Assessment of Artemia franciscana as a probable vector for WSSV transmission to Macrobrachium idella idella (Hilgendorf, 1898)

Transcription

1 International Journal of Chemical and Analytical Science ISSN: 0910 Research Article Assessment of Artemia franciscana as a probable vector for WSSV transmission to Macrobrachium idella idella MA Badhul Haq, H Ashraf Ali, AR Nazar, Upasana Ghosh, R Shalini, Somnath Chakraborty* Faculty of Marine Sciences, Center of Advanced Study in Marine Biology, Annamalai University, Parangipettai 08 0, Tamil Nadu, India. The present research work is carried out to confirm that the White Spot Syndrome Virus (WSSV) is an important pathogen of penaeid shrimp. For this work, WSSV infected farm reared Penaeus monodon were collected from Marakkanam, Pondicherry. The experimental animals were formerly tested for WSSV using WSSV test strip kit. The brine shrimp Artemia franciscana cysts were obtained from Andhra Pradesh. The cysts were hatched in filtered seawater (0 ppt salinity) at 8 0ºC. The Artemia naupli were exposed to WSSV inoculations of dosage (0µl, µl and 10µl) in three separate tanks along with a control tank maintained with WSSV negative P. monodon haemolymph. Post larvas of Macrobrachium idella idella were obtained from the Vellar estuary, Parangipettai. The M. idella idella were fed with Artemia naupli exposed to WSSV inoculations. After few days the infected M. idella idella showed white patches on their body and finally the hemolymph was collected from the moribund animals and subjected to nested PCR analysis for WSSV detection. The nested PCR showed the maximum positive result for 10µl of WSSV inoculations. Therefore, it can be concluded that the WSSV infected meat and Artemia is acting as a reservoir or carrier of WSSV for fresh water prawn Macrobrachium idella idella. Key words: Artemia franciscana, vector, WSSV, Macrobrachium idella idella INTRODUCTION White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimps are cultured, as well as in the Gulf of Mexico and SE USA. In most countries viral diseases have been the most important cause of economic losses. In order to give some idea about the rate at which viral diseases are increasing, Lightner et al., (1990) reported six viruses affecting penaeid shrimps. By 199, the list of known viruses had increased to 1 (Lightner 199a) and today, around 1 viruses have been reported in shrimps, some of which have several strains. Of these, viruses, Taura syndrome virus, yellow head virus and white spot virus, have been responsible for the most severe losses. During the last decade, white spot syndrome virus (WSSV) has emerged as the major shrimp pathogen causing epizootics and heavy crop failures across the world (Rosenberry 001).The virulence of geographic isolates of WSSV was compared using Litopenaeus vannamei post larvae. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. Even though nearly 10 of the known shrimp viruses have been found in cultured penaeid shrimp in the Western Hemisphere, at least four viruses caused pandemics that have adversely affected the global penaeid shrimp farming industry since These viruses in the approximate order of their discovery are Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV), and White Spot Syndrome Virus (WSSV). The socioeconomic impact of the diseases caused by these viruses have been so severe in some shrimp producing countries of Asia and the western hemisphere (Americas) that they were listed by the World Animal Health Organization (or Office International des Epizootics OIE) as posing a significant disease threat to cultured and wild crustaceans as a consequence of international trade or movement of infected crustaceans. White spot syndrome virus (WSSV) is an extremely virulent causative agent of white spot syndrome, an acute, contagious disease of shrimp. WSSV was first reported from farmed Marsupenaeus japonicus in Japan in 199 (Inouye et al., 199, 199, Nakano et al., 199, Momoyama et al., 199, 199) and called as the penaeid rodshaped DNA virus (PRDV) or rodshaped nuclear virus of M. japonicus (RVPJ) (Takahashi et al., 199, Momoyama et al., 199, Kimura et al.,199, Inouye et al., 199, 199). Similar rodshaped viruses from elsewhere in Asia were called as hypodermal and hematopoietic necrosis baculovirus (HHNBV) (Huang et al.199a,b) or Chinese baculovirus (CBV) (Lu et al., 199a,b, Loh et al., 1998) for China, White spot baculovirus (WSBV) for Taiwan (Chou et al., 199, Wang et al.,199,199a, Lo et al. 199a, 199), and systemic ectodermal and mesodermal baculovirus (SEMBV) or PmNOBII in Thailand (Wongteerasupaya et al., 199, 199). Lightner (199) has grouped these viruses in a single white spot syndrome virus (WSSV) complex. WSSV is a dsdna virus originally considered related to nonoccluded baculoviruses, but its relationship with other viruses must await more detailed genome analysis. The status of work on WSSV has been reviewed (Lightner 199, Flegel 199, Flegel et al., 199). Fry obtained from wild broodstock stocked in the rearing ponds are known to carry WSSV, as they are numerous than other crustaceans and perhaps even aquatic insect larvae (Lo et al., 199a, 199, Flegel 199, Kanchanaphum et al., 1998, Supamattaya et al., 1998), but massive mortality usually occurs with juvenile shrimp in rearing ponds, possibly precipitated by environmental factors. White Spot Syndrome Virus (WSSV), a doublestranded genome with size between 9 and 0 kilobase pairs (kbp) (Van Hulten et al., 001, Yang et al., 001, Chen et al., 00). The genus Whispovirus within the family Nimaviridea (Mayo 00). Corresponding Author: Somnath Chakraborty, Faculty of Marine Sciences, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 080, Tamil Nadu, India Received 11010; Revised 10011; Accepted 0011 September, 011 International Journal of Chemical and Analytical Science, 011, (9),

2 White spot syndrome causes high mortality and it also seriously affect most of the commercially cultured marine shrimp species, not just in Taiwan and Asia but globally also (Chou et al. 199, Flegel 199, Spann et al. 199). WSSV infects many types of ectoderm and mesodermal tissues, including the cuticular epithelium, connective, nervous, muscle, lymphoid and haematopoietic tissues. The virus also severely damages the stomach, gills, antennal gland, heart and eyes. During later stages of infection, these organs are destroyed and most of the cells are lysed. The shrimp then show reddish colouration of the hepatopancreas and the characteristic 1 mm diameter white spots (inclusions) on their carapace, appendages and inside surfaces of the body. They also show lethargic behaviour and cumulative mortality typically reaches percent within two to seven days of infection (GSMFC Web site). White spot disease (WSD) has proved to be the most serious epidemic in cultured shrimp throughout Asia (Wang et al., 199, Wongteerasupaya et al., 199, Lo et al., 199a, Flegel 199, Park et al., 1998, Sudha et al., 1998, Magbanua et al., 000) and, more recently, in America (Calderon et al., 000). Recently, disease outbreaks have caused mass mortality among cultured penaeid shrimps in Asian countries. Outbreaks of WSSV were initially restricted to the Asian countries, but they have now been reported from the USA (Loh et al., 1998), Central American and South America. Using molecular biological techniques it has been shown that WSSV from these outbreaks is identical or closely related (Lo et al., 199a, 1999, Loh et al.,1998, Takahashi et al., 199, Wang et al., 199, 199b, Wongteerasupaya et al., 199). In India, the pathogen was first identified as systemic ectodermal and mesodermal baculovirus (SEMBV) following the designation in Thailand (Wongteerasupaya et al., 199). Subsequently, many reports have been published (Karunasagar et al., 199; Mohan et al., 1998; Rajendran et al., 1998, 1999), and it has been generally agreed that the pathogen responsible for the epizootic is WSSV. Since 199, white spot syndrome virus (WSSV) has emerged as the most serious disease of cultured penaeid shrimp in the Eastern and Western hemispheres (Chou et al., 199; Flegel, 199; Huang et al., 199; Inouye et al., 199; Lightner, 199; Wand Q., et al., 1999; Wang, Y.G., et al., 1999). WSSV, as with most viral diseases, is not thought to be truly vertically transmitted, because disinfection of water supplies and the washing and /or disinfection of the eggs and nauplius are successful in preventing its transmission from positive broodstock to their larvae (horizontal transmission). Instead, it is generally believed that the virus sticks to the outer shell of the egg, since, if it gains entry to the egg, it is rendered infertile and it will not hatch. WSSV has such a wide host range that extends not only to several shrimp species but also too many other crustaceans (Lo et al., 199b). WSSV is also capable of infecting or contaminating the brine shrimp Artemia (Crustacea, Anostraca), that was used worldwide for feeding fish and crustaceans (Bardach et al., 19, Sorgeloos & Persoone 19, Sorgeloos 1980, Kim et al., 199, Naessens et al., 199). For example, Artemia nauplii are routinely used in penaeid shrimp larviculture from mysis to post larval (PL) stages (Cook & Murphy 199, Beard et al., 19, Emmerson 198). Thus, if infected or contaminated with WSSV, Artemia would constitute a serious threat to the shrimp industry. WSSV has not hitherto been positively identified either in Artemia or in Artemia cysts (sometimes mistakenly called brine shrimp eggs ). In aquaculture operations, feed is an important input and accounts for about 0 0% of the recurring investment. Live feed organisms play an important role in the dietary regimen of cultivable fish and shellfish, particularly in the larval stages. Among the live feed organisms, the brine shrimp Artemia comes in for prime consideration because of its nutritional and operational advantages Sorgeloss et al., 198. Artemia, however, are also considered as a possible vector for the introduction of viruses and bacteria into the rearing systems; live feeds are thought to be responsible for the source of viral and bacterial infections Tatani et al., 198; Muroga et al., 1989; Nicolas et al., 1989; Mortensen et al., 199; Skliris and Richards, Badhul Haq (199) and Srinivasan et al., (1999) had reported about the status on white spot disease in shrimp farm in Vellar Estuary. Sarathi (008) conducted a course experimental challenge of WSSV to examine the clearance of the former in Macrobrachium rosenbergii and the consequent immunological changes. Kallaya Sritunyalucksana (00) also studied the comparison of PCR testing methods for white spot syndrome virus (WSSV) infections in penaeid shrimp. The simultaneous presence of Monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in apparently healthy postlarvae of Penaeus monodon from different hatcheries in India was studied by nested polymerase chain reaction (PCR) Otta et al., (00). Shahadat Hossain et al., (001) had indicated previously that wild caught asymptomatic marine shrimp such as Metapenaeus dobsoni, Parapenaeopsis stylifera, Solenocera indica and Squilla mantis carry WSSV. The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvolike virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV Sivakumar et al., (009). The present study indicated that the shrimp pathogen WSSV, considered to be the most serious virus disease of farmed shrimps, could be transmitted through Artemia to infect Macrobrachium idella idella post larvae (PL). Artemia nauplii accumulated the viral pathogen in through immersion method. The PCR primer pairs to screen Artemia nauplii from several different doses for the presence of WSSV. And also used PCR tests to screen post larva animal from WSSV PCR positive Artemia nauplii and to examine M. idella idella experimentally fed with Artemia nauplii derived from WSSV PCR positive Artemia nauplii. The objective of present investigation is to assess the level of pathogenicity of WSSV for different doses of Artemia and investigate the possibility that the Artemia may act as a reservoir and carrier of white spot syndrome virus to infect fresh water prawn Macrobrachium idella idella. MATERIAL AND METHODS Shrimp Penaeus monodon WSSVinfected farm reared P. monodon (mean body weight = 10. ± 1. g) were collected from Marakkanam, Pondicherry, (Fig 1) that were maintained under captivity. The shrimps used in the experiments were previously tested for WSSV using WSSV test strip kit, as described below. The shrimps were fed with a commercial pellet feed twice daily, during the acclimation and experimental period. September, 011 International Journal of Chemical and Analytical Science, 011, (9),

3 Fig. 1: Location of the shrimp hatchery in Pondicherry Interpretation The results were interpreted by reading the appearance of pink/purple lines on the strip. Negative result was appeared at location C (control). Animal were not infected by WSSV or were infected with virus below the limit of detection of the strip. Positive result (infected by WSSV) was appeared at C (control) and T (test) respectively (Fig. ). Fig. : Preliminary test of WSSV Artemia Artemia franciscana The brine shrimp Artemia franciscana cysts were obtained from THE WATER BASE TED, Andhra Pradesh. The cysts were hatched in filtered seawater (0 ppt salinity) at 8 0ºC. After hrs of incubation, hatched instar I nauplii were separated from the unhatched and empty cysts, and stocked in l epoxy coated tank with fresh filtered seawater and continuous aeration to keep the food particles in suspension and to ensure oxygenation. The nauplii were fed on Chaetoceros sp. Next day the Artemia nauplii were transferred into epoxy experimental tank at per l capacity in four experimental bases. Each tank were stocked in 1/ml of Artemia nauplii. First three tanks was maintained different WSSV viral inoculation (0µl, µl and 10µl). The fourth tank was maintained in control with WSSV negative shrimp haemolymph. Macrobrachium idella idella Post larva of Macrobrachium idella idella were obtained from the Vellar estuary near to Faculty of Marine Science, CAS in Marine Biology, Parangipettai and maintained in four 0 l epoxy tanks at room temperature (8 0ºC) with salinity between and 0 ppt for 1 week to acclimatize prior to initiate the experiments. The animals were fed on artificial powdered feed manufactured by CP AQUA FEED LTD. Filtered and sterilized estuarine water was used for all the experiments. The experimental animals Artemia, M. idella idella ( 10) were randomly selected and screened for the WSSV by PCR using the primers designed by IQ000 kit. Origin and purity of WSSV DNA used as challenging and transmission studies P. monodon were collected from farms undergoing with a WSSV outbreak in Marakkanam, Pondicherry. The animals were individually screened for WSSV using field test strip and nested PCR. WSSV positive animals were selected for viral inoculum preparation. Hemolymph was drawn out directly from the anterior region of the rostrum or heart of infected shrimp using sterile syringes. The pooled hemolymph was centrifuged at 000g for 0 min at ºC. The supernatant was recentrifuged at 8000g for 0 min at ºC, and the final supernatant fluid was filtered through a 0.mm filter. The filtrate was then stored at 0ºC for infectivity studies. The total protein in hemolymph was determined according to the method of Lowry et al., (191). The presence of WSSV in the hemolymph and tissue was checked by PCR using primers designed by IQ000. Total DNA was extracted (Lysis Method) from hemolymph (µl) and tissues ( g) of the collected animals and analyzed by nested PCR to detect WSSV DNA. Animals infected with WSSV alone were used to prepare a WSSV inoculum. The inoculum was used to challenge WSSV negative animals (Artemia fransiscana and M. idella idella) under experimental conditions. All challenged animals displayed symptoms of WSSV infection thus proving the presence of infectious WSSV white patch particles (Fig. ). Fig. : Animal showing WSSV infection Procedure for WSSV preliminary test strip Shrimp tissue sample: Add 0. ml of sample buffer in the tube provided. Place small (approximately mm0 piece of tissue (gills, hepatopancreas, epithelial tissue, muscle and pleopods) into the tube containing the sample buffer. Homogenize the tissue in the tube with the grinder provided. Subsequent to homogenization, run the centrifuged (10,000 rpm for 1 minutes). Place ml of supernatant into the well on the test strip (Fig. ). After sample deposited, observed result interpretation. September, 011 International Journal of Chemical and Analytical Science, 011, (9),

4 WSSV transmission in A. fransiscana The pathogenicity of WSSV on different viral quantity experimental tanks of Artemia nauplii was carried out by immersion challenge method. An aliquot (0,, 10µl/litre) of the WSSV inoculums was used for Artemia nauplii in different doses at three experimental tanks. WSSV negative hemolymph (10µl) was used in fourth experimental tank as control inoculums. The Artemia nauplii were followed by immersion method described above for 1 week. After the exposures, the animals were collected and given oral feed in respective volume. Dead and moribund animal were collected and stored at 0 C. These were then used to detect WSSV using PCR. WSSV challenging in M.idella idella (Feeding of hatched WSSVpositive Artemia nauplii to M. idella idella) Uninfected M. idella idella larvae from a WSSV free (step PCRnegative) post larva were used for this challenge test. In the immersion challenge, immersion & oral batches of healthy post larva of M. idella idella were stocked separately in sterilized aerated seawater. The post larva of M. idella idella ( per tank) were divided into four groups (experimental and control) and maintained separately in epoxy resin coated fibre glass tanks ( l capacity) at 0 C. To validate the pathogenicity of the WSSV inocula, challenge tests were carried out with respective group of 00 members of M. idella idella (0. to 0. g) maintained in experimental set up. Challenge and control protocols were the same as those for the post larva. In group A, the PL was fed on Artemia nauplii (group B and C) exposed to WSSV by immersion. The inoculum of WSSV was introduced to the water at a volume equal to 0.1% of the total stocked animal (1 ml / l) (Venegas et al., 1999; Chen et al., 000). In group B, the animals were used in WSSV positive meat exposed to WSSV by oral route. The third experimental tank was used in both the methods (immersion and oral) in 0.1% of the total stocked animal (1 ml/l) and 10% of body wt. In group C, the animals were inoculated on viral hemolymph and fed (10% of their body weight) on meat of WSSV infected shrimp. This group were exposed to WSSV by immersion and oral method. The D was control groups were exposed (immersion method) to the hemolymph (0.1%) collected from WSSV free healthy shrimp and the animals were fed ( method) on WSSV free Artemia nauplii. The experiment was terminated on the 9th day and remaining animals were individually tested for WSSV using step PCR. Each trial was conducted in triplicates (Table.). The post larvae of M. idella idella were followed by oral and immersion method described above for 1 week. After the exposures, the experimental shrimps were collected, washed with sterile seawater and stored at 0 ºC. These were then used to detection of PCR by nested method (Fig. ). Fig.. PCR analysis samples PL (M. idella idella) Sample preparation and PCR analysis Moribund M. idella idella exhibited red coloration or discoloration and white patch on the body. Each random sample was considered as a separate individual sample in day 1,,,,,,, 8 and 9 intervals for the PCR analysis and was processed separately using disposable eppendorf apparatus to avoid crosscontamination. WSSV infection was determined by nested PCR from gills, hepatopancreas and pleopods (0 µg) individually collected from experimental shrimps at above described interval after inoculation. PCR viral detection was analyzed at the time of hand on training carried out at NK Marine PCR Laboratory, Marakkanam, Pondicherry. The assorted portion of the selected animal was taken for DNA extraction, performed by Lysis method. Briefly, each moribund sample of 10 PL was taken in a 1. ml eppendorf tube and thoroughly homogenized with 00µl of DNA extraction solution (Lysis Buffer) in a sterile mortar pestel. The prepared sample was incubated at 9ºC for 10 minutes, and then centrifuged at 1000 rpm for 10 minutes. 00µl of the upper clear solution was transferred to a fresh 1. ml eppendorf tube with 00µl of 9% ethanol. Vortexed briefly, centrifuged at 1000 rpm for minutes, then the ethanol was decanted and the pellet was dried and dissolved in TE buffer and immediately kept at ºC until used. The PCR was performed using the method of step WSSV diagnostic nested PCR, described by IQ000 Farming IntelliGene Tech. Corp, Taipei, Taiwan using first PCR primer for the preliminary amplification and the nested PCR primer for the second nested amplification. The first PCR profile were carried out in.µl reaction master mixture containing µl of template DNA (approximately ηg) and 0.µl of IQzyme DNA Polymerase and nested PCR were carried out in 1µl of reaction mixture containing 1µl IQzyme DNA Polymerase and make up µl final volume. Amplification was performed in a thermocycler (PCR Express) using the following protocol: 1 cycle at 9ºC for min, then 9 C for 0 sec; C for 0 sec; C for 0 sec, repeatedly 1 cycles were run, then C for 0 sec; 0 C for 0 sec at the end of the final cycle. The second PCR profile was carried out in 9 C for 0 sec, C for 0 sec; C for 0 sec, repeat 0 cycles, then C for 0 sec 0 C for 0 sec at the end of the concluding cycle, followed by a final extension for min at C. Electrophoresis was executed by loading 1µl of the amplified product and µl DNA molecular markers onto 1.% agarose gel with 1 TBE (Trizma, boric acid, EDTA) buffer. The gel was stained using ethidium bromide solution (1 µg ml 1) for 0 min, and the bands were visualized by UV transillumination and GelDoc system. Accomplished WSSV negative and positive result interpretation with help of performed gel, under UV exposure GelDoc System. PCR controls: All PCRs were conducted according to PCR laboratory setup specifications (Kwok & Higuchi 1989, Dieffenbach et al. 199) to avoid contamination. The steps in the PCR such as sample preparation, pre PCR reagent preparation, post PCR, and step PCR were physically separated and performed in different rooms. DNA samples and PCR reagents were handled with positive displacement pipettes and tips with filter barriers to prevent carryover of aerosols. Separate sets of equipment were used for sample preparation, preamplification, postamplification and step PCR work. Utmost care was taken to prevent cross September, 011 International Journal of Chemical and Analytical Science, 011, (9),

5 contamination of samples. A negative control was always included in each PCR run; that contained all the PCR reagents except the template DNA and was processed and loaded immediately after the positive control at each step. This allowed a check on any carry over or crosscontamination. Fig.. Survival & PCR assay results of WSSV in A. Franciscana RESULTS Challenge of Artemia nauplii with WSSV More than 0% of Artemia nauplii exposed to WSSV inoculums proved to be PCR positive for WSSV after the days experiment (Table.1). Of the experimental tanks out of that were PCRpositive, only 1 was negative, indicating a low level of contamination. Table 1. Survival and PCR test results of WSSV in A. Franciscana Groups Days step PCR Negative step PCR positive Artemia exposed to WSSV (0µl/ l) Group A Artemia exposed to WSSV (µl/ l) Group B Artemia exposed to WSSV (10µl/ l) Group C Artemia unexposed to WSSV (10µl/ l) Group D (control) Survival (%) Challenge of M. idella idella post larva with WSSV contaminated Artemia nauplii A high level of infection (up to 9%) was noticed in M. idella idella post larva fed with WSSV infected Artemia nauplii (Table. & ). Out of experimental tanks fed with infected Artemia nauplii and WSSV positive meat, were proven to be step PCR positive whereas control groups were negative. Twostep WSSV PCR tests of Artemia and M. idella idella Examples of the PCR detection results with the IQ 000 primer set in the first tested experimental groups and last one was control group, Artemia results are shown in Fig.. In total, experimental tanks out of experimental tanks samples of B group and C group gave WSSV positive reactions in the second step of the step PCR while B and C each provided positive result in the th day. However, the primer IQ000 gave negative results for day 1 to day in every tested sample of these groups. WSSV negative results were also obtained with a group and control D group of Artemia nauplii tested (Table.1). The cumulative mortality in different viral inoculums of WSSV infected hemolymph (0, and 10µl ranged in per litre culture water in immersion challenge route) is presented in Table.. These ranges were within the expected range for normal rearing conditions. However, there were no differences between control and experimental A groups. The WSSV stock used in previous study was prepared and titrated by immersion or oral inoculations as described previously (EscobedoBonilla et al., 00). The median virus titer of infection was10. SID0 ml 1 by immersion route and 10. SID0 ml 1 by oral route. Earlier study reported the cumulative mortality rates in all the developmental stages ranged from.8% to.% in immersion challenge route and..8% in oral route. These mortality values were within the predicted limits that could be seen in normal rearing. In both the challenge experiments the naupliar stage was negative by PCR while all the other developmental stages were positive for HPV on day of post challenge by single step PCR. PCR assay for HPV was negative in all the developmental stages of Artemia in control groups. The PCR detection of WSSV revealed no positive results in any of the 0µl viral inoculums of Artemia immersed in WSSV suspension (Table 1, Fig. ). Animals in experimental A group and control groups were healthy and showed no symptoms of WSSV disease. The mortality was occurred in group B and C in th & rd day onwards. WSSV positive indications were shown in group B and C. After rd day the WSSV positive were presented 9 bp (sample of very light WSSV infection) and the end of the day (Fig. & 8), the positive band showed moderate WSSV infection i.e., 0 bp. The group A and group D (control) presented negative band at 88 bp. Negative samples showed only one band (88bp), which was the product of house keeping gene (Fig. & 9). The molecular weight markers were recorded as 88 bp, 0 bp and bp respectively (Fig. ). The challenge test against M. idella idella post larvae treated with infected meat and Artemia exposed to the WSSV immersion exposed to the WSSV were compared with control group (uninfected hemolymph and fresh live A. franciscana). Due to cannibalistic behaviour mortality observed 1% / day subsequently on, and 9 days respectively in groups D (Table.) control tank and group C. The PCR analysis showed a negative result for the entire control group. September, 011 International Journal of Chemical and Analytical Science, 011, (9),

6 Table. Survival and diagnostic data for each experimental group of M.idella idella from a transmission trial with white spot syndrome virus (WSSV). Treatment Group A Group B Group C Method of inoculums route & route Day to collected PCR sample Determined by PCR SI SI Replicate 1 Replicate Determined Determined by by Test strip PCR Group D 19 SI SI Determined by Test strip SI: Severe infection of WSSV; : Moderate infection of WSSV; : Light infection of WSSV; : Negative for test strip and : Positive for test strip Similarly, in group C, M. idella idella was transmitted with WSSV inoculums resulted 0% mortality within days post inoculums (Table.&). Whereas % mortality was observed in group C after 8 days of oral and immersion inoculums of the WSSV infected shrimp (hemolymph and meat). By oral challenge (group B), the initial mortality taken place at days post inoculum and 9% at days respectively. By immersion challenge, the first departed M. idella idella were perceived at days post inoculum and mortality was 11% at days post inoculum (Table.). The group A and B were recorded 9% and % of mortality in the end of 9 th day. Moribund PL lay on their backs waving their pleopods for several hours prior to death. No other clinical signs of disease were observed. In oral challenges, the infected prawn tissue was rapidly consumed. In this replicate, average 101 dead animal was detected on each of Days to 8 d post challenges and PL were survived at Group A, immersion method in trial completion at 9 days (9% mortality). The control group, PCR was negative for the Artemia nauplii fed with WSSV free hemolymph and positive for all other groups. The control samples were presented as 88 bp and group C showed severe WSSV positive band in 910 bp, 0 bp and 9 bp (Fig. 11 & 1). In group A and B light and moderate WSSV infection and positive bands were observed having 0 bp and 9 bp (Fig.10). Overall the experimental survival was presented as 1.%. Survival in the control group was 9% on completion of the trial (Table. and ). Test strip analyses were made in day onwards (Table.). WSSV positive was found on the end of day in group A and B. In group C, WSSV positive were recorded day 9. Negative results were presented in group D control tank. Similarly, Sarathi et al., (008) studied the post larvae of Macrobrachium rosenbergii injected with WSSV, the samples of gills, pleopods, soft tissue of head and hemolymph were collected at different intervals of post infection. The moribund shrimps showed WSSV positive and the existing shrimps showed negative by PCR. Fig.. PCR results for Group A September, 011 International Journal of Chemical and Analytical Science, 011, (9),

7 Table. Cumulative percentage mortality levels in method of viral challenges of Artemia exposed to WSSV by immersion, route and & route Fig. 8. PCR results for Group C Expe rime ntal Days Method of challenge and and and and and and and and and Number of shrimp (M. idella idella) used Cumulative mortality (%) Detection WSSV by PCR Fig. 9. PCR results for Group D Fig.. PCR results for Group B September, 011 International Journal of Chemical and Analytical Science, 011, (9),

8 Fig. 10. PCR results showing WSSV and bands in Group A & B. Fig. 11. PCR results showing WSSV bands in Group C. Fig. 1. PCR results showing WSSV bands in Group D. Fig. 1. Cumulative percent mortality in M. idella idella exposed to WSSV September, 011 International Journal of Chemical and Analytical Science, 011, (9),

9 DISCUSSION AND CONCLUSION WSSV has been widely reported in cultured and wild susceptible animals, such as shrimps, crabs, and other arthropods (Lo et al., 199, Chakraborty et al., 00, Hossain et al., 00, Lightner, 00, De la RosaVélez and Bonami, 00, and EscobedoBonilla et al., 008). Possible routes for WSSV transmission include cannibalism of moribund shrimps (Wu et al., 001 and Lotz and Soto, 00), vertical transmission from infected spawners to stocked postlarvae (SánchezMartínez et al., 00), and horizontal transmission from batches of infected post larvae in a pond and subsequent spread of the pathogen to a neighboring pond or to another farm (Wu et al., 001 and Lightner, 00). The WSSV has been found to be a highly pathogenic virus for the penaeid shrimp and transmitted to cultured shrimp via contaminated water and ingestion of WSSV infected shrimp meat (Kasornchandra et al., 199). Evidence of transmission of WSSV among the shrimp by cohabitation in the shrimp farming environment also exists (Flegel, 199; Flegel et al., 1998). The studies indicate that the most common marine and freshwater decapods including uncultured arthropods can be infected by the WSSV and can act as a reservoir or carrier for WSSV (Lo et al., 199; Chang et al., 1998; Lo and Kou, 1998; Maeda et al., 1998; Supamattaya et al., 1998; Wang et al., 1998). A detailed experimental study on the pathogenicity of WSSV on Artemia and their possibility of acting as a reservoir or carrier of WSSV in the shrimp hatcheries and farms is lacking from the above studies. Presently an invigorative emphasis is made to fill this gap as Artemia is an important cohabitant and live feed organism for marine shrimp in the hatcheries. The cumulative mortality data obtained from the pathogenicity experiments from the current investigation showed that the existing WSSV can infect the different dosages of WSSV viral inoculums by the immersion challenge route. The PCR results also confirmed the above surveillance. The exact mechanism of resistance to WSSV was not known at present in control groups. A steady trend of mortalities noticed in the post larva M. idella idella treated with infected meat and A. francisca exposed to WSSV, whereas % mortality occurred in group C that was fed with WSSV infected shrimp meat and immersed by infected hemolymph. These observations clearly indicate that A.francisca is acting as a reservoir or mechanical carrier for WSSV. This observation was further inveterated by PCR (Figs. and 1). The WSSV caused.% and.% mortality in M. lamerrae and M. idella, respectively, by immersion method and.% and.% mortality in M. lamerrae and M. idella, respectively, by oral route. This virus caused % mortality in M. idella, M. lamerrae, P. indicus and P. monodon when the animals were injected with WSSV intramuscularly Sahul Hameed et al., (000). In the challenge tests by intramuscular injection, C. destructor albidus displayed a similar level of susceptibility to white spot disease (WSD) as Penaeus monodon (i.e. % mortality in d). In one oral challenge test where C. destructor albidus was subjected to significant temperature stress, over 0% died due to severe WSD within 1 days post challenge (Brett F. Edgerton, 00). The outcome of pathogen challenge is determined by many interacting factors including host species, biology, age and health. However, based on the results herein and on published data from trials conducted with several decapods, it appears that freshwater crayfish may have a lower level of susceptibility to WSD than many other decapods such as penaeid prawns (Edgerton 00). Freshwater prawns are also less susceptible to WSD when compared to penaeid prawns (Sahul Hameed et al., 000, Pramod Kiran et al., 00). The finding of cells infected by WSSV in the gonads of Cherax destructor albidus suggests the potential for vertical transmission, as it has been reported for penaeid prawns (Hsu et al., 1999). There is limited information in the literature concerning natural viral infection of Artemia, but there is a report of infection caused by IPNV and the role of Artemia as a reservoir and mechanical vector for IPNV to fish (Mortensen et al., 199). In our investigation, the WSSV presented to infect any transmission methods of Artemia and M. idella idella fed with infected and Artemia exposed to WSSV by the immersion challenge and oral route. Therefore, it can be concluded that WSSV infected meat and Artemia is acting as a reservoir or carrier of WSSV for fresh water prawn Macrobrachium idella idella, under these experimental conditions. 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