EP0209599A1 - A 987p fimbriae-producing microorganism, a vaccine for the immunization of pigs as well as a method for production of the vaccine - Google Patents

A 987p fimbriae-producing microorganism, a vaccine for the immunization of pigs as well as a method for production of the vaccine

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Publication number
EP0209599A1
EP0209599A1 EP86901359A EP86901359A EP0209599A1 EP 0209599 A1 EP0209599 A1 EP 0209599A1 EP 86901359 A EP86901359 A EP 86901359A EP 86901359 A EP86901359 A EP 86901359A EP 0209599 A1 EP0209599 A1 EP 0209599A1
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EP
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Prior art keywords
fimbriae
vaccine
microorganism
production
coli
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EP86901359A
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German (de)
French (fr)
Inventor
Jesper Kja Er Pedersen
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Slagteriernes Forskningsinstitut
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Slagteriernes Forskningsinstitut
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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:

Abstract

Un micro-organisme producteur de fibrilles 987P a une quantité de fibrilles 987P égale à cinq pourcent ou davantage en poids de sa teneur en protéines. Par exemple, 10 pourcent en poids de fibrilles 987P purifiées peuvent être extraits d'E. coli NCTC 11.665, calculés sur la base de la teneur totale en protéines des cellules. Le micro-organisme est utilisé sous la forme de cellules entières et/ou de matériau des parois cellulaires pour produire un vaccin utilisé pour immuniser des cochons, des truies ou des porcelets de lait par exemple, contre des souches pathogènes d'E. coli. Le vaccin est produit en cultivant les micro-organismes producteurs de fibrilles 987P. Si on le veut, le micro-organisme est ensuite tué, et le matériau de la paroi cellulaire, contenant les fibrilles 987 P, est isolé puis mélangé à un excipient et/ou à un adjuvant.A 987P fibril-producing microorganism has an amount of 987P fibrils equal to five percent or more by weight of its protein content. For example, 10 percent by weight of purified 987P fibrils can be extracted from E. coli NCTC 11.665, calculated on the basis of the total protein content of the cells. The microorganism is used in the form of whole cells and / or cell wall material to produce a vaccine used to immunize pigs, sows or suckling pigs, for example, against pathogenic strains of E. coli. The vaccine is produced by growing the 987P fibrillary microorganisms. If desired, the microorganism is then killed, and the cell wall material, containing the 987 P fibrils, is isolated and mixed with an excipient and / or an adjuvant.

Description

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.
Pigs, especially sucking pigs, often suffer from diarrhoea caused by enterophathogenic coli bacteria. Even in herds where the pig breeder aims at a healthy environment for the pigs it is common for the breeder to experience a considerable loss of sucking pigs on account of diarrhoea. It is, for example, common for the pigs of the so-called SPF status to suffer From diarrhoea caused by E. coli strains.
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.
R.E. Isaacson and P. Richter have in J. Bact. 146, p. 784-789, (1981) described the production of purified 987P fimbriae which may be used in such a vaccine for the immunization of pigs against pathogenic coli bacteria of seroype 987P, but the resultant production of fimbriae is low so that the vaccine becomes expensive. It has now quite surprisingly been found that there are E. coli strains which produce 987P fimbriae in several times higher quantities than the strains known so far. It has thus become possible to product the above- mentioned vaccine against pathogenic coli bacteria in a considerably less cost-consuming way. Besides, the E. coli strains dealt with can in some cases be used directly in the crude form as vaccine because as compared to the amount of Fimbriae they contain many times smaller amounts oF toxins.
Accordingly, 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.
According to the invention 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.
It is possible through genetic engineering to transfer genes from a chromosome to plasmids which in turn are transferred to a non-toxic cell. 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.TU1Ε 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. By experiment 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.
Accordingly, 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. Accordingly, 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.
Accordingly, 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.
SUBSTITUTE SHEET As a rule, sows will be able to resist a toxin-containing vaccine and transfer the antibodies formed to their sucking pigs via the colostrum.
On the other hand, sucking pigs will not be able to resist a toxin-containing vaccine. In this case 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.
Oral or parenteral administration into sows will cause formation of antibodies which via the sow's colostrum are transferred to the sucking pigs which are protected against coli diarrhoea. In order to produce the said formation of antibodies 2.5-5 mg of fimbria protein can be injected intramuscularly into sows six and three weeks before farrowing. In the case of re-vaccination it will only be necessary to vaccinate the sows three weeks before farrowing.
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.
The invention is explained in greater detail in connection with the following examples.
Example 1
Isolation of Fim+ colonies
A test tube with 10 ml BHI (Brain Heart Infusion Broth, Difco) is seeded with an 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.
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.
Accordingly, 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.
A 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.
After repeated re-seedings of agglutinating colonies and of pellicle layers in BHI and culturing at 37°C for two to three days without shaking, cultures are obtained with an increasingly higher frequency of bacteria in the Fim+ phase.
After approx. 1 1/2-2 months colonies are isolated which after re-seeding on a fresh blood agar plate every day remain in the Fim+ phase during the whole of the subsequent test period of approx. three months.
S E Obtainment oF NCTC 11,665
Culturing of the Fim+-stable colonies is continued in BHI under the same conditions as described above, and the pellicle which has Formed is spread and cultured on blood agar plates.
In order to examine the fimbria production of the colonies 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). On the basis of the 987P fimbria band (23,000 Dalton) it is possible to make an estimate of the fimbria production of the cells. There are none or only weakly coloured bands corresponding to a low fimbria production of the colonies.
It is tried to separate coloured bands further using Western blotting which confirms that the protein in the band is 987P fimbriae.
After many re-seedings without major changes, a colony appears unexpectedly which gives a strongly coloured band by electrophoresis. It is a special coli strain which produced 987P fimbriae in an exceptiunally high amount as the fimbria protein constitutes a much higher proportion of the total protein content of the cell, evaluated from the degree of colouring of the protein bands.
The strongly fimbriae-producing coli strain was deposited with the NCTC wider No. 11,665.
Example 2
Culturing
1 litre of BHI bouillon is seeded with the NCTC 11,665 strain, and incubation takes place at 37°C for approx. 16 hours during shaking.
Extraction of 987P fimbriae 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). The suspension is homogenised in an Omni mixer for approx. 10 minutes, and the homogenised suspension is centrifuged at 10,000 rev./min. for 10 minutes. The supernatant is decanted and its pH is adjusted to 3.9 using 1 N acetic acid. After shirring For half an hour 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.
An equal volume of a crystallization buffer is added to the supernatant. The buffer consists of 0.09 M MOPS (pH 7.2), 0.2 M MgCl2 and 1.7% NaCl. During stirring on an ice bath for half an hour, the 987P fimbriae are precipitated and collected by ceπtrifugation 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 σbtained is centrifuged at 12,000 rev./min. for 20 minutes at 4°C. The supernatant contains the 987P fimbriae in a dissolved state.
The above described procedure consisting of addition of a crystallization buffer to the supernatant, collection of precipitated material by centrifugation, dissolution of the precipitate in MOPS, and centrifugation of the solution for obtainment of a supernatant solution of 987P fimbriae is repeated at least four times.
The last crystallization results in a pure white precipitate of 987P fimbriae which after freeze-drying weighs 250 mg corresponding to approx.10% of the total protein content of the collected cells. Purity and yield
The purity and yield of the individual stages are determined by SDS-page electrophoresis according to Laemmli, Nature, 227, 680-685, (1970). For the electrophoresis a 15% low bis-polyacrylamide-gel is used which consists oF 11.9 mg H20, 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-H2O, (pH 8.8). A 6% stacking gel is used which consists oF 9.5 ml H2O, 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.
At a molecular weight of 23,000 Dalton there are very strong bands of 987P fimbriae in both culture medium, intermediate products and final products, compared to the bands from the previously isolated 987P fimbriae-producing E. coli bacteria.
In a similar way, yield and purity are followed using Western blotting. 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 Na2HPO4 (20 ul 40% H2O2 is added just before use). The colour development is stopped with a stop buffer.
Strong colourings are again seen opposite to the band corresponding to the 987P fimbriae protein, and the purity of the 987P fimbria product proves to be high since there are no other bands with the homologous antiserum used.
The purity of the product is confirmed by determination of the aminσ acid composition as described by Klemm in Eur. 3. Biochem., 117, p. 617-627, (1981).
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).
Amino acid sequencing
Manual Edman degradation was carried out on the obtained 9B7P protein as described by Klemm in the 3. Biochem., 117, p.617-627 (1981). The following sequence was found: Ala - Pro - Ala - Glu - Asn - Asn - Thr - Ser - Glx - Ala - Asx - Leu.
Example 3
Production of oral test vaccine
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
150 mg of the Fimbria product obtained in Example 3 ia suspended in a solution consisting of 240 ml 0.9% NaCl solution and 60 ml Alhydrogel.
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:
0 = normal firm to semi-soft faeces
+ = soft, mucous faeces ++ = soft to watery faeces +++ = profuse watery diarrhoea, dehydration
It can be seen from the results in Tables 1 and 2 that the vaccine according to the invention administered to sows before farrowing reduces the occurrence of diarrhoea and the mortality of the sucking pigs exposed to toxic 987P E. coli bacteria.
It also appears from Tables 1 and 2 that the vaccine according to the invention is more effective against diarrhoea than the commercial vaccine.
International Application No: PCT/ D K86/00012
MICROORGANISMS
Optional Sheet in connection with the microorganism referred to on page- -, Hne- 5-6 . of the description >
A. IDENTIFICATION OF DEPOSIT *
Further deposits are Identified on an additional sheet Q >
Name of depositary Institution *
National Collection of Type Cultures
Address of depositary Institution (including postal code and country) *
Central Public Health Laboratory 61 Colindale Aυenue London NW9 5HT, England
Pate of deposit * Accession Number *
1st June 1984 11.665
ADDITIONAL INDICATIONS » (leave blank if not applicable). This Information is continued on a separate attached sheet Q
C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE » O the indications are not for all designated States)
D. SEPARATE FURNISHING OF INDICATIONS « (leave blank If not applicable)
The indications listed below will be submitted to the International Bureau later - (Specify the general nature of the indications e.g.. " Accession Number of Deposit ")
E. Q This sheet was received with the international application when filed (to be checked by the receiving Office)
(Authorized Officer)
St The date of receipt (from the applicant) by the International
... 26 MAY 1986 26. 05, 86

Claims

C l a i m s
1. A 987P fimbriae-producing microorganism, c h a r a c t e r i z e d by 987P fimbriae in an amount corresponding to five percent or more by weight of its protein content.
2. A microorganism as claimed in claim 1, c h a r a c t e r i z e d in that it is a 987P fimbriae-producing E. coli bacterium.
3. A microorganism as claimed in claims 2-3, c h a r a c t e r i z e d by 987P fimbriae in an amount corresponding to 5-15 percent by weight of its protein content.
4. A microorganism as claimed in claims 1-3, c h a r a c t e r i z e d in that it contains in the chromosome the entire gene or a fragment of the gene responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in the NCTC.
5. A microorganism as claimed in claims 1-4, c h a r a c t e r i z e d in that it contains a plasmid with a fragment of the gene responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in the NCTC.
6. A vaccine for the immunization of pigs, c h a r a c t e r i z e d 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.
7. A vaccine as claimed in claim 6, c h a r a c t e r i z e d in that the microorganism is a 987P fimbriae-producing E. coli bacterium.
8. A vaccine as claimed in claims 6-7, c h a r a c t e r i z e d in that the microorganism has 987P fimbriae in an amount corresponding to 5-15 percent by weight of its protein content..
9. A vaccine as claimed in claims 6-8, c h a r a c t e r i z e d in that the microorganism in the chromosome contains the entire gene or a fragment of the gene responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in the NCTC. .
SUBSTITUTE SHEET
10. A vaccine as claimed in claims 6-9, c h a r ac t e r i z e d in that the microorganism contains a plasmid with a fragment of the gene responsible for the fimbria production of an E. coli strain, desposited under No. 11,665 in the NCTC.
11. A method for production of the vaccine as claimed in claim 6, c h a r a c t e r i z e d in that a microorganism is cultured which produces 987P fimbria in an amount corresponding to five percent or more by weight of its protein content and if desired, the microorganism is killed and the cell wall material comprising 987P fimbriae is isolated, whereupon it is mixed with a carrier substance and/or an adjuvant.
12. A method as claimed in claim 11, c h ar ac t e r i zed in that a 987P fimbriae-producing E. coli bacterium is used.
13. A method as claimed in claims 11-12, c h a r a c t e r i z e d in that an organism is used having 987P fimbriae in an amount corresponding to 5-15 percent by weight of its protein content.
14. A method as claimed in claims 11-13, c h a r a c t e r i z e d in that an organism is used containing in the chromosome the entire gene or a fragment of the gene responsible for the fimbria production of an E. coli strain deposited under No. 11,665 in the NCTC.
EP86901359A 1985-01-31 1986-01-31 A 987p fimbriae-producing microorganism, a vaccine for the immunization of pigs as well as a method for production of the vaccine Withdrawn EP0209599A1 (en)

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DK418/85 1985-01-31
DK41885A DK41885A (en) 1985-01-31 1985-01-31 987P-FIMBRIE-PRODUCING MICROORGANISM, VACCINE FOR IMMUNIZATION OF PIGS, AND PROCEDURE FOR PREPARING THE VACCINE

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EP0314224B1 (en) * 1987-10-26 1992-05-27 Akzo N.V. Vaccine against e.coli septicaemia in poultry
SE9100556D0 (en) * 1991-02-26 1991-02-26 Holmgren Jan PREPARATION AND USE OF FORMALIN-KILLED COLONIZATION FACTOR ANTIGEN (CFA) EXPRESSING A COLI ORGANISMS FOR VACCINATION AGAINST ENTERIC INFECTION / DIARRHEA CAUSED BY ENTEROTOXIGENIC AND COLI BACTERIA IN HUMANS
ES2076895B1 (en) * 1994-02-04 1996-08-16 Ind Farma Especial FIMBRIAS-ADHESINAS OF TYPE 1 AND P ISOLATED FROM NEW STRAINS OF E. COLI, PROCEDURE FOR THEIR OBTAINING AND APPLICATIONS.
US6471966B1 (en) 1994-02-04 2002-10-29 Industrial Farmaceutica Y De Especialidades, S.A. Type 1 and type p fimbriae-adhesins isolated from novel e. coli strains, process for their preparation and uses thereof
US6355859B1 (en) 1997-05-20 2002-03-12 Biotechnology Research And Development Corporation Interactions between genotype and diet in swine that prevent E. coli associated intestinal disease
DK0985052T3 (en) 1997-05-20 2003-10-27 Us Agriculture Methods and preparations for identifying pigs genetically resistant to F18-E. coli-associated diseases
WO2004048606A2 (en) 2002-11-25 2004-06-10 Den Kgl. Veterinær-Og Landbohøjskole Porcine polymorphisms and methods for detecting them

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US4237115A (en) * 1977-11-23 1980-12-02 Bactex, Inc. Method of immunization against enterotoxogenic infection by Escherichia coli
NZ199866A (en) * 1981-03-09 1984-11-09 Cetus Corp Vaccine for prevention of gastroenteric disease:non-pathogenic strain genetically engineered to comprise genes for adhesin and/or toxoids
EP0107845A3 (en) * 1982-10-29 1985-07-03 Bactex Incorporated Vaccine for immunizing cattle against infectious bovine keratoconjunctivitis by moraxella bovis

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