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Chapter 3

Designing a Biosecurity Plan at the facility level: Criteria, Steps and Obstacles
T.W. Flegel
Introduction

Starting from 8,987 metric tons in 1970, production from shrimp aquaculture reached 3,146,918 metric tons in 2006, an increase of 350 times. By contrast, the capture fishery started from 1,108,567 tons in 1970 and reached 3,460,003 tons in 2006, an increase of only 3 times. The trend indicates that aquaculture production will eventually surpass that of the capture fishery and that it will be the main supplier for increased future demand as the world population grows. The spectacular increase in cultivated shrimp production has occurred despite our relatively poor basic knowledge of the cultivated species and despite estimated losses of approximately US$15 billion since the early 1990’s due to disease (Flegel et al., 2008). Disease losses have been caused mostly by viral pathogens (approximately 60%) and bacterial pathogens (approximately 20%), while fungi, parasites and other diseases have been much less important (Fig. 1)(Flegel, 2006a). Although the greatest proportion of the disease losses have been caused by viral pathogens, little work was published on shrimp-viral interaction before 2000 see the reviews (Bachère et al., 2000; Söderhäll, 1999; Sritunyalucksana and Söderhäll, 2000). Most of the published literature on shrimp and other crustaceans up to that time focused on responses to bacterial pathogens.

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Figure 1. Estimated losses to various pathogens in shrimp aquaculture according to the Global Aquaculture Alliance survey (Flegel, 2006a).

Most studies on shrimp response to viral pathogens have been done in the laboratory and most have been directed towards gaining a better understanding of the shrimp-viral interaction at the molecular level (Flegel and Sritunyalucksana, 2009). However, a primary aim of the work has been to find new ways of preventing or reducing the economic impact of viral pathogens for shrimp farmers and this review will focus on research outcomes that have reduced the impact or have promise to do so. Since the focus will be at the application level, those interested in a deeper understanding of the underlying issues may refer to the article cited above as an entry to the relevant literature.

Scientific names of cultivated shrimp

Most of the cultivated shrimp species are classified in the family Penaeidae and they have been included in the genus Penaeus for more than 100 years (Table 1). During the interval 1969–1972 the genus Penaeus was divided into sub-genera (Burukovsky, 1972; Pérez Farfante, 1969; Tirmizi, 1971) (Table 1) based on some common morphological features. This did not affect the traditional usage of names since the sub-genus names were rarely used. However, in 1997 Pérez Farfante and Kensley (1997) published a monograph in which they unilaterally elevated the sub-genus names to generic rank without providing any explanation as to why the move was necessary and justified or what benefits would result from the change. I personally disagreed with the change and could find no reason why discarding the practice of the previous 100 years was justified. However, I discovered while attending aquaculture meetings and meetings of shrimp disease specialists that many disagreed with the proposed name change but felt they were obliged by the international rules of zoological nomenclature to accept it. When I explained that the international rules did not oblige people to accept the change, but that they had the right to refuse it, I was asked whether there was any reference to support my point of view. As a result, I wrote an article explaining the issues and encouraged readers to make their own decisions based on the reasoned assessment of arguments for and against the change (Flegel, 2007b). I followed this with a rebuttal to criticism of my article, in which I presented additional information and reaffirmed the lack of rules obligating people to accept the proposed name change. I still encourage individual scientists and practitioners to use their own judgment in accepting or rejecting the proposed change (Flegel, 2008). Since I disagree with the proposed change, I will follow the 100-year tradition and use the genus Penaeus rather than the proposed new genus names, but I provide Table 1 as a ready reference to sub-genus names. This table was derived from the publication by Holthius (1980) that is available for free download from the FAO web-site. FAO and the World Organization for Animal Health still use the traditional species names given by Holthius.

Shrimp defenses against pathogens

Before specifically examining the shrimp response to viral pathogens, it would be worthwhile to give a brief overall summary of the shrimp response to pathogens. We may begin with external barriers to infection such as the shrimp cuticle, a non-living layer that covers the body. It contains

Table 1. Traditional species names for the penaeid shrimp together with their date of naming (Holthuis, 1980).
Genus Sub-genus Species Authority Year
Penaeus Farfantepenaeus (1969) aztecus Ives 1891
brasiliensis Latreille 1817
brevirostris Kingsley 1878
californiensis Holmes 1900
duorarum Burkenroad 1939
notialis Pérez-Farfante 1967
paulensis Pérez-Farfante 1967
subtilis Pérez-Farfante 1967
Fenneropenueus(1969) chinensis Osbeck 1765
indicus Milne Edwards 1837
merguiensis De Man 1888
penicillatus Alcock 1905
Litopenaeus (1969) occidentalis Streets 1871
schmitti Burkenroad 1936
setiferus Linnaeus 1767
stylirostris Stimpson 1874
vannamei Boone 1931
Marsupenaeus (1971) japonicus Bate 1888
Melicertus (1969) canaliculatus Olivier 1811
kerathurus Forskål 1775
latisulcatus Kishinouye 1896
longistylus Kubo 1943
marginatus Randall 1840
plebejus Hess 1865
Penaeus (1969) esculentus Haswell 1879
monodon Fabricius 1798
semisulcatus De Haan 1844

chitin hardened by calcium except for areas (e.g., between abdominal segments and in the stomach) where flexibility is required to allow movement. The cuticle surface is lubricated by mucus that is released from very small canals (around 1 micron in diameter) that arise from tegumental glands in the underlying connective tissue. During shrimp cultivation in grow-out ponds, the cuticle is replaced at approximate 10 day intervals by the process of molting. The cuticle forms a physical barrier to pathogens, and molting allows for the regular discard of damaged cuticle or any attached bacteria, protozoans or other foreign material. In addition, healthy shrimp constantly use their appendages for preening and cleaning to remove foreign material from the body surface. If pathogens or foreign material manage to penetrate the cuticle, they face an array of defenses in the shrimp blood serum (hemolymph) and from shrimp blood cells (hemocytes). For example, the shrimp have a very effective and rapid clotting mechanism that can quickly plug leaks that might arise from physical penetration of the cuticle. It results from cross-linking of clotting protein in the hemolymph by an enzyme called transglutaminase produced by hemocytes. The hemolymph also contains high levels of agglutinins (lectins) that can cause bacteial cells to clump together to prevent spread and aid ingestion by hemocytes.

Unlike vertebrates, shrimp and other arthropods do not produce antibodies, and most people believe that they are incapable of specifically induced, adaptive immunity and immune memory such as that induced in vertebrates by the use of vaccines. However, they do possess a powerful innate immune response system that is based on molecules in the hemolymph called pattern recognition proteins or PRP. PRP respond to pathogen-associated molecular patterns (PAMP) that are unique to various types of microbial pathogens. Examples are the peptidoglycan cell-wall component of Gram-positive bacteria, the lipopolysaccharide cell-wall component of Gram-negative bacteria and the beta-glucan cell-wall component of fungi. If PRP in the shrimp hemolymph bind with matching PAMP molecules, they form a complex that binds, in turn, to receptors on the membrane of granular shrimp hemocytes to stimulate the release of their granules into the hemolymph. Granule release initiates a complex set of biochemical reactions called the pro-phenoloxidase (ProPO) cascade that involves a variety of antimicrobial elements. These include production of chemicals toxic to microbes and molecules that attach to foreign material to stimulate hemocyte aggregation and ingestion (i.e., phagocytosis) of foreign material. In extreme cases, the hemocytes surround and isolate invaders (i.e., form granules) to separate them from adjacent tissue. In cases of sufficiently heavy infections, the ProPO response results in formation of melanin that is grossly apparent as brown or blackened lesions in shrimp tissues or in the cuticle. A typical reaction to bacteria is shown in Fig. 2, where a Vibrio infection of the hepatopancreas has caused hemocytes to aggregate in large numbers to form a melanized granule. PRP in the shrimp hemolymph can also stimulate hemocytes to produce a large aray of antimicrobial proteins, some of which have broad spectrum antimicrobial activity and others more group specific activity to Gram-negative or Gram-positive bacteria, for example.

With respect to viral pathogens of shrimp, it is difficult to imagine any kind of stable, group-associated PAMP that might be characteristic of viruses in general or even for any particular viral group. This is because viral outer coatings (i.e., capsids or envelopes) are composed of proteins. Indeed, it is well-known that viral capsid and envelope proteins constantly change over time to evade host defenses that have been elicited by previous viral generations. That is the reason yearly changes in the human flu vaccine are required. The absence of a PAMP response to viruses in shrimp is evident by the absence of hemocytic aggregation and melanization, even in heavy viral infections. An example is shown in Fig. 3 where a heavy infection of a parvovirus in the hepatopancreas of the black tiger shrimp Penaeus monodon does not show massive hemocyte aggregation and melanization similar to that seen with the Vibrio infection in Fig. 2.

Another important feature of shrimp viral infections is that they may be divided into two types as acute infections leading to severe disease with mortality and persistent infections with reduced severity or absence of disease (see Flegel, 2007a; Flegel and Pasharawipas, 1998) for reviews). This is an important distinction, since individual viral pathogens may sometimes cause severe disease outbreaks but may also be carried as active infections for long periods (sometimes for a shrimp lifetime) without any visible signs of disease. Curiously, the massive outbreaks experienced in shrimp cultivation ponds have not, so far, been reported from natural populations, even in areas where severe pond outbreaks have occurred (Flegel, 2009). This is despite the common detection of causative viruses in grossly normal shrimp captured from wild populations (e.g., Lo et al., 1996).

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Figure 2. Hematoxylin and eosin stained tissue section of the hepatopancreas of the black tiger shrimp Penaeus monodon shrimp specimen heavily infected with Vibrio. Note the massive aggregation of hemocytes and the melanized granule formed by encapsulating hemocytes.

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Figure 3. Hematoxylin and eosin stained tissue section of the hepatopancreas of the black tiger shrimp Penaeus monodon shrimp specimen heavily infected with a parvovirus formerly called hepatopancreatic parvovirus (HPV) but now called a densovirus in the proposed genus Hepanvirus (Tijssen and 2008). Note the lack of massive hemocyte aggregation and melanization despite the large number of viral infected cells characterized by large basophilic intranuclear inclusions.

Since shrimp are able to survive for long periods with persistent infections, it is important in shrimp viral challenge tests to determine whether grossly normal test animals are free of viral pathogens prior to challenge and whether or not survivors are infected with the challenge virus post-challenge. There would be three possible outcomes: individuals that are “uninfected”, “infected but not diseased” or “infected and diseased”. The interactive response leading to each of these outcomes would be different and might vary with viral isolate type and shrimp target species or shrimp life stage within a single species (Peng et al., 1998b; Spann et al., 2000). Despite the importance of these distinctions, survivors in shrimp viral challenge tests are sometimes simply labeled “resistant”.

The serious threat from viral pathogens, particularly white spot syndrome virus (WSSV) was probably the reason for the rapid increase in work on shrimp-viral interaction since 2000. For example, of approximately 120 references in a recent review on shrimp molecular responses to viral pathogens (Flegel and Sritunyalucksana, 2009), approximately 80% dealt with WSSV and about 10% each with Taura syndrome virus (TSV) and yellow head virus (YHV). By contrast, very little has been done with the remainder of approximately 20 known shrimp viruses (Flegel et al., 2008; Lightner, 1996b), many of which are still poorly characterized and whose numbers are steadily rising (Fig. 4). Thus, caution may be in order with respect to generalizations about shrimp responses based on results arising mostly from three viruses, and of the three, 89% from the single pathogen WSSV.

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Fig. 4. Number of shrimp viruses described since 1974
(Couch, 1974; Lightner, 1988, 1993; Lightner and Redman, 1998).

In summary, the topic of shrimp response to viral pathogens at this time as like a large picture puzzle with only a small number of pieces currently available to be linked together into a full image. With so much unknown and so many investigations remaining to be done, it is difficult to make firm universial conclusions about shrimp-virus interactions. Despite this ambiguity, major progress has been made, and a number of practical measures for prevention have been devised. Some of these have already proven to be effective while others are still in the testing phase. Almost all of the measures proposed are preventative, since no practical treatment method has yet been devised to cure viral disease infections in cultivated shrimp. The following sections of this review will cover major insights that have improved our ability to control and limit the economic losses from viral disease outbreaks in cultivated shrimp. Also covered will be important discoveries that may eventually lead to improved methods of control.

Domesticated SPF shrimp

By far the most significant change in the shrimp culture industry in the past decade or so has been the rapid expansion in the use of domesticated and genetically selected post larvae for stocking of production ponds (Wyban, 2007a, b). As with other animal production industries such as chickens, swine and cattle, this has removed the risks associated with rearing wild animals of uncertain disease status, and it is the reason for the steep increase in world shrimp production since 2001 (Fig. 5). In areas where captured broodstock or post-larvae are used for stocking ponds, disease risks are much higher, particularly for viral pathogens. Although measures can be taken to reduce the risks, the ultimate objective for all cultivated species should be the development of domesticated stocks selected for absence of a specified list of pathogens. Such stocks are referred to as specific pathogen-free or SPF stocks, and they obtain their status by successful cultivation in high quarantine facilities for 2 or more years in the absence of any signs of the listed pathogens. Once shrimp leave an SPF facility, they may be reared onward in facilities of lower quarantine status and should then be referred to as high-health stocks rather than SPF stocks. Shrimp farmers are encouraged to be skeptical of claims of SPF or high-health status for marketed post larvae and should demand proof of post larval origin, even to the point of visiting production facilities if they are nearby. Farmers should also form local or national associations and keep records of results from post-larval suppliers to ascertain which are reliable and which are not.

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Figure 5. Graph of world shrimp production by capture and aquaculture showing a steep increase in production from around 2001 with the widespread use of domesticated and genetically selected shrimp (Source FAO).

Cultivated shrimp species

Asia has always been the dominant world producer of cultivated shrimp. Before 2002, world shrimp production from aquaculture was dominated by the Asian black tiger shrimp or prawn Penaeus monodon followed by the American whiteleg shrimp Penaeus vannamei. However, success with rearing of imported SPF P. vannamei in Taiwan and China the late 1990’s led to its widespread adoption in Asia as the cultivated species of choice. Thus, the situation is now reversed with P. vannamei dominating world production followed by P. monodon. Another species is Penaeus indicus that is cultivated particularly in middle-eastern countries because it tolerates high salinity better than P. monodon. Similarly, the more northern species Penaeus chinensis is more suitable for cultivation at low temperatures than tropical species. In this review, the emphasis will be on the tropical species P. monodon and P. vannamei.

Important shrimp viruses

At this time, approximately 20 shrimp viruses have been described (see Fig. 4). However, several types with differing virulence have been described for some of these and this effectively expands the list. The reason for the expansion in number of shrimp viruses described since 1974 (Couch, 1974) is probably due to the fact that shrimp cultivation has grown rapidly, that more disease specialists are looking and that shrimp are being moved to non-native areas for cultivation where they may encounter local pathogens to which they have not been previously exposed (Flegel and Fegan, 2002; Flegel et al., 2004). Another problem is that grossly healthy shrimp can carry unknown viruses that do not affect them, but can be transported for cultivation in new geographical locations where the virus may be lethal for local species (Flegel et al., 2004). Much care must be taken to avoid this possibility in the transboundary movement of shrimp and other crustacean stocks.

Fortunately, of the 20 or so shrimp viruses known, only a few are of great danger in shrimp aquaculture and severity depends on the species and strain of shrimp cultivated. For example, some SPF stocks have been selected for tolerance to specific viruses. In addition, measures to prevent outbreaks for all of these dangerous viruses are very similar, and when they are in place, they are also effective in reducing the risk of outbreaks caused by all of the others. In the following sections, the viruses of major concern will be described briefly, mostly for the purpose of indicating features important for implementation of control measures, but also as a means to direct readers to more detailed literature, if they require more information. White spot syndrome virus (WSSV) is the most important of all of the shrimp viruses in terms of overall production losses to date and in number of cultivated species (all) that are susceptible to outbreaks with high mortality. The next most severe pathogen is probably yellow head virus (YHV) in terms of propensity for high and rapid mortality with major cultivated species such as the black tiger shrimp Penaeus monodon and the American whiteleg shrimp Penaeus vannamei, but recent work suggests that there are 5 or 6 geograpical types of YHV and that the most virulent type (YHV-1) has been reported only from Thailand and Taiwan. Two other deadly viruses, infectious myonecrosis virus (IMNV) and Taura syndrome virus (TSV) are of importance for Penaeus vannamei but not for P. monodon. Another important virus is infectious hypodermal and hematopoietic necrosis virus (IHHNV) that can cause high mortality in the American blue shrimp Penaeus stylirostris and stunted growth in P. vannamei, but has little, if any, affect on P. monodon. Two viruses associated with stunted growth in P. monodon are a densovirus previously called hepatopancreatic virus (HPV) but now called Peneaeus monodon densovirus (PemoDNV) and Laem Singh virus (LSNV). Finally, I will briefly cover one baculovirus from P. monodon, not because it is a serious pathogen but because it is a good marker for proficiency of hatcheries that produce post larvae from captured broodstock.

Importance of persistent viral infections in shrimp

Although we do not understand the basis for it (Flegel, 2007a), it is well known that shrimp often carry one or more viruses without showing any gross signs of disease. For example, an early report on lethal yellow head virus (YHV) infections from Thailand showed that the YHV-infected shrimp had underlying infections of two other viruses (a parvovirus and a baculovirus)(Chantanachookin et al., 1993). Even as a dual infection, these 2 viruses would not have killed the shrimp. It is also well-known that surviving shrimp from severe viral disease outbreaks are often infected with the relevant virus although they show no signs of disease (Flegel, 2007a). The virus is usually present in the survivors at low levels, but is active and can cause lethal infections in naïve shrimp if they are co-cultivated with the survivors. This phenomenon was first discovered for Penaeus stylirostris densovirus (PstDNV) (formerly called infectious hypodermal and hematopoietic necrosis virus or IHHNV) infections in the blue shrimp P. stylirostris, but it was subsequently found that it probably occurs with all shrimp viruses, including major pathogens such as WSSV, YHV and TSV (Flegel, 2007a; Flegel and Pasharawipas, 1998).

Retrospective analysis of the initial PstDNV disease outbreaks in the Americas revealed that the virus originated from grossly normal P. monodon broodstock imported from Asia to America for experimental aquaculture (Lightner, 1996a). The virus was unknown before it jumped from the imported P. monodon to the native-American species P. stylirostris. This incident was a good warning that shrimp can carry one or more viruses without gross signs of infection (Flegel et al., 2004) and that it is dangerous to move living shrimp and other crustaceans for aquaculture from one geographical region to another without proper quarantine procedures, even if they are SPF for a list of known pathogens (Flegel, 2006b). In the case of the IHHNV example, the virus was unknown at the time of shrimp translocation so it would not have been possible to have it on an SPF list. Likewise, SPF stocks of P. vannamei from the Americas that are certified free of a list of known viral pathogens may still be carriers of unknown viurses that are inocuous for P. vannamei but could be lethal for other shrimp or crustaceans in other geographical regions.

Unfortunately, the lesson about viral infections in grossly normal shrimp was not well taken and succeeding shrimp viruses such as WSSV, TSV and IMNV were spread across geographical regions by careless transport of broodstock and/or PL for aquaculture (Flegel, 2006b; Flegel and Fegan, 2002; Senapin et al., 2007). For the broad picture, shrimp farmers in any particular country should be aware of the dangers of importing exotic crustaceans of any kind for aquaculture without going through the recommended quarantine procedures, combined with tests for unknown viruses that might be a danger to local species (Flegel, 2006b). This process should be applied even to domesticated stocks that are SPF for a list of known pathogens. Indeed, the best option for any country for a long term cultivation stragegy with exotic stocks would be to invest in establishment of local breeding centers comprised of properly vetted stocks for continuous supply of broodstock and of post-larvae for stocking ponds. This would avoid the continual risk of unknown pathogens that would be associated with continuous importation and direct use of exotic stocks, even from a foreign breeding center that produces SPF stocks.

An allied issue is the co-cultivation of one shrimp species with another shrimp species or crustacean species. For example, rearing of captured P. monodon and exotic SPF P. vannamei in an Asian shrimp hatchery would be a good way to transfer PstDNV from P. monodon to P. vannmei. In another example, it has recently been shown that Macrobrachium rosenbergii nodavirus (MrNV) (the cause of white muscke disease in Macrobrachium rosenbergii) can be transmitted from M. rosenbergii to larvae of Penaeus monodon and P. indicus and result in white muscle disease with high mortality (Ravi et al., 2009), even though it does not cause mortality in challenged juvenile shrimp of the same species (Sudhakaran et al., 2006). Thus, there is good reason to avoid mixed cultures of shrimp or other crustaceans unless one is very, very certain that negative viral interchanges are not possible.

In summary, shrimp, other crustaceans and arthropods in general are capable of carrying one or more viral pathogens (even lethal ones) as persistent infections for long periods (up to a lifetime) without gross signs of disease. The viruses can be passed on to other shrimp or crustaceans that may become diseased or they can be passed from the broodstock shrimp to their larvae and post-larvae, either naturally or in a hatchery. To avoid cross-transfer of viruses, it is important to rear individual species separately and to prevent the entry of potential carrier species into shrimp cultivation ponds by adopting appropriate biosecurity measures.

Biology and control of white spot syndrome virus

White spot syndrome virus (WSSV) is a circular double-stranded DNA virus with a genome of approximately 300 kb that was first described from Japan as penaeid rod shaped DNA virus (PRDV) (Inouye et al., 1994; Inouye et al., 1996) and later from other Asian countries under various other names (Wongteerasupaya et al., 1995). In some cases, it was named because of its shape (Wongteerasupaya et al., 1995). However, this was a mistake, because of its unique nature, it was eventually classified in a new virus family Nimaviridae and genus Whispovirus (Vlak et al., 2005). It can cause high mortality for all the commonly cultivated penaeid shrimp species but it also infects a wide range of other crustaceans, many of which do not die from infection (Flegel, 2001; Flegel, 2006c; Lo et al., 1996). Readers can consult the chapter on shrimp diseases in this book for details about diagnosis of WSSV infections.

During the period of dominant culture of Penaeus monodon and before the widespread use of domesticated stocks of SPF Penaeus vannamei, the main avenue of WSSV entry into shrimp cultivation ponds in Thailand was grossly normal post larvae (PL) that were infected with WSSV (Withyachumnarnkul, 1999; Withyachumnarnkul et al., 2003). These PL became infected with WSSV via WSSV-infected broodstock captured from the wild and used in hatcheries because they appeared healthy and showed no gross signs of WSSV infection (Lo et al., 1996). Two Thai studies showed that false negative PCR test results for WSSV and not horizontal transfer from the shrimp pond environment were the main reason for WSSV disease outbreaks, so long as good pond biosecurity was maintained (Chanratchakool and Limsuwan, 1998; Withyachumnarnkul, 1999). Once this was known, improved WSSV testing by PCR in the hatchery and before PL stocking (Thakur et al., 2002) helped to reduce the frequency of WSSV outbreaks. However, even with improved testing systems, lack of uniform test protocols and inherent test limitations meant that not all infected PL batches could be eliminated, and WSSV outbreaks continued to occur at unacceptable frequency. The introduction and widespread use of domesticated SPF stocks of P. vannamei rather than captured stocks of P. monodon reduced losses to WSSV to a minimum and led to a steep increase in total production of cultivated shrimp, as described above. This example shows how important it is that domesticated SPF stocks be developed for any shrimp species that is targeted for sustainable aquaculture.

If shrimp farmers are dependent on captured broodstock as a source of post-larvae for stocking ponds, a number of preventative measures can be taken to reduce the probability of producing post-larvae (PL) infected with WSSV. The first measure would be to screen broodstock specimens for WSSV before entry to the hatchery and to eliminate any shrimp that test positive. This first screening may give false negative results if the level of WSSV infection is below the detection limit of the method used, so it is necessary to check the females for WSSV again after spawning (Hsu et al., 1999). By holding and spawning the females and their offspring separately and testing the females again after spawning for the presence of WSSV, any positive specimens can be discarded together with their nauplii. In cases where it is difficult or impossible to find captured broodstock negative for WSSV, transmission from the broodstock to the larvae will be reduced by the usual practice of thorough washing of the eggs and/or nauplii with or without disinfectant (Hsu et al., 1999; Kasornchandra et al., 2000). This is because WSSV is present mostly in cells around the eggs in the ovary and rarely in the eggs themselves (Hsu et al., 1999; Kasornchandra et al., 2000). When the eggs are infected, they are usually damaged sufficiently to make them non-viable (Hsu et al., 1999).

Thus as above, using individual spawning followed by individual rearing of their larvae, any larval batches that test positive for WSSV during the subsequent rearing interval can be discarded. However, even with these precautions, PCR testing of the PL is recommended again before they are used to stock ponds (Thakur et al., 2002). It should be kept in mind that this screening process cannot guarantee that PL will be free of WSSV. All tests are based on representative samples and subject to sensitivity limits of the test methods being used. The most that a PL test report can state, for example, is that the test was negative for a specified level of prevalence and a specified sensitivity limit. Indeed, every negative test result reported should be accompanied by the target level of prevalence and the test sensitivity limit so the recipient will know there is still some possibility that the PL are infected even though the test results are negative. Despite this limitation, the testing routine can reduce the probability that batches of PL are infected with WSSV upon stocking, and this can improve the probability of avoiding WSSV disease outbreaks. Again, the best assurance can be achieved by the use of certified SPF stocks.

Before stocking ponds with WSSV-negative or SPF PL, ponds should be prepared in a manner that eliminates all possible natural, crustacean carriers of WSSV, since there are many (Flegel, 2006c; Lo et al., 1996). This can be done simply by filtration at 300 microns or less followed by storage of the fileterd water for at least 4 days before stocking. Alternatively, some farmers treat the pond water with disinfectants or short-lived insecticides to remove crustacean carriers. If these precautions are followed with biosecurity measures including prior filtration and storage of water before water exchange, together with other measures to prevent entry of WSSV carriers, the probability of WSSV outbreaks can be significantly reduced.

For regular monitoring during pond cultivation, it may be convenient to devise a practical testing schedule for pre-patent WSSV infections similar to that previously recommended (Lo et al., 1998; Tsai et al., 1999) and based on the concept that presence of low level WSSV infections even at high prevalence does not exclude the possibility of successful harvests (Tsai et al., 1999; Withyachumnarnkul, 1999). This could employ convenient and inexpensive immunochromatographic test strips (Fig. 6) with sensitivity close to that of 1-step nested PCR to check pooled swimming leg (pleopod) clippings from 10 shrimp randomly collected from each test pond. A positive result would indicate pre-patent WSSV infections at the level of 26% prevalence or more in the pond population (i.e., assuming a pond population of 100,000 or more) and would suggest that emergency response measures be invoked. A negative result would not exclude the possibility of pre-patent WSSV infections below 26% prevalence or WSSV infections below the pre-patent level, even at higher prevalence.

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Figure 6. Example of a lateral-flow, immunochromatographic strip test for the presence of white spot syndrome virus and yellow head virus at pre-patent or patent levels of infection.

For ponds with shrimp infected with WSSV at carrier levels, it is possible to reach successful harvests under good rearing conditions (Chanratchakool and Limsuwan, 1998; Withyachumnarnkul, 1999) but physical stress (Peng et al., 1998a) and environmental stress such as low temperature (Guan et al., 2003; Montgomery-Brock et al., 2007; Vidal et al., 2001) or rapid changes in salinity (Liu et al., 2006) can result in conversion of low level infections to patent infections and disease outbreaks. Other stressful conditions may also lead to outbreaks. Although the molecular mechanisms underlying the transition from stable persitent infections to outbreak level infections is not yet understood, it is clear that maintaining stable pond conditions near the optimum for shrimp growth and survival can reduce the probability of disease outbreaks, even if shrimp are infected. When seasonal weather is unstable and things like rapid changes in temperature and salinity cannot be controlled, the best strategy may be to delay cultivation until favorable, stable weather conditions prevail.

Biology and control of yellow head virus (YHV)

YHV was first described from Thailand (Boonyaratpalin et al., 1993; Chantanachookin et al., 1993) but is now known to exist as several different geographical types (Wijegoonawardane et al., 2008) and their recombinants (Gangnonngiw et al., 2009; Wijegoonawardane, 2008; Wijegoonawardane et al., 2004). They are now classified in the new family Roniviridae under the genus Okavirus (Walker et al., 2005). The original type (YHV-1) described from Thailand causes severe mortality in natural infections of P. monodon and P. vannamei cultivated in Thailand but has not been confirmed from other countries escept Taiwan. A second type (YHV-2), first called lymphoid organ virus (LOV) (Spann et al., 1995) but later considered identical to gill associated virus (GAV) (Cowley et al., 1999; Spann et al., 1997) is less virulent (Wijegoonawardane et al., 2008). The other geographical types are apparently non-virulent (Wijegoonawardane et al., 2008). YHV is a rod-shaped, single-stranded, positive sense RNA virus with a spiked envelope and a genome of approximately 27 kb (Cowley and Walker, 2002; Sittidilokratna et al., 2008; Sittidilokratna et al., 2002).

As with WSSV, YHV can be transmitted from broodstock to larvae in the hatchery (Cowley et al., 2002) but it is the cells around the eggs and sperm that are infected and not the reproductive cells themselves. Thus, measures to prevent YHV entry and transmission in the hatchery when captured broodstock are used are the same as those described above for WSSV (i.e., RT-PCR testing before and after spawning, individual spawning and larval rearing, washing of nauplii, etc.). Similar also are limitations of the screening methods and the possibility that PL might be infected despite negative test results. Before and after stocking the PL in rearing ponds, preventative measures against horizontal transmission of YHV from natural carriers such as Palaemonid shrimp (Longyant et al., 2006) must also be invoked. In addition, recent work (unpublished) in an area in Thailand prone to YHV outbreaks suggests that there may be an insect carrier state for YHV.

Specifically, 3 sets of 8 rearing ponds were prepared such that 4 ponds in each set were completely covered with netting of mesh equivalent to window screens while the 4 adjacent ponds were not. Ponds were stocked with either P. mondon or P. vannamei that tested negative for YHV and other important shrimp viruses. After two months of cultivation, one of the uncovered ponds experienced a severe YHV outbreak on 22 November 2008 and all of the other uncovered ponds followed with severe YHV outbreaks within the succeeding 4 weeks (18 December 2008). By contrast, all of the covered ponds were unaffected and went through to normal harvests. Since all of the ponds were stocked with shared post-larvae and were handled under identical management routines, it was concluded that the original outbreak pond on 22 November 2008 was infected by horizontal transfer via the airborne route.

After that, further outbreaks could have occurred from the same original source or by viral transfer within the farm from the 1st and subsequent outbreak ponds to uninfected ponds. In support of the latter proposal, occasional, whole dead shrimp were found on top of the mosquito netting of many of the covered ponds, indicating that birds flocking to eat dying shrimp in the uncovered outbreak ponds sometimes dropped them into adjacent uncovered ponds. Knowing that YHV can be spread rapidly by cannibalism, this could have been another effective way of spread. However, covering the ponds with fine-mesh netting prevented the entry of both insects and dropped shrimp. For all subsequent crops, the manager of the farm decided to cover all of the ponds with fine netting since he said that the covers lasted 3 years and that their cost would be easily covered by improved production.

To get some idea of the potential rapidity of spread by cannibalism, imagine one heavily YHV-infected (moribund) shrimp being dropped into an adjacent uninfected pond. Imagine further the appendages of this shrimp being eaten by 20 shrimp followed by an incubation period of 3 to 5 days before they too became moribund and were cannibalized in a second cycle by another 20 shrimp each. At the end of the 3rd cycle (9-15 days) there would be 203 = 8,000 infected shrimp in the pond and at the end of the 4th cycle (12-20 days) 204 = 160,000. If 2 infected shrimp were dropped into an uninfected pond, the number for the 3rd cycle would be 403 = 64,000 and if three were dropped, the number for the 3rd cycle would be 216,000. Increasing or decreasing the number of cannibals or the incubation period has a big effect on the rate of spread, and these factors can be affected by such things as stocking density and level of shrimp stress, respectively. In any case, it is clear that dropped, moribund shrimp pose a serious risk for disease spread, not only for YHV but also for WSSV and other shrimp pathogens.

Biology and control of Taura syndrome virus (TSV)

Taura syndrome was first described from the Americas in 1992 (Jimenez, 1992) but the etiological agent (TSV) was not described until 1995 (Hasson et al., 1995). TSV is classified in the family Dicistroviridae near cricket paralysis virus (Cripavirus) (Mari et al., 2002) as a non-enveloped, icosahedral virus approximately 31-32 nm in diameter. It has a single-stranded, positive-sense RNA genome of approximately 10 kb (Bonami et al., 1997; Hasson et al., 1995). Severe disease losses from TSV have been reported only for Penaeus vannamei, first from the Americas but later from Asia after P. vannamei became widely cultivated due to the availability of genetically selected, specific pathogen free (SPF), domesticated stocks (Wyban, 2007a, b). However, current difficulties with TSV have declined sharply due to the availability of genetically tolerant domesticated stocks, despite the report of escape mutants (Erickson et al., 2005) and geographical, genetic variation (Côté et al., 2008; Nielsen et al., 2005; Tang and Lightner, 2005). It is worthy of note that these SPF tolerant stocks often test positive for TSV in Asian cultivation ponds, indicating that they become infected without signs of disease after stocking.

The spread of TSV to Asia by importation of exotic P. vannamei for aquaculture was initially regarded with serious concern about potential impact on native shrimp and other crustacean species. However, at the time of writing, it has been 10 years since its first introduction to Asia and so far there have been no reports of serious losses caused by TSV in native crustacean species. This is despite the fact that the penaeid shrimp P. monodon, P. japonicus, and P. chinensis and the freshwater prawn Macrobrachium rosenbergii (Nielsen et al., 2005) together with local crab species (Kiatpathomchai et al., 2008) have been reported susceptible to infection.

Since TSV is problematic only for P. vannamei and since the latter is now widely available as highly TSV-tolerant, SPF stocks, transmission from infected broodstock to larvae in the hatchery is no longer a problem, and control measures focus on prevention of horizontal spread from natural carriers to reared SPF stocks. These prevention measures are the same as those for WSSV and YHV described above and are, in fact, maintained more for the prevention of WSSV and YHV than TSV because of the availability of SPF stocks highly tolerant to TSV.

Biology and control of infectious myonecrosis virus (IMNV)

Infectous myonecrosis virus (IMNV) is an unenveloped, cytoplasmic dsRNA virus of 40 nm diameter with a genome of 7650 bp (Poulos et al., 2006). It is aligned with viruses in the Family Totiviridae near the genus Giardiavirus. It was first reported to cause disease outbreaks in exotic P. vannamei cultivated in Brazil in 2002 (Lightner et al., 2004) and we may conjecture that it was a local virus that caused no notable problem with local crustaceans but was lethal when it jumped to P. vannamei. It caused gross signs of whitening of the abdominal muscle of the shrimp accompanied by slow mortality that persisted throughout culture and reached up to 70% in some ponds. In experimental infections, P. monodon showed no mortality but similar histopathology (Tang et al., 2005). Outbreaks of IMNV disease were confined to the Americas until 2006 when affected ponds were reported from Indonesia (Senapin et al., 2007). Based on genetic identity to IMNV from Brazil, it was proposed that the outbreak originated from grossly normal P. vannamei imported for Aquaculture from Brazil.

Curiously, the mortality reported from Indonesia was more severe and rapid than that reported from Brazil, but the reason for this is unknown. Careful control over the import of P. vannamei and other crustaceans for aquaculture from Brazil and now Indonesia would be the best way to prevent outbreaks of this virus elsewhere. SPF stocks developed in the Americas before the advent of IMNV were free of IMNV and it is possible to keep them so by maintaining appropriate quarantine measures.

Although nothing is known about the native reservoir for IMNV in Brazil, it is likely, as with other shrimp viruses, that a number of carrier species will eventually be found and that they might show no visible signs of disease. Work should be done in Indonesia to determine whether native crustaceans in the area of the outbreaks are susceptible to IMNV disease outbreaks or whether they can serve as potential carriers and if so whether any carriers have become established. Other countries in Asia where IMNV has not been reported should add the virus to their watch lists and try to prevent its entry.

Whitened muscles are caused in P. vannamei not only by IMNV but also by muscle cramps and by Penaeus vannamei nodavirus (PvNV) previously reported only from Colombia (Tang et al., 2007). PvNV does not cause high mortality, but shows gross signs of muscle whitening and histological lesions similar to those seen with IMNV infections. Muscle cramps are easily induced by stress in P. vannamei and can cause mortality by shrimp immobilization if the cramps are not quickly resolved. Histopathology of cramps consists of muscle coaggulation in the absence of hemocytic aggregation and cytoplasmic viral inclusions seen with IMNV and PvNV (Fig. 7).

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Figure 7. Comparison of histopathology for IMNV (left) and muscle cramp (right) showing the absence of hemocyte aggregation and viral inclusions in muscle cramp.

Another virus that causes whitened muscle in shrimp is Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV) (Bonami et al., 2005; Qian et al., 2003). XSV is probably a sattelite virus (Sri Widada and Bonami, 2004) while MrNV is probably the major cause of what is called white tail disease (WTD) in M. rosenbergii (Zhang et al., 2006). In 2006, it was reported that 3 juvenile penaeid shrimp species (P. indicus, P. japonicus and P. monodon) could be experimentally infected with MrNV/XSV but without mortality (Sudhakaran et al., 2006). Later (Ravi et al., 2009) reported that these viruses could cause white muscle disease and high mortality in post-larvae of P. monodon and P. indicus in Indian hatcheries that were in close proximity to culture activities with M. rosenbergii. As a result, we examined archived material of white muscle P. vannamei from several countries in Asia that we had previously tested and found negative for IMNV and PvNV. We discovered that some of the specimens were positive for MrNV alone or in combination with XSV (unpublished). Although we cannot yet confirm that MrNV was the cause of white muscle and mortality in the disease outbreak ponds, it is probably wise to put MrNV on the watch list for cultivated P. vannamei in Asia.

The danger of MrNV is particularly disturbing because it has been found to be transmitted not only from broodstock to their offspring (Sudhakaran et al., 2007b) but also by infected insect carriers (Sudhakaran et al., 2007a; Sudhakaran et al., 2008). Since domesticated SPF stocks of P. vannamei are available, preventing the spread of MrNV to cultivated P. vannamei would involve biosecurity measures to prevent M. rosenbergii or insects and their aquatic larvae from contacting P. vannamei at all stages of the cultivation cycle from broodstock production facilities to hatcheries and growout ponds.

Biology and control of Laem Singh virus (LSNV)

Laem Singh virus (LSNV) was first reported from Thailand (Sritunyalucksana et al., 2006b) from shrimp ponds exhibiting signs of monodon slow growth syndrome (MSGS). The latter has a pond-based case definition as follows. The suspected population should be RT-PCR positive for Laem-Singh virus and must have a coefficient of variation (CV = Standard deviation/Mean) of more than 35% by weight and absence of hepatopancreatic parvovirus (HPV) (now called Penaeus monodon densovirus or PemoDNV) or of other severe hepatopancreatic infections by known agents while also complying with any 3 out of the 5 following gross signs: 1. Unusually dark color; 2. Average daily weight gain of less than 0.1 g/day at 4 months; 3. Unusually bright yellow markings; 4. “Bamboo-shaped” abdominal segments; 5. Brittle antennae.

LSNV is an RNA virus that has not yet been completely characterized. Based on the sequence of its RNA-dependent RNA-polymerase (RdRp) gene and Northern blot hybridization of total RNA extracts, it has a genome size of approximately 5 kb and it most closely related (but at low sequence identity) to viruses in the insect-associated plant virus family Sobemoviridae. At first, it was assumed that LSNV was not linked to MSGS because positive RT-PCR results were obtained not only from MSGS ponds but also from shrimp ponds exhibiting normal growth (Sritunyalucksana et al., 2006b). However, the situation was reassessed when it was discovered later that retinopathy associated with strong LSNV in situ hybridization reactions was present in stunted shrimp but not normal size shrimp from MSGS ponds, even though both were positive for LSNV by RT-PCR (Figs. 8 & 9). Similarly, LSNV positive shrimp from normal growth ponds did not show LSNV-associated retinopathy. Thus, LSNV was sometimes associated with MSGS and sometimes not, and in epidemiological terminology, it was defined as a necessary but not sufficient cause of MSGS. The currently mystery is what factor(s)

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Figure 8. Photomictographs of hematoxylin and eosin stained eye tissue sections showing retinopathy in small shrimp but not normal size shrimp from MSGS ponds, despite the fact that both are positive for LSNV by RT-PCR assay.

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Figure 9. Photomicrographs of in situ hybridization test results for the presence of LSNV in eye tissue sections of small LSNV-infected shrimp from MSGS ponds compared to large LSNV-infected shrimp from the same MSGS pond and to large LSNV-negative shrimp from a normal growth pond. Note the positive (dark brown) reaction indicative of LSNV only in the eye tissue from the small shrimp from the MSGS pond.

must be combined with LSNV to cause MSGS, and that issue is the topic of hot research. An altenate explanation, also being explored, is the possibility that there are two or more types of LSNV, not all dangerous, that cannot be distinguished by the current RT-PCR test being used.

While these issues are being sorted out, it is important to screen captured P. monodon broodstock and their PL for LSNV by RT-PCR and to eliminate positive batches from the shrimp cultivation system as described above for WSSV and YHV. LSNV should also be added to the list of pathogens for inclusion in the SPF list in any program for the development of domesticated SPF stocks of P. monodon.

Since P. vannamei positive for LSNV by RT-PCR, injected with LSNV preparations from P. monodon or co-cultivated with P. monodon in MSGS ponds show no negative effects (unpublished), we assume that P. vannamei is unaffected by LSNV. Thus, it need not be on the list of excluded pathogens for SPF P. vannamei. On the other hand, the fact that it may be an unaffected carrier of LSNV means that it should always be considered a potential risk for LSNV transfer to P. monodon, particularly in hatcheries, and whenever co-culture of the two species is contemplated.

Biology and control of Penaeus monodon densovirus (PmDNV)

Penaeus monodon densovirus (PmDNV) was formerly called hepatopancreatic parvovirus (HPV) of Penaeus monodon (Flegel, 2006c; Lightner, 1996b). However, a recent proposal to the International Committee on Taxonomy of Viruses (Tijssen, 2008) recommends that this and related penaeid shrimp parvoviruses be included in a new genus Hepanvirus in the sub-family Densovirinae of the family Parvoviridae as Penaeus monodon densovirus (PmDNV), Penaeus merguiensis densovirus (PmergDNV) and Penaeus chinensis densovirus (PchinDNV). These differ in genome size and organization and target tissue from the other penaeid shrimp parvovirus included in the sub-family Densovirinae but in the genus Brevidensovirus as Penaeus stylirostris densovirus (PstDNV)(Tattersall et al., 2005). See the previous section of this review covering PstDNV, formerly called infectious hypodermal and hematopoeitic necrosis virus (IHHNV)(Lightner, 1996b). As far as we know, PmDNV does not cause severe mortality in P. monodon but is associated with severe growth retardation (Flegel, 2006c; Flegel et al., 1999). Thus, it can cause significant economic losses in proportion to the number of infected shrimp in a pond.

Like all of the other viruses covered so far, PmDNV can be transferred from infected broodstock to their PL in the hatchery. However, viral-infected cells are confined to the shrimp hepatopancreas and eggs and sperm are uninfected. Thus, viral transmission to the larvae takes place via broodstock feces in spawning tanks and preventative measures against PmDNV for captured broodstock are the same as those for all the other viruses previously covered (i.e., PCR testing before and after spawning, individual spawning and larval rearing, washing of nauplii, etc.). In addition, females may be starved for 2-4 hours before spawning to reduce the amount of feces left in spawning tanks. Also similar to other viruses are limitations of the screening methods and the possibility that PL might be infected despite negative PCR test results. Before and after stocking the PL in rearing ponds, preventative measrues against horizontal transmission of PmDNV from natural carriers must also be invoked, even though little work has been done to establish what these might be. However, work carried out in Thailand with aquatic insects (unpublished) revealed that some samples of back swimmers, water boatmen and water skaters collected from PmDNV infected shrimp ponds tested positive for PmDNV by PCR (Phromjai et al., 2002). Although it was not established whether these insects were mechanical or infected carriers of PmDNV, shrimp hatchery operators were advised to take appropriate action to prevent hatchery and nursery tanks being populated by these insects. The precaution was deemed particularly important for outdoor nursery tanks, and especially if they were illuminated at night. Due to the high density of PL in hatchery and nursery tanks, rates of viral spread could be especially high. Although the limited data in hand is anecdotal, it appears that transmission and spread of PmDNV in grow-out ponds is not a serious problem.

With respect to P. vannamei, it is known to be capable of PmDNV infection (Lightner, 1996b). However, the virus is on the list of pathogens excluded from domesticated SPF stocks, and this eliminates the possibility of its introduction into the cultivation system if such stocks are used. If these stocks have become infected with PmDNV in grow-out ponds since the widespread use of P. vannamei in Thailand in 2002, it has not caused a problem significant enough to have raised farmer attention or resulted in any published outbreak report. The same is true for China and other Asian countries where P. vannamei is now extensively reared. In any case, the need to cover hatchery and nursery tanks of P. vannamei to prevent entry of MrNV via insect carriers (see above) would also protect them from entry of PmDNV by the same route.

Biology and control of Penaeus monodon polyhedrovirus (PemoNPV)

Penaeus monodon polyhedrovirus (PemoNPV) was formerly called monodon baculovirus (MBV) (Lightner, 1996b) but is now tentatively inlcuded as a species of the genus Nucleopolyhedrovirus in the family Baculoviridae (Theilmann et al., 2005). It is a large, bacilliform virus with a double-stranded DNA genome and it produces granules of polyhedral protein (polyhedrin) in the nuclei of hepatopancreatic and midgut cells of P. monodon. Viral particles are embedded in the polyhedrin granules to protect them from being degraded after they are sloughed into shrimp feces. Thus, transmission of PemoNPV occurs when uninfected shrimp ingest polyhedrin granules containing the virus. Since these granules range from a few microns up to around 10 microns in diameter, they are about the same size as the algae consumed by naupliar and zoeal stages of P. monodon and this is the reason that PemoNPV infections can spread very rapidly in larval rearing tanks in a hatchery. Experience in Thailand indicates that the virus does not spread effectively with the larger sizes of shrimp in growout ponds. PemoNPV may sometimes cause high mortality in the shrimp hatchery, but it seems to cause only growth retardation in the proportion of infected shrimp at the late stages of pond cultivation (Flegel et al., 1999). Thus, the most significant economic loss probably results from the extended cultivation time required to obtain profitable shrimp sizes when infection rates are high.

As with PmDNV, shrimp larvae become infected with PemoNPV via broodsock feces in spawning tanks. Thus, measures to prevent PemoNPV entry and transmission in the hatchery when captured broodstock are used are the same as those described above for other viruses (e.g., PCR testing before and after spawning, individual spawning and larval rearing, washing of nauplii, etc.). However, due to its less severe nature, many hatchery operators skip PCR screening of the broodstock and concentrate on thorough washing of eggs and/or nauplii before they begin feeding. As with HPV, starving of the females for 2-4 hours before spawning may also help to reduce the amount of feces left in spawning tanks.

A good feature of PemoNPV is that the polyhedrin granules containing the virus can be seen easily with a light microscope in broodstock feces and in larvae from zoeal stages onward in the hatchery. With a little experience, whole mounts (not squashed) with a coverglass are sufficient to reveal these intranuclear inclusions with a 40x objective in unstained zoea, mysis and PL up to PL4 or 6. With later PL stages, hepatopancreatic squash mounts stained with malechite green are usually employed (see the chapter on disease diagnosis). With a little experience and a simple, inexpensive microscope, any shrimp farmer can screen PL for the presence of PemoNPV polyhedrin granules in squash mounts, or can have a daughter, son, etc. trained to do so. Since the virus can be easily eliminated from hatcheries by proper washing of eggs and/or nauplii, a check for PemoNPV prevalence in several batches of PL from any hatchery using captured P. monodon broodstock will quickly reveal whether the hatchery has a good viral prevention system or not. Basically, no batch should be positive for PemoNPV and the more batches that are positive, the worse the proficiency of the hatchery. When we further consider for all other viruses above that proper washing of eggs and/or nauplii was also an effective way of reducing transmission from broodstock to their offspring, it is clear that PemoNPV is an easy and inexpensive way for farmers to personally assess hatchery proficiency in the production of low risk PL from captured P. monodon. Antibody strip tests similar to that shown in Fig. 7 for WSSV and YHV would make it even easier for farmers to do their own test. Any hatchery that produces most or many PL batches positive for PemoNPV is likely producing PL coinfected with other more serious viruses that require more expensive or more sophisticated methods of detection. In summary, PemoNPV is not a very serious threat to P. monodon production, but it provides an easy, inexpensive and convenient way for shrimp farmers to personally evaluate the proficiency of hatcheries in producing low risk PL from captured P. monodon.

With respect to P. vannamei, PemoNPV is on the list of excluded pagthogens for SPF stocks and similar to PmDNV, there have been no reports of PemoNPV outbreaks in farmed P. vannamei since its widespread use in Asia since 2002.

Summary on the biology and control of major shrimp viruses

A few common facts can be derived from the discussion of the 7 viruses described above. One is that all the viruses can exist in their shrimp hosts and probably other crustacean hosts in active states, in company with other viruses, and with or without visible signs of disease. The mechanism for the switch from the non-disease to disease state is not yet understood, but various types of stress appear to be triggers for it. There are several important consequences arising from these facts. The first is that translocation of exotic shrimp to new locations for aquaculture is accompanied by two risks. One is the possibility of transferring known (or unknown) exotic viruses to new locations together with the shrimp. The other is the possibility that known (or unknown) viruses will jump to the exotic imported shrimp from local shrimp or other crustaceans. Precautions must be taken to avoid these possibilities. A second consequence is that use of captured wild shrimp as broodstock for PL production to stock rearing ponds is always accompanied by high risk that the broodstock will carry one or more known or unknown viruses without showing any sign of infection. Since there is a high probability that they will transmit these viruses to their offspring in shrimp hatcheries, precautions must be taken to reduce the probability of transmission to the minimum possible by testing for known viruses and using individually spawned and reared larvae. However, that probability will never be zero, and this is the reason for paramount need to develop domesticated SPF stocks for any shrimp species targeted for sustainable industrial production. The success of this approach has been amply proven by the spike in shrimp production that followed widespread use of domesticated SPF P. vannamei.

If a secure supply uninfected PL can be obtained for stocking shrimp ponds, the next biggest issue for viral disease prevention is to maintain strict biosecurity to prevent viral transmission from natural carriers to shrimp in rearing ponds. In most cases, this involves exclusion of potential shrimp and other crustacean carriers during pond preparation before stocking and during rearing after stocking. Although this can be accomplished simply by filtration and storage of water before it is used in rearing ponds, some farmers elect to use short-lived insecticides or disinfectants to treat water before it is used. Physical barriers (e.g., low fences) are often used to limit crab entry over land. We might now also add the recommendation that ponds be covered, when possible, with fine netting (i.e., equivalent to mosquito netting) to prevent the transmission of viruses via insect carriers and dropped, moribund shrimp from outbreak ponds. If the ponds cannot be covered, then at a minimum, aquatic insects must be excluded from hatchery and nursery tanks where high shrimp density would promote high viral transmission rates.

Some promising research directions

Probiotics. Many shrimp farmers add preparations of living bacterial cells called “probiotics” to their cultivation ponds prompted by advertising and by sales personnel who assure them that these preparations will improve water quality or prevent bacterial and viral diseases. Sometimes the sales campaigns are supported by positive results from properly controlled laboratory tests, but there are no reports of statistically significant production improvement (p?0.05) in properly controlled field tests on a large commercial scale. Most of the work done has been focused on bacteria and the only properly controlled field tests on probiotics revealed no significant effect of their use on measured water quality parameters (Boyd and Gross, 1998). Until proper studies have been carried out at the commercial production scale, the question of probiotic efficacy against viral pathogens must be left open to question. Consult the following reviews for more information (Irianto and Austin, 2002; Balcázar et al., 2006; Wang et al., 2008b).

Immunostimulants. The topic of immunostimulants is often confused with probiotics, but it is a separate issue since immunostimulants are not living cells. They may be crude preparations such as whole, dead microbial cells (e.g., yeasts or bacteria), semi-purified products from plants and microbes or pure chemicals (Raa, 1996). A large number of these products are on the market and some have been tested and shown to be effective for vial control in proper laboratory trials, but few have been tested for efficacy in full-scale field trials where laboratory successes may not be realized (Sritunyalucksana et al., 1999). As with probiotics, the efficacy of immunostimulants must be left open to question until proper studies have been carried out to show statistically significant improvement in production efficiency at the commercial production scale.

Shrimp vaccines. Venegas et al. (2000) first reported that Kuruma shrimp (Penaeus japonicus) survivors from WSSV outbreaks were able to survive a subsequent WSSV challenge with very little mortality when compared to naïve control shrimp. This was later shown to be associated with WSSV neutralizing activity in the shrimp hemolymph (Wu et al., 2002). Other reports have shown that administration of inactivated WSSV (Bright Singh et al., 2005; Flegel, 2006c) or heterologously expressed WSSV coat proteins by either injection or feeding can give some protection against mortality from a subsequent WSSV challenge (Namikoshi et al., 2004; Vaseeharan et al., 2006; Witteveldt et al., 2004a; Witteveldt et al., 2004b; Xu et al., 2006). Although these reports refer to the process as “vaccination”, the underlying mechanisms have not been elucidated and shrimp are not known to possess antibodies. In addition, no specificity tests were carried out using a second virus such as YHV or TSV, so it is not known whether the phenomenon is a specific or general response against viruses. Even if it is specific, it may be that free VP28 from shrimp feed can bind to viral receptors on shrimp cells and block their subsequent binding to WSSV viral particles. The short term effectiveness of VP28 supports this proposal.

In another approach, a shrimp protein (PmRab7) that binds to WSSV envelope protein VP28 was discovered in Penaeus monodon and it was found that injection of PmRab7 produced in bacteria could reduce shrimp mortality after WSSV challenge (Sritunyalucksana et al., 2006a). In subsequent work, the PmRab7 gene was transferred to and successfully expressed in the yeast Pichia pastoris and the whole yeast was added in a small amount to shrimp feed. This also reduced mortality caused by WSSV (unpublished data). These results suggested that direct interaction between PmRab7 and WSSV viral particles was required for infection. Later, it was demonstrated that injection of dsRNA-PmRab7 (see the section on RNAi below) specifically silenced PmRab7 and prevented mortality caused not only by WSSV but also by YHV (Ongvarrasopone et al., 2008). It is assumed that addition of PmRab7 to shrimp feed leads to the binding of free PmRab7 to WSSV particles and blocks their binding to PmRab7 in shrimp cells. In effect, it may be the reverse of using free VP28 added to shrimp feed. If this proposed mechanism is correct, we could call both of these free proteins antiviral reagents, but not vaccines. Since the results reported so far are from laboratory trials, further tests are needed to determine whether such reagents will be efficacious in large scale commercial applications.

An even more recent development is the use of “DNA vaccines” to protect shrimp from viral pathogens (Rout et al., 2007, Rajeshkumar et al., 2009, Ning et al., 2009), but again, the underlying mechanisms have not been elucidated and no specificity tests have been carried out. More molecular work is needed on the subject of antiviral reagents and so called “vaccines”. Some of the relevant issues are discussed in a recent review by Johnson et al. (2008).

RNA interference. RNA interference (RNAi) is another advanced technology that has recently been used in the laboratory to protect shrimp from viral diseases (Robalino et al., 2004; Robalino et al., 2005; Tirasophon et al., 2005; Tirasophon et al., 2007; Westenberg et al., 2005; La Fauce and Owens, 2009; Xu et al., 2007; Yodmuang et al., 2006). The subject has been recently reviewed (Robalino et al., 2007). Knockdown of shrimp Dicer-1, a key gene in the RNAi pathway, resulted in more rapid mortality and higher viral loads in shrimp experimentally challenged a type of yellow head virus (YHV) (Su et al., 2008) confirming that RNAi is important in the shrimp response to viral pathogens.

Using RNAi to protect shrimp from viral diseases consists of making small fragments of double-stranded RNA (dsRNA) with sequences that match those of viral genes and injecting them into shrimp to achieve disease protection. It was probably via the RNAi system that resistance to TSV infection was achieved in transgenic shrimp expressing an antisense viral construct (Lu and Sun, 2005). It remains to be seen whether the protection from dsRNA administration is long-lived and whether the shrimp remain infected after viral challenge. Although this concept is very interesting, issues of cost, safety and public acceptance remain to be resolved.

In addition to its role in response to viruses, the RNAi pathway is increasingly being used to study or confirm the roles of candidate immune-related molecules by dsRNA knockdown (de la Vega et al., 2008; Liu et al., 2007; Maningas et al., 2008). This is usually done by injecting dsRNA to knock-down specific shrimp or viral genes to determine the effect of their loss on the outcome or course of viral infections. We believe the insights gained on the mechanisms of host-viral interactions in shrimp will lead to new discoveries and new methods for viral control that do not use dsRNA reagents.

Interaction between different viruses

It has been reported that prior infection with Penaeus stylirostris densovirus (PstDNV) (formerly called infectious hypodermal and hematopoietic necrosis virus or IHHNV) can afford some protection against death when shrimp are challenged with WSSV (Bonnichon et al., 2006; Melena et al., 2006; Tang et al., 2003) This seems to be related to reduction in WSSV replication (Montgomery-Brock et al., 2007). Since both wild and cultivated shrimp are commonly infected simultaneously with two or more viruses (Flegel, 2006b; Flegel et al., 1997; Flegel et al., 2004), it would be interesting to determine whether similar interactions occur with other known or likely shrimp virus combinations. It would also be worthwhile to understand the molecular mechanisms behind any protective effects to see if they could be induced without infections.

Viral sequences in the shrimp genome

When non-infectious sequences of PstDNV were discovered to be inserted into the chromosomal DNA of Penaeus mondon from East Africa and Australia (Tang and Lightner, 2006), a new detection method for the infectious form had to be developed (Tang, 2007 #4365). WSSV-like sequences have also been reported from the genome of P. monodon from Australia (de la Vega, 2006) and many viral-like sequences have been found in a Fosmid clone library constructed from the P. monodon genome (Huang et al., 2008). A similar phenomenon has been reported from insects (Lin et al., 1999; Crochu et al., 2004). Since these viruses are not retroviruses, one can raise the question as to whether shrimp and other arthropods have a mechanism for integration of viral sequences into their genomes and whether this might play some role in their subsequent ability to tolerate infections without signs of disease.

Persistent infections and viral accommodation

Since shrimp and other arthropods have the capacity to carry single to multiple viral infections without gross signs of disease, a theoretical viral accommodation model has been proposed to account for the phenomenon, and directions for testing the model have been proposed (Flegel, 2007a; Flegel and Pasharawipas, 1998). Briefly, the updated model (Flegel, 2007a) proposes that shrimp and other arthropods have an active (adaptive) mechanism for accommodation of viral pathogens in a manner that leads to persistent infections without signs of disease, that blocks viral-triggered apoptosis and that also provides some protection against mortality upon subsequent superinfection with the same and possibly other viruses.

An initially unknown memory mechanism was an essential element of the viral accommodation model (Flegel and Pasharawipas, 1998), but memory was later proposed to reside in the persistent viral infections themselves (Flegel, 2007a). From the evidence in the section above, however, we may possibly add viral sequences inserted into the host genome. This could be a potential mechanism for heritable memory. It raises the possibility for existence of a natural process for insertion of viral genome fragments into the shrimp host genome in a manner that leads to production of antisense mRNA transcripts capable of protecting against mortality upon challenge by the same virus. As described in the section on RNAi, artificial antisense constructs of TSV were effective in protecting against TSV in transgenic shrimp (Lu and Sun, 2005). If such a natural process does exist, it would constitute a new type of adaptive response to viral pathogens, perhaps unique to arthropods in the animal kingdom. A requisite for such a system, at least for RNA viruses that are not retroviruses, would be a host reverse transcriptase (RT). As it turns out, several candidate RT sequences can be found by a quick search of the P. monodon and P. vannamei databases. Thus, we might imagine a process based on reverse transcription of mRNA sequences that would allow for a common response pathway to cover both RNA and DNA viruses. Such a process might allow the host to specifically target viral mRNA sequences based on some kind of non-self recognition mechanism. The variety of RT-like sequences that can be found in the shrimp databases supports the possibility. If this speculation proved to be correct, understanding the process would allow its manipulation in a directed manner (much in the way that protective antigens are selected for vaccine preparation in vertebrates) to insure that protective viral seqences were inserted into the shrimp genome by the natural shrimp mechanism. Ideas such as these suggest avenues of research that can lead to new disease control applications.

A corollary to the accommodation model is that failure of its mechanism will lead to massive apoptosis and death of the infected shrimp. To distinguish massive apoptosis leading to death from that normally referred to in work on metazoan development and on protective antiviral responses in vertebrates, we have proposed the term “kakoapoptosis” (Flegel and Sritunyalucksana, 2009) derived by combining the Greek “kako” for bad or “detrimental” with apoptosis. This distinguishes kakoapotosis clearly from beneficial apoptosis associated with development and maintenance of homeostasis, since that must also occur in shrimp, as it does in all other multicellular animals.

The viral accommodation concept is controversial, particularly with respect to kakoapoptosis, although the latter has been reported to occur in both WSSV and YHV infections (Sahtout et al., 2001; Khanobdee et al., 2002; Sahul Hameed et al., 2006; Wongprasert et al., 2003). In addition, knock-down of shrimp caspase-3 with dsRNA was reported to reduce mortality in P. vannamei challenged with WSSV (Rijiravanich et al., 2008). By contrast, other publications claim that apoptosis (not kakoapotosis) is part of the antiviral response to protect shrimp from WSSV in P. vannamei (Granja et al., 2003) and P. japonicus (Wang et al., 2008a; Wang and Zhang, 2008). Yet another study report claimed that apoptosis did not play an important role in survival of WSSV-challenged P. japonicus that were tolerant to WSSV (Wu and Muroga, 2004). At this time, differences in test shrimp, viral inocula and test protocols that have been used make comparisons among the various reports difficult. However, if kakoapoptosis does turn out to be a major cause of massive shrimp mortality, understanding it may allow for simple interventions that will prevent its occurrence in viral-infected shrimp. Again such ideas suggest avenues of research that may lead to new disease control applications.

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