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MOLECULAR DETECTION & CLONING FOR RIICKETTSIA-LIKE BACTERIA OF MILKY HAEMOLYMPH DISEASE OF SPINY LOBSTER Panulirus spp.

Isti Koesharyani*, Lila Gardenia & Ni Luh Anggra Lasmika
Centre for Aquaculture Research and Development
Jl. Rangunan No. 20, Pasar Minggu
Jakarta Selatan 12540
istisugama@yahoo.com

Spiny lobster (Panulirus homarus and Panulirus ornatus) are important commodities in Indonesia. The aquaculture of lobster is susceptible for several diseases like parasite, fungi, bacteria and virus. Among those diseaes, Milky Hemolymph Disease (MHD) is often seen as a symptomp to mass mortality occured at lobster farms in Gerupuk Bay of Lombok. The aim of this study is to determine the lobster diseases on cage culture in Grupuk Bay of Lombok, the study was undertaken from Januari to March 2015. The determination of diseases was by application of   molecular plat-form, polymerase chain reactin (PCR) with designation of specific primer for MHD  (254F/R), 254F: 5'-cga-gga-cca-gag-atg-gac-ctt-3' and 254R: 5'-gct-cat-tgt-cac-cgc-cat-tgt-3' with PCR size product of 254 bp. and for cloned the patogen was use TA-cloning  Invitrogen for the DNA plasmid as positive control for other analysis. Several tissue samples i.e hepatopancreas, Haemolymph, part of muscle Hepatopancreas P. homarus and P. ornatus were taken from cage culture farms at Gerupuk Bay then preserved on 90% Ethanol for further analysis  by PCR and then the amplificated DNA were cloned into pCR®2.1 plasmid and transformed into competent E. coli.

In the present study almost all lobster samples from Gerupuk Bay were positive  by MHD, as the results of PCR amplification whereaas the band appear at 254 bp. (Table. 1). Milky haemolymph Disease (MHD) is a descriptive name for diseases affecting spiny lobsters. The syndrome is given name according to the symptoms. the hemolymph of the affected lobster appear to be  turbid or faculent and turns to a milky liquid. Milky liquid can be recognize in infected lobsters. The turbidity is caused by huge number of rickettsia-like bacteria (RLB) circulating in the hemolymph (Fig 1)   




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