Search Results (48)

Environmental DNA (eDNA) offers a promising, non-invasive approach for monitoring infectious agents in aquaculture. While molecular techniques for detecting shrimp pathogens are well established in host tissues, there is a lack of standardized protocols for pathogen detection from environmental samples using conventional PCR. In this study, we developed and validated a universal conventional PCR protocol for monitoring DNA from major viral and bacterial shrimp pathogens within pond water and sediment samples. The method was applied to two commercial shrimp farms in Mexico, where eDNA was extracted from field-collected water and sediment. Using published primer sets, we successfully amplified DNA sequences corresponding to six key pathogens—Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Baculovirus penaei (BP), Monodon baculovirus (MBV), Shrimp hemocyte iridescent virus (SHIV), Candidatus Hepatobacter penaei (NHP-B), and Acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio spp.—in environmental samples. Sequencing of PCR amplicons confirmed 93–100% identity to previously reported pathogen strains, highlighting the method’s reliability. Pathogen detection rates varied by site, sample type, and date, with the percentage of positive samples ranging from 11.1% to 77.7%. Notably, this is the first report of SHIV DNA detection from environmental samples in the Americas, highlighting its value for pathogen surveillance even in the absence of documented outbreaks. This protocol offers a cost-effective and scalable tool for pathogen surveillance in shrimp aquaculture, enhancing early disease detection and contributing to improved biosecurity and risk assessment frameworks. Full article

This 90-day trial evaluated the integrated benefits of biofloc technology (BFT) in lined ponds for intensive Penaeus vannamei culture, comparing it with a conventional water-exchange (WE) system. The BFT system maintained favorable water quality with a 68.4% reduction in cumulative water exchange. Concentrations of toxic total ammonia–nitrogen (TAN) and nitrite–nitrogen (NO₂⁻-N) were better controlled, and total suspended solids (TSS) stabilized within a beneficial range (150–200 mg L⁻¹). Microbial analysis indicated that BFT significantly increased total bacterial abundance in both culture water and shrimp hepatopancreas while reducing the total Vibrio-to-bacteria ratio in culture water to below 6%, significantly lower than in the WE system (>18%). Moreover, BFT significantly lowered the loads of specific pathogens, acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus, and Enterocytozoon hepatopenaei (EHP) in both culture water and shrimp hepatopancreas. Regarding production performance, BFT significantly enhanced shrimp survival rate (82.4% vs. 71.5%), yield (3460 vs. 2948 kg pond⁻¹), and water productivity (0.85 vs. 0.28 kg m⁻³), while lowering the feed conversion ratio (1.16 vs. 1.33). In conclusion, BFT achieves stable water quality, effective pathogen suppression, and enhanced production efficiency through microbial regulation, offering a viable water-saving, environmentally sound, and disease-resilient strategy for intensive P. vannamei culture. Full article

Concerning shrimp diseases, including acute hepatopancreatic necrosis disease (AHPND), hepatopancreatic parvovirus (HPV) infection and Enterocytozoon hepatopenaei (EHP) microsporidiosis negatively impact shrimp aquaculture through acute mortality, chronic growth retardation or compromised health that increases susceptibility to concurrent infections. All three diseases damage hepatopancreas, a vital organ for nutrient absorption and growth, though their clinical outcomes differ: AHPND is typically associated with rapid, high mortality, EHP primarily causes chronic production losses and HPV, while currently of lower pathogenic significance, may still impair health under certain conditions. Outbreak severity is often intensified by poor water quality, inadequate farm management, antibiotic misuse and pathogen vectors, leading to substantial economic losses. Timely and accurate diagnosis is therefore critical for effective disease management. This study investigates two convolutional neural network (CNN) architectures, EfficientNet and MobileNet. A curated and preprocessed dataset was used to fine-tune both models with a standardized custom classification head, ensuring a controlled backbone comparison. Experimental results show both architectures achieving over 95% accuracy, with MobileNet providing faster inference suitable for on-site deployment. These findings demonstrate the practical feasibility of lightweight CNN-based diagnostics tools for real-time, scalable, and cost-efficient health monitoring in shrimp aquaculture, bridging the gap between the laboratory-grade performance and field-level usability. Full article

Acute hepatopancreatic necrosis disease (AHPND), caused by the bacterium Vibrio parahaemolyticus, is a major threat to global shrimp aquaculture. In this study, we evaluated the therapeutic effects of phage therapy in Litopenaeus vannamei challenged with AHPND-causing Vibrio parahaemolyticus. Phage application at various concentrations significantly improved shrimp survival, with the 1 ppm group demonstrating the highest survival rate. Enzymatic assays revealed that phage-treated shrimp exhibited enhanced immune enzyme activities, including acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). In addition, antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and total antioxidant capacity (T-AOC) significantly improved, accompanied by reduced malondialdehyde (MDA) levels. Serum biochemical analyses demonstrated marked improvements in lipid metabolism, particularly reductions in triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL), alongside higher levels of beneficial high-density lipoprotein (HDL). Transcriptomic analysis identified 2274 differentially expressed genes (DEGs), notably enriched in pathways involving fatty acid metabolism, peroxisome functions, lysosomes, and Toll-like receptor (TLR) signaling. Specifically, phage treatment upregulated immune and metabolic regulatory genes, including Toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MYD88), interleukin-1β (IL-1β), nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor (PPAR), indicating activation of innate immunity and antioxidant defense pathways. These findings suggest that phage therapy induces protective immunometabolic adaptations beyond its direct antibacterial effects, thereby providing an ecologically sustainable alternative to antibiotics for managing bacterial diseases in shrimp aquaculture. Full article

Florfenicol (FF) is one of the common antimicrobials used to control bacterial disease in shrimp aquaculture. This study aimed to determine the pharmacokinetics (PK) parameters of FF in white leg shrimp plasma, hepatopancreas and muscle as well as its residue depletion in shrimp muscle and the impact on shrimp hepatopancreas histology during and after FF medication. In the PK experiment, shrimp were fed once at 10 mg FF/kg body weight (bw) via oral in-feed administration to determine PK parameters in plasma, hepatopancreas and muscle. The maximum concentration (C_max) of 60.56 μg/L in plasma was observed after 1.77 h (T_max). In muscle, a C_max of 11.76 μg/kg was attained after 0.20 h, while in hepatopancreas, the C_max was higher (386.92 μg/kg) and was rapidly obtained (T_max = 0.19 h). The C_max values in shrimp plasma were below the minimum inhibitory concentration (MIC) against Vibrio parahaemolyticus, known to cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. Therefore, it can be concluded that to ensure the effectiveness of this treatment, the dose should be higher than 10 mg FF/kg bw. FF depletion in white leg shrimp muscle and its histological impact on hepatopancreas were determined after feeding FF-medicated feed once-a-day or twice-a-day for 3 consecutive days with a dose of 10 mg FF/kg bw. The residues in shrimp muscle were rapidly eliminated and fell below the limit of quantification at 24 h after stopping medication. The withdrawal time of FF in shrimp muscle was 27.9 degree-days (2 days at 26.5 °C) according to the maximum residue limit (MRL) of 100 µg/kg set by the European Commission and Korean Ministry and when feeding FF twice-a-day for 3 days. The results from histological analysis showed that there was no negative effect on shrimp hepatopancreas after stopping medication in both once- and twice-a-day treatments. Full article

This study evaluated the genetic parameters for growth and Vibrio parahaemolyticus (Vp_AHPND) resistance in both the introduced MK strain and the self-constructed GK strain of Penaeus vannamei, investigating the impact of genotyped female parents on trait estimates under a single-parent nested mating design. A total of 32 families from the MK strain and 44 families from the GK strain were analyzed. Fifty-four female parents from both strains were genotyped using the “Yellow Sea Chip No. 1” containing 10.0 K SNPs. In the MK strain, heritability estimates ranged from 0.439 to 0.458 for body weight (Bw) and from 0.308 to 0.489 for survival time (ST) and survival rates at 36 h (36 SR), 50% mortality (SS₅₀), and 60 h (60 SR). In the GK strain, heritability for Bw ranged from 0.724 to 0.726, while ST, 36 SR, SS₅₀, and 60 SR had heritability estimates between 0.370 and 0.593. Genetic correlations between Bw and ST were 0.601 to 0.622 in the MK strain and 0.742 to 0.744 in the GK strain. For Bw and survival rates, correlations ranged from 0.120 to 0.547 in the MK strain and from 0.426 to 0.906 in the GK strain. The genetic correlation between ST and survival rates was not significantly different from 1 (p > 0.05) in both strains. High Pearson correlations (0.853 to 0.997, p < 0.01) were observed among survival rates at different points. Predictive accuracies for Bw, ST, and survival rates using single-step genomic best linear unbiased prediction (ssGBLUP) were comparable to pedigree-based best linear unbiased prediction (pBLUP) in the MK strain, while in the GK strain, ssGBLUP improved predictive accuracies for Bw, ST, and SS₅₀ by 0.20%, 0.32%, and 0.38%, respectively. The results indicate that both growth and Vp_AHPND resistance have significant breeding potential. Although the genetic correlation between weight and resistance varies across different populations, there is a positive genetic correlation between these traits, supporting the feasibility of multi-trait selection. To enhance genetic accuracy, breeding programs should include more genotyped progeny. These findings also suggest that infection frequency and observation time influence resistance performance and breeding selection, emphasizing the need for a tailored resistance evaluation program to improve breeding efficiency and reduce costs. Full article

The microbiome of the shrimp’s digestive tract shows differences between healthy and acute hepatopancreatic necrosis disease (AHPND)-affected shrimp. The present study aimed to evaluate the impact of probiotic consumption on the microbial community in experimentally AHPND-infected shrimp. Effective probiotics (EPs) Vibrio alginolyticus (Va32A), V. campbellii (VcHA), and Bacillus pumilus (BPY100) and non-effective probiotics (NEPs) B. pumilus (Bp43, and BpY119), were employed in bioassays with Penaeus vannamei and challenged with AHPND-causing V. parahaemolyticus (Vp_AHPND). Stomach (Sto), intestine (Int), and hepatopancreas (Hep) were analyzed by metabarcoding (16S rRNA gene) to characterize the microbiome and biomarkers. Hep-VcHA showed the highest alpha diversity (Shannon index = 5.88; 166 ASVs), whereas the lowest was for Hep-Bp43 (2.33; 7 ASVs). Proteobacteria, Actinobacteria, Bacteroidetes, and Saccharibacteria were the most abundant phyla. The relative abundance of Vibrio sp. was the highest in the Hep and Int of Bp43, BPY119 and the positive control, followed by Rhodobacteraceae in the EP group. Principle coordinate analysis (PCoA) showed a cluster grouped negative (Sto and Hep) control with almost all organs in the EP group causing 28.79% of the variation. The core microbiome of EP was mainly represented by Rhodobacteraceae, Caldilineaceae, Celeribacter indicus, Illumatobacter, Microbacterium, Ruegeria atlantica, Saccharibacteria sp., Shimia biformata, and Thalassobius mediterraneus, whose relative abundance was enriched by probiotics, which may explain their protective roles against Vp_AHPND, whereas the low survival in the NEP group was associated with a higher diversity of Vibrio spp. Our results present an ecosystem-friendly alternative based on beneficial microorganisms to prevent and control AHPND and probably other bacterial diseases in shrimp farming. Full article

Postlarval shrimp frequently face threats from acute hepatopancreatic necrosis disease (AHPND). Although AHPND affects both postlarval and adult shrimp, abiotic and biotic factors are distinct between life stages, such as rearing water nutrient levels and host life stage-dependent microbiota. The response of postlarvae-associated microbiota to AHPND, however, remains largely unexplored compared with its effects on juvenile and adult shrimp. To address this knowledge gap, a comparative analysis of postlarvae-associated microbiota and the ecological processes underlying AHPND progression was performed by sequencing the bacterial V3–V4 hypervariable region of the 16S rRNA gene. AHPND infection was validated by high copies of pirAB genes (Toxin 1) in diseased shrimp hepatopancreas. Advanced AHPND significantly altered the structure of the postlarvae-associated microbiota, with significant enrichment of Bacilli and Bdellovibrionia species in healthy larvae compared with matched AHPND-infected cohorts, although gut microbiota recovery was observed at the late disease stage, corresponding with the cessation of postlarval mortality. AHPND infection explained 11.0% (p < 0.001) of the variance in community structures, whereas postlarvae days post hatching also significantly influenced bacterial communities (7.1% variance, p < 0.001). AHPND-infected shrimp exhibited reduced homogeneous selection and increased dispersal limitation and drift governing their microbiota. These changes were primarily driven by specific microbial lineages, including enriched Bin36 Rhodobacteraceae and Bin11 Flavobacteriaceae, and suppressed Bin63 Vibrio and Bin9 Bacillus in AHPND-infected shrimp. After excluding shrimp age effect, 13 AHPND-discriminatory taxa were identified, accurately distinguishing infected shrimp from healthy individuals with 100% precision. Furthermore, AHPND outbreak weakened the network complexity and stability, which was driven by the suppressed keystone taxa that were positively associated with network robustness. Collectively, our findings deepen the understanding of the inextricable interplay between postlarval shrimp health, microbiota dynamics, and survival, as well as the underlying ecological mechanisms over AHPND progression. Full article

To study the factors affecting Penaeus vannamei production in small-scale greenhouse ponds, four ponds in Jiangmen, Guangdong Province, China were selected. This study investigated the variation in the characteristics of bacterial communities and pathogens in pond water and shrimp intestines, as well as water quality factors during the culture stage. Multivariate linear regression equations were used to analyse the potential factors affecting production. The nitrite concentration reached its peak in the mid-culture stage, with a maximum of 16.3 mg·L⁻¹, whereas total nitrogen and salinity were highest in the late culture stage, reaching 48.4 mg·L⁻¹ and 26, respectively. The dominant bacteria in the pond water were Marivita and Rhodobacteraceae, whereas in the shrimp intestines, they were Bacillus and Candidatus Bacilloplasma. The nitrifying bacteria in the pond water were dominated by Nitrosomonas and Nitrobacter. Pathogens detected in the pond water included acute hepatopancreatic necrosis disease (AHPND), Enterocytozoon hepatopenaei (EHP), and white spot syndrome virus (WSSV). The counts of EHP and the relative abundance of Ardenticatenales_norank and Marivita in the pond were the main factors affecting the shrimp production (p < 0.01). This study indicates that establishing optimal bacterial communities, such as Marivita, Nitrobacter, and Rhodobacteraceae, and controlling the counts of EHP and AHPND pathogens is crucial for regulating the pond environment and enhancing production. Full article

Specific strains of Vibrio parahaemolyticus can cause Acute Hepatopancreatic Necrosis Disease (AHPND), a critical issue in shrimp aquaculture despite the application of several control strategies. The use of antibiotics is now restricted due to increasing bacterial resistance and overuse. Silver nanoparticles (AgNPs) have shown potential in shrimp aquaculture, with applications in boosting immunity against certain types of pathogens, promoting growth, and improving survival rates. However, an economically viable solution that protects the organisms has not been found, which is why the search for nanoparticles synthesized with plant extracts is necessary to generate environmentally friendly control strategies. In this study, we synthesized AgNPs from Larrea tridentata extract and administered them orally with feed over a 35-day period. Shrimps fed with AgNP-enriched diets showed a significant increase (p < 0.05) in mRNA expression of immune-related genes (CTL-5, MNK, SR, and GILT), particularly within the first 24–48 h. No significant differences were observed in growth rates, but survival rates in a challenge against V. parahaemolyticus exceeded 85%, higher than the control group. Based on our findings and previous literature, L. tridentata can effectively promote the synthesis of AgNPs and shows potential as an antimicrobial agent, without affecting the growth or survival of treated shrimp. Full article

Acute hepatopancreatic necrosis disease (AHPND) is one of the most important diseases in the global shrimp industry. The emergence of mutant AHPND-associated V. parahaemolyticus (Vp_AHPND) strains has raised concerns regarding potential misdiagnosis and unforeseen pathogenicity. In this study, we report the first emergence of a type II (pirA⁻, pirB⁺) natural mutant, Vp_AHPND (strain 20-082A3), isolated from cultured Penaeus vannamei in Korea. Phenotypic and genetic analyses revealed a close relationship between the mutant strain 20-082A3 and the virulent Korean Vp_AHPND strain 19-021-D1, which caused an outbreak in 2019. Detailed sequence analysis of AHPND-associated plasmids showed that plasmid pVp_20-082A3B in strain 20-082A3 was almost identical (>99.9%) to that of strain 19-021-D1. Moreover, strains 20-082A3 and 19-021-D1 exhibited the same multilocus sequence type (ST 413) and serotype (O1:Un-typeable K-serogroup), suggesting that the mutant strain is closely related to and may have originated from the virulent strain 19-021-D1. Similar to previous reports on the natural mutant Vp_AHPND, strain 20-082A3 did not induce AHPND-related symptoms or cause mortality in the shrimp bioassay. The emergence of a mutant strain which is almost identical to the virulent Vp_AHPND highlights the need for surveillance of the pathogen prevalent in Korea. Further investigations to elucidate the potential relationship between ST 413 and recent Korean Vp_AHPND isolates are needed. Full article

Shrimp aquaculture is facing a serious disease, acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio paraheamolyticus (Vp_APHND). For sustainable shrimp aquaculture, massive losses of shrimp infected with Vp_APHND must be prevented. Research and selection of shrimp tolerant to Vp_APHND infection is a sustainable approach to reducing the risk of AHPND. This study focused on the identification and development of potential DNA markers associated with AHPND using DArT sequencing (DArTSeq) and a genome-wide association study. Three populations of post-larval Litopenaeus vannamei were immersed in Vp_APHND to collect susceptible (D) and tolerant (S) samples. The 45 D and 48 S shrimp had their genotypes analyzed using DArTSeq. A total of 108,983 SNPs and 17,212 InDels were obtained from the DArTseq data, while the biallelic 516 SNPs and 2293 InDels were finally filtered with PIC < 0.1, MAF < 0.05, and a call rate ≥ 80%. The filtered variants were analyzed for their association with AHPND tolerance. Although there were no significantly associated SNPs and InDels above the Bonferroni correction threshold, candidate variants, four SNPs and 17 InDels corresponding to p < 0.01, were provided for further validation of the AHPND tolerance trait. The candidate SNPs are located on an exon of the zinc finger protein 239-like gene, an intron of an uncharacterized gene, and in intergenic regions. Most of the candidate InDels are in the intergenic regions, with fewer in the intronic and exonic regions. This study provides information on SNPs and InDels for white shrimp. These markers will support the variant database of shrimp and be useful in shrimp aquaculture for breeding selection. Full article

Acute hepatopancreatic necrosis disease, caused by Vibrio parahaemolyticus strains carrying the pirA and pirB toxin genes (VpAHPND), has been causing great economic losses in Asia and America in the shrimp farming industry. Numerous strains are resistant to antibiotics. However, supplementation with probiotic antagonists has become a more desirable treatment alternative. Fourteen strains of microorganisms were assessed for their potential to inhibit VpAHPND in vitro activity. The bacteria with the highest activity were challenged with VpAHPND-infected Pacific white shrimp Litopenaeus vannamei. Furthermore, the genomic characteristics of probiotic bacteria were explored by whole-genome sequencing. We identified the Sonora strain as Bacillus pumilus, which possesses positive proteolytic and cellulolytic activities that may improve shrimp nutrient uptake and digestion. Challenge trials showed a low cumulative mortality (11.1%). B. pumilus Son has a genome of 3,512,470 bp and 3734 coding sequences contained in 327 subsystems. Some of these genes are related to the biosynthesis of antimicrobial peptides (surfactins, fengycin, schizokinen, bacilibactin, and bacilysin), nitrogen and phosphorus metabolism, and stress response. Our in vitro and in vivo findings suggest that B. pumilus Sonora has potential as a functional probiotic. Full article

A high-performance liquid chromatography method coupled to tandem mass spectrometry was validated in order to study the pharmacokinetics of cefotaxime in shrimp hepatopancreases and plasma, as well as its withdrawal time related to a maximum residue limit (MRL) in shrimp muscle. Pharmacokinetics parameters were investigated through oral medication at a single dose of 25 mg/kg shrimp body weight and subsequent hepatopancreas and plasma cefotaxime concentration measurements at 0.5, 1, 2, 4, 8, 12 and 24 h after shrimp were fed with medication. The maximum concentration of cefotaxime was observed after one hour in the hepatopancreas (C_max, 19.45 ± 2.10 mg/kg) and 4 h in plasma (0.184 ± 0.061 mg/L). Based on a minimum inhibitory concentration (MIC) of cefotaxime of 4.13 mg/L against Vibrio parahaemolyticus (known to cause acute hepatopancreatic necrosis disease (AHPND) in white leg shrimp), it was observed that the time during which the hepatopancreas cefotaxime concentration was above the MIC was 23 h. An every 24 h cefotaxime treatment could thus be effective in fighting against this bacterium in shrimp. The withdrawal time of cefotaxime was determined after shrimp were fed with medicated feed once a day and twice a day for three consecutive days. Shrimp muscle was collected on day 1 and day 3 during medication and 1, 3, 7, 14 and 21 days after medication was stopped. Considering an MRL of 50 μg/kg, the withdrawal times were 8.5 degree-days (corresponding to 6.9 h at 29.5 °C) after shrimp were fed with medicated feed once a day for 3 days and 95.5 degree-days (77.7 h at 29.5 °C) after shrimp were fed with medicated feed twice a day for 3 days. Moreover, histological analysis revealed that feeding shrimp with cefotaxime at the given dose in once- or twice-a-day treatments did not negatively impact the shrimp hepatopancreas. Full article

In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of Penaeus vannamei. Observations of fresh mounts revealed structures resembling lipid droplets, similar to those seen in a condition known as “las bolitas syndrome”. Routine histopathological examinations identified detached cells and tissues in the digestive tracts of affected zoea, contrasting with the typical algal cell contents seen in healthy zoea. Polymerase chain reaction (PCR) testing for over 20 known shrimp pathogens indicated minimal differences between diseased and healthy batches. Both groups tested negative for acute hepatopancreatic necrosis disease (AHPND) but positive for Vibrio species and Rickettsia-like bacteria in the diseased samples. Histological analyses of the affected zoea revealed characteristic tissue degeneration in the hepatopancreas, forming spheres that eventually migrated into the upper gut, midgut, and midgut caeca, a pathology identified as bolitas syndrome (BS). Microbiological assessments revealed Vibrio species at concentrations of 10⁶ CFU zoea/g in affected zoea, approximately two orders of magnitude higher than in healthy zoea. Bacterial isolation from both healthy and BS-affected zoea on thiosulphate–citrate–bile salts–sucrose (TCBS) agar and CHROMagar™ (Paris, France), followed by identification using API 20E, identified six strains of Vibrio alginolyticus. Despite similarities to “las bolitas syndrome” in fresh mounts, distinct histopathological differences were noted, particularly the presence of sloughed cells in the intestines and variations in hepatopancreatic lobes. This study highlights the critical need for further research to fully understand the etiology and pathology of bolitas syndrome in zoea stage 2 of P. vannamei to develop effective mitigation strategies for hatchery operations. Full article