Indonesian Aquaculture Journal, 11 .
, 2016, 81-86 Available online at: http://ejournal-balitbang.
id/index.
php/iaj MOLECULAR DETECTION AND CLONING FOR RICKETTSIA-LIKE BACTERIA OF MILKY HAEMOLYMPH DISEASE OF SPINY LOBSTER Panulirus spp.
Isti Koesharyani*)#.
Lila Gardenia**), and Ni Luh Anggra Lasmika***) **) Centre for Aquaculture Research and Development Institute for Freshwater Aquaculture Research and Development ***) Fish Quarantine and Inspection Agency Mataram
ABSTRACT
Spiny lobster (Panulirus homarus and Panulirus ornatu.
are important commodities for Indonesia.
The aquaculture of lobster is susceptible for several diseases like parasite, fungi, bacteria, and virus.
Among those diseases, milky haemolymph disease (MHD) is often seen as a symptom to mass mortality occurred at lobster farms in Gerupuk Bay of Lombok.
The purpose of this study was to determine the lobster diseases on cage culture in Gerupuk Bay of Lombok.
West Nusa Tenggara.
The study was undertaken from January to March 2015.
Diseases status was determined by application of molecular plat-form, polymerase chain reaction (PCR) with designation of specific primer for MHD .
F/R), 254F: 5Ao-CGA-GGA-CCA-GAG-ATGGAC-CTT-3Ao and 254R: 5Ao-GCT-CAT-TGT-CAC-CGC-CAT-TGT-3Ao with PCR size product of 254 for cloned the pathogen was used 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 pCRA2.
1 plasmid and transformed into competent E.
The result showed almost all lobster samples from Gerupuk Bay were positive infected by MHD, as the results of PCR amplification whereas the band appeared at 254bp.
Also MHD plasmid has been successfully cloned and will be used for further examination.
Histopathologically in hepatopancreas infection have seen necrosis that contain numerous of rickettsia-like bacteria.
KEYWORDS:
MHD.
INTRODUCTION
Indonesia mainly in Lombok.
West Nusa Tenggara (NTB) is a major seed lobster producer and grow-out of wild lobster which have been exported to around the world.
The price of lobster is high in both the domestic and export markets.
Within a year.
NTB produced 78.
5 tons of lobster worth IDR 55.
25 billion.
While the economic value of lobster seed sales reached IDR 16 billion per year (Petersen et al.
, 2.
There are many species of lobster in Indonesia, such as Panulirus homarus.
Panulirus ornatus.
Panulirus versicolor.
Panulirus longipes.
Panulirus polyphagus, and Panulirus penicillatus.
Among those species Panulirus homarus were 90% as a grow-out cultured lobster in cage (Jones, 2015.
Mustafa, 2.
Intensive capture Correspondence: Centre for Aquaculture Research and Development.
Gedung Balitbang KP.
II.
Jl.
Pasir Putih II.
Ancol Timur.
Jakarta Utara 14430.
Indonesian.
Phone: .
64700928 E-mail: istisugama@yahoo.
can lead to a decline in natural population while the lobster can not be cultured.
So it needs the proper techniques for handling the seed, density, feed, and disease prevention during grow-out in cage.
However, along the grow-out of lobster even with the right technique there always have problem of emerging disease.
The disease that commonly attack lobster farming include, rickettsia-like bacteria, true bacteria (Vibrio alginolyticu.
, protozoa, black gill disease kind of fungi (Fusarium sp.
), virus (Panulirusargus V1/ Pa.
, parasite, and big head syndrome.
Those pathogens have had a major impact on the lobster culture (Diggles et al.
, 2000.
Shields & Behringer, 2004.
Behringer, 2012.
OIE, 2007.
Kiryu et al.
, 2009.
Shield, 2011.
Lightner & Redman, 2010.
Stentiford et al.
Stentiford et al.
, 2012.
Jones, 2.
Among the pathogens, milky haemolymph disease or MHD often infects to several species of cultured spiny lobster such as Panulirus homarus.
Panulirus ornatus, and Panulirus polyphagus at all life stages and could cause mortality that reached 70%-100% (Jones, 2.
Milky Copyright @ 2016.
Indonesian Aquaculture Journal, p-ISSN 0215-0883, e-ISSN 2502-657 Molecular detection and cloning for rickettsia-like bacteria .
(Isti Koesharyan.
haemolymph disease (MHD) is a descriptive name for disease affecting spiny lobsters.
The syndrome is given name according to the symptoms of the hemolymph of the affected a lobster appeared to be turbid or feculent/cloudy and turned to a milky liquid.
The turbidity is caused by huge number of rickettsialike bacteria (RLB) circulating in the hemolymph.
MHD
was reported also attack to black tiger shrimp Penaeus monodon farmed in East Africa and Madagascar (Nunan et al.
, 2003.
, wild European shore crabs Carcinus maenas (Eddy et al.
, 2.
and in cultured spiny lobsters Panulirus spp.
in Vietnam (Lightner & Redman.
According information of pathogens that affect to lobster culture farming and associated with the mass mortality that occurred in Lombok, so the disease status was determined through standard identification by polymerase chain reaction (PCR) plat-form and DNA MHD of lobster will cloned by using vector plasmid for other research purpose.
The purpose of this study was to determine the lobster diseases on cage culture in Gerupuk Bay of Lombok through molecular detection and rickettsia-like bacteria cloning.
MATERIALS AND METHODS
Samples Lobster samples with disease symptom were collected from Gerupuk Bay of Lombok.
Indonesia in periods of Januari-March 2015.
A hepatopancreas, haemolymph, and muscle of P.
homarus and P.
were transported by keeping them in 90% ethanol for molecular investigation and in formalin buffer for histopathological purposes.
Polymerase Chain Reaction (PCR) and Cloning Analysis Total DNA extraction was done by Kit DNAzol.
total of 25-30 mg tissue sample and or 100 AAL of haemolymph from naturally infected lobster in 1.
5 mL clean micro tube was homogenized in 1 mL DNAzol.
Homogenate was centrifuged at 12.
000 x g for 10 at room temperature.
Supernatant was then transferred into new tube.
This step removed insoluble tissue fragment.
RNA and excess polysaccharides from the homogenate.
DNA was precipitated from the homogenate with 0.
5 mL of 100% ethanol per 1 mL DNAzol reagent used for the isolation.
Mix sample by inversion and store them at room temperature .
AC to 30AC) for 1 to 3 min.
DNA should quickly become visible as a cloudy precipitate.
DNA
precipitate was centrifuged at 8.
000 x g for 3 min.
room temperature.
carefully decant the supernatant leaving the DNA pellet.
Wash the DNA precipitate twice with 0.
5 mL of 95% ethanol.
Washing was done by inverting the tubes 2-6 times.
The tube was stored vertically for 0.
5 to 1 min.
to allow the DNA to settle to the bottom of the tubes and removed the ethanol by pipetting or decanting.
Air dries the DNA by storing it in an open tube for 5 to15 second after removing the ethanol (If the DNA is exposed to air for more than a few seconds, it will be much more difficult to dissolv.
Dissolve the DNA in 100-200 AAL Triss EDTA (TE) or Water.
Dnase.
RNase free (Deionized water treated with 0.
001 DEPC filtered through 0,2 micron filter and autoclave.
, and as template DNA for PCR amplification and other investigation.
PCR amplification was done in Speed STARTM HS TAKARA RRO 70A contained: Buffer I, dNTPs.
SpeedSTARS HS DNA polymerase each 1 AAL reverse and forward primers 10 pMol and nuclease-free water.
Primers were designed from geographic origin of lobster from Vietnam.
Primer set designation: 254F/ : 254F: 5Ao-CGA-GGA-CCA-GAG-ATG-GAC-CTT-3Ao and 254R: 5Ao-GCT-CAT-TGT-CAC-CGC-CAT-TGT-3Ao the expected PCR product size was 254 bp (OIE, 2.
The optimation cycle condition program was pre-denaturation for 3 sec.
at 96AC, 40 cycles for denaturation for 5 secat 96AC.
annealing/elongation for 30 sec.
65AC then followed by an extension period for 3 sec.
at 72AC after the last cycle.
Reaction PCR mixes prepared in 20 AAL were amplified in an automatic thermal cycler (AB.
AB.
applied biosystem, verity.
96 well fas.
Analysis of PCR products or amplification was running on a 1.
5% gel electrophoresis in 1x TAE buffer and stained with ethidium bromide, the target product was measured using a fast ruler low range DNA ladder .
1,500 b.
under UV-Trans illuminator.
Cloning, the PCR amplicons were ligated with TA cloning.
DNA PCR product were ligase using the vector plasmid pCRA2.
1-TOPO system (INVITROGEN).
Ligation transformed into competent E.
coli were plated onto Luria-Bertani (LB) media containing 100 AAg antibiotic ampicillin and containing Xgal .
-bromo4-chloro-3-indolyl-AAD-galactosid.
and IPTG .
sopropyl-1-thio-A-D-galactosid.
Plates were incubated overnight at 37AC.
White and single colonies were isolated then scratched at LB plate for mass culture in LB both, cultured overnight at 37AC put on a shaker.
DNA was extracted from the cultures byusing Plasmid Mini Kit (Gene Ai.
Plasmid DNA concentrations and purity were estimated by measuring the 260/280 optical density ratios by Nanodrop.
Ligation of correct products was confirmed by restriction enzyme digestion EcoRI .
AAL 10x Buffer RE.
5 AAL EcoRI enzyme.
5 AAL H2O.
6 AAL DNA plasmi.
and analysis of products on agarose gels.
Right Plasmid DNA were store in mix 0.
85 mL of culture with 0,15 mL sterile Glycerol and transfer to cryovial and store at -80AC.
Copyright @ 2016.
Indonesian Aquaculture Journal, p-ISSN 0215-0883, e-ISSN 2502-6577 Indonesian Aquaculture Journal, 11 .
, 2016, 81-86 Histopathology analysis.
Infected tissue (Hepatopancreas orga.
were fixed in DavidsonAos solution for 24 hours then transferred for refixation process in 70% ethanol solution for further histopathologycal Samples of lobster were analyzed at the Division of Veterinary Pathology Section.
Faculty of Veterinary Medicine.
Bogor Agricultural University.
RESULTS AND DISCUSSION
Mortality of spiny lobsters P.
homarus and ornatus with similar MHD symptom were found in grow-out floating sea cages culture at Gerupuk Bay Lombok.
The symptoms or gross sign of naturally infected spiny lobster were milky appearance in abdomen, in tail muscle, necrosis on hepatopancreas, lethargy, poor feed response, and significant mortality (Figure .
Severe infected lobster was characterized by special sign not only marked with white color like milk on the abdomen but also on part of hepatopancreas releasing a milky liquid, severely affected lobster become lethargic, lose appetite then moribund (Nunan et al.
, 2.
Extracted DNA from infected spiny lobsters .
epatopancreas, haemolymph, and both muscle of P.
homarus and P.
from Gerupuk Bay were amplified using specific primer pairs for MHD (OIE.
The thermal cycling PCR program have been optimized as shown in part material and method All samples were positive by rickettsia-like bacteria (Table .
Infection, as evidenced from PCR examination, suggested that the intracellular Figure 1.
rickettsia-like bacterium (RLB) was the cause of disease and resultant mortalities in the samples of lobster from Lombok Island.
The causative agent of the disease was a genus Rikettsia Gram-negative bacterium obligate intracellular parasite that could not be cultured in artificial nutria environments.
Attempts to isolate and grow the potential causative agent on a wide range of agar-based culture media failed despite the use of long incubation periods and a range of dilutions of the infected haemolymph (Eddy et al.
Rikettsia was grown either in tissue or embryo culture i.
chicken embryo.
Rickettsia is obligate intracellular pathogenic bacteria that grow only within the cytoplasm, and occasionally the nucleus, of eukaryotic cells (Frutos et al.
, 1994 in Nunan et al.
Amplification fragment from all tissue hepatopancreas, haemolymph, muscle of P.
homarus and P.
was ilustrated on a single 254 bp of PCR.
Amplification product was run on 1.
5% agarose gel and staning with ethidium bromide is shown in Figure 2.
Additional data of artificial infection of MHD by injection pathway, showed that the clinical symptom appear on the day fourth after injection with antigen of MHD-spiny lobster and infected lobster were began loss of appetite in day ten then finally mass mortality occurred at day fourteen (Lasmika, 2.
This is in conformity with the opinion of Nunan et al.
which MHD-SL highly virulent and could cause mass mortality of up to 100%.
Lobster infected by MHD, (Black arro.
in part of abdomen looks white color like milk .
were found in grow-out floating sea cages culture in Gerupuk Bay.
Lombok Copyright @ 2016.
Indonesian Aquaculture Journal, p-ISSN 0215-0883, e-ISSN 2502-657 Molecular detection and cloning for rickettsia-like bacteria .
(Isti Koesharyan.
Table.
Diagnostic amplification PCR result of MHD in natural infection of spiny lobster samples from Gerupuk Bay.
Lombok Organ PCR tested Hepatopancreas of Panulirus homarus Haemolymph of Panulirus homarus Muscle tissue of Panulirus homarus Muscle tissue of Panulirus ornatus Positive Positive Positive Positive 1500 bp 850 bp 400 bp 254 bp 200 bp Figure 2.
Amplification PCR product of natural infection MHD of spiny lobster from different tissue in 1.
5% agarose gel, 1= hepatopancreas.
2= haemolymph.
3= muscle Panulirus 4= muscle Panulirus ornatus.
M= marker FastRuler Low Range DNA Ladder .
1,500 b.
Several species of mollusc, fish, and crustacean were detected as infected with Rickettsia like Organism/RLO (Galvan et al.
, 2.
Black tiger shrimp Penaeus monodon.
European shore crab Carcinus maenas and spiny lobster Panulirus spp.
affected by milky haemolymph syndrome (Nunan et al.
, 2010 and Nunan et al.
, 2003.
The result of experimental infection of P.
vannamei by a rickettsia-like bacterium (RLB) originating from P.
monodon, showed that P.
vannamei developed RLB infection when injected with either RLB infected P.
monodon or P.
vannamei tissue homogenates (Nunan et al.
, 2003.
Consequently, it is should be paid attention because the disease can infect to shrimps by artificial injection of homogenated RLO which shrimps were huge culture in Indonesia.
This condition will be more dangerous because P.
monodon is also very sensitive to WSSV infections that harmful virus.
Therefore, prevention needs to be done for transmission route the MHD to other aquaculture by shorter cropping period, prophylactic antibiotics in feed, use of vitamins, immunostimulants, and use probiotics in feed.
Cloning DNA of MHD.
coli bacteria bring the recombinant plasmid DNA of MHD were mass cultured in Luria Berani Broth .
% trypton, 0.
5% yeast extract, 1% NaCl in pH 7 containing 100 AAg/mL of ampicilli.
and further purified.
Confirmation of the success of the cloning was done by restriction enzyme EcoRI and PCR test against recombinant plasmid DNA purified.
Plasmids of MHD were stored in 80AC as positive control and for further experiment, sequencing (Figure .
The histopathology analysis of MHD in spiny lobster.
RLB appeared visible on the hepatopancreas tissue.
Photomicrograph-histopathology of hepatopancreas organ .
x magnificatio.
in ephitel tubulus hepatopancreas have seen necrosis and part of the lumen of hepatopancreas contain huge number of Rickettsia-like bacteria.
Deparaffinized slide sections were stained with Haematoxyln-Eosin/H & E (Figure .
Furthermore studies on this pathogen are needed to classify this organism that was found from Indonesia.
Comparison from the same gene in other rickettsia like-bacteria will be analyzed and placed in a phylogenic tree.
Molecular diagnostic procedures developed in this study will be a standard after validate the specificity and potential utility of these techniques.
CONCLUSION
Milky haemolymph desease (MHD) was found in spiny lobster from Gerupuk Bay.
Lombok.
West Nusa Copyright @ 2016.
Indonesian Aquaculture Journal, p-ISSN 0215-0883, e-ISSN 2502-6577 Indonesian Aquaculture Journal, 11 .
, 2016, 81-86 254 bp Figure 3.
White colonies then scratched at LB plate for mass culture in LB broth and PCR product cloned DNA plasmid of MHD Figure 4.
Photomicrograph-histopathology of hepatopancreas organ .
x magnificatio.
, .
ephitel tubulus hepatopancreas with necrosis .
lack arro.
and the lumen of hepatopancreas contain huge number of bacteria .
lack triangl.
, .
normal tissue.
slide sections were stain with haematoxyln-eosin (H & E) Tenggara.
Indonesia.
Histopathological analysis showed necrosis in hepatopancreas and huge number of Rickettsia-like bacteria in lumen of tubulus in hepatopancreas of infected lobster.
Cloned of MHD from spiny lobster can be used for further experiment.
ACKNOWLEDGEMENTS
We thank to Grit Mrotzek as a senior technician at HKI Germany for their assistance in molecular diagnosis and to Mr.
Johan Afandi as technician at Fish Health Laboratory, for their help in PCR and cloning Further thanks is extended to Mrs.
Dewi Ratih Agungpriyono DVM Devision of Veterinary Pathology Section.
Faculty of Veterinary Medicine.
Bogor Agricultural University for her helping in providing histopathology analysis of MHD mortality at the experimental site.
REFERENCES