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

Abstract

For the first time, a total of 14 Mycoplasma isolates were cultured from 24 moribund prawns investigated during an outbreak of mid-crop mortality syndrome in northern Queensland, Australia. Mycoplasma were isolated from the gill appendages, brains and eyes of the prawns. Mycoplasma growth occurred between 20 and 37°C with or without CO2 in modified Frey's medium containing 0.5 to 3.0% sodium chloride and 20% foetal bovine serum. Optimal growth was observed at 37°C with 5 % COz. All strains were size filtered and cloned, and their morphology, biochemical and biomolecular characteristics were compared with the characteristics of previously described Mycoplasma species. The results showed that these strains belonged to 2 new species, for which the temporary names Mycoplasma P1 (MPI) and Mycoplasma P 2 (MP2) were designated. Both Mycoplasma fermented most of the tested carbohydrates 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 and had no phosphatase activity. Both species lysed sheep erythrocytes. A genomic library (Mbol digested) was prepared from MP1 DNA and cloned into pUC19. Colony hybridisation, using a probe prepared from purified MPI, was used to identify colonies of interest. MP1 DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridisation analysis with immobhsed DNA from MP1, MP2, M. bovis, M. dispar, M. agalactiae, M. bovjyenitalium, M. ovipneumonjae, Mycoplasma Group 7, M. aryinini and bacteria belonging to different genera. The probe reacted with genornic DNA from MP1 only. To further enhance the sensitivity, an MP1 specific polymerase chain reaction (PCR) assay was designed and produced a 254 bp amplicon which discriminated MP1 from all other Mycoplasma DNA tested. Using the DNA probe and PCR assay, most of the Mycoplasma isolated from the diseased prawns could be designated as strain MP1 (11/14, -80%).