infectious diseases and control strategies in shrimp

Infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) or Penaeus stylirostris densovirus (PstDNV), was first reported in 1981 in Hawaii affecting the species Litopenaeus stylirostris and L. vannamei. This pathogen rapidly spread to other countries in America (Mexico, Panama, Colombia, Ecuador and Argentina), Asia (Indonesia, Malaysia, Philippines, Singapore and Thailand) and French Polynesia. Its genome is organised into three open reading frames (ORFs) encoding a non-structural protein, an unknown protein and a capsid protein, respectively.

IHHNV causes infection to several shrimp species. It appears that Penaeus monodon is not affected by IHHNV infection since no clinical signs and no differences in size, weight or fertility was found in IHHNV-positive animals. Clinical signs of IHHNV infection depend on the species age and size, being the early juvenile stages more susceptible to the disease.

In L. stylisrostris, acute IHHNV infection includes reduced feeding and locomotion, erratic swimming and death. In L. vannamei, acute IHHNV infection showed reduced growth rate, marked size differences within a pond and deformity of the rostrum, antennae and/or cuticle which is known as 'runt deformity syndrome' (RDS). Histological lesions are Cowdry-type A inclusion bodies in infected animals. This virus became the main pathogen both in shrimp fisheries and aquaculture in the 1980s in Mexico.

It was estimated that its economical impact was between 0.5 and 1 billion US dollars. This virus is still present in wild and farmed shrimp in Mexico and other countries.

Taura syndrome virus (TSV) First reported in shrimp farms near Taura river, Ecuador in

1992, TSV soon spread to several countries in South, Central, North America and Hawaii. Since 1999, TSV was also detected in Asia (Taiwan, Thailand and Korea) which imported stocks of L.

INFECTIOUS DISEASES AND CONTROL STRATEGIES IN SHRIMP

A brief overview of infectious diseases impacting farmed shrimp and some experimental strategies on disease control

by César Marcial Escobedo-Bonilla, Instituto Politécnico Nacional - Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Sinaloa

Modern shrimp aquaculture began in 1933 in Japan with the induced spawning and hatching of Marsupenaeus japonicus larvae.

This technology allowed the production of shrimp larvae in hatcheries instead of using larvae from the wild to stock grow-out ponds.

Shrimp farming is an important activity in several low-income countries in Asia, America and Africa as it generates employment and wealth. Nonetheless, intensification of shrimp culture increased the appearance of infectious diseases due to deviations in environmental and physiological factors.

Infectious diseases caused by viruses or bacteria represent the biggest threat to development of shrimp farming due to high mortalities. Pathogens that have caused severe epizootics and high mortalities to different stages and species of shrimp include Baculoviruses, Parvo-like viruses, Dicistrovirus, Ronivirus, Nimavirus and more recently, a bacterium Vibrio parahaemolyticus (Figure 1).

24 | January | February 2016 - International Aquafeed

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vannamei from South America. Its genome consists of a single, positive-sense RNA strand

around 10 kilobases long. It has two ORFs separated by a shorty non-coding intergenic region. ORF 1 encodes a putative non-structural polyprotein with several domains such as a helicase, a protease and a RNA-dependent RNA polymerase; ORF 2 encodes three structural proteins VP2, VP1 and VP3.

In specific pathogen-free L. vannamei, larger animals are more susceptible to infection and mortality than early juveniles. TSV infection has three clinical stages. In the acute stage (three – five days after onset of infection) animals display soft exoskeleton, melanised multifocal necrosis and expanded chromatophores. Here, animals become weak, have empty gut and often die (75 – 95 percent) during moulting. Cellular lesions include pyknosis, karyorrhexis and necrosis in epithelia of cuticle, digestive tract, gills, antennal gland and haematopoietic tissues.

The transition stage (four - eight days after onset of infection) shows a reduction in cellular lesions and melanisation, indicating the onset of the chronic phase. Here, surviving shrimp (eight days after infection) show wound repair and regeneration of epithelial tissues in affected organs. Mortality ceases and

SIZE 10MAXUM

80

160 0

NORGREN

MAX.

MIN.

393.31[9990]

391.31[9939]

48.00[1219]

52.19[1325]

DCCInlet

CYL.Disch.

End ofHead

BINInlet

53.25[1353]

57.69[1465]

102.13 [2594]

111.12 [2822]

2

18.00 [457]

1.93 [49]

256T060

1545

30

195.72 [4971]

F085

SHIM

PO

64.83[1647]

108.59[2759]

15.00[381]

36.91[937]

31.19[792]

29.19[741]

MAX.

MIN.

19.16[487]

30.00[762]

39.00[991]

Ă12.00[305]

2.00 NPT [WATER]

2.00 NPT [STEAM]

1.00 NPT [STEAM]

66.50[1689]

12.56 [319]

24.59 [625]15.88 [404]

284.00[7214]

1.00 NPT

2.00 NPT3/4 NPT

101.44[2577]

30.38[772]

88.00[2236]

108.28[2750]

278.03[7062]

199.38[5064]

269.88[6855]

67.28[1709]

15.00[381]

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Figure 1 - (A) IHHNV, (B)TSV, (C) YHV, (D) WSSV and (E) V. parahaemolyticus

b

C

D

e

a

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surviving shrimp molt shedding the necrotised cuticle. Several shrimp species are susceptible to TSV infection except

for shrimp from the genus Farfantepenaeus (F. aztecus and F duorarum) which seem to be resistant to TSV infection upon experimental infection.

The economic impact of TSV during 1992 to 1996 was estimated between 1.2 to 2 billion US dollars.

Yellow-head virus (YHV) It appeared in 1992 in Thailand and later it spread to other

countries in Asia (Taiwan, Indonesia, Malaysia, China, Philippines, India), Australia and America (USA and probably Mexico).

YHV has up to six different genotypes including the gill-associated virus (GAV) from Australia. Due to its morphology, YHV first was thought to be a granulosis-like virus (Baculoviridae). Later, its genome was found to be a RNA molecule.

The YHV genome is a single linear (positive strand) RNA molecule of 26652 nucleotides. It is organised into four distinct ORFs. ORF1a has a 3C-like protease motif; ORF1b has a “SDD” polymerase metal ion binding domain helicase. ORF2 encodes putative nucleocapsid proteins (g7 and g2) and ORF3 encodes putative surface glycoproteins (p18/20, p33 and g2.1). ORF4 is very small and it has no known product.

Many shrimp species are susceptible to YHV. Clinical signs include pale yellow body colouration, especially in hepatopancreas and gills in P. monodon. Other clinical signs are erratic swimming near pond shores and cumulative mortality up to 100 percent within three - five days after onset of clinical signs.

YHV causes systemic infection and replicates in organs such as gills, foregut, lymphoid organ, connective tissues of nerves, eyestalk, hepatopancreas and muscle. Cellular lesions include pyknosis and kariorrhexis in epithelial cells in gills, connective tissues and hematopoietic tissues.

The estimated losses caused by YHV from 1990 to 2007 are 500 million US dollars.

White spot syndrome virus (WSSV) The virion is bacilliform, non-occluded, enveloped, with a tail-

like appendage at one end. WSSV has one of the largest genomes (292 - 308 Kilobasepairs)

recorded for viruses. It contains up to 683 ORFs encoding peptides from 51 to 6077 aminoacids representing 92 percent of the total genome information.

WSSV was first recorded in Taiwan and soon after it spread to several countries in Asia and America. It has a broad host range including several penaeid shrimp, caridean shrimp, lobsters, crayfish, crabs and other decapod crustaceans.

Clinical signs include white spots in the inner surface of cuticle, probably formed by calcium carbonate accumulation due to dysfunction of epithelial cells; reddish discoloration of the body due to expansion of chromatophores; reduced feeding, lethargy and delayed hemolymph clotting.

Cumulative mortality (100%) occurs three- ten days after onset of clinical signs. Histopathology shows hypertrophied nuclei of WSSV-infected tissues with intranuclear amphophilic inclusions and marginated chromatin.

Since it first appeared in 1992 the economic impact of WSSV on shrimp aquaculture is well over eight billion US dollars and still is the most damaging viral pathogen for farmed shrimp worldwide.

Bacterial infections are common in shrimp aquaculture. Many diseases caused by Vibrio bacteria may kill wild and farmed shrimp both from hatcheries and grow-out ponds. Worldwide, several Vibrio species exist in the marine environment.

Recently, a novel disease known as early mortality syndrome

Figure 2 - Mortality curves from shrimp treated with (a) a diet supplemented with Spirulina platensis, and (b) inoculated with cidofovir. A slight delay in mortality was observed in the Spirulina treatment, whereas a better antiviral effect was shown in the cidofovir-treated shrimp. Nonetheless, either of these treatments prevented WSSV infection or shrimp mortality. From Rahman et al., (2006) Aquaculture 255: 600-605.

Figure 3 - Mortality curves from shrimp treated by hyperthermia. (a) shrimp inoculated with a low dose of WSSV by oral route. (b) shrimp inoculated with a high dose of WSSV by intramuscular route. In both experiments, the effect of hyperthermia greatly reduced mortality in WSSV-infected shrimp. From Rahman et al., (2006) Aquaculture. 261: 842-849.


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(EMS) or acute hepatopancreatic necrosis syndrome (AHPNS), is caused by a singular type of Vibrio parahaemolyticus. This bacterium contains an extrachromosomic plasmid (pVPA3-1) encoding two toxins (PirA/PirB), related to Photorhabdus luminiscens that are responsible for shrimp mortality.

EMS can produce high mortalities to affected shrimp early after pond stocking (10 - 40 days). Surviving shrimp might undergo stunting. EMS was first reported in 2009 in China and since then, it has caused massive mortalities to farmed shrimp in Asian countries such as Vietnam (2011), Thailand (2012) and Malaysia (2012).

In 2013, EMS was also recorded in Mexico where it caused severe production losses (up to 80 percent of total production in Sinaloa, Sonora and Nayarit).

Several strategies have been developed and tested under experimental conditions to tackle the negative impact of infectious diseases caused by viruses or bacteria. These include:

Immunostimulants Substances (peptidoglycans, β-glucans or lipopolysaccharides)

extracted from cell walls of bacteria (Bacillus sp.), fungi (Saccharomyces cerevisiae, Schizophyllum commune), algae (Sargassum polycystum) or herbs, which activate humoral (antibacterial activity, agglutinins, cytokine-like factors, modulators, clotting factors) and cellular (prophenoloxidase

system, encapsulation, nodule formation, phagocytosis) defense responses in shrimp.

Experimental animals fed with immunostimulants before or during challenge with bacteria or viruses showed reduced mortality compared to untreated controls.

Antivirals Substances from plants, algae or even

synthetic, have been tested in shrimp with variable results. A diet containing Spirulina platensis showed no antiviral effect but only slightly delayed mortality in WSSV-challenged shrimp. In contrast, an extract from Indian Cynodon dactylon supplemented to feed (2 percent), showed 100 percent protection upon a per os WSSV infection.

An antiviral (bis [2-methylheptyl] phthalate) extracted from the Indian plant Pongamia pinnata fed before and during a per os WSSV challenge showed a dose-dependent reduction of mortality (60 to 20 percent). A synthetic antiviral (cidofovir) showed higher efficacy than the Spirulina-supplemented diet to reduce and delay mortality of treated shrimp. Nonetheless, cidofovir did not prevent WSSV infection (figure 2).

Induction of a “quasi-immune” response and virus neutralisation

This strategy is based on the rationale that some shrimp surviving a virus outbreak may become resistant to a subsequent pathogen infection. Therefore it indicates a sort of “memory” in these animals.

Several studies evaluated the protective effect of inactive viral particles or recombinant viral envelope proteins administered to shrimp to prime their innate defense system. Results showed reduced mortality of treated animals. In addition, monoclonal or polyclonal antibodies directed against viral envelope proteins have been used to inactivate viral particles through virus neutralisation assays.

Three concentrations (10-1, 10-2 and 10-3) of a WSSV stock each mixed with an equal volume of a purified monoclonal antibody against WSSV VP28 showed a dose-dependent neutralisation effect. Shrimp inoculated only with WSSV showed 100 percent

mortality at seven days post inoculation (dpi). Shrimp treated with neutralised virus

concentrations 10-1 and 10-2 showed a slight delay in time to mortality (100 percent at 11 dpi). Animals treated with the 10-3 neutralised concentration showed 20 percent mortality at 25 dpi.

Recombinant subunit peptides displayed 20 – 40 percent shrimp mortality depending on time of WSSV challenge [3 to 21 days post treatment (dpt), respectively]. Other experiments reported mortalities between 48 percent with VP292 to 30 – 5 percent with VP28 as these recombinant peptides were administered twice during the experiments.

DNA vaccines These tools are plasmids encoding WSSV envelope

proteins (VP15, VP28, VP35 and VP281). Shrimp

Figure 4 - Mortality curve from shrimp treated with dsRNA against WSSV genes vp26 or vp28 using a high WSSV dose. RNAi against WSSV vp28 or vp26 effectively reduced WSSV infection and shrimp mortality compared to an unrelated dsRNA (LacZ) and controls. From Mejía-Ruíz et al., (2011) J Inv. Pathol. 107: 65-68.

Low water temperature is also effective to inhibit virus replication in species living in temperate or cold water. In shrimp M. japonicus, water temperature at 15 ºC showed better WSSV inhibition than 33 ºC


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International Aquafeed - January | February 2016 | 29

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P. monodon treated with a vp28 DNA vaccine by intramuscular route showed 10 percent mortality when challenged with WSSV seven days post vaccination (dpv).

Mortality increased to 20, 80 and 95 percent when WSSV challenge was done at 14, 25 and 50 dpv, respectively. A plasmid containing WSSV vp28 gene and injected to WSSV-challenged P. monodon showed 10, 24, 33 and 44 percent mortality at 7, 14, 21 and 30 dpv, respectively.

Oral delivery of a plasmid containing the WSSV gene vp28 expressed in attenuated S. typhimurium adsorbed into commercial feed showed protection against WSSV challenge in crayfish Cambarus clarkii. Crayfish mortality at 7, 15 and 25 dpv was 17, 33 and 43 percent, respectively.

Water temperature Using high water temperature (hyperthermia) at 32 ºC before,

just after or even until 18 hours after WSSV inoculation reduced virus replication and shrimp mortality (0 – 30 percent) compared to controls at 27 ºC (100 percent) (figure 3). The route of WSSV inoculation did not influence hyperthermia efficacy. Using hyperthermia in alternate periods of 18 hours is still effective against WSSV (0 – 40 percent mortality). Although hyperthermia reduced virus replication in shrimp and crayfish, animals remain infected as determined by PCR.

Low water temperature is also effective to inhibit virus replication in species living in temperate or cold water. In shrimp M. japonicus, water temperature at 15 ºC showed better WSSV inhibition than 33 ºC. Likewise, crayfish species (Pacifactacus leniusculus Astacus astacus and P. clarkii) maintained at 4, 10 or 12 ºC showed zero percent mortality upon WSSV infection. In contrast, WSSV-infected animals maintained at 22 - 24 ºC had 100 percent mortality.

The mechanism of inhibiting virus replication is still unknown, but it has been suggested that hyperthermia may induce

apoptosis of infected cells. Alternatively, another hypothesis is that hyperthermia may impair the activity of cellular enzymes essential for virus replication, thus inhibiting replication but animals remain infected.

RNA interference (RNAi) First described in the nematode Caenorhabditis elegans, RNE

interference is also found in fungi, plants and animals. In plants, one biological function of RNAi was antiviral. RNAi can be a useful tool against viral infections in animals.

Several RNAi studies have been done against shrimp viruses. Sequence-specific RNAi has been used to inhibit replication of TSV, IHHNV, YHV and WSSV. Double-stranded (ds)RNA against a TSV protease strongly inhibited TSV replication (11 percent mortality) whereas controls had 100 percent mortality.

The antiviral effect of RNAi against IHHNV was demonstrated to be both preventative and therapeutic, since dsRNA against ORF1/2 or ORF3 administered either 12 hours before or 24 hours after IHHNV challenge, effectively inhibited IHHNV replication. RNAi treatment (dsRNA) against YHV protease in vivo showed zero percent mortality at ten days post challenge.

In contrast, controls had greater than 90 percent mortality. Several RNAi studies have been done against WSSV since this is the most lethal pathogen in shrimp aquaculture. Variable efficacy against WSSV replication (0 – 66 percent mortality) has been reported depending on the genes targeted by dsRNA.

The antiviral effect against WSSV lasts up to 10 dpt (Figure 4). Duration of antiviral effect reduced as time between treatment and WSSV challenge increased. Antiviral effect was extended up to 30 days post challenge through continuous re-infection of treated shrimp. Alternatively, continuous dsRNA administration through feed may also increase the duration of the antiviral effect in cultured shrimp.

Bacterial infections are common in shrimp aquaculture. Many diseases caused by Vibrio bacteria may kill wild and farmed shrimp both from hatcheries and grow-out ponds.


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