Abstract:
The present invention relates generally to molecular compounds which encode the protective M-like protein of  Streptococcus equi  (SeM), the amino acid compound which is thereby encoded, and compositions of matter which incorporate either the encoding compounds or the cellular components for which they encode. For instance, vaccines which utilize the amino acid compounds or vectors and cell lines useful to make the amino acid compounds described herein are subjects of the present invention. The present invention provides methods to stimulate  S. equi -specific immune response in horses. It also provides diagnostic assays for  Streptococcus equi.

Description:
This application claims priority to U.S. Provisional Patent Application No. 60/050,577, filed Jun. 24, 1997. 
    
    
     This invention was developed under a grant from the U.S. Government: USDA-NRICGP Number 95-01837, and therefore, the U.S. Government may have certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates generally to molecular compounds which encode the protective M-like protein of  Streptococcus equi  (SeM), the amino acid compound which is thereby encoded, and compositions of matter which incorporate either the encoding compounds or the cellular components for which they encode. For instance, vaccines which utilize the amino acid compounds or vectors and cell lines useful to make the amino acid compounds described herein are subjects of the present invention. The present invention provides methods to stimulate  S. equi -specific immune response in horses. It also provides diagnostic assays for  Streptococcus equi.    
       Streptococcus equi,  a Lancefield group C streptococcus, causes strangles, a highly contagious disease of the nasopharynx and draining lymph nodes of Equidae. The 58 kDa antiphagocytic M-like protein (SeM) is a major virulence factor and protective antigen and functions by limiting deposition of C3b on the bacterial surface and by directly binding fibrinogen. Boschwitz and Timoney, 17  Microbiol. Pathogenesis  121 (1994) and Boschwitz and Timoney, 62  Infect. Imnun.  3515 (1994). 
     In the recent past,  S. equi  outbreaks on horse farms have been avoided and treated by quarantine of suspect animals; antiseptic handling of food, bedding and housing; and antibiotics when indicated. Vaccines comprising avirulent  S. equi  or fractions thereof have been described, but success rate has been low. U.S. Pat. No. 5,183,659 describes a vaccine which stimulated a nasopharyngeal antibody response in horses, but the vaccine had a limitation of many such vaccines, which is risk of reversion to virulence and occasional abcess development in vaccinated horses. 
       S. equi  is shed in nasal discharges and pus from lymph nodes of affected animals. Routine laboratory detection of the bacterium involves bacteriologic culture of nasal swabs, nasal washes and pus from abscesses and is often difficult because of background contamination, small numbers of the organism, or the presence of  S. zooepidemicus  and other β-hemolytic streptococci. Completion of culture and identification usually takes 2 to 3 days, an excessively lengthy interval given the highly contagious nature of strangles and the need to quickly identify shedding horses so that they may be isolated in the early stages of an outbreak. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide molecular compounds which encode SeM and compositions of matter which incorporate either the encoding compounds or the cellular components for which they encode. 
     It is therefore an object to provide vectors, cell lines and cell membrane preparations using the compounds disclosed. 
     It is yet another object to provide a method to provide assays for detection of  Streptococcus equi.    
     Other objects and features of the present invention win be apparent from the following detailed description, examples and claims. 
     Definitions 
     As used herein, the following terms shall have the corresponding meaning set forth. All other terms are intended to have the meaning as understood commonly by those in the relevant field of art. 
     “Biological Sample” means nasal or oral mucus sample or blood sample. 
     “Transformation” and “transfection” mean to cause nucleic acid to enter a cell, whether or not the nucleic acid incorporates into the genome. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The invention will be further described in conjunction with the accompanying drawings, in which: 
     FIG. 1 (SEQ ID NO:10) is the nucleotide and deduced amino acid sequence of SeM. Base and amino acid positions are shown on the left. Putative promoter and ribosomal binding sites (RBS) are boxed, and signal and membrane anchor sequences are shown in bold type. Repeats are underlined. 
     FIG. 2 is an immunoblot showing reactions of a lysate of  E. coli  BL21, SeM02 and a mutanolysin extract of  S. equi  with antisera 216 and 963 to SeM and recombinant SeM respectively. 
     FIG. 3 is an immunoblot showing the reactions of mutanolysin extracts of a series of temporally and geographically separated isolates of  S. equi  with antisera to recombinant SeM. Estimated molecular masses are shown to the right of the figure. 
     FIG. 4 is an immunoblot showing linear epitopes recognized by IgA in nasal washes of convalescent (8 weeks) horses. 
     FIG. 5 is graph illustrating the regions of SeM reactive with antibodies in horse sera taken 8 weeks after recovery from strangles. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides nucleic acid compounds comprising a compound of the following sequence: 
     
       
         
               
               
               
             
           
               
                 1 
                 AGCTTTCTGTCACCTGATGGTCCTTATCAAATACTGTAATTGATAACTTCAAACAGCCCT 
                   
               
               
                   
               
               
                 61 
                 GTAGAGATTTTACTAACGACATAGTATCCATGCTAAGCGTCACCCCCTTCATAATCCTCA 
               
               
                   
               
               
                 121 
                 CGGTATCTTATTCTATCTTAAAATTTAAGAAAAGCAAGGATATGCACTTATAATGAAAAA 
               
               
                   
               
               
                 181 
                 ATAGACATAAAAAACAATAATATACATTCTTGCTTATTAAATAAAAATGACAATGTACTG 
               
               
                   
               
               
                 241 
                 CATAAAGAAGTTCCTGTCATTAAAATAAAAGTGCCATGAGGTTATAATAGTATGGTAAAA 
               
               
                   
               
               
                 301 
                 CAAAAAAGTGTGCCCATAACGGGTAGAGAGGAATTGACATATGTTTTTGAGAAATAACAA 
               
               
                   
               
               
                 361 
                 GCCAAAATTTAGCATCAGAAAACTAAGTGCCGGTGCAGCATCAGTATTAGTTGCAACAAG 
               
               
                   
               
               
                 421 
                 TGTGTTGGGAGGGACAACTGTAAAAGCGAACTCTGAGGTTAGTCGTACGGCGACTCCAAG 
               
               
                   
               
               
                 481 
                 ATTATCGCGTGATTTAAAAAATAGATTAAGCGATATAGCCATAAGTGGAGATGCCTCATC 
               
               
                   
               
               
                 541 
                 AGCCCAAAAAGTTCGAAATCTTCTAAAAGGCGCCTCTGTTGGGGATTTACAGGCATTATT 
               
               
                   
               
               
                 601 
                 GAGAGGTCTTGATTCAGCAAGGGCTGCGTATGGTAGAGATGATTATTACAATTTATTGAT 
               
               
                   
               
               
                 661 
                 GCACCTTTCATCGATGTTAAATGATAAACCTGATGGGGATAGAAGACAATTAAGTTTGGC 
               
               
                   
               
               
                 721 
                 TTCATTACTTGTAGATGAAATTGAAAAGCGGATTGCTGATGGAGATAGGTATGCAAAACT 
               
               
                   
               
               
                 781 
                 TCTTGAGGCTAAACTTGCAGCTATTAAATCTCAACAAGAAATGCTTAGAGAAAGAGATTC 
               
               
                   
               
               
                 841 
                 CCAACTTCGAAATCTAGAGAAGGAGAAAGAACAAGAGCTCACAAAAGCTAAAGATGAGCG 
               
               
                   
               
               
                 901 
                 TCAAGCTCTTACCGAATCATTCAACAAAACTTTATCAAGATCAACAAAAGAGTATAATAA 
               
               
                   
               
               
                 961 
                 ACTAAAAACAGAACTTGCAAAAGAAAAAGAAAAAGCAGCTAAGATGACTAAGGAATTAGC 
               
               
                   
               
               
                 1021 
                 AGATAAGCTAAGCAATGCTGAAGCAAGTCGTGATAAAGCCTTTGCAGTATCAAAAGATTT 
               
               
                   
               
               
                 1081 
                 AGCAGATAAACTAAGTAGTGCTGAAGCAAGTCGTGATAAAGCTTTTGCAGTATCAAAAGA 
               
               
                   
               
               
                 1141 
                 TTTAGCAGATAAATTGGCAGCTAAAACAGCAGAAGCTGAAAAGTTAATGGAAAACGTTGG 
               
               
                   
               
               
                 1201 
                 TAGTCTAGACCGCTTGGTAGAGTCTGCAAAACGTGAAATGGCTCAAAAATTAGCAGAAAT 
               
               
                   
               
               
                 1261 
                 TGATCAATTAACTGCTGATAAGGCTAAGGCTGATGCAGAGCTTGCAGCTGCAAATGACAC 
               
               
                   
               
               
                 1321 
                 CATTGCATCACTTCAAACAGAGCTAGAAAAAGCTAAGACAGAGCTTGCTGTTTCAGAGCG 
               
               
                   
               
               
                 1381 
                 TTTGATTGAATCAGGCAAACGTGAAATTGCTGAGCTACAAAAACAAAAAGATGCTTCTGA 
               
               
                   
               
               
                 1441 
                 TAAGGCTTTAGTAGAATCACAAGCTAATGTAGCAGAGCTTGAAAAACAAAAAGCAGCATC 
               
               
                   
               
               
                 1501 
                 AGATGCTAAGGTAGCAGAGCTTGAAAAAGAAGTTGAAGCTGCTAAAGCTGAGGTTGCAGA 
               
               
                   
               
               
                 1561 
                 TCTTAAAGCACAATTAGCTAAGAAAGAAGAAGAGCTTGAAGCCGTTAAGAAGGAAAAAGA 
               
               
                   
               
               
                 1621 
                 AGCGCTTGAAGCTAAGATTGAAGAGCTCAAAAAAGCTCATGCTGAGGAACTTTCAAAACT 
               
               
                   
               
               
                 1681 
                 TAAAGAAATGCTTGAGAAGAAAGACCATGCAAATGCAGATCTTCAAGCAGAAATCAATCG 
               
               
                   
               
               
                 1741 
                 CTTGAAGCAAGAGCTAGCTGACAGGATTAAGTCATTGTCACAAGGTGGTCGTGCTTCACA 
               
               
                   
               
               
                 1801 
                 AACAAACCCAGGCACTACAACTGCTAAAGCAGGTCAATTGCCATCTACTGGTGAGTCTGC 
               
               
                   
               
               
                 1861 
                 TAACCCATTCTTCACTATTGCAGCTCTTACTGTCATCGCTGGTGCTGGAATGGCTGTGGT 
               
               
                   
               
               
                 1921 
                 GTCTCCTAAACGCAAAGAAAACTAAGCTATTTCCTCTTTCCCCAATGGACAATAGCCGAA 
               
               
                   
               
               
                 1981 
                 ATAATAGAGCGACTATCGTTCTAACACAAAAGCAACAGTCTCCTGTCTGTTGCTTTTTGT 
               
               
                   
               
               
                 2041 
                 GATATTAGGGCTCATCAGTCTAGGCTAATGGTTTTCTGCGCTTTATCTGCA 
               
             
          
         
       
     
     [SEQ ID NO. 1 (M-protein (SeM) gene of  S. equi )]. The open reading frame starts at 341 and continues to base 1934. Preferably, the compound is SEQ ID NO 1. However, also provided are compounds which are complementary to SEQ ID NO 1 or complementary to portions of SEQ ID NO 1. A preferred portion of SEQ ID NO 1 is that described above as base number 341 to 1943, inclusive (SEQ ID NO 3). Moreover, the compound defined by bases number 452 through approximately 1331 are also preferred (SEQ ID NO 4). 
     As skilled artisans can appreciate, the DNA compounds exemplified above can readily instruct one as to useful, complementary RNA sequences. Any such RNA sequences are also considered within the scope of the present invention. In the present disclosure, this group of DNA and RNA compounds are termed “nucleic acid compounds”. 
     Moreover, cells transfected with a (or multiple copies of a) nucleic acid compound of the present invention are provided. Preferred is a cell transfected with SEQ ID NO 1 or a portion thereof. Such cells may be prokaryotic or eukaryotic. Preferred cells include:  E. coli, S. cerevisiae,  and Salmonella spp. Also provided are vectors transfected with a (or multiple copies of a) nucleic acid compound of the present invention. Preferred vectors include pox viruses, adenoviruses or other viral vectors. 
     The nucleic acid compounds may be obtained by PCR using primers as is skill of the art, or from Gen Bank, accession number U73162. Vectors and cell lines can also be obtained as is skill of the art. 
     Moreover, also provided is the amino acid compound: 
     
       
         
               
               
               
             
           
               
                   
                       5        10        15        20        25        30 
                   
               
               
                   
               
               
                 1 
                 M F L R N N K P K F S I R K L S A G A A S V L V A T S V L G 
               
               
                   
               
               
                 31 
                 G T T V K A N S E V S R T A T P R L S R D L K N R L S D I A 
               
               
                   
               
               
                 61 
                 I S G D A S S A Q K V R N L L K G A S V G D L Q A L L R G L 
               
               
                   
               
               
                 91 
                 D S A R A A Y G R D D Y Y N L L M H L S S M L N D K P D G D 
               
               
                   
               
               
                 121 
                 R R Q L S L A S L L V D E I E K R I A D G D R Y A K L L E A 
               
               
                   
               
               
                 151 
                 K L A A I K S Q Q E M L R E R D S Q L R N L E K E K E Q E L 
               
               
                   
               
               
                 181 
                 T K A K D E R Q A L T E S F N K T L S R S T K E Y N K L K T 
               
               
                   
               
               
                 211 
                 E L A K E K E K A A K M T K E L A D K L S N A E A S R D K A 
               
               
                   
               
               
                 241 
                 F A V S K D L A D K L S S A E A S R D K A F A V S K D L A D 
               
               
                   
               
               
                 271 
                 K L A A K T A E A E K L M E N V G S L D R L V E S A K R E M 
               
               
                   
               
               
                 301 
                 A Q K L A E I D Q L T A D K A K A D A E L A A A N D T I A S 
               
               
                   
               
               
                 331 
                 L Q T E L E K A K T E L A V S E R L I E S G K R E I A E L Q 
               
               
                   
               
               
                 361 
                 K Q K D A S D K A L V E S Q A N V A E L E K Q K A A S D A K 
               
               
                   
               
               
                 391 
                 V A E L E K E V E A A K A E V A D L K A Q L A K K E E E L E 
               
               
                   
               
               
                 421 
                 A V K K E K E A L E A K I E E L K K A H A E E L S K L K E M 
               
               
                   
               
               
                 451 
                 L E K K D H A N A D L Q A E I N R L K Q E L A D R I K S L S 
               
               
                   
               
               
                 481 
                 Q G G R A S Q T N P G T T T A K A G Q L P S T G E S A N P F 
               
               
                   
               
               
                 511 
                 F T I A A L T V I A G A G M A V V S P K R K E N 
               
             
          
         
       
     
     which is SEQ ID NO 2. The present invention also includes portions of the above sequence. The most preferred portion of the above sequence is: residues 37 to 330 (SEQ ID NO 5) however, one skilled in the art recognizes that any especially antigenic portion is commercially significant, and is included in the scope of the present invention. 
     The amino acid compounds can be obtained either by overexpression and purification in microorganisms, or, in some cases, by conventional peptide synthesis. 
     Therefore, a vaccine for  S. equi  is provided by the present invention. A vaccine which is administered intranasally or orally is a preferred embodiment. A vaccine of the present invention can comprise the entire SEQ ID NO 2, or portions thereof. 
     The vaccines of the present invention can be of any pharmaceutically-acceptable formulation. For example, SEQ ID NO 5 can be incorporated into bilayer vesicles (liposomes), in an aqueous medium according to known procedures, such as that described by Debs et al., 265  J. Biol. Chem.  10189 (1990). Any available carrier or liposome-forming lipid may be utilized in any formulation which delivers SeM antigen, for example, poly-DL-Lactide-co-glycolide may be utilized in an intranasal spray formulation which comprises SEQ ID NO 5. Formulations which include adjuvants which enhance the delivery of antigen to the mucosa, such as small amounts of the Bsubunit of the cholera toxin are also within the scope of the present invention. 
     The present invention also provides methods to stimulate a  S. equi -specific immune response in horses comprising administering a compound of SEQ ID NO 2 or portions thereof. Introduction of the antigen nasally or orally is preferred. 
     Lastly, the present invention provides methods to determine the presence of  S. equi  in horses via polymerase chain reaction. The polymerase chain reaction diagnostic assay of the present invention can be accomplished according to known methods so long as primers for the sequence herein disclosed are used as some of the starting materials. Methods for PCR can be found in many journals and books, for instance, the PCR diagnostic methods can be accomplished according to  Techniques in PCR, PCR, Current Protocols in Molecular Biology or Maniatis.  As skilled artisans are aware, preferred primers are those which are at least 50% GC content, ideally 19 to 23 base pairs in length, and are not capable of annealing to duplicate sections of the target DNA. 
     EXAMPLES 
     Example 1 
     Cloning, Sequencing and Expression of SeM 
     Chromosomal DNA of  S. equi  CF32 was partially digested with Tsp 5091 (New England Biolabs Inc., Beverly Mass.) and 3-8 kb fragments ligated to λ ZAPII digested with EcoRI (Stratagene, LaJolla, Calif.). After packaging (Gigapack II) (Stratagene, LaJolla, Calif.) and transfection into  E. coli  XLI-Blue MRF′ (Stratagene, LaJolla, Calif.) , the library was plated, amplified and stored at −70° C. in 7% DMSO. The library was screened on duplicate nitrocellulose discs by using rabbit 216 antiserum (1:4000 dilution) to the acid extracted 41 kDa fragment of SeM. Several reactive plaques were screened until all plaques gave a positive signal. Proteins in these phage lysates were separated by SDS PAGE and immunoblotted with serum 216. A plasmid containing a 3.5 kb fragment encoding SeM was excised from a positive phage and the resulting plasmid designated pSeM01. Nucleotide sequencing was performed on HindIII, Pvu II and Hind III-Pvu II fragments of the  S. equi  insert in pSK by automated cycle sequencing. Sequences were aligned and connected by DNASIS (Hitachi Software Engineering America, Ltd., San Diego, Calif.). SeM without its signal sequence was subcloned into the Bam H1 site of the pET15b (Novagen, Madison, Wis.) expression vector using the polymerase chain reaction (PCR) with pSeM01 as template and primers SeM-F (gcggatcCGAACTCTGAGGTTAGTCGT) (SEQ ID NO 6) and SeM-R (gcggatccATAGCTTAGTTTTCTTTGCG) (SEQ ID NO 7). The resulting plasmid was designated pSeM02 and transformed into  E. coli  BL21 (DE3). Recombinant SeM was isolated from a lysate of BL21 by affinity chromatography on His-Bind Resin (Novagen, Madison, Wis.). 
     Analysis of the connected sequences revealed the presence of one open reading frame of 1605 nucleotides encoding the seM gene (FIG.  1 ). Translation of seM revealed a basic (CH+4.5) preprotein of 535 amino acids of calculated molecular mass 58,251 and pI of 8.67. N terminal amino acid sequence (residues 37-52) was identical to that obtained by direct microsequencing of the 41 kDa SeM fragment purified from an acid extract of  S. equi.  The predicted amino acid sequence exhibits features typical of streptococcal surface proteins. The signal sequence is 36 residues. The N terminus of the mature protein has a net positive charge. The anchor membrane spanning region and charged tail sequences are similar to those of other group A and C streptococcal sequences. Two direct repeats (21 residues) are located between residues 226 and 267. Other shorter direct repeats varying in length from 3 to 6 residues occur in the carboxy terminal half of the molecule. Analysis of the secondary structure of the translated protein shows an extensive region of alpha helix extendnng approximately from residue 120 to 480. The secondary structure prediction shows turns in the vicinity of residues 120 and 480 to 500. 
     Example 2 
     Amino Acid Sequence Determination 
     Acid extracted SeM (2 mg) purified as described in Example 3 was loaded onto a 2 cm wide slot on a 1.5 mm thick acrylamide (9%) gel for preparative SDS PAGE. The gel was prerun with 0.1 mM thioglycolic acid for 40 mins at 100 V before loading the protein. Following electrophoresis, and electrophoretic transfer to Immobilon P (Millipore), the major 41 and 46 kDa fragments of SeM were identified by staining for 1 min. with 0.025% Coomassie blue in 40% methanol and 5% glacial acetic acid followed by destaining for 2 min in 30% methanol and 5% glacial acetic acid. The 41 kDa band was excised and microsequence analysis performed on a model 477A pulse liquid phase sequencer (Applied Bio Systems) at the University of Kentucky Macromolecular Synthesis Laboratory. 
     Example 3 
     Protein Extraction 
     M-like protein was extracted from an overnight culture (18 h) of  S. equi  by hot acid extraction (Lancefield and Perlmann. 96  J. Exp. Med.  71 (1952) and adsorbed to a column of hydroxyapatite in 10 mM phosphate buffer pH 7.2. The M-like proteins were eluted in 0.2 M Na2HPO4, desalted on Sephadex G25 and lyophilized. The pellet was dissolved in 25% acetonitrile+0.5% trifluoroacetic acid and loaded on a phenyl RP reverse phase column (Bio-Rad, San Francisco Calif.) connected to a Waters 650 protein purification system (Waters, Marlborough, Mass.). Protein was eluted using a linear gradient of 25-65% acetonitrile+0.5% TFA. A dot immunoblot on cellulose nitrate using SeM specific rabbit antiserum was used to screen peaks. The peak containing SeM protein eluted at a concentration of approximately 42% acetonitrile. Positive peaks from several runs were pooled and further purified on the same column. Purified protein was lyophilized, resuspended in PBS and stored in aliquots at −20° C. Mutanolysin extracts of  S. equi  strains were performed as described previously (Galán and Timoney, 26  J. Clin. Microbiol.  1142 (1988). 
     Example 4 
     Antisera 
     Antiserum was raised against SeM purified by a combination of hydroxyapatite and reverse-phase chromatography. A New Zealand white rabbit (216) was injected subcutaneously with 50 μg SeM in complete Freund&#39;s adjuvant followed at 3 week intervals by 2 similar doses emulsified in incomplete Freund&#39;s adjuvant. Serum was harvested at 8 weeks. Rabbit 963 was similarly hyperimmunized with recombinant SeM from  E. coli  sonicate. Rabbit Ec was immunized with a lysate of  E. coli  NovaBlue containing plasmid pT7 Blue without an insert. Adult ICR mice were immunized with 25 μg SeM purified from  E. coli  sonicate by HIS-Tag chromatography. Purified SeM (25 μg) was mixed with 5 μg mycolic dipeptide (MDP) and alhydrogel (30%) and administered subcutaneously in a dose volume of 100 ml. Two subcutaneous booster doses containing 25 μg SeM but no MDP were administered 10 and 20 days later. Mice were bled at 28 days. All antisera were stored at −70° C. until used. 
     Example 5 
     Immunoblotting 
     Proteins in streptococcal extracts or purified from  E. coli  sonicate were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE) and electroblotted to a sheet of nitrocellulose and incubated in the appropriate antiserum diluted 1:200 in PBS and then in peroxidase-conjugated protein G (1:4000). Reactive bands were visualized by using 4-chloro-1-naphthol (0.5 mg/ml) as substrate. 
     Rabbit 963 antiserum to recSeM reacted with a 58 kDa protein in a mutanolysin extract of  S. equi  and with a slightly larger 60 kDa protein expressed by  E. coli  BL21 containing pSeM02 (FIG.  2 ). The same protein bands were recognized by rabbit 216 antiserum to the 41 kDa fragment of SeM. FIG. 3 shows the immunoblot profiles of mutanolysin extracts of a series of  S. equi  isolates collected at different times in the US and Europe. Antiserum to recSeM recognized two protein bands of 58 and 56 kDa in all the extracts. 
     Example 6 
     ELISA 
     An acid extracted fragment (41 kDa) of SeM purified by preparative electrophoresis on agarose was used to coat wells (2.5 mg/well) of polystyrene ELISA plates (Costar, 25880, Corning Glass Company, Corning N.Y.). After washing and blocking in 0.1 M phosphate buffered saline (PBS) containing 0.05 Tween 20 and 1% bovine serum albumin, mouse or rabbit sera diluted 1:80 and 1:200 respectively in PBS were added in triplicate to the wells (100 ml/well). After incubation for 3 hours at 37° C., bound IgG was detected with either peroxidase conjugated protein G (1:4000) or rabbit anti-mouse IgG followed by O-phenylene diamine (0.0001 mM) solution. Mean OD values of triplicate readings were blanked against wells containing antigen and PBS. 
     Example 7 
     Opsonic Assay 
     Equine neutrophils were separated from freshly collected heparinized horse blood with a discontinuous Percoll gradient Pycock et al., 42  Res. Vet. Sci.  411 (1987). Neutrophils from 7 ml of blood were suspended in RPM1 medium (Gibco, Grand Island, N.Y.) and 80 ml aliquots (6×105 cells) added in triplicate to wells of a 24 well cell culture cluster (Costar, Cambridge, Mass.). Each well contained a circular glass coverslip (12 mm diameter). The cell cluster was incubated for 2 h at 37° C. in 5% CO2 and the cells washed once with PBS to remove non-adherent neutrophils. The test organisms ( S. equi  CF32 and  S. zooepidemicus  W60) were grown overnight at 37 C. in THB with 0.2% yeast extract to an OD of 0.6. Twenty ml of culture was added to 25 ml serum and 450 ml RPMI added. After the plate was gently shaken for 30 minutes at 37 C. the coverslips were washed once with PBS (pH 7.2) fixed in 10% formalin and stained with Giemsa The numbers of neutrophils with associated streptococci per 100 cells were then counted for each serum and expressed as a percentage. All assays were performed in triplicate. The differences in the opsonic activities of immune and control sera were evaluated statistically by a Student t-test (Unpaired Observations) based on the means of three experiments. 
     Sera from mice immunized with purified recombinant SeM showed 15 times greater (p&lt;0.01) opsonic activity for  S. equi  than non-immune mouse sera. These sera also showed strong antibody responses by ELISA to the 41 kDa fragment of SeM (Example 6). 
     Example 8 
     Fibrinogen Binding Assay 
     Equine fibrinogen (0.5 mg/well) was bound to wells of 96 well polystyrene ELISA plates (Costar). After washing and blocking, recombinant SeM (0.4 mg/well) was added in triplicate to separate wells and incubated for 2 hours at 37 C. After washing, 1:80 dilutions of rabbit antisera to the 41 kDa fragment of SeM was added to the appropriate wells and incubated at 37 C. for 2 hours. Control wells consisted of wells from which fibrinogen was omitted and wells treated with sera from the same rabbits before immunization. Amounts of specific rabbit antibody that bound to SeM fixed to fibrinogen were detected as described under ELISA. 
     SeM showed strong binding to equine fibrinogen immobilized on wells of ELISA plates. Mean ELISA value (±SD) for SeM bound to fibrinogen after correction for non-specific binding of the protein to blocked well surfaces was 0.9±0.1. The corrected value was 0.1±0.1 when preimmune sera were used to assay for binding of the streptococcal protein. 
     Example 9 
     Nucleotide Sequence Accession Numbers 
     The Gen Bank accession number for the nucleotide sequence of seM is U73162. 
     Example 10 
     Homologies 
     With the exception of signal and membrane anchor sequences, no homology of SeM with group A or G M protein sequences in the GenBank database was detected. SeM showed some homology between its signal (39% identity) and membrane anchor (66% identity) sequences with those in the database. 
     Example 11 
     Presence of SeM-binding Antibodies in Convalescent Horses 
     Regions of SeM reactive with antibodies in horse sera taken 8 weeks after recovery from strangles are shown in FIG.  3 . Most horses showed reponses to epitopes on the central region of SeM (residues 170 to 270). Responses of individual horses to the N terminal third and carboxy terminal regions of SeM were much more variable. No horse responded to peptide 151-166 alone. Linear epitopes recognized by IgA in convalescent (8 weeks) nasal washes are found on the same region reactive with serum antibody (FIG.  4 ). Multiple epitopes are targeted and, as in the case of serum antibodies, there is considerable variation in the responses of individual horses. 
     Example 12 
     Immunizations of Non-exposed Yearling Ponies 
     Two groups of 3 yearling Welsh ponies were immunized with microencapsulated recombinant fusion peptide of the M-protein (SeM, amino acids 231-330) produced in  E. coli  BL21 and with a microencapsulated extract of the host  E. coli  alone. Encapsulated protein (100 μg) was sprayed into each nostril on day 1 using a nasal atomizer. Booster doses of 150 and 350 μg were given on days 7 and 42, respectively. Serum and nasal washes were collected at days 1, 7, 21 and 42 and assayed for SeM-specific IgG in serum and IgA in nasal washes, 
     Specific mucosal IgA responses to SeM were evident at day 21 in 2 of the 3 ponies and in all ponies at day 49. None of the 3 control ponies immunized with  E. coli  extract alone responded to SeM. No serum antibody responses were detected in any pony. These studies demonstrate the feasibility of selectively-inducing specific mucosal antibody responses in horses by using a microencapsulated streptococcal peptide. 
     Example 13 
     PCR Diagnostic Assay 
     Nasal swabs (Precision Dynamic Corp. San Fernando, Calif.; Culturette, Baxter Healthcare Corp., Deerfield, Ill.) were collected from affected and exposed in-contact horses 1 to 5 days after clinical diagnosis of strangles on farms A, B, C, and D. Some horses were sampled more than once over the following 3 weeks. Nasal washes were collected from horses at UK farm 15 and 85 days following commingling exposure to 2 horses with clinical strangles. All these horses developed strangles within 17 days of commingling exposure. Nasal washes were collected by instilling 50 ml phosphate buffered saline (pH 7.2) via a 8 mm diameter latex tube inserted 15 cm into the nostril and collecting the fluid that drained out. The fluid was centrifuged at 3000 g and the pelleted debris separated for culture and PCR. Swabs and nasal wash pellets were cultured on Columbia CNA horse blood agar and incubated for 18 hours at 37° C. Beta hemolytic colonies were subcultured and their fermentation behavior tested in lactose, sorbitol and trehalose broths. Mucoid beta-hemolytic colonies that did not ferment any of these sugars were identified as  S. equi.    
     DNA for PCR from nasal swabs and washes was prepared as follows: Swab tips were placed in 300 μl of sterile water, vortexed, the tips removed and the fluid centrifuged at 14,000 g for 10 minutes. The sediments were resuspended in 20 μl of K-buffer (1× Gen Amp Buffer II, Perkin Elmer, 0.5% Tween 20, 100 μg/ml Proteinase K). Nasal wash pellets were suspended in an equal volume of K-buffer. The suspensions were incubated for 30 minutes at 55° C., boiled for 5 minutes and then centrifuged for 5 minutes at 14,000 g. The reaction mix for PCR in a total volume of 30 μl was prepared in Gen Amp Buffer II and contained 2 mM MgCl 2 , 0.2 mM dNTP, 0.5 units Taq polymerase, 0.25 μM SeM6 and SeM7 primers, and 2-5 μl sample. The primer sequences were 5′-TGCATAAAGAAGTTCCTGTC (SeM7-forward (bases 239-258) SEQ ID NO 8) and 5′-GATTCGGTAAGAGCTTGACG (SeM reverse(bases 899-918) SEQ ID NO 9). Mineral oil (30 μl) was added to seal the reaction mix. Cycling was performed as follows: −92° C. for 2 minutes; 92° C. for 1 minute; 58° C. for 1 minute; 72° C. for 1 minute (30 times); 72° C. for 5 minutes; 4° C. final. The PCR products were separated by ethidium bromide agarose gel electrophoresis. The SeM fragment amplified with primers SeM6 and 7 was 679 bp. 
     The PCR detected a 679 bp DNA fragment in 37 specimens including 14 of 15 that were positive by culture. The sensitivity of PCR appears to be much greater than culture. 
     Although the present invention has been fully described, it is to be noted that various changes and modifications are apparent to those skilled in the art Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims.