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• • 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
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0258—Escherichia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• a 987P fimbriae-producing microorganism a vaccine for the immunization of pigs as well as a method for production of the vaccine.
• the present invention relates to a 987P fimbriae-producing microorganism.
• the pathogenic coli strains are characteristic by producing toxins and also by carrying type K88, K 99 or 987P fimbriae which fix the bacterium to receptors on the epithelium of the intestine.
• These fimbriae are threadlike protein structures which consist of amino acid chains with a molecular weight of between 16,000 and 28,000 Dalton.
• the enterophathogenic coli strains lose their pathogenic characteristics when they no longer express their adherence antigen, also even if they still produce toxins. E. coli diarrhoea in pigs can, therefore, be avoided by preventing the bacteria from adhering to the intestinal wall.
• Administration of antibodies against K88 fimbriae counteracts e.g. colonization of K88 fimbriae-producing strains on the intestinal wall, and the enteropathogenic coli bacteria can, therefore, no longer exercise their toxic effect in the interstinal tract.
• 987P fimbriae-producing coli bacteria have not been examined to the same extent as K88 and K99 fimbriae-producing bacteria, but in vitro experiments have shown that the adherence of 987P fimbriae-producing coli bacteria to the intestinal epithelium of pigs can be impeded by purified Fab-fragments of specific 987P antibodies, just as it has been proved that purified 987P fimbriae impede the adherence of the bacteria to the intestinal epithelium. It means that 987P fimbriae can serve for the immunization of pigs against pathogenic E. coli bacteria of serotype 987P.
• a 987P Fimbriae-producing microorganism has been obtained according to the invention, which microorganism has 987P Fimbriae in an amount corresponding to five percent or more by weight of its protein content.
• Different protein determination methods can be used for determination of the 987P fimbriae-production of a microorganism, e.g. the Lowry method (see J. Biol. Chem., 193, 265, (1951)) or electrophoresis, e.g. SDS-page electrophoresis.
• the microorganism may be a 987P-producing E. coli bacterium. It may be a natural toxin-producing coli bacterium or an artificially produced coli bacterium from which the toxin-producing characteristic has been removed or weakened, e.g. by genetic engineering.
• the bacterium may also be a naturally non-toxic coli bacterium whose chromosome has been replaced partially, e.g. by a genetic engineering technique, by parts of the chromosome from the 987P fimbriae-producing E. coli bacterium in question. Chiefly, only the gene responsible for the 987P fimbriae production has been transferred , but on the other hand not the gene which is responsible for the toxin production. Such a bacterium without toxin-producing characteristics has the advantage of being directly applicable as a vaccine, with no risk of harming the vaccinated animal.
• the organism according to the invention may, therefore, also be a non-toxic E. coli bacterium which contains a plasmid with the above mentioned gene for production of 987P fimbriae.
• SUBST.TU1 ⁇ SHSET The microorganism according to the invention produces 987P fimbriae e.g. in an amount corresponding to 5-15 percent by weight of its protein content.
• an E. coli strains has thus been produced which produces 987P fimbriae in an amount corresponding to approx. 10 percent by weight of its protein content.
• This strain is deposited with National Collection of Type Cultures (NCTC), England, under No. 11,665.
• the microorganism according to the invention in the chromosome may contain the entire gene or a fragment of the gene responsible for the fimbria production of the E. coli strain deposited under No. 11,665 in the NCTC.
• the organism according to the invention may thus be the deposited strain itself or an organism which through genetic engineering has obtained its 987P fimbriae-producing characteristic from the said strain.
• the gene for 987P fimbria production of the said strain can also be transferred to a plasmid of another organism through genetic engineering.
• the microorganism according to the invention may contain a plasmid with a fragment responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in the NCTC.
• the microorganism according to the invention can be used for production of a vaccine for the immunization of pigs against pathogenic 987P fim-briae-carrying E. coli bacteria.
• the invention also relates to a vaccine for the immunization of pigs.
• the vaccine according to the invention is characteristic in that it contains whole cells and/or cell wall material of a microorganism which produces 987P fimbriae in an amount corresponding to five percent or more by weight of its protein content together with a carrier substance and/or an adjuvant.
• the vaccine may thus contain living or dead whole cells of the microorganism. If the organism has no toxin-producing gene the vaccine can be administered without hesitation. If on the other hand the organism is toxin-producing, like the above-mentioned E. coli strain NCTC No. 11,665, the vaccine should be adminstered with caution in order to avoid disease- causing attacks of diarrhoea.
• sucking pigs will not be able to resist a toxin-containing vaccine.
• a vaccine of non-toxic whole cells can be used, or a vaccine of non-toxic parts of the cell wall material from toxic cells, e.g. the purified fimbriae of the cells.
• Ordinary feed and aqueous solutions can be used as carrier substances, e.g. a physiological NaCl solution.
• Aluminium hydroxide such as "Alhydrogel” from Superfos can be used as an adjuvant.
• the vaccine according to the invention can be injected parenterally, e.g. subcutaneously or intramuscularly, into the animal so that antibodies are formed which prevent natural toxic E. coli bacteria from adhering to the intestinal epithelium where they might otherwise give off diarrhoea-causing toxins.
• a vaccine of living cells can be administered orally which is an economic advantage e.g. with sucking pigs.
• the fimbriae of the vaccine will occupy the receptors of the intestinal wall, which will not be accessible as a place of adherence for natural toxic E. coli bacteria.
• Oral administration of the vaccine may also cause formation of antibodies in the intestinal wall which will amplify the protective effect which is obtained by occupation of the intestinal receptors for 987P fimbriae.
• a microorganism in the form of a 987P fimbriae-producing E. coli bacterium can be used for the vaccine. Chiefly an organism is used which has 987P fimbriae in an amount corresponding to 5-15 percent by weight of its protein content.
• the vaccine according to the invention can be produced on the basis of a microorganism which in the chromosome contains all the genes or a fragment of the gene responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in NCTC.
• the vaccine according to the invention can also be produced on the basis of a microorganism which contains a plasmid with a fragment of the gene responsible for the fimbria productiion of an E. coli strain deposited under No. 11,665 in NCTC.
• the vaccine according to the invention can also be made on the basis of a the E. coli bacterium NCTC No. 11,665 itself or an organism which has been given the 987P fimbriae-producing characteristics by genetic engineering.
• the invention also relates to a method for production of the above-mentioned vaccine, and this method is characteristic in that a microorganism which produces 987P fimbriae in an amount corresponding to five percent by weight of its protein content is cultured. If desired, the microorganism is then killed and the cell wall material comprising 987P fimbriae is isolated whereupon it is mixed with a carrier substance and/or an adjuvant.
• test tube with 10 ml BHI Brain Heart Infusion Broth, Difco
• E. coli strain, serotype 09: K103: 987P received from the State Serum Institute, Copenhagen. Culturing is carried out for 14 days at 37°C without shaking.
• a bacterium If a bacterium is fimbriated, it fills more relative to its weight, and fimbriated bacteria cells will, therefore, have a tendency to seek towards the surface during culturing without shaking.
• samples are only drawn from the surface layer of the seeded tube. They are spread on blood agar plates consisting of BHI with 2% agar and 5% red blood cells from horses and cultured for 16 hours at 37°C. The white colonies are tested with an antiserum which agglutinates with 987P fimbriae (serotype 020:987P). Few colonies show agglutination during this first culturing.
• test tube with 10 ml BHI is seeded with samples from the surface layer in the tubes which show the strongest agglutination and they are cultured for five to eight days without shaking at 37°C.
• Samples from the surface layer in the test tubes are seeded into new test tubes with 10 ml BHI and cultured without shaking at 37°C. After two to three days pellicles appear on the surface in some of the tubes. Samples of the pellicles are spread on blood agar plates, and the white colonies are tested for agglutination. Considerably more agglutinating colonies are now obtained.
• the cells are boiled in a buffer containing 3% SDS (Sodium dodecylsulphate) and 1%2-mercaptoethanol, and the protein material obtained is examined by electrophoresis.
• a drop of the material is mixed with a drop of a 987P fimbria preparation on an SDS polyacrylamide-gel and developed with Coomaasie Brilliant Blue G (see Example 2 for further details).
• 987P fimbria band (23,000 Dalton) it is possible to make an estimate of the fimbria production of the cells.
• the strongly fimbriae-producing coli strain was deposited with the NCTC wider No. 11,665.
• the 987P fimbriae-carrying cells formed during culturing are extracted, and the fimbriae are purified according to the method described by Issacson and Richter in J. Bact. 146, p. 874-789 (1981).
• the culture medium is centrifuged at 10,000 rev./min. for approx. 10 minutes at 4°C, and the precipitate is suspended in 100 ml of 0.01 M MOPS (3-N-morpholino) propane-sulphonic acid), (pH 7.2).
• MOPS 3-N-morpholino propane-sulphonic acid
• the supernatant is decanted and its pH is adjusted to 3.9 using 1 N acetic acid.
• the fimbriae are precipitated, and the suspension is centrifuged for 20 minutes at 12,000 rev./min.
• the precipitate is dissolved in 50 ml MOPS during stirring on an ice bath for at least three hours, stands for 16 hours at 4°C, and is stirred on an ice bath for one hour.
• the solution is centrifuged at 12,000 rev./min . for 20 minutes to remove undissolved material.
• the buffer consists of 0.09 M MOPS (pH 7.2), 0.2 M MgCl 2 and 1.7% NaCl. During stirring on an ice bath for half an hour, the 987P fimbriae are precipitated and collected by ce ⁇ trifugation at 10,000 rev./min. for 10 minutes.
• the precipitate is re-suspended during stirring in MOPS, and the suspension is treated in the same way as described above.
• the solution ⁇ btained is centrifuged at 12,000 rev./min. for 20 minutes at 4°C.
• the supernatant contains the 987P fimbriae in a dissolved state.
• the purity and yield of the individual stages are determined by SDS-page electrophoresis according to Laemmli, Nature, 227, 680-685, (1970).
• electrophoresis a 15% low bis-polyacrylamide-gel is used which consists oF 11.9 mg H 2 0, 15 ml 40% acrylamide, 2 ml 1.5% bis-acrylamide, 400 ul 10% SDS, 400 ul 10% glycerol, 300 ul 10% ammonium persulphate solution, and 20 ul TEMED in 10 ml 1.5 M Tris-H 2 O, (pH 8.8).
• a 6% stacking gel which consists oF 9.5 ml H 2 O, 3 ml 40% acrylamide, 2.1 ml 1.5% bis-acrylamide, 200 ul 10% SDS, 100 ul 10% glycerol, 150 ul ammonium persulphate and 20 ul TEMED in 5 ml 0.5 M Tris-HCl (pH 6.8).
• the sample is boiled For two minutes in a buffer containing 3% SDS and 1% 2-mercaptoethanol and transferred to the gel.
• 25 mM Tris-HCl and 250 mM glycine in a 1% SDS solution are used as electrophoresis buffer.
• the electrophoresis is carried oud at 85 V for 14 hrs (corresponding to approx. 1200 volt hours).
• the gel is developed by Coomassie Brilliant Blue G.
• the protein material is fractionated by means of the above-mentioned SDS- page electrophoresis, but instead of the 15% low bis-polyacrylamide-gel a 15% high bis-polyacrylamide-gel is used.
• the fractionated protein substances are blotted onto nitrocellulose paper at a voltage of 25 V for 16 hours.
• the paper or filter is washed in water for approx. one minute, in Tween buffer (10 mM Tris-HCl, 155 mM NaCl in 0.5% Tween 20 (pH 7.4)) for 30 minutes, and again in water for one minute.
• the filter is placed in a large glass tube, and rabbit antiserum (09:K103:987P) dissolved in Tween buffer is added.
• the tube is closed and rotated for 60 minutes.
• the filter is washed in water for one minute, in Tween buffer for half an hour, again in Tween buffer for half an hour, and then in water for one minute.
• SUBSTITUTE SHEET The filter is incubated in a glass tube with protein A conjugated with peroxidase (from Sigma Laboratories) and washed twice with Tween buffer for half an hour.
• the filter is placed on a glass plate for development of colour and is then covered with a freshly made substrate solution (80 mg dioctylsodium sulphosuccinate (DONS) and 24 mg tetramethylbenzidine (TMB) dissolved in 10 ml ethanol added to 30 ml mM citric acid and 20 mM Na 2 HPO 4 (20 ul 40% H 2 O 2 is added just before use).
• DONS dioctylsodium sulphosuccinate
• TMB tetramethylbenzidine
• the product proves to be more than 98% pure which is in accordance with the SDS-page electrophoresis.
• the analysis for amino acids is in accordance with the fact that the N-terminal amino acid in 987P fimbriae is alanine (Isaacson and Richter, 1981).
• a culture of the NCTC 11, 665 bacterium is spread on a blood agar plate and cultured at 37°C for 16 hours. White colonies are seeded into 10 ml BHI and incubated at 37°C for 16 hours. 0.1 ml of the culture medium obtained is inoculated into 200 ml UHT milk which is kept at 37°C without shaking for 16 hours. Production of intramuscular test vaccine
• Production of a challenge bacterial culture 10 ml BHI is inoculated with three different heterologous toxic bacterial strains, all of serotype 987P. Culturing is carried out For 16 hours at 37°C during shaking. The culture medium obtained is poured into 100 ml BHI, and cultured for 16 hours at 37°C during shaking.
• Vaccination test A total of 28 pregnant sows are used for the test. A control group of eight sows is given a normal feed. Another group of sows is given a feed into which 200 ml of the oral test vaccine is mixed on three subsequent days, at the latest 10 days before farrowing.
• a third group of sows is given a normal feed, but they have 5 ml of the intramuscular vaccine injected six and three weeks before farrowing.
• a Fourth group of sows is also given normal feed, but they receive the recommended dose of 2 ml of another 987P fimbriae-containing vaccine intramuscularly six and three weeks before farrowing.
• the udders of all the sows are swabbed morning and evening with the challenge bacterial culture from one day before farrowing till one day after farrowing.
• the faeces of the sucking pigs are examined one day after farrowing by means of a swab test and evaluated according to the following scale:

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• 1985
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• 1986
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