Scientific Article Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster J Fitzmaurice *§ , M Sewell * , L Manso-Silván † , F Thiaucourt † , WL McDonald * and JS O’Keefe * Abstract AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to al- low differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. my- coides subsp mycoides large colony (MmmLC), M. mycoides sub- sp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspe- cies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the as- says. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detec- tion limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respec- tively. The M. mycoides subspecies assay had a detection limit of 10 3 (SD 10 2 ) cfu/ml milk, 10 4 (SD 10 4 ) cfu per swab, and 10 3 (SD 10 3 ) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the mem- bers of the M. mycoides cluster. KEY WORDS: Mycoplasma mycoides cluster, real-time PCR, di- agnostic assays Introduction The M. mycoides cluster is comprised of M. mycoides subsp my- coides large colony (MmmLC), M. mycoides subsp capri (Mmc), M. mycoides subsp mycoides small colony (MmmSC), M. capri- colum subsp capricolum (Mcc), M. capricolum subsp capripneumo- niae (Mccp), and Mycoplasma sp bovine group 7 (BG7) (Taylor et al 1992). Infection with Mmc in goats causes similar clinical signs to that caused by MmmLC, including pneumonia, polyar- thritis, mastitis, keratoconjunctivtis and septicaemia (DaMassa et al 1992; Taylor et al 1992), and the two cannot be distinguished using biochemical tests. DNA-DNA hybridisation studies on MmmLC and Mmc have reported homology ranging from 75% to 94% (Askaa et al 1978; Christiansen and Erno 1982), and sequencing of the 16S rRNA genes revealed that the two were 99.9% identical (Pettersson et al 1996). Phylogenetic studies based on the sequencing of a putative membrane protein gene did not separate MmmLC and Mmc, and it has been proposed that these two subspecies be considered as a single species (Thiaucourt et al 2000). MmmSC is the causative agent of contagious bovine pleuropneumonia, a severe respiratory disease of cattle, and of considerable economic importance (Anonymous 2000). It is a listed communicable animal disease of the Office International des Épizooties (OIE). Mcc has been reported to cause mastitis, arthritis, keratoconjunctivitis, pneumonia and vulvovaginitis in goats (Bölske et al 1988). Contagious caprine pleuropneumonia (CCPP) is an infectious respiratory disease affecting goats, and is also a listed disease of the OIE; the causative agent of CCPP is Mccp (Thiaucourt and Bölske 1996), which has previously been referred to as Mycoplasma F38 (Leach et al 1993). The final mem- ber of the mycoides cluster, BG7, has been reported as the cause of mastitis (Connole et al 1967), polyarthritis (Shiel et al 1982) and abortion in cattle (Hum et al 2000), and pneumonia in calves (Alexander et al 1985). In New Zealand in 2001, an outbreak of MmmLC occurred in goat kids on a dairy-goat farm and in calves fed unpasteurised goat milk and colostrum from the affected dairy-goat herd. This outbreak gave rise to the practical problems of differentiating en- demic MmmLC, using biochemical testing, from the other exotic members of the M. mycoides cluster. Diagnostic testing for members of the M. mycoides cluster poses difficulties because of the similarities in disease symptoms caused by each species and the high degree of similarity between them both phenotypically and genetically. In addition, intra-species heterogeneity has been observed in MmmLC/Mmc and Mcc, * Investigation and Diagnostic Centre, Biosecurity New Zealand, Ministry of Agriculture and Forestry, PO Box 40742, Upper Hutt, New Zealand. † CIRAD EMVT Santé Animale, TA30/G Campus International de Baillarguet, 34398 Montpellier Cedex 5, France. § Author for correspondence. Email: justine.fitzmaurice@maf.govt.nz BG7 Mycoplasma sp bovine group 7 CCPP Contagious caprine pleuropneumonia Mccp Mycoplasma capricolum subsp capripneumoniae Mcc Mycoplasma capricolum subsp capricolum Mmc Mycoplasma mycoides subsp capri MmmLC Mycoplasma mycoides subsp mycoides large colony MmmSC Mycoplasma mycoides subsp mycoides small colony OIE Office International des Épizooties PBS Phosphate buffered saline SBA Sheep blood agar New Zealand Veterinary Journal 56(1), 40-47, 2008 40