Prevalence and distribution of the insertion element ISMag1 in Mycoplasma agalactiae

Abstract

In characterising Mycoplasma agalactiae strains from various European countries and from Africa, a new insertion sequence (IS), ISMag1, which is related to IS of the family of IS30 insertion elements, has been identified by DNA sequence analysis and Southern blot hybridisation. ISMag1 has a size of 1515 bp, and contains inverted repeats of 3 bp and a gene encoding the putative transposase on a single open reading frame. ISMag1 is present only in the rarely isolated serotypes E, F, G and H of M. agalactiae, where it is found in 1 to approximately 30 copies. The different patterns obtained by hybridisation of a labelled probe of ISMag1 to genomic DNA cut with various restriction enzymes correlate to some extent to the different serotypes and to variations of the nucleotide sequences of the uvrC genes of the different strains. Based on uvrC sequences, the strains of M. agalactiae carrying ISMag1 form a cluster, separate from the other strains. IS patterns obtained with ISMag1 allow a fine subtyping of the serotypes E, F, G and H of M. agalactiae for epidemiological studies. The potential role of ISMag1 and of its copy numbers on virulence and persistence of the respective strains requests further studies.

Introduction

Mycoplasma agalactiae is the causative agent of contagious agalactia (CA), a world-wide spread syndrome affecting ovine and caprine species, mainly characterised by mastitis, arthritis and keratoconjunctivitis. In lactating females, CA-induced mastitis leads to a rapid and severe reduction in milk secretion. The economic losses caused by these low milk yields are critical in regions depending on ewe or goat dairy products, such as the Mediterranean basin and eastern Europe, where control of CA is of particular importance (DaMassa et al., 1992, Bergonier et al., 1997). During natural infections, acute forms of the disease with marked clinical signs like agalactia are often observed, but chronic forms with only mild (or no) clinical signs also occur. Furthermore, experimental evidence confirmed that field strains of M. agalactiae may have various degrees of virulence in lactating ewes (Sanchis et al., 2000). The complete or partial lack of symptoms in certain infected animals makes the control of spread of M. agalactiae difficult. It is still unclear whether the major differences in virulence observed in M. agalactiae infected animals are due to variations between individual bacterial strains or due to variations in the physiological status of the individual hosts. This lack of clarity is mainly due to insufficient basic knowledge of the molecular mechanisms of pathogenicity and molecular genetics of this pathogen. Therefore, accurate and rational methods to subtype M. agalactiae for epidemiological purpose are of particular interest. Currently, the sole subtyping method of M. agalactiae is serotyping, based on a small set of monoclonal antigens targeting non-variable antigens of M. agalactiae (Bergonier et al., 1996). Serotypes A, B, C and D are most frequently encountered, while serotypes E, F, G and H are rarely isolated (Bergonier et al., 1996).

Recently, analysis of patterns of insertion sequences (IS) has been used extensively for epidemiological purposes (van Embden et al., 1993, Stanley et al., 1993, Small et al., 1994, Frey, 1998). Bacterial IS elements are mobile, transposable genetic elements of about 800–2500 bp which are often present in multiple copies on bacterial genomes (Galas and Chandler, 1989). Transposition of IS elements can cause deletions, insertions and inversions of genomic loci and thus contribute to the genetic variability of bacteria (Lewin, 1997). In mycoplasmas, however, there are only a few IS elements currently known (Bhugra and Dybvig, 1993, Frey et al., 1995, Calcutt et al., 1999, Vilei et al., 1999) but they contributed significantly to epidemiological studies of mycoplasmal diseases such as contagious bovine pleuropneumonia (Cheng et al., 1995, Vilei et al., 1999, March et al., 2000).

In this study, we report the discovery of a new IS element, named ISMag1, of M. agalactiae and describe its prevalence in the various serotypes.