Journal of Invertebrate Pathology 110 (2012) 166–173

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Journal of Invertebrate Pathology

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Historic emergence, impact and current status of shrimp pathogens in Asia

Timothy W. Flegel ⇑Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, ThailandNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Klong 1, Klong Luang, Pratum Thani 12120, Thailand

a r t i c l e i n f o

Article history:Available online 10 March 2012

Keywords:ShrimpPathogensAsiaReviewVirusesBacteriaParasites

0022-2011/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jip.2012.03.004

⇑ Address: Center of Excellence for Shrimp Molecul(Centex Shrimp), Faculty of Science, Mahidol Univer10400, Thailand. Fax: +66 2 3547344.

E-mail address: sctwf@mahidol.ac.th

a b s t r a c t

It is estimated that approximately 60% of disease losses in shrimp aquaculture have been caused by viralpathogens and 20% by bacterial pathogens. By comparison, losses to fungi and parasites have beenrelatively small. For bacterial pathogens, Vibrio species are the most important while for viral pathogensimportance has changed since 2003 when domesticated and genetically selected stocks of the Americanwhiteleg shrimp Penaeus (Litopenaeus) vannamei (Boone 1931) replaced the formerly dominant giant tigeror black tiger shrimp Penaeus (Penaeus) monodon (Fabricius 1798) as the dominant cultivated species. Forboth species, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal. Nextmost important for P. vannamei is infectious myonecrosis virus (IMNV), originally reported from Brazil,but since 2006 from Indonesia where it was probably introduced by careless importation of shrimp aqua-culture stocks. So far, IMNV has not been reported from other countries in Asia. Former impacts of Taurasyndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) on this specieshave dramatically declined due to the introduction of tolerant stocks and to implementation of good bio-security practices. Another problem recently reported for P. vannamei in Asia is abdominal segment defor-mity disease (ASDD), possibly caused by a previously unknown retrovirus-like agent. Next most importantafter WSSV and YHV for P. monodon is monodon slow growth syndrome (MSGS) for which componentcauses appear to be Laem Singh virus (LSNV) and a cryptic integrase containing element (ICE). Hepatopan-creatic parvovirus (HPV) and monodon baculovirus (MBV) may be problematic when captured P. monodonare used to produce larvae, but only in the absence of proper preventative measures. Since 2009 increasinglosses with P. vannamei in China, Vietnam and now Thailand are associated with acute hepatopancreaticnecrosis syndrome (AHPNS) of presently unknown cause. Despite these problems, total production of cul-tivated penaeid shrimp from Asia will probably continue to rise as transient disease problems are solvedand use of post larvae originating from domesticated SPF shrimp stocks in more biosecure settings expands.

� 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1662. Overview of major shrimp viruses in Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1683. Overview of new and major non-viral pathogens in Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1684. Major viral pathogens of P. vannamei in Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705. Major viral pathogens of P. monodon in Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1716. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

1. Introduction

The history of Asian shrimp culture in relation to disease issueshas been reviewed detail in a number of previous publications

ll rights reserved.

ar Biology and Biotechnologysity, Rama 6 Road, Bangkok

(Flegel, 2006a; Flegel et al., 2008; Fulks and Main, 1992). Here willbe presented a brief overall review of major issues only. In a reviewof diseases in the shrimp aquaculture industry up to 2005 (Flegelet al., 2008), it was estimated that global production losses dueto disease over the preceding 15 years had amounted to approxi-mately US$15 billion, with about 80% occurring in Asia. Accordingto a survey conducted by the Global Aquaculture Alliance, approx-imately 60% of disease losses in shrimp aquaculture could beattributed to viral diseases and approximately 20% to bacterial

Fig. 1. Production of cultivated penaeid shrimp in Asia (1979–2009). Source: FAOFishBase Plus

Fig. 2. Example of steadily increasing production of cultured Penaeid shrimp inThailand despite periodic disease setbacks. Note the recovery due to wide use of SPFstocks of P. vannamei from 2002 onward.

T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173 167

diseases (Flegel, 2006b) (Flegel et al., 2008). Thus, 80% of the dis-ease losses were attributed to only two pathogen groups, withviruses having approximately four times more negative impacton production than bacteria. The remaining 20% of losses wereattributed to a variety of other pathogens (e.g., parasites and fungi)and to abiotic or unknown causes. In most cases, diseases causedby bacterial pathogens and parasites can be prevented by appropri-ate management of shrimp cultivation (i.e., appropriate manipula-tion of parameters such as biosecurity, water quality, stockingdensity, aeration, feed quality, feed quantity and seed quality).Therefore, most of the information covered in this review will con-cern issues related to shrimp viruses and only a little with issuesrelated to bacteria and parasites.

After the publication of the Bangkok Declaration and the pro-ceedings from the conference on Aquaculture in the 3rd Millenium(Kongkeo and Jiansan, 2000; Subasinghe et al., 2000), a major rev-olution in shrimp aquaculture occurred, with the widespreadadoption of domesticated and genetically improved whitelegshrimp Penaeus (Litopenaeus) vannamei as the species of choiceover the formerly dominant species Penaeus (Penaeus) monodon(Wyban, 2007a,b). This has fulfilled one of the recommended inter-ventions of the Bangkok Declaration (i.e., ‘‘developing and utilizingimproved domestication and broodstock management practicesand efficient breeding plans to improve production in aquatic ani-mals’’). The resulting increase in Asian shrimp aquaculture produc-tion output from approximately 0.9 million metric tons in 2004 to2.9 million metric tons (more than triple) in 2009 (Fig. 1, FAO, Fish-Base Plus) is a testament as to how effective such interventions canbe. On the other hand, it should not be assumed that this increasein production was due solely to introduction of the new stocks,since it was accompanied by a suite of other global aquaculturemanagement practices (GAP), particularly regarding biosecurity.

The economically devastating pandemic caused by white spotsyndrome virus after its first appearance in China in 1992 (Flegeland Alday-Sanz, 1998; Flegel, 1997; Nakano et al., 1994) served asa major stimulus for more intensive research on shrimp–virus inter-actions and as a major impetus for the widespread adoption of SPFstocks of P. vannamei. The benefits gained from research on shrimpdiseases and their contribution to increased production during the1990s were reviewed up to 2005 (Flegel et al., 2008). Despiteperiodic setbacks such as occurred in Thailand (Fig. 2), recoveryhas always followed, and production has continued to rise. Now,the main problem for farmers appears to be low farm-gate pricesresulting from competition in the global market and not losses dueto disease. On the other hand, these price factors are driving compe-tition based on production efficiency, so continual reduction in dis-ease losses to the absolute minimum remains an important goal.Thus, as summarized earlier (Flegel et al., 2008), there is still need

for improvement in many areas, including the need for: (1) develop-ment of domesticated and genetically improved, specific pathogen-free (SPF) stocks for all cultivated species; (2) more widespread useand standardization of diagnostic tests; (3) wider application andimprovement in biosecurity; (4) better control over transboundarymovement of living crustaceans for culture; (5) investigation ofthe efficacy of probiotics, immunostimulants and so-called‘‘vaccines’’ in full-scale field trials; (6) full understanding of thehost–pathogen interaction in shrimp; (7) more work on shrimp epi-demiology; (8) more studies on molecular ecology (i.e., metagenom-ics) and biochemical engineering to control the microbial dynamicsin shrimp ponds and hatchery tanks.

The paramount need for SPF domesticated shrimp stocks for sus-tainable shrimp aquaculture is based on a major biosecurity issuefor shrimp and other crustaceans that differs markedly from verte-brate species. The latter are often capable of clearing viral patho-gens from their systems during suitable periods of quarantine. Bycontrast, crustaceans often carry (and share among species) oneor more viral pathogens (even lethal ones) as persistent infectionsfor long periods (up to a lifetime) without showing any gross signsof disease (Flegel, 2007; Flegel et al., 2004). Although these virusesare often present in at low levels, they are not latent but active andcan be passed on to other naïve shrimp or crustaceans to cause mor-tality. They can also be passed from broodstock to their grossly nor-mal larvae and post-larvae (PL), either naturally or in a hatchery,and this may lead to subsequent disease outbreaks in rearing pondsstocked with the infected PL. This propensity of grossly normalcrustaceans to carry known and unknown viral pathogens meansthat special precautions are needed whenever living crustaceansdestined for aquaculture are translocated over large geographicaldistances, and especially to areas outside their natural range (Flegel,2006c; Flegel and Fegan, 2002).

Unfortunately, disregard for this propensity has resulted in sev-eral major shrimp virus epidemics (epizootics), most notably forinfectious hypodermal and hematopoietic necrosis virus or IHHNV(also called Penaeus stylirostris densovirus or PstDNV) (Tattersallet al., 2005) in the blue shrimp Penaeus (Litopenaeus) stylirostrisand the whiteleg shrimp P. vannamei in the Americas (Lightnerand Redman, 1991), white spot syndrome virus (WSSV) in all cul-tivated shrimp in Asia and the Americas (Flegel, 2006b), Taura syn-drome virus in P. vannamei cultivated in Asia (Nielsen et al., 2005;Tu et al., 1999) and most recently infectious myonecrosis virus(IMNV) in P. vannamei cultivated in Indonesia (Senapin et al.,2007). Every country should be wary of importing exoticcrustaceans of any kind for aquaculture without going throughthe recommended quarantine procedures, combined with testsfor unknown viruses that might be a danger to local species (Flegel,2006c). This process should be applied even to exotic domesticated

168 T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173

stocks that are SPF for a list of known pathogens. To reduce risks tothe minimum, any country that imports exotic stocks for aquacul-ture should invest in establishment of local breeding centers com-prised of properly vetted stocks that could be used for ongoingsupply of broodstock and of post-larvae to stock cultivation ponds.This would avoid the continual risk of importing unknownpathogens that might be associated with continuous importationand direct use of exotic stocks, even from a foreign breeding centerthat produces SPF stocks.

An allied issue concerns the co-cultivation of one shrimp spe-cies with one or more other shrimp species or with other crusta-cean species. For example, rearing of captured P. monodon andexotic SPF P. vannamei in an Asian shrimp hatchery would be agood way to transfer endemic IHHNV from P. monodon to P. vann-mei at the larval stage. In another example, it has recently beenshown that Macrobrachium rosenbergii nodavirus (MrNV) (thecause of white muscle disease in M. rosenbergii) can infect larvaeof P. monodon and Penaeus (Fenneropenaeus) indicus and result inhigh mortality from white muscle disease (Ravi et al., 2009), eventhough it does not cause mortality in challenged juvenile shrimp ofthe same two species (Sudhakaran et al., 2006). This was discov-ered in hatcheries co-cultivating M. rosenbergii and penaeidshrimp. In summary, there are good reasons to avoid mixed cul-tures of shrimp or other crustaceans unless it is certain that nega-tive viral interchanges are not possible.

2. Overview of major shrimp viruses in Asia

Although approximately 20 shrimp viruses have been de-scribed, some with sub-types that differ in virulence, only afew pose serious threats to shrimp farmers, and the list of seri-ous viruses differs according to shrimp species and country ofcultivation. White spot syndrome virus (WSSV) is the most seri-ous pathogen in terms of overall production losses because it islethal for all cultivated species and because mortality can be highand rapid (Flegel, 2006a). The next most severe pathogen is prob-ably yellow head virus (YHV) (Dhar et al., 2004; Sittidilokratnaet al., 2008; Walker et al., 2005) that causes high and rapid mor-tality with both P. monodon (Boonyaratpalin et al., 1993; Chanta-nachookin et al., 1993) and P. vannamei (Senapin et al., 2010;Sittidilokratna et al., 2009). However, recent work (Wijegoona-wardane et al., 2008) has shown that there are five or six geo-graphical types of YHV and that the most virulent type (YHV-1) has been reported to cause serious disease outbreaks only inThailand.

Two other deadly viruses, infectious myonecrosis virus (IMNV)(Poulos et al., 2006) and Taura syndrome virus (TSV) (Dhar et al.,2004; Hasson et al., 1995; Lightner, 2011) are of importance forP. vannamei but not for P. monodon (Tang et al., 2005). Anotherimportant virus is infectious hypodermal and hematopoieticnecrosis virus (IHHNV) that can cause high mortality in the Amer-ican blue shrimp P. stylirostris and stunted growth in P. vannamei(Lightner, 1996). However, it also has little, if any, effect on P. mon-odon (Withyachumnarnkul et al., 2006). Another relatively recentphenomenon in P. vannamei is abdominal segment deformity dis-ease (ASDD) (Sakaew et al., 2008) that causes deformities but notmortality or retarded growth. In P. monodon, Laem Singh virus(LSNV) (Sritunyalucksana et al., 2006) and an associated integrasecontaining element (ICE) (Panphut et al., 2011) are associated withsevere stunting of growth. Less important are a densovirus calledhepatopancreatic virus (HPV) (Flegel et al., 1999) or P. monodondensovirus (PemoDNV) and monodon baculovirus (MBV) alsocalled P. monodon polyhedrovirus (PemoNPV), since they can beeliminated from captured broodstock by proper washing of eggsand/or nauplii in the hatchery.

Since most commercial stocks of P. vannamei used in Asia arenow highly tolerant to TSV and since IHHNV does not usually causedifficulties for this species, even if it becomes exposed in rearingponds (i.e., not as PL), the list of serious viral pathogens for P. vanna-mei currently includes WSSV, YHV Type-1, and IMNV (depending onthe country). Since HPV and MBV can be removed easily by washingeggs and nauplii in the hatchery, the list for P. monodon includesWSSV, YHV Type-1 and LSNV/ICE (again depending on the country).

3. Overview of new and major non-viral pathogens in Asia

As previously reported, the most serious bacterial pathogens forpenaeid shrimp are Vibrio species (e.g., V. harveyi, V. parahaemolyt-icus and V. vulnificus) (Longyant et al., 2008) and rickettsia (Nunanet al., 2003a,b, 2010). As previously stated, serious Vibrio infectionsare usually the result of mismanagement and they will not be dis-cussed here further, except to mention that disease outbreakscaused by Vibrio penaecida (Ishimaru et al., 1995; Saulnier et al.,2000) and Vibrio nigripulcritudo (Goarant et al., 2006; Lemonnieret al., 2006) can be initiated by unmanageable weather conditions,and that infections with a new species Vibrio mediteraneanei (syn-onym = Vibrio shiloi = Vibrio shilonii) have recently been reportedfrom diseased shrimp in Thailand (Longyant et al., 2008).

With respect to rickettsial-like bacteria in penaeid shrimp inAsia, a 3-year study in Thailand (Tardmee, 2009) based on PCRanalysis of normal, diseased (including suspected rickettsial infec-tions) and stressed P. monodon gave a great variety of ampliconssome with homology to the sequence of rickettsial infections re-ported for P. monodon from East Africa. However, none of thesegave positive reactions by in situ hybridization with tissues of theoriginating shrimp, including hepatopancreatic tissues showinghistological signs of bacterial lesions. Nor did any of the samplesgive positive reactions with the IQ2000 kit for detection of thecausative agent of Texas necrotizing hepatopancreatitis (NHP). Bycontrast, all of the shrimp showing signs of bacterial lesions gavepositive reactions for antibodies to various Vibrio species, includingV. harveyi and V. parahaemolyticus. Similarly, none of the samplesreceived by our laboratory from China, Malaysia and Vietnam haveever given positive results for NHP using the IQ2000 kit (FarmingIntelligene/GeneReach, Taipei). A newly described rickettsial path-ogen has been reported for cage-cultured lobsters in Vietnam (Nu-nan et al., 2010), but so far there have been no reports ofwidespread disease outbreaks caused by this or other rickettsialpathogens in cultured peaneid shrimp in Asia.

For parasites, two new pathogens have recently been found,including a microsporidian (Tourtip et al., 2009) from Thailandand a haplosporidian from Indonesia (unpublished). The microspo-ridian (Enterocytozoon hepatopenaei) was originally discovered in P.monodon but not found to be associated with serious disease. How-ever, recent outbreaks of white feces syndrome (WFS) in P. mon-odon in Vietnam (Ha et al., 2011) and P. vannamei in Vietnamand Thailand (Centex Shrimp, unpublished) have been associatedwith serious infections by a microsporidian morphologically simi-lar to E. hepatopenaei. We have found that the parasite shares 95%rRNA gene sequence identity with that of E. hepatopenaei at Gen-Bank (FJ496356) (Centex shrimp unpublished). Thus, the parasiteis probably a variant of the same species or is a new species inthe genus. Curiously, not many of the infected hepatopancreatic(HP) cells produce spores, and most appear to be morphologicallynormal with hematoxylin and eosin staining. Even with a 100�objective, developmental stages are very difficult to distinguishfrom normal cytoplasmic structures of the HP cells (Fig. 3). How-ever, in situ hybridization reveals that HP cells may be heavily in-fected, except for the E-cells. Given the high level of infection, itis reasonable to suggest that the energy demand to support the

Fig. 3. Photomicrographs of hepatopancreatic tissue sections of P. vannamei infected with Enterocytozoon. The left column shows sections stained with H&E while the rightcolumn shows contiguous sections processed for in situ hybridization using the haplosporidian rRNA gene probe.

T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173 169

parasite would have a negative effect on shrimp growth and mightlead to secondary bacterial infections, especially under stressfulrearing conditions. We have developed a PCR detection methodand an in situ hybridization probe (unpublished), and these shouldhelp to identify the source of infections. Once identified, entry tothe shrimp production system can be blocked. For example, thiswas done previously in Thailand for the microsporidian Agmasomapenaei after fish were identified as the probable carriers by PCR(Pasharawipas and Flegel, 1994) and removed from water supplycanals.

Starting from China in 2009 and progressing to Vietnam in 2010and Thailand in 2011, there have been widespread outbreaks ofdisease characterized by massive degeneration of the hepatopan-creas (Centex Shrimp, unpublished) (Fig. 4). Estimated losses forVietnam since October–November 2010 may be in excess ofUS$75 million (David Kawahigashi, Vannamei 101, Talang, Phuket,Thailand, personal communication). D.V. Lightner (University ofArizona, personal communication) has described this as a possibleidiopathic condition with a clear case definition as follows: acuteprogressive degeneration of the hepatopancreas (HP) accompanied

Fig. 4. Photomicrograph of acute hepatopancreatic necrosis in a shrimp specimenfrom Vietnam. Note that the E-cells of the tubules on the left are still intact whilethere is massive sloughing of tubule cells in the central region of the hepatopan-creas on the right.

Fig. 5. Photomicrographs of hepatopancreatic tissue of a diseased P. vannameispecimen stained with hematoxylin and eosin and showing tubule epithelial cellsinfected with the haplosporidian parasite.

170 T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173

by lack of mitotic activity in E cells, medial to distal dysfunction ofB, F and R cells, prominent karyomegaly, sloughing of tubular epi-thelial cells and terminal stages that include intertubular hemocy-tic aggregation and secondary bacterial infections. He has namedthis condition acute hepatopancreatic necrosis syndrome (AHPNS).

The cause is unknown, but our recent in situ hybridization testswith specimens exhibiting these signs of disease (Centex Shrimp,unpublished) were negative using the Enterocytozoon probe de-scribed above. In addition, the histopathology of the Enterocytozooninfections does not fit the case definition given for AHPNS. This isan example of a newly emerging disease of unknown cause, whereinitial investigations are based on histopathology of a limited num-ber of specimens received simultaneously by several laboratories.A clear case definition (such as the one provided here by Dr. Light-ner) is a fundamental, initial requirement for the subsequent,wide-scope, epidemiological and pathological research needed tofind the cause of the disease.

From Indonesia, high mortality of juvenile P. vannamei in hatch-eries and juvenile shrimp less than one month old in growoutponds has occurred since 2007 in association with hapolosproidianinfections of the hepatopancreas (Fig. 5). Prevalence in affectedponds was as high as 30% at around one month after stockingand decreased thereafter to less than 5% at harvest due to progres-sive mortality (overall mortality 60% to 90%). The losses since 2007have been estimated at approximately US$5 million. The grosssigns of disease and histopathology caused by the parasite resem-bled those previously described from the Americas (Dyková et al.,1988; Nunan et al., 2007), and sequences of PCR-amplified rRNAsequences shared 96% identity with those form the American re-port. Given the 4% difference in sequence identity, it is uncertainwhether the parasite is a variant of the previously described Amer-ican species or a new species. Nor is it certain whether the infec-tions originated from imported shrimp stocks or by transmissionfrom local carriers. In any case, elimination of the parasite fromhatchery stocks has been shown to be effective in stemming thedisease outbreaks (unpublished).

4. Major viral pathogens of P. vannamei in Asia

As stated above, the list of serious viral pathogens for P. vanna-mei in Asia currently includes WSSV, YHV Type-1, and IMNV. WSSVis still the most serious threat for all countries and for all cultivatedspecies of shrimp in Asia. There are a number of recent reviewsthat cover aspects of WSSV biology and control measures (Escobe-do-Bonilla et al., 2008; Leu et al., 2009; Lightner, 2011; Liu et al.,2009; Sánchez-Martínez et al., 2007; Sánchez-Paz, 2010). Sincethe widespread use of SPF stocks of P. vannamei in relatively biose-cure rearing ponds in Asia, the impact of WSSV has greatly de-clined, as can be seen from Figs. 1 and 2. However, it stillremains the most serious threat to shrimp farmers. When unin-fected post larvae derived from true SPF stocks are used, outbreaksoccur due to breeches in biosecurity. Since most of the productionstill takes place in uncovered, outdoor ponds with some frequencyof water exchange, this threat will remain, but losses can be sub-stantially reduced by good farmer training and adherence to globalaquaculture practice (GAP) (Walker and Mohan, 2009).

In contrast to WSSV, serious disease outbreaks (i.e., high and ra-pid mortality) caused by YHV Type-1 have been reported sporadi-cally only from Thailand. YHV Type-1 is subdivided into Type-1a(the first to appear in P. monodon in Thailand) and Type-1b (a var-iant that appeared later in P. vannamei in Thailand) (Senapin et al.,2010; Sittidilokratna et al., 2009) . The latter has a 162 nucleic aciddeletion in the viral envelope protein gp116 when compared toType-1a. Despite the large genome deletion, Type-1b is still highlylethal to P. vannamei and its virions have the same morphology asthose of Type-1a by electron microscopy. Fortunately, the deletionis not in the region of the target for commonly used RT-PCR meth-ods for YHV detection. The reason for the curious restriction of YHVType-1 to Thailand is still unknown but is suspected to be linked toan unknown reservoir carrier. The lack of spread within Asia,

T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173 171

despite the massive export of living and fresh frozen shrimp sug-gested that the product does not pose a threat to importing coun-tries when derived from normal harvest ponds (Flegel, 2009). Thiswas supported by subsequent experimental work (Sritunyaluck-sana et al., 2010). In addition, the lack of spread via broodstockand post larvae exported from Thailand supports the suspicion thatthe mode of transmission to growout ponds is usually horizontalfrom environmental sources and that it can be prevented by com-pletely covering ponds with netting of mesh equivalent to thatused in window screens (Flegel, 2010).

In Asia, IMNV first caused disease outbreaks in Indonesia in2006, probably because of the illegal import of shrimp broodstockfrom Brazil (Senapin et al., 2007). As previously reported from Bra-zil (Poulos et al., 2006), losses were cumulative with up to 80%mortality. However, anecdotal information since 2010 indicatesthat some rearing ponds stocked with locally produced post larvaegive survivals up to 70%, despite testing positive for IMNV by RT-PCR. In continual testing of 198 shrimp specimens from severalcountries (China, India, Indonesia, Malaysia, Taiwan, Thailand andVietnam) suspected of IMNV infection since 2006, we have ob-tained positive results by RT-PCR analysis only from Indonesia(Senapin et al., 2011). Thus, up to the time of writing this paper,IMNV was still restricted to Indonesia in Asia and it is unlikely thatit would affect other countries in the region so long as infectedshrimp broodstock or post larvae are not imported for aquaculture(Flegel, 2006c). Most countries in Asia already quarantine importedstocks of P. vannamei and screen them for IMNV. It is further rec-ommended that all Asian countries involved in P. vannamei cultiva-tion set up monitoring and emergency response procedures forrapid containment in the event of an initial outbreak from illegallyimported stocks for aquaculture. Finally, it is clear from Section 3above that the current problems with high shrimp mortality in Chi-na and Vietnam are not caused by IMNV.

In closing this section on P. vannamei, I would like to include abrief mention of recent work on abdominal segment deformity dis-ease (ASDD) (Sakaew et al., 2008) that causes gross distortion ofthe abdominal segments of juvenile P. vannamei in growout ponds.Although there is no negative effect on growth or survival, thefarm-gate value of the proportion of deformed shrimp is usually re-duced by 10%. Recent unpublished work on shrimp with grosssigns of ASDD by W. Sakaew at Centex Shrimp has revealed thata reverse-transcriptase containing retrovius-like agent (RLA) withclosest relationship to non-long terminal repeat (non-LTR) typeelements is present in these shrimp. Indications are that an inac-tive DNA form present in the genome can be activated in stressedfemale broodstock to produce an active RNA form. When this ac-tive form is transmitted to larvae in the hatchery, they show grosssigns of ASDD that can be seen from the zoeal stages onward. Pre-liminary results (unpublished) indicate that the RLA is not presentin the genome of all stocks of P. vannamei. Thus, if the RLA is actu-ally a component cause of the phenomenon and not just associ-ated, it should be possible to use PCR to identify RLA-freeindividuals and thus eliminate the RLA (and associated deformi-ties) in SPF stocks by genetic selection. In the meantime, the PCRand RT-PCR methods that have been developed for its detectioncan be used to monitor broodstock for transmission risk.

5. Major viral pathogens of P. monodon in Asia

As with SPF P. vannamei, WSSV is the main threat for farmerscultivating SPF P. monodon and the preventative methods for SPFstocks of both species are the same. However, in some countries,captured P. monodon are still used as broodstock to produce postlarvae for stocking production ponds. In such cases, screening ofbroodstock and post larvae for WSSV (and other pathogens) by

PCR and discard of positive individuals or batches can help to re-duce the incidence of disease outbreaks, so long as biosecurity sim-ilar to that used for SPF stocks is employed (Withyachumnarnkul,1999). In addition, building on previous work indicating that ele-vated water temperature can prevent WSSV disease in infectedshrimp (Granja et al., 2006; Vidal et al., 2001), work in Thailandhas revealed that holding post larvae infected with WSSV continu-ously at 32 �C for 7 days can clear them of WSSV before stockingponds (Wongmaneeprateep et al., 2010). The mechanism for WSSVinhibition at 32 �C has recently been discovered (Lin et al., 2011).However, use of such methods to improve post larval quality with-out also providing appropriate biosecurity in rearing ponds (e.g.,carrier removal and restricted water exchange) does not signifi-cantly reduce the incidence of disease outbreaks in areas whereWSSV may be prevalent in natural carriers and exchange water(Corsin et al., 2002, 2003).

The next most serious threat for P. monodon is yellow head virus(YHV) that has been adequately covered in Section 6 above. AfterYHV, the most serious threat for P. monodon in Asia is monodonslow growth syndrome (MSGS) (Flegel, 2006a; Sritunyalucksanaet al., 2006). Since the case definition for MSGS is pond-based, indi-vidual shrimp specimens should be referred to as originating froma pond that fits the MSGS case definition, and not as individualshrimp showing gross signs of MSGS. This is important becauseshrimp from MSGS ponds exhibit a range of features that cannotbe explicitly defined for individual specimens. For example, onemight select a random shrimp specimen from the wild or from ashrimp pond and state whether or not it showed gross signs ofWSSV infection. However, this would not be possible for MSGS.Nor could the term MSGS be applied to larvae or post-larvae in ashrimp hatchery. As shown in Fig. 2, MSGS was the main reasonthat Thai shrimp farmers switched from rearing P. monodon torearing P. vannamei derived from SPF stocks. Similar to diseasecaused by YHV Type-1, MSGS has not apparently affected P. mon-odon cultivation in other Asian countries (Rai et al., 2009), andthe reason for this is currently unknown. A new virus calledLaem-Singh virus (LSNV) was found in shrimp from MSGS pondsby shotgun cloning (Sritunyalucksana et al., 2006) and was latershown to be associated with retinopathy in small infected shrimpbut not large infected shrimp from MSGS ponds (Pratoomthai et al.,2008) or infected shrimp from normal ponds. Thus, LSNV was pro-posed to be a necessary but insufficient cause of MSGS, and thesearch continued for other possible component causes. As a result,a second agent called an integrase containing element (ICE) wassubsequently found in shrimp from MSGS ponds, also by shotguncloning (Panphut et al., 2011). Although LSNV and ICE were shownto occur together in shrimp from MSGS ponds but not in shrimpfrom normal ponds, it has not been possible so far to purify eitherLSNV or ICE to carry out independent and combined challengetests, so a direct causal link between the dual infection and MSGShas yet to be proven. In any case LSNV and ICE should be added tothe list of pathogens to be excluded from SPF stocks.

6. Conclusions

Cultivation of penaeid shrimp in Asia has changed dramaticallysince 2002 because of the widespread adoption of domesticated,SPF stocks of exotic P. vannamei as the source of post larvae tostock rearing ponds that operate under obligatory biosecurity con-ditions and follow global aquaculture practice (GAP). A major re-sult has been a marked decline in the negative impact of majorshrimp pathogens, particularly viral pathogens, and a concomi-tantly marked increase in production. Of the remaining seriousthreats, WSSV is still the most important for all cultivated speciesin all countries. The relative threat from other viral pathogens

172 T.W. Flegel / Journal of Invertebrate Pathology 110 (2012) 166–173

depends on species of shrimp cultivated and geographical locationof the shrimp farms. They are for P. monodon, YHV-type1 andLSNV/ICE and for P. vannamei, IMNV and YHV-type1. Formerthreats for P. vannamei (i.e., IHHNV and TSV) have virtually beeneliminated by use of SPF stocks. Former threats for P. monodon(i.e., HPV and MBV) can been eliminated by use of SPF stocks orby proper hatchery management for systems based on capturedbroodstock. It is expected that more recent threats for P. vannameisuch as Enterocytozoon and a haplosporidian will be quickly elimi-nated by identification and blocking of the reservoir sources. Withrespect to the most recently emerged disease AHPNS, the cause isstill unknown and the impact on overall production remains to beassessed.

Acknowledgments

I would like to thank Mahidol University, the Thai National Cen-ter for Genetic Engineering and Biotechnology (BIOTEC), the ThaiNational Science and Technology Development Agency (NSTDA),the Thai Research Fund and the Center of Excellence on Agricul-tural Biotechnology, Science and Technology Postgraduate Educa-tion and Research Development Office, Office of Higher EducationCommission, Ministry of Education (AG-GIO/PERDO-CHE) whohave variously contributed funding support to Centex Shrimp sinceits founding in 2002.

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