Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains
Introduction
The mycoplasmas are closely related to the gram-positive bacteria but are arranged into a separate class called the Mollicutes. The mycoplasmas lack a rigid cell wall, have a low genomic G+C content and are the smallest organisms capable of self-replication (Razin et al., 1998). In general, the mycoplasmas are regarded as host specific and many of them are pathogenic and, therefore, of great concern in veterinary medicine (Simecka et al., 1992, Ross, 1993, Nicholas, 1998). Mycoplasma agalactiae causes contagious agalactia (CA), a disease of goats and sheep characterised by mastitis, arthritis and keratoconjunctivitis (Lefévre, 1996). In cattle mastitis, arthritis and respiratory disease are caused by Mycoplasma bovis (Ross, 1993). M. agalactiae and M. bovis have been shown to be very closely related on the basis of biochemical and immunological methods (Askaa and Ernø, 1976) and 16S rRNA sequence comparisons (Mattsson et al., 1994, Pettersson et al., 1996). Initially considered to be subspecies of the same species, they were subsequently reclassified as two separate species (Askaa and Ernø, 1976). Because of their importance in veterinary medicine many detection systems for the identification of M. agalactiae and M. bovis have been developed. Their close relationship makes differentiation difficult by traditional methods such as serology, which is one of the reasons why PCR is often the method of choice for identification of these species. A good diagnostic PCR system should be based on a well characterised gene with enough variability to allow the design of specific primers, even for closely related species, but sufficient stability to detect all isolates within a species. The 16S rRNA genes have regions of differing evolutionary variability and are frequently used for phylogenetic studies (Gray et al., 1984, Weisburg et al., 1989, Olsen and Woese, 1993). This gene is, therefore, a preferred choice as diagnostic target for many bacteria. Different detection systems have been developed for the identification of M. agalactiae and M. bovis (Levisohn et al., 1991, Mattsson et al., 1991, Tola et al., 1994, Tola et al., 1996, Subramaniam et al., 1998) and some of them are based on PCR of the 16S rRNA gene (Chavez Gonzalez et al., 1995, Bergonier et al., 1996, Johansson et al., 1996). Both species have been found to have two rRNA operons, as do many mycoplasmas (Christiansen and Ernø, 1990, Pettersson et al., 1996). Sequence differences often exist between the two rRNA operons and the corresponding nucleotide positions are referred to as polymorphisms (Pettersson et al., 1996). Furthermore, sequence differences may also exist between the 16S rRNA genes of homologous operons in different isolates of the same species. Polymorphisms and sequence differences between isolates of the same species are referred to as intraspecific variation (Clayton et al., 1995). Results from analyses by 16S rRNA based PCR indicate that intraspecific variations may occur in M. agalactiae and M. bovis. It has been shown recently that intraspecific variations between isolates exist in M. agalactiae (Subramaniam et al., 1998). The 16S rRNA genes from 16 field isolates of M. agalactiae and seven field isolates of M. bovis of different geographical origins were, therefore, sequenced and analysed together with the sequences of the type strains of the species to investigate the extent of intraspecific variation within the species.
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Mycoplasma isolates and sample preparation
The M. agalactiae and M. bovis isolates and their origins are listed in Table 1. All the isolates were confirmed to be M. agalactiae or M. bovis, either by the supplying laboratory or by the National Veterinary Institute. The isolates examined at the National Veterinary Institute (M. agalactiae isolates 17, 41, 5725, CC2a, CC3a, CC4b, 396/12, 847.121, L9, 5G, 25/95 and M133/87, and M. bovis isolates 148/88 and120/81) were tested by the immunofluorescence test (Rosendal and Black, 1972). The
M. agalactiae isolates
All of the European M. agalactiae isolates, including the type strain PG2T, which was originally isolated in Spain (Edward and Freundt, 1973), had similar polymorphism patterns. One polymorphism, 1032Y, was shared by all the European isolates including the type strain. Two other polymorphic positions, 126M, and 1048R, were shared by 5 and 9 isolates, respectively, including the type strain (Table 2). Among the European isolates, the Hungarian isolate 396/12 had a polymorphism, 375R, that was
Discussion
Diagnostic PCR systems based on the 16S rRNA genes are used to detect and distinguish between M. agalactiae and M. bovis. The reliability and specificity of these PCR systems were dependent on the very few nucleotide differences between the 16S rRNA genes of these species. This study of 17 M. agalactiae and eight M. bovis isolates shows that there is substantial variability between the 16S rRNA gene sequences within these species. Among the 17 analysed M. agalactiae isolates, 21 polymorphic
Acknowledgements
We wish to thank Stefan Jernberg, Virginia Melys and Karin von Schantz for rapid and skillful technical support. We also owe our gratitude to Hywell Ball, Elisabeth Brandão, Soraya Deniz, Al El Ebeedy, Antonio Fernández, Michelle Glew, Èva Kaszanyitzky, Jacques Nicolet, François Poumarat, Konrad Sachse and Kriton Sarris, who have provided us with some of the isolates.
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