PENGEMBANGAN MULTIPLEKS PCR (MPCR) UNTUK MENDETEKSI VIRUS PENYAKIT KERDIL UDANG WINDU DI TAMBAK PADA MUSIM BERBEDA

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1 PENGEMBANGAN MULTIPLEKS PCR (MPCR) UNTUK MENDETEKSI VIRUS PENYAKIT KERDIL UDANG WINDU DI TAMBAK PADA MUSIM BERBEDA MULTIPLEX PCR DEVELOPMENT FOR DETECTION OF MSGS-RELATED VIRUSES OF TIGER SHRIMP IN GROW-OUT PONDS AT DIFFERENT SEASONS Sriwulan 1, Akbar Tahir 2, Alexander Rantetondok 1, Baharuddin 3 1. Jurusan Perikanan Fakultas Ilmu Kelautan dan Perikanan UNHAS 2. Jurusan Ilmu Kelautan Fakultas Ilmu Kelautan dan Perikanan UNHAS 3. Jurusan Hama dan Penyakit Tumbuhan Fakultas Pertanian UNHAS Alamat Korespondensi: Ir. Sriwulan, MP Fakultas Ilmu Kelautan dan Perikanan Universitas Hasanuddin Jl. Perintis Kemerdekaan KM 10. Tamalanrea Makassar sriwulancinga@yahoo.com HP:

2 Abstrak Penyakit kerdil udang windu telah menyebabkan kerugian pada petani tambak karena ukuran udang tidak mencapai ukuran standar sesuai umur udang. Penelitian ini bertujuan menganalisis virus MSGS (MBV, IHHNV dan HPV) di tambak pada musim hujan dan kemarau menggunakan MPCR. Sampel yang digunakan adalah udang windu berumur 3-4 bulan pemeliharaan, berukuran kerdil (6.97± ±1.90 g) dan normal (22.94± ±8.22 g). Primer spesifiik pada MPCR untuk MBV, IHHNV dan HPV masing-masing berukuran 261 bp, 302 bp dan 595 bp mampu mengampilifikasi DNA ketiga virus tersebut. Hasil Chisquare, prevalensi virus MSGS pada musim hujan tidak berbeda dengan musim kemarau, baik pada udang normal maupun udang kerdil (P>0.05) juga tidak ada korelasi antara kualitas air dengan prevalensi MBV, IHHNV dan HPV di udang normal dan kerdil (P>0.05). Pada musim kemarau prevalensi tipe infeksi virus MSGS pada udang kerdil lebih tinggi dari udang normal dan prevalensi udang yang tidak terinfeksi virus MSGS lebih tinggi pada udang normal baik pada musim hujan maupun musim kemarau, sebaliknya prevalensi udang yang terinfeksi virus MSGS lebih tinggi di udang kerdil pada musim kemarau (P<0.05). MPCR dapat mendeteksi virus MSGS dengan cepat dan virus MSGS bertanggungjawab terhadap kekerdilan udang windu di tambak. Kata kunci : multipleks PCR, musim, MSGS, tambak, virus. Abstract Stunted growth phenomenon of shrimp has caused significant lost due to its undersize. This research was aimed to analyze occurrence of viruses MBV, IHHNV and HPV, in shrimp ponds at dry and rainy seasons. Samples were shrimps cultivated for 3 to 4 months. The samples were designated as stunted, size 6.97±2.34 to 16.86±1.90 g, and Normal, size 22.94±4.62 to 40.31±8.22 g. MPCR Specific primers used for detection of MBV, IHHNV and HPV could amplify the three viruses DNA with PCR products of 261 bp, 302 bp, and 595 bp, respectively. Prevalence of MSGS viruses was not different significantly between rainy and dry season in both normal and stunted samples (P > 0.05). No correlation was found between water quality parameters and the prevalence of infection of MBV, IHHNV and HPV in normal and stunted shrimp (P > 0.05). In dry season, prevalence of infection type MSGS-related viruses was higher in stunted shrimp than normal shrimp, and prevalence of uninfected shrimp was higher in normal shrimp in both seasons. On the other hand, prevalence of infected shrimp was higher in stunted shrimp in dry season (P < 0.05). It was concluded that the MPCR can implementation as a fast detection for MSGS viruses and the viruses were responsible for the shrimp stunted growth phenomenon in shrimp ponds. Key words: Growth-out ponds, MPCR, MSGS, Seasons, Viruses.

3 Introduction Stunted growth of shrimp frequently observed in shrimp ponds is widely known as black tiger shrimp monodon slow growth Syndrome (MSGS). The MSGS in South Sulawesi has caused financial lost to many farmers because of the size of the shrimp ponds which can not reach the standard sizes to suit the age of the shrimp. In Thailand, shrimp reared in ponds for 4 months showed stunted growth with a daily growth rate of around 0.07 to 0.15 g / day or just weighed approximately 8.16 g / fish, compared to the normal daily growth rate of about 0.2 g / day weighing approximately 24 g / fish once maintained for 4 months (Chayaburakul et al., 2004). The stunted growth of black tiger shrimp (msgs) may be caused by a number of disease agents such as DNA viruses IHHNV, MBV, HPV and the RNA virus LSNV (Chayaburakul et al., 2004; Sritunyalucksana et al., 2006) as well as parasites intestinal gregarine (Poulpanich and Withyachumnarnkul, 2009). The viruses are agents of shrimp diseases that are very dangerous because theya are transmitted vertically through the broodstock to larva and horizontally through environment in which the reservoir hosts involve all kinds of seawater and freshwater Krustase (Catap and Travina, 2005). In addition, viral infection can not be treated because viruses are intracellular organisms that can not be reached by the circulatory system of the shrimp so that the use of antibiotics for the virus is not suitable. The development of the virus is influenced by several factors such as environmental conditions and condition of host / shrimp. Environmental conditions such as temperature, salinity and ph are greatly influenced by seasons so that conditions can affect the development of the virus and the host shrimp condition. Some research suggests that the development of the virus more quickly in the rainy season due to salinity and low temperature compared to the dry season (Montgomery-Brock et al., 2007; Montgomery-Brock et al., 2004; Karunasagar and Karunsagar, 1997) Early detection of disease agents is needed in the prevention and control of disease. Multiplex PCR (MPCR) as a molecular detection method could be used as a method of early detection because it can detect several types of disease agents simultaneously in a single reaction or simultaneously. MSGS Virus (MBV, IHHNV and HPV) can be detected simultaneously with MPCR so that preventive measures as recommended usage / MSGS virus-free fry stocking can

4 be done to prevent detonation msgs virus populations in ponds and to control measures such as the use of appropriate probitoik and immunostimulan. Based on the description, this study aimed to analyze the MSGS virus MBV, IHHNV and HPV in the pond during the rainy and a dry season with MPCR method. Method Time and Sampling Location The research was conducted in February 2011 until January Sampling location is Takalar and Pinrang at the rainy and dry seasons. Analysis of samples with MPCR were performed at the Fish Parasites and Diseases Laboratory of FIKP UNHAS. Samples The samples in this study were shrimp that have been cultivated for 3-4 months in ponds which consist of two sizes i.e normal and dwarf size (Table 1). Shrimp size is the size of a normal g/day and dwarf is g/day (Chayaburakul et al., 2004). Number of samples are 10 normal and dwarf shrimp respectively on each farm per season DNA Extraction and Amplification Organs such as carapace, gills, and the last segment of the body of shrimp and hepatopancreas were homogenized using tissue grinder and mortar. The results of this were used in the extraction of DNA using DNA Mini Kit QiaAmp kit. Extracted DNA is used as template DNA in MPCR. Specific primers for amplicication DNA template is: HPV 2F/2R 5 -GGAAGCCTGTGTTCCTGACT-3 5 -CGTCTCCGGATTGCTCTGAT-3 (595 bp) (Tang et al., 2008) MBV 261F/R 5 -AATCCTAGGCGATCTTACCA-3 5 -CGTTCGTTGATGAACATCTC-3 (261bp) (Surachetpong et al., 2005) IHHNV F/R 5 -ATTTCTCCAAGCCTTCTCACC-3 5 -TGATGTAAGTAATTCCTCTCTGT-3 (302bp) (Khawsak et al., 2008). Amplification of viral DNA MBV, IHHNV and HPV with MPCR conducted under MPCR is predenaturasi 95 C for 15 min, denaturation 94 o C for 30 seconds, annealing 59 o C for 1 minute 30 seconds, extension 72oC for 1 minute 30 seconds, and final extension of 72oC for 10 minutes, with 35 cycles. Composition MPCR (25 ml): Master Mix 12.5 ml, 2.5 ml primer mix, Q-solution 2.5 ml, RNA-ase free water 5.5 ml and 2.0 ml template. To determine the success of MSGS viral DNA mengamplifikasi MPCR, MPCR product is running on agarose

5 1.5% in the electrophoresis apparatus for 45 minutes or until ¾ of the way DNA gel. The results of running soaked in a solution of the dye DNA ETBR about minutes and then transferred to distilled water about 10 minutes. DNA bands on the gel were visualized using UV transilluminator. Variables measured Variables measured were the type of, msgs virus prevalence, prevalence of infection type msgs and water quality data to support the discussion of the prevalence of the virus msgs berdasakan season. Data on the prevalence of the MSGS virus shrimp from the shrimp ponds is the percentage of each type of virus infected msgs in a shrimp population or in the number of samples per farm in the rainy season and dry season. Type of infection in this study is a model that is a combination of shrimp viruses infect a single infection type (MBV, IHHNV or HPV), doubles (MBV + IHHNV, MBV + + IHHNV HPV or HPV) and triples (MBV + + IHHNV HPV) and non-infected and infected with the virus. Water quality parameters such as temperature, salinity and ph as supporting data measured in situ to assist in analyzing the prevalence of the virus due to differences in the seasons. Data Analyses Analysis of differences in virus prevalence of MBV, IHHNV and HPV infection type (single, double, triple) between normal and dwarf shrimp in the rainy season and dry using nonparametric statistical analysis is Chisquare test and to see the relationship between water quality parameters with the prevalence of the MSGS virus with Spearman correlation analysis with SPSS version Results Type of MSGS Virus Amplified in MPCR The results of MSGS viral DNA amplification with MPCR shows that MPCR with specific primers can amplification viral DNA MBV, IHHNV and HPV simultaneously (Figure 1). MSGS Virus Prevalence in Pond on Rainy and The Dry Season The prevalence of virus MSGS in normal and dwarf shrimp in ponds in both rainy and dry season in this study were obtained from prevalence data Pinrang and Takalar virus. The analysis showed that the prevalence of the MSGS virus during the rainy season is not different

6 from the prevalence in the dry season, both in normal shrimp and shrimp dwarf (P> 0.05) (Figure 2 and 3). The results of correlation analysis also showed no correlation between temperature, salinity and ph of the prevalence of MBV, IHHNV and HPV in normal and dwarf shrimp in the rainy and the dry season (P> 0.05). The Prevalence of Type MSGS Virus Infection in Pond On The Rainy and Dry Season The prevalence of the type of viral infection in the rainy season on normal and dwarf size is not significantly different (P> 0.05), but in the dry season there are different types of virus infection prevalence among normal with shrimp dwarf, where the prevalence of the type of MSGS viral infection in shrimp dwarf higher of normal shrimp (Figures 4 and 5). However, the prevalence of the MSGS virus to uninfected shrimp higher in normal shrimp good in the rainy season and the dry season, whereas the prevalence of MSGS virus-infected shrimp higher in dwarf shrimp in the dry season (P <0.05) (Figure 6). This shows the impact of MSGS viral infections stunt shrimp in ponds and the presence of MSGS viral infections in ponds affected by seasonality. Discussion The Multiplex PCR with specific primers in this study can be used as a method of early and rapid detection, both for the seed to be stocked as well as for monitoring the health of shrimp in ponds so that control measures can be taken. These results show that specific primers for virus IHHNV, MBV and HPV virus can amplification the viruses, simultaneously. The prevalence of the three types of virus infection such as high msgs between dwarf shrimp normally on wet and dry seasons so that infection by all three viruses were allegedly not the only cause of the smallness of the shrimp. Prevalence is an indicator of the level of pathogen infection is the presence of pathogens in the percentage of the population. Value is a high prevalence in this study indicate a condition that is very risky for shrimp cultivation in South Sulawesi, because the existence of the virus in shrimp albeit at a low prevalence rate is already very dangerous. This is because the virus that infect Krustase including transmission or shrimp have a very wide spread levels are vertically from parent to offspring through embryo infection and horizontally through predation environments such as shrimp or Krustase infected, through vector and feces of infected Krustase. In addition to a very wide transmission, the virus in Krustase also has a very wide host range that can infect all types Krustase Krustase both

7 freshwater and seawater (Peng et al., 1990; Lo et al., 1997; Rajan et al., 2000 ). In addition, the three MSGS virus has a high level of pathogenicity can cause high mortality and / or decreased growth on host. MBV virus caused the death of up to 90% of the shrimp larvae (Ramasamy et al., 1995; Manivannan, 2002). Lightner et al. (1983), IHHNV in 1981 has led to more than 90% mortality in the cultivation of P. stylirostris in Hawaii and on P.vannamei and P. monodon causes decreased growth and defect known as runt deformity syndrome (RDS). While the HPV virus causes death and the size of the shrimp larvae shrimp infected with HPV virus significantly shorter than those not infected (Flegel et al., 1999), most HPV-infected shrimp grow very slowly and stopped growing at about 6 cm long with weighing about 5 g (200 shrimp per kg) (Flegel, 2006). The prevalence of the MSGS virus in ponds shows that there is no difference between normal and dwarf shrimp, both in the rainy and the dry season. This indicates that the virus IHHNV, MBV and HPV in the pond is not the only cause of the smallness of the black tiger shrimp, but suspected there were other factors, such as pond bottom soil conditions that are generally overlooked by farmers in South Sulawesi and various other physico-chemical factors, which can cause stress to the shrimp and the existence of a vector as the host reservoir and transmit the virus. Withyachumnarnkul et al. (2006) also obtain IHHNV infection rates did not differ based on shrimp size and to see the real effect of the size of the shrimp IHHNV infection suggested should be the number of samples and sampling locations increased. While Tang and Lightner (2011), the duplex real-time PCR they also found no real differences about MBV and HPV viral load between normal than the dwarf shrimp that concluded that although MBV and HPV has been reported as the cause of dwarf shrimp, but there indication that factors other than viral infection which is also responsible for stunted growth in the sample. Prevalence data type MSGS virus infection in ponds intended to see the combination of the three viruses in infected individuals MSGS shrimp in ponds. This is helpful in analyzing the properties of interfer or co-infection of a virus in an individual shrimp. Khawsak et al. (2008), an individual shrimp can infected by some kind of virus infection in the form of single, double, triple or multipleinfection. Data type MSGS virus infection in South Sulawesi farms in this study shows the interesting thing is that all three MSGS virus can infect a joint or co-infection in an individual shrimp in ponds without interfere. According to Tang and Lightner (2011), MBV and HPV are two viruses that replicate independently on an individual shrimp. HPV infects the cells

8 prefers E (Embryonalzellen) hepatopancreas, also encodes a DNA polymerase activity that replication depends on host cell replication. MBV is a baculovirus that has DNA polymerase and can replicate in B cells (Blasenzellen), F (Fibrenzellen) and R (Restzellen). It is proved that the two viruses may not interact with each other. Neither the virus IHHNV, although IHHNV also is Parpovirus like HPV that do not encode a DNA polymerase that is dependent on the host cell DNA replication machinery and provide structurally similar, but different target tissue infections. HPV infects epithelial cells of the hepatopancreas while IHHNV infects essentially all non-enteric tissues (Lightner et al., 1983, Lightner and Redman, 1985). In addition to the coinfection of three viruses MSGS, also shows that the HPV virus as a single infection never appeared on shrimp and shrimp dwarf normal good in the rainy and dry season. This phenomenon was discovered by Lightner (1996), HPV infections are rarely found to be single so gross sign for HPV is difficult to determine and attack with agents of other diseases causing high mortality in juvenile stages and in 4 weeks to reach %. It is also found by Umesha et al. (2006), HPV is never found as single infection always infected in the form of double infection type (MBV HPV + and HPV + WSSV) or triple infection (HPV + + MBV WSSV). In general, the results of this study showed that the prevalence of the virus MBV, IHHNV and HPV did not differ between normal and dwarf shrimp in both rainy and dry seasons, but the prevalence of the type of virus infection was higher in dwarf shrimp are not infected with the virus and the prevalence was higher in normal shrimp so it can be said that the virus MBV, IHHNV and HPV that infect shrimp in ponds responsible for the smallness of shrimp. In addition, the prevalence of the MSGS virus in ponds during the rainy season did not differ between normal and dwarf shrimp. This suggests that during the rainy season both normal and dwarf shrimp vulnerability and sensitivity to the virus increases. In the rainy season the water quality to be not optimal for shrimp life causing stress. In stressful conditions, Haemocyte Total Count (THC) crustacea decreased, the enzyme activity associated with the disease resinstensi decreased and increased sensitivity to pathogens (Truscott & White, 1990; Vargas-Albores et al., 1998; Le Moullac & Haffner, 2000). M. japonicus shrimp immune reactions at higher temperatures (31 o C) Phenoloksidase activity (PO) and THC higher than the ambient temperature (27 o C). The High THC and PO can reduce viral replication in shrimp (You et al., 2010). This is evidenced by the Montgomery-Brock et al. (2007) that replication of IHHNV in shrimp L. vannamei were maintained at 32.8 ± 1.0 o C warmer temperatures lower than the temperature of

9 24.4 ± 0.5 o C cooler. While Pan and Jiang (2002), a brief change in salinity 10 hours from 30 to 15, nor by fluctuations in ph from 8.5 to 7.0 or 9.5 bacteriolitic activity and antibacterial activity of two shrimp (Fenneropenaeus chinensis and Litopenaeus vannamei) gradually reduced, while the PO activity increased. Changes in salinity can cause a decrease immunocompetens shrimp and followed by a gradual recovery. Pan et al. (2005), changes in salinity should not exceed 5 there are any changes in the ph of no more than 5 scale than optimal. The results of previous studies on the line with our study, where the temperature, salinity and ph is higher in the dry season than the rainy season (Table 2) so that the shrimp avoid the influence of stress will lower the immune system to fight the virus and the virus's ability to replicate a lower. Conclusions and Suggestions The MPCR is molecular detection methods can be used as a method of early and rapid detection. Virus MBV, IHHNV and HPV as the MSGS virus can be detected in the pond simultaneously with MPCR using primers specific for MBV, IHHNV and HPV measuring 261 bp, 302 bp and 595 bp, respectively. MSGS virus in this study are responsible for the smallness of shrimp in ponds because even MSGS virus prevalence in shrimp ponds did not differ based on the size of the shrimp and season, but the prevalence of type MSGS virus infectious were higher in both dwarf shrimp in the rainy season (78%) and the dry season (65%) than normal shrimp in the rainy season (60%) and dry season (43%). Virus MBV, IHHNV and HPV can infect shrimp simultaneously on one individual shrimp (co-infection). Further research on the epidemiology of the virus IHHNV, MBP and HPV needs to be done to determine the virus control measures. Acknowledgement I would like to thanks MITRA BAHARI PROGRAM - COREMAP II and Directorate of Higher Education through LPPM UNHAS for financial support for this study through scholarships and Grant in Writing Doctoral Dissertation, respectively. References Catap, E.S. and Travina, R.D. (2005). Experimental transmission of Hepatopancreatic Parvovirus (HPV) infection in Penaeus monodon postlarvae. In P. Walker, R. Lester and

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12 Tabel 1. Number and size of samples from shrimp ponds Rainy Season Dry Season Lokasi Tambak Normal Kerdil Normal Kerdil Jumlah (ekor) Ukuran (g)±sd Jumlah (ekor) Ukuran (g)±sd Jumlah (ekor) Ukuran (g)±sd Jumlah (ekor) Ukuran (g)±sd Takalar ± ± ± ±4.28 Maros ±3.22 Pangkep ± ±3.36 Barru ±2.66 Pinrang ± ± ± ±3.50 Total SD*: Standar Deviasi Tabel 2. Water quality parameters in shrimp ponds at rainy and dry seasons Figure 1. MPCR sampels from shrimp ponds. M adalah marker 100 bp, lane 1 (IHHNV+HPV)), lane 3 (IHHNV), lane 9, 14 (MBV), lane 8, 15, 16 (MBV+IHHNV), Lane 11, 13, 17 (MBV+IHHNV+HPV).

13 Figure 2. Prevalence of virus MBV, IHHNV and HPV in South Sulawesi (Takalar and Pinrang) in normal and dwarf shrimp at rainy season (P>0.05) Figure 3. Prevalence virus MBV, IHHNV dan HPV in South Sulawesi (Takalar dan Pinrang) in norma and dwarf shrimp in dry season (P>0.05). Figure 4. Prevalence type of infection virus MSGS in ponds in South Sulawesi (Takalar dan Pinrang) in rainy season (P>0.05).

14 Figure 5. Prevalence type of infection virus MSGS in ponds in South Sulawesi (Takalar dan Pinrang) in dry season (P>0.05). Figure 6. Prevalence type of infection, infected and not infected in normal and dwarf shrimp at rainy and dry season (P<0.05).