AACL BI OFLUX
Aqu a cu lt u r e , Aqu a r iu m , Con se r va t ion & Le gisla t ion
I n t e r n a t ion a l Jou r n a l of t h e Bioflu x Socie t y
PCR de t e ct ion of w h it e spot sy n dr om e v ir u s
( W SSV ) fr om fa r m e d Pa cific w h it e sh r im p
(
Lit ope n a e u s va nn a m e i
) in se le ct e d sit e s of t he
Ph ilippin e s
1
Benedict A. Maralit ,
2Christ opher M. A. Caipang,
1Mudj ekeewis D. Sant os,
and
3Mar y Bet h B. Maningas
1
Nat ional Fisheries Research and Dev elopm ent I nst it ut e, Quezon Av enue, Quezon Cit y Philippines 1103; 2Facult y of Biosciences and Aquacult ure, Univ ersit y of Nordland, Bodø 8049, Norw ay; 3Research Cent er for t he Nat ural and Applied Sciences, Thom as Aquinas Research Com plex / Biological Sciences Depart m ent , College of Science, Univ ersit y of
Sant o Tom as, Philippines 1008. Corresponding aut hors: M. B. Maningas, m ary bet hm aningas@gm ail. com and C. M. A. Caipang, cm acaipang@y ahoo. com
Abst r a ct. Gr eat losses caused by whit e spot syndr om e virus ( WSSV) in shr im p cult ur e have been at t r ibut ed t o poor scr eening pr ocedur es in far m s and t he lack of sufficient access t o specific pat hogen fr ee br ood st ock. Thus, ear ly det ect ion of t he vir us is consider ed t he best opt ion for shr im p far m er s. The st udy, t hus, assessed vir al incidence in t he Philippines and par t ially sequenced and char act er ized t he Philippine WSSV isolat e wit h r egar ds t o ot her isolat es in GenBank. Developed pr im er s for PCR can det ect t ar get genes fr om 0. 4 pg of DNA ext r act fr om shr im p sam ples. PCR det ect ion r evealed t hat 6. 67 % ( 1/ 15) of m ar ket sam ples fr om Zam bales ar e infect ed wit h WSSV. Shr im p sam ples fr om a local shop and a public m ar ket in Gener al Sant os Cit y showed 46. 67% ( 7/ 15 ) and 20% ( 3/ 15) WSSV- posit ive sam ples r espect ively. Shr im p sour ces fr om Capiz and Bat angas, however , showed negat ive det ect ion for WSV. No significant differ ence in t he num ber of infect ed sam ples fr om t he sam pling sit es was found. Com bined det ect ions r eveal t hat t he Philippines has a low infect ion r at e of 1 4. 67% . The st udy has par t ially sequenced and char act er ized Philippine isolat e. Dur ing t he sam pling per iod, m ost shr im ps in Gener al Sant os Cit y wer e WSSV- posit ive by PCR det ect ion.
Ke y W or ds: WSV, Philippines, shr im p, Lit openaeus vannam ei.
I n t r odu ct ion. Global product ion of sea foods from aquacult ure has grow n rapidly ov er t he past four decades, cont ribut ing t o 45% of t he w orld’s supply of fish (including crust aceans, m ollusk s, echinoderm s and am phibians) for hum an consum pt ion ( Subasinghe et al 2009) . The shrim p aquacult ure indust ry has grow n rapidly ov er sev eral decades t o becom e a m aj or global indust ry t hat has cont ribut ed significant ly t o socio-econom ic dev elopm ent in m any poor coast al com m unit ies (Walk er & Mohan 2009) . How ev er, t he ecological dist urbances and changes in pat t erns of t rade associat ed wit h t he dev elopm ent of shrim p farm ing hav e unfort unat ely hindered furt her grow t h because of t he em ergence and spread of v iral and bact erial disease.
( Tendencia & Usero 2007) . Recent report s on shrim p done by de la Peña et al ( 2008) det erm ined t he prev alence of m onodon baculov irus ( MBV) in w ild P. m onodon. The lat est report s are done by Tendencia et al ( 2010) ex ploring t he effect of rainfall and at m ospheric t em perat ure on t he prev alence of t he WSSV in pond cult ured P. m onodon. Most of t hese dat a, how ev er, are com prehensiv e only for t he Visay as region of t he Philippines, ev en if t he presence of viruses is cont inuously confirm ed by shrim p farm ers in Luzon.
To dat e, viral disease problem is st ill in full effect largely because t here has not been any significant adv ancem ent in effect iv ely eradicat ing or m inim izing t he effect s of t hese pat hogenic shrim p viruses. The Philippines, in it s effort t o prev ent t he int roduct ion of t hese viruses in t he count ry , has im posed a ban on t he im port at ion of shrim ps from ot her count ries. How ev er, t his ban has been lift ed t o reviv e t he ailing shrim p aquacult ure indust ry t hat w as once included in t he largest producers of shrim p in t he w orld ( Aguiba 2007) .
I n May 2008, during 6t h Philippine Shrim p Congress, t hree k ey fact ors hav e been ident ified in order t o successfully reviv e t he count ry ’s ailing shrim p indust ry. These include 1) t he cult ure of specific- pat hogen free ( SPF) and specific- pat hogen resist ant ( SPR) broodst ock and “ high healt h” fry ; 2) use of best m anagem ent pract ices ( BMPs) such as probiot ics and biosecurit y m easures; and 3) m ark et ing and com pliance t o food safet y regulat ions ( Moj ica 2008) . How ev er, for t hese init iat iv es t o be successful, especially t he dev elopm ent of BMPs and SPF/ SPR brood st ock s and fries, it is im perat iv e t hat recent inform at ion on t he prev alence and det ect ion of pat hogens in t he different regions of t he count ry affect ing t he indust ry be docum ent ed. Ut ilizat ion of current ly av ailable m olecular biology and biot echnology- based applicat ions lik e poly m erase chain react ion ( PCR) can prov ide such im port ant inform at ion and applicat ions. Thus, it is t he aim of t he st udy t o det ect and det erm ine WSSV prev alence in t he count ry t hrough t he select ed sam pling sit es and part ially sequence and charact erize t he Philippine WSSV isolat e wit h regards t o t he WSSV isolat es st ored in Genbank. I t is not ew ort hy t o m ent ion t hat t his is t he first report on WSSV det ect ion in L. v annam ei in t he count ry .
M a t e r ials a n d M e t h ods. Sam ple collect ion of L. v annam ei w as done in Bat angas, Zam bales, Capiz and General Sant os ( Sout h Cot abat o) from Oct ober 2009 and June 2010. During each collect ion, 2 shrim p sam ples of each k ind w ere preserv ed in et hanol t o be used for ident ificat ion. Shrim p sam ple ident ificat ion w as done at t he Nat ional Fisheries Resources Dev elopm ent I nst it ut e ( Quezon Cit y, Philippines) w here t he sam ples w ere ident ified according t o m orphological st andards used in shrim ps.
Each PCR react ion cont ained 1 unit Taq polym erase, 0.6 M each of designed WVF ( 5’ GTA CGG CAA TAC TGG AGG AGG 3’) and WVR prim ers ( 5’ GGA GAT GTG TAA GAT GGA CAA GG 3’) or published prim ers ( Tapay et al 1999) , 0. 2 M dNTP solut ion m ix , and 1x buffer w it h 1. 5 m M MgCl2. React ions w ere cy cled in t he program : init ial denat urat ion
t em perat ure at 95 ° C for 5 m inut es, 35 cy cles of denat urat ion t em perat ure at 95 ° C for 30 seconds, annealing at 60° C for 30 seconds, and ex t ension at 72 ° C for 30 seconds, and Final DNA ex t ension at 72 ° C for 5 m inut es and follow ed by 4 ° C st orage. PCR product s w ere t hen run on a 1% agarose gel and v iew ed under UV light .
Re su lt s an d D iscu ssion. Tw o frozen shrim p sam ples wit h gross signs of WSSV w ere subj ect ed t o PCR det ect ion using LMT prim ers published by Tapay et al ( 1999) . The prim er set w as successful only in det ect ing WSSV in one of t he sam ples ( Figure 1) , t hough success and efficiency in t he am plificat ion of Philippine shrim p WSSV isolat es hav e been confirm ed by Nat ividad et al ( 2006) . These prim ers w ere designed from a cloned fragm ent of t he WSSV genom e w hich m ay hav e a low copy num ber DNA in t he sam ples.
1 2 3 4 5 6 7
Figure 1. PCR det ect ion of 2 shrim p sam ples from t he Philippines using designed ( WVF) and published prim ers ( LMT) . Lane 1: low m ass DNA ladder, lane 2: negat iv e cont rol for
WVF prim ers, lane 3: sam ple 1, lane 4: sam ple 2, lane 5: negat iv e cont rol for LMT prim ers, lane 6: sam ple 1, Lane 7: sam ple 7.
Using designed prim er set s, an approxim at ely 150 bp DNA fragm ent w as am plified from t he t w o sam ples ( Figure 1) . Serial dilut ion rev eals t hat t he designed prim ers, PCR m ix and condit ions hav e a det ect ion lim it of 0. 4 pg. This prot ocol proves t o be m ore sensit iv e t han t hat of Tapay et al ( 1999) w hich has a det ect ion lim it of 10 pg.
Pairwise sequence alignm ent of t he 2 field sam ples show s 100% ident it y wit h each ot her ( Figure 2) .
Global alignm ent using Blast n indicat es t hat t he t arget DNA t em plat e is conserv ed am ong ot her isolat es in Genbank w it h 100% ident it y ( Figure 3) . This rev eals t hat , based on t he DNA fragm ent s am plified, t he sequences of Philippine isolat es are v ery sim ilar t o t he published sequences of China, Taiw an and Thailand isolat es ( Nat ividad et al 2006) . Thus, t his designed prim er set w as used in all subsequent WSSV det ect ions.
Figure 3. Sequences producing significant alignm ent s in GenBank ( blast n).
PCR det ect ion for Luzon rev ealed Bat angas sam ples from a specific pond t o be negat iv e for WSSV, despit e t he presence of a disease out break during t he sam pling period in nearby ponds. According t o t he pond operat or and ow ner, t he non- infect ion of t he pond m ay be caused by get t ing w at er from a source different from t he ones t hat got infect ed. Meanw hile, one ( 1/ 15 or 6. 67% ) m ark et sam ple from Zam bales ret urned a posit iv e result ( Figure 4) .
1 2 3 4 5 6 7 8 9 10 1 1
Figure 4. WSSV det ect ion for Zam bales sam ples, lane 1: ladder, lane 2: negat ive cont rol, lane 3: posit iv e cont rol, lane 4 t o 11: sam ples 1 t o 8.
A v ery low viral load in t he sam ple m ay hav e result ed in a v ery faint posit iv e band in t he PCR det ect ion. On t he ot her hand, all Capiz m arket sam ples are found t o be negat iv e for WSSV, show ing a different scenario in shrim p farm s in Negros, w here WSSV infect ions are observ ed annually ( Tendencia et al 2010) . Sam ples from a farm and a shop in General Sant os Cit y w ere found t o be posit iv e for WSSV. For inst ance, fresh sam ples bought from a local shop in t he cit y w ere found posit ive, 7/ 15 or 46. 67% ( Figure 5) . Meanw hile, 3/ 15 ( 20% ) pond sam ples from sam e sam pling region w ere also found posit iv e for WSSV. This is t he second report for high prev alence of WSSV in t he area ( Leung & Engle 2006) .
1 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16 1 7 18
Figure 6 show s t he sum m ary of WSV incidence in each of t he sam pling sit es. One sam ple t - t est ( SPSS Soft w are Version 13) at 95% confidence int erv al rev eals t hat t here is no significant difference in t he num ber of WSSV posit ive sam ples in t he sam pling sit es. How ev er, m ost of t he WSSV infect ions w ere observ ed in t he Mindanao sam pling sit es. Taking int o account t he result s of WSSV det ect ion from all t he sam pling sit es, t he Philippines has a low ov erall WSSV infect ion of 14. 67% com pared t o t he first report of WSSV out break in 2000 w here an ov erall prev alence of 71. 83% ( 51/ 71) w as observ ed.
Figure 6. Dist ribut ion of WSV posit iv e sam ples based on PCR det ect ion.
I n 1999, t he Philippine shrim p, w het her cult ured or w ild, is st ill WSSV free. How ev er, frozen shrim p im port ed t o t he Philippines w ere found posit ive ( Belak et al 1999) . This m ay be t he cause of t he first WSSV report in 2000 ( Tapay et al 2000) alt hough st rong evidence is lacking as viral dy nam ics of WSSV in t he environm ent is not com plet ely underst ood. Report s in t he following y ears show ed 20. 75% av erage annual WSSV prev alence in shrim p farm s in Negros I sland ( Tendencia & Usero 2007) . This is support ed by Leung & Engle ( 2006) list ing Negros, Cebu and Mindanao, around t he cit ies of Dav ao and General Sant os as sev erely affect ed high densit y P. m onodon farm ing areas. The current st udy confirm s t hat General Sant os Cit y is st ill affect ed by WSSV, t hough not as sev ere as t he previous report . I t is apparent t hat t he virus persist s in localized regions suggest ing t hat cert ain fact ors allow for t he persist ence of t he virus in t he environm ent ov er t he y ears. How ev er, general pract ices som ehow m ak e t he v irus m anageable t o a cert ain ex t ent t hat large m ort alit ies and infect ion in ot her areas, w hich hav e nev er been infect ed, are prevent ed. Subsequent ly , it is alarm ing t hat WSSV infect ion w as det ect ed in
L. v annam ei, w hich are supposed t o be SPF ( shrim p pat hogen free) as t hese w ere im port ed from ot her count ries. These findings hav e im plicat ions in t he current best m anagem ent pract ices of t he shrim p aquacult ure indust ry . The decree on t he “ lift ing of t he ban for t he im port at ion of L. v annam ei” in t he count ry m ight need revisit ing.
Con clusion s. The st udy has part ially sequenced and charact erized Philippine WSSV isolat e. I t concludes t hat t he m ost WSSV- infect ed shrim ps w ere found in General Sant os Cit y during t he sam pling period. On t he ot her hand, no infect ions w ere found in Bat angas and Capiz. No significant difference in t he num ber of infect ions w as found in the sam pling sit es. The st udy suggest s t hat nat ural infect ions of WSSV are m anageable in Philippine shrim p farm s as show n by t he low ov erall det ect ion. I t is alarm ing t hough, t hat som e regions of t he count ry t est ed posit iv ely, since L. v annam ei shrim ps are supposedly clear of any pat hogen ( SPF) as t hey ent er t he Philippine t errit ory.
and t he Graduat e School, Universit y of Sant o Tom as; and t he Genet ic Fingerprint ing Laborat ory , Nat ional Fisheries Research and Dev elopm ent I nst it ut e.
Re fe r en ce s
Aguiba M. M. , 2007 Shrim p indust ry faces st ronger grow t h: DA lift s im port ban on ‘w hit e’ broodst ock . Manila Bullet in. ( ht t p: / / w w w . m b. com .ph/ node/ 2505) Last accessed June 13, 2011.
Albaladej o J. D. , Tapay L. M. , Migo V. P. , Alfafara C. G. , Som ga J. R. , May o S. L. , et al., 1998 Screening for shrim p viruses in t he Philippines. I n: Flegel, T. W. ( Ed. ) , Adv ances in Shrim p Biot echnology. Nat ional Cent er for Genet ic Engineering and Biot echnology , Bangk ok , Thailand.
Belak J., Dhar A. K. , Prim avera J. H., de la Peña L. D. , Pet t it P. , Alciv ar- Warren A., 1999 Prev alence of Viral Diseases ( I HHNV and WSSV) in Penaeus m onodon from t he Philippines and it s associat ion w it h m angrov e st at us and shrim p cult ure sy st em s. I n: Alciv ar- Warren A ( ed) . Proceedings of t he Aquacult ure and Conserv at ion of Marine Shrim p Biodiv ersit y Sy m posium . Tuft s Univ ersit y School of Vet erinary Medicine, Nort h Graft on.
De la Peña L. D. , Lavilla- Pit ogo C. R., Villar C. B. R. , Paner M. G., Capulos G. C. , 2008 Prev alence of m onodon baculovirus ( MBV) in wild shrim p Penaeus m onodon in t he Philippines. Aquacult ure 2 8 5: 19–22.
Leung P. S. , Engle C. , 2006 Shrim p cult ure: econom ics, m ark et and t rade. 1st ed. Black well Publishing Professional, USA, pp 220- 230.
Light ner D. V. , 2003 The Penaeid Shrim p Viral Pandem ics due t o I HHNV, WSSV, TSV and YHV: Hist ory in t he Am ericas and Current St at us. Proceedings of t he 32nd UJNR Aquaculure Panel Sy m posium , Davis and Sant a Barbara, California, USA, Nov em ber 17- 18 and 20, 2003.
Moj ica M. J. , 2008 Shrim p Making Wav es Again. BAR Research and Dev elopm ent Digest
1 0: 3. ( ht t p: / / w w w . bar. gov. ph/ bardigest / 2008/ j ulsep08_w hit e_shrim ps. asp) .
Nat ividad K. D. T., Mat sum ura M., Migo, M. V. P. , Albadej o J. D. , Magbanua J. P. V. , Nom ura N. , 2006 Sim ult aneous PCR det ect ion of t w o shrim p v iruses ( WSSV and MBV) in post larv ae of Penaeus m onodon in t he Philippines. Aquacult ure 2 5 7: 142-149.
Sant os M. D. , Lopez G. V. , Barut N. C. , 2010 A pilot st udy on t he genet ic v ariat ion of East ern lit t le t una (Eut hy nnus affinis) in Sout heast Asia. Phil J Sci 1 3 9: 43- 50.
Subasinghe R. , Sot o D., Jia J. , 2009 Global aquacult ure and it s role in sust ainable dev elopm ent . Rev Aquac 1: 2–9.
Tapay L. M. , Nadala E. C. B. Jr., Loh P. C. , 1999 A poly m erase chain react ion prot ocol for t he det ect ion of v arious geographical isolat es of whit e spot virus. J Virol Met hods 8 2: 39- 43.
Tapay L. M., Magbanua F. O. , Nat ividad K. T., Migo V. P. , Alfafara C. G. , De la Peña F. O. , Miranda R. O. , Albadej o J. D., Nadala E. C. B. Jr. , Loh P. C., 2000 Whit e spot sy ndrom e virus (WSSV) in cult ured Penaeus m onodon in t he Philippines. Dis Aquat Org 4 2: 77- 82.
Tendencia E. A., Bosm a R. H. , Usero R. C. , Verret h J. A. J. , 2010 Effect of rainfall and at m ospheric t em perat ure on t he prev alence of t he w hit e spot sy ndrom e virus in pond cult ured Penaeus m onodon. Aquac Res 4 1: 594- 597.
Tendencia E. A. , Usero R. C. , 2007 Prev alence of w hit e spot sy ndrom e virus in shrim p (Penaeus m onodon) in Negros I sland, Philippines: 2000- 2006. World Aquacult ure Societ y, Asian- Pacific Aquacult ure 2007 Meet ing. Aquacult ure Depart m ent , Sout heast Asian Fisheries Developm ent Cent er. I loilo, Philippines.
Received: 13 June 2 011. Accept ed: 02 July 201 1. Published on line: 02 July 201 1. Aut hor s:
Benedict A. Mar alit , Genet ic Finger pr int ing Labor at or y, Nat ional Fisher ies Resear ch and Developm ent I nst it ut e, Quezon Cit y 110 3, Philippines, benedict _bdek@yahoo. com
Chr ist opher Mar lowe A. Caipang, Facult y of Biosciences and Aq uacult ur e, Univer sit y of Nor dland, Bodø 8 049, Nor way, cm acaipang@yahoo. com
Mudj ekeewis D. Sant os, Mar ine Fisher ies Resear ch Division, Nat ional Fisher ies Resear ch and Developm ent I nst it ut e, Quezon Cit y 110 3, Philippines, m udj iesant os@yahoo. com
Mar y Bet h B. Maningas, Resear ch Cent er for t he Nat ur al and Applied Sciences, Univer sit y of Sant o Tom as, Philippines 1008, m ar ybet hm aning as@yahoo. com
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