Virulence-encoding DNA sequences of Strepococcus suis and related products and methods

The invention provides DNA sequences which code for polypeptides which are characteristic for the virulence of the pathogenic bacterium Streptococcus suis and parts thereof, and polypeptides and antibodies derived therefrom. The sequences code for a polypeptide of 90,000-120,000 daltons or a polypeptide of higher molecular weight containing such a polypeptide, and for a polypeptide of 135,000-136,000 daltons (muramidase released protein), or parts thereof. The sequences themselves, and also the polypeptides and antibodies derived therefrom, are used for diagnosis of and protection against infection by S. suis in mammals, including man.

FIELD OF THE INVENTION 
The invention is in the field of veterinary and human preventive medicine, 
in particular that of the diagnosis of and protection against infection by 
pathogenic strains of the bacterium Streptococcus suis. 
Infections with Streptococcus suis serotype 2 in young pigs at about the 
time of weaning have been a growing problem in the Netherlands since 1983. 
The disease is characterised by meningitis, arthritis, sepsis and death 
(Clifton-Hadley 1983, ref. 6; Vecht et al. 1985, ref. 44; Windsor 1977, 
ref. 50). It is estimated that 5-10 per cent of farms have problems of 
this type. The mortality is estimated at 2.5% and the morbidity in 
affected farms is on average 2-5%. Therapeutic and preventive measures 
have only a limited effect. The economic damage is accordingly 
appreciable. The disease is a zoonosis. Humans are also susceptible to 
this infection, with the risk of sepsis and meningitis with possibly 
permanent side-effects; rare cases of death have been reported (Arends and 
Zanen 1988, ref. 2). This related mostly to cases of people with a skin 
wound coming into contact with infected pork. In particular, pig farmers 
and slaughterhouse staff belong to the risk group. 
There are indications that the increased rate of illness on pig farms in 
the Netherlands since 1983 is to be ascribed to the import of breeding 
animals which are carriers of S. suis type 2. Carriers are often healthy 
adult pigs which harbour the streptococci in the tonsils and mucosa of the 
upper respiratory tract. The infection is transmitted via these carriers 
to susceptible animals, frequently piglets at weaning age. Diagnosis of 
animals which are already sick or have died is based on isolation and 
determination of S. suis type 2 from clinical samples or organs after 
necropsy. Detection of carriers is based on bacteriological examination of 
nose or throat swabs or tonsil biopsies using a selective/elective medium 
(Van Leengoed et al. 1987, ref. 27). On the basis of diagnostic testing to 
detect carriers, it should be possible to set up a control programme. 
However, testing for carriers using the conventional becteriological 
techniques is time-consuming, which complicates the processing of large 
numbers of samples; there is also a risk of false negative results due to 
overgrowth with contaminants. Finally, interpretation of the test demands 
a great deal of experience. Moreover, diagnosis and possible control on 
the basis of diagnosis are further complicated by the occurrence of 
differences in pathogenicity within the S. suis type 2 species. Regular 
testing for carriers within a control programme is sensible only if truly 
virulent strains of S. suis type 2 can be differentiated from avirulent 
strains. Current diagnostic techniques do not make such discrimination. 
Consequently, control based on the detection of carriers of virulent S. 
suis type 2 strains is not yet possible. 
Differences in virulence are ascribed, inter alia, to the presence or 
absence of virulence factors. In 1984, Arends and Zanen (ref. 1) described 
"lysozyme-positive proteins" in human strains. In a study with 
experimental animals it was found that a "lysozyme-positive" strain 
(D-282) was pathogenic for gnotobiotic pigs, in contrast to a 
"lysozyme-negative" strain (T-15) (Vecht et al. 1989, ref. 43). The 
"lysozyme-positive protein " is probably identical to the 
muramidase-released protein (MRP) of strain D-282. 
The pig industry in the Netherlands and many other countries has a pyramid 
structure, with a small number of breeding herds at the top, from where 
animals are distributed to replication herds. These supply a large number 
of fattening herds, supplying animal products to slaughterhouses. A 
control program based on diagnosis (certification of farms, elimination of 
positive carriers, import requirements) should primarily aim at creating 
herds which are free of S. suis type 2 high in this pyramid. A vaccine 
would primarily be useful in affecting herds lower in the pyramid. 
Furthermore, means and methods for diagnosing infections by Streptococcus 
suis in human medicine can be of value. 
SUMMARY OF THE INVENTION 
The object of the invention is to provide methods and means which make it 
possible, in a more effective manner than hitherto, to detect infections 
by Streptococcus suis on the one hand and to prevent such infections by 
elimination of infected pigs and carrier pigs on the other hand. 
This object is achieved by using a DNA sequence from the gene which codes 
for a virulence characteristic of S. suis. In this context, a virulence 
characteristic is defined as a polypeptide whose presence is associated 
with the virulence of an organism, in this case the bacterium S. suis, in 
particular serotype 2. 
Two genes of virulent strains of S. suis type 2 have been found which code 
for two proteins, which are designated MRP (muramidase released protein) 
and EF (extracellular factor) and which appear to be characteristic for 
virulence (virulence factors). MRP and EF are high molecular weight 
proteins. MRP (136kd) is a protein associated with the cell envelope and 
can be released from the cell wall by muramidase. EF (110kD) is an 
extracellular product which is secreted by the bacterium into the growth 
medium. EF has higher molecular weight counterparts which are denoted 
herein as EF* . 
The invention provides new diagnostic methods which are able to 
differentiate between virulent and avirulent strains. These methods are 
based on the genes encoding MRP, EF and EF* and their expression products. 
On the basis of the expression of one or both proteins by said genes, 
three different phenotypes of S. suis type 2 have been found to date: i.e. 
the MRP+ EF+ phenotype, the MRP+ EF- phenotype and the MRP- EF- phenotype. 
77% (N=111) of strains isolated from organs of pigs showing clinical 
symptoms of disease were found to possess the MRP+ EF+phenotype, while 86% 
(n=42) of isolates from tonsils of non-suspect normal slaughter pigs were 
found to possess the MRP- EF- phenotype. The MRP+ EF- phenotype was most 
frequently found (74%) (n=27) in isolates from human patients with 
infections of S. suis type 2 (see FIG. 10). Hence infected animals and 
carriers of virulent strains can be detected, and a vaccine based on MRP, 
EF and/or EF* can be developed. Using the diagnostic methods for detecting 
carriers and infected pig herds and/or using vaccines based on MRP, EF 
and/or EF* , a program for controlling infection by S. suis type 2 in pig 
herds can be developed. 
The invention therefore also relates to the DNA sequence of the gene which, 
apart from coding for specific high molecular weight polypeptides, codes 
for the 90-120 kDa polypeptide which is a characteristic of S. suis 
virulence, which gene, hereinafter designated the ef gene has the 
nucleotide sequence according to SEQ ID No: 1 for S. suis serotype D-282, 
and to equivalent sequences and to parts of said sequences. The nucleotide 
sequence of the entire region coding for EF and the flanking sequences 
have been determined. Analysis of the sequence of the ef gene SEQ ID NO: 
1provides an open reading frame of 2529 nucleotides which codes for a 
polypeptide of 843 amino acids (calculated molecular weight 90,014). 
Monoclonal antibodies generated against the 110 kDa EF protein recognised 
proteins with a higher molecular weight in culture supernatants of all 38 
strains with a MRP+EF-phenotype. This indicates that certain of the 110 
kDA EF and the high molecular weight proteins are identical. None of the 
91 strains with a MRP+ EF+ phenotype was found to produce these high 
molecular weight proteins. At the same time, DNA probes based on the gene 
which codes for the 110 kDa EF were found to hybridise with genes which 
code for the high molecular weight proteins of MRP+ EF- strains. This 
indicates that the 110 kDa EF and the high molecular weight proteins are 
related, which implies that at least part of the ef gene, from strains 
with a MRP+ EF- phenotype, is identical to the ef gene of strains with the 
MRP+ EF+ phenotype. The higher molecular weight counterpart of the protein 
EF is designated herein as EF* , and the gene encoding it as the ef* gene. 
The corresponding nucleotide and amino acid sequences are represented in 
SEQ ID NO: 2 
The invention also relates to the DNA sequence of the gene which codes for 
the 135-136 kDa polypeptide which is also a virulence characteristic of S. 
suis, which gene, hereinafter designated the mrp gene, has the nucleotide 
sequence according to SEQ ID NO: 3 for S. suis serotype 2 strain D-282, 
and to equivalent sequences and to parts of said sequences. The nucleotide 
sequence of the entire region coding for MRP and the flanking sequences 
have been determined. Analysis of the sequence and the flanking sequences 
have been determined. Analysis of the sequence of the mrp gene SEQ ID NO: 
3 shows an open reading frame of 3768 nucleotides which codes for a 
polypeptide of 1256 amino acids (calculated molecular weight 135,794). 
In this context, an equivalent sequence comprises a sequence which is 
essentially the same as the sequence shown but can display slight 
differences, such as point mutations, or other modifications which may be 
caused by substitution, deletion, insertion or additional. Similarly, an 
equivalent sequence also comprises a sequence which, despite any 
differences in nucleotide sequence, hybridises with the sequence shown or 
with its complement, and also comprises a related sequence which means 
that it codes for the same amino acid sequence despite differences in 
nucleotide sequence. 
The invention also relates to a recombinant polynucleotide which contains 
an ef/ef* gene and/or mrp gene sequence as described above, in the 
presence of a regulating sequence. A recombinant of this type, such as a 
virus vector, a plasmid or a bacterium, can be used for expression of the 
gene or of relevant parts thereof in a desired environment, for example 
for the production of immunogenic peptides intended for the diagnosis of 
an infection, or for controlling infection with virulent strains of S. 
suis by vaccination. 
Polynucleotide probes which contain a sequence as described above, derived 
from a gene which codes for a virulence characteristic of S. suis, also 
form part of the invention. A probe of this type in particular corresponds 
with part of the nucleotide sequence of one of the two said genes. The 
probe can be used for direct detection of the presence of sequences of 
virulent strains of S. suis. The probe can also be used as a basis for a 
primer for the multiplication of polynucleotides (for example in a 
polymerase chain reaction) as part of a diagnostic method or a protection 
method. 
A suitable polynucleotide probe was found to be a partial sequence 
containing at least 10 nucleotides, preferably at least 15 nucleotides, up 
to 835 nucleotides from the sequence 1100-1934 of the mrp gene. Another 
suitable polynucleotide probe was found to be a partial sequence 
containing 10-417, in particular 15-417 nucleotides from the sequence 
2890-3306 of the ef* gene. These probes differentiate effectively between 
pathogenic and non-pathogenic strains of S. suis. A combination of such an 
mrp based probe and an ef* based probe is an especially powerful 
diagnostic tool. 
The invention also relates to polypeptides which are derived from a 
polynucleotide sequence described above. A polypeptide of this type is 
either coded by said sequence or obtained by expression of said sequence 
and essentially corresponds to a S. suis protein characteristic of 
virulence, or to a part thereof. A polypeptide of this type can, for 
example, be used as an antigen in an immunoassay, as an immunogen in the 
immunisation of mammals or as an immunogen for the production of 
antibodies for diagnostic purposes. The antibodies generated in this way 
also form part of the invention. Such antibodies can be polyclonal or 
monoclonal and can be conjugated with a marker (enzyme, isotope, 
luminescent substance or complex-forming agent); the antibody can also be 
bound to solid carriers or substrates. 
The invention also relates to methods for the detection of an infection by 
a pathogenic strain or by a non-pathogenic strain of S. suis, in which one 
or more polynucleotide probes, polypeptides and/or antibodies as described 
above are used. "Infection" signifies here the presence of the pathogenic 
organism, both in the case where there are clinical signs of disease 
(infection in a narrow sense) and in the case where there are no clinical 
signs of disease (infection in a broad sense, of contamination). For 
immunoassays, such as a determination of the presence of antigens of 
and/or antibodies against S. suis in a sample or in clinical material, it 
is possible, for example, to use on a microtiter plate a polypeptide (110 
kDa) which is encoded by the ef/ef* gene or a part thereof, and/or an 
antibody which has been generated against such a polypeptide. In addition, 
it is also possible to use a polypeptide (136 kDa) encoded by the mrp gene 
or a part thereof, and/or an antibody which has been generated against 
such a polypeptide. The diagnostic methods can be carried out using 
procedures known per se. Examples are Enzyme-Linked Immunosorbent Assays 
(ELISA) and Double Antibody Sandwich (DAS)-ELISA. 
The methods described above can be carried out with the aid of diagnostic 
kits. A diagnostic kit according to the invention contains, respectively, 
at least one polynucleotide or a polypeptide which corresponds to or is 
derived from a sequence of the ef/ef* gene or mrp gene or a part thereof 
or contains an antibody which has been generated against the polypeptide 
derived from one of the said ef/ef* and mrp sequences. It is also possible 
to use combinations of probes and the like, in particular of ef* 
diagnostic agents and mrp diagnostic agents, or combinations of primers, 
for example for carrying out PCR. The kits can also contain the components 
required for carrying out diagnoses, such as reagents (labelling 
substances, dyes and the like), supports (filters, plates and the like), 
media and calibrating agents as well as a manual for carrying out the 
diagnosis. 
The invention also relates to a method for protecting mammals against 
infection by Streptococcus suis, in which method a polynucleotide, a 
polypeptide or an antibody as described above is used. When an antibody is 
used, the method is a passive immunisation, that is to say there is direct 
provision of antibodies against the pathogenic organism; since antibodies 
which are derived from EF, EF* and MRP are directed against the most 
virulent forms of S. suis, a procedure of this type can be an effective 
method for protecting against, or controlling, infection, especially if 
the animal to be protected is not itself able to produce sufficient 
antibodies, for example if infection has already taken place or in the 
case of young animals. 
Another form of passive immunisation in the case of pigs is the 
administration of antibodies to the piglets via the colostrum from the 
sow. In this case the dam is actively immunised with one or both 
polypeptides during pregnancy, that is to say before the birth of the 
piglets. When a polypeptide or a polynucleotide (optionally in the form of 
a recombinant organism) is used, the procedure is an active immunisation, 
the animal to be protected being stimulated, by means of the immunogenic 
polypeptide which is administered directly or in the form of a gene for 
expression, to produce antibodies. 
Another suitable method of immunisation is the administration of a 
polypeptide from which the activity responsible for virulence has been 
neutralised. Such a polypeptide should then no longer be pathogenic, while 
immunogenic characteristics are retained. It can be obtained, for example, 
by expression of a gene which has been modified with respect to the 
original ef/ef* or mrp gene, such as by means of deletion. 
Vaccines for protecting mammals against an infection by S. suis, which 
vaccines contain a polynucleotide, a polypeptide or an antibody as 
described above, also form part of the invention. 
A particular vaccine according to the invention is a vaccine which contains 
a S. suis material which does not or does not completely bring to 
expression at least one of the polypeptides corresponding to EF and MRP. 
This material can originate from or can be formed by a possible live 
strain which is not virulent or is less virulent. 
The role of virulence factors which are involved in the pathogenesis of S. 
suis type 2 has been studied in vivo by means of gnotobiotic/germ-free 
piglets with S. suis type 2 strains defined in respect of virulence 
factors (MRP and EF). The animal experiments were monitored by means of 
haematological, bacteriological and (histo)pathological analytical 
techniques. 
BRIEF DESCRIPTION OF THE SEQUENCE LISTING 
SEQ ID NO: 1 
Nucleotide sequence of the ef gene and the adjacent sequences and the EF 
amino acid sequence derived therefrom. The presumed ribosome binding site, 
the -35 and -10 regions of the presumed promoters, and the regions with 
complementary symmetry are marked. The possible cleaving site for signal 
peptidase is between nucleotides 498-499. 
SEQ ID NO: 2 
Nucleotide sequence of the fragment encoding the S. suis type 2 ef* gene of 
strain 1890 and the deduced amino acid sequence of the EF* protein of 
class I. The putative ribosome binding site, the -35 and -10 regions of 
the putative promoter sequences, the repetitive regions R1-R11, and the 
putative termination signals are indicated. The region between the 
nucleotides 2859 and 5228 is absent in the gene encoding the 110 kDa EF 
protein. The region between the nucleotides 3423 and 4456 is absent in the 
genes encoding the call IV and class V EF* proteins. 
SEQ ID NO: 3 
Nucleotide sequence of the 4.6 kb EcoRI-HindIII fragment with the mrp gene 
of S. suis type 2 and the MRP amino acid sequence derived therefrom. The 
probable ribosome binding site, the -35 and -10 regions of the presumed 
promoter sequences, the region of complementary symmetry beyond the mrp 
gene, the repeating amino acid sequences and the envelope anchor region 
are indicated.

DETAILED DESCRIPTION OF THE INVENTION 
EXAMPLE 1 
Cloning and nucleotide sequence analysis of the gene encoding the 110 kDa 
extracellular protein of pathogenic Streptococcus suis type 2 strains 
MATERIAL AND METHODS 
Bacterial strains and growth conditions. E. coli strains JM101 (29) and 
LE392 (33) were used as hosts for recombinant plasmids and bacteriophages. 
The pathogenic MRP.sup.+ EF.sup.+ strain D282 of S. suis type 2 (43) was 
used for the isolation of chromosomal DNA. E. coli strains were grown in 
Luria broth (30). Ampicillin was added as needed to a final concentration 
of 50 .mu.g/ml. S. suis strains were grown in Todd-Hewitt broth (Oxoid, 
Ltd., London, England). 
Construction and immunological screening of the DNA library. A DNA library 
of S. suis type 2 strain D282 was constructed in LambdaGEM-11 as 
recommended by the manufacturer of the cloning vector (Promega, Madison, 
USA). Recombinant bacteriophages were plated on E. coli strain LE392 and 
incubated for 16 h at 37.degree. C. 
Nitrocellulose filters (Schleicher and Schuell, Inc., Dassel, Germany) were 
placed on the plaques, and the plates were further incubated for 2 h at 
37.degree. C. Recombinants that produced EF were visualized with 
monoclonal antibodies (Mabs) directed against EF (Example 4). Bound 
antibodies were detected with anti-mouse serum conjugated with alkaline 
phosphatase (Zymed Laboratories, Inc., San Francisco, USA) as described by 
Maniatis et al. (28). Selected EF positive clones were purified by several 
rounds of single plaque isolation and immunological screening. 
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and 
Western blot analysis. Proteins were separated by SDS gel electrophoresis 
in which 4% stacking and 6% separating gels were used (26). The separated 
proteins were transferred to nitrocellulose in a Semi-Dry transfer cell 
(Bio-Rad Laboratories, Richmond, USA). Specific proteins were visualized 
by use of polyclonal antibodies (Pabs, Example 4) or Mabs directed against 
EF and anti-rabbit or anti-mouse sera conjugated with alkaline phosphatase 
(Zymed Laboratories). 
DNA manipulations and nucleotide sequence analysis. Selected restriction 
fragments were (sub)-cloned in the plasmid vector pKUN19 (24) by standard 
molecular biological techniques (28). Progressive unidirectional deletions 
were made with the Erase-a-Base system from Promega (Madison, USA). DNA 
sequences were determined by the dideoxy chain termination method (37). 
DNA and protein sequences were analysed by the software packages PCGENE 
(Intelli-genetics Corp., Mountain View Calif.) and Wisconsin GCG 
(University of Wisconsin). 
RESULTS 
Cloning of the ef gene. A DNA library was constructed by isolating 
chromosomal DNA from strain D282 of S. suis type 2. This DNA was partially 
digested with the restriction enzyme Sau3A and cloned into the 
bacteriophage LambdaGEM11 replacement vector. The library contained 
approximately 5.times.10.sup.5 recombinants per .mu.g of DNA. Two thousand 
plaques of recombinant phages were tested for the presence of antigenic 
determinants of EF by use of a Mab directed against EF. Two plaques were 
positive. The expression of EF by the two selected recombinant 
bacteriophages was studied by Western blotting to analyse the proteins 
eluted from plaques. Both recombinants encoded a protein that comigrated 
with EF secreted by S. suis and that was recognized by Mabs directed 
against EF. Thus both recombinant bacteriophages contained the complete 
genetic information for EF. The genetic information for EF on the 
recombinant bacteriophages was localized using restriction enzyme 
analysis. The two clones shared a DNA region of about 13 kb. Parts of the 
common DNA region were subcloned into plasmid pKUN19 (FIG. 3) and the 
proteins expressed by the recombinant plasmids were analyzed by Western 
blotting. The plasmid containing the 6.8 kb KpnI-SalI fragment (pEF2-19, 
FIG. 3) encoded a protein with a molecular weight identical to EF, that 
was recognized by Mabs directed against EF. Plasmids containing the 5.8 kb 
EcoRV-SalI or the 5.3 kb BglII-SalI fragment, however, did not express EF. 
These data indicate that the EcoRV and the BglII sites are within regions 
required for EF expression. 
Nucleotide sequence of the ef gene. The nucleotide sequence of the fragment 
comprising the EF encoding region was determined. The sequence SEQ ID NO: 
1 showed the presence of 3 major open reading frames (ORFs). ORF1 (from 
nucleotide 361 to 2890), ORF2 (from nucleotide 2856 to 3459) and ORF3 
(from nucleotide 3462 to 4053) encoded polypeptides of 843 amino acids, of 
201 amino acids and of 197 amino acids respectively. ORF1 contained a 
putative ATG start codon that is preceded by a sequence that is similar to 
ribosome binding sites of several types of gram-positive bacteria (17). In 
contrast, neither a start codon, nor a ribosome binding site upstream of 
the ORFs 2 and 3 could be found. The 3' end of ORF1 and the 5' end of ORF2 
are overlapping, albeit in different frames. The ORFs 2 and 3 are 
separated by a single TAA stop codon. Upstream of ORF1 two putative 
promoter sequences were found that resembled the -35 and -10 consensus 
sequences of promoters commonly found in gram-positive bacteria (FIG. 1A). 
Downstream of ORF3, two regions of extended dyad symmetry were present. 
Because both regions contained a stretch of thymidine residues at the end 
of the potential stem-loop structures, these potential transcription 
terminators are likely to be rho-independent (34, 40). Because the 
sequence data did not reveal obvious transcription and translation signals 
upstream of, or within ORF2 and ORF3, it is doubtful that these ORFs 
express proteins. Another possibility is that the entire sequenced region 
contains one large open reading frame. This situation would occur if only 
two sequence errors were present: a +1 base pair frame shift in the region 
2856 to 2892 and an error in the stop codon at position 3459. This 
possibility was excluded by sequencing the ef gene from three additional, 
independently selected clones. Fragments of the initial clones were used 
as hybridization probes in order to isolate these clones from the 
chromosome. The nucleotide sequences of these fragments were identical to 
those presented in SEQ ID NO: 1 
Amino acid sequence of EF. Because only ORF1 was preceded by appropriate 
expression/initiation signals, this ORF probably encodes EF. This was 
confirmed by subcloning two fragments into plasmid pKUN19: a SpeI-SnaBI 
fragment, that contained the entire ORFs 1 and 2 and a SpeI-NarI fragment, 
that contained ORF1 and the 5' end of ORF2 (FIG. 3). The proteins 
expressed by the recombinant plasmids were analysed by Western blotting. 
In E. coli both recombinant plasmids encoded a protein that was recognized 
by a Mab directed against EF and that had a molecular weight identical to 
that of EF secreted by S. suis. Therefore, ORF1 encodes EF. The molecular 
weight of the ORF1 product calculated from the sequence (90,000) differed, 
however, from that of EF estimated from SDS polyacrylamide gels (110,000). 
EF is exclusively found in the supernatant of S. suis cultures, and thus 
the protein is expected to be preceded by a signal peptide. Indeed, the 
first 46 amino acids of the deduced amino acid sequence of EF are 
characteristic of a typical signal peptide. An N-terminal part that 
contained six positively charged amino acids was followed by a 
hydro-phobic core of 21 amino acids and a putative signal peptidase 
cleavage site (45). The hydropathy pattern (25) of the deduced amino acid 
sequence showed that, apart from the signal peptide, the EF protein was 
very hydrophilic and did not contain extended hydrophobic regions (cf. 
MRP, Example 3). No significant similarities were found between the 
deduced amino acid sequence of EF and the protein sequences in the EMBL 
Data Library. 
Although appropriate translation initiation signals upstream of ORF2 and 
ORF3 could not be found, the deduced amino acid sequences of ORF2 and ORF3 
showed some properties which raised doubt to the idea that those frames 
are not expressed. The N-terminus of the putative ORF2 protein showed two 
highly repetative units of 57 amino acids (identity 82%). The C-terminus 
of the putative ORF3 protein is functionally similar to C-terminal regions 
of several cell-envelope located proteins of grampositive bacteria (10, 
12, 13, 16, 41). A hydrophobic region was preceded by the conserved 
sequence Leu-Pro-X-Thr-Gly-Glu and followed by a highly hydrophilic 
region. This similarity suggests that the putative ORF3 protein is 
associated with the cell-envelope. 
EXAMPLE 2 
Cloning and nucleotide sequence analysis of genes encoding extracellular 
proteins of non-pathogenic Streptococcus suis type 2 strains 
MATERIALS AND METHODS 
Bacterial strains and growth conditions. Escherichia coli strain JM101 (29) 
was used as host for recombinant plasmids. Seventeen MRP.sup.+ EF* strains 
of S. suis type 2 were isolated from human patients, five strains from 
tonsils of slaughthered pigs, seven strains from organs of diseased pigs 
and from two strain the origin was unknown (Example 4). The E. coli strain 
was grown in Luria broth (30). Ampicillin was added as needed to a final 
concentration of 50 .mu.g/ml. Streptococcus suis strains were grown in 
Todd-Hewitt broth (Oxoid, Ltd., London, England). 
Genomic DNA and oligonucleotides. Genomic DNA was isolated by lysis in 
proteinase K/SDS solution, extraction with phenol/chloroform and 
precipitation with ethanol (28). The sequences of the oligonucleotides 
used in the polymerase chain reaction (PCR) were: 
5'-ATGTAATTGAATTCTCTTTTTAAGT-3' and 5'-AAACGTCCGCAGACTTCTAGATTAAAAGC-3'. 
These oligonucleotides correspond to the positions 35 to 59 and 4308 to 
4279 in the S. suis type 2 ef gene SEQ ID NO: 1. The underlined sequences 
indicate the recognition sites for the restriction enzymes EcoRI and XbaI. 
DNA manipulations and nucleotide sequence analyses were carried out as 
described in Example 1. 
SDS - PAGE and Western blot analysis were carried out as described in 
Example 1. 
Southern hybridization. DNA was transferred to Gene-Screen Plus membranes 
(New England Nuclear Corp., Dreieich, Germany) as described by Maniatis et 
al. (28). DNA probes were labeled with (.sup.32 P)dCTP (3000 Ci/mMol, 
Amersham Corp., Arlington Heights, USA) by the use of a random primed 
labeling kit (Boehringer GmbH, Mannheim, Germany). The blots were 
hybridized with DNA probes as recommended by the supplier of the 
Gene-Screen Plus membranes. After hybridization the membranes were washed 
twice with a solution of 2.times.SSC (1.times.SSC is 0.15M NaCl plus 
0.015M trisodium citrate, pH 7.0) for 5 min at room temperature and twice 
with a solution of 0.1.times.SSC plus 0.5% SDS for 30 min at 65.degree. C. 
Amplification of genomic DNA fragments by Polymerase Chain Reaction (PCR). 
PCR was used to amplify ef* sequences. Genomic DNA from different 
MRP.sup.+ EF* strains of S. suis type 2 was used as a template. Amplified 
DNA fragments were isolated by agarose gelelectrophoresis and extraction 
from the gel with Gene Clean (Bio101, La Jolla, USA). The purified 
fragments were digested with EcoRI and XbaI and cloned into the plasmid 
pKUN19 (24). To exclude mistakes in the DNA sequences as a result of the 
PCR, six independently choosen clones were mixed prior to the nucleotide 
sequence analyses. 
RESULTS 
Western blot of EF* proteins. Culture supernatants of strains of S.suis 
type 2 belonging to the MRP.sup.+ EF* phenotype contained proteins that 
were recognized by Mabs directed against EF (Examples 4, 6). The molecular 
weights (MW) of these proteins varied and were higher than that of EF. The 
proteins secreted by thirty-one strains of the MRP.sup.+ EF* phenotype 
were compared with those secreted by a strain of the MRP.sup.+ EF.sup.+ 
phenotype. EF* proteins of five different molecular weight classes were 
found. Three strains synthesized an EF* protein of approximately 195 kDa 
(class I); eighteen an EF* of approximately 180 kDa (class II); one an EF* 
of approximately 175 kDa (class III); five an EF* of approximately 160 kDa 
(class IV) and four an EF* of approximately 155 kDa (class V). 
Southern hybridization of ef* genes. The relationship between the genes 
encoding the 110 kD EF and the EF* proteins was studied. Chromosomal DNA 
of different MRP.sup.+ EF* strains (two representatives of each class were 
taken) and of the MRP.sup.+ EF.sup.+ strain D282 (43) was digested with 
the restriction enzyme PstI. The various DNAs were hybridized with a 
.sup.32 P labeled EcoRV-SnaBI fragment containing the entire ef gene (FIG. 
4, see Example 1). The results showed that the DNA digests of the 
MRP.sup.+ EF* as well as the MRP.sup.+ EF.sup.+ strains contained two PstI 
fragments that strongly hybridized with the probe. These data indicated 
that the genes encoding the 110 kDa EF and the EF* proteins are strongly 
related. The length of the largest hybridizing fragment was the same in 
all strains. In contrast, the length of the smallest hybridizing fragment 
differed between the strains. Moreover, the variation in length of the 
smallest hybridizing fragment correlated well with the variation in the 
molecular weight of the EF* proteins secreted by the different strains. 
Since the smallest hybridizing fragment is located at the 3' end of the ef 
gene (FIG. 4, Example 1), these data suggest that the ef and ef* genes 
differed mainly at their 3' ends. 
Cloning of ef* genes. The genes encoding the different EF* proteins were 
obtained using PCR to amplify the ef* containing DNA fragments. Genomic 
DNA of 5 different MRP.sup.+ EF* strains of S. suis type 2 (one 
representative of each class) was used as a template. The amplified 
fragments were digested with restriction enzymes EcoRI and XbaI and cloned 
into E. coli. 
Ef* gene of class I. The nucleotide sequence of a 6.8 kb EcoRI-XbaI 
fragment containing the entire ef* gene of class I and the regions 
flanking it was determined. Analysis of the sequence revealed two 
open-reading frames (ORFs, SEQ ID NO. 2). The first ORF (from nucleotide 
361 to 5827) and the second ORF (from nucleotide 5830 to 6421) encoded 
polypeptides of 1822 amino acids and 197 amino acids respectively. Based 
on its size the first ORF is expected to encode the EF* protein (195 kDa). 
The ORFs were separated by a single TAA stop codon. The first ORF 
contained a putative ATG start codon that was preceded by a sequence 
similar to bacterial ribosome-binding sites (17). In contrast, the second 
ORF was not preceded by an appropriate start codon, nor by a putative 
ribosome-binding site. 
The first 46 amino acids of the deduced amino acid sequence of the EF* 
protein had the characteristics of a typical signal peptide (45). The C 
terminus of the mature part of the protein contained a number of imperfect 
repeats of 76 amino acids. In the EF* protein of class I ten and a half 
repeats were present (denoted as R1 to R11, SEQ ID NO. 2. The first four 
repeats were contiguous as were the last six and a half repeats. The 
fourth and the fifth repeated unit, however, were separated by 113 amino 
acids and the fifth and the six unit by 22 amino acids (FIG. 5). The amino 
acid sequences of the last five and a half unit were highly conserved, 
whereas the sequences of the first five units were more variable. One 
particular amino acid sequence, Asn-Pro-Asn-Leu, was conserved in all 
repeated units. No significant homology was found between the EF* sequence 
of class I and any protein sequence in the EMBL Data Library. 
Ef* genes of class II, III, IV and V. Because the genes encoding the 
various EF* proteins differed mainly at their 3' ends, the nucleotide 
sequences of the small PstI fragments from the genes of class II, III, IV 
and V were determined. Comparison of the nucleotide sequences showed that 
the various ef* genes were highly homologous in this region. The ef* genes 
differed, however, in the number and the arrangement of repeated units 
(FIG. 5). Unlike the ef* gene of class I, the ef* genes of class II and IV 
lacked the R9 and R10 regions; that of class III lacked the R6, R7 and R9 
regions and that of class of IV lacked the R7, R8 and R9 regions. In 
addition, the ef* genes of class IV and V lacked a fragment of 1,032 bp, 
which contained R4, R5 and parts of R3 and R6. The translational reading 
frame of the region located at the 3' end of the missing fragment remained 
the same. The nucleotide sequences at the regions of the left and right 
ends of this 1,032 bp fragment showed direct repeats of 9 bp (FIG. 6A). 
Homology between ef* and ef genes. Because EF* proteins were recognized by 
Mabs directed against the 110 kDa EF protein and because the ef* genes 
strongly hybridized with an ef-probe, the ef (Example 1) and ef* genes are 
assumed to be partly identical. Comparison of the nucleotide sequences of 
the ef and the ef* gene of class I showed that the 2,499 nucleotides 
located at the 5' end of the ef and ef* encoding regions were identical. 
Unlike the gene encoding the EF* protein of class I, the gene encoding the 
110 kDa EF protein lacked a 2,368 bp fragment. As a result of this 
deletion the reading frame was altered and the region located at the 3' 
-end of the 2,368 bp fragment was translated in different frames in ef and 
ef* genes. Consequently, the 110 kDa EF protein will not contain the 
repeated amino acid units. Analysis of the nucleotide sequences at the 
regions of the left and right ends of the 2,368 bp fragment showed direct 
repeats of 10 bp (containing one mismatch) (FIG. 6B). Thus, the gene 
encoding the 110 kDa EF protein could have been the result of a specific 
deletion of 2,368 bp within an ef* gene. This would implicate that a S. 
suis strain that is non-pathogenic can change into a strain that is 
pathogenic. 
EXAMPLE 3 
Cloning and nucleotide sequence of the gene encoding the 136 kDa surface 
protein (MRP) of Streptococcus suis type 2 
MATERIALS AND METHODS 
Bacterial strains and growth conditions. Escherichia coli strain JM 101 
(supE,thi,(lac-proAB.sup.-)[F'traD36, lacI.sup.q Z.DELTA.M15], 29) was 
used as a host for recombinant plasmid DNA. E. coli strain LE392 [F.sup.- 
'hsdR574(rk.sup.- 'mk.sup.-), supE44, supF58, lacY1, or .DELTA.(lac1ZY)6, 
galK2, galT22, mel1. trpR55](33) was used as a host for recombinant 
bacteriophages. The pathogenic MRP.sup.+ EF.sup.+ strain D282 of S.suis 
type 2 (43) was used for isolating chromosomal DNA. E. coli strains were 
grown on LB broth (30). Solid LB medium contained 1.5% agar. Ampicillin 
was added as needed to a final concentration of 50 .mu.g/ml. streptococcus 
suis strains were grown in Todd-Hewitt broth (Oxoid Ltd.) 
Southern hybridization was carried out as described in Example 2. 
Construction and immunological screening of the DNA library were carried 
out as described in Example 1 substituting MRP for EF. 
SDS - PAGE and Western blot analysis were carried out as described in 
Example 1 substituting MRP for EF. 
Nucleotide sequence analysis was carried out as described in Example 1. 
RESULTS 
Construction and screening of the library. Chromosomal DNA isolated from 
strain D282 of S. suis type 2 was partially digested with the restriction 
enzyme Sau3A. A DNA library was then constructed in the bacteriophage 
LambdaGEM11 replacement vector. Approximately 5.times.10.sup.5 
recombinants /.mu.g DNA were obtained. A MAb directed against MRP was used 
to screen 1,400 recombinant plaques for the presence of antigenic 
determinants of MRP. Five recombinant plaques reacted positive. 
Characterization of the immunoreactive recombinants. The expression of MRP 
by the five selected recombinant bacteriophages was studied by Western 
blotting to analyse the proteins eluted from the plaques. All five 
recombinants encoded proteins that were recognized by MAbs directed 
against MRP. These proteins, however, had lower molecular weights (MW) 
than the MRP. Two clones encoded a protein of approximately 70 kDa (clones 
10 and 11); two clones encoded a protein of approximately 80 kDa (clones 9 
and 12), and one clone encoded a protein of approximately 90 kDa (clone 
7). Therefore, it was concluded that the five recombinants did not contain 
the complete genetic information for MRP. Restriction enzyme analysis was 
used to compare the DNA inserts of the five recombinants. All clones 
shared a DNA region of about 17 kb (FIG. 7A). The DNA inserts differed, 
however, at the 3' and 5' ends. The variation in length at the 3' ends of 
the inserts correlated well with the variation in MW of the truncated MRP 
proteins (cf. FIG. 7A). This correlation indicates that MRP encoding 
sequences were located at the 3' end of the DNA inserts. This was 
confirmed by subcloning fragments derived from the 3' end of the DNA 
inserts of clones 7, 9, and 10 (FIG.7B) into plasmid vector pKUN19 (24). 
These construct encoded truncated MRP proteins that were indistinguishable 
from the truncated MRP proteins encoded by the recombinant phages (FIG. 
8). Deletion of the 0.7 kb EcoRI-KpnI fragment from these contructs 
stopped the expression of the truncated MRP proteins. This suggests that 
the expression of mrp is initiated from the 0.7 kb EcoRI-KpnI fragment. 
Cloning of the complete mrp gene. The complete gene for MRP was obtained by 
hybridization of .sup.32 P labeled KpnI-SacI fragment of pMR7-2(FIG. 7B) 
with EcoRI or KpnI digested chromosomal DNA of strain D282 of S. suis type 
2. An EcoRi fragment of 7 kb and KpnI fragment of 7 kb hybridized with the 
probe. Because of its size, the EcoRi fragment was expected to contain the 
complete mrp gene and because the expression of mrp is initiated from the 
0.7 kb EcoRI-KpnI fragment, the KpnI fragment was expected to contain only 
the 30' end of the gene. Fragments ranging from 6 to 8 kb from EcoRI and 
KpnI digested chromosomal DNA were isolated , and ligated into the EcoRI 
or KpnI site of pKUN19, whereafter the ligation mixtures were transformed 
into E. coli JM101. Thirteen out of 50 selected recombinants clones 
obtained with the KpnI fragments hybridized with a MRP probe. All of these 
recombinant clones contained a plasmid (pMR-C) with a 7 kb KpnI insert. In 
contrast, of 2,500 selected recombinant clones obtained with EcoRI 
fragments, none hybridized with the probe. Since the 7 kb EcoRI fragment 
is expected to contain the complete mrp gene, this finding indicates that 
expression of MRP is toxic in E. coli. Nevertheless, a plasmid (pMR11) 
with the entire mrp gene could be constructed by combining the 5' end of 
the mrp gene (isolated from pMR7-2) and the 3' end of the gene (isolated 
from pMR-C) by forced cloning. The copy number of this plasmid appeared to 
be strongly reduced, about 20 times, compared to the copy number of 
pKUN19. The low copy number presumably reduced the toxic effects of 
high-level expression of MRP in E. coli to tolerable levels. The proteins 
produced by E. coli cells containing pMR11, were analysed by Western 
blotting. As expected, these cells produced a 136 kDa protein that 
comigrated with MRP and that was recognized by PAbs directed against MRP. 
Nucleotide sequence of the mrp gene. The nucleotide sequence of a 4.6 kb 
EcoRI-HindIII fragment, containing the entire mrp gene and the regions 
flanking it was determined. Analysis of the sequences, SEQ ID NO: 3, 
revealed an open reading frame of 3,768 nucleotides coding for a 
polypeptide of 1,256 amino acids (with a calculated MW of 135,794). The 
putative ATG start codon is preceded by a sequence that is similar to 
ribosome-biding sites in several types of gram-positive bacteria (17). The 
nucleotide sequence upstream of mrp resembles the -35 and -10 consensus 
sequences of promoters commonly found in gram-positive bacteria. 
downstream of the mrp gene, a region showing extended dyad symmetry can be 
detected. The potential hairpin structure in the corresponding mRNA has a 
12 bp stem separated by a 6 bp loop (.DELTA.G =-15.9 kcal/mol, calculated 
according to the rules of Tinoco et at., 40). Since the region of dyad 
symmetry is not followed by a thymidine-rich region, this potential 
transcription terminator signal appears to be rho-dependent (34). 
Amino acid sequence of MRP. MRP is a cell-envelope V2 associated pro and 
must be translocated across the cytoplasmic membrane. The mature protein 
must therefore contain a signal peptide. Indeed, the first 47 amino acids 
of the MRP have the characteristics of a typical signal peptide. An 
N-terminal part that contains seven positively charged residues is 
followed by a hydrophobic core of 21 amino acids and a putative signal 
peptidase cleavage site (45, vertical arrow in SEQ ID NO: 3). Cleavage of 
the signal peptide would result in a mature protein with an MW of 131.094, 
which is close to the MW (136 kDa) of MRP, estimated from 
SDS-polyacrylamide gels (Example 4). A second hydrophobic region of 20 
amino acids was identified at the C terminus of the protein (FIG. 11). If 
this region is analogous to other envelope associated proteins of 
gram-positive bacteria (10, 11, 12, 13, 16, 20, 38, 39, 46), it is 
probably a cell membrane anchor. A short highly charged region and a 
region with the Leu-Pro-X-Thr-Gly-Glu amino acid sequence, two regions 
that flank the presumed cellmembrane anchor, are also highly conserved 
among surface proteins of gram-positive bacteria (FIG. 12). The amino acid 
sequence Leu-Pro-X-Thr-Gly-Glu is putatively involved in cell-wall 
binding. 
Several other regions were identified in the MRP sequence. The mature form 
of MRP starts with a unique N-terminal sequence of 824 amino acids. This 
region is followed by a stretch of amino acids that is rich in proline 
residues: of 86 amino acids, 26 are proline residues. This region is 
followed by three repeated units of 54 amino acids (FIG. 13). The first 
unit is separated from the second by 77 amino acids, but the second and 
third unit are contiguous. The sequences of the first and the second unit 
are highly conserved, whereas the third varies. The third repeated unit is 
followed by the envelope anchor sequence. There was little homology 
between the MRP sequence and the protein sequences of the EMBL Data 
Library. One subsequence of MRP, amino acid residues 619-985, however, 
shared some similarity (17.2% identity in a 377 amino acids sequence) with 
a sequence of the fibronectin-binding protein of Staphylococcus aureus 
(39). 
EXAMPLE 4 
Identification of two proteins associated with virulence of Streptococcus 
suis type 2 
MATERIAL AND METHODS 
Streptococcal isolates. 180 strains of S. suis type 2 were obtained from 
three different sources. A total of 111 of these strains were obtained 
from four Animal Health Services in the Netherlands. These strains were 
isolated from organs of diseased pigs in the course of routine diagnostic 
procedures. Another 42 strains were isolated from tonsils of healthy pigs 
when they were slaughtered. 27 strains were isolated from human patients 
with S. suis type 2 infections. Tonsillar and human strains were kindly 
provided by J. P. Arends, Streeklaboratorium voor de Volksgezondheid voor 
Groningen en Drente, Groningen, the Netherlands. All strains were typed as 
S. suis type 2 by using biochemical and serological methods, as described 
previously (44). Strain 1 (=D282) had been determined previously to be 
virulent for newborn germfree pigs and produced MRP, whereas strain 2 
(=T-15) was nonvirulent and did not produce MRP (43). Therefore, strains 1 
(MRP.sup.+) and 2 (MRP.sup.-) were used as reference strains. 
Culture conditions. A 1-day-old colony of each bacterial strain was grown 
on Columbia blood agar base (code CM 331; Oxoid, Ltd.) containing 6% horse 
blood and was incubated overnight at 37.degree. C. in Todd-Hewitt broth 
(code CM 189; Oxoid). Early stationary growth phase cultures were obtained 
from the overnight cultures, diluted 10 times in Todd-Hewitt broth, and 
incubated for 4 h at 37.degree. C. 
Cell fractionation. Two cell fractions (protoplast supernatant and culture 
supernatant) were prepared from each of the 180 strains. Two more cell 
fractions (protoplasts and membrane vesicles) were prepared from 23 
strains selected randomly from the 180 strains. The 23 strains were 
isolated from both diseased and healthy pigs, as well as from human 
patients. The four cell fractions were isolated from early stationary 
growth phase cultures in Todd-Hewitt broth. Protoplasts were isolated as 
described by Van der Vossen et al. (47). After centrifugation in an 
Eppendorf centrifuge, the protoplasts and the remaining supernatants 
(protoplast supernatant) were collected. Membrane vesicles were isolated 
as described by Driessen et al. (9). The broth cultures were centrifuged 
at 4,000.times.g for 15 min, and the culture supernatants were collected. 
Preparation of antigens and antisera. After a stationary growth phase 
culture of strain D-282 was centrifuged, the supernatant was harvested, 
concentrated by filtration (type PM30 filters; Amicon Corp., Danvers, 
Mass.) to a concentration of 3 mg/ml, and dialysed once against 
Tris-buffered saline (50 mM, pH 7.5). This product was used as an antigen 
for raising polyclonal antibodies (PAb) in rabbits and monoclonal 
antibodies (MAb) in mice. Rabbits were immunized by intramuscular and 
subcutaneous inoculation of 2 mg portions of protein emulsified in equal 
volumes of Freund imcomplete adjuvant. Inoculations were repeated the 
following day without the adjuvant. After 5 weeks the rabbits were given 
intravenous booster inoculations of the same antigen dose, but without the 
adjuvant. After 6 weeks, the rabbits were exsanguinated. The serum of one 
rabbit (rabbit K191) was used as a probe in the Western blot analysis. 
MAbs against the protein EF were raised in BALB/c mice. The mice were 
immunized intraperitoneally with 0.5 ml portions of antigen containing 25 
.mu.g of protein emulsified in equal volumes of Freund imcomplete 
adjuvant; 3 weeks later this procedure was repeated. After 5 weeks, the 
mice were given intravenous booster inoculations of the same antigen dose, 
but without the adjuvant. Hybridoma cell lines were prepared as described 
by Van Zijderveld et al. (51). After 10 to 14 days, hybridomas were tested 
for antibodies against EF by using an enzyme-linked immunosorbent assay. 
Hybridoma culture supernatants (diluted 1:2) were then tested for anti-EF 
MAb on Western blots of culture supernatants from strain D-282. Binding of 
MAb to the 110 kDa protein on the nitrocellulose filters was visualized 
with anti-mouse immunoglobulins conjugated with alkaline phosphatase. The 
positive cells were cloned twice by limiting dilution in microtiter 
plates. The resulting monoclonal cell lines were used to produce ascites 
fluid in pristane-primed male BALB/c mice, as described previously (51). 
Indirect enzyme-linked immunosorbent assay for screening hybridoma culture 
supernatants. Polystyrene microtiter plates (Greiner, Nurtingen, Germany) 
were coated for 16 h at 37.degree. C. with a solution containing the 
concentrated, dialysed culture supernatant from strain D-282 (see above) 
diluted in phosphate-buffered saline (pH 7.2; 0.075 mg of protein per ml), 
and these preparations were incubated for 16 h at 37.degree. C. Twofold 
dilutions of hybridoma culture supernatants were applied and tested as 
described previously (51). Bound antibodies were incubated with anti-mouse 
immunoglobulins (diluted 1:500) that were conjugated with horseradish 
peroxidase (HRPO, Nordic, Tilburg, The Netherlands). 
Electrophoresis and Western blotting. The various cell fractions were 
analysed by SDS-PAGE as described by Laemmli (26) on 6 or 12% 
polyacrylamide. After electrophoresis, the proteins were stained with 
silver (32). For Western blot analysis, the proteins were electroblotted 
onto nitrocellulose by using a Multiphor II Nova Blot system (Pharmacia 
LKB, Uppsala, Sweden). The blots were probed with a 1:500 dilution of 
rabbit K191 PAb or with a 1:300 dilution of mouse MAb. Bound PAb were 
visualized with anti-rabbit immunoglobulins conjugated with alkaline 
phosphatase. Bound MAb were visualized with a 1:1,000 dilution of 
anti-mouse immunoglobulins conjugated with alkaline phosphatase (Zymed). 
RESULTS 
Protein profiles of four cell fractions of 23 selected strains. The protein 
profiles of the protoplast supernatants and membrane vesicle cell 
fractions from two S. suis isolates belonging to each group studied 
(diseased pigs, healthy pigs, and human patients), prepared from the 23 
strains examined were almost identical. In contrast, the protein profiles 
of the culture and protoplast supernatants differed distinctly. The 
protein profiles of isolates obtained from diseased pigs contained two 
protein bands that were absent in the protein profiles of most isolates 
obtained from healthy pigs. One band represented a 136 kDa protein, which 
was identified as MRP (43). In the SDS-PAGE analysis, separating gels 
containing 6% polyacrylamide revealed the presence of MRP in both culture 
and protoplast supernatants (strains 1, 5, 24, and 26). The second band 
represented a 110 kDa protein; because this protein was detected only in 
culture supernatants, it was designated EF. Both MRP and EF were present 
in the culture supernatant of virulent reference strain 1 (=D-282), but 
were absent in all cell fractions of nonvirulent reference strain 2 
(=T-15). The eight strains isolated from diseased pigs contained both MRP 
and EF. Six of the eight strains isolated from healthy pigs lacked these 
proteins. Six of the seven strains isolated from human patients contained 
MRP, but only three of the six also contained EF. 
When rabbit K191 PAb directed against culture supernatants were used as 
probes in the immunoblotting analysis, MRP and EF were clearly detected in 
the cell fractions of S. suis type 2 strains. Protoplast supernatants, 
culture supernatants, and membrane vesicles of strains 1, 5, 24, and 26 
contained the 136-kDa MRP (FIG. 9). Because MRP is a major component of 
protoplast supernatants, this protein must be localized in the cell 
envelope of the bacteria. The culture supernatants of strains 1 and 5 also 
contained the 110 kDa EF. Strains 24 and 26 contained MRP but not EF; 
strains 2 and 13 contained neither of the proteins. 
On the basis of the presence of MRP and EF in culture supernatants, the 
following three phenotypes of S. suis type 2 strains were distinguished: 
MRP.sup.+ EF.sup.+, and MRP.sup.+ EF.sup.-, and MRP.sup.- EF.sup.- (FIG. 
10). Proteins bands at various molecular masses higher than 150 kDa 
reacted with rabbit K191 serum and were visualized in Western blots of 
culture supernatants of strains 17, 24, 25, 26, and 28. As such proteins 
were also recognized by the anti-EF MAb, except in the culture supernatant 
of strain 25, the 110 kDa EF was probably related to these proteins. 
Western blots probed with the mouse anti-EF MAb showed that all of the 
strains with the MRP.sup.+ EF.sup.- phenotype contained higher molecular 
weight proteins in their culture supernatants. However, none of the 
strains with the MRP.sup.+ EF.sup.+ phenotype contained such proteins. 
Probing with rabbit K191 serum revealed high molecular weight proteins in 
culture supernatants of 12 MRP.sup.- EF.sup.- strains, including strain 
25. Immunoblotting with anti-EF MAb showed that these proteins were not 
related to EF. When the four cell fractions were analysed by SDS-PAGE on 
12% slab gels, no low molecular weight proteins associated with virulence 
were detected. 
Protein profiles of culture and protoplast supernatants of 180 strains. All 
180 S. suis type 2 strains were analysed for the occurrence of the three 
phenotypes in culture and protoplast supernatants by using 6% slab gels. 
Eighty percent of the strains isolated from the organs of diseased pigs 
had the MRP.sup.+ EF.sup.+ phenotype (Table 1). 
TABLE 1 
______________________________________ 
Prevalence of MRP and EF phenotypes in 180 streptococcal 
strains isolated from diseased pigs, from healthy pigs when 
they were slaughtered, and from human patients. 
No. (%) of strains isolated from: 
S. suis type 2 
Organs of Tonsils of Human 
phenotype diseased pigs 
healthy pigs 
patients 
______________________________________ 
MRP.sup.+ EF.sup.+ 
86 (77) 1 (2) 4 (15) 
MRP.sup.+ EF.sup.- 
13 (12) 5 (12) 20 (74) 
MRP.sup.- EF.sup.- 
12 (11) 36 (86) 3 (11) 
______________________________________ 
In contrast, only 2% of the strains isolated from tonsils of healthy pigs 
had this phenotype; 86% of these strains were MRP.sup.- EF.sup.-. Only 15% 
of the strains isolated from human patients had the MRP.sup.+ EF.sup.+ 
phenotype. Among the S. suis type 2 strains tested, far more human strains 
(74%) than porcine strains (12%) had the MRP.sup.+ EF.sup.- phenotype; 
89% of the human strains were MRP.sup.+. The MRP.sup.- EF.sup.+ phenotype 
was not detected. 
EXAMPLE 5 
Virulence of Streptococcus suis type 2 strains in new-born germ-free pigs. 
MATERIALS AND METHODS 
Pigs. Fifty-two germ-free pigs, cross-breeds of Great Yorkshire and Dutch 
Landrace, were obtained from four sows by caesarian sections. Sows in both 
experiments were full sisters. Pigs were allotted to 12 groups each 
consisting of 4 or 5 pigs. Each group was housed in a sterile stainless 
steel incubator. Housing and feeding were as described before (43). 
Inocula. Ten S. suis type 2 strains belonging to either phenotype MRP+EF+, 
MRP+EF-, or MRP-EF- were obtained from three sources: from a pig with 
meningitis, from healthy pigs at slaughter, and from human patients (Table 
2). The strains were biochemically and serologically typed as described 
earlier (44). Strains were stored as stock suspensions on glass beads in 
Nutrient Broth with 15% glycerol at -70.degree. C. A one-day-old colony of 
each strain, grown on Columbia blood agar base (Code CM 331, Oxoid) 
containing 6% horse blood, was incubated overnight at 37.degree. C. in 
Todd-Hewitt broth (Code CM 189, Oxoid). Early stationary growth phase 
cultures were obtained by diluting the overnight cultures in Todd-Hewitt 
broth (1:10) and incubated them at 37.degree. C. Incubation was stopped 
after approximately 4 h, when the optical density at 600 nm was 0.5. 
Cultures containing approximately 1 to 3.times.10.sup.9 CFU/ml were then 
centrifuged at 4000.times. g for 15 min. The supernatant was analysed for 
MRP and EF. Then the pellets were washed and suspend at an A.sub.600 =1 in 
a solution of phosphate-buffered saline (PBS), 139.89 mM NaCl, 2.68 mM 
KCl, 8.1 mM Na.sub.2 HPO.sub.4, 2.79mM KH.sub.2 PO.sub.4, ph 7.2, and then 
used as inoculum. Bordetella bronchiseptica strain 92932, isolated from 
the nose of a pig with atrophic rhinitis, was used to predispose pigs to 
S. suis infection (23, 43). The strain was kept on Dorset egg medium. The 
inoculum was prepared by culturing a 48 hour old colony from sheep blood 
agar in brain heart infusion broth. After 18 h of incubation at 37.degree. 
C., this medium contained approximately 10.sup.9 CFU/ml. The brain heart 
infusion broth was diluted (1:100) in PBS to prepare the inoculum. 
Electrophoresis and Western blotting. The MRP/EF phenotypes of the S. suis 
strains used as inocula and of the isolates recovered at the end of the 
experiments were determined. SDS-PAGE as described by Laemmli (26) (6% 
polyacrylamide) and Western blotting were used to analyse cell culture 
supernatants of isolates recovered from nasopharynx of all pigs, and from 
inflamed tissue such as meninges or joints of affected pigs. After 
electrophoresis the proteins were stained with silver (32). For Western 
blot analysis, the proteins were electroblotted onto nitrocellulose by the 
Multiphor II Nova Blot system, according to the recommendations of the 
manufacturer (Pharmacia LKB). Nitrocellulose filters were incubated either 
with a 1:1 mixture of mouse anti-MRP monoclonal antibodies (MAb) (11.3 
mg/ml) and anti-EF MAb (8.4 mg/ml) each in a 1:200 dilution, or with a 
1:500 dilution of polyclonal anti-MRP/EF rabbit serum (K191) (8.2 mg/ml) 
(Examples 4, 6). Filters were incubated with a 1:1000 dilution of 
anti-mouse immunoglobulins conjugated with alkaline phosphatase (AP) or a 
1:3000 dilution of AP conjugated anti-rabbit immunoglobulin 
g(.gamma.+.kappa.) (Zymed). Bound antibodies were visualized by adding the 
substrate bromochloroindolyl phosphate (Sigma, St. Louis, Mo.--nitro blue 
tetrazolium (Merck, Darmstad, Germany) in phosphatase buffer (100 mM NaCl, 
5 mM MgCl.sub.2, 100 mM diethanolamine; ph 9.5). 
Experimental design. The study consisted of two experiments with an 
interval of five months. Five day old germ-free pigs were inoculated 
intranasally with a plastic disposable syringe filled with a suspension of 
B. bronchiseptica strain 92932 in brain heart infusion broth. The inocula 
contained 0.84.times.10.sup.7 CFU in experiment I and 1.0.times.10.sup.7 
CFU in experiment II. Two days post inoculation (pi) the pigs were 
similarly inoculated inside the sterile incubator with one of the ten S. 
suis type 2 strains (Table 2). 
The mean (.+-.SD) inoculum size of these strains was 1.4 
(+0.60).times.10.sup.6 CFU. All inoculations consisted of a 0.5 ml 
bacterial suspension in each nostril during the inspiratory phase of 
breathing. In both experiments strain 3 (MRP+EF+) was used as positive 
control and strain 12 (MRP-EF-) was used as negative control (see Results 
section). Pigs were killed either when they became mortally ill or at the 
end of the experiment (3 to 4 weeks pi), and they were subsequently 
necropsied. 
TABLE 2 
______________________________________ 
Experimental design. 
S. Suis Source.sup.1 of 
Dosage.sup.2 
No. of 
strain 
S. suis S. suis of S. suis 
pigs 
no. phenotype isolation inoculation 
inoculated 
______________________________________ 
3 MRP+EF+ meninges pig 
1.84 5 
3 MRP+EF+ meninges pig 
1.96 4 
10 MRP+EF+ tonsil pig 1.52 5 
22 MRP+EF+ human 2.93 4 
17 MRP+EF- tonsil pig 1.26 4 
24 MRP+EF- human 1.22 4 
28 MRP+EF- human 1.23 4 
12 MRP-EF- tonsil pig 1.05 5 
12 MRP-EF- tonsil pig 0.98 4 
16 MRP-EF- tonsil pig 0.70 4 
18 MRP-EF- tonsil pig 1.10 4 
25 MRP-EF- human 0.97 4 
______________________________________ 
.sup.1 Strain 3 was isolated during routine diagnostic procedures from a 
pig with meningitis. Strains 10, 12, 16, and 18 were isolated at slaughte 
from the tonsils of healthy pigs. Strains 22 (no. 830544), 24 (no. 
740113), 25 (no. 821021) and 28 (no 760366) were isolated from human 
patients with S. suis type 2 meningitis. (Numbers between parentheses 
refer to those by J. P. Arends and H. C. Zanen (2)). 
.sup.2 .times. 10.sup.6 CFU. 
Disease monitoring . Pigs were monitored daily for clinical signs of 
disease, such as fever, dysfunction of the CNS and lameness. Blood samples 
from each pig were collected three times weekly by venipuncture of the 
cranial vena cava. White blood cells were counted with a conducting 
counter (Contraves A.G., Zurich, Switerland) (18 ). The number of 
neutrophils was calculated after differential count of Giemsa-stained 
blood smears. Swabs specimens of nasopharynx and feces were collected 
daily and plated directly onto Columbia agar containing 6% horse blood. 
The presence of S. suis type 2 and of B. bronchiseptica was confirmed by 
slide agglutination test in which a suspension of the monocultures was 
mixed with the appropiate hyperimmune rabbit serum (DLO-Central Veterinary 
Institute, Lelystad, NL). After pigs were killed, they were examined for 
pathologic changes. Tissue specimens of the CNS, serosae, liver, spleen, 
and tonsils were bacteriologically and histologically examined as 
described before (43). 
RESULTS 
Electrophoresis and Western blotting. When immunoblots were used to analyse 
culture supernatants of the S. suis strains before inoculation, three 
phenotypes were distinguish. Strains 3, 10, and 22 belonged to the MRP+EF+ 
phenotype, strains 17, 24, and 28 were of the MRP+EF- phenotype, and 
strains 12, 16, 18, and 25 belonged to the MRP-EF- phenotype. The rabbit 
polyclonal antibodies (PAb) recognized proteins that were greater than 150 
kDa in the culture supernatants of the MRP+EF - strains. These high 
molecular weight proteins were also detected by the anti-EF MAb, 
indicating that the 110 kDa EF and the &gt;150 kDa proteins share epitopes. 
In both the SDS-PAGE and Western blot, the phenotypes of the S. suis 
strains used as inocula were identical to the phenotypes of the isolates 
collected at the end of both experiments from tonsils and inflamed tissues 
of infected pigs. 
Clinical signs of disease. In both experiments, rectal temperatures of all 
pigs inoculated with strains of the MRP+EF+ phenotype increased from day 2 
pi onwards, with peaks at 41.8.degree. C. between days 4 and 9. Rectal 
temperatures of ten pigs inoculated with strains of the MRP+EF- phenotype 
were higher than 40.degree. C. for short periods of 24 to 96 h between 
days 2 and 22. Frequency of fever was highest in the MRP+EF+ groups (40%) 
(Table 3). The frequency of increased polmorphous leucocytes (PML) in 
blood was highest in the MRP+EF+ groups (Table 3). Analysis of variance 
was performed on the log of PML counts in blood samples of pigs inoculated 
with strains of the three phenotypes. Three days before inoculations no 
significant differences were found between the geometric mean PML counts 
of the three groups. From day one pi onwards, the means of numbers of PML 
in blood samples of pigs inoculated with strains of the MRP+EF+ phenotype 
were significantly higher (p&lt;0.01) then in either the MRP+EF+ groups or 
the MRP-EF- groups. On day 20 pi, the means in the MRP+EF+ and MRP+EF- 
groups did not differ significantly from each other, but those means 
differed significantly (p&lt;0.01) from the means in the MRP-EF- groups. 
Morbidity in pigs inoculated with strains of the MRP+EF+ phenotype was 
100%. From day 2 onwards, non-specific signs of systemic disease, such as 
depression, recumbency, lack of appetite, and fever were observed. During 
the following days, pigs showed more specific signs of disease, such as 
ataxia, circular movements, opisthotonus, recumbency with paddling, and 
lameness. The frequency of specific signs of diseases in the MRP+EF+ 
groups was 57% (Table 3). Nine pigs died in the course of the experiment, 
and three were killed in the terminal stages of disease. The mortality 
rate in these groups was thus 12/18 (67). Nine pigs inoculated with 
strains of the MRP+EF- phenotype developed fever or granulocytosis or 
showed other nonspecific signs of disease, but did not show specific 
clinical signs, such as nervous disorders or lameness. Pigs in the MRP-EF- 
groups did not develop clinical signs of disease (Table 3) 
TABLE 3 
______________________________________ 
Frequency of three parameters of disease observed in pigs 
inoculated with S. suis type 2 (10 strains belonging to three 
phenotypes) 
Frequency.sup.1 (%) of 3 parameters of disease 
PML 
S. suis Fever in blood Clinical signs of disease 
phenotype 
&gt;40.degree. C. 
&gt;10.sup.10 /L 
specific.sup.2 
non-specific.sup.3 
______________________________________ 
MRP+EF+ 40 78 57 21 
MRP+EF- 5 16 0 5 
MRP-EF- 0 3 0 0 
______________________________________ 
.sup.1 Number of positive records/total number of records 
.sup.2 Lameness and nervous disorders such as ataxia, circular movements, 
opisthotonus, and recumbency with paddling. 
.sup.3 Depression, lack of appetite, and recumbency. 
Pathologic findings are summarized in Table 4. Severe and frequent 
inflammations of the CNS, serosa, and joints were only detected in pigs 
inoculated with strains of the MRP+EF+ phenotype. Pneumonia and bronchitis 
were observed in various forms. Follicle formation in B cell areas and 
blast cell formation in T cell areas of the white pulp of the 
spleen--signs of active immune response--were more frequently observed in 
pigs inoculated with strains of the MRP+EF- phenotype (50%) than in pigs 
inoculated with strains of the MRP-EF- phenotype (22%) or strains of the 
MRP+EF+ phenotype (11%) (Table 4). Some pigs inoculated with MRP+Ef+ 
showed lymphocytolysis in the germinal centres, while the marginal zone 
surrounding the white pulp was inflamed, signs of acute septichaemia in 
young animals (42). Active follicles in tonsils were also more often seen 
in pigs inoculated with strains of the MRP+EF- or MRP-Ef- phenotype. 
TABLE 4 
______________________________________ 
Pathologic lesions detected in various tissues of pigs 
inoculated with S. suis type 2 (10 strains of three 
phenotypes 
No. of pigs with pathologic lesions 
phenotype phenotype phenotype 
Tissue and MRP+EF+ MRP+EF- MRP-EF- 
pathologic (no. (no. (no. 
lesions tested = 18) 
tested = 12) 
tested = 22) 
______________________________________ 
CNS 
Meningitis.sup.1 
12 0 0 
Encephalitis.sup.1 
10 1 0 
Choroiditis 7 0 0 
Malacia 5 0 0 
Serosae/joints 
Peri-/epicarditis 
11 1 1 
Pleuritis 5 1 0 
Peritonitis 14 6 0 
Polyarthritis.sup.2 
15 0 0 
Lungs 
Cath. 1 1 1 
broncho-pneumonia 
Fibrinous 3 0 0 
pneumonia 
Interstitial 
7 5 5 
pneumonia 
Bronchitis/ 2 2 3 
Peribronchiolitis 
Liver 
Periportal and/or 
11 8 3 
intralobular foci 
Spleen 
Active white pulp 
2 6 5 
Active red pulp 
4 0 2 
Tonsil 
Active follicles 
3 9 12 
Exudation in crypts 
1 5 6 
______________________________________ 
.sup.1 Affecting cerebrum, cerebellum, pons, mesencephalon and medulla 
oblongata in various combinations. 
.sup.2 Affecting carpal, metacarpal, tarsal, metarsal, knee, elbow, 
shoulder and hip joints in various combinations. 
Bacteriologic findings. From day 1 one pi to the end of the experiment, the 
streptococcal strains and B. bronchiseptica were isolated daily from 
naso-pharyngeal and fecal swab specimens of all pigs. A Bacillus species 
was also isolated from day six pi onwards from pigs inoculated with strain 
16 (experiment I) and from day 19 pi onwards from pigs inoculated with 
strain 24 (experiment II). Pigs in the other groups remained free from 
contaminating bacteria. 
At necropsy, S. suis type 2 was mostly isolated from organs and tissues 
(CNS, serosae, and joints) that also showed pathologic changes (Table 5). 
B. bronchiseptica was only isolated from lungs and tonsils. Both S. suis 
and B. bronchiseptica were also isolated from the tonsils of all pigs. 
TABLE 5 
______________________________________ 
Isolation of streptococci from various tissues of pigs 
inoculated with S. suis type 2 (10 strains of three 
phenotypes). 
No. of pigs from which S. suis 
was isolated at necropsy 
phenotype phenotye phenotype 
MRP+EF+ MRP+EF- MRP-EF- 
(no. (no. (no. 
Tissue tested = 18) 
tested = 12) 
tested = 22) 
______________________________________ 
CNS 14 0 0 
Serosae 9 2 0 
Joints 13 2 0 
Lungs 6 (9) 0 (2) 2 (8) 
______________________________________ 
.sup.1 Numbers in parentheses indicate number of pigs from which B. 
bronchiseptica was also isolated. 
EXAMPLE 6 
Discrimination between Virulent and Nonvirulent Streptococcus suis type 2 
Strains by Enzyme-Linked Immunosorbent Assay 
MATERIALS AND METHODS 
Bacteria. 179 strains of S. suis type 2 obtained from three sources were 
examined: from organs of diseased pigs in the course of routine diagnostic 
procedures, from tonsils of healthy pigs at slaughter, and from human 
patients suffering from S. suis type 2 infection. SDS-PAGE and Western 
blotting techniques were used in an earlier study to detect MRP and EF in 
culture supernatants, and on the basis of these results strains were 
categorized into three phenotypes: MRP+EF+, MRP+EF-, and MRP-EF- (Example 
4). Also tested were 22 strains of S. suis serotypes 1 to 22 (15), 22 
other streptococci, 20 bacterial strains of 15 different species, and one 
yeast (DLO-Central Veterinary Institute, Lelystad) (Table 6). 
TABLE 6 
______________________________________ 
List of microorganisms 
Group Microorganisms Microorganisms 
______________________________________ 
A Streptococcus pyogenes humanis 
Other bacterial species: 
B Streptococcus agalactiae 
Staphylococcus aureus 
C Streptococcus equi Staphylococcus 
epidermidis 
Streptococcus equisimilis porcine 
Staphylococcus hyicus 
Streptococcus dysgalactiae 
Aerococcus viridans 
Streptococcus zooepidemicus 
Actinomyces pyogenes 
D Enterococcus faecalis 
Escherichia coli (3x) 
Enterococcus faecium 
Klebsiella oxytoca 
Enterococcus liquefaciens 
Klebsiella pneumoniae 
Streptococcus bovis (2x) 
Micrococcus strain 3551 
Streptococcus zymogenes 
Micrococcus Luteus 
E Streptococcus group E 
Pasteurella multocida 
G Streptococcus group G (2X) 
(4x) 
L Streptococcus group L (2X) 
Proteus vulgaris 
p Streptococcus group P 
Salmonella typhimurium 
Q Streptococcus group Q 
Serratia liquefaciens 
Streptococcus milleri III 
Yeast: 
Streptococcus sanguis 
Cryptococcus laurentii 
Streptococcus uberis 
______________________________________ 
Culture condition and antigen preparation. A 1 day old colony of the 
bacteria grown overnight on Columbia blood agar base (code CM 331. Oxoid 
Ltd.) containing 6% horse blood was inoculated into Todd-Hewitt broth 
(code CM 189, Oxoid). After overnight growth at 37.degree. C., cultures 
were centrifuged at 4000.times. g for 15 min. At 600 nm the optical 
densities of the 20 hour cultures were found to vary from 0.60 to 1.04. 
Some species had lower densities, these were Bordetella bronchiseptica 
(0.23). Micrococcus species (0.08 to 0.15). Streptoccoccus equinus (0.36). 
Cryptococcus neoformans (0.05). Twofold serial dilutions of untreated 
culture supernatants were used as test samples in the two DAS-ELISAs. 
Culture supernatant of S. suis type 2 strain D.sub.282 (MRP+EF) was 
concentrated and partially purified by ultrafiltration (type PM30 filters, 
Amicon Cooperation). It was diluted in phosphate-buffered saline (PBS) 
(136.89 mM NaCl. 2.68 mM KCl. 8.1 mM Na.sub.2 HPO.sub.4. 2.79 mM KH.sub.2 
PO.sub.4. pH 7.2), to a final protein concentration of 75 .mu.g/ml. This 
product was used as coating antigen for the selection of different 
monoclonals in the direct competition ELISA and for screening hybridoma 
culture supernatants in the indirect ELISA. 
Preparation of polyclonal and monoclonal antibodies. Rabbit (Ra) polyclonal 
antibodies (PAb) directed against MRP and EF (Ra K.sub.191) and three 
different MAbs directed against EF were prepared as described in Example 
4. MAbs that specifically recognize MRP were prepared essentially the same 
as MAbs that recognize EF. Antigen production and immunization procedures 
in female BALB/c mice have been described (Example 4). Hybridoma cell 
lines were prepared as described (52). After 10 to 14 days, hybridoma 
culture supernatants were tested for antibodies against MRP in an indirect 
ELISA (see below). Hybridoma culture supernatants (diluted 1:2) were then 
tested on Western blots of culture supernatants of strain D-282 for 
antibodies directed against MRP. Bound MAb to the 136 kDa protein were 
visualized by using anti-mouse immunoglobulins conjugated to alkaline 
phosphatase and the substrate described below. Five supernatants were 
found positive, and the cells from these wells were cloned twice by 
limiting dilution in microtiter plates. 
The five cell lines that were positive for anti-MRP antibodies and the 
three cell lines that were positive for anti-EF antibodies were used to 
produce ascites fluid in pristane-primed male BALB/c mice. MAbs directed 
against MRP and EF were purified from ascites fluid by ammonium sulphate 
precipitation (50% saturation) and dialysed against PBS. The five anti-MRP 
MAbs were designated: MRP.sub.1 to MRP.sub.5, the three anti-EF MABs were 
designated: EF.sub.1 to EF.sub.3. The immunoglobulin isotype of all MAbs 
was IgG.sub.1 and was determined by double immunodiffusion with mouse 
isotype-specific antisera (Nordic) in gels of 1% agarose in PBS. The PAbs 
and MAbs were stored at -20.degree. C. 
Indirect ELISA for screening hybridoma culture supernatants. Polystyrene 
microtiter plates (Greiner, Nurtingen, Germany) were coated for 16 h at 
37.degree. C. with the solution of concentrated and dialysed culture 
supernatant of strain D-282 (see above). They were then diluted in PBS, pH 
7.2 (75 .mu.g/ml protein). Twofold dilutions of hybridoma culture 
supernatants were added to the wells according to the procedure described 
by Van Zijderveld et al. (51). After the plates were washed, antimouse 
immunoglobulins (diluted 1:500) conjugated with horse radish peroxidase 
(HRPO, Nordic) were added. After incubation for 1 h at 37.degree. C. and 
five washings, the bound HRPO-antibody was then detected by the addition 
of substrate, 0.1% (w/v) solution of recrystallized 5-aminosalicylic acid 
(5-AS) (Merck) in 0.01M phosphate buffer, pH 5.95, containing 0.01M sodium 
EDTA to which H.sub.2 O.sub.2 had been added, immediately before use to an 
end concentration of 0.005% (wt/vol). After 2 h incubation at room 
temperature, the absorbance was measured at 450 nm with a Titertek 
Multiskan photometer (Flow Labs). 
Direct competition ELISA. MAbs were selected with the direct competition 
ELISA and were used to develop the MRP and EF double antibody sandwich 
(DAS) ELISAs. Purified anti-MRP and anti-EF MAbs and rabbit PAbs were 
conjugated to HRPO (Boehringer Mannheim, Germany) with the periodate 
method of Wilson and Nakane (49). Conjugated immunoglobulins were stored 
at -20.degree. C. in 50% glycerol. Conjugate solutions were made in PBS-Tw 
containing 5% fetal calf serum and 0.5% sodium chloride. 50 .mu.l of 
nonconjugated anti-MRP MAbs in serial twofold dilutions (range 1:20 to 
1:10,240) were added to the wells of polystyrene microtiter ELISA plates 
(Greiner) that had been coated with the culture supernatant of strain 
D.sub.282 that had been partially purified in PBS (75 .mu.g/ml protein). 
The plates were then incubated for 30 min at 37.degree. C. To allow the 
nonconjugated MAb to compete with the MAb conjugates, 50 ml of the optimal 
dilution of each of the five anti-MRP MAbs conjugated to HRPO were added. 
After incubation for 1 h at 37.degree. C., plates were washed and the 
bound HRPO antibody was then detected by the addition of the substrate 
5-AS H.sub.2 O.sub.2 as described above. After 2 h incubation at room 
temperature, the absorbance was read. The titers of competition were 
expressed as the highest dilution showing an A.sub.450 of 50% of the mean 
absorbance of wells to which only conjugate was added. The epitope 
specificity of the three anti-EF MAbs was determined with a competition 
ELISA similar to the one described for the anti-MRP MAbs. 
SDS-PAGE and Western blotting techniques. Culture supernatants of the 22 S. 
suis serotypes and the other microrganisms (Table 6) were separated by 
SDS-PAGE on 6% polyacrylamide. For Western blot analysis, the proteins 
were electroblotted onto nitrocellulose by the Multiphor II Nova Blot 
system according to the recommendations of the manufacturer (Pharmacia 
LKB). The blots were probed with a 1:300 dilution of mouse MAb. Bound MAbs 
were visualized with a 1:1000 dilution of anti-mouse immunoglobulins 
conjugated with alkaline phosphatase (Zymed). 
RESULTS 
Direct competition ELISA. The five anti-MRP clones and the three anti-EF 
clones were tested for competition. Some anti-MRP clones competed with 
each other. The five anti-MRP MAbs were directed against at least three 
different epitopes: the first was recognized by MRP.sub.1 and MRP.sub.2, 
the second by MRP.sub.3, and the third by MRP.sub.4 and MRP.sub.5. Because 
all three anti-EF clones competed, they are probably directed against the 
same epitope. 
MRP double antibody sandwich ELISA. In an MRP DAS-ELISA using MRP.sub.3 as 
catching antibody and HRPO-MRP.sub.1 as conjugate, each well of the 
polystyrene microtiter ELISA plates was coated with 100 .mu.l containing 
2.3 .mu.g MRP.sub.3 per well in 0.05M carbonate buffer, pH 9.6. After 
adsorption for 16 h at 37.degree. C., coated plates were used immediately 
or stored at -20.degree. C. Twofold serial dilutions of 100 .mu.l culture 
supernatants, ranging from 1:1 to 1:128 in PBS containing 0.05% (wt/vol) 
Tween 80, of strains to be tested, were added to the wells. After 1 h 
incubation at 37.degree. C., plates were washed five times with 0.05% 
Tween 80 in tap water, and 100 .mu.l 7.2. was added to each well. Using 
checker-board titrations, the optimal dilution of catching antibody and 
conjugate was determined. After 1 h incubation at 37.degree. C., the 
substrate 5-AS H.sub.2 O.sub.2 was added as described above. Wells with an 
A.sub.450 .gtoreq.0.2 were scored positive. To each plate a positive 
control was added, consisting of 100 .mu.l of undiluted culture 
supernatant of the virulent S. suis type 2 strain 4005 (MRP+EF+). A 
negative control was also added, consisting of 100 .mu.l of undiluted 
culture supernatant of the non-virulent strain T-15 (MRP-EF-) (43). 
The MRP DAS-ELISA was used to test 179 strains of S. suis type 2 belonging 
to the three phenotypes MRP+EF+, MRP+EF-, and MRP-EF-, as was previously 
determined by SDS-PAGE and Western blot. Most strains scored in the ELISA 
the same as they did in the Western blot (Table 7). All MRP+EF+ strains 
were MRP-positive in the ELISA. One MRP+EF- strain scored false negative. 
Three of the MRP-EF- strains (6%) scored false positive. The sensitivity 
(TP/TP+FN) (TP=true positive, FN=false negative) of the MRP DAS-ELISA was 
99% (130 out of 131 strains), the specificity (TN/TN+FP) (TN=true 
negative, FP=false positive) was 94% (45 out of 48 strains), and the 
predictive value (TP/TP+FP) was 98% (130 out of 133 strains). The MRP 
DAS-ELISA discriminated well between the MRP-positive and MRP-negative 
strains of S. suis type 2. 
TABLE 7 
______________________________________ 
Results of 179 strains of S. suis type 2 (three phenotypes) 
tested in the MRP and EF DAS-ELISAs. 
MRP DAS ELISA EF DAS ELISA 
No. No. No. No. 
phenotype 
strains + strains - strains + 
strains - 
______________________________________ 
MRP+EF+ 92 (100%) 0 92 (100%) 
0 
MRP+EF- 38 (97%) 1 (3%) 0 39 (100%) 
MRP-EF- 3 (6%) 45 (94%) 0 48 (100%) 
______________________________________ 
Titration curves of culture supernatants of strains belonging to three 
phenotypes of S. suis type 2, after testing in the MRP DAS-ELISA, were 
recorded. The mean (.+-. standard deviation) of the absorbances obtained 
from the undiluted culture supernatants of the 92 MRP+EF+ isolates was 
1.2259 (.+-.0.1165), the mean absorbance of the 39 MRP+EF- isolates was 
1.2129 (.+-.0.2076), and the mean absorbance of the 48 MRP-EF- isolates 
was 0.1180 (.+-.0.2546). Therefore plates can be read visually instead of 
having to be measured photometrically to discriminate MRP-positive strains 
(phenotypes MRP+EF+ or MRP+EF-) from MRP-negative strains (phenotype 
MRP-EF-). 
Culture supernatants of 18 of the 21 reference S. suis strains of other 
serotypes had absorbances lower than 0.2. Three serotypes were positive 
and had the following absorbance values: undiluted culture supernatant of 
serotype 3 had A.sub.450 =0.731; culture supernatant of serotype 5 had 
A.sub.450 =0.587, and culture supernatant of serotype 15 (former 
Lancefield group T) had A.sub.450 =0.516. These serotypes were also 
positive in the Western blot; MRP.sub.3 apparently recognized proteins of 
higher molecular weight than 150 kDa in the culture supernatants of these 
serotypes. Absorbances of all other microorganisms listed in Table 6 were 
&lt;0.2. 
EF Double Antibody Sandwich ELISA. In a DAS ELISA that recognizes a 
specific antigen in the test sample, two different MAbs were used, one as 
catching antibody and the other as conjugate, and each recognizing 
different epitopes on the antigen, as was done for the MRP DAS-ELISA. In 
the Western blot the EF MAbs recognize a high molecular form of EF (&gt;150 
kDa) in the culture supernatants of all strains belonging to the 
MRP+EF-phenotype (Example 4). Therefore it is unlikely that an ELISA with 
EF.sub.2 as catching antibody can discriminate between MRP+EF+ and MRP+EF- 
strains. Moreover, because the three EF MABs blocked each other, we had to 
use EF.sub.2 as catching antibody and the polyclonal rabbit serum 
(K.sub.191) as conjugate. Some ELISAs were tested using EF.sub.1 as 
catching antibody and EF.sub.2 or EF.sub.3 as conjugates, and indeed these 
MAbs blocked each other completely. 
The procedure of the EF DAS-ELISA was essentially as that described for the 
MRP DAS-ELISA. Each well of the microtiter ELISA plates was coated with 
100 ml containing 3.3 .mu.g of EF.sub.2 in 0.05M carbonate buffer, pH 9.6. 
After adsorption, coated plates were used immediately or stored at 
-20.degree. C. Twofold serial dilutions of 100 .mu.l culture supernatants 
ranging from 1:1 to 1:128 were used. After incubation and washings, 100 
.mu.l containing 2.7 .mu.g polyclonal Ra K.sub.191 HRPO conjugate in PBS, 
pH 7.2, was added to each well. After 1 h incubation at 37.degree. C., the 
plates were developed with substrate 5-AS H.sub.2 O.sub.2 as described 
above. Wells with an A.sub.450 .gtoreq.0.4 were scored positive. The same 
controls as mentioned above were used on each plate. 
The 179 S. suis type 2 strains with a predetermined protein profile were 
tested in the EF DAS-ELISA. Surprisingly, none of the 39 MRP+EF- strains 
scored positive in this ELISA, whereas all 92 MRP+EF+ strains did (Table 
7). All 48 MRP-EF- strains were negative in the EF DAS-ELISA. Since no 
other false positive or false negative results were detected, the EF 
DAS-ELISA apparently discriminated reliably between the high and the low 
molecular form of EF, and hence between S. suis type 2 strains belonging 
to the MRP+EF+ and MRP+EF- phenotypes. 
Since the direct competition ELISA had shown that the three anti-EF MAbs 
blocked each other, MAb EF.sub.2 was used as catching antibody and the 
polyclonal Ra K.sub.191 serum as conjugate. Streptococcus suis type 2 
strains belonging to the MRP+EF- phenotype, however, produce a 
high-molecular weight (&gt;150 kDa) form of EF (example 4). Because MAb 
EF.sub.2 does not discriminate between the 110-kDa EF and this 
high-molecular weight form in the Western blot, it was unlikely to do so 
in the EF DAS-ELISA. Surprisingly Mab EF.sub.2 captured the 110-kDa EF in 
the culture supernatant of all MRP+EF+ strains but apparently not the 
higher-molecular weight form in the MRP+EF- strains (Table 7). Some 
MRP-EF- strains gave signals between 0.2 and 0.4, which were still lower 
than 50% of the maximal absorbance values and thus not high enough to be 
interpreted as positive. Treating the culture supernatants with SDS before 
blotting may uncover epitopes of the higher-molecular weight form of EF 
that are not accessible to the EF.sub.2 MAb in its undenaturated form. 
Because all MRP+EF- strains and other S. suis serotypes showed no false 
negative or false positive reactions in this ELISA, the sensitivity and 
specificity of the test were considered to be 100%. 
Titration curves of culture supernatants of strains belonging to three 
phenotypes of S. suis type 2 were recorded after testing in the EF 
DAS-ELISA. The mean (.+-. standard deviation) of the absorbances obtained 
from the undiluted culture supernatants of the 93 MRP+EF+ strains was 
0.8204 (.+-.0.149), the mean absorbance of the 39 MRP+EF- strains was 
0.1551 (.+-.0.046), and the mean absorbance of the 48 MRP-EF- strains was 
0.1061 (.+-.0.0371). Thus, as for the MRP DAS-ELISA, plates can be read 
visually to discriminate between EF-positive strains (phenotype MRP+EF+) 
and EF-negative strains (phenotypes MRP+EF- or MRP-EF-). 
None of the 21 reference S. suis strains with a serotype other than type 2 
were EF-positive in the ELISA. Some other bacterial species had positive 
absorbance values: Streptococcus Lancefield group G (A.sub.450 =0.445), 
group L (A.sub.450 =0.348), Streptococcus equi (A.sub.450 =0.671), and 
Staphylococcus aureus (A.sub.450 =0.718). 
EXAMPLE 7 
Differentiation between pathogenic and non-pathogenic strains of 
Streptococcus suis type 2 by using polymerase chain reaction (PCR). 
MATERIALS AND METHODS. 
Bacteria and growth conditions. Thirteen strains of S. suis type 2 were 
selected to examine whether the Polymerase Chain Reaction (PCR) method 
(36) could be useful to differentiate between the three phenotypes of S. 
suis type 2. Pathogenicity and the expression of the MRP and EF proteins 
of these strains were determined in Examples 4 and 5. Strains were grown 
overnight at 37.degree. C. on Columbia blood agar base (code CM 331, 
Oxoid) containing 6% horse blood. S. suis type 2 colonies were inoculated 
in 10 ml Todd-Hewitt broth (code CM 189, Oxoid), and grown overnight at 
37.degree. C. 
DNA Isolations. DNA of overnight grown cultures was isolated as described 
by Maniatis et. al (28). DNA was diluted to 10 ng/.mu.l in distilled water 
before use in the PCR. 
Clinical specimens. Nose swabs and tonsillar tissues were obtained post 
mortem from sows at slaughter. Nose swabs were inoculated on blood plates. 
S. suis type 2 strains were isolated from tonsils as described before 
(27). 
Sample preparation. Clinical specimens for the PCR were prepared by the 
method described by Boom et. al (4), with some minor modifications: The 
specimens were added to 900 .mu.l L6 lysis buffer plus 40 .mu.l diatom 
earth solution in an Eppendorf tube [L6 buffer is 100 ml 0.1M TRIS HCl (pH 
6.4) plus 120 g guanidine (iso)thiocyanate (GuSCN, Fluka cat nr. 50990) 
plus 22 ml 0.2M EDTA (pH 8.0) plus 2.6 g Triton X-100. Diatom earth 
solution is 10 g Diatom earth (Janssen Chimica Cat. nr. 17.346.80) in 50 
ml distilled water plus 500 .mu.l 32% (w/v) HCl]. The clinical specimens 
were incubated overnight in L6 buffer in the dark at room temperature. 150 
.mu.l of the solution was pipetted in wells of microtiter plates 
containing Durapore membranes (Multiscreen MAHV N45, Millipore). The 
microtiter plate was put on the vacuum manifold (MAVM 09600, Millipore), 
and the samples were washed 5 times with 200 .mu.l L2 washing solution (L2 
buffer is 100 ml 0.1M Tris-HCl (pH 6.4) plus 120 g GuSCN), 5 times with 
200 .mu.l 70% ethanol, and once with 200 .mu.l aceton. The filters were 
not allowed to run dry between the wash steps. The bottom of the 
microtiter plate was dried on a tissue and the samples were dried 
completely for 15 minutes at 56.degree. C. 75 .mu.l PCR buffer (see below) 
was added to the individual wells. The plate was incubated for 15 minutes 
at 56.degree. C. The microtiter plate was again put on the vacuum 
manifold, with a standard microtiter plate (Micronic) beneath the Durapore 
plate. Vacuum was applied, and the PCR buffer, containing the DNA was 
collected in the lower microtiter plate, whereas the diatom earth remained 
on the Durapore filters. 
PCR assay. The PCR contained 10 ng purified DNA or 25 .mu.l clinical 
specimen in a total volume of 50 .mu.l. The reaction mixtures contained 10 
mM Tris-HCl (pH 9.0), 2 mM MgCl.sub.2, 50 mM KCl, 0.01% gelatin, 0.2 mM of 
each of the four deoxynucleotide triphosphates, 1 .mu.M of each of the 
four primers and 0.5 U of Amplitaq polymerase (Perkin Elmer Cetus, 
Norwalk, Conn.), and was overlaid with 2 drops of paraffine oil. DNA 
amplification was carried out in a Perkin Elmer Thermal Cycler for 25 or 
40 cycles: 1 minute 94.degree. C., 1 minute 55.degree. C., and 2 minutes 
72.degree. C. Ten to 20 .mu.l of the amplified DNA was analysed on a 1.5% 
agarose gel, that contained ethidium bromide. 
PCR primers. The sequences of the oligonucleotides used in the PCR were: 
p15. SEQ ID NO 3 base pairs 1403-1425: 5'-GGT ATA CCT TGC TGG TAC GGT TC 
-3', p16: SEQ ID NO: 3 base pairs 1914-1934: 5'-AGT CTC TAC AGC TGT AGC 
TGG -3'(which correspond to the complement), p-34: SEQ ID NO: 2 base pairs 
2890-2908: 5'-GTT GAA AAC AAA GCA TTC G -3', and p-35: SEQ ID NO 2 base 
pairs 3229-3249: 5'- CTT CGA CAA AAT GTC AGA TTC -3'. The oligonucleotides 
p-15 and p-16 correspond to the indicated positions in the S. suis type 2 
mrp gene (Example 3, SEQ ID NO:3). The oligonucleotides p-34 and p-35 
correspond to the indicated positions and in the S. suis type 2 ef* gene 
(Example 2, SEQ ID NO:2). Primers were synthesized on an Applied Biosystem 
synthesizer type 381A following the manufacturers protocol. 
RESULTS 
Specificity of PCR. Within the mrp and ef* genes (cf. Examples 3 and 2), 
two regions (designated as m-VI and e-V) were determined that could be 
used to differentiate between the three phenotypes of S. suis type 2 
strains (see also Example 8). Primers based on the m-VI region (p-15 and 
p-16), and the e-V region (p-34 and p-35) were used in a PCR. The primers 
p-15 and p-16 amplified a 532 bp fragment in the m-VI region. The primers 
p-34 and p-35 amplified a 360 bp fragment in the e-V region. Chromosomal 
DNA of 4 MRP.sup.+ EF.sup.+, 4 MRP.sup.+ EF* and 5 MRP.sup.- EF.sup.- 
strains was used in a PCR with these primers (see FIG. 15). After 25 cycli 
the amplified fragments were analysed on an agarose gel. A 532 bp fragment 
was amplified from DNA of MRP.sup.+ EF.sup.+ strains. A 532 bp fragment as 
well as a 360 bp fragment were amplified from DNA MRP.sup.+ EF* strains. 
In contrast, neither the 532 bp nor the 360 bp fragment was amplified from 
DNA of MRP.sup.-EF.sup.- strains. These data show that this PCR can be 
used to differentiate between the three phenotypes of S. suis type 2. 
The phenotypes of 82 strains of S. suis type 2, isolated from the tonsils 
of 37 healthy sows at slaughter, were determined by Western blotting 
(Example 4), ELISA (Example 6), hybridization experiments with DNA probes 
m-VI and e-V (Example 8), and by PCR. 79 strains, isolated from 36 of the 
37 sows were classified identical by the four methods (96.3%). 3 strains, 
isolated from one sow, were classified as MRP.sup.+ EF* by the PCR and DNA 
hybridization experiments and as MRP.sup.- EF.sup.- by Western blotting 
and ELISA. These results indicate that the PCR is a useful alternative to 
determine the phenotype of a S. suis type 2 strain. 
Sensitivity of PCR. Purified chromosomal DNA of a MRP.sup.+ EF* S. suis 
type 2 strain was diluted in distilled water and used directly in the PCR. 
After 40 cycli of PCR, 25 fg DNA was detected. This indicates that DNA of 
14 cells, after amplification by PCR, could be detected on an agarose gel, 
based on data that a Streptococcal cel contains about 1.75 fg DNA (35). 
The sensitivity of the PCR on whole cells was determined. Therefore, 
MRP.sup.+ EF* cells were diluted in phosfate buffered saline (PBS (pH 
7.2); 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na.sub.2 HPO.sub.4, 2.8 mM KH.sub.2 
PO.sub.4) and prepared for PCR as described above. Amplified fragments 
could still be detected in samples that contained about 50 cells prior to 
the PCR (40 cycli). 
The PCR can be used directly on clinical material. Serial dilutions of S. 
suis type 2 cells were added to nose swabs. It was found that amplified 
fragments can still be detected in samples that contain about 50 cells 
prior to the PCR. 
EXAMPLE 8 
Differentiation between pathogenic and non-pathogenic strains of S. suis 
type 2 using DNA probes. 
MATERIALS AND METHODS 
Bacteria. Thirteen strains of S. suis type 2 (4 MRP.sup.+ EF* strains and 5 
MRP.sup.- EF.sup.- strains) were selected to examine whether regions of 
the mrp, ef, and ef* genes could be useful to differentiate between the 
three phenotypes of S. suis type 2. Except for strain 16, pathogenicity of 
these strains was tested in an infection experiment of piglets (Example 
5). 
170 strains of S. suis type 2 were obtained from three sources: From organs 
of diseased pigs (103 strains), from tonsils of healthy pigs at slaughter 
(40 strains) and from human patients (27 strains). Reference strains of S. 
suis serotypes 1 to 22 (15), 21 other Streptococci species and 45 other 
bacterial strains (38 different species, DLO Central Veterinary Institute, 
Table 8) were used to test the specificity of the mrp and ef probes. 
Media. E. coli JM101 strains were grown in LB broth (30). Ampicillin was 
added as needed to a final concentration of 50 .mu.g/ml. All other 
bacterial strains were grown overnight at 37.degree. C. on Columbia blood 
agar base (code CM 331, Oxoid) containing 6% horse blood. Overnight grown 
colonies were incubated in 10 ml Todd-Hewitt broth (code CM 189, Oxoid), 
and grown overnight at 37.degree. C. 
DNA isolations and manipulations. Chromosomal DNA isolations and routine 
DNA techniques were performed as described by Maniatis et al (28). Crude 
lysates were made as follows: overnight grown cultures were centrifuged at 
4000.times. g for 10 minutes, and the pellet fraction was resuspended in 
500 to 1000 .mu.l TEG-lysozym buffer (25 mM TRIS.Cl pH 8.0, 10 mM EDTA, 50 
mM glucose and 1 mg/ml lysozym). After 30 minutes at 25.degree. C., the 
samples were used in the dot-blot assay. 
Probes. The plasmids pMR11, pEF2-19 and pEF17-7 (cf. Examples 1, 2, 3) were 
used to generate subclones into pKUN19 (24). Fragments of appropriate 
subclones were isolated from preparative agarose gels with the gene-clean 
kit (Bio 101 Inc., La Jolla, USA). Purified fragments were subsequently 
labeled with .alpha.-.sup.32 P dCTP (3000 Ci/mMol, Amersham) with the 
random primed labeling kit (Boehringer GmbH) following the manufacturers 
protocol and used as probes. 
Southern hybridizations. Chromosomal DNA of the 13 selected S. suis 2 
strains (1 .mu.g DNA) was spotted on Gene-screen nylon membrane 
(New-England Nuclear Corp., Boston, USA). The membranes were incubated 
with the .sup.32 P-labeled mrp and ef probes as recommended by the 
manufacturer. After overnight hybridization, the filters were washed twice 
with 2.times. SSC for 5 minutes at room temperature, and twice with 
0.1.times. SSC plus 0.5% (SDS) for 30 minutes at 65.degree. C. (1.times. 
SSC=0.15M NaCl plus 0.015M Sodium Citrate). For the group of 170 S. suis 2 
strains, the 22 reference strains of S. suis type 1 to 22, and the group 
of other Streptococci and other bacteria, 20 .mu.l of a DNA or crude 
lysate sample was dotted on Zeta probe nylon membrane (Biorad) with a dot 
blot apparatus (Bethesda Research Laboratories). 
The membranes were incubated with the .sup.32 P-labeled mrp and ef probes 
as recommended by the manufacturer. After overnight hybridization, the 
membranes were washed twice in 40 mM Na phosphate buffer, pH 7.2 plus 5% 
SDS plus 1 mM EDTA for 30 minutes at 65.degree. C. and twice in 40 mM Na 
phosphate buffer, pH 7.2 plus 1% SDS plus 1 mM EDTA for 30 minutes at 
65.degree. C. All (pre)hybridizations were carried out in a hybridization 
oven (Hybaid). 
RESULTS 
Mrp probes. Chromosomal DNA of the 3 phenotypes of S. suis type 2 was 
hybridized to different regions of the mrp gene. Six different mrp probes 
were used (schematically shown in FIG. 14a). The EcoRI-SnaBI fragment, 
m-I, contained the entire mrp encoding region. The m-II, m-III, m-IV and 
m-V probes contained different regions of the mrp gene (see FIG. 16). The 
MRP.sup.+ EF.sup.+ and the MRP.sup.+ EF* strains strongly hybridized with 
all mrp probes. In addition, the m-I, m-II, m-IV and m-V probes strongly 
hybridized with 4 of the 5 MRP.sup.- EF.sup.- strains. One MRP.sup.- 
EF.sup.- strain (strain 25) did not hybridize with any of the mrp probes. 
These data indicate that 4 MRP.sup.- EF.sup.- strains contained large 
regions homologous to the mrp gene of strain D282, whereas strain 25 
lacked the entire mrp gene. These 4 MRP.sup.- EF.sup.- strains, however, 
hybridized only weakly with probe m-III, indicating that only a small part 
of probe m-III was homologous to their DNA. A probe m-VI was constructed 
by removing 385 bp at the 5', and 325 bp at the 3' ends of probe m-III. 
The 5 MRP.sup.- EF.sup.- strains did not hybridize at all with probe 
m-VI, indicating that these strains lacked the region homologous to the 
m-VI probe. Therefore, probe m-VI can be used to differentiate between 
MRP.sup.+ and MRP.sup.- strains. 
Ef and ef* probes. Chromosomal DNA of the 3 phenotypes S. suis type 2 was 
hybridized to different regions of the ef gene. Four different ef probes 
(schematically shown in FIG. 14b) were used. All MRP.sup.+ EF.sup.+ and 
MRP.sup.+ EF* strains and 1 MRP.sup.- EF.sup.- strain hybridized with all 
ef probes. In contrast, 4 MRP.sup.- EF.sup.- strains did not hybridize 
with any of the ef probes. These data indicate that most of these 
MRP.sup.- EF.sup.- strains lacked the entire region homologous to the ef 
gene, whereas 1 MRP.sup.- EF.sup.- strain seemed to contain the entire 
region homologous to the ef gene. Therefore, the probes e-I to e-IV could 
not been used to differentiate between the 3 phenotypes. 
Since the gene encoding the EF* proteins contain a DNA fragment which is 
absent in the gene encoding the EF protein, part of this extra DNA was 
selected as a probe (FIG. 14c, probe e-V). Probe e-V hybridized with all 
MRP.sup.+ EF* strains. On the contrary, none of the MRP.sup.+ EF.sup.+ 
and MRP.sup.- EF.sup.- strains hybridized with the e-V probe. These data 
suggest that the MRP.sup.+ EF.sup.+ and MRP.sup.- EF.sup.- strains 
lacked the region homologous to e-V. Probe e-V is thus specific for 
MRP.sup.+ EF* strains. 
Therefore, if m-VI and e-V are used in complementary hybridization studies, 
a differentiation between the three phenotypes of S. suis type 2 will be 
possible. If S. suis type 2 strains hybridize with probe m-VI and e-V, 
these strains belong to the MRP.sup.+ EF* phenotype. If S. suis type 2 
strains hybridize with m-VI but not with e-V, these strains belong to the 
MRP.sup.+ EF.sup.+ phenotype, and finally if strains do not hybridize 
with m-VI and e-V, these strains belong to the MRP.sup.- EF.sup.- 
phenotype. 
The mrp, ef and ef* probes were tested on 170 other strains of S. suis type 
2. 88 strains had a MRP.sup.+ EF.sup.+ phenotype, 37 strains a MRP.sup.+ 
EF* phenotype and 45 strains had a MRP.sup.- EF.sup.- phenotype. In 
accord with the data presented above, all MRP.sup.+ EF.sup.+ strains 
hybridized with the probes m-I to m-VI and e-I to e-IV, but none 
hybridized with probe e-V. Moreover, all the 37 MRP.sup.+ EF* strains 
hybridized with all the probes. Only two of the 45 MRP.sup.- EF.sup.- 
strains, however, hybridized with probe m-VI and e-V and would therefore 
wrongly be classified as MRP.sup.+ EF* strains. Therefore, by using m-VI 
and e-V, the phenotype of a S. suis type 2 strain can be predicted with a 
very high probability (168/170; 98.8%). 
Specificity of the m-VI and e-V probes. DNA of the reference strains of S. 
suis serotype 1 to serotype 22 was tested for hybridization with probes 
m-VI and e-V. It was found that S. suis type 2 (strain 735), 4, 5 and 14 
hybridized with the m-VI probe and that type 1/2, 2, 4, 5, 6, 14 and 15 
hybridized with the e-V probe. These data suggest that the mrp and ef 
genes are not specific for S. suis type 2, but that homologous sequences 
are present in several serotypes. Based on these data, serotypes 2, 4, 5 
and 14 would be classified as MRP.sup.+ EF* strains, whereas serotypes 
1/2, 6 and 15 would be classified as MRP.sup.- EF.sup.- strains. 
Chromosomal DNA from swine pathogens and several common bacteria was tested 
with the probes m-I, m-VI, e-III and e-V. The species tested are listed in 
Table 8. Although some species hybridized with probe m-I (Escherichia 
coli, Klebsiella oxytoca, K. pneumoniae and Salmonella typhimurium), none 
hybridized with the probes m-VI, e-III and e-V. These data shown that 
although in some species parts of the mrp genes are found, the probes m-VI 
and e-V are specific for S. suis. Hence, the probes m-VI and e-V have 
potential diagnostic value. 
TABLE 8 
______________________________________ 
List of other species on which the probes were tested for 
specificity. 
______________________________________ 
Streptococcus species 
S. agalactiae S. equi 
S. equisimilis porcine 
S. zooepidemicus 
S. dysgalactiae Enterococcus faecalis 
E. liquefaciens E. zymogenes 
E. faecium S. group E 
S. milleri III S. bovis 
S. pyogenes humanis 
S. uburis 
S. animale G S. group G 
S. group L biotype I 
S. group L biotype II 
S. group P S. group Q 
S. sanguis 
Other Bacteria 
Actinobacillus pleuropneumoniae 
Actinobacillus viridans 
Actinobacillus suis 
Aeromonas hydrophila 
Actinomyces pyogenes 
Bacillus licheniformis 
Bacillus cereus Bordetella bronchiseptica 
Bacillus subtilis Brucella suis biotype II 
Brucella suis biotype I 
Campylobacter faecalis 
Campylobacter coli 
Candida albicans 
Campylobacter jejuni 
Erysipelothrix rhusiopathiae 
Clostridium perfringens 
Klebsiella oxytoca 
A non-toxic 
Clostridium perfringens 
Listeria monocytogenes 
A toxic 
Escherichia coli Micrococcus luteus 
Haemophilus parasuis 
Mycoplasma hyopneumoniae 
Klebsiella pneumoniae 
Mycoplasma hyosynoviae 
Micrococcus strain 3551 
Pasteurella multocida 
Mycobacterium avium serovar2 
Salmonella typhimurium 
Mycoplasma hyorhinis 
Staphylococcus aureus 
Pseudomonas aeruginosa 
Staphylococcus hyicus 
Pasteurella vulgaris 
hyicus 
Serratia liquefaciens 
Staphylococcus epidermidis 
Yersinia enterocolitica 
______________________________________ 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4376 base pairs 
(B) TYPE: Nucleic acid with corresponding amino acids 
(C) STRANDEDNESS: single stranded 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Streptococcus suis type II (pathogenic) 
(ix) FEATURE: 
(D) OTHER INFORMATION: Extracellular protein factor (EF) gene 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 66 to 71 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 89 to 94 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 153 to 158 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 176 to 181 
(ix) FEATURE: 
(A) NAME/KEY: ribosome binding site 
(B) LOCATION: bp 350 to 356 
(ix) FEATURE: 
(A) NAME/KEY: signal peptide 
(B) LOCATION: bp 361 to 498 
(ix) FEATURE: 
(A) NAME/KEY: mature peptide 
(B) LOCATION: bp 499 to 2890 
(ix) FEATURE: 
(A) NAME/KEY: dyad symmetry regions 
(B) LOCATION: from bp 4186 to 4198 and from bp 4203 to 4215 
(ix) FEATURE: 
(A) NAME/KEY: dyad symmetry regions 
(B) LOCATION: from bp 4243 to 4257 and from bp 4263 to 4276 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTGAACAACTTAAAACTAGTTAGTTTTGTTTAAAATGTAATTGAATTGTCTTTTTAAGTA60 
GGCTGTTTACACGATATTTGTCTTCCTTTATATAAATATGATAGATTTTCAGTAAATTTT120 
TCAAAAAAACCTCAAAAATAACAGATTTTTTCTTGTATCTTTGAGGCATAAGGAGTATAA180 
TGGTGACGGTATTCAAGTAGAAATTTTATATACTCTTGATGAAAACATTCTGTCTACTTT240 
AAAATAAATAATCTACTGGGTATCCTTCTGCTAAGTTTTTAAAGCAGGAGGTGTGTTTTT300 
GTACATGGTGTTACAGGAACCAGAAATGATCGATTCGCCAGTAAAATATAGGAGGATATC360 
ATGTCTTATAAAGATATGTTCAGAAAAGAACAACGTTTTTCTTTTCGT408 
MetSerTyrLysAspMetPheArgLysGluGlnArgPheSerPheArg 
45-40-35 
AAATTTAGCTTTGGTCTAGCTTCGGCAGTCATTGCAAACGTTATTTTG456 
LysPheSerPheGlyLeuAlaSerAlaValIleAlaAsnValIleLeu 
30-25-20-15 
GGAGGAGCAATCGCAAACAGCCCTGTTGTTCATGCTAACACAGTGACA504 
GlyGlyAlaIleAlaAsnSerProValValHisAlaAsnThrValThr 
10-51 
GAAGCAGAGACAGCTGTAGCACCAGCTAACCAAGACCTTGGAAATGAG552 
GluAlaGluThrAlaValAlaProAlaAsnGlnAspLeuGlyAsnGlu 
51015 
ACTAAAACGGAAGAAGAACCCAAGGAACCAATCGAAGCAGTTCGCACG600 
ThrLysThrGluGluGluProLysGluProIleGluAlaValArgThr 
202530 
GACATGGAAAACCGTGCAGCTGAAATCTTGCCGGAGGCGCTGAATGCT648 
AspMetGluAsnArgAlaAlaGluIleLeuProGluAlaLeuAsnAla 
35404550 
AGTGTAACAAACCAAGCACCAGTTATTCCGACTATTGGAGATCTTCCT696 
SerValThrAsnGlnAlaProValIleProThrIleGlyAspLeuPro 
556065 
AAAGATGCGAGTGGTCAGAATGTTCATGGTAAGGCAACGGATAATAAG744 
LysAspAlaSerGlyGlnAsnValHisGlyLysAlaThrAspAsnLys 
707580 
ATTTATCGTGTTGTATACGTTTTTGGTAATGTAGCAGGGACTACGGAG792 
IleTyrArgValValTyrValPheGlyAsnValAlaGlyThrThrGlu 
859095 
ACAGAAGATGGTAAACAAAATGTTGCTCCAACATTTAACAGAAATGAT840 
ThrGluAspGlyLysGlnAsnValAlaProThrPheAsnArgAsnAsp 
100105110 
GCAACTAAAACTTTTCCAATCACAGATCCAGATAGCGACATTCAAACT888 
AlaThrLysThrPheProIleThrAspProAspSerAspIleGlnThr 
115120125130 
ATTTCATACGAAGTTCCAGCTGATATTGCAAGCTATACCTTGGATGAT936 
IleSerTyrGluValProAlaAspIleAlaSerTyrThrLeuAspAsp 
135140145 
CCAAACTCAATTGTTACTAATGGCACCTCACCTGGTCCAGTATCTTAC984 
ProAsnSerIleValThrAsnGlyThrSerProGlyProValSerTyr 
150155160 
TTAGATGGTCCAAATGGGTCAGCCACTCTCACACAAGATGGTTATCTA1032 
LeuAspGlyProAsnGlySerAlaThrLeuThrGlnAspGlyTyrLeu 
165170175 
ACAGGAAGTTTCCCTTGGGGAGCAGGAGACCTAGCTGGTCGTCGGATT1080 
ThrGlySerPheProTrpGlyAlaGlyAspLeuAlaGlyArgArgIle 
180185190 
AAAGTGACGGATGCCACTGGTAATACTACTAAGAGTAATCCGTTCTAT1128 
LysValThrAspAlaThrGlyAsnThrThrLysSerAsnProPheTyr 
195200205210 
ATGGTTGCATATACAGTCAAGCCAGTAGATGATAAACCTCTAGCAGTA1176 
MetValAlaTyrThrValLysProValAspAspLysProLeuAlaVal 
215220225 
TCAAACTCTTCTGAGCTGACGGAACAGGCTATTTTTGATAAGTTGGTT1224 
SerAsnSerSerGluLeuThrGluGlnAlaIlePheAspLysLeuVal 
230235240 
GTCGATAAGTCTGCTAAAACAACTTCAAATAGCGCTCTTGTAATTGAT1272 
ValAspLysSerAlaLysThrThrSerAsnSerAlaLeuValIleAsp 
245250255 
TCTAGCAACTACAAACATTCAATTGCAGGTTATCGTACCGTAAATTCT1320 
SerSerAsnTyrLysHisSerIleAlaGlyTyrArgThrValAsnSer 
260265270 
GATGGCACAAAAACAGAAACAGTAGAGGAAACAAATCTATCTGATTTC1368 
AspGlyThrLysThrGluThrValGluGluThrAsnLeuSerAspPhe 
275280285290 
CCAACTGAAGGTAAATACGAAGTTCGAGTAAAAACAACCAATGTTTAC1416 
ProThrGluGlyLysTyrGluValArgValLysThrThrAsnValTyr 
295300305 
GGTCAAACTATCTACAACTGGATTCCTGTAAATGCCTATAAGTTGGAC1464 
GlyGlnThrIleTyrAsnTrpIleProValAsnAlaTyrLysLeuAsp 
310315320 
ACAGCGAAGGATGCTGAAATTCGGAAGTATACAGACAACCAAGCCCCA1512 
ThrAlaLysAspAlaGluIleArgLysTyrThrAspAsnGlnAlaPro 
325330335 
ATTCATGCTATAATGCAAATTGGTCAAGCTGGAGAAAAGGCAGCAGTT1560 
IleHisAlaIleMetGlnIleGlyGlnAlaGlyGluLysAlaAlaVal 
340345350 
ATATTGAAGGATATTCCATCCGATTTCAGTATTGAAAACTTCAATTTG1608 
IleLeuLysAspIleProSerAspPheSerIleGluAsnPheAsnLeu 
355360365370 
AAAGATGGTGTAGCAGATGAGCTTGCTAAACGTAACTTGGAATTTGTA1656 
LysAspGlyValAlaAspGluLeuAlaLysArgAsnLeuGluPheVal 
375380385 
AGAAATGATGCAGTGGCGACAACTGATACTGATGGAGATGGCGCCAAA1704 
ArgAsnAspAlaValAlaThrThrAspThrAspGlyAspGlyAlaLys 
390395400 
GAAGGAATTGTTGGATATATTCAACCAAAAACTGGCGGTGCAAACAGT1752 
GluGlyIleValGlyTyrIleGlnProLysThrGlyGlyAlaAsnSer 
405410415 
GGGGTAGCCACTTATACAGGATCAAATAATCTTACTTATGGCTTCACT1800 
GlyValAlaThrTyrThrGlySerAsnAsnLeuThrTyrGlyPheThr 
420425430 
TACAAAGCTGTTGAGACAAAAGATAAGGCGAATGCCACAGAGGCTAAA1848 
TyrLysAlaValGluThrLysAspLysAlaAsnAlaThrGluAlaLys 
435440445450 
ACTCTCGAATTAGATTACACCATCTTATTCATAGATACTAAAGCACCA1896 
ThrLeuGluLeuAspTyrThrIleLeuPheIleAspThrLysAlaPro 
455460465 
GTCATGACACCTAAATCAGAGTACATCCGTTTTGTTGGTGAAGAGTAT1944 
ValMetThrProLysSerGluTyrIleArgPheValGlyGluGluTyr 
470475480 
ACGGTTAGCGTCCCAGGTACGGATAACGCCTTCCTTAATACCGGCAAA1992 
ThrValSerValProGlyThrAspAsnAlaPheLeuAsnThrGlyLys 
485490495 
CTAAATGGAACTCTCTCAATTTTGAAAGATGGAGAGTCAGGTTCTCTT2040 
LeuAsnGlyThrLeuSerIleLeuLysAspGlyGluSerGlySerLeu 
500505510 
GTATCATCAGACTTAGGTACAAACACTAAGATTACTTCAGAACTGGAT2088 
ValSerSerAspLeuGlyThrAsnThrLysIleThrSerGluLeuAsp 
515520525530 
CCTACGGGAGCAACTGCAAACCAAGGAGATGACGGTCAATCTTCAACT2136 
ProThrGlyAlaThrAlaAsnGlnGlyAspAspGlyGlnSerSerThr 
535540545 
AAGTTTAACGTTAAGATTACAGGTACCGGACCTGCTACAGAAGGTACC2184 
LysPheAsnValLysIleThrGlyThrGlyProAlaThrGluGlyThr 
550555560 
GGCACTTATAAGCTTCGTGTTGGAGAAGATAACTATCCTTTTGGTCCA2232 
GlyThrTyrLysLeuArgValGlyGluAspAsnTyrProPheGlyPro 
565570575 
GAGGGGAAACTTGTTGATGGAAATAAACCAGAAAATGTAGGTTTGACA2280 
GluGlyLysLeuValAspGlyAsnLysProGluAsnValGlyLeuThr 
580585590 
TCTGTAAAAGTTACCTTCGTAAAACATGCTACGGTGTCAACACCAGTT2328 
SerValLysValThrPheValLysHisAlaThrValSerThrProVal 
595600605610 
TCTGTTGAAAATCCAGCTAACTTAACGCCAGAAGAAAAAGCCGCAGTT2376 
SerValGluAsnProAlaAsnLeuThrProGluGluLysAlaAlaVal 
615620625 
ATTGCTCAAATCAAGAAAGACAACGCAGACAACGAAAGATTGAAGGGC2424 
IleAlaGlnIleLysLysAspAsnAlaAspAsnGluArgLeuLysGly 
630635640 
TTGCCAGATTCAGCATTTACAGTTAACTCAGATGGTACTGTGTCAGTT2472 
LeuProAspSerAlaPheThrValAsnSerAspGlyThrValSerVal 
645650655 
GACTACAGTGCCGGTGGTGTCAATGTTGATGGTGCGACAGACATTATT2520 
AspTyrSerAlaGlyGlyValAsnValAspGlyAlaThrAspIleIle 
660665670 
AAGAATGCTACCACAAACTTGGCAGATACACGGAATGAAGCAAAAGCA2568 
LysAsnAlaThrThrAsnLeuAlaAspThrArgAsnGluAlaLysAla 
675680685690 
GAAATCGACACAAAATTAGCTGAACATAAAAAAGCTATCGAAGCAAAA2616 
GluIleAspThrLysLeuAlaGluHisLysLysAlaIleGluAlaLys 
695700705 
CGGGATGAAGCGTTTTCTAAAATTGATGATGACATTTCCTTGAGAGCA2664 
ArgAspGluAlaPheSerLysIleAspAspAspIleSerLeuArgAla 
710715720 
GAACAGAGACAGGCTGCTAAGGATGCCGTTGCTGCAGCTGCTGGGGAT2712 
GluGlnArgGlnAlaAlaLysAspAlaValAlaAlaAlaAlaGlyAsp 
725730735 
GCTTTGAAAGAATTAGACAACAAGGCGACAGAAGCAAAAGAAAAAATT2760 
AlaLeuLysGluLeuAspAsnLysAlaThrGluAlaLysGluLysIle 
740745750 
GATAAAGCTACGACGGCCTCAGAAATCAATGATGCTAAGACTAATGGT2808 
AspLysAlaThrThrAlaSerGluIleAsnAspAlaLysThrAsnGly 
755760765770 
GAGATTAATCTGGACAGTGCAGAAGCAGTAGGCGAAAAAGCTATTAAC2856 
GluIleAsnLeuAspSerAlaGluAlaValGlyGluLysAlaIleAsn 
775780785 
CAGTCGAAGCGCAATCGGCAGAGGACAAAGGCGTAGGTTCAATCGCC2903 
GlnSerLysArgAsnArgGlnArgThrLysAla 
790795 
CAAGATGTTCTTGACGCAGCGAAACAAGATGCTAAGAATAAGATTGCT2951 
GlnAspValLeuAspAlaAlaLysGlnAspAlaLysAsnLysIleAla 
800805810 
AAAGAATCCGACGCTGCTAAGTCAGCCATTGACGCGAATCCAAACTTG2999 
LysGluSerAspAlaAlaLysSerAlaIleAspAlaAsnProAsnLeu 
815820825 
ACAGATGCAGAGAAGGAATCAGCTAAGAAAGCGGTAGATGCAGATGCT3047 
ThrAspAlaGluLysGluSerAlaLysLysAlaValAspAlaAspAla 
830835840845 
AAAGCTGCGACAGATGCAATTGATGCTTCAACAAGTCCAGTCGAAGCG3095 
LysAlaAlaThrAspAlaIleAspAlaSerThrSerProValGluAla 
850855860 
CAATCGGCAGAGGACAAAGGCGTAGGCGCCATCGCCAAAGACATTCTT3143 
GlnSerAlaGluAspLysGlyValGlyAlaIleAlaLysAspIleLeu 
865870875 
GATGCCGCGAAACAAGATGCTAAGAACAAGATTGCTAAAGAGGCAGAA3191 
AspAlaAlaLysGlnAspAlaLysAsnLysIleAlaLysGluAlaGlu 
880885890 
TCCGCTAAGTCAGTCATTGACTCCAATCCGAACTTGACAGATGCAGCT3239 
SerAlaLysSerValIleAspSerAsnProAsnLeuThrAspAlaAla 
895900905 
AAGGAAGCGGCTAAATCTGAAATTGATAAAGCTGTTGAGGAAGCGATT3287 
LysGluAlaAlaLysSerGluIleAspLysAlaValGluGluAlaIle 
910915920925 
GTTTTAATCAATGGTGTTAGAACTTATCAAGAGTTGGAAAAAATCAAA3335 
ValLeuIleAsnGlyValArgThrTyrGlnGluLeuGluLysIleLys 
930935940 
CTTCCAATGGCAGCTCTAATTAAACCAGCTGCGAAAGTAACACCAGTG3383 
LeuProMetAlaAlaLeuIleLysProAlaAlaLysValThrProVal 
945950955 
GTTGATCCAAATAACTTGACTGAAAAAGAAATTGCTCGTATCAAGGCA3431 
ValAspProAsnAsnLeuThrGluLysGluIleAlaArgIleLysAla 
960965970 
TTCCTTAAAGAGAACAATAACCTCCCATAAGGAACAGAGATTAATGTT3479 
PheLeuLysGluAsnAsnAsnLeuProGlyThrGluIleAsnVal 
975980985 
TCTAAAGATGCTTCAGTGACAATTAAATATCCAGATGGAACTATTGAT3527 
SerLysAspAlaSerValThrIleLysTyrProAspGlyThrIleAsp 
9909951000 
TTGCTATCACCAGTAGAAGTTGTGAAGCAGGCAGATAAAACTGCTCCT3575 
LeuLeuSerProValGluValValLysGlnAlaAspLysThrAlaPro 
1005101010151020 
ACGGTCGCAAATGATGGCAAAGGTAATATTGTGATTGTACCGTCTGAA3623 
ThrValAlaAsnAspGlyLysGlyAsnIleValIleValProSerGlu 
102510301035 
AAAGCTGTTGAGCTTGTTGTTTCATACGTAGATAACAATGGTAAGTCG3671 
LysAlaValGluLeuValValSerTyrValAspAsnAsnGlyLysSer 
104010451050 
CAAACTGTAGTTGTTACGAAAGGTACGGATGGTTTATGGACAGCAAGT3719 
GlnThrValValValThrLysGlyThrAspGlyLeuTrpThrAlaSer 
105510601065 
AATACAGTGGTGATTGTGGACCCTGTGACTGGGCAAGTAATCGTTCCA3767 
AsnThrValValIleValAspProValThrGlyGlnValIleValPro 
107010751080 
GGTTCTGTTATTAAGCCAGGTACAGTTGTTACAGCATACTCTAAAGAC3815 
GlySerValIleLysProGlyThrValValThrAlaTyrSerLysAsp 
1085109010951100 
GAGGTTGGAAATAGTTCTGATTCAGCAGAAGCTGAAGTTGTAGCAGTA3863 
GluValGlyAsnSerSerAspSerAlaGluAlaGluValValAlaVal 
110511101115 
GACGAAAATAATTCTGCAGCAGGAGTGAAAGTTAAATCAGTTACTACA3911 
AspGluAsnAsnSerAlaAlaGlyValLysValLysSerValThrThr 
112011251130 
AATGCTAATAATGTTGAGAAGAAAGCTAAGCAATTACCGAATACTGGT3959 
AsnAlaAsnAsnValGluLysLysAlaLysGlnLeuProAsnThrGly 
113511401145 
GAGGAAGCAAATTCAGCAACTTCACTCGGATTAGTAGCTCTTGGACTC4007 
GluGluAlaAsnSerAlaThrSerLeuGlyLeuValAlaLeuGlyLeu 
115011551160 
GGATTAGCACTTCTTGCAGCAAAGAGAAGAAGAGACGAAGAAGCTTAA4055 
GlyLeuAlaLeuLeuAlaAlaLysArgArgArgAspGluGluAla 
116511701175 
GATAAGCTCTTCCTCAGAACTCTTTTGGAAGCCGCAATTTTCCTAGAAGATAGTAGTATG4115 
ATACTCTTTCATAGCAAGGAAATTCCCTCGCTATGATTGGTAGGTATCAGTTATTATCTA4175 
TCGAACCCCCAAAATCCAAAGTCATTCGACTTTGGATTTTTTTGATACGACATGCTCGTC4235 
ATACCTAAAAAACAGCCTTCTCTTGCCGAGAGGCTGTTTTTCATGCTTTTAATCTAAAAG4295 
TCTGCGGACGTTTTTTCAATAAAATCCAGTAACCGATGCTAACATAGGCAATCATAGCTA4355 
GGGAAACCAGCAGGATATAGG4376 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6744 base pairs 
(B) TYPE: Nucleic acid with corresponding amino acids 
(C) STRANDEDNESS: single stranded 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Streptococcus suis type II (pathogenic) 
(ix) FEATURE: 
(D) OTHER INFORMATION: Extracellular factor related protein 
(EF*) gene 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 66 to 71 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 89 to 94 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 153 to 158 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 176 to 181 
(ix) FEATURE: 
(A) NAME/KEY: ribosome binding site 
(B) LOCATION: bp 350 to 356 
(ix) FEATURE: 
(A) NAME/KEY: signal peptide 
(B) LOCATION: bp 361 to 498 
(ix) FEATURE: 
(A) NAME/KEY: start of repetitive units R1-R11 
(B) LOCATION: bp 2869, 3097, 3292, 3520, 4087, 4381, 4609, 
4837, 5065, 5293, 5521: 
(ix) FEATURE: 
(A) NAME/KEY: start of repetitive Asn--Pro--Asn--Leu sequences 
(B) LOCATION: bp 2932, 3160, 3355, 3583, 4150, 4444, 4672, 
4900, 5128, 5356, 5584: 
(ix) FEATURE: 
(A) NAME/KEY: dyad symmetry regions 
(B) LOCATION: from bp 6554 to 6566 and from bp 6571 to 6583 
(ix) FEATURE: 
(A) NAME/KEY: dyad symmetry regions 
(B) LOCATION: from bp 6611 to 6625 and from bp 6631 to 6644 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TTGAACAACTTAAAACTAGTTAGTTTTGTTTAAAATGTAATTGAATTGTCTTTTTAAGTA60 
GGCTGTTTACACGATATTTGTCTTCCTTTATATAAATATGATAGATTTTCAGTAAATTTT120 
TCAAAAAAACCTCAAAAATAACAGATTTTTTCTTGTATCTTTGAGGCATAAGGAGTATAA180 
TGGTGACGGTATTCAAGTAGAAATTTTATATACTCTTGATGAAAACATTCTGTCTACTTT240 
AAAATAAATAATCTACTGGGTATCCTTCTGCTAAGTTTTTAAAGCAGGAGGTGTGTTTTT300 
GTACATGGTGTTACAGGAACCAGAAATGATCGATTCGCCAGTAAAATATAGGAGGATATC360 
ATGTCTTATAAAGATATGTTCAGAAAAGAACAACGTTTTTCTTTTCGT408 
MetSerTyrLysAspMetPheArgLysGluGlnArgPheSerPheArg 
45-40-35 
AAATTTAGCTTTGGTCTAGCTTCGGCAGTCATTGCAAACGTTATTTTG456 
LysPheSerPheGlyLeuAlaSerAlaValIleAlaAsnValIleLeu 
30-25-20-15 
GGAGGAGCAATCGCAAACAGCCCTGTTGTTCATGCTAACACAGTGACA504 
GlyGlyAlaIleAlaAsnSerProValValHisAlaAsnThrValThr 
10-51 
GAAGCAGAGACAGCTGTAGCACCAGCTAACCAAGACCTTGGAAATGAG552 
GluAlaGluThrAlaValAlaProAlaAsnGlnAspLeuGlyAsnGlu 
51015 
ACTAAAACGGAAGAAGAACCCAAGGAACCAATCGAAGCAGTTCGCACG600 
ThrLysThrGluGluGluProLysGluProIleGluAlaValArgThr 
202530 
GACATGGAAAACCGTGCAGCTGAAATCTTGCCGGAGGCGCTGAATGCT648 
AspMetGluAsnArgAlaAlaGluIleLeuProGluAlaLeuAsnAla 
35404550 
AGTGTAACAAACCAAGCACCAGTTATTCCGACTATTGGAGATCTTCCT696 
SerValThrAsnGlnAlaProValIleProThrIleGlyAspLeuPro 
556065 
AAAGATGCGAGTGGTCAGAATGTTCATGGTAAGGCAACGGATAATAAG744 
LysAspAlaSerGlyGlnAsnValHisGlyLysAlaThrAspAsnLys 
707580 
ATTTATCGTGTTGTATACGTTTTTGGTAATGTAGCAGGGACTACGGAG792 
IleTyrArgValValTyrValPheGlyAsnValAlaGlyThrThrGlu 
859095 
ACAGAAGATGGTAAACAAAATGTTGCTCCAACATTTAACAGAAATGAT840 
ThrGluAspGlyLysGlnAsnValAlaProThrPheAsnArgAsnAsp 
100105110 
GCAACTAAAACTTTTCCAATCACAGATCCAGATAGCGACATTCAAACT888 
AlaThrLysThrPheProIleThrAspProAspSerAspIleGlnThr 
115120125130 
ATTTCATACGAAGTTCCAGCTGATATTGCAAGCTATACCTTGGATGAT936 
IleSerTyrGluValProAlaAspIleAlaSerTyrThrLeuAspAsp 
135140145 
CCAAACTCAATTGTTACTAATGGCACCTCACCTGGTCCAGTATCTTAC984 
ProAsnSerIleValThrAsnGlyThrSerProGlyProValSerTyr 
150155160 
TTAGATGGTCCAAATGGGTCAGCCACTCTCACACAAGATGGTTATCTA1032 
LeuAspGlyProAsnGlySerAlaThrLeuThrGlnAspGlyTyrLeu 
165170175 
ACAGGAAGTTTCCCTTGGGGAGCAGGAGACCTAGCTGGTCGTCGGATT1080 
ThrGlySerPheProTrpGlyAlaGlyAspLeuAlaGlyArgArgIle 
180185190 
AAAGTGACGGATGCCACTGGTAATACTACTAAGAGTAATCCGTTCTAT1128 
LysValThrAspAlaThrGlyAsnThrThrLysSerAsnProPheTyr 
195200205210 
ATGGTTGCATATACAGTCAAGCCAGTAGATGATAAACCTCTAGCAGTA1176 
MetValAlaTyrThrValLysProValAspAspLysProLeuAlaVal 
215220225 
TCAAACTCTTCTGAGCTGACGGAACAGGCTATTTTTGATAAGTTGGTT1224 
SerAsnSerSerGluLeuThrGluGlnAlaIlePheAspLysLeuVal 
230235240 
GTCGATAAGTCTGCTAAAACAACTTCAAATAGCGCTCTTGTAATTGAT1272 
ValAspLysSerAlaLysThrThrSerAsnSerAlaLeuValIleAsp 
245250255 
TCTAGCAACTACAAACATTCAATTGCAGGTTATCGTACCGTAAATTCT1320 
SerSerAsnTyrLysHisSerIleAlaGlyTyrArgThrValAsnSer 
260265270 
GATGGCACAAAAACAGAAACAGTAGAGGAAACAAATCTATCTGATTTC1368 
AspGlyThrLysThrGluThrValGluGluThrAsnLeuSerAspPhe 
275280285290 
CCAACTGAAGGTAAATACGAAGTTCGAGTAAAAACAACCAATGTTTAC1416 
ProThrGluGlyLysTyrGluValArgValLysThrThrAsnValTyr 
295300305 
GGTCAAACTATCTACAACTGGATTCCTGTAAATGCCTATAAGTTGGAC1464 
GlyGlnThrIleTyrAsnTrpIleProValAsnAlaTyrLysLeuAsp 
310315320 
ACAGCGAAGGATGCTGAAATTCGGAAGTATACAGACAACCAAGCCCCA1512 
ThrAlaLysAspAlaGluIleArgLysTyrThrAspAsnGlnAlaPro 
325330335 
ATTCATGCTATAATGCAAATTGGTCAAGCTGGAGAAAAGGCAGCAGTT1560 
IleHisAlaIleMetGlnIleGlyGlnAlaGlyGluLysAlaAlaVal 
340345350 
ATATTGAAGGATATTCCATCCGATTTCAGTATTGAAAACTTCAATTTG1608 
IleLeuLysAspIleProSerAspPheSerIleGluAsnPheAsnLeu 
355360365370 
AAAGATGGTGTAGCAGATGAGCTTGCTAAACGTAACTTGGAATTTGTA1656 
LysAspGlyValAlaAspGluLeuAlaLysArgAsnLeuGluPheVal 
375380385 
AGAAATGATGCAGTGGCGACAACTGATACTGATGGAGATGGCGCCAAA1704 
ArgAsnAspAlaValAlaThrThrAspThrAspGlyAspGlyAlaLys 
390395400 
GAAGGAATTGTTGGATATATTCAACCAAAAACTGGCGGTGCAAACAGT1752 
GluGlyIleValGlyTyrIleGlnProLysThrGlyGlyAlaAsnSer 
405410415 
GGGGTAGCCACTTATACAGGATCAAATAATCTTACTTATGGCTTCACT1800 
GlyValAlaThrTyrThrGlySerAsnAsnLeuThrTyrGlyPheThr 
420425430 
TACAAAGCTGTTGAGACAAAAGATAAGGCGAATGCCACAGAGGCTAAA1848 
TyrLysAlaValGluThrLysAspLysAlaAsnAlaThrGluAlaLys 
435440445450 
ACTCTCGAATTAGATTACACCATCTTATTCATAGATACTAAAGCACCA1896 
ThrLeuGluLeuAspTyrThrIleLeuPheIleAspThrLysAlaPro 
455460465 
GTCATGACACCTAAATCAGAGTACATCCGTTTTGTTGGTGAAGAGTAT1944 
ValMetThrProLysSerGluTyrIleArgPheValGlyGluGluTyr 
470475480 
ACGGTTAGCGTCCCAGGTACGGATAACGCCTTCCTTAATACCGGCAAA1992 
ThrValSerValProGlyThrAspAsnAlaPheLeuAsnThrGlyLys 
485490495 
CTAAATGGAACTCTCTCAATTTTGAAAGATGGAGAGTCAGGTTCTCTT2040 
LeuAsnGlyThrLeuSerIleLeuLysAspGlyGluSerGlySerLeu 
500505510 
GTATCATCAGACTTAGGTACAAACACTAAGATTACTTCAGAACTGGAT2088 
ValSerSerAspLeuGlyThrAsnThrLysIleThrSerGluLeuAsp 
515520525530 
CCTACGGGAGCAACTGCAAACCAAGGAGATGACGGTCAATCTTCAACT2136 
ProThrGlyAlaThrAlaAsnGlnGlyAspAspGlyGlnSerSerThr 
535540545 
AAGTTTAACGTTAAGATTACAGGTACCGGACCTGCTACAGAAGGTACC2184 
LysPheAsnValLysIleThrGlyThrGlyProAlaThrGluGlyThr 
550555560 
GGCACTTATAAGCTTCGTGTTGGAGAAGATAACTATCCTTTTGGTCCA2232 
GlyThrTyrLysLeuArgValGlyGluAspAsnTyrProPheGlyPro 
565570575 
GAGGGGAAACTTGTTGATGGAAATAAACCAGAAAATGTAGGTTTGACA2280 
GluGlyLysLeuValAspGlyAsnLysProGluAsnValGlyLeuThr 
580585590 
TCTGTAAAAGTTACCTTCGTAAAACATGCTACGGTGTCAACACCAGTT2328 
SerValLysValThrPheValLysHisAlaThrValSerThrProVal 
595600605610 
TCTGTTGAAAATCCAGCTAACTTAACGCCAGAAGAAAAAGCCGCAGTT2376 
SerValGluAsnProAlaAsnLeuThrProGluGluLysAlaAlaVal 
615620625 
ATTGCTCAAATCAAGAAAGACAACGCAGACAACGAAAGATTGAAGGGC2424 
IleAlaGlnIleLysLysAspAsnAlaAspAsnGluArgLeuLysGly 
630635640 
TTGCCAGATTCAGCATTTACAGTTAACTCAGATGGTACTGTGTCAGTT2472 
LeuProAspSerAlaPheThrValAsnSerAspGlyThrValSerVal 
645650655 
GACTACAGTGCCGGTGGTGTCAATGTTGATGGTGCGACAGACATTATT2520 
AspTyrSerAlaGlyGlyValAsnValAspGlyAlaThrAspIleIle 
660665670 
AAGAATGCTACCACAAACTTGGCAGATACACGGAATGAAGCAAAAGCA2568 
LysAsnAlaThrThrAsnLeuAlaAspThrArgAsnGluAlaLysAla 
675680685690 
GAAATCGACACAAAATTAGCTGAACATAAAAAAGCTATCGAAGCAAAA2616 
GluIleAspThrLysLeuAlaGluHisLysLysAlaIleGluAlaLys 
695700705 
CGGGATGAAGCGTTTTCTAAAATTGATGATGACATTTCCTTGAGAGCA2664 
ArgAspGluAlaPheSerLysIleAspAspAspIleSerLeuArgAla 
710715720 
GAACAGAGACAGGCTGCTAAGGATGCCGTTGCTGCAGCTGCTGGGGAT2712 
GluGlnArgGlnAlaAlaLysAspAlaValAlaAlaAlaAlaGlyAsp 
725730735 
GCTTTGAAAGAATTAGACAACAAGGCGACAGAAGCAAAAGAAAAAATT2760 
AlaLeuLysGluLeuAspAsnLysAlaThrGluAlaLysGluLysIle 
740745750 
GATAAAGCTACGACGGCCTCAGAAATCAATGATGCTAAGACTAATGGT2808 
AspLysAlaThrThrAlaSerGluIleAsnAspAlaLysThrAsnGly 
755760765770 
GAGATTAATCTGGACAGTGCAGAAGCAGTAGGCGAAAAAGCTATTAAC2856 
GluIleAsnLeuAspSerAlaGluAlaValGlyGluLysAlaIleAsn 
775780785 
CAGGCGAAGGAAAAAGAACTGGCAAAAGCAGAAGTTGAAAACAAAGCA2904 
GlnAlaLysGluLysGluLeuAlaLysAlaGluValGluAsnLysAla 
790795800 
TTCGAGGCATTGGAAAAAGTTAACAATAATCCAAACTTGTTAGAAGAA2952 
PheGluAlaLeuGluLysValAsnAsnAsnProAsnLeuLeuGluGlu 
805810815 
GAGAAAAAAGCATACTTTGATGATATTAAAGAATCTAAAGAAGTTGCA3000 
GluLysLysAlaTyrPheAspAspIleLysGluSerLysGluValAla 
820825830 
GTTGAGAAAATCAATAATGCTGAAAATACTGCTGAAATTACGGCAGCA3048 
ValGluLysIleAsnAsnAlaGluAsnThrAlaGluIleThrAlaAla 
835840845850 
ATTGACGAAGCGGAAATTGCATACAATGAAGATGTTATTAACGCAGCC3096 
IleAspGluAlaGluIleAlaTyrAsnGluAspValIleAsnAlaAla 
855860865 
CAACTTGATGCTTTGAATAAGCTTGAAAAAGATAGCGAAGAAACTAAG3144 
GlnLeuAspAlaLeuAsnLysLeuGluLysAspSerGluGluThrLys 
870875880 
GCAGCTATTGATGCTAATCCAAACTTAACTCCGGAAGAGAAAGCGAAA3192 
AlaAlaIleAspAlaAsnProAsnLeuThrProGluGluLysAlaLys 
885890895 
GCTATTGCTAAGGTAGAAGAGCTTGTTAATAATGCTGAATCTGACATT3240 
AlaIleAlaLysValGluGluLeuValAsnAsnAlaGluSerAspIle 
900905910 
TTGTCGAAGCCTACCCCAGAAACAGTTCAAGCAGTGGAGGATAAGGCT3288 
LeuSerLysProThrProGluThrValGlnAlaValGluAspLysAla 
915920925930 
GACAAAGATCTTGCCAAAGTAGAACTTCAAGCAGCAGCAGACGGTGCG3336 
AspLysAspLeuAlaLysValGluLeuGlnAlaAlaAlaAspGlyAla 
935940945 
AAGAAAGGCATTGAAGCAAATCCGAATTTGACTCCAGAAGAGAAAGAT3384 
LysLysGlyIleGluAlaAsnProAsnLeuThrProGluGluLysAsp 
950955960 
GTAGCTAAGAAGGCAGTAGAAGACGCGGTTAAGGTGGCGACAGACGCT3432 
ValAlaLysLysAlaValGluAspAlaValLysValAlaThrAspAla 
965970975 
ATTGATAAGGCGTCAACTCCAACCGAAGTTGACACAGCGACAAGCGAT3480 
IleAspLysAlaSerThrProThrGluValAspThrAlaThrSerAsp 
980985990 
GGAGTGAAGGCTATTGATGCAGAAGAGTTTAAAGCTACTCAGAAAGAT3528 
GlyValLysAlaIleAspAlaGluGluPheLysAlaThrGlnLysAsp 
995100010051010 
GCTAAGAACAAGATTGCCAAAGAAGCAGAATCAGCTAAGAAAGCGATT3576 
AlaLysAsnLysIleAlaLysGluAlaGluSerAlaLysLysAlaIle 
101510201025 
GACGACAATCCAAACTTGACTCCAGATGAGAAGGAATCAGCTAAGAAT3624 
AspAspAsnProAsnLeuThrProAspGluLysGluSerAlaLysAsn 
103010351040 
GCAGTGGAAGAGGCGGCTAAGGTAGCAACAGCCGCTATTGATAAAGCA3672 
AlaSerGluGluAlaAlaLysValAlaThrAlaAlaIleAspLysAla 
104510501055 
TCAACTCCAGATGCAGTTCAAGTAGAAGAGGACAAAGGTGTAGCAGCT3720 
SerThrProAspAlaValGlnValGluGluAspLysGlyValAlaAla 
106010651070 
ATCAATTTGATTACTGCCAAGGCAGATGCTAAAGGTGTCATTGCTGCT3768 
IleAsnLeuIleThrAlaLysAlaAspAlaLysGlyValIleAlaAla 
1075108010851090 
AAGTTGGCAGATGAAATCAAGAAGCTCGAAGATAAGCAAGCAGAAGCA3816 
LysLeuAlaAspGluIleLysLysLeuGluAspLysGlnAlaGluAla 
109511001105 
GAAAAAGCTATCGATGCGTCAACTATGACTAATGAGGAGAAAGCAATC3864 
GluLysAlaIleAspAlaSerThrMetThrAsnGluGluLysAlaIle 
111011151120 
GCTAAGAAGGCTCTTCAAGATGTTGTAGATAAAGGAAAAGCAGAGCTT3912 
AlaLysLysAlaLeuGlnAspValValAspLysGlyLysAlaGluLeu 
112511351135 
GAAGACGCAGCTAGGGTAGCAACAAATGAGATTCATGAAGCTACTACT3960 
GluAspAlaAlaArgValAlaThrAsnGluIleHisGluAlaThrThr 
114011451150 
ACAGAAAAAGCGAAAGCGGCGGAACTTGCTGGCGAAAAGAGCTTGACA4008 
ThrGluLysAlaLysAlaAlaGluLeuAlaGlyGluLysSerLeuThr 
1155116511651170 
GACACAGGTAAAGAAGCTAGAGATGCAGTTGAATTGGCTAAGGATAAA4056 
AspThrGlyLysGluAlaArgAspAlaValGluLeuAlaLysAspLys 
117511801185 
GAATTAGCTAAGGAAGCAATCCGAACAGAAGAAGAAGAAGCTACTAAA4104 
GluLeuAlaLysGluAlaIleArgThrGluGluGluGluAlaThrLys 
119011951200 
ATAGTAGAGAAACTTGCAGAAGATACGCGCAAAGCTATCGAGGACAAT4152 
IleValGluLysLeuAlaGluAspThrArgLysAlaIleGluAspAsn 
120512101215 
CCAAACTTGTCAGATGAAGATAAGCAAGCGGAAATTAAAAAGCTAACT4200 
ProAsnLeuSerAspGluAspLysGlnAlaGluIleLysLysLeuThr 
122012251230 
GACGCTGTGGCAAAAACTTTAGCAACCATTCGTGACAATGCAGATAAG4248 
AspAlaValAlaLysThrLeuAlaThrIleArgAspAsnAlaAspLys 
1235124012451250 
CGTACGCAAGAAGCAGAAAAAGCTCAAGCCCTAGCAGATCTTGAAAAA4296 
ArgThrGlnGluAlaGluLysAlaGlnAlaLeuAlaAspLeuGluLys 
125512601265 
GCTAAAGAAACACAGAAAATTGCAGATAAAGCTGCGATTGATAGGTTG4344 
AlaLysGluThrGlnLysIleAlaAspLysAlaAlaIleAspArgLeu 
127012751280 
ACTATACTTGTGAAAGATGGTGAGCTTGAAGCTACTAAACAAGATGCT4392 
ThrIleLeuValLysAspGlyGluLeuGluAlaThrLysGlnAspAla 
128512901295 
AAGAACAAGATTGCTAAAGATGCAGCCGCTGCTAAAGAAGCAATTGCA4440 
LysAsnLysIleAlaLysAspAlaAlaAlaAlaLysGluAlaIleAla 
130013051310 
AGCAATCCAAACTTGACAGACGCAGAGAAGAAAACCTTCACCGATGCG4488 
SerAsnProAsnLeuThrAspAlaGluLysLysThrPheThrAspAla 
1315132013251330 
GTAGATGCAGAAGTAGCCAAAGCTAACGACGCAATTTCAGCTGCAACC4536 
ValAspAlaGluValAlaLysAlaAsnAspAlaIleSerAlaAlaThr 
133513401345 
AGCCCAGCAGATGTTCAAAAAGAAGAGGATGCAGGTGTTGCAGCCATT4584 
SerProAlaAspValGlnLysGluGluAspAlaGlyValAlaAlaIle 
135013551360 
GCAGAAGATGTTCTTGACGCAGCTAAACAAGATGCTAAGAATAAGATT4632 
AlaGluAspValLeuAspAlaAlaLysGlnAspAlaLysAsnLysIle 
136513701375 
GCTAAAGATGCAGCCGCTGCTAAAGAAGCAATTGGCTCCAATCCAAAC4680 
AlaLysAspAlaAlaAlaAlaLysGluAlaIleGlySerAsnProAsn 
138013851390 
TTGACAGACGCAGAGAAGAAAACCTTCACCGATGCGGTAGATGCAGAA4728 
LeuThrAspAlaGluLysLysThrPheThrAspAlaValAspAlaGlu 
1395140014051410 
GTAGCCAAAGCTAACGACGCAATTTCAGCTGCAACCAGCCCAGCAGAT4776 
ValAlaLysAlaAsnAspAlaIleSerAlaAlaThrSerProAlaAsp 
141514201425 
GTTCAAAAAGAAGAGGATGCAGGTGTTGCAGCCATTGCAGAAGATGTT4824 
ValGlnLysGluGluAspAlaGlyValAlaAlaIleAlaGluAspVal 
143014351440 
CTTGACGCAGCTAAACAAGATGCTAAGAATAAGATTGCTAAAGAATCC4872 
LeuAspAlaAlaLysGlnAspAlaLysAsnLysIleAlaLysGluSer 
144514501455 
GACGCTGCTAAGTCAGCCATTGACGCGAATCCAAACTTGACAGATGCA4920 
AspAlaAlaLysSerAlaIleAspAlaAsnProAsnLeuThrAspAla 
146014651470 
GAGAAGGAATCAGCTAAGAAAGCAGTTGATGCTGATGCTAAAGCTGCG4968 
GluLysGluSerAlaLysLysAlaValAspAlaAspAlaLysAlaAla 
1475148014851490 
ACAGATGCAATTGATGCTTCAACAAGTCCAGTCGAAGCGCAATCGGCA5016 
ThrAspAlaIleAspAlaSerThrSerProValGluAlaGlnSerAla 
149515001505 
GAGGACAAAGGCGTAGGTTCAATCGCCCAAGATGTTCTTGACGCAGCG5064 
GluAspLysGlyValGlySerIleAlaGlnAspValLeuAspAlaAla 
151015151520 
AAACAAGATGCTAAGAACAAGATTGCCAAAGAAGTTGCCGCAGCTAAA5112 
LysGlnAspAlaLysAsnLysIleAlaLysGluValAlaAlaAlaLys 
152515301535 
GAAGCAATTGATGCCAATCCGAACTTATCAGATGCAGAGAAGGAAGCT5160 
GluAlaIleAspAlaAsnProAsnLeuSerAspAlaGluLysGluAla 
154015451550 
TCTAAGAAAGCGGTAGATGCAGATGCTAAAGCTACGACAGATGCAATT5208 
SerLysLysAlaValAspAlaAspAlaLysAlaThrThrAspAlaIle 
1555156015651570 
GATGCTTCAACAAGTCCAGTCGAAGCGCAATCGGCAGAGGACAAAGGC5256 
AspAlaSerThrSerProValGluAlaGlnSerAlaGluAspLysGly 
157515801585 
GTAGGTTCAATCGCCCAAGATGTTCTTGACGCAGCGAAACAAGATGCT5304 
ValGlySerIleArgGlnAspValLeuAspAlaAlaLysGlnAspAla 
159015951600 
AAGAATAAGATTGCTAAAGAATCCGACGCTGCTAAGTCAGCCATTGAC5352 
LysAsnLysIleAlaLysGluSerAspAlaAlaLysSerAlaIleAsp 
160516101615 
GCGAATCCAAACTTGACAGATGCAGAGAAGGAATCAGCTAAGAAAGCG5400 
AlaAsnProAsnLeuThrAspAlaGluLysGluSerAlaLysLysAla 
162016251630 
GTAGATGCAGATGCTAAAGCTGCGACAGATGCAATTGATGCTTCAACA5448 
ValAspAlaAspAlaLysAlaAlaThrAspAlaIleAspAlaSerThr 
1635164016451650 
AGTCCAGTCGAAGCGCAATCGGCAGAGGACAAAGGCGTAGGCGCCATC5496 
SerProValGluAlaGlnSerAlaGluAspLysGlyValGlyAlaIle 
165516601665 
GCCAAAGACATTCTTGATGCCGCGAAACAAGATGCTAAGAACAAGATT5544 
AlaLysAspIleLeuAspAlaAlaLysGlnAspAlaLysAsnLysIle 
167016751680 
GCTAAAGAGGCAGAATCCGCTAAGTCAGTCATTGACTCCAATCCGAAC5592 
AlaLysGluAlaGluSerAlaLysSerValIleAspSerAsnProAsn 
168516901695 
TTGACAGATGCAGCTAAGGAAGCGGCTAAATCTGAAATTGATAAAGCT5640 
LeuThrAspAlaAlaLysGluAlaAlaLysSerGluIleAspLysAla 
170017051710 
GTTGAGGAAGCGATTGTTTTAATCAATGGTGTTAGAACTTATCAAGAG5688 
ValGluGluAlaIleValLeuIleAsnGlyValArgThrTyrGlnGlu 
1715172017251730 
TTGGAAAAAATCAAACTTCCAATGGCAGCTCTAATTAAACCAGCTGCG5736 
LeuGluLysIleLysLeuProMetAlaAlaLeuIleLysProAlaAla 
173517401745 
AAAGTAACACCAGTGGTTGATCCAAATAACTTGACTGAAAAAGAAATT5784 
LysValThrProValValAspProAsnAsnLeuThrGluLysGluIle 
175017551760 
GCTCGTATCAAGGCATTCCTTAAAGAGAACAATAACCTCCCATAA5829 
AlaArgIleLysAlaPheLeuLysGluAsnAsnAsnLeuPro 
176517701775 
GGAACAGAGATTAATGTTTCTAAAGATGCTTCAGTGACAATTAAATATCCAGATGGAACT5889 
ATTGATTTGCTATCACCAGTAGAAGTTGTGAAGCAGGCAGATAAAACTGCTCCTACGGTC5949 
GCAAATGATGGCAAAGGTAATATTGTGATTGTACCGTCTGAAAAAGCTGTTGAGCTTGTT6009 
GTTTCATACGTAGATAACAATGGTAAGTCGCAAACTGTAGTTGTTACGAAAGGTACGGAT6069 
GGTTTATGGACAGCAAGTAATACAGTGGTGATTGTGGACCCTGTGACTGGGCAAGTAATC6129 
GTTCCAGGTTCTGTTATTAAGCCAGGTACAGTTGTTACAGCATACTCTAAAGACGAGGTT6189 
GGAAATAGTTCTGATTCAGCAGAAGCTGAAGTTGTAGCAGTAGACGAAAATAATTCTGCA6249 
GCAGGAGTGAAAGTTAAATCAGTTACTACAAATGCTAATAATGTTGAGAAGAAAGCTAAG6309 
CAATTACCGAATACTGGTGAGGAAGCAAATTCAGCAACTTCACTCGGATTAGTAGCTCTT6369 
GGACTCGGATTAGCACTTCTTGCAGCAAAGAGAAGAAGAGACGAAGAAGCTTAAGATAAG6429 
CTCTTCCTCAGAACTCTTTTGGAAGCCGCAATTTTCCTAGAAGATAGTAGTATGATACTC6489 
TTTCATAGCAAGGAAATTCCCTCGCTATGATTGGTAGGTATCAGTTATTATCTATCGAAC6549 
CCCCAAAATCCAAAGTCATTCGACTTTGGATTTTTTTGATACGACATGCTCGTCATACCT6609 
AAAAAACAGCCTTCTCTTGCCGAGAGGCTGTTTTTCATGCTTTTAATCTAAAAGTCTGCG6669 
GACGTTTTTTCAATAAAATCCAGTAACCGATGCTAACATAGGCAATCATAGCTAGGGAAA6729 
CCAGCAGGATATAGG6744 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4118 base pairs 
(B) TYPE: Nucleic acid with corresponding amino acids 
(C) STRANDEDNESS: single stranded 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Streptococcus suis type II (pathogenic) 
(ix) FEATURE: 
(D) OTHER INFORMATION: Muramidase released protein (MRP) gene 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 4 to 9 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 29 to 34 
(ix) FEATURE: 
(A) NAME/KEY: promoter -35 region 
(B) LOCATION: bp 40 to 45 
(ix) FEATURE: 
(A) NAME/KEY: promoter -10 region 
(B) LOCATION: bp 63 to 68 
(ix) FEATURE: 
(A) NAME/KEY: ribosome binding site 
(B) LOCATION: bp 147 to 152 
(ix) FEATURE: 
(A) NAME/KEY: signal peptide 
(B) LOCATION: bp 159 to 299 
(ix) FEATURE: 
(A) NAME/KEY: mature peptide 
(B) LOCATION: bp 300 to 3926 
(ix) FEATURE: 
(A) NAME/KEY: proline rich region 
(B) LOCATION: from bp 2757 to 3014 
(ix) FEATURE: 
(A) NAME/KEY: repetitive units 
(B) LOCATION: from bp 3015 to 3176, 3423 to 3584 and 3585 to 
3743 
(ix) FEATURE: 
(A) NAME/KEY: membrane anchor sequence 
(B) LOCATION: from bp 3825 to 3926 
(ix) FEATURE: 
(A) NAME/KEY: dyad symmetry regions 
(B) LOCATION: from bp 4069 to 4080 and from bp 4087 to 4098 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAATTCATAATGTTTTTTTGAGGAATTTTATAATATTACTTGGCATTTAAAGTTATTTGT60 
AGTATAATACCTCGAATGATTGCGGGAGTTTTCAAGGCTTTGATACAAAGAGTAGAAAAT120 
TTGTGTAATTAAATTAATATTTATATGGGGGATTTTTT158 
ATGCGTAGATCAAATAAAAAATCATTTGACTGGTACGGTACGAAACAA206 
MetArgArgSerAsnLysLysSerPheAspTrpTyrGlyThrLysGln 
45-40-35 
CAATTTTCGATTCGTAAGTATCATTTTGGGGCAGCAAGCGTTTTGCTT254 
GlnPheSerIleArgLysTyrHisPheGlyAlaAlaSerValLeuLeu 
30-25-20 
GGTGTGTCGTTAGTTTTAGGTGCTGGTGCACAGGTTGTTAAGGCTGAT302 
GlyValSerLeuValLeuGlyAlaGlyAlaGlnValValLysAlaAsp 
15-10-51 
GAAACTGTTGCTTCATCAGAACCAACTATTGCCAGTAGTGTAGCGCCT350 
GluThrValAlaSerSerGluProThrIleAlaSerSerValAlaPro 
51015 
GCTTCAACAGAAGCGGTTGCAGAAGAAGCAGAAAAAACAAATGCTGAA398 
AlaSerThrGluAlaValAlaGluGluAlaGluLysThrAsnAlaGlu 
202530 
AATACGAGTGCAGTAGCTACGACTTCAACAGAAGTTGAAAAAGCGAAA446 
AsnThrSerAlaValAlaThrThrSerThrGluValGluLysAlaLys 
354045 
GCTGTTCTTGAACAGGTAACATCAGAATCACCACTTTTGGCTGGTCTT494 
AlaValLeuGluGlnValThrSerGluSerProLeuLeuAlaGlyLeu 
50556065 
GGTCAAAAAGAGTTGGCTAAAACTGAAGATGCAACTCTTGCAAAAGCT542 
GlyGlnLysGluLeuAlaLysThrGluAspAlaThrLeuAlaLysAla 
707580 
ATAGAGGATGCTCAAACAAAACTTGCAGCAGCTAAGGCAATTTTGGCT590 
IleGluAspAlaGlnThrLysLeuAlaAlaAlaLysAlaIleLeuAla 
859095 
GACTCAGAAGCAACTGTTGAGCAAGTTGAAGCGCAAGTCGCAGCGGTT638 
AspSerGluAlaThrValGluGlnValGluAlaGlnValAlaAlaVal 
100105110 
AAAGTAGCCAACGAGGCGCTAGGGAATGAATTGCAAAAATACACTGTA686 
LysValAlaAsnGluAlaLeuGlyAsnGluLeuGlnLysTyrThrVal 
115120125 
GATGGTCTCTTGACAGCGGCTCTTGATACAGTAGCACCTGATACAACT734 
AspGlyLeuLeuThrAlaAlaLeuAspThrValAlaProAspThrThr 
130135140145 
GCATCAACATTGAAAGTTGGTGATGGCGAAGGTACCCTTCTAGATAGC782 
AlaSerThrLeuLysValGlyAspGlyGluGlyThrLeuLeuAspSer 
150155160 
ACTACAACAGCAACGCCTTCAATGGCTGAGCCAAATGGTGCAGCAATT830 
ThrThrThrAlaThrProSerMetAlaGluProAsnGlyAlaAlaIle 
165170175 
GCTCCACATACACTTCGAACTCAAGATGGAATTAAAGCGACATCAGAG878 
AlaProHisThrLeuArgThrGlnAspGlyIleLysAlaThrSerGlu 
180185190 
CCAAATTGGTATACTTTTGAATCGTACGATTTGTACTCATATAATAAA926 
ProAsnTrpTyrThrPheGluSerTyrAspLeuTyrSerTyrAsnLys 
195200205 
AATATGGCTAGCTCAACTTATAAAGGAGCTGAAGTTGATGCCTACATT974 
AsnMetAlaSerSerThrTyrLysGlyAlaGluValAspAlaTyrIle 
210215220225 
CGTTACTCTTTGGATAATGATTCGTCAACAACTGCTGTTTTAGCAGAG1022 
ArgTyrSerLeuAspAsnAspSerSerThrThrAlaValLeuAlaGlu 
230235240 
TTGGTAAGTAGGACAACTGGTGATGTGTTAGAGAAATATACGATTGAA1070 
LeuValSerArgThrThrGlyAspValLeuGluLysTyrThrIleGlu 
245250255 
CCGGGCGAGAGTGTTACGTTTTCACATCCGACAAAAGTTAATGCTAAT1118 
ProGlyGluSerValThrPheSerHisProThrLysValAsnAlaAsn 
260265270 
AATAGCAATATAACTGTGACTTATGATACCTCATTAGCTTCTGCTAAT1166 
AsnSerAsnIleThrValThrTyrAspThrSerLeuAlaSerAlaAsn 
275280285 
ACTCCTGGAGCATTGAAATTCTCTGCTAATGATGATGTTTATTCAACA1214 
ThrProGlyAlaLeuLysPheSerAlaAsnAspAspValTyrSerThr 
290295300305 
ATTATTGTACCTGCTTATCAGATTAATACAACTCGTTACGTCACTGAA1262 
IleIleValProAlaTyrGlnIleAsnThrThrArgTyrValThrGlu 
310315320 
AGTGGCAAAGTTTTGGCAACCTATGGTCTTCAAACTATTGCAGGACAG1310 
SerGlyLysValLeuAlaThrTyrGlyLeuGlnThrIleAlaGlyGln 
325330335 
GTAGTTACTCCATCTTCTGTTCGTGTATTTACTGGGTATGATTATGTG1358 
ValValThrProSerSerValArgValPheThrGlyTyrAspTyrVal 
340345350 
GCAACTACAACTAAAGCCGTTCAAGGTCCATATCCAAAGGGAACGGTA1406 
AlaThrThrThrLysAlaValGlnGlyProTyrProLysGlyThrVal 
355360365 
TACCTTGCTGGTACGGTTCAAAAGGATACAGTACAATATAAAGTTATT1454 
TyrLeuAlaGlyThrValGlnLysAspThrValGlnTyrLysValIle 
370375380385 
CGTGAAATTGTGGAGAACGACCAAGCAGTTCTTAAATTCTATTATTTA1502 
ArgGluIleValGluAsnAspGlnAlaValLeuLysPheTyrTyrLeu 
390395400 
GATCCTACCTATAAGGGTGAAGTAGATTGGAGAGGAACTGATACGACT1550 
AspProThrTyrLysGlyGluValAspTrpArgGlyThrAspThrThr 
405410415 
GGGTTTATTGAGTTGCTTACAACTTCCCCAACAACCTATAAAGTTGGT1598 
GlyPheIleGluLeuLeuThrThrSerProThrThrTyrLysValGly 
420425430 
ACTATATACGATTACAATATTAATTCAAAAATTACAGCTCCATTTACT1646 
ThrIleTyrAspTyrAsnIleAsnSerLysIleThrAlaProPheThr 
435440445 
ATTGATCCTACCAAGAATGTTATGGTTTTCAAGGAAAGTGAACAGAAC1694 
IleAspProThrLysAsnValMetValPheLysGluSerGluGlnAsn 
450455460465 
GAGCAAGGTAGCAAATATCGCGTCATTGCTCAATGGTCAGGAGATGAA1742 
GluGlnGlySerLysTyrArgValIleAlaGlnTrpSerGlyAspGlu 
470475480 
ACCACTAAAGGTATATATGGAAAAATCTATATCGCTACTCAGGTTTGG1790 
ThrThrLysGlyIleTyrGlyLysIleTyrIleAlaThrGlnValTrp 
485490495 
ACGACTAAATTGGGAACAAACGAGTGGGGATGGTTTGACTATTCTGAT1838 
ThrThrLysLeuGlyThrAsnGluTrpGlyTrpPheAspTyrSerAsp 
500505510 
GACCAAGCTGGTATAAAATTTAATAACAAAGGTTTTTGGCCGGCAGGT1886 
AspGlnAlaGlyIleLysPheAsnAsnLysGlyPheTrpProAlaGly 
515520525 
GTTCAAAATACACTTCGAAATGCTACTCCAGCTACAGCTGTAGAGACT1934 
ValGlnAsnThrLeuArgAsnAlaThrProAlaThrAlaValGluThr 
530535540545 
ACTTATATCTACAAAGAAAGTTCCAAGTATGGTGATGTCATTGTTGAG1982 
ThrTyrIleTyrLysGluSerSerLysTyrGlyAspValIleValGlu 
550555560 
TACTACGATACTGACGGAAAACAAATTGTAAATTCAGTTGTAGATACT2030 
TyrTyrAspThrAspGlyLysGlnIleValAsnSerValValAspThr 
565570575 
CCTAAGTCAGCTCTTGGCACAGAGTATAATACAGATGTGGACCGTAGA2078 
ProLysSerAlaLeuGlyThrGluTyrAsnThrAspValAspArgArg 
580585590 
CCAGCCAGCTTGGTTGCTGCTGATGGGACAGTCTACTTCTACAAAGAA2126 
ProAlaSerLeuValAlaAlaAspGlyThrValTyrPheTyrLysGlu 
595600605 
GTTAAGTCTGATTCAGCTAAGACAACCGGTACAGTAGTTGCAGGTACG2174 
ValLysSerAspSerAlaLysThrThrGlyThrValValAlaGlyThr 
610615620625 
ACAACTGTTAAGTATGTTTACGAAAAAGCTGGTAGCGTTAATGTTAAC2222 
ThrThrValLysTyrValTyrGluLysAlaGlySerValAsnValAsn 
630635640 
TTCGTTGACATCAATGGTAAAGTAATCAAAGCTCCTGTTTCAGATGAA2270 
PheValAspIleAsnGlyLysValIleLysAlaProValSerAspGlu 
645650655 
AAAGATGCGAAACCTGGTTACAATTATGATACCGACTTGGATCAGAAA2318 
LysAspAlaLysProGlyTyrAsnTyrAspThrAspLeuAspGlnLys 
660665670 
TTAGCTTCCATCACTTTTGAAGGCAAGGAATACAAACTTGTTCCTGCT2366 
LeuAlaSerIleThrPheGluGlyLysGluTyrLysLeuValProAla 
675680685 
GGTGATTATCCGGTTGGTAAAGTTGGCAAGGGAAATAACTTGATTGAA2414 
GlyAspTyrProValGlyLysValGlyLysGlyAsnAsnLeuIleGlu 
690695700705 
GTTGGTAATAATACTGCGAAAGGTATTGACCCAACAACAGGCAAAATT2462 
ValGlyAsnAsnThrAlaLysGlyIleAspProThrThrGlyLysIle 
710715720 
GAAGCCGGTGTTAACAAAGAAGTTACCTATGTCTATAGAGCAGTGACA2510 
GluAlaGlyValAsnLysGluValThrTyrValTyrArgAlaValThr 
725730735 
GGTTCTGTAGTTGTAAATTACAAAGATACAGAAGGTAATGTGATTAAA2558 
GlySerValValValAsnTyrLysAspThrGluGlyAsnValIleLys 
740745750 
GATCCAGAAACGGATGTGTCTGATGCACCGGTTGGAGATGCTTATACT2606 
AspProGluThrAspValSerAspAlaProValGlyAspAlaTyrThr 
755760765 
ACAACTGACAAGAAACCAAACGAAATCATCACAAAAGATGGATCACGC2654 
ThrThrAspLysLysProAsnGluIleIleThrLysAspGlySerArg 
770775780785 
TATGTTCTTGTTCCATCTAAGACAGATGGTGAGGAAAATGGTAAAGTT2702 
TyrValLeuValProSerLysThrAspGlyGluGluAsnGlyLysVal 
790795800 
ATCGAAGGAACAATCACAGTAACTTATGTTTACCAGAAAGTTGCAAAC2750 
IleGluGlyThrIleThrValThrTyrValTyrGlnLysValAlaAsn 
805810815 
TGGATTCCAGAGATTCCAAATGTACCAGAAACAGACCGTCCAAAAGTA2798 
TrpIleProGluIleProAsnValProGluThrAspArgProLysVal 
820825830 
CCTTACCCATTTGACCCAACAGAGCCAGACGAGCCAATCGATCCAACG2846 
ProTyrProPheAspProThrGluProAspGluProIleAspProThr 
835840845 
ACACCAGGAACAAATGGCGAGGTTCCAAATATTCCTTACGTTCCAGGA2894 
ThrProGlyThrAsnGlyGluValProAsnIleProTyrValProGly 
850855860865 
TATACACCGGTTGATCCTAAGGATAACACGCCGTTGAAACCAATTGAT2942 
TyrThrProValAspProLysAspAsnThrProLeuLysProIleAsp 
870875880 
CCAAATGATCCAGGTAAGGGTTATGTACCACCAACACCAGAAAATCCA2990 
ProAsnAspProGlyLysGlyTyrValProProThrProGluAsnPro 
885890895 
GGTGTTGATACACCAATTCCTTATGTTCCAGTTAAAAAAGTCGTAACT3038 
GlyValAspThrProIleProTyrValProValLysLysValValThr 
900905910 
AACCACGTTGATGAAGAGGGTAACCCTATTGCACCGCAAGAAGAGGGA3086 
AsnHisValAspGluGluGlyAsnProIleAlaProGlnGluGluGly 
915920925 
ACAAAACCAAACAAATCAATCCCAGGTTACGAGTTCACAGGTAAAACT3134 
ThrLysProAsnLysSerIleProGlyTyrGluPheThrGlyLysThr 
930935940945 
GTTACTGACGAAGATGGCAACACAACTCACATCTACAAGAAAACACCA3182 
ValThrAspGluAspGlyAsnThrThrHisIleTyrLysLysThrPro 
950955960 
GAAGTTAAGAATGGTACAGTTGTTGTTAACTATGTAACAGAAGATGGC3230 
GluValLysAsnGlyThrValValValAsnTyrValThrGluAspGly 
965970975 
ACAGTTATCAAGGAACCTGTAACAGATACACCAACTTCTCCAGAAGGC3278 
ThrValIleLysGluProValThrAspThrProThrSerProGluGly 
980985990 
ACACCATACGACACTACAGACAACAAACCTAAGACAATCACTTTCAAA3326 
ThrProTyrAspThrThrAspAsnLysProLysThrIleThrPheLys 
99510001005 
GGTGAAGAGTATGAATTGGTTCGTGTTGACGGTACAGAAAACGGTAAA3374 
GlyGluGluTyrGluLeuValArgValAspGlyThrGluAsnGlyLys 
1010101510201025 
GTTGTAGAAGGTGAAACAGTTGTGACTTACGTTTACCGTAAAGTCGAA3422 
ValValGluGlyGluThrValValThrTyrValTyrArgLysValGlu 
103010351040 
ACACCTGCTAAGAAAGTTGTAACTAACCACGTTGATGAAGAGGGTAAC3470 
ThrProAlaLysLysValValThrAsnHisValAspGluGluGlyAsn 
104510501055 
CCTGTTGCGCCGCAAGAAGAGGGAACAAAACCAAACAAATCAATCCCA3518 
ProValAlaProGlnGluGluGlyThrLysProAsnLysSerIlePro 
106010651070 
GGTTACGAATTTACAGGTAAAACTGTTACTGACGAAGATGGCAACACA3566 
GlyTyrGluPheThrGlyLysThrValThrAspGluAspGlyAsnThr 
107510801085 
ACTCACATCTACAAGAAAACACCTGCTAAGAAAGTTGTGACTAACCAC3614 
ThrHisIleTyrLysLysThrProAlaLysLysValValThrAsnHis 
1090109511001105 
GTTGATGAAGAAGGTAACCCTATTGCTCCACAAGAGGATGGGACAACA3662 
ValAspGluGluGlyAsnProIleAlaProGlnGluAspGlyThrThr 
111011151120 
CCAAAACGTCAAATTTCAGGTTACGAGTATGTGCGTACTGTAGTTGAT3710 
ProLysArgGlnIleSerGlyTyrGluTyrValArgThrValValAsp 
112511301135 
GAAGAAGGTAACACGACACATATTTATCGCAAACTTTCTAATAAACCA3758 
GluGluGlyAsnThrThrHisIleTyrArgLysLeuSerAsnLysPro 
114011451150 
ACAACACCTGAGAAGGAAACTCCTGCAAAACCTCAAGCAGGTAAAACC3806 
ThrThrProGluLysGluThrProAlaLysProGlnAlaGlyLysThr 
115511601165 
GCTTCAGGTAAAGCTCAATTGCCAAATACTGGTGAGGCTTCATCTGTG3854 
AlaSerGlyLysAlaGlnLeuProAsnThrGlyGluAlaSerSerVal 
1170117511801185 
GCAGGTGCGCTTGGTACAGCAATGCTTGTCGCAACACTTGCGTTTGCA3902 
AlaGlyAlaLeuGlyThrAlaMetLeuValAlaThrLeuAlaPheAla 
119011951200 
AGAAAACGTCGTCGTAACGAAGATTAGTCAAAATTCTTTATACAGAC3949 
ArgLysArgArgArgAsnGluAsp 
1205 
TTTATTCCCCCACATAGAAAGTATAAGAATTGTACGTAACATGCAGGATTGCCTTTCCGA4009 
AAAAATGAGGCTGGGCAAAAAGTCCAGAGTTACATCTTAGAGTTCGCTCCATTTCCAACC4069 
TCCAACAGTCACTACTCTGACTGTTGGAGCTGTGTGGGGGTGGGAGACG4118 
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