SALMONELLA POLYNUCLEOTIDE SEQUENCE

Abstract: DNA comprising a sequence characteristic of certain serotypes of the genus Salmonella is provided and used as polymerase chain reaction and hybridization targets for the identification of said serotypes. The DNA, in recombinant form, e.g. as plasmids, is used to transform suitable host cells to make them capable of expressing amino acid sequences characteristic of said serotypes. Test kits are provided comprising probes targeted at the characteristic sequences and amino acid sequences expressed by the transformants may also be used in immunological test kits for the serotypes.

Description: SALMONELLA POLYNUCLEOTIDE SEQUENCE This invention relates to polynucleotides (DNA) comprising a sequence characteristic of certain serotypes of the genus Salmonella; to the use of sequences comprising the characteristic sequence as polymerase chain reaction and hybridization targets for the identification of said serotypes and to test kits for this; to the use of polynucleotides comprising the sequence to transform suitable host cells to make them capable of expressing amino acid sequences characteristic of said strains; to said amino acid sequence when so expressed and kits containing them; and to plasmids and transformed cells containing said polynucleotide sequences. Organisms of the genus Salmonella, in particular S. enteritidis. S. dublin and S. typhimurium are responsible for infective food poisoning caused by their ingestion in contaminated food. Infection with Salmonella may also occur as a result of contact with contaminated materials. Once ingested, Salmonella is able to establish itself in the gut and multiply rapidly, resulting in the appearance of clinical symptoms several days after the initial ingestion. It is therefore highly desirable to provide test methods by means of which Salmonella organisms may be detected. In recent years immunological tests have been devised in which specific antibodies, particularly monoclonal antibodies ("MABs"), to specific antigens are raised and which by exploitation of the antigen - antibody specific binding reaction the presence of the antigen can be detected. Such tests are fast and very specific. It is known that Salmonella organisms have fimbria like structures on their surface (Duguid; J. P and R. R. Gillies (1958) J. Pathol. Bacteriol. 25:519-520) and published evidence (Clegg, S., and G. F. Gerlach (1987) J. Bacteriol. 1&9.:93-4-938. ) suggests that there are antigenically distinct types of fimbriae, ie. possessing specific epitopes on the fimbrial antigens. The possibility of immunogenic tests for Salmonella, at least S. enteritidis. based upon these fimbrial antigens has been suggested (MAFF, Central Veterinary Laboratory "Animal Health" (1989):33). Methods of raising MABs to antigens on the surface of micro-organisms such as Salmonella are generally known. Unfortunately known methods for raising antibodies to Salmonella surface antigens only go part way toward providing an immunological test for Salmonella. The basis of all these tests is to isolate micro-organisms from a sample suspected of harbouring Salmonella, then to grow the micro-organisms in vitro in a suitable culture medium until a quantity of the Salmonella sufficient to detect by such a test is believed to be present in the medium, and then applying the test. A problem occurs in that although Salmonella micro-organisms produce their fimbrial antigen when they grow in vivo, eg. in the gut, in animal tissues or fluids, in food products and in some natural environments, many of the fimbrial antigens are not produced when they are grown in vitro. The present inventors have determined the polynucleotide sequence responsible for producing a characteristic fimbrial antigen. Salmonella enteritidis fimbrial antigen (SEFA) . SEFA has an amino acid sequence forming an epitope on the fimbria 'in vivo' which is specifically found encoded by the DNA of the species S. enteritidis and some strains of the species S. dublin and S. Moscow but which is apparently absent in virtually all other serotypes. The identification and recognition of the significance of this sequence provides the basis for a number of determinative tests for the presence of the particular organisms or DNA/RNA derived from them and provides a method for production of transformed organisms capable of expressing SEFA or epitopic parts of SEFA. The amino acid sequence of SEFA is provided below; it is of course to be expected that allelic variation will occur in some organisms. AMINO ACID SEQUENCE OF SALMONELLA ENTERITIDIS FIMBRIAL ANTIGEN M L I V D F W R F C N M R K S A S A V A V L A L I A C G S A H A A G F V G N K A E V Q A A V T I A A Q N T T S A N W S Q D P G F T G P A V A A G Q K V G T L S I T A T G P H N S V S I A G K G A S V S G G V A T V P F V D G Q G Q P V F R G R I Q G A N I N D Q A N T G I D G L A G W R V A S S Q E T L N V P V T T F G K S T L P A G T F T A T F Y V Q Q Y Q N The codes above are standard codes, read amino- terminal to carboxy -terminal, left to right, M to N, according to the following key: Thus in its broadest form the present invention relates to DNA which forms all or part of the coding sequence for the SEFA sequence above or to allelic variants of that sequence, which carry the codons for its characteristic epi topes. A first preferred aspect of the present invention provides recombinant DNA comprising the sequences I and II: Sequence I 5'- G CTCAGAATAC AACATCAGCC AACTGGAGTC AGGAT -3' 3'- C GAGTCTTATG TTGTAGTCGG TTGACCTCAG TCCTA -5' 230 240 250 Sequence II 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTT -3' AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAA -5' 370 380 390 400 410 sequences degenerately equivalent thereto, or sequences encoding for allelic variants of the part of SEFA for which the sequences I and II encode. The numerals below each ten base pair sequence in sequence I and II above are those designating the position of the individual base pairs in a larger characteristic sequence that comprises the entire SEFA antigen coding polynucleotide sequence. By 'degenerately equivalent' is meant that substitute codons are present, these being codons which though they differ in their nucleotide base sequence from the codons identified in sequences I and II above, still code for the same amino acid, as will be understood by a man skilled in the art. Preferred recombinant DNA of the invention, comprising sequences I and II, is that comprising sequences III and IV: Sequence III 5'- ATGCTAAT AGTTGATTTT TGGAGATTTT GTAATATGCG TAAATCAGCA 3'- TACGATTA TCAACTAAAA ACCTCTAAAA CATTATACGC ATTTAGTCGT 80 90 100 110 120 TCTGCAGTAG CAGTTCTTGC TTTAATTGCA TGTGGCAGTG CCCACGCAGC TGGCTTTGTT AGACGTCATC GTCAAGAACG AAATTAACGT ACACCGTCAC GGGTGCGTCG ACCGAAACAA 130 140 150 160 170 180 GGTAACAAAG CAGAGGTTCA GGCAGCGGTT ACTATTGCAG CTCAGAATAC AACATCAGCC CCATTGTTTC GTCTCCAAGT CCGTCGCCAA TGATAACGTC GAGTCTTATG TTGTAGTCGG 190 200 210 220 230 240 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTTC AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAAG 370 380 390 400 410 420 CGTGGGCGTA TTCAGGGAGC CAATATTAAT GACCAAGCAA ATACTGGAAT TGACGGGCTT GCACCCGCAT AAGTCCCTCG GTTATAATTA CTGGTTCGTT TATGACCTTA ACTGCCCGAA 430 440 450 460 470 480 GCAGGTTGGC GAGTTGCCAG CTCTCAAGAA ACGCTAAATG TCCCTGTCAC AACCTTTGGT CGTCCAACCG CTCAACGGTC GAGAGTTCTT TGCGATTTAC AGGGACAGTG T GGAAACCA 490 500 510 520 530 540 AAATCGACCC TGCCAGCAGG TACTTTCACT GCGACCTTCT ACGTTCAGCA GTATCAAAAC -3' TTTAGCTGGG ACGGTCGTCC ATGAAAGTGA CGCTGGAAGA TGCAAGTCGT CATAGTTTTG - ' 550 560 570 580 590 600 sequences degenerately equivalent thereto or sequences which encode for allelic variants of SEFA. The significance of sequences III and IV is that when they run contiguously together, such that the -3' end of the top strand of sequence III is immediately followed by the top strand 5'- end of sequence IV, they consist of the polynucleotide sequence that encodes the amino acid sequence for SEFA (said upper strand) . Thus polynucleotide sequence encoding SEFA is on the upper strand as shown above beginning ATGCTAATAG on III and ending GTATCAAAAC on IV. Further sequences which comprise suitable flanking sequences for control of amino acid sequence expression may be produced by genetic engineering techniques from this continuous sequence. The invention further provides recombinant DNA comprising sequence III and IV, in the form of that comprising sequences V and VI: Sequence V 5'- GATCCTTGTT TTTTTTCTTA AATTTTTAAA ATGGCGTGAG TATATTAGCA TCCGCACAGA 3'- CTAGGAACAA AAAAAAGAAT TTAAAAATTT TACCGCACTC ATATAATCGT AGGCGTGTCT 10 20 30 40 50 60 TAAATTGTGC GAATGCTAAT AGTTGATTTT TGGAGATTTT GTAATATGCG TAAATCAGCA ATTTAACACG CTTACGATTA TCAACTAAAA ACCTCTAAAA CATTATACGC ATTTAGTCGT 70 80 0 100 110 120 TCTGCAGTAG CAGTTCTTGC TTTAATTGCA TGTGGCAGTG CCCACGCAGC TGGCTTTGTT AGACGTCATC GTCAAGAACG AAATTAACGT ACACCGTCAC GGGTGCGTCG ACCGAAACAA 130 140 150 160 170 180 GGTAACAAAG CAGAGGTTCA GGCAGCGGTT ACTATTGCAG CTCAGAATAC AACATCAGCC CCATTGTTTC GTCTCCAAGT CCGTCGCCAA TGATAACGTC GAGTCTTATG TTGTAGTCGG 190 200 210 220 230 240 Sequence VI 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTTC AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAAG 370 380 390 400 410 420 CGTGGGCGTA TTCAGGGAGC CAATATTAAT GACCAAGCAA ATACTGGAAT TGACGGGCTT GCACCCGCAT AAGTCCCTCG GTTATAATTA CTGGTTCGTT TATGACCTTA ACTGCCCGAA 430 440 450 460 470 480 GCAGGTTGGC GAGTTGCCAG CTCTCAAGAA ACGCTAAATG TCCCTGTCAC AACCTTTGGT CGTCCAACCG CTCAACGGTC GAGAGTTCTT TGCGATTTAC AGGGACAGTG TTGGAAACCA 490 500 510 520 530 50 AAATCGACCC TGCCAGCAGG TACTTTCACT GCGACCTTCT ACGTTCAGCA GTATCAAAAC TTTAGCTGGG ACGGTCGTCC ATGAAAGTGA CGCTGGAAGA TGCAAGTCGT CATAGTTTTG 550 560 570 580 590 600 TAATTTAATT TAAACTTTAT AAATGCCCTC AATATGAGCG AGTTTGGATA ATTTTATTAT ATTAAATTAA ATTTGAAATA TTTACGGGAG TTATACTCGC TCAAACCTAT TAAAATAATA 610 620 630 640 650 660 TTTAAAAATA TCTATTTTGA ATAGATAGGT TTTATGCTTC CATGCAAAAA CTTAAAGAGG AAATTTTTAT AGATAAAACT TATCTATCCA AAATACGAAG GTACGTTTTT GAATTTCTCC 670 680 690 700 710 720 GATTATGTAT ATTTTGAATA AATTTATACG TAGAACTGTT ATCTTTTTCC TTTTTTTTGC CTAATACATA TAAAACTTAT TTAAATATGC ATCTTGACAA TAGAAAAAGG AAAAAAAACG 730 740 750 760 770 780 TACCTTCCAA TTGCTTCTTC GGAAAGTAAA AAAATTGAGC AACCATTATT AACACAAAAA ATGGAAGGTT AACGAAGAAG CCTTTCATTT TTTTAACTCG TTGGTAATAA TTGTGTTTTT 790 800 810 820 830 840 TATTATGGCC TAAGATTGGG CACTACACGT GTTATTTATA AAGAAGATGC TCCATCAACA ATAATACCGG ATTCTAACCC GTGATGTGCA CAATAAATAT TTCTTCTACG AGGTAGTTGT 850 860 870 880 890 900 AGTTTTTGGA TTATGAATGA AAAAGAATAT CCAATCCTTG TTCAAACTCA AGTATATAAT TCAAAAACCT AATACTTACT TTTTCTTATA GGTTAGGAAC AAGTTTGAGT TCATATATTA 910 920 930 940 950 960 GATGATAAAT CATCAAAAGC TCCATTTATT GTAACACCAC CTATTTTGAA AGTTGAAAGT CTACTATTTA CTAGTTTTCG AGGTAAATAA CATTGTGGTG GATAAAACTT TCAACTTTCA 970 980 990 1000 1010 1020 AATGCGCGAA CAAGATTGAA GGTAATACCA ACAAGTAATC TATTCAATAA AAATGAGGAG TTACGCGCTT GTTCTAACTT CCATTATGGT TGTTCATTAG ATAAGTTATT TTTACTCCTC 1030 1040 1050 1060 1070 1080 TCTTTGTATT GGTTGTGTGT AAAAGGAGTC CCACCACTAA ATGATAATGA AAGCAATAAT AGAAACATAA CCAACACACA TTTTCCTCAG GGTGGTGATT TACTATTACT TTCGTTATTA 1090 1100 1110 1120 1130 1140 AAAAACAACA TAACTACGAA TCTTAATGTG AATGTGGTTA CGAATAGTTG TATTAAATTA TTTTTGTTGT ATTGATGCTT AGAATTACAC TTACACCAAT GCTTATCAAC ATAATTTAAT 1150 1160 1170 1180 1190 1200 ATTTATAGGC CTAAAACTAT AGACTTAACG ACAATGGAGA TTGCAGATAA ATTAAAGTTA TAAATATCCG GATTTTGATA TCTGAATTGC TGTTACCTCT AACGTCTATT TAATTTCAAT 1210 1220 1230 1240 1250 1260 GAGAGAAAAG GAAATAGTAT AGTTATAAAG AATCCAACAT CATCATATGT GAATATTGCA CTCTCTTTTC CTTTATCATA TCAATATTTC TTAGGTTGTA GTAGTATACA CTTATAACGT 1270 1280 1290 1300 1310 1320 AATATTAAAT CTGGTAATTT AAGTTTTAAT ATTCCAAATG GATATATTGA GCCATTTGGA TTATAATTTA GACCATTAAA TTCAAAATTA TAAGGTTTAC CTATATAACT CGGTAAACCT 1330 1340 1350 1360 1370 1380 TATGCTCAAT TACCTGGTGG AGTACATAGT AAAATAACTT TGACTATTTT GGATGATAAC ATACGAGTTA ATGGACCACC TCATGTATCA TTTTATTGAA ACTGATAAAA CCTACTATTG 1390 1400 1410 1420 1430 1440 GGCGCTGAAA TTATAAGAGA ATTATTAGTT TAAGGTGTAA AACAAATGAA GAAAACCACA CCGCGACTTT AATATTCTCT TAATAATCAA ATTCCACATT TTGTTTACTT CTTTTGGTGT 1450 1460 1470 1480 1490 1500 ATTACTCTAT TTGTTTTAAC CAGTGTATTT CACTCTGGAA ATGTTTTCTC CAGACAATAT TAATGAGATA AACAAAATTG GTCACATAAA GTGAGACCTT TACAAAAGAG GTCTGTTATA 1510 1520 1530 15^0 1550 1560 AATTTCGACT ATGGAAGTTT GAGTCTTCTC CCGGTGAGAA TGCATCTTTT CTAAGTGTTG TTAAAGCTGA TACCTTCAAA CTCAGAAGAG GGCCACTCTT ACGTAGAAAA GATTCACAAC 1570 1580 1590 1600 1610 1620 AAACGCTTCC CTGGTAATTA TGTTGTTGAT GTATATTTGA ATAATCAGTT AAAAGAAACT TTTGCGAAGG GACCATTAAT ACAACAACTA CATATAAACT TATTAGTCAA TTTTCTTTGA 1630 1640 1650 1660 1670 1680 ACTGAGTTGT ATTTCAAATC AATGACTCAG ACTCTAGAAC CATGCTTAAC AAAAGAAAAA TGACTCAACA TAAAGTTTAG TTACTGAGTC TGAGATCTTG GTACGAATTG TTTTCTTTTT 1690 1700 1710 1720 1730 1740 CTTATAAAGT ATGGGATCGC CATCCAGGAG CTTCATGGGT TGCAGTTTGA TAATGAACAA GAATATTTCA TACCCTAGCG GTAGGTCCTC GAAGTACCCA ACGTCAAACT ATTACTTGTT 1750 1760 1770 1780 1790 1800 TGCGTTCTCT TAGAGCATTC TCCTCTTTAA ATATACTTAT AACGCGGCTA ACCAAAGTTT ACGCAAGAGA ATCTCGTAAG AGGAGAAATT TATATGAATA TTGCGCCGAT TGGTTTCAAA 1810 1820 1830 1840 1850 i860 GCTTTTAAAT GCACCATCTA AAATTCTATC TCCAATAGAC AGTGAAATTG CTGATGAAAA CGAAAATTTA CGTGGTAGAT TTTAAGATAG AGGTTATCTG TCACTTTAAC GACTACTTTT 1870 1880 1890 1900 1910 1920 TATCTGGGAT GATGGCATTA ACGCTTTTCT TTTAAATTAC AGAGCTTAAT TATTTGCATT ATAGACCCTA CTACCGTAAT TGCGAAAAGA AAATTTAATG TCTCGAATTA ATAAACGTAA 1930 1940 1950 I960 1970 1980 CTAAGGTTGG AGGAGAGAGA TTCATACTTT GGTCAAATTC AACCTTGGTT TTAATTTTGG GATTCCAACC TCCTCTCTCT AAGTATGAAA CCAGTTTAAG TTGGAACCAA AATTAAAACC 1990 2000 2010 2020 2030 2040 TCCCTGGCGG CTAAGGAATC TATCATCTTG GCAAAACTTG TCAAGCGAAA AAAAATTTGA AGGGACCGCC GATTCCTTAG ATAGTAGAAC AGTTTTGAAC AGTTCGCTTT TTTTTAAACT 2050 2060 2070 2080 2090 2100 ATCAGCATAT ATTTATGCTG AGCGAGGTTT AAAAAAAATA AAGAGCAAAC TAACAGTTGG TAGTCGTATA TAAATACGAC TCGCTCCAAA TTTTTTTTAT TTCTCGTTTG ATTGTCAACC 2110 2120 2130 2140 2150 2160 GGACAAATAT ACCAGTGCAG ATTTATTCGA TAGCGTACCA TTTAGAGGCT TTTCTTTAAA CCTGTTTATA TGGTCACGTC TAAATAAGCT ATCGCATGGT AAATCTCCGA AAAGAAATTT 2170 2180 2190 2200 2210 2220 TAAAGATGAA AGTATGATAC CTTTCTCACA GAGAACATAT TATCCAACAA TACGTGGTAT ATTTCTACTT TCATACTATG GAAAGAGTGT CTCTTGTATA ATAGGTTGTT ATGCACCATA 2230 2240 2250 2260 2270 2280 TGCGAAAACC AATGCGACTG TAGAAGTAAG ACAAAATGGA TACTTGATAT ATTCTACTTC ACGCTTTTGG TTACGCTGAC ATCTTCATTC TGTTTTACCT ATGAACTATA TAAGATGAAG 2290 2300 2310 2320 2330 2340 AGTCCCCCCC GGGCAATTCG AGATAGGTAG AGAACAAATT GCTGATC -3' TCAGGGGGGG CCCGTTAAGC TCTATCCATC TCTTGTTTAA CGACTAG - ' 2350 2360 2370 2380 or sequences degeneratively equivalent thereto. For the purposes of expressing SEFA polypeptide or epitopic parts thereof the paired sequences I and II; III and IV; or V and VI run contiguously with each other without intervening base pairs between the two, in each case. These contiguous sequences are designated sequence VII, VIII and IX respectively. For the purpose of expressing SEFA it will be realised by the skilled man that all the sequences above may comprise degenerate codons instead of those listed above. It is not envisaged that such use will necessarily provide any advantage as preparation would be probably be more lengthy, but some transformed microorganisms may express SEFA more readily with certain codons in degenerate form suited to them. The present invention provides novel recombinant plasmids, comprising the recombinant DNA comprising either paired sequences selected from I and II, III and IV, or V and VI or the contiguous sequences VII, VIII and IX, the degenerative or allelic equivalents of any of these; said plasmids being capable of expressing polypeptides characteristic of SEFA when used to transform suitable microorganisms. These recombinant plasmids may then be used to transform a host, such as E coli or yeast, whereby use of cloning and selection methods provides clones which contain the particular sequence or suitably flanked antigen encoding portion having expression enabling sequences with it. Convenient tools for the selection of these clones are the aforementioned sequences themselves as modified in known ways to provide probes, ie. by radiolabelling. Such probe sequences are readily provided by use of the polymerase chain reaction on native SEFA sequence template or by DNA synthesizer techniques; radiolabelling being achieved using standard techiques to tag on 32 P. Preferred microorganisms for transformation are E. coli and yeasts; a particularly preferred microorganism being E. coli DH5alpha. Thus preferred plasmids will be those known to the man skilled in the art as suitable for transforming such organisms. Particularly preferred plasmids are accordingly pBR322, pACYCl84 and, most preferred, pUClδ. The polynucleotides sequences above may be combined with any of these known plasmids for the purposes of providing the novel plasmids of the invention. Particularly preferred will be plasmids into which polynucleotides consisting of the contiguous sequences VIII or IX have been inserted as these will be readily provided from cultured S. enteritidis or S. dublin by use of restriction endonucleases and encode for the entire SEFA amino acid sequence. In this respect use of antibodies targeted for SEFA allows facile recognition of transformed organisms which is particularly useful for selecting expressing organisms from a background population. Such antibodies are the subject of copending MAFF patent application (PCT GB 91 . our reference P0958) of inventor C J Thorns). (See Tables I and II). For example, the contiguous sequence IX may be blunt-ended using Klenow polymerase infilling and then ligated into a plasmid such as pUClδ. Alternatively total genomic DNA is extracted from S. enteritidis or a strain of S. dublin possessing said fimbrial antigen, as determined using the monoclonal antibodies and techniques disclosed in the applicants copending application referred to above, and then partially digested with SauIIIA restriction endonuclease to leave large fragments, some of which contain the sequences referred to above, which are then ligated into the plasmid vectors above. The vectors of the present invention have further utility in so far as the contiguous sequences VII, VIII and IX all comprise a single BamHl restriction endonuclease recognition site into which foreign peptide encoding DNA may be ligated by which it is sited within the reading frame of the transformant transcription system. This site is at the junction between the two sequences that make up the contiguous sequence; that occuring between base pair 255 and 2 6 in the numbering system applied at the bottom of each 10 base pairs above. Thus the present invention provides plasmids and transformants comprising the sequences I and II, or III and IV, or V and VI, or their degenerative or allelic equivalents, which have been augmented with further sequences. The invention provides a method for preparing these plasmid and transformants which inserts the further sequences into plasmids comprising the contiguous sequences VII, VIII or IX the at that BamHl site. Such augmented transformants are potentially capable of expression of mixed epitopic polypeptides comprising epitopes of SEFA together with further 'foreign' peptides. This opens the way to recombinantly produced peptides that are not easily expressed by other means. The fact that SEFA is a polypeptide that is passed to the exterior of the Salmonella cell of advantage in the recovery of such expressed polypeptides. The 'foreign' peptides may be further SEFA epitopes. Thus the invention also provides micro-organisms, eg E.coli or yeasts, which have been transformed by insertion of one or more of the aforementioned sequences eg, by use of said plasmids. Use of the micro-organisms provided by the invention gives a method of expression of the antigenic amino acid sequence SEFA referred to above and epitopic parts thereof which might be used as antigenic activity, that is having the ability to evoke production of antibodies in animal bodies. In addition to use of the transformant expressed SEFA or epitopic parts thereof for immunological test purposes and kits for such, the recognition of the significance of the DNA sequences defined above provides methods of determination of DNA or RNA as being derived from the S. enteritidis or S. dublin serotypes in other, DNA/RNA based, tests. TABLE I 264 Salmonella strains examined with monoclonal antibody MAB69/25 Serogroup Serotype Serogroup Serotype (No. strains tested) (No. strains tested) B S. agama (1) Dl S. gallinarium ( ) S. agona (1) S. moscow (1) S. bredeney (1) S. ouakam (1) S. derby (1) S. panama (1) S. heidelberg (1) S. pullorum (3) S. indiana (1) S. wangata (1) S. reading (1) El S. anatum (1) S. schwarzengrund (1) S. give (1) S. Stanley (1) S. lexington (1) S. typhimurium (64) S. london (1) Cl S. bareilly (1) S. meleagridis (1) S. infantis (1) S. nchanga (1) S. lille (1) S. orion (1) S. livingstone (1) E2 S. binza (1) S. mbandaka (1) S. drypool (1) S. montevideo (1) S. manila (1) S. ohio (1) S. newington (1) S. oranienburg (1) E4 S. taksony (1) S. oslo (1) S. senftenberg (1) S. thompson (1) F S. aberdeen (1) S. virchow (1) Gl S. havana (1) C2 S. goldcoast (1) S. worthington (1) S. hadar (1) G2 S. a iobo (1) S. newport (1) S. kedougou (1) C3 S. albany (1) K S. cerro (1) S. kentucky (2) N S. urbana (1) S. tado (1) 0 S. adelaide (1) Dl S. berta (1) S. ealing (1) S. canastel (1) R S. johannesburg (1) S. dublin (36) S S. offa (1) S. durban (1) T S. gera (1) S. enteritidis (58) TABLE l± Direct binding of MAB 69/25 to Salmonella strains Number Monoclonal antibody Serotype Examined MAB 69/25 abound S. enteritidis PT 1 2 S. enteritidis PT 4 22 S. enteritidis 6 S. enteritidis 1 S. enteritidis 1 S. enteritidis 1 S. enteritidis 12 S. enteritidis 4 S. enteritidis 7 S. enteritidis PT 30 1 S. enteritidis untypable 1 S. dublin 12 S. dublin 24 S. moscow 1 Other Salmonella strains'" 169 a Mean percentage of antibody binding relative to binding to high control (see text) b Range of binding c Serotypes listed in Table I PT = Phage type The present invention further provides methods for determining the presence of microorganisms having DNA or RNA polynucleotide sequence encoding for SEFA or an epitopic part thereof, or such DNA or RNA itself, comprising: (a) providing a sample suspected of containing said encoding polynucleotide sequence; (b) determining the presence of said sequence by monitoring hybridization of SEFA targeted polynucleotide probes to it. Such hybridization technique is carried out by methods that are now conventional in the art, using probes which are comprised of sequences complementary to a significant part of the target sequence and using temperature conditions suitable to achieved a desired stringency dependent on the degree of match of the probe to the target. In a preferred form of this method the invention further provides methods for determining the presence of microorganisms having DNA or RNA polynucleotide sequence encoding for SEFA or an epitopic part thereof, or such DNA or RNA itself, comprising: (a) providing a sample suspected of containing said encoding polynucleotide sequence; (b) subjecting said sample to conditions under which polynucleotide sequences comprising sequences (I) and (II) are replicated by use of the polymerase chain reaction; (c) determining the presence of any sequence produced. Conveniently the sequence produced is detected, in both cases, by use of a hybridization probe suitably specific thereto which comprises any of the aforementioned sequences, more specifically being one of the sequences in a suitably labelled form eg. being labelled in some way as will be known to a man skilled in the art. Most conveniently the label will incorporate radioactive phophorous ( 32 P) . A preferred such method comprises a PCR step (b) which employs primer pairs comprising one primer selected from groups (A) and the other from group(B): Group A: Group B: Al: 5' -GTGCGAATGCTAATAGTTGA- 3' Bl: 5' -AAAACAGGCTGTCCTTGTCCA- 3' A2: 5' -TGCGTAAATCAGCATCTGCA- 3' B2: 5' -TTAGCGTTTCTTGAGAGCTGG- 3' A3: 5' -TCTGCAGTAGCAGTTCTTGC- 3' B3: 5' -TTTTGATACTGCTGAACGTAG- 3' A4: 5' -GCTCAGAATACAACATCAGCCAA- 3' The primers are numbered Al to A4 and Bl to B3 for the purposes of identification later in this specification. Any of the possible pairs selected in this way will identify the characteristic sequences VI, VII or IX sufficiently specifically enough for serotype determination purposes, ie: for determination of a Salmonella as being a SEFA encoding serotype and thus of one of the serotypes listed above. As will be understood by a man skilled in the art, sequences which will specifically hybridize with sequence (VII) will include sequence (VII) itself, those having 75^ ° r more, preferably 0% or more conformity to that sequence, and sequences comprising either strand of the two complementary sequences of any of these. Thus the step (c) of the method of this aspect of the invention may be carried out using a variety of hybridization probes that combine sufficiently specifically with the characteristic 'target' sequence comprising sequence (VII) . For most purposes the primer sequences selected from those of groups (A) and (B) will be sufficiently specific to give reliable determination of the characteristic sequence, especially if a different 'primer' sequence is used for the probe of step (c) than those used for step (b) . The step (b) is carried out using the enzyme Taq polymerase as is now conventional in the art. The necessary conditions are those as described in EP-A-0201184 or EP-A-0200362 (both Cetus Corpn.) In such reaction, the appropriate primers derived from the sequences act as initiators for synthesis of large quantities of DNA identical to, or substantially identical to the initial double stranded DNA sequence. In this way substantially larger quantities of the DNA sequence may be made from the small quantities which may be available by isolation from the S. enteritidis or S. dublin thus increasing the amount of sequence available to be detected. The mere opresence of increased amount of DNA may be used in this case to signify presence of target sequence. The genetically transformed organisms of the invention and their use to produce SEFA and SEFA containing sequences of the invention will now be described by way of example only, the examples including use of the detection methods of the invention for confirming presence of transformants: Example At Preparation and cloning of S. enteritidis fimbrial antigen genes. Step Al. Total genomic DNA was extracted from S. enteritidis using the method described in J B Goldberg & D E Ohman, (1984) J Bact 1 8 1115-1121. Step A2. The DNA from step Al was partially digested with SauIIIA restriction endonuclease to yield fragments with an size range between 5 and 10 kb. 2ug of genomic DNA in a Tris-HCl based buffer of pH 7.4 were mixed with 0.25 units of SauIIIA and incubated at 37°C. Step A3. Cloning vector pUClβ was digested to completion with BamHl, giving compatible cohesive ends with SauIIIA, and was dephosphorylated with calf intestinal phosphatase. Step A4. S. enteritidis DNA was ligated with vector pUClδ using T4 DNA ligase supplied by Bethesda Research Laboratories Life Technologies Inc. (Cat. No. 5224SB/SC ). The supplier's instructions for use in ligation were followed. Step A-ό. The recombinant plasmid from step A4 was used to transform commercially available E.coli DH5alpha supplied by Befesda Labs (see above) as Library Efficiency (RTM) DH5alpha Competent Cells (Cat. No. 8263SA) using the supplier's instructions to produce a genomic library. Step A6. Transformants were transferred to the surface of HYBOND-C filters by replica plating for Western Blotting. Standard Western Blotting procedures using the S. enteritidis fimbrial antigen specific monoclonal antibody MAB 69/25, de r ived by standard techniques from hybridoma cells deposited under Accession No.90101101 on 11 October 1990 at the European Collection of Animal Cell Cultures, PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, as described and claimed in copending application No (PCT GB91 ;our ref P0958WOD) , were done to identify transformant colonies expressing SEFA and thus containing the aforementioned sequences (VI), (VII) and (IX). Step A7. The recombinant plasmids from fimbrial antigen positive transformants were extracted and used in confirmatory tests to prove the insert encoded said fimbrial antigen. At the end of stage A7 it is possible to probe the DNA of said transformants to show the presence of the sequences and then to analyse said sequence by known sequencing methods. EXAMPLE B: Presentation of epitopes within the SEFA antigen bv insertion of foreign DNA. in frame, into the SEFA encoding sequence. As stated above, the present invention further provides the prospect of exploitation of the polynucleotide sequences of the present invention having with sequences encoding for desired foreign protein or peptide products to produce transformants having ability to secrete the desired product. SB10 epitope of Mvcobacterium bovis secreted antigen, MPB70 (Radford et al, (1990), J. Gen. Micro. 13 : 265-272) consists of the amino acid sequence as encoded for below: Q D P V encoded amino acid 5'- CAG GAC CCG GTC ~3' coding/master strand 3'- GTC CTG GGC CAG -5' complimentary strand Synthetic oligonucleotides encompassing this sequence and providing BamHl cohesive ends were made using an ABI PCR MATE EP model 391 DNA synthesizer following the manufacturer's methods. The oligonucleotides were as follows: SB10.1 5'- GAT CAG GAC CCG GTC GCT -3' SB10.2 3'- TC CTG GGC CAG CGA CTA G -5' The two oligonucleotides, SBlO.l and SB10.2 were allowed to anneal to form a double stranded (duplex) molecule by heating to 95°C and then cooling to room temperature over a two hour period. Annealing was assessed by comparing rate of migration of the duplex molecule compared with the rate of migration of the two single oligonucleotides when run through % agarose in TBE buffer. A marginal retardation in migration rate was observed and suggested near 100# annealing. A lambda EMBL library was prepared from S.enteritidis strain 1246 providing a 9 to 23 kilobase library which was probed with the SEFA sequence IX (consisting of sequences V and VI run contiguously) . Hybridizing fragments were subcloned into pUClδ and a suitable vector comprising the SEFA antigen gene flanked by adjacent contiguous chromosomal DNA was selected on its ability to transform E.coliDH- alpha to a SEFA expressing form: all general methods as conventional to the art (see eg. Maniatis). The pUClδ vector so obtained was digested with BamHl and agarose gel electrophoresis demonstrated that the DNA was cut once at the unique BamHl site within the SEFA gene. Cut vector and duplex oligonucleotide (SBlO.l plus SB10.2) were mixed together (1:10 ratio) and ligated using T4 ligase (Life Technologies) using the manufacturers methods. The saturating amounts of duplex oligonucleotide increased rate of insertion and the lack of terminal phosphate groups on the duplex prevented multiple insertion. The ligated construct was designed to be as follows: Q D Q D P V A D P amino acid 5*- CAG GAT CAG GAC CCG GTC GCT GAT CCT -3' coding/master strand 3'- GTC CTA GTC CTG GGC CAG CGA CTA GGA -5' complimentary strand The ligated construct lacks the GGATCC BamHl recognition sequence. Thus prior to transforming the construct into E.coli DH5 alpha, the ligated DNA was cut with BamHl to linearise any of the vector which lacked insert. The ligated DNA was then used to transform E.coli using standard procedures. Recombinants were picked directly into a Polymerase Chain Reaction mixture in which the primers were designed to flank the insertion site to yield a product of 219 base pairs without insert or 237 base pairs with insert. PCR products were sized by gel lectrophoresis and those shown to be 237 base pairs were tested by digestion with BamHl to ensure loss of the site. A sample (δul) was taken from the aqueous phase of the PCR reaction mixture and made 20ul by addition of HPLC grade water, X10 reaction buffer and 5U BamHl. The PCR product was digested for 3 hours at 37°C. Control experiments using the 219 base pair product were performed to demonstrate digestion. The entire reaction mixtures were loaded onto agarose gels and the DNA products resolved; those PCR products shown to be 237 base pairs did not cut with BamHl giving evidence for insertion of the oligonucleotide duplex. To confirm the presence of the insert and determine its orientation, PCR experiments were set up in which the primers were SB10.2 and a series of primers from primer group A above (see page lδ) toward the proximal (5') end of the SEFA antigen gene. Of twelve recombinants tested, five gave the desired sized product and were, therefore, shown to have the insert in the correct orientation. To confirm that the insert was encoding the SB10 epitope and was 'in frame' with the SEFA antigen sequence, double stranded DNA sequencing using standard protocols was done on the five positive clones identified above. The primers used were: 5'- TCTGCAGTAGCAGTTCTTGC -3' for the coding strand and 5'- AAAACAGGCTGTCCTTGTCCA -3' for the complimentary strand. The DNA sequence of both strands across the insert site was established and was as predicted above. E. coli recombinants harbouring the constructs, designated SEFA::SB10. 1 to 5 were tested immunologically for the production of SEFA. Western blots of whole E. coli cells harbouring each of the SEFA: :SB10 constructs demonstrated the presence of a protein of about 15kDal (and a less intense protein band of about lδ.5 kDal) when using anti-SEFA polyclonal and anti-SEFA monoclonal antibody 69/25- In control experiments, E. coli recombinants harbouring the vector gave protein bands of l4.5kDal and lδkDal in Western blot experiments using the same antibodies. This data clearly demonstrates that the SEFA polynucleotide sequence may be modified to express additional amino acids within its primary structure without the loss of reactivity to one SEFA epitope specific antibody. The complete sequence of the largest of the sequences of the invention, sequence IX, is given below with the sequences I, II, Ill, IV, V, VI, VII and VIII being indicated together with the probe sequences from probe groups A and B. These sequences are marked by reference to their 5' and 3' ends: eg. 1-5'. 1-3' etc. The numberini given below each 10 base pairs of the sequences I to VI above being related to their positions in this sequence IX. Sequence IX V-5 « '- CATCCTTGTT TTTTTTCTTA AATTTTTAAA ATGGCGTGAG TATATTAGCA TCCGCACAGA '- CTAGGAACAA AAAAAAGAAT TTAAAAATTT TACCGCACTC ATATAATCGT AGGCGTGTCT 10 20 30 40 50 60 TAAATCAGCA ATTTAACACG CTTACGATTA TCAAC1AAAA ACCTCTAAAA CATTATACGC ATTTAGTCGT 70 δO 90 100 110 120 A3-5' TCTGCAGTAG CAGTTCTTGC TTTAATTGCA TGTGGCAGTG CCCACGCAGC TGGCTTTGTT AGACGTCATC GTCAAGAACG AAATTAACGT ACACCGTCAC GGGTGCGTCG ACCGAAACAA 130 140 150 160 170 180 A4-5' GGTAACAAAG CAGAGGTTCA GGCAGCGGTT ACTATTGCAS CTCAGAATAC AACATCAGCC CCATTGTTTC GTCTCCAAGT CCGTCGCCAA TGATAACGTC. GAGTCTTATG TTGTAGTCGG 190 200 210 220 230 240 A4-3' I,III and V -3':II,IV and VI-5' AACTGGAGTC AGGAICCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT TTGACCTCAG TCCTASGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 250 j 260 270 280 290 300 BamHl site. ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTG I T I T _ T 3 TlC AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAAG 370 380 390 400 410 4£0 Bl-3' Bl-5' CGTGGGCGTA TTCAGGGAGC CAATATTAAT GACCAAGCAA ATACTGGAAT TGACGGGCTT GCACCCGCAT AAGTCCCTCG GTTATAATTA CTGGTTCGTT TATGACCTTA ACTGCCCGAA 430 440 450 460 470 480 GCAGGTTGGC GAGTTGCCAG CTCTCAAGAA ACGCTAAATG TCCCTGTCAC AACCTTTGGT CGTCCAACCG CTCAACGGT" GAGAGTTCTT TGCGATTTAC AGGGACAGTG TTGGAAACCA IV-3' AAATCGACCC TGCCAGCAGG TACTTTCACT GCGACCTTCT ACGTTCAGCA GTATCAAAAC TTTAGCTGGG ACGGTCGTCC ATGAAAGTGA CGCTGGAAGA TGCAAGTCGT CATAGTTTTG 550 560 570 580 590 600 B3-3' B3-5' TAATTTAATT TAAACTTTAT AAATGCCCTC AATATGAGCG AGTTTGGATA ATTTTATTAT ATTAAATTAA ATTTGAAATA TTTACGGGAG TTATACTCGC TCAAACCTAT TAAAATAATA 610 620 630 640 650 660 TTTAAAAATA TCTATTTTGA ATAGATAGGT TTTATGCTTC CATGCAAAAA CTTAAAGAGG AAATTTTTAT AGATAAAACT TATCTATCCA AAATACGAAG GTACGTTTTT GAATTTCTCC 670 680 690 700 710 720 GATTATGTAT ATTTTGAATA AATTTATACG TAGAACTGTT ATCTTTTTCC TTTTTTTTGC CTAATACATA TAAAACTTAT TTAAATATGC ATCTTGACAA TAGAAAAAGG AAAAAAAACG 730 740 750 760 770 780 TACCTTCCAA TTGCTTCTTC GGAAAGTAAA AAAATTGAGC AACCATTATT AACACAAAAA ATGGAAGGTT AACGAAGAAG CCTTTCATTT TTTTAACTCG TTGGTAATAA TTGTGTTTTT 790 800 810 820 830 840 TATTATGGCC TAAGATTGGG CACTACACGT GTTATTTATA AAGAAGATGC TCCATCAACA ATAATACCGG ATTCTAACCC GTGATGTGCA CAATAAATAT TTCTTCTACG AGGTAGTTGT 850 860 870 8δo 690 900 AGTTTTTGGA TTATGAATGA AAAAGAATAT CCAATCCTTG TTCAAACTCA AGTATATAAT TCAAAAACCT AATACTTACT TTTTCTTATA GGTTAGGAAC AAGTTTGAGT TCATATATTA 910 920 930 940 950 960 GATGATAAAT CATCAAAAGC TCCATTTATT GTAACACCAC CTATTTTGAA AGTTGAAAGT CTACTATTTA GTAGTTTTCG AGGTAAATAA CATTGTGGTG GATAAAACTT TCAACTTTCA 970 9δO 990 1000 1010 1020 AATGCGCGAA CAAGATTGAA GGTAATACCA ACAAGTAATC TATTCAATAA AAATGAGGAG TTACGCGCTT GTTCTAACTT CCATTATGGT TGTTCATTAG ATAAGTTATT TTTACTCCTC 1030 1040 1050 1060 1070 ιoδo TCTTTGTATT GGTTGTGTGT AAAAGGAGTC CCACCACTAA ATGATAATGA AAGCAATAAT AGAAACATAA CCAACACACA TTTTCCTCAG GGTGGTGATT TACTATTACT TTCGTTATTA 1090 1100 1110 1120 1130 1140 AAAAACAACA TAACTACGAA TCTTAATGTG AATGTGGTTA CGAATAGTTG TATTAAATTA TTTTTGTTGT ATTGATGCTT AGAATTACAC TTACACCAAT GCTTATCAAC ATAATTTAAT 1150 1160 1170 nδo 1190 1200 ATTTATAGGC CTAAAACTAT AGACTTAACG ACAATGGAGA TTGCAGATAA ATTAAAGTTA TAAATATCCG GATTTTGATA TCTGAATTGC TGTTACCTCT AACGTCTATT TAATTTCAAT 1210 1220 1220 1240 1250 1260 GAGAGAAAAG GAAATAGTAT AGTTATAAAG AATCCAACAT CATCATATGT GAATATTGCA CTCTCTTTTC CTTTATCATA TCAATATTTC TTAGGTTGTA GTAGTATACA CTTATAACGT 1270 12δ0 1290 1300 1310 1320 AATATTAAAT CTGGTAATTT AAGTTTTAAT ATTCCAAATG GATATATTGA GCCATTTGGA TTATAATTTA GACCATTAAA TTCAAAATTA TAAGGTTTAC CTATATAACT CGGTAAACCT 1330 1340 1350 1360 1370 1380 TATGCTCAAT TACCTGGTGG AGTACATAGT AAAATAACTT TGACTATTTT GGATGATAAC ATACGAGTTA ATGGACCACC TCATGTATCA TTTTATTGAA ACTGATAAAA CCTACTATTG 1390 1400 1410 1420 1430 1440 GGCGCTGAAA TTATAAGAGA ATTATTAGTT TAAGGTGTAA AACAAATGAA GAAAA; ACA CCGCGACTTT AATATTCTCT TAATAATCAA ATTCCACATT TTGTTTACTT CTTTTGGTGT 1450 1460 1470 1480 1490 1500 ATTACTCTAT TTGTTTTAAC CAGTGTATTT CACTCTGGAA ATGTTTTCTC CAGACAATAT TAATGAGATA AACAAAATTG GTCACATAAA GTGAGACCTT TACAAAAGAG GTCTGTTATA 1510 1520 1530 1540 1550 1560 AATTTCGACT ATGGAAGTTT GAGTCTTCTC CCGGT3AGAA TGCATCTTTT CTAAGTGTTG TTAAAGCTGA TACCTTCAAA CTCAGAAGAG GGCCACTCTT ACGTAGAAAA GATTCACAAC 1570 1580 1590 1600 1610 1620 AAACGCTTCC CTGGTAATTA TGTTGTTGAT GTATATTTGA ATAATCAGTT AAAAGAAACT TTTGCGAAGG GACCATTAAT ACAACAACTA CATATAAACT TATTAGTCAA TTTTCTTTGA 1630 1640 1650 1660 1670 1680 ACTGAGTTGT ATTTCAAATC AATGACTCAG ACTCTAGAAC CATGCTTAAC AAAAGAAAAA TGACTCAACA TAAAGTTTAG TTACTGAGTC TGAGATCTTG GTACGAATTG TTTTCTTTTT 1690 1700 1710 1720 1730 1740 CTTATAAAGT ATGGGATCGC CATCCAGGAG CTTCATGGGT TGCAGTTTGA TAATGAACAA GAATATTTCA TACCCTAGCG GTAGGTCCTC GAAGTACCCA ACGTCAAACT ATTACTTGTT 1750 1760 1770 1780 1790 1800 TGCGTTCTCT TAGAGCATTC TCCTCTTTAA ATATACTTAT AACGCGGCTA ACCAAAGTTT ACGCAAGAGA ATCTCGTAAG AGGAGAAATT TATATGAATA TTGCGCCGAT TGGTTTCAAA 1810 1820 1830 1840 1850 i860 GCTTTTAAAT GCACCATCTA AAATTCTATC TCCAATAGAC AGTGAAATTG CTGATGAAAA CGAAAATTTA CGTGGTAGAT TTTAAGATAG AGGTTATCTG TCACTTTAAC GACTACTTTT 1870 ιδδo 1690 1900 1910 1920 TATCTGGGAT GATGGCATTA ACGCTTTTCT TTTAAATTAC AGAGCTTAAT TATTTGCATT ATAGACCCTA CTACCGTAAT TGCGAAAAGA AAATTTAATG TCTCGAATTA ATAAACGTAA 1930 1940 1950 I960 1970 19δO CTAAGGTTGG AGGAGAGAGA TTCATACTTT GGTCAAATTC AACCTTGGTT TTAATTTTGG GATTCCAACC TCCTCTCTCT AAGTATGAAA CCAGTTTAAG TTGGAACCAA AATTAAAACC 1990 2000 2010 2020 2030 2θ4θ TCCCTGGCGG CTAAGGAATC TATCATCTTG GCAAAACTTG TCAAGCGAAA AAAAATTTGA AGGGACCGCC GATTCCTTAG ATAGTAGAAC AGTTTTGAAC AGTTCGCTTT TTTTTAAACT 2050 2060 2070 2060 2090 2100 ATCAGCATAT ATTTATGCTG AGCGAGGTTT AAAAAAAATA AAGAGCAAAC TAACAGTTGG TAGTCGTATA TAAATACGAC TCGCTCCAAA TTTTTTTTAT TTCTCGTTTG ATTGTCAACC 2110 2120 2130 2l4θ 2150 2160 GGACAAATAT ACCAGTGCAG ATTTATTCGA TAGCGTACCA TTTAGAGGCT TTTCTTTAAA CCTGTTTATA TGGTCACGTC TAAATAAGCT ATCGCATGGT AAATCTCCGA AAAGAAATTT 2170 2180 2190 2200 2210 2220 TAAAGATGAA AGTATGATAC CTTTCTCACA GAGAACATAT TATCCAACAA TACGTGGTAT ATTTCTACTT TCATACTATG GAAAGAGTGT CTCTTGTATA ATAGGTTGTT ATGCACCATA 2230 2240 2250 2260 2270 2280 TGCGAAAACC AATGCGACTG TAGAAGTAAG ACAAAATGGA TACTTGATAT ATTCTACTTC ACGCTTTTGG TTACGCTGAC ATCTTCATTC TGTTTTACCT ATGAACTATA TAAGATGAAG 2290 2300 2310 2320 2330 2340 vι-3' AGTCCCCCCC GGGCAATTCG AGATAGGTAG AGAACAAATT GCTGATG ~3' TCAGGGGGGG CCCGTTAAGC TCTATCCATC TCTTGTTTAA CGACTAG "5' 2350 2360 2370 2380

Claims: CLAIMS 1. Recombinant DNA encoding for the Salmonella enteritidis fimbrial antigen amino acid sequence: M L I V D F W R F C N M R K S A S A V A V L A L I A C G S A H A A G F V G N K A E V Q A A V T I A A Q N T T S A N W S Q D P G F T G P A V A A G Q K V G T L S I T A T G P H N S V S I A G K G A S V S G G V A T V P F V D G Q G Q P V F R G R I Q G A N I N D Q A N T G I D G L A G W R V A S S Q E T L N V P V T T F G K S T L P A G T F T A T F Y V Q Q Y Q N for an epitopic part thereof or for alleles of either. 2. Recombinant DNA as claimed in Claim 1 comprising the sequences I and II: Sequence I 5'- G CTCAGAATAC AACATCAGCC AACTGGAGTC AGGAT -3' 3*- C GAGTCTTATG TTGTAGTCGG TTGACCTCAG TCCTA ~5' 230 240 250 Sequence II 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 3^0 350 36O TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTT -3' AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAA -5' 370 380 390 400 410 sequences degenerately equivalent thereto, or sequences encoding for allelic variants of the part of SEFA for which the sequences I and II encode. 3. Recombinant DNA as claimed in Claim 1 or 2 wherein the sequence comprising sequences I and II comprises sequences III and IV: Sequence III 5'- ATGCTAAT AGTTGATTTT TGGAGATTTT GTAATATGCG TAAATCAGCA 3'- TACGATTA TCAACTAAAA ACCTCTAAAA CATTATACGC ATTTAGTCGT 80 90 100 110 120 TCTGCAGTAG CAGTTCTTGC TTTAATTGCA TGTGGCAGTG CCCACGCAGC TGGCTTTGTT AGACGTCATC GTCAAGAACG AAATTAACGT ACACCGTCAC GGGTGCGTCG ACCGAAACAA 130 140 150 160 170 180 GGTAACAAAG CAGAGGTTCA GGCAGCGGTT ACTATTGCAG CTCAGAATAC AACATCAGCC CCATTGTTTC GTCTCCAAGT CCGTCGCCAA TGATAACGTC GAGTCTTATG TTGTAGTCGG 190 200 210 220 230 240 AACTGGAGTC AGGAT -3' TTGACCTCAG TCCTA -5' 250 Sequence IV 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTTC AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAAG 370 380 390 400 410 420 CGTGGGCGTA TTCAGGGAGC CAATATTAAT GACCAAGCAA ATACTGGAAT TGACGGGCTT GCACCCGCAT AAGTCCCTCG GTTATAATTA CTGGTTCGTT TATGACCTTA ACTGCCCGAA 430 440 450 460 470 480 GCAGGTTGGC GAGTTGCCAG CTCTCAAGAA ACGCTAAATG TCCCTGTCAC AACCTTTGGT CGTCCAACCG CTCAACGGTC GAGAGTTCTT TGCGATTTAC AGGGACAGTG TTGGAAACCA 490 500 510 520 530 540 AAATCGACCC TGCCAGCAGG TACTTTCACT GCGACCTTCT ACGTTCAGCA GTATCAAAAC -3' TTTAGCTGGG ACGGTCGTCC ATGAAAGTGA CGCTGGAAGA TGCAAGTCGT CATAGTTTTG -5' 550 560 570 580 590 600 sequences degenerately equivalent thereto or sequences which encode for allelic variants of SEFA. 4. Recombinant DNA as claimed in any one of the preceding claims wherein suitable flanking sequences for control of amino acid sequence expression are provided. 5. Recombinant DNA as claimed in any one of the preceding claims wherein the sequences I and II are provided in sequences comprising sequences V and VI respectively: Sequence V 5'- GATCCTTGTT TTTTTTCTTA AATTTTTAAA ATGGCGTGAG TATATTAGCA TCCGCACAGA 3'- CTAGGAACAA AAAAAAGAAT TTAAAAATTT TACCGCACTC ATATAATCGT AGGCGTGTCT 10 20 30 40 50 60 TAAATTGTGC GAATGCTAAT AGTTGATTTT TGGAGATTTT GTAATATGCG TAAATCAGCA ATTTAACACG CTTACGATTA TCAACTAAAA ACCTCTAAAA CATTATACGC ATTTAGTCGT 70 80 90 100 110 120 TCTGCAGTAG CAGTTCTTGC TTTAATTGCA TGTGGCAGTG CCCACGCAGC TGGCTTTGTT AGACGTCATC GTCAAGAACG AAATTAACGT ACACCGTCAC GGGTGCGTCG ACCGAAACAA 130 140 150 160 170 180 GGTAACAAAG CAGAGGTTCA GGCAGCGGTT ACTATTGCAG CTCAGAATAC AACATCAGCC CCATTGTTTC GTCTCCAAGT CCGTCGCCAA TGATAACGTC GAGTCTTATG TTGTAGTCGG 190 200 210 220 230 240 Sequence VI 5'- CCTGG CTTTACAGGG CCTGCTGTTG CTGCTGGTCA GAAAGTTGGT 3'- GGACC GAAATGTCCC GGACGACAAC GACGACCAGT CTTTCAACCA 260 270 280 290 300 ACTCTCAGCA TTACTGCTAC TGGTCCACAT AACTCAGTAT CTATTGCAGG TAAAGGGGCT TGAGAGTCGT AATGACGATG ACCAGGTGTA TTGAGTCATA GATAACGTCC ATTTCCCCGA 310 320 330 340 350 360 TCGGTATCTG GTGGTGTAGC CACTGTCCCG TTCGTTGATG GACAAGGACA GCCTGTTTTC AGCCATAGAC CACCACATCG GTGACAGGGC AAGCAACTAC CTGTTCCTGT CGGACAAAAG 370 380 390 400 410 420 CGTGGGCGTA TTCAGGGAGC CAATATTAAT GACCAAGCAA ATACTGGAAT TGACGGGCTT GCACCCGCAT AAGTCCCTCG GTTATAATTA CTGGTTCGTT TATGACCTTA ACTGCCCGAA 430 440 450 460 470 480 GCAGGTTGGC GAGTTGCCAG CTCTCAAGAA ACGCTAAATG TCCCTGTCAC AACCTTTGGT CGTCCAACCG CTCAACGGTC GAGAGTTCTT TGCGATTTAC AGGGACAGTG TTGGAAACCA 490 500 510 520 530 540 AAATCGACCC TGCCAGCAGG TACTTTCACT GCGACCTTCT ACGTTCAGCA GTATCAAAAC TTTAGCTGGG ACGGTCGTCC ATGAAAGTGA CGCTGGAAGA TGCAAGTCGT CATAGTTTTG 550 560 570 580 590 600 TAATTTAATT TAAACTTTAT AAATGCCCTC AATATGAGCG AGTTTGGATA ATTTTATTAT ATTAAATTAA ATTTGAAATA TTTACGGGAG TTATACTCGC TCAAACCTAT TAAAATAATA 610 620 630 640 650 660 TTTAAAAATA TCTATTTTGA ATAGATAGGT TTTATGCTTC CATGCAAAAA CTTAAAGAGG AAATTTTTAT AGATAAAACT TATCTATCCA AAATACGAAG GTACGTTTTT GAATTTCTCC 670 680 690 700 710 720 GATTATGTAT ATTTTGAATA AATTTATACG TAGAACTGTT ATCTTTTTCC TTTTTTTGC CTAATACATA TAAAACTTAT TTAAATATGC ATCTTGACAA TAGAAAAAGG AAAAAAAACG 730 7^0 750 760 770 780 TACCTTCCAA TTGCTTCTTC GGAAAGTAAA AAAATTGAGC AACCATTATT AACACAAAAA ATGGAAGGTT AACGAAGAAG CCTTTCATTT TTTTAACTCG TTGGTAATAA TTGTGTTTTT 790 800 810 820 830 840 TATTATGGCC TAAGATTGGG CACTACACGT GTTATTTATA AAGAAGATGC TCCATCAACA ATAATACCGG ATTCTAACCC GTGATGTGCA CAATAAATAT TTCTTCTACG AGGTAGTTGT 850 860 870 880 890 900 AGTTTTTGGA TTATGAATGA AAAAGAATAT CCAATCCTTG TTCAAACTCA AGTATATAAT TCAAAAACCT AATACTTACT TTTTCTTATA GGTTAGGAAC AAGTTTGAGT TCATATATTA 910 920 930 940 950 960 GATGATAAAT CATCAAAAGC TCCATTTATT GTAACACCAC CTATTTTGAA AGTTGAAAGT CTACTATTTA GTAGTTTTCG AGGTAAATAA CATTGTGGTG GATAAAACTT TCAACTTTCA 970 980 990 1000 1010 1020 AATGCGCGAA CAAGATTGAA GGTAATACCA ACAAGTAATC TATTCAATAA AAATGAGGAG TTACGCGCTT GTTCTAACTT CCATTATGGT TGTTCATTAG ATAAGTTATT TTTACTCCTC 1030 1040 1050 1060 1070 1080 TCTTTGTATT GGTTGTGTGT AAAAGGAGTC CCACCACTAA ATGATAATGA AAGCAATAAT AGAAACATAA CCAACACACA TTTTCCTCAG GGTGGTGATT TACTATTACT TTCGTTATTA 1090 1100 1110 1120 1130 1140 AAAAACAACA TAACTACGAA TCTTAATGTG AATGTGGTTA CGAATAGTTG TATTAAATTA TTTTTGTTGT ATTGATGCTT AGAATTACAC TTACACCAAT GCTTATCAAC ATAATTTAAT 1150 1160 1170 nδo 1190 1200 ATTTATAGGC CTAAAACTAT AGACTTAACG ACAATGGAGA TTGCAGATAA ATTAAAGTTA TAAATATCCG GATTTTGATA TCTGAATTGC TGTTACCTCT AACGTCTATT TAATTTCAAT 1210 1220 1220 1240 1250 1260 GAGAGAAAAG GAAATAGTAT AGTTATAAAG AATCCAACAT CATCATATGT GAATATTGCA CTCTCTTTTC CTTTATCATA TCAATATTTC TTAGGTTGTA GTAGTATACA CTTATAACGT 1270 1280 1290 1300 1310 1320 AATATTAAAT CTGGTAATTT AAGTTTTAAT ATTCCAAATG GATATATTGA GCCATTTGGA TTATAATTTA GACCATTAAA TTCAAAATTA TAAGGTTTAC CTATATAACT CGGTAAACCT 1330 1340 1350 1360 1370 1380 TATGCTCAAT TACCTGGTGG AGTACATAGT AAAATAACTT TGACTATTTT GGATGATAAC ATACGAGTTA ATGGACCACC TCATGTATCA TTTTATTGAA ACTGATAAAA CCTACTATTG 1390 1400 1410 1020 1430 1440 GGCGCTGAAA TTATAAGAGA ATTATTAGTT TAAGGTGTAA AACAAATGAA GAAAACCACA CCGCGACTTT AATATTCTCT TAATAATCAA ATTCCACATT TTGTTTACTT CTTTTGGTGT 1450 1460 1470 1480 1490 1500 ATTACTCTAT TTGTTTTAAC CAGTGTATTT CACTCTGGAA ATGTTTTCTC CAGACAATAT TAATGAGATA AACAAAATTG GTCACATAAA GTGAGACCTT TACAAAAGAG GTCTGTTATA 1510 1520 1530 1540 1550 1560 AATTTCGACT ATGGAAGTTT GAGTCTTCTC CCGGTGAGAA TGCATCTTTT CTAAGTGTTG TTAAAGCTGA TACCTTCAAA CTCAGAAGAG GGCCACTCTT ACGTAGAAAA GATTCACAAC 1570 1580 1590 1600 1610 1620 AAACGCTTCC CTGGTAATTA TGTTGTTGAT GTATATTTGA ATAATCAGTT AAAAGAAACT TTTGCGAAGG GACCATTAAT ACAACAACTA CATATAAACT TATTAGTCAA TTTTCTTTGA 1630 1640 1650 1660 1670 1680 ACTGAGTTGT ATTTCAAATC AATGACTCAG ACTCTAGAAC CATGCTTAAC AAAAGAAAAA TGACTCAACA TAAAGTTTAG TTACTGAGTC TGAGATCTTG GTACGAATTG TTTTCTTTTT 1690 1700 1710 1720 1730 1740 CTTATAAAGT ATGGGATCGC CATCCAGGAG CTTCATGGGT TGCAGTTTGA TAATGAACAA GAATATTTCA TACCCTAGCG GTAGGTCCTC GAAGTACCCA ACGTCAAACT ATTACTTGTT 1750 1760 1770 1780 1790 1800 TGCGTTCTCT TAGAGCATTC TCCTCTTTAA ATATACTTAT AACGCGGCTA ACCAAAGTTT ACGCAAGAGA ATCTCGTAAG AGGAGAAATT TATATGAATA TTGCGCCGAT TGGTTTCAAA 1810 1620 lδ30 1840 1850 i860 GCTTTTAAAT GCACCATCTA AAATTCTATC TCCAATAGAC AGTGAAATTG CTGATGAAAA CGAAAATTTA CGTGGTAGAT TTTAAGATAG AGGTTATCTG TCACTTTAAC GACTACTTTT 1870 ιδδo 1690 1900 1910 1920 TATCTGGGAT GATGGCATTA ACGCTTTTCT TTTAAATTAC AGAGCTTAAT TATTTGCATT ATAGACCCTA CTACCGTAAT TGCGAAAAGA AAATTTAATG TCTCGAATTA ATAAACGTAA 1930 1940 1950 i960 1970 I960 CTAAGGTTGG AGGAGAGAGA TTCATACTTT GGTCAAATTC AACCTTGGTT TTAATTTTGG GATTCCAACC TCCTCTCTCT AAGTATGAAA CCAGTTTAAG TTGGAACCAA AATTAAAACC 1990 2000 2010 2020 2030 2040 TCCCTGGCGG CTAAGGAATC TATCATCTTG GCAAAACTTG TCAAGCGAAA AAAAATTTGA AGGGACCGCC GATTCCTTAG ATAGTAGAAC AGTTTTGAAC AGTTCGCTTT TTTTTAAACT 2050 2060 2070 20δ0 2090 2100 ATCAGCATAT ATTTATGCTG AGCGAGGTTT AAAAAAAATA AAGAGCAAAC TAACAGTTGG TAGTCGTATA TAAATACGAC TCGCTCCAAA TTTTTTTTAT TTCTCGTTTG ATTGTCAACC 2110 2120 2130 2140 2150 2160 GGACAAATAT ACCAGTGCAG ATTTATTCGA TAGCGTACCA TTTAGAGGCT TTTCTTTAAA CCTGTTTATA TGGTCACGTC TAAATAAGCT ATCGCATGGT AAATCTCCGA AAAGAAATTT 2170 2160 2190 2200 2210 2220 TAAAGATGAA AGTATGATAC CTTTCTCACA GAGAACATAT TATCCAACAA TACGTGGTAT ATTTCTACTT TCATACTATG GAAAGAGTGT CTCTTGTATA ATAGGTTGTT ATGCACCATA 2230 2240 2250 2260 2270 2260 TGCGAAAACC AATGCGACTG TAGAAGTAAG ACAAAATGGA TACTTGATAT ATTCTACTTC ACGCTTTTGG TTACGCTGAC ATCTTCATTC TGTTTTACCT ATGAACTATA TAAGATGAAG 2290 2300 2310 2320 2330 2340 AGTCCCCCCC GGGCAATTCG AGATAGGTAG AGAACAAATT GCTGATC ~3' TCAGGGGGGG CCCGTTAAGC TCTATCCATC TCTTGTTTAA CGACTAG -5' 2350 2360 2370 2380 or sequences degeneratively equivalent thereto. 6. Recombinant DNA as claimed in Claim 2 wherein the sequences I and II are comprised within a contiguous sequence VII (as described herein) . 7. Recombinant DNA as claimed in Claim 3 wherein the sequences III and IV are comprised within a contiguous sequence VIII (as described herein) . 8. Recombinant DNA as claimed in Claim 5 wherein the sequences V and VI are comprised within a contiguous sequence IX (as described herein) . 9. Recombinant DNA as claimed in Claim 3 or Claim 7 wherein the amino acid sequence encoded is all or part of an allele of SEFA. 10. Recombinant DNA as claimed in any one of claims 1 to 5 further comprising a sequence encoding for a further amino acid sequence. 11. Recombinant DNA as claimed in Claim 10 wherein tae further amino acid sequence comprises additional SEFA antigen or epitopic parts thereof. 12. Recombinant DNA as claimed in Claim 10 wherein the further amino acid sequence comprises a non-SEFA epitopic sequence. 13- Recombinant DNA as claimed in Claim 12 wherein the non-SEFA epitopic sequence comprises SB10 epitope of Mvcobacterium bovis. 14. A novel plasmid comprising recombinant DNA as claimed in any one of Claims 1 to 13. 15. A plasmid as claimed in Claim 14 comprising a plasmid suitable for transformation of E.coli or yeast into which the recombinant DNA has been inserted. 16. A plasmid as claimed in Claim 14 or 15 comprising pBR322, pACYCl84 or pUClδ into which the recombinant DNA has been inserted. 17. A transformant microorganism comprising a plasmid as claimed in any one of claims l4, 15 or 16. lδ. A microorganism as claimed in Claim 17 wherein the plasmid host is a yeast or an E.coli. 19. A microorganism as claimed in Claim lδ wherein the plasmid host is an E. coli DH5alpha. 20. A plasmid as claimed in any one of Claims 14, 15 or 16 wherein the recombinant DNA sequences are produced by extracting total genomic DNA from an S. enteritidis or a SEFA expressing S. dublin: partially digesting the genomic DNA with SauIIIA restriction endonuclease to provide fragments in the size range to 10 kilobases; ligating the fragments into a plasmid pBR322, pACYClδ4 or pUClδ and selecting desired plasmids for their ability to express SEFA, a part thereof or an allele of either. 21. A plasmid as claimed in Claim 20 wherein a further DNA sequence has been ligated into the BamHl site in sequence I, III, V, VII, VIII or IX. 22. A plasmid as claimed in Claim 20 wherein the further DNA sequence is in frame with the SEFA expressing sequence. 23. A transformant microrganism as claimed in any one of Claims 17 to 19 wherein the plasmid is that as claimed in any one of Claims 20 to 22. 24. A polypeptide or oligopeptide comprising SEFA, an epitopic part thereof or alleles of either as expressed by a transformant as claimed in any one of Claims 17, 18, 19 or 23. 25. A test kit for the identification of microorganisms as being of either serotype S. enteritidis or S. dublin comprising a polypeptide as claimed in Claim 24. 26. A method for the determination of the presence of microorganisms having DNA or RNA polynucleotide sequence encoding for SEFA, an epitopic part thereof or alleles of either, or such DNA or RNA itself, comprising: (a) providing a sample suspected of containing said encoding polynucleotide sequence; (b) determining the presence of said sequence by monitoring hybridization of SEFA sequence targeted polynucleotide hybridization probes with said DNA or RNA. 27- A method as claimed in Claim 26 wherein the polynucleotide probes are targeted to any one of the sequences VII, VIII or XI. 2δ. A method as claimed in Claim 27 wherein the polynucleotide probe consists of sequence VII, VIII or XI. 29. A method for determining the presence of microorganisms having DNA or RNA polynucleotide sequence encoding for SEFA or an epitopic part thereof, or such DNA or RNA itself, comprising: (a) providing a sample suspected of containing said encoding polynucleotide sequence; (b) subjecting said sample to conditions under which polynucleotide sequences comprising sequences (I) and (II) are replicated by use of the polymerase chain reaction; (c) determining the presence of any sequence produced. 30. A method as claimed in Claim 29 wherein the step (c) is carried out using a polynucleotide hybridization probe. 31. A method as claimed in either of Claim 29 or Claim 30 wherein step (b) employs primer pairs comprising one primer selected from group (A) and the other from group (B) : Group A: Group B: 5' -GTGCGAATGCTAATAGTTGA- 3' 5' -AAAACAGGCTGTCCTTGTCCA- 3' 5' -TGCGTAAATCAGCATCTGCA- 3' 5' -TTAGCGTTTCTTGAGAGCTGG- 3* 5' -TCTGCAGTAGCAGTTCTTGC- 3' 5' -TTTTGATACTGCTGAACGTAG- 3' 5' -GCTCAGAATACAACATCAGCCAA- 3* 32. A method as claimed in any one of Claims 29 to 31 wherein the step (c) is carried out using an oligonucleotide probe selected from sequences of either of groups A or B (as described herein) which is different to that of either of the primers used for step (b) . 33 * A test kit for performing the method of any one of Claims 26 to 28 comprising polynucleotide hybridization probes targeted at sequence VII, VIII. 34. A test kit as claimed in Claim 33 wherein the probes comprise sequences comprising sequence VII or VIII. 35- A test kit for performing the method of any one of Claims 29 to 32 comprising primers and probes having sequences selected from the groups (A) and (B) .

Inventors: WOODWARD MARTIN JOHN, WOODWARD, MARTIN, JOHN
Applicants: MINI AGRICULTURE & FISHERIES, THE MINISTER FOR AGRICULTURE, FISHERIES AND FOOD I
Application year: 1991
Main classification: C12Q1
IPC codes: C07K7, C07K14, C12N15, C12P21, C12Q1, C12R1, G01N33