DK162509B - Vaccine against diseases caused by mycoplasms, its preparation, and mycoplasma membranes as the active principle - Google Patents

Description

DK 162509 B

The invention relates to a vaccine against diseases caused by mycoplasmas, its preparation and mycoplasma membranes as an active principle.

5 Diseases caused by microorganisms that have a membrane like outer envelopes, such as gram-negative bacteria and mycoplasmas, are very widespread, especially in livestock breeding, where they are responsible for a significant loss of income. This is particularly the case in intensive pig breeding, where enzootic pneumonia in pigs caused by Mycoplasma hyopneumoniae is very frequent.

A recent article on modern pig breeding, W. Pond, Pour la Science 66, 60-68 (1983), describes the significance that this type of breeding currently has in the world. Pigs in particular give approx. 25% of the energy and 9% of the protein of animal origin. In breeding, pigs produce a greater yield than beef cattle, sheep and poultry.

Moreover, pork has excellent nutritional qualities and is appreciated by consumers. It is therefore not surprising that pig farming has a not insignificant place in the economy of several countries in North and South America,

Europe, Asia and Oceania. This applies to both highly industrialized countries and less industrialized countries. In France, especially in Brittany, pig breeding is an essential one

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part of the agricultural economy.

The methods of breeding pigs have been greatly improved, both in terms of yields obtained and the conditions of breeding, especially the hygienic conditions. However, the improvements have not made it possible to protect animals 30 from a certain number of diseases, including mycoplasmosis. Among diseases affecting the respiratory system of pigs, pneumonia is very common, especially in intensive breeding. In a three-year epidemiological study conducted in Brittany, Mycoplasma hyopneumoniae has been detected in 35 to 42% of lung lesions in pigs aged 8-12 weeks.

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On the other hand, anti-M. hyopneumoniae antibodies have been detected by indirect hemagglutination in 801 of the herds (M. Kobisch et al., Journées de Recherche Porcine and France (1977), pages 161-164; M. Kobisch et al., 5 Rec. Med. Vét. 154 (1978), pages 847-852; and JP Tillon,

Journées de Recherche Porcine and France (1980), pages 361-380). The primary etiological role of M. hyopneumoniae in swine enzootic pneumonia has been demonstrated by R.F.W. Goodwin et al., British Vet. TJ_ (1973), pages 10 456-464, and reviewed by W.P. Switzer et al., Diseases of

Swine (1975), 4th ed., Dunne and Lemans Eds., Iowa University Press.

Respiratory diseases are responsible for a significant decrease in yield in several pig herds. It is possible to fight bacteria using antibiotics, but mycoplasmas pose a particular problem related to their simple structure. Since they have no cell wall, these microorganisms are completely insensitive to any antibiotic whose target is precisely the cell wall, such as penicil-20 lin, cephalosporin and bacitracin. On the other hand, the plasma membrane of mycoplasmas will be impermeable to a large number of antibiotics whose targets are intracellular (S. Razin, Microbiol. Rev. (1978), pages 414-470).

It appears that vaccination will be an effective and profitable way to fight mycoplasmas.

There is currently no vaccine for diseases caused by mycoplasmas on the market. Admittedly, vaccines have been proposed for these diseases, such as live attenuated strains (U.S. Patent No. 30,343,136) or vaccines containing complete myoplasmic cells, which are doped with heat, with an agent such as formaldehyde, sodium ithyl mercuric salicylate or β-propiolactone, or by ultrasound treatment (FR Patent Application No. 2,201,878). However, for various reasons, none of these 35 vaccines are satisfactory or commonly used.

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It has been found, according to the invention, that the plasma membrane of a mycoplasm having a membrane as an outer envelope may constitute a particularly effective active principle of a vaccine against diseases caused by the mycoplasm when the plasma membrane is separated from the rest of the cell.

The invention therefore relates to a vaccine for protection against diseases caused by a mycoplasma, which vaccine is characterized in that it contains as active principle 10 plasma membranes of the mycoplasma concerned, which are at least largely released from the other components of the mycoplasma.

Mycoplasms include: - in poultry pathology: Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma meleagridis; - in meat cattle pathology: Mycoplasma mycoides. Mycoplasma bovis, Mycoplasma bovirhinis and Mycoplasma bovigenitalium; - in sheep and goat pathology: Mycoplasma 20 agalacticae, Mycoplasma ovipneumoniae, Mycoplasma capricolum and Mycoplasma mycoides subspecies capri; - in equine pathology: Mycoplasma equigenitalium, Mycoplasma equirhinis, Acholeplasma heppikon and Acholeplasma equiletale; and 25 - in the field of swine pathology: Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.

According to a preferred embodiment, the vaccine according to the invention takes the form of a suspension of membranes in a so-called physiological saline solution, ie. a solution of 30 NaCl at 8.5 g / liter, in the presence of an adjuvant which ensures gradual release of the immunogen.

According to a particularly preferred embodiment, the adjuvant is aluminum hydroxide, agarose or anti-membrane antibodies of the mycoplasm.

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Other adjuvants, especially oily, may be used but are not preferred.

The concentration of proteins derived from membranes in the vaccine is preferably in the range 2-3 g / liter 5 and is advantageously 2.5 g / liter. The concentration of aluminum hydroxide is preferably in the range of 12-13 g / liter depending on the concentration of proteins. It is advantageously 12.5 g / liter at a concentration of proteins of 2.5 g / liter.

Agarose is generally used in the form of a very diluted gel, preferably at a concentration in the range 1.5-2 g / liter depending on the concentration of proteins. It is advantageously 1.7 g / liter at a concentration of proteins of 2.5 g / liter.

According to a particularly preferred embodiment, the invention relates to a vaccine for the protection of pigs from enzootic pneumonia, which is characterized in that it consists essentially of a suspension of membranes of Mycoplasma hyopneumoniae which are liberated - at least in large - for the other constituents of the my-20 plasma, in an amount corresponding to a weight of proteins of 2-3 g / liter, in an aqueous NaCl solution of 8.5 g / liter, in the presence of 12-13 g / liter aluminum hydroxide as adjuvant.

According to another particularly preferred embodiment, the invention relates to a vaccine for the protection of pigs against enzootic pneumonia, which is characterized in that it consists essentially of a suspension of membranes of Mycoplasma hyapneumoniae which - at least to a large extent - are liberated from the other constituents of the mycoplasm, in an amount corresponding to a weight of proteins 30 of 2-3 g / liter, in an aqueous solution of NaCl at 8.5 g / liter, in the presence of 1.5-2 g / liter liter of agarose as adjuvant.

The invention also relates to a method for preparing the vaccine described above, which is characterized in that it comprises substantially the following steps:

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a) culturing the mycoplasm for which a vaccine is desired to be produced until the end of the exponential growth phase; 5 b) harvesting the cells by centrifugation; c) lysing the harvested cells by ultrasound at 20-28 kHz, for several d) sedimentation of the membranes by centrifugation, e) washing the membrane sediment several times with a buffer 10 using centrifuges and dispersions, and f) formulating the membranes into a form useful for vaccination .

The culture of the cells is advantageously carried out at 37 ° C on a liquid medium such as Friis medium (N.F. Friis, 15 North. Vet. Med. 27, 337-339 (1975)).

The cells, which are centrifuged (at about 10,000 g), are washed with a buffer of low basic pH, e.g.

0.1 M sodium phosphate buffer with a pH of 7.5 containing 8.5 g NaCl per ml. liter.

The cells are then dispersed, e.g. by means of a Potter cylinder so that a dense suspension is obtained in a weakly basic buffer, e.g. tris-HCl buffer, 0.1M, pH 8.0. Ultrasonic lysis is performed in several steps, advantageously at increasing frequencies within the range indicated above, in the vicinity of 0 ° C.

After thorough centrifugation (at about 40,000 g for about 1 hour) of the lysate, the membrane sediment is washed thoroughly (several times with buffer, e.g., tris-HCl buffer, 0.1M, pH 8.0) and dispersed and centrifuged several times again.

The membranes thus assembled are ready for use in the preparation of the vaccine. They can be stored at -70 ° C in the form of a concentrated suspension in an aqueous solution of NaCl at 8.5 g / liter, e.g. with 20 mg of protein per day. ml, or in freeze-dried form.

Thus, the invention also relates to new industrial products as new properties: 6

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a concentrated suspension in an aqueous NaCl solution with 8.5 g / liter of membranes of a mycoplasm, at least largely liberated from the other constituents of the mycoplasma, for use as an active principle in a vaccine and - membranes of a mycoplasm, at least largely liberated from the other constituents of the mycoplasm, in freeze-dried form for use as active principle 10 in a vaccine.

The vaccine of the present invention, which does not contain live cells, is free of toxicity and therefore does not present the risk to the treated animal that live vaccines with attenuated strains do, and is at the same time more effective and easier to use than vaccines which simply contain killed cells, without separation of the plasma membranes, such as disclosed in FR Patent Application No. 2,201,878.

Thus, in contrast to what might be expected, the vaccines prepared according to the invention are also active when administered subcutaneously or intramuscularly, while the vaccines disclosed in FR Patent Application No. 2,201,878 have only a preventive effect on the treated animal when inoculated. in trachea by injection or atomization. In addition, when used in pregnant females, the vaccines prepared according to the invention also provide effective protection of the unborn litters, which is a very important benefit to the health of industrial breeding facilities.

Thus, it is possible to effectively protect a litter of pigs from diseases caused by Mycoplasma hyopneumoniae 30 by giving the pregnant sow a subcutaneous or intramuscular injection of a vaccine as defined above 7-8 weeks before birth and then again 2 weeks before the latter.

According to a particularly preferred embodiment, the invention relates to such a vaccine as a unit dose which is characterized in that it consists essentially of a vaccine.

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Suspension of membranes of Mycoplasma hyopneumoniae, which is at least largely liberated from the other constituents of the mycoplasma, in an amount corresponding to a weight of proteins of 4-6 mg, preferably 5 mg, in 2 ml of an aqueous 5 0.35% NaCl solution in the presence of 1.20-1.30%, preferably 1.25%, aluminum hydroxide or 0.15-0.20%, preferably 0.17% agarose, or also a saturating amount for the swine antibody membranes against Mycoplasma hyopneumoniae membranes.

The invention is further explained in the following description of a purely illustrative example of the application of the invention and of the results obtainable thereby.

1. ~ _Production_of_vaccine_mod_MycoDlasma_hyopneumoniae.

1) Cultivation of the mycoplasma.

Mycoplasma hyopneumoniae (strain BQ 14: M. Kobisch et al., Rec. Med. Vét. 152, 817-827 (1976)) is grown at 37 ° C on the liquid medium described by Friis. The cells are collected at the end of the exponential growth phase by centrifugation (10,000 g, 15 minutes at 4 ° C) and washed twice with 0.1 M sodium phosphate buffer, pH 7.5, containing 8.5 g NaCl per ml. liter.

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The cells are dispersed by a Potter cylinder to give a dense suspension in tris-HCl buffer, 0.1M, pH 8.0). They are then illuminated by ultrasound: 2x1 minute at 20 kHz and 1 minute at 28 kHz. The process is carried out at 0 ° C, allowing the material to rest for 1 minute between two consecutive cycles.

The cell lysate is centrifuged for 1 hour at 40,000 g and 4 ° C to settle the membranes. They are washed 4 times with 0.1M Tris-HCl buffer, pH 8.0, by successive dispersion and centrifugation.

The prepared material is stored at -70 ° C, either in the form of a concentrated suspension (20 mg protein per ml) or in lyophilized form.

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s 3) Manufacture_of_vaccine_.

The vaccines consist of membranes of M. hyopneumoniae prepared as described above in suspension in a solution of NaCl at 8.5 g / liter. The concentration of proteins is 2.5 mg / ml. The adjuvants used are: 1.25% aluminum hydroxide (vaccine I), 0.17% agarose (vaccine II) and swine antibody against membranes of M. hyopneumoniae which precipitate the membranes (vaccine III).

10 II - Use of the vaccines.

The vaccines, whose preparation is described above, are used to vaccinate first-born pregnant sows from the protected pig stables at the Station de Pathologie Porcine de Ploufragan (Cotes du Nord, France). The herd 15 of these stables consists of animals born by hysterectomy and kept protected from contamination. The sows used in the trial are placed in sheltered booths 10 weeks before the expected time of birth.

7 or 8 weeks before birth, 20 sows are vaccinated and an intramuscular booster injection is given 2 weeks before birth. Each vaccine portion contains 2 ml of membrane suspension, ie. 5 mg of proteins.

Two test series are conducted: 1_. .Suggestion series: 25 ~ one sow (10043) receives vaccine in, - one sow (10044) receives vaccine II, - two sows (controls 071 and 1010) are not vaccinated.

2. Trial series: - two sows (20008 and 200012) receive vaccine II, 30 - one sow (20013) receives vaccine III, - two sows (controls 20005 and 20007) are not vaccinated.

The sows give birth to the number of pigs listed in the table below: 35

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__§Ο_ηΓΛ__ _ ^} tal_grise___ 10043 9 10044 6 071 11 5 1010 6 20008 11 20012 4 20013 10 10 20005 9 20007 9

All pigs, when 2, 3, 4 and 5 days old, receive a dose of 0.5 ml per day. nostrils of a suspension of

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M. hyopneumoniae with 10 colony forming units per ml. The strain used is the same as that used for the preparation of the vaccine.

The pigs are examined every day. The rectal temperature 20 is taken, the clinical signs are noted daily and blood punctures are performed every week in all the pigs. They are weighed and their feed consumption is determined weekly.

The pigs are killed incrementally in each group within 5 to 10 weeks after infection. After macroscopic examination of the lungs, bacterial and mycoplasma controls as well as histological examinations are performed. Serum antibodies are detected by passive hemagglutination, cf. M. Kobisch et al., Rec. With.

Vet. 154, 847-852 (1978).

30 III - Results ^ 1) Condition_of_2rise_born_of_not-treated_sows.

The results of the first series of experiments confirm the previous observations. The experimentally induced infection with M. hyopneumoniae manifested clinically at 35 hyperthermia and cough, which appeared the day after the infection and continued during the induction period of the lung lesions.

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M. hyopneumoniae inhibited the growth of pigs (compared to pigs born of vaccinated sows). The lung lesions were found to be very extensive at the time of the first kills (5 weeks after infection) and until the end of the experiment (10 weeks after infection). Contrary to what was observed in the previous experiments, no scars corresponding to areas of atelectasis were noted that indicate scarring development. In this experiment, the presence of serum antibodies detected by passive hemagglutination was preceded by the recurrence of the lesions. At the anatomicopathological level, the presence of a pneumonia characterized by lymphoid infiltrates confirmed the macroscopic observations.

The results of the second series are less clear. The pathogenicity of M. hyopneumoniae was less evident, especially in pigs from sow 20007, who had health problems at birth and received antibiotics on days D3 to D6 after birth. M. hyopneumoniae induced cough in all the infected animals but had little effect on growth and body temperature. The lung lesions were unconstrained in pigs from sow 2000. It does not appear that M. hyopneumoniae propagated in the lungs of pigs in this litter within 60 days, although it is present in trachea at the first killing. M. hyopneumoniae was found again in the lungs of 66% of pigs from sow 20005 (in trachea mucus in 88%) and in the lungs in 55% of pigs from sow 20007 (in trachea mucus in 100%) . These results indicate that microbiological determinations of M. hyopneumoniae should be performed not only in the lungs but also in trachea-30 mucus, which proves to be a good indicator of the presence of the mycoplasm.

The level of serum antibodies detected in the pigs did not reach the significance threshold at the end of the experiment.

Regarding the pigs of sow 20005, it should be noted that colostrum antibodies were transferred, which apparently had an effect on the synthesis of active antibodies,

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o which usually follows an infection. The reduced propagation of M. hyopneumoniae during the first stages of the experiment explains the low level of antibodies in pigs from sow 20007.

5 2) Assessment_of_merabranernes_yaccinationsgotentiel.

In the experimental model used, the protective effect of vaccine I (membranes + aluminum hydroxide) on the pigs is 100%. In the case of vaccine II (membranes + agarose) it is 100% in the first test series and 87% in the -ίο second test series. The efficacy of vaccine III (membranes precipitated with antibodies) is only 70% (when excluding the mesothelioma that does not correspond to the lesions described for M. hyopneumoniae infection).

It is noted that the vaccines used elicit 15 serum antibodies in the sows. The pigs born of these sows, after ingestion of colostrum, have antibodies derived from the mother, and their level decreases rapidly with time, reaching a mean value below the significance threshold in the weeks following birth. Six weeks after the infection of the pigs, the 2o sows have an increase in the level of circulating antibodies. Finally, M. hyopneumoniae is only found to a small extent in pigs born of vaccinated sows (13-26%).

3) Conclusion ^.

The experiments carried out show that membranes of M.

25 hyopneumoniae injected subcutaneously and intramuscularly into pregnant sows has a significant protective effect on the pigs (absence of pneumonia and decreased growth). This is especially true when aluminum hydroxide or agarose is used as an adjuvant.

Since the antibodies derived from the maternal animal have a life of the order of 3-4 weeks and the animals are highly susceptible to infection between the 8th and the 12th week, it may in some cases be advantageous to carry out a further vaccination of 6 weeks old pigs , ie at the time point at which the passive antibodies have disappeared and do not prevent vaccination, giving a booster vaccination 3 weeks later, ie. when the pigs are 9 weeks old.

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Incidentally, in a previous trial, a vaccination of 12-week-old pigs and a re-vaccination was performed when the pigs are 14 weeks old. Hereby it was found that the animals respond to the vaccination by synthesizing serum antibodies (passive hemaglutination assay).

These results show that the vaccine of the invention, at very reduced cost due to its simple preparation, enables effective control of diseases caused by mycoplasmas, and especially respiratory diseases that affect a large number of pig stables and significantly reduce their yield.

It should be noted that Friis medium has the following composition: 15 ^ Hank's saline solution 500 ml. distilled water 750 ml

A

C "Bacto brain heart infusion" (Difco) 8.2 g "Bacto PPLO Broth" (Difco) 8.7 g 20 yeast extract 60 ml 1% phenol red 4 ml bacitracin 500 mg (50,000 5% thallium acetate (Merck) 3 ml a horse serum 320 ml and that Hank's saline solution has the following composition: 35

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0 r / NaCl 80 g KC1 4 g solution. a MgSO4'7H2 ° Ig / MgCl-2 / 6H20 1 g 5 / solution in 400 ml distilled water In CaCl2, anhydrous 1.4 g V distilled water, up to 500 ml ^ Na2HPO4.12H20 1.5 g 10 solution B solution9 in 400 ml distilled water In KH2 PO4 0.6 g distilled water up to 500 ml 15 20 25 30 35

Claims (10)

1. A vaccine for protection against diseases caused by a mycoplasma, characterized in that it contains as active principle plasma membranes of the relevant mycoplasma which are at least largely liberated from the other components of the mycoplasma. Vaccine according to claim 1, characterized in that it is in the form of a suspension of the membranes in a NaCl solution at 8.5 g / liter, in the presence of an adjuvant which ensures the gradual release of the immunogen. Vaccine according to claim 2, characterized in that the adjuvant is aluminum hydroxide, agarose or anti-membrane antibodies for the mycoplasm. Vaccine according to one of claims 1-3, characterized in that the protein concentration is in the range 2 to 3 mg / ml. Vaccine according to claim 1 for the protection of pigs from enzootic pneumonia, characterized in that it consists essentially of a suspension of membranes 20 of Mycoplasma hyopneumoniae, which is at least largely liberated from the other constituents of the mycoplasma. an amount corresponding to a protein weight of 2 to 3 g / liter in an aqueous NaCl solution at 8.5 g / liter in the presence of 12 to 13 g / liter of aluminum hydroxide as adjuvant. Vaccine according to claim 1 for the protection of pigs from enzootic pneumonia, characterized in that it consists essentially of a suspension of membranes of Mycoplasma hyopneumoniae, which is at least largely liberated from the other constituents of the mycoplasma, in a 30 amount corresponding to a protein weight of 2 to 3 g / liter in an aqueous solution of NaCl with 8.5 g / liter in the presence of 1.5 to 2 g / liter of agarose as adjuvant. A method of producing a vaccine according to any one of claims 1-6, characterized in that it comprises essentially the following steps: a) culturing the mycoplasm for which it is desired to prepare a vaccine against the completion of said vaccine; (b) harvesting the cells by centrifugation; (c) illuminating the harvested cells with ultrasound at 20 to 28 kHz 5 in several steps for a total of 2 to 5 minutes; (d) sedimentation of the membranes by centrifugation; (e) washing the membrane sediment more times with a buffer using centrifuges and dispersions, and f) formulating the membranes in a form useful for vaccination. 8. Concentrated suspension in an aqueous solution of NaCl with 8.5 g / liter of membranes of a mycoplasm, at least largely liberated from the other constituents of the mycoplasma, for use as an active principle in a vaccine according to claim first Membranes of a mycoplasm, at least largely liberated from the other constituents of the mycoplasm, in freeze-dried form for use as an active principle in a vaccine according to claim 1. The unit dose of a vaccine according to claim 1 for the protection of pigs and unborn litters of pregnant sows against diseases caused by Mycoplasma hyopneumoniae, characterized in that it consists essentially of a suspension of membranes of Mycoplasma hyopneumoniae comprising at least 25 is largely liberated from the other constituents of the mycoplasm, in an amount corresponding to a weight of proteins of 4 to 6 mg, preferably 5 mg, in 2 ml of an aqueous 0.85% NaCl solution in the presence of 1 , 20 to 1.30%, preferably 1.25%, aluminum hydroxide or 0.15 to 0.20%, preferably 0.17%, agarose or also a saturating amount of porcine antibodies to Mycoplasma hyopneumoniae membranes.