Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ تاثیر برخی غذاهای وارداتی مراکز تکثیر میگو با آزمایش واکنش زنجیره‌ای پلیمراز (Real time PCR) مثبت ویروس لکه سفید و توانایی آنها در آلودگی پست‌لاروها The effect of some imported foods from shrimp breeding centers on positive real-time PCR tests for white spot virus and their ability to infect post-larvae بهداشت و بيماريهاي آبزيان بهداشت بيماريها پژوهشي Research پرورش میگو به عنوان یکی از ارکان آبزی‌پروری به دلیل ارزش غذایی بالا، مورد استقبال آبزی‌پروران، تجار و مردم قرار گرفته است. طی سال‌های اخیر توسعه صنعت میگو در ایران، به دلیل عدم دسترسی کافی نهاده‌های اولیه تولید غذا از خارج از کشور، با مشکل جدی مواجه شده است. بررسی‌های اخیر نشان داده است که عمده غذاهای تکثیر میگو وارداتی است. در بررسی‌های اولیه کارشناسان که مبتنی بر آزمایش‌های واکنش زنجیره‌ای پلیمراز کمی (Real time PCR) بوده، مشخص گردیده است که برخی از غذاهای وارداتی مراکز تکثیر میگو آلوده به ویروس لکه سفید میگو هستند. برخی دیگر از کارشناسان معتقدند که وجود قطعاتی از ژنوم ویروس موجب می‌گردد تا آزمایش‌های واکنش زنجیره‌ای پلیمراز مربوطه مثبت گردد. بنابراین، بر اساس همین ابهام که آیا مثبت بودن آزمایش واکنش زنجیره‌ای پلیمراز غذا، می‌تواند دال بر وجود ویروس فعال باشد و این‌که این غذا کانونی برای ایجاد و انتقال ویروس گردد، تحقیقی تدوین گردید که مشتمل بر 4 گروه تحقیقاتی شامل یک گروه کنترل و 3 گروه تیمار در 3 تکرار با 90 قطعه میگو در هر گروه (30 قطعه در هر تکرار) در آکواریوم‌های شیشه‌ای 150×30×50 بوده است. گروه شاهد با غذای عاری از ویروس لکه سفید (Real time منفی) و گروه‌های بعدی با نتایج Real time  مثبت به‌ترتیب گروه‌های 2، 3 و 4 با غذاهای تجاری  MPZ،MEF و Flak تغذیه شدند. پس از گذشت 20 روز از غذادهی، ردیابی ویروس لکه سفید در نمونه میگوها در آزمون‌های Real time ، Nested-PCR (PCR آشیانه‌ای) و در نهایت از نظر هیستوپاتولوژی منفی گردید. نتایج این تحقیق نشان داد، انتقال عفونت لکه سفید میگو از طریق غذاهای پلت وارداتی مراکز تکثیر محتمل نیست. Introduction The growing global population and increasing demand for healthy protein have made aquaculture, particularly shrimp farming, a crucial solution. However, rapid industry expansion has led to disease outbreaks like White Spot Syndrome Virus (WSSV), causing $20 billion in Asian losses over two decades (Davies, 2016). Global farmed shrimp production surged from 50,000 tons (1975) to 4.5 million tons (2018) (FAO, 2018), with Iran's production reaching 60,000 tons in 2022 and targeting 150,000 tons. Intensive farming practices (up to 1.5 million PLs/0.8 ha) and excessive feeding (20-25% unused protein) exacerbate disease risks. WSSV already affects 10-14% of Iranian farms (Madani, 2021), causing annual losses of 5,000 tons (~$17 million) - potentially doubling by 2023. This study evaluates WSSV transmission through contaminated imported feed using real-time PCR, comparing results with nested PCR, histopathology, and control groups to address this critical biosecurity gap. Methodology This experimental study evaluated the potential transmission of White Spot Syndrome Virus (WSSV) through imported shrimp feeds using four test groups: one control group fed WSSV-free feed (RT-PCR negative) and three treatment groups fed RT-PCR positive commercial feeds (MPZ, MEF, and Flak). Each group consisted of 90 PL5 shrimp (30 per replicate, 3 replicates) maintained in 150×30×50 cm glass aquariums containing 60% sterilized coastal water with continuous aeration. The trial lasted 20 days with four daily feedings, alternate-day siphoning, and 20% water exchange. Survival rates were calculated and compared statistically. For molecular analysis, samples from moribund shrimp underwent DNA extraction using commercial kits, followed by TaqMan RT-qPCR with specific WSSV primers (50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C/15 sec and 60°C/1 min) and nested-PCR using the IQ2000 WSV kit. Histopathological examination involved fixing six PLs per group in Davidson's solution, followed by standard ethanol-xylene processing, paraffin embedding, 4μm sectioning, and H&E staining. Data were analyzed using one-way ANOVA with Tukey's post-hoc test (α=0.05). Results The experimental feeds for groups 2-4 tested positive for WSSV by RT-PCR at an accredited veterinary laboratory, with Ct values consistently above 30, indicating low viral genome loads (Figures 2-4). Survival rates after 20 days showed no significant differences (p>0.05) between groups: control (95.55%), MPZ (95.55%), MEF (97.77%), and Flak (94.44%) (Figure 5). Although the Flak group had slightly lower survival, statistical analysis revealed no significant differences among groups. All shrimp samples tested negative for WSSV infection through both RT-PCR (Table 2) and nested-PCR confirmation (Figure 6) using the IQ2000 kit. Histopathological examination of hepatopancreas, intestine, muscle and gill tissues showed no viral inclusion bodies in any experimental group (Figures 7-10). The study results clearly demonstrate that while traces of WSSV genetic material were detected in the imported shrimp feeds, these findings do not indicate any actual disease risk. The high Ct values (above 30) from PCR testing show the viral material present was minimal and likely non-infectious fragments rather than live virus. Most importantly, after 20 days of feeding, comprehensive testing revealed no evidence of WSSV infection in any shrimp across all experimental groups. All shrimp samples tested negative through both molecular methods (real-time PCR and nested PCR) and histological examination, with no viral inclusion bodies found in any tissues. The survival rates across all groups remained consistently high (94.44-97.77%) with no statistically significant differences, confirming that the PCR-positive feeds performed just as well as the control feed. This strongly suggests that the presence of viral genetic material in these imported feeds does not translate to actual disease transmission or negative impacts on shrimp health and survival. The consistent negative results across all diagnostic methods provide robust evidence that these feeds do not pose a WSSV transmission risk under normal aquaculture conditions. These findings have important implications for the shrimp farming industry, indicating that proper PCR testing can effectively distinguish between non-infectious viral fragments and genuine disease threats in imported feeds. The findings conclusively show that the presence of WSSV genetic material in imported feeds did not lead to actual infection or affect shrimp survival under these experimental conditions. Discussion and conclusion This study aimed to assess the potential pathogenicity of shrimp hatchery feed that tested positive in real-time PCR for white spot syndrome virus (WSSV). The results confirmed trace amounts of WSSV genetic material in imported larval-stage feed (Figures 2-4), though high Ct values indicated low viral genome levels, suggesting contamination rather than active infection. The detected genetic material likely originated from natural shrimp-based pigments or antioxidants added during feed production. While some researchers claim WSSV is inactivated by heat or freezing, inconsistent global measures (sometimes conflicting with OIE standards) have perpetuated uncertainty (Durand et al., 2000). Deadly viruses can spread via: <ol> <li class="ds-markdown-paragraph" style="text-align:justify">Transport of infected live stocks (Schnurrenberger et al., 1987),</li> <li class="ds-markdown-paragraph" style="text-align:justify">Bird vectors (Garza et al., 1997),</li> <li class="ds-markdown-paragraph" style="text-align:justify">Import/reprocessing of frozen food (Humphrey, 1995).</li> </ol> Durand et al. (2000) found WSSV survives freezing/cold storage, prompting this study to evaluate processing methods beyond freezing. WSSV has been detected in post-larvae (PL) of various Penaeus species across different regions (Withyachumnarnkul et al., 2003), with multiple potential transmission routes proposed. Vertical transmission was confirmed by Mohan et al. (1997), who observed WSSV inclusions in reproductive organs and eggs of P. monodon. Momoyama et al. (1998) reported WSSV survival after freezing, while Durand et al. (2000) suggested that block freezing could degrade WSSV DNA, attributing viral inactivation to ice crystal damage or repeated freeze-thaw cycles during processing. Contradictory findings exist: Hasson et al. (2006) and Sritunyalucksana et al. (2010) noted WSSV viability in frozen shrimp. However, Aranguren Caro et al. (2020) demonstrated that boiling WSSV-infected shrimp for 1–30 minutes eliminated infectivity, despite qPCR detecting viral DNA (with no significant Ct differences). Nested PCR and histopathology confirmed the loss of infectious WSSV in boiled samples, with no pathological lesions observed—aligning with this study’s results (Figures 7–10), which suggest heat-treated feed poses no infection risk to broodstock. Experimental WSSV infection via feeding contaminated shrimp tissue has been demonstrated (Chou et al., 1998; Hameed et al., 2002; Momoyama et al., 1998). However, wild shrimp/crabs of unknown WSSV status are still used as feed in some systems (Corsin et al., 2001). Few studies evaluate feeding practices' impact on WSD outbreaks. While MPEDA/NACA found no link (Corsin et al., 2005), Vietnam associated higher feed amounts with WSD incidence—possibly due to water quality or stocking density (Corsin et al., 2001). Contrary to this study’s conclusion (no transmission via feed), Maeda et al. (1998) suggested commercial feed could influence WSSV positivity. Similarly, Corsin et al. (2001) linked specific feed brands to WSSV presence in harvested shrimp, though without disease correlation. In India, 43% of ponds used WSSV-positive feed, with some brands showing higher contamination—likely due to raw materials or milder processing (Corsin et al., 2002). However, no direct feed-WSD relationship was found, implying low-quality feed may weaken immunity, exacerbating natural infections. Pongmaneerat et al. (2001) observed no WSSV in hemolymph after feeding WSSV-positive feed, despite oral transmission being highly effective (Soto and Lotz, 2001). This study concludes that hatchery feed cannot transmit WSSV to post-larvae; real-time PCR likely detects non-infective viral fragments from raw materials. Conflict of interest The authors declare no conflict of interest. پست لارو میگو, غذای لاروی, بیماری لکه سفید میگو, مراکز تکثیر میگو, تشخیص Shrimp, Hatchery, Larval feed, WSSV, Diagnosis 27 39 http://isfj.ir/browse.php?a_code=A-10-553-3&slc_lang=fa&sid=1 S. Kakoolaki شاپور کاکولکی bsh443@gmail.com 100319475328460040081 100319475328460040081 Yes مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران B. Ghaednia بابک قائدنیا babak.ghaednia@gmail.com 100319475328460040082 100319475328460040082 No مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران S.A.M. Bahari Meymandi سید امیر مختار بهاری میمندی amir.bahari@yahoo.com 100319475328460040083 100319475328460040083 No سازمان دامپزشکی ایران، اداره کل هرمزگان، هرمزگان، ایران A. Sepahdari ابوالفضل سپهداری asepahdari@yahoo.com 100319475328460040084 100319475328460040084 No مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران I. Sharifpour عیسی شریف پور isharifpour@yahoo.com 100319475328460040085 100319475328460040085 No مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران M. Ahangarzade مینا آهنگرزاده rkbs_kh@yahoo.com 100319475328460040086 100319475328460040086 No پژوهشکده آبزی پروری جنوب کشور، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، اهواز، ایران A. Hemati عاطفه همتی atefeh.hemati2543@gmail.com 100319475328460040087 100319475328460040087 No مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران M.K. Pazir محمد خلیل پذیر dr.pazir@gmail.com 100319475328460040088 100319475328460040088 No پژوهشکده میگوی کشور، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، بوشهر، ایران