Isolation, characterisation and DNA analysis of Mycoplasma spp. from moribund prawns Penaeus monodon cultured in Australia

For the first time, a total of 14 Mycoplasma isolates were cultured rom 24 moribund prawns investigated during an outbreak of mid-crop mortality syndr ome in northern Queensland, Australia. Mycoplasma were isolated from the gi ll appendages, brains and eyes of the prawns. Mycoplasma growth occurred be tween 20 and 37 degrees C with or without CO2 in modified Frey's medium con taining 0.5 to 3.0% sodium chloride and 20% foetal bovine serum. Optimal gr owth was observed at 37 degrees C with 5% CO2. All strains were size filter ed and cloned, and their morphology, biochemical and biomolecular character istics were compared with the characteristics of previously described Mycop lasma species. The results showed that these strains belonged to 2 new spec ies, for which the temporary names Mycoplasma P1 (MP1) and Mycoplasma P2 (M P2) were designated. Both Mycoplasma fermented most of the tested carbohydr ates but did not hydrolyse arginine and urea. MP1 produced films and spots and had high phosphatase activity, but MP2 did not produce films or spots a nd had no phosphatase activity. Both species lysed sheep erythrocytes. A ge nomic library (Mbo1 digested) was prepared from MP1 DNA and cloned into pUC 19. Colony hybridisation, using a probe prepared from purified MP1, was use d to identify colonies of interest. MP1 DNA fragments were retrieved from r ecombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridisation analysis with immobilised DNA from MP1, MP2, M, bovis, M. dispar, M. agalactiae, M. bovig enitalium, M. ovipneumoniae, Mycoplasma Group 7, M. arginini and bacteria b elonging to different genera. The probe reacted with genomic DNA from MP1 o nly. To further enhance the sensitivity, an MP1 specific polymerase chain r eaction (PCR) assay was designed and produced a 254 bp amplicon which discr iminated MP1 from all other Mycoplasma DNA tested. Using the DNA probe and PCR assay, most of the Mycoplasma isolated from the diseased prawns could b e designated as strain MP1 (11/14, similar to 80 %).