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Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates
Veterinary Microbiology 152 (2011) 205–211
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Short communication
Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates L.M. Marques a,b, M. Buzinhani a, A.M.S. Guimaraes a, R.C.P. Marques a, S.T. Farias c, R.L. Neto a, M. Yamaguti a, R.C. Oliveira a, J. Timenetsky a,* a b c
Instituto de Cieˆncias Biome´dicas, Departamento de Microbiologia, Universidade de Sa˜o Paulo, Brazil Instituto Multidisciplinar em Sau´de, Nu´cleo de Tecnologia em Sau´de, Universidade Federal da Bahia, Brazil Centro de Cieˆncias Exatas e da Natureza, Departamento de Biologia Molecular, Universidade Federal da Paraı´ba, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2010 Received in revised form 7 April 2011 Accepted 8 April 2011
Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Ureaplasma diversum Intraspecific variation 16S rRNA gene
1. Introduction Ureaplasmas colonize the respiratory and urogenital tract of animals and humans. These organisms were initially isolated from the reproductive tract of a bovine by Taylor-Robinson et al. (1967); 11% of clinically healthy cows had ureaplasmas in their reproductive tract. Subsequent studies on detection of these organisms identified several isolates in the genital tract of males and females without reproductive problems. For this reason, these organisms were initially considered non-pathogenic. Nevertheless, Doig et al. (1980a,b) experimentally infected
* Corresponding author at: Instituto de Cieˆncias Biome´dicas, Av. Professor Lineu Prestes, 1374, CEP 05508 900, Sa˜o Paulo, SP, Brazil. Tel.: +55 11 3091 7297; fax: +55 11 3091 7354. E-mail address: [email protected] (J. Timenetsky). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.04.007
cows and observed granular vulvovaginitis. Similar studies with U. diversum strains inoculated in the mammary gland of sheep and cattle have indicated variable virulence in different strains, justifying the presence of ureaplasmas in healthy animals (Ball and Mackie, 1985; Howard et al., 1973). Bovine and human ureaplasmas were initially differentiated through serology identification (Ogata et al., 1979). Sequencing of the 16S rRNA gene provided basis for their classification later on. Organisms isolated from bovine were named U. diversum (Mycoplasmataceae family, Mycoplasmatales order). Ureaplasma are considered more recently evolved than genera Acholeplasma and Spiroplasma and have one or two copies of the 16S rRNA gene. The complete genome sequences of U. parvum and U. urealyticum have two copies of 16S rRNA each. Robertson et al. (2002) sequenced the 16S rRNA, urease and multiple-banded antigen gene sequences from all 14 serotypes of U. urealyticum associated with repiratory and
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reproductive diseases in humans. Then these mollicutes were reclassified in two species: U. urealyticum and U. parvum. Accordingly, the aim of this study was to sequence and analyze the 16S rRNA partial sequences of different isolates of U. diversum recovered from bovine.
DNA sequences was constructed using the ClustalW (Thompson et al., 1997). A phylogenetic tree was obtained using the neighbor-joining method with Tajima–Nei correction. The data set was resampled 1000 times to generate bootstrap values.
2. Materials and methods
3. Results
2.1. Ureaplasma strains and culture conditions
3.1. Intra-specific analysis of the 16S rRNA gene
Ureaplasma diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. The field isolates comprised 24 ureaplasmas isolated from the vulvovaginal mucus of female cattle with reproductive alterations and 10 ureaplasmas isolated from bovine semen. Females were from seven farms from four different States in Brazil, including Sao Paulo (Farm 1: strains A203, 64 and 59; Farm 2: Alva, Gota, 98, 91, 93, 94, 95, 100 and 102; Farm 4: 77, 84, 72, 73 and 74; Farm 7: 19V, 16V, 7V and 9V), Mato Grosso do Sul (Farm 3: 1664 and 805), Minas Gerais (Farm 6: Ud11, Ud18 and Ud3), and Alagoas (Farm 5: 112). The sampling was conducted from 1999 to 2005. The semen isolates were obtained from a previous study (Marques et al., 2009), all from five different Artificial Insemination Centers from Sao Paulo State. These organisms were identified as U. diversum using a previously described PCR (Cardoso et al., 2000). These organisms were cultivated in UB liquid medium (Ruhnke, 1994) at 37 8C for 2 days.
The complete sequence of the 16S rRNA of U. diversum (GenBank accession number D78650) was compared to the sequences obtained in the present study (Harasawa and Cassell, 1996) using the multiple sequence alignment tool ClustalW. A fragment containing four hypervariable regions were selected for the sequence variation analyses according to Harasawa and Cassell (1996). This fragment comprised the nucleotides between positions 99 to 694. Nucleotide (Adenine, A; Cytosine, C; Guanine, G; Thymine, T) polymorphisms were identified based on their position within the 16S rRNA gene and indicated as Y = T/C, R = A/G, M = A/C, K = G/T, W = A/T and S = G/C (International Union of Biochemistry and Molecular Biology, IUBMB). Forty-four single nucleotide polymorphisms (SNP) were detected (Table 1). Deletions and insertions were only observed in the isolate 112, from Farm 5. The transversion A/C (M) was found in most of the isolates at positions 160 and 238 compared to the GenBank reference sequence and the reference strains sequenced in this study. Interestingly, the semen isolates S1, S6, S7 and S8 showed a transversion A/T (W) at these positions. SNPs were also observed for most of the isolates at positions 295 (18/36, 50%), 298 (33/36, 91.7%), 406 (28/36, 77.8%), 448 (25/36 69.4%), 459 (26/36 72.2%), 515 (33/36 91.7%) and 534 (34/36 94.4%). Unique SNPs were observed in isolates from the same farms as shown in Table 1. SNPs in positions 133, 212 and 213 were restricted to isolates from farm ‘‘TOURO2’’, P6 and P7. Isolates from farm P1 and P3 showed distinct polymorphic patterns when compared. Transversions were observed in 33 nucleotide positions, whereas transitions were observed in 21 nucleotide positions. Transversion A/T (W) and transition A/G (R) were observed in 11 nucleotide positions each; transition T/C (Y) was observed in 10 nucleotide positions and transversions G/C (S) and G/T (K) were observed in 9 nucleotide positions. Transversion A/C (M) was observed only in 4 nucleotide positions. The average distance between the strains was 0.0966. Surprisingly, isolate 112 showed a distance of 0.2280. The reference strains and the isolates showed minimum similarity of 96%, except for isolate 112, which showed 88% similarity. A 0.03 distance cut-off was used in the intra-specific analysis for cluster definition, implying a similarity of at least 97% between isolates (Fig. 1). The dendogram showed 7 clusters, A to G. Cluster A comprised 18 isolates from four farms: two from the Sao Paulo State (Farm 2 and Farm 7), two from Mato Grosso do Sul State (Farm 3) and one from Minas Gerais State (Farm 6). Cluster B comprised the isolate A203 from Farm 1 and cluster C comprised the semen isolates from farm TOURO2. The
2.2. DNA extraction and PCR for the amplification of the 16S rRNA Cultures of ureaplasma in 200 ml of UB medium were used for DNA extraction according to the method described by Boom et al. (1990). The specific primer set F16S and R16 amplifies nearly the entire 16s rDNA sequence of Mollicutes and was used as described by Harasawa et al. (1996). PCR products (10 ml) were then electrophoresed in 1% agarose gel with 10 mg/ml of ethidium bromide and visualized under UV light (Vilber Lourmat, Germany). 2.3. DNA purification, quantification and sequencing After confirmation of the only band in the electrophoresis, PCR products were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham) and quantified using a Low Mass DNA Ladder (Invitrogen). Purified PCR products were sequenced according to the MegaBACE 1000 protocol, using the DYEnamic ET Dye Terminator kit (with Thermo Sequenase II DNA Polymerase). Sequences were analyzed in the Sequence Analyzer software, Base Caller Cimarron 3.12. 2.4. Phylogenetic analyses The 16S rRNA gene sequences were initially compared to sequences deposited in the GenBank databases using the BLASTn algorithm (Benson et al., 2009). Phylogenetic analyses were performed using MEGA4 software (Tamura et al., 2007). Briefly, a multiple sequence alignment of the
Table 1 Polymorphism and differences in the 16S rRNA gene sequence of U. diversum isolates, reference strains and U. diversum 16S rRNA GenBank sequence. Position 118
119
133
134
159
160
162
171
177
196
197
212
213
238
249
253
255
284
285
295
298
299
300
314
345
GenBank 5T 7T 7V 9V 10T 13T 15T 16V 18T 19V 59 64 72 73 74 77 84 91 93 94 95 98 100 102 112 805 1664 49782 49783 A203 Alva Gota S1 S6 S7 S8 Ud3 Ud11 Ud18
D78650 1437325/5T 1437325/7T 1437325/7V 1437325/9V 1437325/10T 1437325/13T 1437325/15T 1437325/16V 1437325/18T 1437325/19V 1437325/59 1437325/64 1437325/72 1437325/73 1437325/74 1437325/77 1437325/84 1437325/91 1437325/93 1437325/94 1437325/95 1437325/98 1437325/100 1437325/102 1437325/112 1437325/805 1437325/1664 1437325/82 1437325/83 1437325/A203 1437325/Alva 1437325/Gota 1437325/S1 1437325/S6 1437325/S7 1437325/S8 1437325/Ud3 1437325/Ud11 1437325/Ud18
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
T W W – – W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A R R – – R R R – R – – – – – R R R – – – – – – – – – – – – – – – – – – – – – –
C – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – Y Y Y
A M M M M M M M M M M W W M M M M M M M M M M M M M M M – – M M M W W W W M M M
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S – – – – – –
C – – – – – – – – – – – – – – – – – – – – – – – – Y – – – – – – – Y Y Y Y – – –
T – – – – – – – – – – – – – – Y – Y – – – – – – – – – – – – – – – Y Y Y Y – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
A R R – – R R R – R – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
G K K – – K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A M M M M M M M M M M W W M M M M M M M M M M M M M M M – – M M M W W W W M M M
C – – – – – – – – – – – – – – – – – – – – – – – – Y – – – – – – – Y Y Y Y – – –
A – – – – – – – – – – – – – – – – – – – – – R R R – – – – – – R – – – – – – – –
T – – – – – – – – – – – – – – Y – Y – – – – – – – – – – – – – – – Y Y Y Y – – –
A R R – – R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
G R R – – R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
A – – W W – – – W – W – – – – – – – W W W W W W W – W W – – – W W – – – – W W W
C Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y – Y Y – – Y Y Y Y Y Y Y Y Y Y
T Y Y – – Y Y – Y – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
C Y Y – – Y Y – Y – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A – – – – – – – – – – – – – – – – – – – – – W W W – – – – – – – W – – – – – – –
C – – Y Y – – – Y – Y – – – – – – – Y Y Y Y Y Y Y – Y Y – – – Y Y – – – – Y Y Y
Strain
Accession number
Position 355
394
401
404
405
406
408
416
417
427
428
439
444
448
459
463
473
487
488
499
500
515
529
531
534
GenBank 5T 7T 7V
D78650 1437325/5T 1437325/7T 1437325/7V
T – – –
C Y Y –
T – – K
G – – R
A R R –
G R R R
A M M –
T W W –
A R R –
T – – –
A – – –
C – – –
A – – –
A – – W
A W W W
T – – K
C S S –
T W W –
A W W –
G S S –
T K K –
G – – S
T K K –
G K K –
G R R K
207
Accession number
L.M. Marques et al. / Veterinary Microbiology 152 (2011) 205–211
Strain
208
Table 1 (Continued ) Accession number
Position 355
394
401
404
405
406
408
416
417
427
428
439
444
448
459
463
473
487
488
499
500
515
529
531
534
9V 10T 13T 15T 16V 18T 19V 59 64 72 73 74 77 84 91 93 94 95 98 100 102 112 805 1664 49782 49783 A203 Alva Gota S1 S6 S7 S8 Ud3 Ud11 Ud18
1437325/9V 1437325/10T 1437325/13T 1437325/15T 1437325/16V 1437325/18T 1437325/19V 1437325/59 1437325/64 1437325/72 1437325/73 1437325/74 1437325/77 1437325/84 1437325/91 1437325/93 1437325/94 1437325/95 1437325/98 1437325/100 1437325/102 1437325/112 1437325/805 1437325/1664 1437325/82 1437325/83 1437325/A203 1437325/Alva 1437325/Gota 1437325/S1 1437325/S6 1437325/S7 1437325/S8 1437325/Ud3 1437325/Ud11 1437325/Ud18
– – – – – – – – – K K K K K – – – – – – – – – – – – – – – – – – – – – –
– Y Y Y
K – – – K – K – – – – – – – K K K K K K K – – – – – – K K – – – – K K K
R – – – R – R – – – – – – – R R R R R R R – – – – – – R R – – – – R R R
– R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
R R R R R R R – – R R R R R R R R R R R R R R R – – R R R – – – – R R R
– M M M – M – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
– – – – – – – W W W W W W W – – – – – – – – – – – – – – – W W W W – – –
– – – – – – – R R R R R R R – – – – – – – – – – – – – – – R R R R – – –
– – – – – – – – – M M M M M – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – W W – – – – – – – – – – – – – – – – – – – – W W W W – – –
W – – – W – W – – W W W W W W W W W W W W W W W – – – W W – – – – W W W
W W W W W W W – – – – – – – W W W W W W W W W W – – – W W – – – – W W W
K – – – K – K – – – – – – – K K K K K K K – – – – – – K K – – – – K K K
– S S S – S – – – – – – – – – – – – – – – – – – – – S – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – Y – – – – – – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– S S S – S – – – – – – – – – – – – – – – – – – – – S – – – – – – – – –
– K K K – K – – – – – – – – – – – – – – – – – – – – K – – – – – – – – –
S S – – S – S S S S S S S R S S S S S S S S S S – – S S S S S S S S S S
– K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
K R R R K R K K K R R R R R K K K K K K K R K K – K K K K – – – – K K K
Y – – – Y Y Y Y Y – – – – – – – – – – – – – – – – – – – – – –
Polymorphisms are indicated by letters: Y, R, M, K, W, S.
L.M. Marques et al. / Veterinary Microbiology 152 (2011) 205–211
Strain
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Fig. 1. Dendrogram based on 16S rRNA fragments sequences amplified by U. diversum isolates, reference strains and U. diversum 16S rRNA GenBank sequence. Phylogenetic tree was obtained using the neighbor-joining method with Tajima–Nei correction. The data set was resampled 1000 times to generate bootstrap values.
reference strains U. diversum ATCC 48782, ATCC 49783 and U. diversum deposited in the GenBank were grouped in cluster D. Cluster E comprised isolates from farms 1 and 4 from Sao Paulo State and cluster F grouped semen isolates from TOURO1. Cluster G comprised isolate 112 from Alagoas State and showed a distance of approximately 12%.
4. Discussion In the present study, the 16S rRNA gene fragment sequence of ureaplasmas from healthy and unhealthy animals showed a significant number of SNPs compared to the U. diversum sequence deposited in the GenBank. This
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indicates that intraspecific variability exists within these organisms. Interestingly, we identified an isolate with only 88% similarity to the reference U. diversum 16S rRNA gene fragment. Buzinhani et al. (2007a,b) previously identified this isolate as U. diversum by PCR methodology and metabolic inhibition test with hyperimmune serum 49782 and 49783. Nevertheless, serologic cross-reactivity has been described for Mollicute organisms and the genotypic analysis of this study suggests that this organism is likely to be a novel Ureaplasma species. The presence of SNPs has been described in organisms of the Mollicute class (Buim et al., 2008; Ko¨nigsson et al., 2002). Ko¨nigsson et al. (2002) sequenced the U1 and U8 regions of the 16S rRNA of 17 isolates of M. agalactiae and 8 isolates of M. bovis and identified 21 and 12 SNPs, respectively (Pettersson et al., 1996). Within the M. mycoides cluster, strains of M. capripneumoniae have shown the greatest degree of intraspecific variability, with 11 SNPs along the entire 16S rRNA gene (Heldtander et al., 2001; Pettersson et al., 1998). The difference between reference and field strains observed herein also correlates with the 16S rRNA gene fragment analysis of Brazilian M. synoviae isolates, in which SNP variability as high as six has been reported between vaccine and field strains (Buim et al., 2008). The reasons for and consequences of the 16S rRNA gene variability in biological functions of ureaplasmas are unknown. Several factors may be related to the presence of SNPs, such as rRNA gene organization, DNA repair mechanism failure and presence of repetitive sequences (Amikam et al., 1984). SNP phenomena among the seven rDNA copies of Escherichia coli have been related to the presence of multigenic organization from divergent taxon, stabilization of random mutation, gene conversion among the multigenes, or lateral gene transfer (Cilia et al., 1996). Similarly, the 16S rRNA polymorphism of Mollicutes has been suggested to be related to the presence of the two copies of rRNA operons (Pettersson et al., 1994, 1996; Heldtander et al., 1998). However, in direct sequencing of PCR products, a double peak for individual bases in the DNA sequencing chromatograph would indicate the presence of two or more different copies of the 16 SrRNA gene that were amplified together. Double peaks were not observed in any samples tested herein, which indicates that U. diversum is likely to have one copy or identical copies of the 16S rRNA. The DNA repair mechanism of Mollicutes may be related to the high frequency of SNPs. These organisms present a higher percentage of mutations compared to other bacteria (Woese et al., 1985). It is believed that the reductive evolution of mycoplasmas led to loss of important genes of DNA repair, which can explain mutations (Ko¨nigsson et al., 2002). In fact, the main DNA repair systems of bacteria are absent in Mollicutes, such as the MutSLH system (Rocha and Blanchard, 2002). Although the consequences of SNPs in rDNA are unknown, the sequence divergence may have had significant impact on the taxonomy and epidemiology of mycoplasmas. Mollicute species taxonomic classification has relied on the 16S rRNA sequences, especially in cases where the organism cannot be cultivated. This sequence
divergence, which can be as high as 4% as described in this study, can lead to erroneous reporting of new species or subspecies. Once again, the importance of a complex combination of phenotypic and genotypic characteristics for species definition is highlighted. On the other hand, molecular epidemiologists can take advantage of intraspecific variability of rRNA genes. In the present study, the presence of SNPs of the 16S rRNA allowed a discriminatory analysis among the isolates from different farms. Isolates were divided into seven clusters according to their origin. Similarly, Pettersson et al. (1998) and Heldtander et al. (2001) have described the discriminatory power of the 16S rRNA gene sequence of different M. capripneumoniae strains from several regions in Africa. Some studies have also shown that hemotrophic mycoplasmas may cluster according to origin or animal species (Hoelzle, 2008; Willi et al., 2007). Our dendogram results partially corroborate with previous PFGE and AFLP analyses of isolates from frames 1, 2, 3, 4 and 5 (Buzinhani et al., 2007a,b). The isolates from Farm 2 showed to be clustered by PFGE and AFLP as well as by our dendogram analysis. Isolates from Farm 3 and 4 were clustered in different groups in Buzinhani studies when compared with results obtained in the present study. Isolates from Farm 1 were grouped among different clusters in the present analysis. It is noteworthy that a correlation between 16S rRNA gene sequence of the isolates and animal health could not be a related, as well as an association of sequences of the isolates and virulence factors, such as phospholipase, capsule (data not shown) and invasion in non-phogocytic cells (Marques et al., 2010). In conclusion, the genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. The low-gene sequence similarity of isolate 112 indicates that currently available primer sets and hyperimmune serum for the identification of U. diversum may be cross-reacting with this novel organism.
Acknowledgments This study was supported by FAPESP (grant 06/568550). We thank Aricelma P. Franc¸a for valuable technical assistance, as well as AcademicEnglishSolutions.com for revising the English. References Amikam, D., Glaser, G., Razin, S., 1984. Mycoplasmas (Mollicutes) have a low number of rRNA genes. J. Bacteriol. 158, 376–378. Ball, H.J., Mackie, D.P., 1985. The ovine mammary gland as in experimental model to determine the virulence of animal ureaplasmas. J. Hyg. Camb. 95, 375–382. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2009. GenBank. Nucleic Acids Res. 37, 26–31. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-Van Dillen, P.M., Van Der Noordaa, J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503.
L.M. Marques et al. / Veterinary Microbiology 152 (2011) 205–211 Buim, M.R., Buzinhani, M., Yamaguti, M., Oliveira, R.C., Mettifogo, E., Timenetsky, J., Ferreira, A.J., 2008. Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing. Comp. Immunol. Microbiol. Infect. Dis. 33, 15–23. Buzinhani, M., Buim, M.R., Yamaguti, M., Oliveira, R.C., Mettifogo, E., Timenetsky, J., 2007a. Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis. Vet. J. 173, 688–690. Buzinhani, M., Mettifogo, E., Buim, M.R., Moreno, A.M., Paixa˜o, R., Timenetsky, J., 2007b. Isolates of Ureaplasma diversum genotyped by single-enzyme amplified length polymorphism. Braz. J. Microbiol. 38, 28–32. Cardoso, M.V., Scarcelli, E., Grasso, L.M.P.S., Teixeira, S.R., Genovez, M.E., 2000. Ureaplasma diversum and reproductive disorder in Brazilian cows and heifers; first report. Anim. Reprod. Sci. 63, 137–143. Cilia, V., Lafay, B., Christen, R., 1996. Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level. Mol. Biol. Evol. 13, 451–461. Doig, P.A., Ruhnke, H.L., Palmer, N.C., 1980a. Experimental bovine genital ureaplasmosis. I. Granular vulvitis following vulvar inoculation. Can. J. Comp. Med. 44, 252–258. Doig, P.A., Ruhnke, H.L., Palmer, N.C., 1980b. Experimental bovine genital ureaplasmosis. II. Granular vulvitis, endometriti and salpingites following uterine inoculation. Can. J. Comp. Med. 44, 259–266. Harasawa, R., Cassell, G.H., 1996. Phylogenetic analysis of genes coding for 16S rRNA in mammalian u\reaplasmas. Int. J. Syst. Bacteriol. 46, 827– 829. Harasawa, R., Lefkowitz, E.J., Glass, J.I., Cassell, G.H., 1996. Phylogenetic analysis of the 16S-23S rRNA intergenic spacer regions of the genus Ureaplasma. J. Vet. Med. Sci. 58, 191–195. Heldtander, M., Pettersson, B., Tully, J.G., Johansson, K.E., 1998. Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas: Mycoplasma adleri, Mycoplasma auris Mycoplasma cottewii and Mycoplasma yeatsii. Int. J. Syst. Bacteriol. 48, 263–268. Heldtander, M., Wesonga, H., Bo¨lske, G., Pettersson, B., Johansson, K.E., 2001. Genetic diversity and evolution of Mycoplasma capricolum subsp. Capripneumoniae strain from Eastern Africa assessed by 16S rDNA sequence analysis. Vet. Microbiol. 78, 13–28. Hoelzle, L.E., 2008. Haemotrophic mycoplasmas: recent advances in Mycoplasma suis. Vet. Microbiol. 130, 215–226. Howard, C.J., Gourlay, R.N., Brownlie, J., 1973. The virulence of T-mycoplasmas isolated from various animal species, assayed by intramammary inoculation in cattle. J. Hyg. Camb. 71, 163–170. Ko¨nigsson, M.H., Bo¨lske, G., Johansson, K.E., 2002. Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains. Vet. Microbiol. 85, 209–220. Marques, L.M., Buzinhani, M., Neto, R.L., Oliveira, R.C., Yamaguti, M., Guimara˜es, A.M., Timenetsky, J., 2009. Detection of Ureaplasma
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diversum in bovine semen straws for artificial insemination. Vet. Rec. 165, 572–573. Marques, L.M., Ueno, P.M., Buzinhani, M., Cortez, B.A., Neto, R.L., Yamaguti, M., Oliveira, R.C., Guimara˜es, A.M., Monezi, T.A., Braga, A.C., Machado-Santelli, G.M., Timenetsky, J., 2010. Invasion of Ureaplasma diversum in Hep-2 cells. BMC Microbiol. 17, 83. Ogata, M., Kotani, H., Yamamoto, K., 1979. Serological comparison of bovine ureaplasmas. Nippon Juigaku Zasshi 41, 629–637. Pettersson, B., Bo¨lske, G., Thiaucourt, F., Uhle´n, M., Johansson, K., 1998. Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes. J. Bacteriol. 180, 2350–2358. Pettersson, B., Johansson, K.E., Uhle´n, M., 1994. Sequence analysis of 16S rRNA from Mycoplasma by direct solid-phase DNA sequencing. Appl. Environ. Microbiol. 60, 2456–2461. Pettersson, B., Uhle´n, M., Johansson, K.E., 1996. Phylogeny of some Mycoplasma from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group. Int. J. Syst. Bacteriol. 46, 1093–1098. Robertson, J.A., Stemke, G.W., Davis, J.W., Harasawa, R., Thirkell, D., Kong, F., Shepard, M.C., Ford, D.K., 2002. Proposal of Ureaplasma parvum sp. nov. and emended description of Ureaplasma urealyticum (Shepard et al. 1974) Robertson et al. 2001. Int. J. Syst. Evol. Microbiol. 52, 587– 597. Rocha, E.P., Blanchard, A., 2002. Genomic repeats, genome plasticity and the dynamics of mycoplasma evolution. Nucleic Acids Res. 30, 2031– 2042. Ruhnke, H.L., 1994. Mycoplasmas associated with bovine genital tract infections. In: Whitford, H.W., Rosenbusch, R.F., Lauerman, L.H. (Eds.), Mycoplasmosis in Animals: Laboratory Diagnosis. Iowa State University Press, Ames, Iowa, pp. 56–62. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Taylor-Robinson, D., Haig, D.A., Williams, M.H., 1967. Bovine T-strain mycoplasma. Ann. N. Y. Acad. Sci. 143, 517–518. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Willi, B., Boretti, F.S., Tasker, S., Meli, M.L., Wengi, N., Reusch, C.E., Lutz, H., Hofmann-Lehmann, R., 2007. From Haemobartonella to hemoplasma: molecular methods provide new insights. Vet. Microbiol. 125, 197– 209. Woese, C.R., Stackebrandt, E., Ludwig, W., 1985. What are mycoplasmas: the relationship of time and mode in bacterial evolution. J. Mol. Evol. 21, 305–316.
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Short communication
Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates L.M. Marques a,b, M. Buzinhani a, A.M.S. Guimaraes a, R.C.P. Marques a, S.T. Farias c, R.L. Neto a, M. Yamaguti a, R.C. Oliveira a, J. Timenetsky a,* a b c
Instituto de Cieˆncias Biome´dicas, Departamento de Microbiologia, Universidade de Sa˜o Paulo, Brazil Instituto Multidisciplinar em Sau´de, Nu´cleo de Tecnologia em Sau´de, Universidade Federal da Bahia, Brazil Centro de Cieˆncias Exatas e da Natureza, Departamento de Biologia Molecular, Universidade Federal da Paraı´ba, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2010 Received in revised form 7 April 2011 Accepted 8 April 2011
Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Ureaplasma diversum Intraspecific variation 16S rRNA gene
1. Introduction Ureaplasmas colonize the respiratory and urogenital tract of animals and humans. These organisms were initially isolated from the reproductive tract of a bovine by Taylor-Robinson et al. (1967); 11% of clinically healthy cows had ureaplasmas in their reproductive tract. Subsequent studies on detection of these organisms identified several isolates in the genital tract of males and females without reproductive problems. For this reason, these organisms were initially considered non-pathogenic. Nevertheless, Doig et al. (1980a,b) experimentally infected
* Corresponding author at: Instituto de Cieˆncias Biome´dicas, Av. Professor Lineu Prestes, 1374, CEP 05508 900, Sa˜o Paulo, SP, Brazil. Tel.: +55 11 3091 7297; fax: +55 11 3091 7354. E-mail address: [email protected] (J. Timenetsky). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.04.007
cows and observed granular vulvovaginitis. Similar studies with U. diversum strains inoculated in the mammary gland of sheep and cattle have indicated variable virulence in different strains, justifying the presence of ureaplasmas in healthy animals (Ball and Mackie, 1985; Howard et al., 1973). Bovine and human ureaplasmas were initially differentiated through serology identification (Ogata et al., 1979). Sequencing of the 16S rRNA gene provided basis for their classification later on. Organisms isolated from bovine were named U. diversum (Mycoplasmataceae family, Mycoplasmatales order). Ureaplasma are considered more recently evolved than genera Acholeplasma and Spiroplasma and have one or two copies of the 16S rRNA gene. The complete genome sequences of U. parvum and U. urealyticum have two copies of 16S rRNA each. Robertson et al. (2002) sequenced the 16S rRNA, urease and multiple-banded antigen gene sequences from all 14 serotypes of U. urealyticum associated with repiratory and
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reproductive diseases in humans. Then these mollicutes were reclassified in two species: U. urealyticum and U. parvum. Accordingly, the aim of this study was to sequence and analyze the 16S rRNA partial sequences of different isolates of U. diversum recovered from bovine.
DNA sequences was constructed using the ClustalW (Thompson et al., 1997). A phylogenetic tree was obtained using the neighbor-joining method with Tajima–Nei correction. The data set was resampled 1000 times to generate bootstrap values.
2. Materials and methods
3. Results
2.1. Ureaplasma strains and culture conditions
3.1. Intra-specific analysis of the 16S rRNA gene
Ureaplasma diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. The field isolates comprised 24 ureaplasmas isolated from the vulvovaginal mucus of female cattle with reproductive alterations and 10 ureaplasmas isolated from bovine semen. Females were from seven farms from four different States in Brazil, including Sao Paulo (Farm 1: strains A203, 64 and 59; Farm 2: Alva, Gota, 98, 91, 93, 94, 95, 100 and 102; Farm 4: 77, 84, 72, 73 and 74; Farm 7: 19V, 16V, 7V and 9V), Mato Grosso do Sul (Farm 3: 1664 and 805), Minas Gerais (Farm 6: Ud11, Ud18 and Ud3), and Alagoas (Farm 5: 112). The sampling was conducted from 1999 to 2005. The semen isolates were obtained from a previous study (Marques et al., 2009), all from five different Artificial Insemination Centers from Sao Paulo State. These organisms were identified as U. diversum using a previously described PCR (Cardoso et al., 2000). These organisms were cultivated in UB liquid medium (Ruhnke, 1994) at 37 8C for 2 days.
The complete sequence of the 16S rRNA of U. diversum (GenBank accession number D78650) was compared to the sequences obtained in the present study (Harasawa and Cassell, 1996) using the multiple sequence alignment tool ClustalW. A fragment containing four hypervariable regions were selected for the sequence variation analyses according to Harasawa and Cassell (1996). This fragment comprised the nucleotides between positions 99 to 694. Nucleotide (Adenine, A; Cytosine, C; Guanine, G; Thymine, T) polymorphisms were identified based on their position within the 16S rRNA gene and indicated as Y = T/C, R = A/G, M = A/C, K = G/T, W = A/T and S = G/C (International Union of Biochemistry and Molecular Biology, IUBMB). Forty-four single nucleotide polymorphisms (SNP) were detected (Table 1). Deletions and insertions were only observed in the isolate 112, from Farm 5. The transversion A/C (M) was found in most of the isolates at positions 160 and 238 compared to the GenBank reference sequence and the reference strains sequenced in this study. Interestingly, the semen isolates S1, S6, S7 and S8 showed a transversion A/T (W) at these positions. SNPs were also observed for most of the isolates at positions 295 (18/36, 50%), 298 (33/36, 91.7%), 406 (28/36, 77.8%), 448 (25/36 69.4%), 459 (26/36 72.2%), 515 (33/36 91.7%) and 534 (34/36 94.4%). Unique SNPs were observed in isolates from the same farms as shown in Table 1. SNPs in positions 133, 212 and 213 were restricted to isolates from farm ‘‘TOURO2’’, P6 and P7. Isolates from farm P1 and P3 showed distinct polymorphic patterns when compared. Transversions were observed in 33 nucleotide positions, whereas transitions were observed in 21 nucleotide positions. Transversion A/T (W) and transition A/G (R) were observed in 11 nucleotide positions each; transition T/C (Y) was observed in 10 nucleotide positions and transversions G/C (S) and G/T (K) were observed in 9 nucleotide positions. Transversion A/C (M) was observed only in 4 nucleotide positions. The average distance between the strains was 0.0966. Surprisingly, isolate 112 showed a distance of 0.2280. The reference strains and the isolates showed minimum similarity of 96%, except for isolate 112, which showed 88% similarity. A 0.03 distance cut-off was used in the intra-specific analysis for cluster definition, implying a similarity of at least 97% between isolates (Fig. 1). The dendogram showed 7 clusters, A to G. Cluster A comprised 18 isolates from four farms: two from the Sao Paulo State (Farm 2 and Farm 7), two from Mato Grosso do Sul State (Farm 3) and one from Minas Gerais State (Farm 6). Cluster B comprised the isolate A203 from Farm 1 and cluster C comprised the semen isolates from farm TOURO2. The
2.2. DNA extraction and PCR for the amplification of the 16S rRNA Cultures of ureaplasma in 200 ml of UB medium were used for DNA extraction according to the method described by Boom et al. (1990). The specific primer set F16S and R16 amplifies nearly the entire 16s rDNA sequence of Mollicutes and was used as described by Harasawa et al. (1996). PCR products (10 ml) were then electrophoresed in 1% agarose gel with 10 mg/ml of ethidium bromide and visualized under UV light (Vilber Lourmat, Germany). 2.3. DNA purification, quantification and sequencing After confirmation of the only band in the electrophoresis, PCR products were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham) and quantified using a Low Mass DNA Ladder (Invitrogen). Purified PCR products were sequenced according to the MegaBACE 1000 protocol, using the DYEnamic ET Dye Terminator kit (with Thermo Sequenase II DNA Polymerase). Sequences were analyzed in the Sequence Analyzer software, Base Caller Cimarron 3.12. 2.4. Phylogenetic analyses The 16S rRNA gene sequences were initially compared to sequences deposited in the GenBank databases using the BLASTn algorithm (Benson et al., 2009). Phylogenetic analyses were performed using MEGA4 software (Tamura et al., 2007). Briefly, a multiple sequence alignment of the
Table 1 Polymorphism and differences in the 16S rRNA gene sequence of U. diversum isolates, reference strains and U. diversum 16S rRNA GenBank sequence. Position 118
119
133
134
159
160
162
171
177
196
197
212
213
238
249
253
255
284
285
295
298
299
300
314
345
GenBank 5T 7T 7V 9V 10T 13T 15T 16V 18T 19V 59 64 72 73 74 77 84 91 93 94 95 98 100 102 112 805 1664 49782 49783 A203 Alva Gota S1 S6 S7 S8 Ud3 Ud11 Ud18
D78650 1437325/5T 1437325/7T 1437325/7V 1437325/9V 1437325/10T 1437325/13T 1437325/15T 1437325/16V 1437325/18T 1437325/19V 1437325/59 1437325/64 1437325/72 1437325/73 1437325/74 1437325/77 1437325/84 1437325/91 1437325/93 1437325/94 1437325/95 1437325/98 1437325/100 1437325/102 1437325/112 1437325/805 1437325/1664 1437325/82 1437325/83 1437325/A203 1437325/Alva 1437325/Gota 1437325/S1 1437325/S6 1437325/S7 1437325/S8 1437325/Ud3 1437325/Ud11 1437325/Ud18
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
T W W – – W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A R R – – R R R – R – – – – – R R R – – – – – – – – – – – – – – – – – – – – – –
C – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – Y Y Y
A M M M M M M M M M M W W M M M M M M M M M M M M M M M – – M M M W W W W M M M
G – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – S – – – – – –
C – – – – – – – – – – – – – – – – – – – – – – – – Y – – – – – – – Y Y Y Y – – –
T – – – – – – – – – – – – – – Y – Y – – – – – – – – – – – – – – – Y Y Y Y – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
G – – – – – – – – – – – – – – – – – S S S – S S S – – – – – – – S – – – – – – –
A R R – – R R R – R – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
G K K – – K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A M M M M M M M M M M W W M M M M M M M M M M M M M M M – – M M M W W W W M M M
C – – – – – – – – – – – – – – – – – – – – – – – – Y – – – – – – – Y Y Y Y – – –
A – – – – – – – – – – – – – – – – – – – – – R R R – – – – – – R – – – – – – – –
T – – – – – – – – – – – – – – Y – Y – – – – – – – – – – – – – – – Y Y Y Y – – –
A R R – – R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
G R R – – R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
A – – W W – – – W – W – – – – – – – W W W W W W W – W W – – – W W – – – – W W W
C Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y – Y Y – – Y Y Y Y Y Y Y Y Y Y
T Y Y – – Y Y – Y – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
C Y Y – – Y Y – Y – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
A – – – – – – – – – – – – – – – – – – – – – W W W – – – – – – – W – – – – – – –
C – – Y Y – – – Y – Y – – – – – – – Y Y Y Y Y Y Y – Y Y – – – Y Y – – – – Y Y Y
Strain
Accession number
Position 355
394
401
404
405
406
408
416
417
427
428
439
444
448
459
463
473
487
488
499
500
515
529
531
534
GenBank 5T 7T 7V
D78650 1437325/5T 1437325/7T 1437325/7V
T – – –
C Y Y –
T – – K
G – – R
A R R –
G R R R
A M M –
T W W –
A R R –
T – – –
A – – –
C – – –
A – – –
A – – W
A W W W
T – – K
C S S –
T W W –
A W W –
G S S –
T K K –
G – – S
T K K –
G K K –
G R R K
207
Accession number
L.M. Marques et al. / Veterinary Microbiology 152 (2011) 205–211
Strain
208
Table 1 (Continued ) Accession number
Position 355
394
401
404
405
406
408
416
417
427
428
439
444
448
459
463
473
487
488
499
500
515
529
531
534
9V 10T 13T 15T 16V 18T 19V 59 64 72 73 74 77 84 91 93 94 95 98 100 102 112 805 1664 49782 49783 A203 Alva Gota S1 S6 S7 S8 Ud3 Ud11 Ud18
1437325/9V 1437325/10T 1437325/13T 1437325/15T 1437325/16V 1437325/18T 1437325/19V 1437325/59 1437325/64 1437325/72 1437325/73 1437325/74 1437325/77 1437325/84 1437325/91 1437325/93 1437325/94 1437325/95 1437325/98 1437325/100 1437325/102 1437325/112 1437325/805 1437325/1664 1437325/82 1437325/83 1437325/A203 1437325/Alva 1437325/Gota 1437325/S1 1437325/S6 1437325/S7 1437325/S8 1437325/Ud3 1437325/Ud11 1437325/Ud18
– – – – – – – – – K K K K K – – – – – – – – – – – – – – – – – – – – – –
– Y Y Y
K – – – K – K – – – – – – – K K K K K K K – – – – – – K K – – – – K K K
R – – – R – R – – – – – – – R R R R R R R – – – – – – R R – – – – R R R
– R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
R R R R R R R – – R R R R R R R R R R R R R R R – – R R R – – – – R R R
– M M M – M – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– R R R – R – – – – – – – – – – – – – – – R – – – – – – – – – – – – – –
– – – – – – – W W W W W W W – – – – – – – – – – – – – – – W W W W – – –
– – – – – – – R R R R R R R – – – – – – – – – – – – – – – R R R R – – –
– – – – – – – – – M M M M M – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – W W – – – – – – – – – – – – – – – – – – – – W W W W – – –
W – – – W – W – – W W W W W W W W W W W W W W W – – – W W – – – – W W W
W W W W W W W – – – – – – – W W W W W W W W W W – – – W W – – – – W W W
K – – – K – K – – – – – – – K K K K K K K – – – – – – K K – – – – K K K
– S S S – S – – – – – – – – – – – – – – – – – – – – S – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – Y – – – – – – – – – – – – – –
– W W W – W – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– S S S – S – – – – – – – – – – – – – – – – – – – – S – – – – – – – – –
– K K K – K – – – – – – – – – – – – – – – – – – – – K – – – – – – – – –
S S – – S – S S S S S S S R S S S S S S S S S S – – S S S S S S S S S S
– K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– K K K – K – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
K R R R K R K K K R R R R R K K K K K K K R K K – K K K K – – – – K K K
Y – – – Y Y Y Y Y – – – – – – – – – – – – – – – – – – – – – –
Polymorphisms are indicated by letters: Y, R, M, K, W, S.
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Fig. 1. Dendrogram based on 16S rRNA fragments sequences amplified by U. diversum isolates, reference strains and U. diversum 16S rRNA GenBank sequence. Phylogenetic tree was obtained using the neighbor-joining method with Tajima–Nei correction. The data set was resampled 1000 times to generate bootstrap values.
reference strains U. diversum ATCC 48782, ATCC 49783 and U. diversum deposited in the GenBank were grouped in cluster D. Cluster E comprised isolates from farms 1 and 4 from Sao Paulo State and cluster F grouped semen isolates from TOURO1. Cluster G comprised isolate 112 from Alagoas State and showed a distance of approximately 12%.
4. Discussion In the present study, the 16S rRNA gene fragment sequence of ureaplasmas from healthy and unhealthy animals showed a significant number of SNPs compared to the U. diversum sequence deposited in the GenBank. This
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indicates that intraspecific variability exists within these organisms. Interestingly, we identified an isolate with only 88% similarity to the reference U. diversum 16S rRNA gene fragment. Buzinhani et al. (2007a,b) previously identified this isolate as U. diversum by PCR methodology and metabolic inhibition test with hyperimmune serum 49782 and 49783. Nevertheless, serologic cross-reactivity has been described for Mollicute organisms and the genotypic analysis of this study suggests that this organism is likely to be a novel Ureaplasma species. The presence of SNPs has been described in organisms of the Mollicute class (Buim et al., 2008; Ko¨nigsson et al., 2002). Ko¨nigsson et al. (2002) sequenced the U1 and U8 regions of the 16S rRNA of 17 isolates of M. agalactiae and 8 isolates of M. bovis and identified 21 and 12 SNPs, respectively (Pettersson et al., 1996). Within the M. mycoides cluster, strains of M. capripneumoniae have shown the greatest degree of intraspecific variability, with 11 SNPs along the entire 16S rRNA gene (Heldtander et al., 2001; Pettersson et al., 1998). The difference between reference and field strains observed herein also correlates with the 16S rRNA gene fragment analysis of Brazilian M. synoviae isolates, in which SNP variability as high as six has been reported between vaccine and field strains (Buim et al., 2008). The reasons for and consequences of the 16S rRNA gene variability in biological functions of ureaplasmas are unknown. Several factors may be related to the presence of SNPs, such as rRNA gene organization, DNA repair mechanism failure and presence of repetitive sequences (Amikam et al., 1984). SNP phenomena among the seven rDNA copies of Escherichia coli have been related to the presence of multigenic organization from divergent taxon, stabilization of random mutation, gene conversion among the multigenes, or lateral gene transfer (Cilia et al., 1996). Similarly, the 16S rRNA polymorphism of Mollicutes has been suggested to be related to the presence of the two copies of rRNA operons (Pettersson et al., 1994, 1996; Heldtander et al., 1998). However, in direct sequencing of PCR products, a double peak for individual bases in the DNA sequencing chromatograph would indicate the presence of two or more different copies of the 16 SrRNA gene that were amplified together. Double peaks were not observed in any samples tested herein, which indicates that U. diversum is likely to have one copy or identical copies of the 16S rRNA. The DNA repair mechanism of Mollicutes may be related to the high frequency of SNPs. These organisms present a higher percentage of mutations compared to other bacteria (Woese et al., 1985). It is believed that the reductive evolution of mycoplasmas led to loss of important genes of DNA repair, which can explain mutations (Ko¨nigsson et al., 2002). In fact, the main DNA repair systems of bacteria are absent in Mollicutes, such as the MutSLH system (Rocha and Blanchard, 2002). Although the consequences of SNPs in rDNA are unknown, the sequence divergence may have had significant impact on the taxonomy and epidemiology of mycoplasmas. Mollicute species taxonomic classification has relied on the 16S rRNA sequences, especially in cases where the organism cannot be cultivated. This sequence
divergence, which can be as high as 4% as described in this study, can lead to erroneous reporting of new species or subspecies. Once again, the importance of a complex combination of phenotypic and genotypic characteristics for species definition is highlighted. On the other hand, molecular epidemiologists can take advantage of intraspecific variability of rRNA genes. In the present study, the presence of SNPs of the 16S rRNA allowed a discriminatory analysis among the isolates from different farms. Isolates were divided into seven clusters according to their origin. Similarly, Pettersson et al. (1998) and Heldtander et al. (2001) have described the discriminatory power of the 16S rRNA gene sequence of different M. capripneumoniae strains from several regions in Africa. Some studies have also shown that hemotrophic mycoplasmas may cluster according to origin or animal species (Hoelzle, 2008; Willi et al., 2007). Our dendogram results partially corroborate with previous PFGE and AFLP analyses of isolates from frames 1, 2, 3, 4 and 5 (Buzinhani et al., 2007a,b). The isolates from Farm 2 showed to be clustered by PFGE and AFLP as well as by our dendogram analysis. Isolates from Farm 3 and 4 were clustered in different groups in Buzinhani studies when compared with results obtained in the present study. Isolates from Farm 1 were grouped among different clusters in the present analysis. It is noteworthy that a correlation between 16S rRNA gene sequence of the isolates and animal health could not be a related, as well as an association of sequences of the isolates and virulence factors, such as phospholipase, capsule (data not shown) and invasion in non-phogocytic cells (Marques et al., 2010). In conclusion, the genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. The low-gene sequence similarity of isolate 112 indicates that currently available primer sets and hyperimmune serum for the identification of U. diversum may be cross-reacting with this novel organism.
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