Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri
Introduction
While the animal pathogens now known as Mycoplasma have been studied for more than a century [6], [7], their affiliations and taxonomy did not begin to be resolved until the 1950s [15], [16]. Those early authors formalized the genus name Mycoplasma Nowak (1928) [16], with M. mycoides as the type species [6], [16], [30]. The strains of this species were classified into two subspecies, Mycoplasma mycoides subsp. mycoides, pathogenic to cattle, with strain PG1 as the representative strain, and M. mycoides subsp. capri, causing infections in goats, with strain PG3 as the representative strain [16]. Subsequently, M. mycoides subsp. mycoides (Mmm) was subdivided into two morphotypes, one of which produced large colonies (MmmLC), and the other small colonies (MmmSC), with strain PG1 being assigned as representative of MmmSC [14]. Most strains of MmmLC and MmmSC were serologically indistinguishable from each other by the growth inhibition test [2], [44], but as well as their differing growth characteristics, they were distinguished by their biochemical and physiological properties, and by LC strains being goat pathogens, and SC strains causing disease in cattle [13], [14], [44].
Many studies have shown that most strains of M. mycoides subsp. mycoides (MmmLC) and M. mycoides subsp. capri (Mmc) are serologically distinct from each other (see [2] for the earlier literature). Serological and metabolic studies of numerous putative strains of each subspecies by Al-Aubaidi et al. [2] identified strain PG3 as the neotype strain for Mmc, and proposed strain Y-goat as the representative strain for MmmLC. Evidence has, however, accumulated for more than 30 years that the serovars MmmLC and Mmc are actually very similar, perhaps taxonomically identical [9], [12], [21], [27], [29], [37], [38]. This led increasingly to suggestions that the two subspecies might be regarded as a single taxon [8], [25], [32], [38], [46], and to the formal proposal that they should be amalgamated as strains of M. mycoides subspecies capri [28]. We provide new evidence to support this proposal, using several taxonomic criteria, applied to 22 strains of MmmLC and eight strains of Mmc. To date, the taxonomic evidence in the literature, and our new study, has been derived from work on at least 112 strains (about 85 MmmLC and 27 Mmc), originating from 17 countries on several continents. We present new data on our 30 strains, 21 of which have not previously been used in comparative studies, and summarize all the key experimental evidence for the amalgamation of the two subspecies.
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Materials and methods
Mycoplasma strains used in this study are listed in Table 1. All strains were grown at 37 °C in broth medium containing tryptose, yeast extract, glucose, glycerol, heat-inactivated porcine serum, HEPES and fresh yeast extract [42]. Mycoplasma DNA was extracted by the method of Bashiruddin [4]. The cluster-specific primers MC323 and MC358, derived from the sequence of CAP-21 [5] were used for the polymerase chain reaction on all the DNA samples.
Restriction endonuclease analysis (REA) of genomic
Results and discussion
Numerous comparative criteria have been applied to more than 100 mycoplasma strains by us and earlier workers (Table 2), which show that MmmLC and Mmc are in fact essentially indistinguishable (Tables 2 and 3). Serological methods have been widely used in the diagnosis of animals infected with members of the M. mycoides cluster, and is one approach that does enable some distinction of MmmLC and Mmc strains (Table 3). Data presented cover the properties and analysis of their DNA and proteins, as
PCR analysis and 16S RNA gene sequencing for the M. mycoides cluster
A single distinct and intense band of 1.5 kb was seen as expected after agarose gel electrophoresis and ethidium bromide staining of PCR products from the 16S rRNA gene from all 30 strains. Partial sequencing of the 16S rRNA gene products from the strains showed >99% sequence identity among them [38] and full-length sequencing (up to 1524 nucleotides) of the 16S rRNA gene from 17 of the strains (12 MmmLC and five Mmc strains) showed all strains to be 99.9% identical to each other. Two
Restriction enzyme analysis of 16S rRNA PCR gene products
As expected from the sequencing results, all the MmmLC and Mmc strains gave similar digestion patterns with six of the endonuclease enzymes tested (AluI, ClaI, HindIII, Sau3AI, RsAI and DraI), and thus did not differentiate the MmmLC and Mmc strains from one another.
Restriction endonuclease analysis (REA) of whole genomic DNA
REA of the genomic DNA of the Mmm LC strains and Mmc strains with HindIII, and PstI showed DNA cleaved to produce a complex of 20–30 bands: patterns for Mmc strains Pendik, BQT, G169, G105/A1, G108 and N108 were identical; MmmLC strains 1141, FR1645, SP80, SP266 and Y-goat® formed one cluster with 60% similarity; strains Pendik, BQT, G169, G105/A1 and FR755 formed a cluster with 65% similarity; and strains N108, G108 and JM formed another cluster showing more than 85% similarity. Thus, this
One-dimensional SDS-PAGE profiles of total cellular proteins
All the strains tested (Table 2) showed very similar and highly reproducible patterns of 15–25 polypeptide bands, but the patterns did not allow discrimination between the MmmLC and Mmc strains (Table 2). All the strains formed cluster groupings of 62–100% similarity, within which some pairs of MmmLC and Mmc strains showed over 80% similarity, which exceeded the similarity between some strains of each type individually. This is entirely consistent with the early observations on other strains
Analysis of the MmmLC and Mmc strains using RAPD
The RAPD technique using arbitrarily-primed-PCR allows detection of specific polymorphisms in the genomic fingerprints of related strains by amplification of random segments of their genomic DNA, produced using random primer sets, constructed without specific nucleotide sequence information [35]. RAPD using the M. mycoides cluster-specific primers, Mlip1 and Mlip4 [34], [35], produced diverse genomic fingerprints showing high genomic polymorphism among the strains, but did not differentiate
Serological differentiation by growth inhibition tests
As expected, the growth of most of 16 strains of MmmLC tested was not inhibited by antiserum to Mmc, and 5–6 Mmc strains tested were not inhibited by either of the MmmLC antisera (Table 3). MmmLC strains FR1645 and SP152 were unaffected by any of the antisera; while MmmLC strain IT247 showed a 2 mm inhibition zone with Mmc antiserum but no inhibition by either of the MmmLC antisera. Mmc strain G169 was inhibited by both Y-goatR and F-30 LC antisera (5 and 2.5 mm zones of inhibition), but was
Disease profiles defining the “mycoides cluster”
The M. mycoides cluster of mycoplasmas cause some serious diseases in ruminants, the most severe of which are the notifiable contagious caprine pleuropneumonia (CCPP), and contagious bovine pleuropneumonia (CBPP). CCPP and CBPP are caused specifically by M. capricolum subsp. capripneumoniae and MmmSC, respectively (for literature, see [28], [31], [46]). The most recently defined distinct species in the cluster is Mycoplasma leachii[28], the causative agent of mastitis and polyarthritis in
Acknowledgement
The work reported in this paper was initiated by the late Dr. Roger J. Miles.
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Present address: National Blood Service, Microbiology Reference Laboratory, Colindale, London NW9 5BG, UK.