Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix

Definitions

• Vaccine against diseases caused by microorganisms such as mycoplasmas, its preparation and microorganism membranes as an active ingredient.
• L 1 oour invention provides a vaccine against mala ⁇ animal dies caused by microorganisms having the outermost shell membrane, and more par ⁇ ticularly against diseases caused by mycoplasmas and a method for its preparation .
• pork has excellent nutritional qualities and is appreciated by consumers. It is therefore not surprising that pig farming occupies a significant place in the economy of many countries in America (North and South), Europe, Asia and Oceania. This concerns both highly industrialized countries and countries that are much less so. In France, especially in Brittany, the raising of pigs is an essential component of the agricultural economy. Pig breeding techniques have greatly improved both in terms of yields obtained and in terms of breeding conditions, hygiene conditions in particular. These advances do not, however, make it possible to shelter farms from a certain number of diseases including ycoplasmosis. Among the diseases which affect the respiratory system of pigs, pneumonia is very common, in particular in the context of intensive farming.
• these microorganisms are completely insensitive to any antibiotic whose target is precisely the wall, such as, for example, penicillin, cephalosporin, and bacitracin.
• the Q mycoplas- plasmigue membrane my be impermeable to a number of antibiotics whose target is intracellular [S. Razin, Microbiol. Rev.
• the membrane plasmigue a microorganism having envelope for the outermost membrane, in particular a mycoplasma may be, when it is separated SOE 0 of the rest of the cell, particularly effective active ingredient of a vaccine against diseases caused by the microorganism concerned.
• the subject of the invention is therefore a vaccine intended to protect against diseases caused by a microorganism having the outermost envelope one, membrane -, in particular a mycoplasma, characterized in that it contains, as active principle, plasma membra ⁇ nes of the microorganism concerned, rid, at least to a large extent, of the other constituents of said microorganism.
• the m ⁇ croorganisms concerned by the invention are in particular gram negative bacteria and especially my ⁇ coplasms. Among these we can cite:
• Mycoplasma mycoids Mycoplasma bo is, Mycoplasma bovirhinis and Mycoplasma bovigenitalium;
• Mycoplasma agalactiae Mycoplasma ovipneumoniae, Mycoplasma caoricolum, and Mycoplasma mycoids subspecies capri;
• the vaccine according to the invention is in the form of a suspension of mem ⁇ branes in a so-called physiological saline solution, that is to say a NaCl solution at 8.5 g / l , in the presence of an adjuvant which provides a gradual release of the immunogen.
• the adjuvant consists of aluminum hydroxide, agarose or anti-membrane antibodies of the microorganism concerned.
• Other adjuvants, in particular oily, can be used; however, they are not preferred.
• the protein concentration from the membranes in the vaccine is preferably in the range of 2 to 3 g / 1; it is advantageously 2.5 g / l.
• the concentration of aluminum hydroxide is preferably in the range of 12 to 13 g / l, depending on the concentration of proteins; it is advantageously 12.5 g / l, for a protein concentration of 2.5 g / l.
• Agarose is generally used in the form of a very dilute gel, preferably at a concentration in the range of 1.5 to 2 g / 1 depending on the protein concentration; it is advantageously 1.7 g / 1 for a protein concentration of 2.5 g / 1.
• the invention relates to a vaccine intended to protect pigs against enzootic pneumonia, characterized in that it essentially consists of a suspension of membranes of Mycoplasma hyopneumoniae free, at least to a large extent, of the other constituents of the microorganism, corresponding to a protein weight of 2 to 3 g / 1, in an aqueous NaCl solution at 8.5 g / l in the presence of 12 13 g / l aluminum hydroxide as an adjuvant.
• the invention relates to a vaccine intended to protect pigs against enzootic pneumonia, charac ⁇ terized in that it consists essentially of a suspension of membranes of Mycoplasma hyopneumoniae cleared, at to a large extent less of the other constituents of the microorganism, corresponding to a weight of proteins of 2 to 3 g / l, in an aqueous NaCl solution at 8.5 g / l in the presence of 1.5 to 2 q / 1 agarose as an adjuvant.
• the invention further relates to a process for the preparation of the vaccine described above, characterized in that it essentially comprises the steps consisting in: a ) cultivating the microorganism against leguel, it is desired to prepare a vaccine until the end of the exponential growth phase; b) harvest the cells by * centrifugation; c) lyse the cells harvested by ultrasound, at 20 to 28 kHz, in several stages. for 2 to 5 minutes in total; d) sedimenting the membranes by centrifugation, and e) introducing them into a form which can be used for vaccination.
• the cell culture is advantageously carried out at 37 ° C. on a liquid medium such as the FRIIS medium *
• the centrifuged cells (at approximately 10,000 g) are washed with a buffer having a slightly raised pH, for example 0.1 M sodium phosphate buffer, pH 7.5, containing 8.5 g / NaCl. 1.
• a buffer having a slightly raised pH for example 0.1 M sodium phosphate buffer, pH 7.5, containing 8.5 g / NaCl. 1.
• the cells are then dispersed, for example by means of a Potter cylinder, so as to obtain a dense suspension in slightly basic ton, for example 0.1 M Tris-HCl buffer, pH 8.0.
• the lysis by ultrasound takes place in several stages, advantageously at increasing frequencies, in the range indicated above, in the vicinity of 0 ° C.
• the sedimented membranes are washed thoroughly (several times with buffer, for example 0.1 M Tris-HCl buffer, pH 8.0) and dispersed and then centrifuged again several times.
• buffer for example 0.1 M Tris-HCl buffer, pH 8.0
• the membranes thus collected are ready to be used to prepare the vaccine. They can be stored at -70 ° C. in the form of a concentrated suspension in an aqueous NaCl solution at 8.5 g / l, for example at 20 mg protein / ml, or in lyophilized form.
• a further subject of the invention is therefore, as new industrial products: - a suspension concentrated in an aqueous NaCl solution at 8.5 g / l of membranes of a microorganism having a membrane, in particular on the outer shell a mycoplasma, rid, at least to a large extent, of the other constituents of the microorganism, for use as an active ingredient in a vaccine; and - membranes of a microorganism having an outer membrane as a particular of a mycoplasma, free, at least to a large extent ⁇ of the other constituents of the microorganis ⁇ me, in lyophilized form, for use as a guue active ingredient of a vaccine.
• the vaccine according to the invention which does not contain any living cells is devoid of toxicity and therefore does not present, for the animal treated, the risks of live vaccines using attenuated strains, while being more effective and more easy vaccines using simply killed cells, without separation of the plastic membrane, such as those described in patent application FR 2 201 878.
• the vaccines obtained according to the invention are active even when they are administered subcutaneously or intramuscularly, whereas the vaccines described in patent application FR 2 201 878 do not have a preventive effect on the treated animal when they are inoculated into the trachea by injection or spraying.
• the vaccine obtained according to 1 'tion inventors are inoculated to a pregnant female, also effectively protect the unborn * scope, that mistletoe is a very important advantage for the safety of factory farms.
• Mycoplasma hyopneumoniae by injecting the pregnant sow subcutaneously or intramuscularly, an inoculum such as defined below, 7 to 8 weeks before parturition, then again 2 weeks before it.
• the invention relates to such an inoculum, or unit dose, mistletoe is characterized in that it is essentially constituted by a suspension of membranes of Mycoplasma hyopneumoniae cleared, at least in to a large extent, of the other constituents of the microorganism, and corresponding to a weight of proteins of 4 to 6 mg, preferably 5 mg, in 2 ml of an aqueous solution of NaCl at 0.85%, in the presence of 1, 20 to 1.30%, of this 1.25% preferen ⁇ .d'hydroxyde aluminum or 0.15 to 0.20%, preferably 0.17% agarose, or a saturating amount for pig antibody membranes anti ⁇ Mycoplasma hyopneumonia membrane.
• the cells are dispersed using a Potter cylinder so as to obtain a dense suspension in 0.1 M Tris-HCl buffer, 8.0 OH. They are then lysed by ultrasound: 2 times 1 minute at 20kHz and once a minu ⁇ te at 28kHz, The operation is carried out at 0 ° C by letting the material rest for one minute between two consecutive cycles The cell lysate is centrifuged for 1 hour 0 to 40,000 xg, at 4 ° C., so as to sediment the membranes These are washed four times with 0.1 M Tris-HCl buffer, pH 8.0 by successive dispersions and centrifugations,
• the material obtained is stored at -70 ° C, either in the form of a concentrated suspension (20 mg protein / ml), either in lyophilized form.
• the vaccines are made up of _M membranes. hyopneumoniae obtained as described above suspended in an NaCl solution at 8.5 g / l. The protein concentration is 2.5 mg / ml.
• the adj . uvants used are: aluminum hydroxide at 1.25% (vaccine I), agarose at 0.17% (vaccine II) and pig antibody anti ⁇ membrane of _M_. hyopneumoniae which precipitate the membranes (vaccine III).
• the vaccines have been used to vaccinate nulliparous gestating sows coming from the protected pigsty of the Ploufragan Pork Pathology Station (Côtes du
• the herd of this pigsty is made up of animals obtained by hysterectomy and kept safe from contaminants.
• the sows subjected to the tests were placed in protected animal houses ten weeks before the expected date of birth.
• the sows were inoculated 7 or 8 weeks before parturition, subcutaneously; a booster was given 2 weeks before parturition, by the intramuscular route.
• Chague inoculum contained 2 ml of membrane suspension, i.e. 5 mg of protein.
• All piglets received at 2, 3, 4 and 5 days of age, at a rate of 0.5 ml per nostril, a suspension of M. hyog tire oniae grading 10 CFU / ml (CFU Colony forming units) .
• the piglets were examined daily. The rectal temperature was recorded, clinical records were noted daily and sanitary punctures performed weekly in all piglets. They were weighed and their food consumption was assessed weekly.
• the piglets were sacrificed in a staggered fashion in each group, 5 to 10 weeks after infection. After the macroscopic observation of the lungs, bacteriological and ycoplasmic controls as well as histological examinations were undertaken. Seric antibodies were detected by passive hemagglutination [M.Kobisch et al., Rec. Med. Vét, 154 (1978), p. 847-852], III - Results
• hyopneumoniae has multiplied in the lungs of the pigs lets this litter before 60 days when it was present, at the level of the trachea from the first sacrifices. . " hyopneumoniae was found in the lungs of 66% of 20005 sow piglets (in 88% tracheal mucus) and in the lungs of 55% of 20007 sow piglets (in 100% tracheal mucus). results show that microbiological research for _m. hyopneumoniae must be conducted from the lungs but also from the tracheal mucus, which proves to be a good witness to the presence of the mycoplasma.
• sows Piglets from these sows, after colostral intake, have antibodies of maternal origin, the rate of which decreases rapidly as a function of time and reaches an average value below the level of significance in the weeks following birth.
• the sows show a rise in the level of circulating antibodies.
• M_. hyopneumoniae is rarely found in piglets from vaccinated sows (13 to 26%).
• the antibodies of maternal origin having a lifespan of the order of 3 to 4 weeks and on the other hand the animals being very susceptible to infection between the 8th and 12th week, it may in some cases be advantageous to perform additional vaccination p orcelets at the age of 6 weeks is to say when the passive antibodies have disappeared and do not prevent a vaccine taken with a booster vaccination 3 weeks later, i.e. at 9 weeks of age.
• pigs aged 12 weeks were vaccinated with a booster vaccination at 14 weeks and it has been established that the animals react to vaccination by synthesizing serum antibodies - (study by passive haemagglutination).
• the vaccine according to the invention makes it possible, with a very reduced cost due to the simplicity of its preparation, to effectively fight against the diseases caused by microorganisms having for their outermost envelope a membrane, in particular mycoplasmas, and especially against respiratory diseases that affect many pig farms and significantly reduce their yield.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (2)

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Publications (1)

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Cited By (12)

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Citations (1)

• 1984
  • 1984-06-15 FR FR8409422A patent/FR2565825B1/fr not_active Expired - Lifetime
• 1985
  • 1985-06-14 WO PCT/FR1985/000152 patent/WO1986000019A1/fr not_active Ceased
  • 1985-06-14 DE DE8585902582T patent/DE3574109D1/de not_active Expired
  • 1985-06-14 JP JP60502608A patent/JPS61502466A/ja active Pending
  • 1985-06-14 EP EP19850902582 patent/EP0185042B1/fr not_active Expired
• 1986
  • 1986-02-14 DK DK071686A patent/DK162509C/da not_active IP Right Cessation

Patent Citations (1)

Non-Patent Citations (1)

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