A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

A specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP).

Contagious caprine pleuropneumonia is a severe infectious disease of goats in Africa and the Middle East. It is caused by a fastidious mycoplasma, Mycoplasma capricolum subsp. capripneumoniae, a member of the "M. mycoides cluster". Members of this cluster share genomic and antigenic features, which result in common biochemical and serological properties, complicating species identification. Two species of this cluster, M. mycoides subsp. capri and M. mycoides subsp. mycoides large colony biotype, are very often isolated from clinical cases resembling contagious caprine pleuropneumonia. Furthermore, in the laboratory, M. capricolum subsp. capripneumoniae can be easily confused with the closely related capricolum subspecies. Considering these constraints and the scarcity of available methods for identification, a specific polymerase chain reaction was developed. A DNA fragment of 7109 bp containing genes coding for the arginine deiminase pathway (ADI) was chosen as target sequence for the selection of a specific primer pair. The full ADI operon from M. capricolum subsp. capripneumoniae strain GL100 was sequenced. Polymorphism within this locus was analyzed by comparison with the sequence from the closely related IPX strain (M. capricolum subsp. capricolum). It varied from 0.6% to 3.5%. The highest divergence was found in a region coding for arcD. Therefore, this gene was chosen as target for the specific amplification of a 316 bp-long DNA fragment. The specificity of this PCR was validated on 14 M. capricolum subsp. capripneumoniae strains and 27 heterologous strains belonging to the "M. mycoides cluster" and M. putrefaciens. This new PCR will be a valuable tool for the surveillance of contagious caprine pleuropneumonia.

A specific PCR for the detection of Mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats.

Mycoplasma putrefaciens is listed as one of the etiologic agents of the contagious agalactia syndrome by the world organisation for animal health. This species has been characterized only recently, 1974, and the number of outbreaks caused by this microorganism so far is very scarce. It induces mastitis in infected goats although other symptoms such as arthritis in adults and septicaemia in kids are also frequently described. Up to now, the identification of M. putrefaciens relied on classical isolation and identification techniques which present a number of limitations. Specific primers for PCR have been designed based on sequence comparisons of the ArcB gene among the 'Mycoplasma mycoides cluster' and related species such as Mycoplasma cottewii and Mycoplasma yeatsii. Sequence alignments confirmed the taxonomic position of M. putrefaciens, which is related to the 'M. mycoides cluster' but also very close to M. yeatsii. The polymorphism observed amongst the different ArcB sequences allowed the determination of a primer pair yielding a specific amplification of a 316 bp-long DNA fragment by PCR. This PCR was validated in two different laboratories with a variety of mycoplasma strains isolated from goats. This new PCR technique will be very useful for a quicker determination of M. putrefaciens strains as well as a better understanding of the prevalence of M. putrefaciens infections.