System. App!. Microbio!. 21, 408-418 (1998) _©_G_us_ta_vF_is_ch_er_Vl_er_lag_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
SYSTEMATIC AND APPLIED MICROBIOLOGY
Differentiation of Actinobacillus pleuropneumoniae Strains by Sequence Analysis of 16S rDNA and Ribosomallntergenic Regions, and Development of a Species Specific Oligonucleotide for in situ Detection VIVIAN FUSSING l >" BRUCE]. PASTER2, FLOYD E. DEWHIRST2, and LARS K. POULSEN 3 1 Department of Microbiology, Danish Veterinary Laboratory, Copenhagen V, Denmark Present address: Dept. Gast.-Int. Infect., Statens Serum Institut, Copenhagen S, Denmark 2 Department of Molecular Genetics, Forsyth Dental Center, Boston, Massachusetts 3 Department of Microbiology, BId 221, Technical University of Denmark, Lyngby, Denmark Present address: BioImage, Novo Nordisk AlS, S0borg, Denmark
Received June 24,1998
Summary The aims of this study were to characterize and determine intra species and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus /ignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding I1e-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs. Key words: Actinobacillus pleuropneumoniae - Ribosomal operons - Ribosomal intergenic region 16S rRNA - in situ detection
Introduction Actinobacillus pleuropneumoniae is the etiological agent of contagious porcine pleuropneumonia, causing economical losses in pig industry worldwide (NICOLET, 1992). Previous studies have indicated that strains of A. pleuropneumoniae are closely related. Until recently, two biotypes and 14 serotypes have been described; some of which appear to be nearly identical, except for minor antigenic differences (NIELSEN et al. 1996; NIELSEN et al. 1997; NIELSEN, 1986; NIELSEN, 1988; FODOR et al. 1989). Toxin genotyping has distinguished 5 groups of A. pleuropneumoniae, which correlate to grouping by serotyping (BECK et al. 1994). Restriction endonuclease
fingerprinting of DNA from A. pleuropneumoniae could discriminate among reference strains representing different serotypes, whereas isolates of the same serotype were genetically very similar (MACINNES et al. 1990). Furthermore, multi locus enzyme electrophoresis (MEE) studies have shown a clonal population structure of the species (M0LLER et al. 1992; HAMPSON et al. 1993; MUSSER et al. 1987). A. pleuropneumoniae is closely related to Actinobacillus lignieresii. Based on comparative sequence analysis of their 165 rRNA genes interestingly, these species are nearly indistinguishable (DEWHIRST et al. 1992), whereas DNA-DNA reassociation values of 60 to
Characterization of A. plcuropllcumolliac by sequence analysis
79% (STACKEBRANDT and GOEBEL, 1994; POHL et al. 1983; BORR et al. 1991) and phenotypic characteristics (MANNHEIM, 1984; MUTTERS et al. 1984) discriminates between the two species. A. lignieresii has been isolated from ruminants (PHILLIPS, 1967; PHILLIPS, 1965) and there are to our knowledge no reports of this species isolated from pigs. Both species has been isolated from lambs (KILIAN et al. 1978; PHILLIPS, 1967). In most bacteria, the ribosomal operon consists of the ribosomal genes in the following order: 16S rRNA, 23S rRNA and 5S rRNA, although some exceptions have been reported (TASCHKE et al. 1986; AMIKAN and KUHN, 1987). Comparative sequence analysis of 16S and 23S rDNA ribosomal genes have been useful in describing phylogenetic relatedness among different species (WOESE, 1987; DEWHIRST et al. 1992). The intergenic region between the 16S and 23S genes, termed the ribosomal intergenic sequence (RIS), is generally more variable than the conserved sequences of the rRNA genes (KOSTMAN et al. 1992; JENSEN et al. 1993). Previous studies have shown that RIS often contains one or more tRNA sequences as well as ribonuclease III and RNase P cleavage sites (YOUNG et al. 1979; MORGAN et al. 1977; KING et al. 1986). Other parts of the region are not known to encode a function and are therefore assumed to accumulate random mutations. A new approach to determine clonal variation of species involves analysis of RIS, either by length polymorphism, restriction fragment length analysis (RFLP), or direct sequencing. As an example, a study of length polymorphism of RIS in Burkholderia (Pseudomonas) cepacia (KOSTMAN et al. 1992) and a RFLP study of the RIS of Rochalimaea henselae showed significant strain diversity (MATAR et al. 1993). Sequence analysis of RIS from Actinobacillus actinomycetemcomitans showed that epidemiologically unrelated strains could be easily discriminated (LEYS et al. 1994). Furthermore, a significant level of length and sequence polymorphism across genus, species and subspecies levels has been demonstrated (GURTLER and STANISICH, 1996; GURTLER and BARRIE, 1995; MATAR et al. 1993; JENSEN et al. 1993). To our knowledge, there are no published works on the RIS's of A. pleuropneumoniae and A. lignieresii. In this study, the relatedness of different serotypes and biotypes of A. pleuropneumoniae and the closely related A. iignieresii as well as few strains of the related Actinobacillus suis for comparison, was determined based on sequence analysis of 16S rDNA genes and ribo-
.. 9F
16SrDNA
409
somal intergenic regions. Furthermore, it was the aim to find a species-specific area in the 16S rRNA for detection of A. pleuropneumoniae directly in diseased porcine tissue samples by a fluorescently-labeled oligonucleotide probe.
Materials and Methods Bacterial strains: Twenty-four strains of A. plcuropllcumoniae, four strains of A. lignieresii, and the type strain of A. suis were included in this study (Table 1). The A. pleuropneumoniae strains included the 13 international reference strains, representing the known serotypes of biotype 1, one biotype 2 (NAD-independent) reference strain, and 10 field strains isolated from cases of pleuropneumonia in Denmark during 1987-1993. These Danish field strains represented the 8 serotypes of biotype 1 and 2 strains of biotype 2. Culturing and storage of strains: A. pleuropneumoniae was cultured on PPLO agar plates containing: PPLO agar (DIFCO, Germany), 30 gIl; glucose, 0.1 gIl; horse serum, 5%; and yeast extract 2.5% (NICOLET, 1971), or in Brain Heart Infusion broth (BHI, DIFCO, UK), 37 gil supplemented with 0,1 % NAD incubated at 37°C in atmospheric air. A. lignieresii and A. suis were cultured on 5% calf blood agar plates (Columbia agar base, Oxoid, UK) or in BHI broth incubated at 37°C in atmospheric air. Cultures of all species were stored at -80°C in BHI broth supplemented with 10% glycerol. Isolation of chromosomal DNA, PCR amplification and purification: DNA was extracted as described by Christensen et al. (1993) and amplified using the GeneAmp kit (Perkin-Elmer Cetus, Conneticut). PCR was carried out according to the manufacturers guideline, using 2.5 U of AmpliTaq DNA polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3 mM MgCI 2, deoxyribonucleotides (each at a final concentration of 250 pM), 150 picomol of each of the 2 primers, and 50 ng chromosomal DNA. Primers 9F and 1493R were used for amplification of 16S rDNA, and primers 1337F and 115R that were selected from the adjacent highly conserved areas of 16S and 23S genes were used for amplification of the ribosomal intergenic sequence (Table 2), (LANE, 1991; DEWHIRST et al. 1992). A schematic representation of the ribosomal operon and the position of the above mentioned primers, are depicted in Figure 1. PCR was performed using a hot start technique including ampliwax PCR Gems, according to the manufacturer's guidelines (Perkin Elmer Cetus). All amplifications were performed using a Perkin Elmer DNA thermal cycler, model 480. The following conditions were used for amplification: denaturation at 94°C for 45 sec, annealing at 50 °C for 45 sec, and elongation at 72 °C for 90 sec with 1 additional sec added for each cycle. A total of 25 cycles was performed followed by a final elongation step
RIS
.... .. ..
23SrDNA
5SrDNA
•
1493 R
1337F
1541F
436 R
115 R
Fig. 1. Schematic presentation of a ribosomal operon. Arrows indicate primer site for PCR amplification of the ribosomal intergenic sequence (RIS), 16S and 23S rDNA, and primer site for sequencing of RIS.
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V. FUSSING et al.
Table 1. Bacterial strains investigated. Strain
A. pleuropneumoniae Reference strains ATCC 27008 T (1) ATCC 27089 (2) ATCC 27090 (3) ATCC 33378 (4) ATCC 33377 (Sa) L20 (5b) ATCC 33590 (6) WF83 (7) CCM 3803 (8) CCM 3888 (9) CCM 6279 (10) 56153 (11) CCM 6311 (12) P597 (2)'· Danish strains 2302 (1) 634 (2) 585 (Sa) 1565 (6) 7949 (7) 11498 (8) 658 (10) 761 (12) 785 (-)" 3906 (14)"
Genbank accession No
Source
16S rDNA
Small RIS
Large RIS
pleuropneumonia, pig, 1964, Argentina pleuropneumonia, pig, 1968, Switzerland periarticular abscess, pig, 1968, Switzerland pig with pneumonia, 1978, USA arthritis, lamb, 1978, USA pleuropneumonia, pig, USA pleuropneumonia, pig, 1978, Denmark pleuropneumonia, pig, Canada pleuropneumonia, pig, Ireland pleuropneumonia, pig, Holland pleuropneumonia, pig, 1987, Denmark pleuropneumonia, pig, Holland pleuropneumonia, pig, 1985, Denmark pleuropneumonia, pig, Switzerland
M75074
AF033075 AF033076 AF033078 AF033079 AF033080 AF033080 AF033081 AF033082 AF033083 AF033081 AF033084 AF033085 AF033081 AF033086
AF033061 AF033062 AF033063 AF033064 AF033061 AF033061 AF033063 AF033065 AF033066 AF033061 AF033067 AF033061 AF033068 AF033066
pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig, pleuropneumonia, pig,
M75074"* AF033058 AF033060 AF033060 AF033060 AF033060 AF033059 AF033060
AF033085 AF033087 AF033080 AF033081 AF033082 AF033083 AF033088 AF033089 AF033090 AF033091
AF033061 AF033062 AF033061 AF033061 AF033064 AF033066 AF033069 AF033068 AF033066 AF033070
1993, Denmark 1993, Denmark 1993, Denmark 1993, Denmark 1987, Denmark 1988, Denmark 1991, Denmark 1993, Denmark 1994, Denmark 1993, Denmark
A. lignieresil ATCC 49236 T NCTC4985 P670 P671
glands, cattle, 1933, USA bovine lesions, 193b, England expectorat, human, 1980, Japan expectorat, human, 1980, Japan
M75068
AF033092 AF033092 AF033092 AF033093
AF033071 AF033072 AF033073 AF033077
A. suis ATCC 33415 T
septicemia, pig, 1962, Holland
M75071
AF033094
AF033074
Serotype is indicated in (brackets), -: indicates new serotype, not named (pers. comm. Ragnhild Nielsen). " Indicate biotype 2. "" 16S rDNA sequence identical to previously published 16S rDNA sequence (M75074) of the type strain of A. pleuropneumoniae (7).
ATCC - American type culture collection. NTCT - National collection of type cultures and pathogenic fungi. CCM - Czechoslovak collection of microorganisms. Table 2. Primers used for PCR-amplification and sequencing. at 72 °C for 15 min. The ribosomal intergenic amplicons were separated in a 1 % low melting agarose gel (SeaPlaque, FMC, USA). The DNA fragments were extracted with phenol from the gel as described previously (Maniatis et al. 1987). Cycle sequencing: Direct cycle sequencing of the purified 16S rDNA and RIS were carried out with TAQuence Cycle-Sequencing Kit (United States Biochemical, USA). The oligonucleotide primers used for sequencing of 16S rDNA (344R, 519R, 786R, 1057R, 1337F, and 1493R) and the RIS (1337F, 1541F, 436R and 115R) are listed in Table 2 (LANE, 1991; DEWHIRST et al. 1992). Primers were 5'-end labeled with [y_33P]_ ATP as specified by USB. Approximately 20 ng of PCR product and 2 pmol of the 5' end-labeled primer were used for each sequencing reaction, which was performed as described by the
5'end position of primer
Sequence (5'-3')
9F (16S rDNA) 344R (16S rDNA) 519R (16S rDNA) 786R (16S rDNA) 1057R (16S rDNA) 1337F (16S rDNA) 1493R (16S rDNA) 1541F (16S rDNA) 436R (RIS) 115R (23S rDNA)
AGA GTT TGA T(CT)(CA) TGG CT ACT GCT GCC TCC CGT CAG CAG CCG CGG TAA TAC CTA CCA GGG TAT CTA ATC GGC ACG AGC TGA CGA CAG CC GGA ATC GCT AGT AAT CG TAC GG(Cff) TAC CTT GTT ACG ACT GG(Cff) TGG A(AfT)C ACC TCC TT TTA GCT GCT TTT GTT CAG GGG TT(C/Gff) CCC CAT TCG G
Characterization of A. pleuropneumoniae by sequence analysis manufacturer. Thermocycler parameters were as follows: 50 cycles of 94°C, 30 sec, 55°C, 30 sec, and 72 °C, 90 sec. Sequence analysis: Nucleotide sequences were read manually. Programs used for data entry were written in Microsoft QuickBASIC, and DNASIS was used for editing, sequence alignments, tRNA search, and secondary structure prediction. Fixation of bacterial cells for whole cell hybridization. Bacteria were fixed in 3 % para formaldehyde as previously described (Stahl and Amann, 1991) and kept at -20°C in storage buffer (50% ethyl alcohol, 10 mM Tris pH 7.5, 0.1% Nonidet PAO) until use. Fixation of lung tissue for in situ hybridization experiments. 1 cm 3 tissue from porcine lungs suspected to be infected with A. pleuropneumoniae, was fixed in 3 % formaldehyde at 4 °C for 36 hours (POULSEN et al. 1994). The tissue was dehydrated in ethanol before embedded in paraplast (Prohosp, Denmark) and sliced in thin sections of 3-5 ]lm. Prior to hybridization, the sections were deparaffinized in xylene for 5-10 min at room temperature and subsequently submerged twice in 96% ethanol for 3 min. Oligonucleotide probes. The specificity of the A. pleuropneumoniae probe (Acb186: 5'-CAC CCT TTA ATC CAT AG3') was evaluated against the ribosomal database (release 3.1) by using the CHECK-PROBE program at the Ribosomal Database Project, Illinois (MAIDAK et al. 1997). The probe was labeled with fluorescein at the 3'-end during synthesis (POULSEN et al. 1994). A nearly conserved, universal bacterial probe, Eub338 (STAHL and AMANN, 1991) was synthesized and labeled with rhoda min as previously described (POULSEN et al. 1994). Both probes were subsequently purified by reverse phase liquid chromatography as previously described (KROGFELT et al. 1993). Whole cell hybridization: Bacterial cells from pure cultures were spotted onto 6-well Teflon slides coated with poly L-Iysine (Sigma Chemical, USA) and hybridized by adding 10 ]ll hybridization solution (0.9M NaCl, 0.1 % SDS, O.lM Tris pH 7.2, 25 ng probe) to each well. Slides were kept in a humid chamber for 16 h at 37°C during hybridization (AMANN et al. 1990). The slides were then rinsed in distilled H 2 0, incubated in 100 ml prewarmed washing solution (0.9M NaCl, O.lM Tris) for 15 min at 37°C, rinsed in distilled H 2 0, and air dried. Prior to microscopic analysis, the slides were mounted in Citiflour (Citifluor Ltd., UK). Thin section hybridization: A DAKO pen (DAKO, Denmark) was used to apply a hydrophobic substance around the section in order to keep the hybridization solution on the thin sections. The section was hybridized as described above for whole cell hybridization. The sections were rinsed in ddH 2 0, after which they were incubated in washing solution I (0.9 M NaCl, 0.1 % SDS, 0.1 M Tris pH 7.2) for 20 min at 37°C, followed by incubation in washing solution II (0.9M NaCl, 0.1 M
411
Tris) for 15 min at 37°C. Finally, the sections were rinsed in ddH 2 0 at room temperature. Microscopy and image analysis: An axioplan epi-fluorescence microscope (Carl Zeiss, Germany) was used to visualize the bacteria as previously described (PASTER et al. 1996). The microscope was fitted with a slow scan CCD (Charged Coupled Device) camera for capturing digitized images (PASTER et al. 1996). For image analysis, the bit range of interest was linearly scaled to 8 bit files in the PMIS software (version 2.11, Photometrics, USA) and exported to Photos hop (Adobe, USA) for final analysis. Genbank accession numbers: The sequence data are available from the GenBank, USA and the accession numbers for all strains sequenced are listed in Table 1.
Results 16S rDNA sequencing Sequence analysis of 16S rDNA genes, position 25 to 1493, from the eight Danish strains revealed only minor differences compared with the type strain ATCC 27088 (2 to 5 bases out of the 1468 bases sequenced). The type strain (serotype 1) of A. pleuropneumoniae and the Danish serotype 1 strain had identical sequences. The Danish serotypes 5, 6, 7, 8, and 12 had identical 16S rDNA sequences, while the Danish strains of serotypes 2 and 10 each had unique sequences (Table 3). Comparison of 16S rDNA sequences from the type strain of A. lignieresii (DEWHIRST et al. 1992) with the type strain of A. pleuropneumoniae showed differences in 3 positions. Two of these were located next to each other at position 188 and 189 (Table 3). Sequencing of RIS PCR amplification of RIS with the universal primers 1337F and 115R resulted in two amplicons of 400-440 and 520-550 base pairs, showing the existence of at least two different ribosomal operons in all strains investigated. The RIS of the type strains of A. pleuropneumoniae, A. lignieresii, and A. suis, are shown in Table 4 and 5. In all three species, the smaller RIS contained a Glu-tRNA gene (92% homology to Glu-tRNA gene of E. coli), and the larger RIS contained two tRNA genes, Ile-tRNA and Ala-tRNA gene (94% and 92% homology, respectively, to tRNA genes of E. coli). Both amplicons contained a
Table 3. Base differences in 16S rDNA between the type strain of A. pleuropneumoniae ATCC 27088\ eight Danish strains of A. pleuropneumoniae and the type strain of A. lignieresii, ATCC 49236 T (DEWHIRST et al. 1992). Bacterial strains
A. A. A. A. A. A.
pleuropneumoniae ATCC 27088 T pleuropneumoniae Serotype 1 pleuropneumoniae Serotype 2 pleuropneumoniae Serotypes 5, 6, 7, 8, 12 pleuropneumoniae Serotype 10 lignieresii ATCC 49236 T
Base differences in 16S rDNA sequences at position (E. coli numbering) 80
188
189
257
264
269
838
843
845
1451
G G A G G G
A A A A A T
T T T T T A
T T T T A T
A A A A C A
G G G G T G
G G A A A A
T T G G G T
A A G G G N
A A A A A C
412
V.
FUSSING
et al.
Table 4. Ribosomal intergenic sequences of the smaller PCR fragments from the type strains of A. pleuropneumoniae, A. lignieresii and A. suis. Ribonuclease III cleavage site (Rn, III cl.site) and Glu-tRNA are marked below the sequence. The Glu-tRNA sequence of Escherichia coli are shown. 1
2
3
4
A.p A.l A.s
CCAGAAATTG AGCGACASSG GTGTTCACAC AGATTGTTTC ATGAATGAAT GAGCGTTTAG . . . . . . . . . . . . . . . . . . . . . TGTTCACAC AGATTGTTTG.ATGAATGAAT GAGCGTTTAG . . . . . . . . . . . . . . . . . . . . GTGTTCACAC AGATTGTTTG ATGAATNAAT AAGCGTTTGC 1---Rn.III cl.site--*--I * **
A.p A.l A. s
GTTAAACGAG TCGGAAAGCG ATTGAAAAAC GGCACGATTT AACGTAAAAT ATTGCGCTCA GTTAAACGAG TCGGAAAGCG ATTGANAAAC GGCACGATTT AACGTAAAAT ATTGCGCTCA GTTAGATGGN GANANAAGTT ATTNNNAAAT AACGNAATTT AACGTAGAAT AATGCGCTCA * ** * * ** * * * ** * * *
A.p A.l A.s
AATGGCAAAG TGGAGAGCAT CTTTAAATGT TGTCCCCATC GTCTAGAGGC CTAGGACATC AATGGCAAAG CGGAGAGCAT CTTTAAATGT TGTCCCCATC GTCTAGAGGC CTAGGACATC AATGGCAAAG TGGAGAGCAT CTTTNAATGT TGTCCCCATC ~TCTAGAGGC CTAGGACATC
5
6
7
8
9
10
1------------------------------
* E.c: A.p A.l A.s E.c:
GTCCCCTTC GTCTAGAGGC CCAGGACACC NCCCTTNNAC GGCGGTAACC GGGGTTCGAA TCCCCGTGGG GACGCCATTT AAAGATGACT GCCCTTTCAC GGCGGTAACC GGGGTTCGAA TCCCCGTGGG GACGCCATTT AAAGATGACT GCCCTTTCAC GGCGGTAACC GGGGTTCGAA TCCCCGTGGG GACGCCATTT AAAGATGACT ----Glu-tRNA--------------------------------------1 GCCCTTTCAC GGCGGTAACA GGGGTTCGAA TCCCCTGGGG GACGCCA 11
A.p A.l A.s
A.p A.l A.s
A.p A.l A.s
-------14--------------
---------CAAGTTCGTT ---------**********
----CGAAAG AAAGCGAAAG -----GAAAG *****
------17------------GTAATTATCT ------------------********** 18
A.p A.l A.s
12
13
-
TTTGTTGTCT GAATTGTTCT TTAAAAAATT GGAAACAAGC TGAAAA-CTG AGAGATT--TTTGTTGTCT GAATTGTTCT TTAAAAAATT GGAAACAAGC TGAAAA-CTG AGAGATTTTT TTTGTTGTCT TAATTGTTCT TTNAAAAATT GGAAACAAGC TGAAAAACTG AGAGATTTTN *** * *
TGAAAATCTT ---AAATCTT --AAAATCTT ***
------------15---------------------TGCTAACGAA ---------**********
16
-------AAA GTCTGAGTAG TAAAAGATAA TACATTGAAA GTCTGAGTAG TTAAA-----------AAA GTCTGAGTAG TTAAA----******* ***** *
AGCTGAACAA AAGCAGCTAA GTGTTTAGTT GAATAAAGTA AGCTGAACAA AAGCAGCTAA GTGTTTAGTT GAATAAAGTA GACTGAACAA AAGCAGTCAA GTGTTTNGTT GAATAAAATA ** ** *
19
TCGCGTTGAA TGCGTTCAAA TAAAATTTNA AAATATTTGA TCGCGTTGAA TGCATTCAAA TAAAATTTGA AAATATTTGA CAGCATTGAA TGGATTTGAA TAAGATTTGA AAA------** ** ******* ** * *
AAACATTTGA GGTTGTAT AAACATTTGA GGTTGTAT ---CATTTGA GGTTGTAT ***
,. indicates differing base positions within these sequences. Small numbers above bases indicate variability among A. pleuropneumoniaestrains specified below. Danish serotype 12 strain: C All strains except reference serotype 1: G One Danish biotype 2 (785): G All serotype 10 strains Danish serotype 2 and all serotype 5 strains: T One Danish biotype 2 strain (785), one base insert: C Reference serotype 3 strain: A All serotype 5 strains: G Reference serotype 3 strain: T Danish serotype 1 strain and reference serotype 11 strain: C 11 - All biotype 2 and serotype 7 strains: A 1 2 3 4 5 6 7 8 9 10
-
12 - Reference serotype 10 strain, one base deletion 13 - All strains except reference serotype 1 strain, two bases insert: TT 14 - One Danish biotype 2 strain (3906), 15 bases insert: TCTAGTTTGTTANAT 15 - One Danish biotype 2 strain (3906), 17 bases insert: TGATAGCGAATATATTG 16 - All serotype 2,7 and 8 and 2 biotype 2 strains (785, P597): T 17 - All serotype 2, 7 and 8 strains and 2 biotype 2 strains (785, P597: 18 bases deletion 18 - All serotype 2 m 3, 4 and 7 strains: A 19 - All serotype 10 strains: A
Table 5. Ribosomal intergenic sequences of the larger peR fragments from A. pieuropneumoniae, A. lignieresii and A. suis. Ribonuclease III cleavage site (Rn.III cl.site), IIe-tRNA and Ala-tRNA are marked below the sequence. The Ile-tRNA and Ala-tRNA sequences of Escherichia coli are shown.
A.p A.1 A.s
CCAGAAATTG AGCGACASSG GTGTTCACAC AGATTGTTTG ATAGAA-AGA AGACGAAAAC . . . . . . . . . . . . . . . . . . . . GTGTTCACAC AGATTGTTTG ATAGAN-AGA NGACGAAAAC GTGTTCACAC AGATTGTTTG ATAGAAGAGA AGACGAAAAC
*
1----Rn.III c1.site----1 1
A.p A.1 A.s
GGATATAATC CGGCATCCTT TTGGGTCTGT AGCTCAGGTG GTTAGAGCGC ACCCCTGATA GGATATAATC CGACATCCTT TTGGGTCTGT AGCTCAGGTG GTTAGAGCGC ACCCCTGATA GGACATNATC CGGCATCCTT TTGGGTCTGT AGCTCAGGTG GTTAGAGCGC ACCCCTNATA
*
E.c
1------------------------------I1e-tRNA--
*
AGGCTTGT AGCTCAGGTG GTTAGAGCGC ACCCCTGATA 1
2
A. P A.1 A. s
AGGGTGAGGT CGGTGGTTCA AGTCCACTCA GACCCACCAC TCAAAACGAG TGAAAGCTGA AGGGTGAGGT CGGTGGTTCA AGTCCACTCA GACCCACCAC TCAAAACGAG TGAAAGCTGA AGGGTGAGGT CGGTGGTTCA AGTCCACTCA GACCCACCAC TCTAAACGAG TNANAGCTNA
E.c
AGGGTGAGGT CGGTGGTTCA AGTCCACTCA GGCCTACCA
A.p A.1 A. s
AAGGTGTGAT TATATTGAGT TATGATGTAT GGGGATATAG CTCAGCTGGG AGAGCGCCTG AAGGTGTGAT TATATTGAGT TATGATGTAT GGGGATATAG CTCAGCTGGG AGAGCGCCTG NAGGTGAGAT TATGTTCAGT CATGATGTAT GGGGATATAG CTCAGCTGGG AGAGCGCCTG
-----------------------------------------1
*
3
*
E.c
* *
1-------------------------------
*
GGGGCTATAG CTCAGCTGGG AGAGCGCCTG
4
A. P A.1 A. s
CCTTGCACGC AGGAGGTCAG CGGTTCGATC CCGCTTATCT CCACCAATCA TCATGACTAA CCTTGCACGC AGGAGGTCAG CGGTTCGATC CCGCTTATCT CCACCAATCA TCATGACTAA CCTTGCACGC AGGAGGTCAG CGGTTCGNTC CCGCTTNTCT CCACCAATCA TCATGACTAA
E.c
CTTTGCACGC AGGGGGTCTG CGGTTCGATC CCGCATAGCT CCACCA
A.p A.1 A.s
GTGAATAAGT ------GAAA GAT---TTGT TTNTTTAGTC ATGATGATAA GTCAAATTNT GTGAATAGAT ATTGTTGATA GATATTTTGT TTATTTAGTC ATGATGATAA GTCAAATTAT GTGAATAAG- -----TNNAA GAT---TTGT TTATTTAGTC ATGATGATAA GTCAAATTAT
---Ala-tRNA--------------------------------------1 5
*** ****** A.p A.1 A.s
*
***
TGTCTTNAAT TGTTCTTTNA AAAATTGGAA ACAAGCTGAA AA-CTGAGAG ATTTTCGAAA TGTCTTAAAT TGTTCTTTAA AAAATTGGAA ACAAGCTGAA AA-CTGAGAG ATTTTTCAAG TGTCTTAAAT TGTTCTTTAA AAAATTGGAA ACAAGCTGAA AAACTGAGAG ATTTTCGAAA
**
* A.p A.1 A.s
6 ------------------------7 -------------------G--AAAG TCTGAGTAGT AAAAGATA-- -----AGT-- AATTNTCTTG AAAATCTTAG TTCGTTAAAG CGAAAGTGCT AACGAATACA TTGAAAGTCT GAGTAGTTAA AAAATCTTAG ---G--AAAG TCTGAGTAGT TAAA------ ---------- ---------- AAAATCTTGA
*** **
****
**
* ******** ********** ********** 8
A.p A.1 A.s
** 9
CTGAACAAAA GCAGCTAAGT GTTTAGTTGA ATAAAGTATC GCGTTGAATG CGTTCAAATA CTGAACAAAA GCAGCTAAGT GTTTNGTTGA ATAAAGTATC GCGTTGAATG CATTCAAATA CTGAACAAAA GCAGTCAAGT GTTTAGTTGA ATAAAATACA GCATTGAATG GATTTGAATA
** A.p A.1 A.s
*
* **
*
**
**
AAATTTGAAA ATATTTGAAA ACATTTGAGG TTGTAT AAATTTGAAA ATATTTGAAA ACATTTGAGG TTGTAT AGATTTGA-- --------AA ACATTTGAGG TTGTAT
*
** ********
,:- Indicates differing base positions within these sequences. Small number above bases indicate variability among A. pleuropneumoniae strains specified below. 1 2 3 4 5
-
Reference serotype 7 strain: A Danish biotype 2 strain (3906): A All serotype 12 strains: G Danish serotype 10 strain: G All serotype 2 strains: A
6 - All serotype 2, 4, 7 and 8 strains and 2 biotype 2 strains (785, P597): T 7 - All serotype 2, 4, 7 and 8 strains and 2 biotype 2 strains (785, P597), 18 bases deletion 8 - All serotype 2, 3, 4 and 7 strains and reference serotype 6: A 9 - All serotype 10 strains: A
V.
414
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et al.
region resembling the ribonuclease III cleavage site found in E. coli (KING et al. 1986). The putative ribonuclease III cleavage site and tRNA encoding regions were highly conserved among the three species in contrast to the remaining regions, which showed variations as indicated by asterisk in Table 4 and 5. The number of base differences, insertions and deletions in the larger RIS easily differentiated the type strains of A. pleuropneumoniae, A. lignieresii, and A. suis, as A. pleuropneumoniae and A. lignieresii differed at 30 positions, A. pleuropneumoniae and A. suis differed at 36 positions and finally A. lignieresii and A. suis differed at 55 positions (Figure 2). The smaller RIS showed less variability among type strains of A. pleuropneumoniae and A. lignieresii as the strains only differed at six positions (Table 4). Relatively few in-
traspecies differences were found among RIS's of the 24 A. pleuropneumoniae strains investigated as only one base differences in either the smaller or larger RIS discriminated the majority of strains (Table 4 and 5). The larger RIS showed the less variability as only 10 different sequences was found. The smaller RIS showed greater intraspecies variability with findings of 16 different sequences among the 24 strains of A. pleuropneumoniae (Figure 3).
In situ hybridization of A. pleuropneumoniae Probe Ap186 targeted the region in 16S rRNA where two differences was found between A. pleuropneumoniae and A. lignieresii (position 186-202, E. coli number-
A.lignie il A. pleuropneumoniae A. suis 100% Homology 1-10 base difference
11-20 base difference 21-30 base difference 31-40 base difference 41-50 base difference 51-60 base difference
Sequence I Sequence 2 Sequence 3 Sequence 4 Sequence S Sequence 6 Sequence 7 Sequence 8 Sequence 9 Sequence 10 Sequence II Sequence 12 Sequence 13 Sequence 14 Sequence 15 Sequence 16 Sequence I: equence 2: Sequence 3: Sequence 4: equence 5: Sequence 6: Sequence 7: Sequence 8: Sequence 9:
Fig. 2. Matrix ba ed on the number of ba e difference in the larger RI of the type train of A. plellropnellmoniae, A. lignieresii and A. sllis.
100% Homology
1 base difference 2 b se difference 3 base difference 4 base difference 5 base difference 6 base difference 7 base difference 8 base difference Dani h biotype 2 strain, unknown serotype Biotype 2 strain, serotype 2 AII serotype 8 strains Reference strain serotype 2 AII serotype 7 strains Danish strain, serotype 2 Reference strain serotype 4 Reference strain serotype 3 Reference strains serotypes 6, 9, and 12 and a Danish strain, serotype 6
equence 10: Danish strain, serotype 12 equence 11 : Reference strain serotype II and a Danish strain, serotype t Sequence 12: Reference strain serotype 1 Sequence 13: All serotype S strains Sequence 14: Danish biotype 2 strain, serotype 14 equence 15: Danish strain, serotype 10 Sequence 16: Rererence strain serotype 10
Fig. 3. Matrix based on the number of base differences in the smaller RIS of strains of A. pleuropneumoniae.
Characterization of A. pleuropneumoniae by sequence analysis
ing). This region was found to be unique to A. pleuropneumoniae by comparisons to the published 165 ribosomal sequences. Whole cell hybridization of probe Ap186 labeled with fluorescein hybridized only to cells of A. pleuropneumoniae ATCC 27088\ and not to cells of A . lignieresii ATCC 49236 T or A. suis ATCC 3341ST. To demonstrate probe specificity, fixed cells of A. pleuropneumoniae ATCC 27088 T and A. lignieresii ATCC
A
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49236 T were mixed and hybridized with probe Ap186 labeled with fluorescein and the universal probe Eub338 labeled with rhodamine. As shown in Figure 4, all bacterial cells hybridized with probe Eub338, but only cells of A. pleuropneumoniae hybridized with probe Ap186. Cells of A. pleuropneumoniae were detected directly in biopsies of diseased porcine lungs and confirmed by culturing. The lung biopsy (Figure SA) showed lesions typi-
B
Fig. 4. Whole cell hybridization of a mixed suspension of A. pleuropneumoniae and A. lignieresii with a mixture of Eub338 rhodamine labeled probe and Ap186 fluorescein labeled probe. (A) Fluorescence specific for rhodamine detection (B) Fluorescence specific for fluorescein detection. Ap186 hybridized only with A. pleuropneumoniae.
Fig. 5. In situ detection of A. pleuropneumoniae in porcine lung tissue section. (A) Differential interference contrast microscopy image of section. Boxed area is shown in B; epifluorescence microscopy of A. pleuropneumoniae detected with Ap 186.
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cal of A. pleuropneumoniae infections including fibrinous pleuritis and necrotic and hemorrhagic lung lesions (INZANA, 1991). Bacterial cells within the tissue hybridized with probe Ap186 and often appeared as distinct colonies (Figure 5B).
Discussion The structural and conserved properties of the ribosomal operon make this region a valuable tool for phylogenetic studies of both inter- and intraspecies relationships (GURTLER and STANISICH, 1996; WOESE, 1987) and this region was chosen for further characterization of strains of A. pleuropneumoniae and A. lignieresii. Several studies have shown that strains of A. pleuropneumoniae are closely related and difficult to discriminate (NIELSEN, 1988; BECK et al. 1994; MACINNES et al. 1990; M0LLER et al. 1992). These findings were supported by comparisons of the 16S rDNA sequences of Danish strains representing eight different serotypes of A. pleuropneumoniae (Table 3). Despite preferences for different hosts, except a few cases of isolation of A. pleuropneumoniae from lambs, and differences in phenotypic traits, interestingly, sequences of 16S rDNA from A. pleuropneumoniae and A. lignieresii are only differing at three base positions. RIS represents a valuable tool for determining relatedness of bacterial strains and closely related species (GURTLER and BARRIE, 1995; LEYS et al. 1994) and might furthermore represent a useful typing method for epidemiological studies. Amplification of the region between 23S and 16S rDNA from A. pleuropneumoniae, A. lignieresii and A. suis showed the existence of at least two different ribosomal operons. Sequencing of these amplicons revealed the presence of t-RNA's and ribonuclease III cleavage sites (Table 4 and 5). The smaller amplicon contained a Glu-tRNA gene, while the sequence of the larger RIS contained both an Ala-tRNAand an Ile-tRNA gene. The regions encoding these genes were highly conserved, while the other parts exhibited more variability. tRNA genes and a ribonuclease III cleavage site are present in the RIS of all 7 ribosomal operons of E. coli (KING et al. 1986). Furthermore, the sequences flanking the 16S and 23S in E. coli are highly conserved with long inverted repeats, providing the potential of forming a double helix structure where initial cleavage with ribonuclease III can occur (KING et al. 1986). This could also be the case for the ribosomal operon of A. pleuropneumoniae, although some variability at the ends of the A. pleuropneumoniae RIS was observed (Table 4 and 5). In order to examine the relatedness of strains of A. pleuropneumoniae, the RIS of 24 strains were analyzed. The smaller RIS showed the greatest intra species variability (Figure 3), although both the smaller and larger amplicon demonstrated a marked degree of genomic homology among strains of A. pleuropneumoniae. By a combination of the variability of the two RIS it might be possible to develop a typing system for A. pleuropneumoniae similar to that described for A. actinomycetemcomitans, in which bacteria are detect-
ed and strains identified by simple peR reactions and sequencing procedures (LEYS et al. 1994). The three species were clearly discriminated on basis of the larger RIS (Figure 2). Less differences were found among A. pleuropneumoniae and A. lignieresii based on the smaller RIS. Both results are consistent with previous findings; a high relatedness of A. pleuropneumoniae and A. lignieresii based on 16S rRNA genes (DEWHIRST et al. 1992), and a clear discrimination of the two species based on DNA-DNA hybridization (BORR et al. 1991; POHL et al. 1983). Sequence analysis of 16S rDNA genes of A. pleuropneumoniae revealed a unique and conserved region of the gene, which could serve as target for a DNA oligonucleotide probe. The fluorescently labeled probe was demonstrated to discriminate A. pleuropneumoniae from the closely related A. lignieresii in whole cell hybridizations (Figure 4). The applicability of the probe was confirmed by readily detection of cells of A. pleuropneumoniae directly in infected lung tissue (Figure 5). This procedure was simple and results was obtained within 2 working days, with the protocol presented in this study. In conclusion, strains of A. pleuropneumoniae were found to be genetically closely related based on analyses of the ribosomal operons. The RIS of A. pleuropneumoniae, A. lignieresii and A. suis contained genes encoding t-RNA's and ribonuclease III cleavage sites as has been found in E. coli. The closely related A. pleuropneumoniae and A. lignieresii could be discriminated by the larger RIS, whereas the smaller RIS showed a higher degree of similarity between the two species. Finally, a species-specific probe for A. pleuropneumoniae targeting the 16S rDNA was used for in situ detection of this species in porcine lung tissue. Acknowledgement This work was supported in part by Public Health Service grants DE-08303 and DE-10374 from the National Institute of Dental Research and by grants from The Danish Veterinary Laboratory and Cowi Consult NS, Denmark. We thank Annie Ravn, Vila Andreasen, Gayle Lafleche, and Jennifer Belcher for technical assistance. We are grateful for the kind donation of A. pleuropneumoniae strains by Dr. R. NIELSEN, and strains of A. lignieresii and A. suis by Dr. W. Frederiksen and Dr. M. Bisgaard.
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YOUNG, R. A., MACKLIS, R., STEITZ, J. A.: Sequence of the 16S23S spacer region in two ribosomal RNA operons of Escherichia coli. J. BioI. Chern. 254, 3264-3271 (1979). Corresponding author: VIVIAN FUSSING, Dept. of Gastrointestinal Infections, Statens Serum Institut, Artillerivej, DK-2300 Copenhagen S, Denmark Phone: +45-3268 3644, Fax: +45-3268 8124, e-mail:
[email protected]