 1 hour. In the examples, the PRIT-5 culture medium was 700 ml of Hank's solution, 1000 ml of distilled water, 11.5 g of bacterial brain heart mixture, and 12 g of bacterial PPL.
5. O broth, 90 ml yeast extract,
The pH was adjusted to 7.5 with 5 g of HEPES buffer and 450 ml of porcine serum.

The tissue of mycoplasma is extremely variable, with particular species showing a tendency to grow in one of a variety of tissue shapes, but the composition of the culture medium influences that shape to predominate. To do. In particular, the tissues of Mycoplasma hyonoimoniae have been shown to have non-negligible variants associated with, for example, the binding of serum from different animal species of growth media (Wen, 1989, J.C.
shine Soc. Vet. Sci. 15:41
-46). Mycoplasma hyonoemoniae organisms grew primarily as a single unit of media, such as Friis medium containing horse and pig serum or dialyzable medium containing only pig serum. In contrast, Mycoplasma hyonoimoniae developed into aberrant forms such as filaments, rings, and long spherical chains when grown in an unfavorable medium, such as rabbit muscle infusion fluid culture medium (Friis, 1977, Acta Vet. S
cand. 18 : 168-176). Furthermore, when cultivated in the Friis culturing medium, the mycoplasma was contaminated separately and had a rod-like appearance with dense stains with Giemsa Stain on both ends. This is in contrast to mycoplasma suspended in other culture media that appeared clustered with irregular tissues. In addition, Mycoplasma hyonoimoniae cultured in Friis medium showed enhanced antigenicity and higher growth rate than those cultured in other media.
The advantages provided by this culture medium are that it facilitates the preparation of the medium, stabilizes the PH of the medium, increases the antigenic durability of mycoplasma, and facilitates the visual perception of biological tissues.

According to the present invention, the antigenically active substance, the drug-accepting base and the vaccine composition are provided for administration to pigs to stimulate the immune response. Antigenic actives include surface suspensions from cultures of Mycoplasma hyonoimoniae that have an antigenic efficacy of at least about 1024 in the antimetabolite test. In the examples, the antigenically active substance is 1 from a culture of Mycoplasma hyonoimoniae PRIT-5.
Includes 10,000 × g-16,000 × g surface suspension. Whole killed mycobacteria may be used to provide an adjuvant action. Inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate may be added to elicit a greater biological response.

In general, the vaccine is about 130 per dose.
-Contains 160 μg of active substance. Vaccines against Mycoplasma hyonoimoniae previously prepared in the art are generally similar for each dose of vaccine,
Alternatively, higher amounts of antigenic active substance were used. For example, Folds (EP 0,283,840) contains at least 5 μg and preferably 100 μg per dose.
It teaches vaccines containing μg of antigenic protein, while Buzzband (WO 90/07935) teaches a vaccine containing 21.65 mg (wet weight) of antigenic protein per dose. In this example, lowering the antigen level in the vaccine required to elicit an immune response in pigs substantially substantiates the antigenic active substance's potency antigenicity of the culture surface suspension of the invention. Furthermore, the antibody titer (titer) of 6 immunized piglets was greater than 256 when compared to control.

As described above, for example, strain PRIT-5, the antigenically active substance of the vaccine of the present invention is a mycoplasma containing a polypeptide and a peptide in order to show the ability antigenicity of Mycoplasma hyonoimoniae. It includes the surface antigen of Hyonoimoniae and its glycosyl group derivative. It should be understood that these peptides or peptide fragments include chemically modified or synthetic derivatives thereof, as is known in the art. All that is required is that the final polypeptide exhibit the potency antigenicity of Mycoplasma hyonoimoniae, as defined above. Such alterations at the amino acid level may be used to improve benefit or antigenicity, or to improve the compatibility (compatibility) of various purification regimens, adjuvants and modes of administration. good.

The present invention also provides a method of stimulating an IgG response in an animal, comprising at least about 1 in a metabolic inhibition test.
Comprising administering to said animal a vaccine composition comprising a surface suspension from a culture of Mycoplasma hyonoimoniae having an antigenic efficacy of 024 and an antigenically active substance comprising a drug-accepting base. The vaccine is administered in an amount sufficient to elicit antibody formation. A study of antibody levels in each age group of piglets showed that antibody levels rose again at about 4 weeks of age, and the best time to immunize piglets was at about 4 weeks of age. There is. The present invention also provides a vaccine method for protecting pigs from mycoplasma pneumonia by the vaccine composition of the present invention, wherein the lesions of the lungs are under laboratory infection-induced conditions as in the commercial population under natural infection conditions. Vaccination in an experimental population of
As shown in Tables 2 and 3, in the laboratory and field experiments, the vaccinated population showed a mean lung score that was statistically significantly lower than that of the control population.

The present invention provides a vaccine for use in pigs, which provides effective immunity without undesirable side effects. The vaccine immediately impacted the pig industry and the study reported about 12 pigs slaughtered with a market weight of about 85 Kg.
It has been shown that a decrease in growth rate in the range of% -17% occurred (Pointon et al. (1985) Aust. Ve.
t. J. 62 : 13, Goodwin (1963) B
r. Vet. J. 119 : 298, and Hetz and Beveridge (1953) Vet. Rec. 65:51
5). The present invention provides a method of administering a vaccine to protect pigs from Mycoplasma pneumonia, said pigs exhibiting no weight loss as a result of said vaccine. The assertive evaluation of field vaccines for pneumonia control is generally accepted as slaughter weight (Husband WO 90/0).
7935, International filing date January 19, 1990). In the examples of the present invention, the average weight of the vaccinated pigs was 2 kg higher than the weight at slaughter or market weight, that is, about 90 kg of control pigs. This is based on Husband (WO 90/07935, International Filing Date 1990 1
In contrast to the results of the field experiment obtained by (19th month), no significant weight gain is seen in the vaccinated population immediately before sacrifice.

Applicant's study investigating weight gain in pigs with mycoplasma pneumonia investigated the relationship between lung lesion score and body weight. Pigs with mild lung lesions lost about 4.86 kg, pigs with severe lung lesions lost about 11.7 kg, and the rate of weight gain was affected by lung lesions of pneumonia (Example 5). And Table 5). Pigs vaccinated by the method of the present invention showed reduced or negligible lung lesions and no weight loss or, in the examples, protection against weight gain mycoplasma pneumonia.

It will be appreciated by those skilled in the art that various changes and modifications can be made other than as described for the methods of preparation and administration of the invention described herein and the vaccines specifically described herein. It is to be understood that this invention includes all such changes and modifications that come within the spirit and scope thereof. The following examples are given by way of illustration, without limiting the scope of the invention. Example 1: Mycoplasma hyonoimoniae PR
Culturing of IT-5 Mycoplasma hyonoimoniae PRIT-5 was isolated from lung diseased tissue of 83 pigs showing mycoplasma pneumonia. Strain PRIT-5 is derived from other strains of Mycoplasma hyonoimoniae, such as "Taylor Robinson et al. (1969) J. Gen. Mi.
It is distinguished by anti-metabolism test and proliferation test based on the procedure of crobiol 55 : 127-137 ".
The cells were cultured at 37 ° C in PRIT-5 culture medium having the following composition. Hank's solution 600-800 ml Distilled water 900-1100 ml Bacte brain heart infusion 10-13 gm Bacterial PPLO fluid medium 11-13 gm yeast extract 80-100 ml HEPES buffer 5-8 gm Porcine serum 400-500 ml Porcine serum was incubated at 56 ° C for 30 minutes without shaking, then at 10,000 xg for 1 hour to remove the precipitate. The cells were incubated at 37 ° C for 48 hours before being subjected to centrifugation. The pH of the culture medium was adjusted to 7.4-7.6 and then the medium was filtered through sterile filter paper.

The metabolism inhibition test was conducted by Taylor Robinson et al. (1969) J. Gen. Microbiol.
55 : 127-137 ", a 24-hour culture of Mycoplasma hyonoimoniae was used as an antigen. The tissue specimen, Giemsa.
The stain process was performed as follows. Smear (Sear
s) was allowed to settle in methyl alcohol for 3 hours and diluted for 1 hour with guimus solution (2 in dilute phosphate buffer).
Drops of stock Giemsa stai
n) is dropped, and slide down (slide) by PH6.8)
stain) was applied so that (down). After staining, the smears were washed with two changes of the same dilution buffer solution and dried in air. Example 2: Vaccine preparation PRIT-5 strains were cultivated by continuous shaking for 48-72 hours at a low temperature of 37 ° C., at which time colonies were 10 ⁹ -10 ¹⁰ ccu /
reach ml). The culture is then 10,000 ×
g, or preferably 16,000 × g, 4
Centrifugation was carried out by frozen centrifuge at 1 ° C for 1 hour. The surface suspension was filtered through an Amicocampani filter to remove components with high molecular weight. The protein concentration of the filtrate is 28 nm with an ultraviolet-spectrophotometer.
It was found to be about 40-60 μg / ml. Bacteria in which the water-soluble antigen was rapidly precipitated were mixed and treated with 0.1-0.2% formalin.
It was allowed to rest overnight at ° C. The suspension was neutralized with NaHSO ₃ on the second day. The formalin-inactivated vaccine was then mixed with aluminum hydroxide gel in a 3: 1 ratio and stored at 4 ° C. This vaccine is 1.5 × 10 ⁹ -10 ¹⁰ ccu
Used as a 2 ml dose containing organisms at a density of 1 / ml and water-soluble antigen at a concentration of 65-80 μg / ml. Each pig received two doses intramuscularly. The first dose is 4
The second dose was given at the age of 6 weeks, and the second dose was given at the age of 6 weeks. The vaccine was mixed well before use.

Standard methods known to those skilled in the art are used in preparing the vaccines of the invention for administration to pigs. For example, selected antigens are lysed with sterile saline. For long-term storage, the antigen is lyophilized and then liquefied with sterile saline solution shortly before administration. Preservative and other standard additives such as providing bulk, eg glycine or sodium chloride, may be added prior to lyophilization. A compatible adjuvant may be administered with the vaccine.

Vaccines according to the invention may be prepared using antibodies raised against the antigen of the invention in laboratory animals such as rabbits. This "passive" vaccine can be given to pigs to protect them from infection with Mycoplasma hyonoimoniae. Example 3: Immune Response to Vaccine (a) Serum Test Three serum methods were used to detect the antibody response of porcine Mycoplasma hyonoimoniae, ie:
"Wen, CN (1985) Ph.D Disse.
rotation, Wotson College, C
Ambridge, UK, pp. 95-109 ";" Wen et al. (1991) Biotechnol. Pro
g. 7 : 69-71 ";" Polar et al. (1977) The.
Zoological Society of Lo
ndon: Regent Park, Englan
d ";" Freeman et al. (1984) Can. J. Com
p. Med. 48 : 202-207 ", indirect hemoglobin agglutination test (IHA), single basal immunohemolysis (SRH), and enzyme-linked immunosorbent assay (ELISA). Compared with others. And SR
H is highly sensitive to IgG and less sensitive to IgM and IgA. After infection, the first antibody to appear was IgM, followed by IgG,
On the other hand, a small amount of IgA was observed. IgM then disappeared leaving only IgG. The vaccine given intramuscularly generated secretory IgG preferentially in the respiratory system. However, the major antibody generated by pigs naturally or artificially infected was respiratory IgA.
IHA was employed to observe changes in antibody levels in pigs of each age group. The antibody dropped to a low level of 6% at the age of 4 weeks and rose again at the age of 6 weeks. It is therefore concluded that the best time to immunize pigs is about 4 weeks of age. (B) Immune Response Suppressing Effect Mycoplasma hyonoimoniae has an immune suppressing effect on infection as described in “Wen and Lin (1988) J. Ch.
inse Vet. Sci. 14 : 267-273 "
Was reported by the inventor of. Regarding the immunosuppressive action of T cells and B cells, it was observed that the host was infected with Mycoplasma hyonoimoniae. This meant the introduction of a mixed infection by the secondary pathogen. T cells are Co-6
It was believed that pigs played an even more important role in Mycoplasma hyonoimoniae infection if exposed to 0 or if B cells were banned by cyclophosphamide. Example 4: Effect of vaccination on the induction of mycoplasma pneumonia (a) Safety test 4 SPF pigs received 2 doses of vaccine at the age of 5 weeks and received 2 boosters 2 weeks later. Two weeks after the booster administration, the two pigs were euthanized,
Four weeks later the other two were euthanized.
The major organs (lung, liver, kidney and spleen) were examined microscopically and pathological cuts showed no abnormal changes or negative pathogen isolation. Furthermore, no hypersensitivity (anaphylaxis) or abnormal findings were observed. (B) Determination of vaccine efficacy (potency) In a pilot study, 9 Caesarean section SPF pigs were divided into 3 groups, the first group was immunized (vaccinated, induced) and the second group. Group was positive control (non vaccinated, induced) and the third group was negative control (non vaccinated, uninduced). The results of this experiment are shown in Table 1.

[Table 1] Each experimental group contained 3 pigs. IT is intratracheal, IM is intramuscular, MH is mycoplasma hyonoimoniae. A total of 18 pigs were tested in the extension (expand) repeat experiment. Serum antibody measurements were performed 2 weeks before and after each experiment. The results of this experiment are shown in Table 2.

The effect of Mycoplasma hyonoimoniae is shown in Table 2 and shows good immunoprotection. Descriptive statistics calculated for lung scores indicate that the mean lung score in the vaccinated group is significantly lower (P <0.01) than that of the control group. The lung lesions of slaughtered pigs were recorded graphically,
The evaluation value is “Goodwin and Whittlestone (1973) Br. Vet. J. 129 : 456-4.
Calculated by the method of 62 ".

[Table 2] It was revealed that the three immunized pigs had no gross lesions in the lungs (Fig. 1) and only minor abnormalities were found in the other three pigs. In contrast, in the positive control group, each lobe of the lung was severely damaged (Fig. 2). Large differences existed between the two groups of pathological fragments. Figure 3 shows normal lung tissue of immunized pigs. FIG. 4 shows the lung tissue of the positive control group in which severe changes in mycoplasma pneumonia were found. In addition, the precipitate from the centrifuged respiratory lavage showed significant differences under the electron microscope. As shown in FIG. 5, immunized porcine respiratory macrophages (macrophages) appeared to phagocytose many mycoplasma showing increased activity of immunized porcine macrophages. . In contrast, in the positive control pigs, only one mycoplasma was swallowed by respiratory macrophages (FIG. 6), whereas many mycoplasmas appeared to be still outside the macrophage.

FIG. 7 shows the antibody response after vaccination. The average efficacy of antibody in 6 immunized pigs was greater than 256X for 4 weeks only. This means that the vaccine was strongly antigenic. Example 5: Evaluation of field tests of the protective effect of vaccination (a) Mycoplasma myonoplasma pneumonia in pigs Field test with Vaccine Mycoplasma pneumonia in pigs is an economically important disease.
Efforts have been made to control the illness with drugs or administrative measures, but they are still widespread. Therefore, efforts are being made to develop vaccines that prevent it. Experimental data provide protection against experimental induction of Mycoplasma hyonoimoniae. However, it is unpredictable whether such experimental studies will be relevant to field experiments.
This study evaluated the protective effect of vaccination under field conditions. Two pig farms (one breeding farm and one pig farm) were used for this field experimental study. 200 piglets were selected from each pig farm and randomly divided into two groups. Group 1 was vaccinated at the age of 4 weeks. Group 2 was injected with saline as a control. All animals were raised to market weight and gross lung lesions were evaluated at the slaughterhouse. The results are shown in Tables 3 and 4. Table 3 shows the statistics of lung evaluation values, the mean evaluation value of lung lesions in the vaccinated groups of beef pigs and breeding pigs is significantly smaller than that of the control group.

As recorded in Table 4, the average weight of vaccinated pigs was 2 kg heavier than the control pigs,
The value of lung lesions for vaccinated pneumonia was less severe than that of control pigs. FIG. 8 shows the normal lung of vaccinated pigs, in contrast FIG. 9 shows the severe lung lesions of control pigs. Again, the contrast between Figures 8 and 9 shows the protection afforded by the vaccine.

[Table 3]

[Table 4] To understand the effect of weight gain in pigs with endemic pneumonia, the relationship between lung lesion rating and body weight was investigated. A total of 143 pigs were used in the study. As shown in Table 5, the average daily increase was 0.9 in 39 pneumonia-free lung lesions.
It was 543 Kg. ADG was 0.516K in the group with low lung lesions of 31 pneumonia (evaluation value range 1-5)
It was g. In 41 mild pneumonia lesion groups (evaluation value 6-20), ADG was 0.507 Kg. In the group of 27 severe lung lesions, ADG
Was 0.478 Kg. The data described here indicate that the rate of weight gain is affected by lung lesions of pneumonia. 3. Compared to control pigs without mild lung lesions for pigs raised for 180 days.
Calculated as weight loss of 86 kg. Also, pigs with severe lung lesions (evaluation value 21-55) lose 11.7 kg of body weight as compared to control pigs without lung lesions. These results again show that mycoplasma pneumonia in pigs is a major disease of the pig industry.

[Table 5]

[Brief description of drawings]

FIG. 1 shows the appearance of the lungs of post-challenge vaccinated pigs. The lesion is not clear.

FIG. 2 shows the appearance of lungs of control pigs after induction. Severe lung lesions are shown.

FIG. 3 shows the histology of post-induction vaccinated pig lung tissue. The appearance is normal. HE stain, 40X.

FIG. 4 shows histology of lung tissue of control pig lungs after challenge. Lung tissue exhibits severe histopathology. HE stain, 40X.

FIG. 5 shows phagocyosis of Mycoplasma hyonoimoniae by macrophages of the respiratory system of pigs vaccinated after induction.

FIG. 6 shows Mycoplasma hyonoimoniae present outside macrophages of the respiratory system of control pigs after induction. A few Mycoplasma hyonoimoniae were phagocytosed by respiratory macrophages.

FIG. 7 shows antibody response of vaccinated pigs as a function of time (weeks) post vaccination. ↑ ↓ Trigger, ---
--- Vaccination group, --- Positive control group,-
... Negative control group. Each antibody response represents the average response of 6 pigs in each group.

FIG. 8 shows the appearance of vaccinated lungs in a field experiment. No lung lesions are observed.

FIG. 9 shows the appearance of lungs of control pigs in the field experiment.
Severe lung lesions are shown.

[Procedure amendment]

[Submission date] November 11, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 1]

[Fig. 2]

[Figure 3]

[Figure 4]

[Figure 5]

[Figure 6]

[Figure 7]

[Figure 8]

[Figure 9]

Claims (19)

[Claims] 1. Metabolic Inhibiti
on Test) with at least about 1024 antigenic potency Mycoplasma hyo
pneumoniae) culture surface suspension (supernatant),
A vaccine for protecting pigs against Mycoplasma pneumoniae, which comprises a drug-accepting carrier. 2. The surface suspended matter is 10,000 × g and 1
The vaccine for protecting pigs against mycoplasma pneumonia according to claim 1, which is a surface suspension obtained by centrifugation at a speed between 6,000 xg. 3. The culture of Mycoplasma hyonoimoniae is 10 ⁹ -10 per milliliter.
A vaccine for protecting pigs against Mycoplasma pneumoniae according to claim 1 having a density of ¹⁰ color changing units. 4. The vaccine for protecting pigs against mycoplasma pneumonia according to claim 1, wherein said Mycoplasma hyonoimoniae is Mycoplasma hyonoimoniae strain PRIT-5. 5. A vaccine for protecting pigs against Mycoplasma pneumoniae according to claim 1, wherein said vaccine comprises about 130 μg of culture surface suspension protein per dose of vaccine. 6. (a) At least 1 in the metabolism inhibition test.
A surface suspension having a water-soluble antigenic substance of a culture of Mycoplasma hyonoimoniae having an antigenicity of 024 is prepared, and (b) the surface suspension is mixed with an adjuvant (adjuvant) and a drug-accepting base. A method for producing a vaccine for protecting pigs against Mycoplasma pneumonia, which comprises the steps of: administering the vaccine to pigs elicits an immune response. 7. The surface suspended matter is 10,000 × g and 1
7. A vaccine for protecting pigs against Mycoplasma pneumoniae according to claim 6, which is a surface suspension obtained after centrifuging the culture of Mycoplasma hyonoimoniae at a rate of between 6,000 xg. Method. 8. A vaccine for protecting pigs against mycoplasma pneumonia according to claim 6, wherein the culture of Mycoplasma hyonoimoniae has a density of 10 ⁹ -10 ¹⁰ color changing units per milliliter. Method. 9. The Mycoplasma hyonoimoniae strain is a Mycoplasma hyonoimoniae strain.
The method for producing a vaccine for protecting pigs against Mycoplasma pneumonia according to claim 6, which is PRIT-5. 10. At least 102 in the metabolism inhibition test.
Comprising the step of administering to a pig an effective dose of a vaccine containing a drug-accepting base and a surface suspension from a culture of Mycoplasma hyonoimoniae having an antigenic efficacy of 4.
A vaccination method for protecting pigs against mycoplasma pneumonia, which is characterized by preventing or reducing the severity of mycoplasma pneumonia, which is statistically determined by the evaluation value of lung lesions when an immune response is elicited. 11. The surface suspension is obtained after centrifugation of the culture of Mycoplasma hyonoimoniae at a rate between 10,000 × g and 16,000 × g. A method of vaccination for protecting pigs against Mycoplasma pneumoniae according to Item. 12. The method of vaccination for protecting pigs against mycoplasma pneumonia according to claim 10, wherein the culture of Mycoplasma hyonoimoniae has a density of 10 ⁹ -10 ¹⁰ color changing units per milliliter. 13. The method of vaccination for protecting pigs against mycoplasma pneumonia according to claim 10, wherein said Mycoplasma hyonoimoniae is Mycoplasma hyonoimoniae strain PRIT-5. 14. The method of vaccination for protecting pigs against Mycoplasma pneumoniae according to claim 10, wherein said vaccine comprises at least 130 μg of culture surface suspension protein per dose of vaccine. 15. The method of vaccination for protecting pigs against Mycoplasma pneumoniae according to claim 10, wherein the administration of the pigs does not result in weight loss as a result of the vaccination. 16. The method of vaccination for protecting pigs against mycoplasma pneumonia according to claim 15, wherein said pigs exhibit weight gain as a result of said vaccination. 17. The weight gain is about 2 in market weight.
The method of vaccination for protecting pigs against mycoplasma pneumonia according to claim 16, which is Kg. 18. The method of vaccination for protecting pigs against mycoplasma pneumonia according to claim 10, wherein said Mycoplasma hyonoimoniae is Mycoplasma hyonoimoniae strain PRIT-5. 19. At least 102 in the metabolism inhibition test.
With an antigenic efficacy of 4 and 1 after 48-72 hours at 37 ° C.
A suspension characterized in that it contains Mycoplasma hyonoimoniae growing to a density of 10 ⁹ -10 ¹⁰ color changing units per milliliter.