Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ اثر پروبیوتیک تک‌سل پلاس بر فراوانی باکتری‌های جنس Vibrioدر استخرهای پرورش مولدین میگو سفید غربی (Litopenaeus vannamei) Effect of Tek Cell Plus probiotic on the abundance of Vibrio genus bacteria in western white shrimp (Litopenaeus vannamei) broodstock rearing ponds بهداشت و بيماريهاي آبزيان بهداشت بيماريها گزارش مورد case report در حال حاضر، بیماری‌‌های فراوانی پرورش میگوی سراسر جهان را تحت تاثیر قرار داده‌‌اند. عوامل عفونی باکتریایی مهم‌‌ترین معضل بهداشتی میگوی پرورشی هستند. یک جایگزین مناسب برای عوامل ضد میکروبی شیمیایی برای جلوگیری از بیماری در آبزی پروری میگو، استفاده از پروبیوتیک‌‌هاست. به طور کلی، بیماری‌‌های ناشی ازVibrio spp. ، علت اصلی مرگ‌ومیر در هچری‌‌های میگو به‌ویژه در مراحل اولیه لاروی است. در بررسی حاضر، نمونه‌ آب مورد مطالعه از 3 استخر مزارع مولدسازی در شهرستان کنارک از عمق 30 سانتی‌متری از سطح آب به ‌صورت تصادفی برداشت شد. اطلاعات مدیریتی مزرعه پرورش میگو (نحوه مدیریت آب و غذادهی) و برخی شاخص‌های کیفی آب (شوری، pH و درجه حرارت آب)، اندازه‌گیری و ثبت &lrm;گردید. در آزمایشگاه نمونه‌ها همگن شده و کشت باکتریایی در محیط کشت‌های MYP، TSA و TCBS در سه تکرار به ‌منظور جداسازی اولیه Vibrio  غالب از آب استخر انجام شد. نتایج شمارش کل باکتری‌‌های جنس Vibrio نشان داد که پس از افزودن پروبیوتیک به آب استخرها، رشد باکتری‌‌های Vibrio شناسایی شده، نسبت به کنترل مثبت (تعداد باکتری‌‌های Vibrio قبل از افزودن پروبیوتیک)، به طور معنی‌داری کاهش یافت. تعداد کل باکتری جنس Vibrio و تعداد کل باکتری هتروترف در سه استخر شماره 1، 2 و 3 قبل از افزودن پروبیوتیک به‌ترتیب اعداد 102×2/3، 102×3/4 و 108×5 واحد کلنی بر میلی‌لیتر و بعد از افزودن پروبیوتیک به‌ترتیب اعداد  102×3 ، 102×3/1 و 102×4 واحد کلنی بر میلی‌لیتر نشان داد. نتایج حاصل از تعیین توانایی بازدارندگی رشد به روش انتشار از طریق چاهک بیشترین و کمترین اثر بازدارندگی محصول تجاری پروبیوتک و استفاده از سوسپانسیون آماده شده (C1) به‌ترتیب در A1-MYP (5/7 میلی‌متر) و V.p-TSA (3/6 میلی‌متر) مشاهده شد و در باکتری خالص شده از محصول تجاری پروبیوتیک در محیط کشت اختصاصی (C2) بیشترین و کمترین اثر بازدارندگی مربوط به A1-MYP (5/8 میلی‌متر) و B2-MYP (6 میلی‌متر) مشاهده گردید. بیشترین فعالیت مهار کنندگی پروبیوتیک در تست چاهک گذاری علیه A1 و V.P مشاهده شد. نتایج نشان داد پروبیوتیک تک سل پلاس دارای خاصیت آنتی‌‌باکتریال در برابر Vibrio غالب جداسازی شده از استخر و Vibrio parahaemolyticus بود. Introduction Aquaculture, as one of the most important sources of animal protein, plays an increasingly significant role in global food security, and its importance has grown further due to the depletion of natural stocks and the rising demand for seafood (Madhana et al., 2021). In this context, shrimp farming has expanded rapidly, particularly in tropical countries, and has become a key sector of aquaculture production (HAU and Haryana, 2022). The Pacific white shrimp Litopenaeus vannamei is considered one of the most important cultured species due to its fast growth, high survival rate, relative resistance to diseases, and tolerance to a wide range of salinities, making it especially suitable for the environmental conditions of southern Iran (Chong‑Robles et al., 2014; Laramore et al., 2001). Despite advances in management practices, bacterial diseases remain among the main challenges faced by shrimp farms, with vibriosis caused by Vibrio spp. being a major contributor to mortality and economic losses (Muthu et al., 2024). Vibrios are Gram‑negative pathogenic bacteria that not only cause diseases in aquatic animals but can also lead to foodborne infections in humans (Bintsis, 2017). The excessive use of antibiotics to control these diseases has resulted in the emergence of antibiotic‑resistant bacteria and disruption of the microbial balance in aquaculture environments (Kesarcodi‑Watson et al., 2008). In this regard, environmentally friendly and preventive approaches, such as the use of probiotics as suitable alternatives to antibiotics, have gained increasing attention (Defoirdt et al., 2011; Proespraiwong et al., 2023). Probiotics contribute to disease reduction and improved survival by enhancing water quality, strengthening host immunity, and competing with pathogenic bacteria, particularly Vibrio spp. (Lahay et al., 2023; Verschuere et al., 2000). The efficacy of these compounds is commonly evaluated through in vitro antagonism assays (Kesarcodi‑Watson et al., 2008).The native probiotic “Tek Cell Plus,” which was isolated from shrimp farms in Bushehr Province and has reached industrial-scale production following global registration and extensive testing (Ghaednia et al., 2024; Mahjoub et al., 2019), was evaluated in the present study for its ability to reduce the bacterial load of vibrios in the pond water of Pacific white shrimp broodstock culture systems, as well as for its inhibitory effects on vibrio growth under laboratory conditions. Methodology Aqueous samples were procured from three discrete broodstock production ponds in the Konarak district utilizing stratified random sampling techniques at a standardized depth of 30 cm below the water surface interface. Comprehensive aquaculture management parameters were documented, including water quality management protocols, nutritional regimes, and physicochemical variables (salinity, pH, and thermal gradient). Laboratory analyses incorporated sample homogenization followed by selective and differential microbiological cultivation on MYP, TSA, and TCBS media, with triplicate inoculations to ensure statistical validity. The antagonistic properties of the commercial probiotic against indigenous Vibrio isolates were evaluated through quantitative inhibition zone assays using the agar well diffusion methodology. Positive controls consisted of pre-intervention Vibrio population densities, allowing for comparative efficacy assessment. Result Microbiological enumeration demonstrated a statistically significant reduction (p<0.05) in Vibrio spp. population densities following probiotic application across all experimental units. Specifically, Total Vibrio Count (TVC) and Total Heterotrophic Bacteria (THB) in ponds 1, 2, and 3 exhibited pre-intervention values of 3.2×10², 4.3×10², and 5×10⁸ CFU/mL, respectively. Post-application analyses revealed substantial reductions to 3×10², 1.3×10², and 4×10² CFU/mL, respectively, indicating a logarithmic decrease in bacterial load, particularly in pond 3. In vitro antagonism assays utilizing the well diffusion technique revealed differential inhibitory capacities. The commercial probiotic suspension (C1) demonstrated maximum and minimum inhibition growth zones against A1-MYP (7.5±0.2 mm) and V.p-TSA (6.3±0.1 mm), respectively. When utilizing purified bacterial isolates from the commercial probiotic (C2), enhanced inhibitory efficacy was observed, with maximum and minimum zones of inhibition registered against A1-MYP (8.5±0.2 mm) and B2-MYP (6.0±0.1 mm), respectively. The most pronounced antimicrobial activity was consistently observed against the A1 isolate and Vibrio parahaemolyticus, suggesting strain-specific antagonistic mechanisms. Discussion and conclusions The present findings demonstrate that the native probiotic Tek Cell Plus, identified as Bacillus vallismortis with the GenBank accession number JQ085958.1, significantly reduced the bacterial load of Vibrio, the dominant bacterial genus in shrimp pond water, relative to the total bacterial population. Similar probiotic effects have been reported for Bacillus cereus and Paenibacillus spp., which were evaluated at concentrations of 10⁴ and 10⁵ CFU mL⁻¹ in post‑larval shrimp ponds. In vivo experiments revealed that these bacteria exerted strong probiotic activity against Vibrio species, resulting in a significant reduction in shrimp larval mortality. The antagonistic activity of B. cereus and Paenibacillus spp. against vibrios has been attributed to the production of bioactive compounds such as zwittermicin‑A and kanosamine (Ravi et al., 2007). These mechanisms are consistent with the outcomes of the present study, wherein the application of B. vallismortis led to a marked reduction in Vibrio abundance in pond water. Supporting evidence is provided by Temario et al. (2022), who demonstrated that dietary supplementation with Bacillus subtilis BF12 significantly increased survival, improved growth performance, and reduced the intestinal abundance of Vibrio parahaemolyticus in Penaeus monodon. Additional studies have emphasized the efficacy of Bacillus-based probiotics in L. vannamei culture systems. In a 45‑day in vivo trial, B. subtilis IPA‑S.51 and Shewanella algae IPA‑S.111 inhibited V. parahaemolyticus growth while also enhancing shrimp growth performance. Agar‑well diffusion assays further confirmed that probiotics, given sufficient contact time, can produce clear inhibition zones against pathogenic vibrios. Ramesh et al. (2014) isolated twelve Bacillus spp. strains from the gut of P. monodon, several of which showed strong inhibitory activity against V. harveyi VSH5, with inhibition zones reaching up to 19.0 ± 0.1 mm. These effects were attributed to pH modification, production of inhibitory metabolites, nutrient competition, and lactonase enzyme activity. Probiotic efficacy has also been demonstrated using other bacterial groups. Studies by Ravi et al. (2007) and Ferreira et al. (2015) confirmed that Bacillus spp. significantly reduced Vibrio abundance in shrimp ponds, while Thompson et al. (2022) showed that lactic acid bacteria, including Lactobacillus curvatus subsp. curvatus, L. plantarum, and Pediococcus acidolactici, inhibited several pathogenic vibrios through the release of antimicrobial compounds. Acute hepatopancreatic necrosis disease (AHPND/EMS), primarily caused by toxigenic strains of V. parahaemolyticus, represents one of the most severe bacterial diseases in shrimp aquaculture. A meta‑analysis by Ghaednia et al. (2024) concluded that direct addition of single‑strain Bacillus-based probiotics to pond water is the most effective application method for AHPND control. The present study aligns with these findings, as water application of B. vallismortis yielded clear suppressive effects on pathogenic vibrios. Finally, bioinformatic analysis identified two Vibrio isolates closely related to Vibrio sagamiensis and Vibrio fortis, both belonging to the class Gammaproteobacteria. Previous studies have reported V. sagamiensis in tropical marine environments (Yoshizawa et al., 2010) and V. fortis as an emerging pathogen in aquatic organisms (Thompson et al., 2003; Wang et al., 2016), suggesting potential pathogenic risks in shrimp farming systems. Overall, the findings confirm the effectiveness of Bacillus vallismortis as a robust probiotic candidate for sustainable control of Vibrio-associated diseases in shrimp aquaculture. Conflict of interest All authors declare that they have no competing interests. Acknowledgment The authors would like to express their sincere appreciation to all those who contributed to this research through their guidance and scientific support. پروبیوتیک تک سل پلاس, Vibrio, Vibrio parahaemolyticus, مولد, میگو, Litopenaeus vannamei Key words: single cell plus bacteria probiotic, Viprio Spp, Vibrio parahaemolyticus, breeder, shrimp breeding, Litopenaeus vannamei, breeding pool 61 73 http://isfj.ir/browse.php?a_code=A-10-2821-1&slc_lang=fa&sid=1 Solmaz Bazdar سولماز بازدار bazdarsolmaz4@gmail.com 100319475328460041517 100319475328460041517 No گروه شیلات، دانشکده علوم دریایی، دانشگاه دریانوردی و علوم دریایی چابهار، چابهار، ایران Mostafa Ghaffari مصطفی غفاری mgmostafaghaffari@gmail.com 100319475328460041518 100319475328460041518 Yes گروه شیلات، دانشکده علوم دریایی، دانشگاه دریانوردی و علوم دریایی چابهار، چابهار، ایران Ashkan Ajdari اشکان اژدری a_arzhan@yahoo.com 100319475328460041519 100319475328460041519 No مرکز تحقیقات شیلاتی آبهای دور، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، چابهار، ایران zeinab kord زینب کرد kord.zeynab97@gmail.com 100319475328460041520 100319475328460041520 No گروه شیلات، دانشکده علوم دریایی، دانشگاه دریانوردی و علوم دریایی چابهار، چابهار، ایران