Shrimp Pathogens Research Articles

Portable DNA extraction and loop-mediated isothermal amplification (LAMP) for the on-site detection of white spot syndrome virus (WSSV)

White spot disease (WSD) has been the most severe and lethal threat to the global shrimp industry for the past three decades. WSD caused by white spot syndrome virus (WSSV) can result in severe economic losses in shrimp culture. The disease can cause rapid and mass mortality of 90–100 % within 3–10 days. Currently, there are no effective treatment measures available for the control of WSD. Strict biosecurity, possible elimination, and early diagnosis can prevent the spread of the pathogen. Several advanced diagnostic methods have been standardised for the early and rapid detection of WSD. However, the field deployable diagnostic methods are limited. LAMP is a cost-effective, alternative nucleic acid amplification method that operates under isothermal conditions. In this study, a novel field-portable LAMP was developed for the diagnosis of WSSV. The WSSV amplification was optimized at an isothermal condition of 65 °C and a reaction time of 55 min. In this closed-tube amplification, the end results were easily visualized with the naked eye by observing the simple color change. This assay is highly sensitive and can detect up to 6 copies of WSSV plasmid DNA and 0.1 fg of WSSV genomic DNA. Additionally, this diagnostic method is highly specific and did not cross-react with other shrimp pathogens such as IHHNV, IMNV, EHP and Vibrio parahaemolyticus or host DNA of Penaeus vannamei, P. monodon, and P. indicus. Furthermore, a field-deployable DNA (FDD) extraction method has been standardised with a simple rotor and dry bath. This visual LAMP-coupled FDD extraction did not require any sophisticated equipment or technical expertise. Thus this robust LAMP assay can potentially be used for routine surveillance, point-of-care testing, early detection, and timely intervention of WSD.

Exploring the antiparasitic and antimicrobial potential of Schinus terebinthifolius Raddi essential oil against fish and shrimp pathogens.

This study aimed to perform invitro antiparasitic and antimicrobial tests with the essential oil (EO) of Schinus terebinthifolius against of fish and shrimp. The chemical composition of the EO of S. terebinthifolius was determined by gas chromatography. For the antiparasitic test, the protozoan Epistylis sp. obtained from parasitized Oreochromis niloticus was used, and exposed to different concentrations of EO (2%, 1%, 0.5%, 0.25%), and control with 1% grain alcohol. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) test with EO of S. terebinthifolius evaluated the antimicrobial potential, with serial dilutions starting at 2% and control with 1% grain alcohol, using the strains of Aeromonas hydrophila (2.2 × 108 CFU mL-1), Edwardsiella tarda, Vibrio parahaemolyticus, V. harveyi, and V. alginolyticus (2.0 × 108 CFU mL-1). Chemical analysis revealed that the major EO compounds of S. terebinthifolius were δ-3-Carene (56.00%) and α-Pinene (16.89%). In the antiparasitic test, the concentration of 2% EO showed 100% efficacy against Epistylis sp. within 5 min. In the antimicrobial tests, the concentration of 2% EO was effective against all bacteria tested. The EO of S. terebinthifolius demonstrated antiparasitic and antimicrobial activity at a concentration of 2%, standing out as an alternative to conventional antibiotics.

Contaminants in shrimp probiotics - a potential emerging threat to food security

Probiotics are used widely in shrimp aquaculture to improve growth and prevent disease; however, for the most part their safety and efficacy remains largely unknown. Shrimp aquaculture may be vulnerable to the negative effects of unsafe and ineffective commercial products, such as the transfer of shrimp and human pathogens and antibiotic resistance genes. Shrimp health may also be adversely affected by the use of these products, potentially leading to crop yield losses. Here, we used 16S amplicon sequencing to identify the bacterial genera present in commercial shrimp probiotics and assess this against their listed product content. We identified the presence of additional genera to the labelled contents including Escherichia/Shigella and Enterococcus that may pose a risk of disease to animals and potentially humans that come into contact with these products, as well as potentially acting as carriers for virulence and antimicrobial resistance genes. Our results suggest that some commercial shrimp probiotics may be misleading to consumers and potentially unsafe for shrimp, people and the environment. We highlight the risks that contaminated commercial probiotics may pose to food security and present a series of safety and efficacy considerations to support the sustainable use of commercial shrimp probiotics.

Detection of antiviral moieties in the leaves of Rhizophora mucronata functional on the shrimp pathogen Penaeus stylirostris penstyldensovirus 1 (PstDVI)/IHHNV as the prospective drugs of choice in aquaculture

Detection of antiviral moieties in the leaves of Rhizophora mucronata functional on the shrimp pathogen Penaeus stylirostris penstyldensovirus 1 (PstDVI)/IHHNV as the prospective drugs of choice in aquaculture

Bimetallic nanoparticles with sulfated galactan eliminate Vibrio parahaemolyticus in shrimp Penaeus vannamei

Bimetallic (Au/Ag) nanoparticles (BNPs) have shown enhanced antibacterial activity compared to their monometallic counterparts. Sulfated galactans (SG) are a naturally occurring polymer commonly found in red seaweed Gracilaria fisheri. They are biocompatible and biodegradable and environmentally friendly. In this study, we utilized SG in combination with BNPs to develop composite materials that potentially enhance antibacterial activity against shrimp pathogens Vibrio parahaemolyticus and Vibrio harveyi, compared to BNPs or SG alone. BNPs were coated with sulfated galactan (SGBNPs) and characterized using UV–vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, zeta potential, and transmission electron microscopy (TEM). UV–vis spectroscopy analysis revealed that the surface plasmon peaks of BNPs and SGBNPs appeared at 530 nm and 532 nm, respectively. Zeta potential measurements showed that SGBNPs had a negative charge of −32.4 mV, while the BNPs solution had a positive charge of 38.7 mV. TEM images demonstrated the spherical morphology of both BNPs and SGBNPs with narrow size distributions (3–10 nm). Analysis of the FTIR spectra indicated that SG maintained its backbone structure in SGBNPs, but some functional groups were altered. Notably, SGBNPs showed superior antimicrobial and antibiofilm activities against V. parahaemolyticus and V. harveyi compared to SG and BNPs. Furthermore, treatment with SGBNPs significantly down-regulated the expression of virulence-related genes (toxR, cpsQ, and mfpA) for V. parahaemolyticus 3HP compared to the respective control, bacteria treated with BNPs or SG. Diets supplemented with SGBNPs, BNPs, or SG showed no detrimental impact on the growth of shrimp Penaeus vannamei. Shrimp fed with SGBNPs-supplemented feed showed significantly higher survival rates than those fed with BNPs-supplemented feed when infected with 3HP after being on the supplemented feed for seven days and a subsequent number of fifteen days. These findings collectively demonstrate the benefit of using SG capped Au–Ag BNPs as an antibacterial agent for the prevention and control of Vibrio sp. Infection in shrimp while reducing the risk of environmental contamination.

Polyculture of Genetically Improved Farmed Tilapia (GIF tilapia) and Penaeus vannamei Using Biofloc Technology – A Review

According to Food and Agriculture Organization of the United Nations (FAO), aquaculture has grown faster and its expansion aimed at meeting the increase of world fish demand, and preserving natural fish stocks. Currently, to produce fish in quantity and quality requires reduction of the environmental impact from aquaculture, through the improvement of culture systems. Disease is the major factor affecting the development and expansion in aquaculture. Losses due to disease in shrimp farming are high. Various approaches to minimize the impact of disease on production are possible. Another approach to keep the pathogen pressure low is polyculture of shrimp and finfish. This practice makes shrimp farming more sustainable by reducing the environmental impact and the incidence of shrimp disease. Antimicrobial peptides in the fish skin kill shrimp pathogens, keeping pathogen pressure of bacteria and viruses low. In polyculture, shrimps can eat tilapia faeces and unused fish feed, while tilapia filter phytoplankton, reducing the risk of low dissolved oxygen levels at night. In addition, shrimp bioturbation at the pond bottom returns nutrients to the water column, enhancing phytoplankton production and consequently the natural feed available for the tilapia. Biofloc technology (BFT) is one of the most applicable and promising systems for sustainable aquaculture development. This technology is essentially based on the recycling of nutrients via microorganisms, primarily (i) heterotrophic bacteria, which convert nitrogen compounds into microbial biomass, in addition to serves as a source of food for aquatic organisms, and (ii) chemoautotrophic bacteria, which convert ammonia to nitrite and nitrate.

Investigating the impact of chlorine dioxide in shrimp-rearing water on stomach microbiome, gill transcriptome, and infection-related mortality in shrimp.

This study aimed to assess the effects of chlorine dioxide (ClO2) in water on whiteleg shrimp Penaeus vannamei, evaluating its impact on stomach microbiota, gill transcriptome, and pathogens. ClO2 was added to the aquarium tanks containing the shrimp. The application of ClO2 to rearing water was lethal to shrimp at concentrations above 1.2 ppm. On the other hand, most of the shrimp survived at 1.0 ppm of ClO2. Microbiome analysis showed that ClO2 administration at 1.0 ppm significantly reduced the α-diversity of bacterial community composition in the shrimp stomach, and this condition persisted for at least 7 days. Transcriptome analysis of shrimp gill revealed that ClO2 treatment caused massive change of the gene expression profile including stress response genes. However, after 7 days of the treatment, the gene expression profile was similar to that of shrimp in the untreated control group, suggesting a recovery to the normal state. This 1.0-ppm ClO2 significantly reduced shrimp mortality in artificial challenges with an AHPND-causing Vibrio parahaemolyticus and white spot syndrome virus, which were added to rearing water. The use of ClO2 at appropriate concentrations effectively eliminates a significant portion of the bacteria in the shrimp stomach and pathogens in the water. The results of this study provide fundamental knowledge on the disinfection of pathogens in water using ClO2 and the creation of semi germ-free shrimp, which has significantly decreased microbiome in the stomach.

Las Bolitas Syndrome in Penaeus vannamei Hatcheries in Latin America.

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.

A Chelex-100-based rapid DNA extraction method and its application in the detection of shrimp pathogens

BackgroundThe Pacific white shrimp is one of the world’s most economically significant aquatic species, being one of the top three species cultured globally. However, the increasing incidence of diseases such as acute hepatopancreatic necrosis disease and hepatopancreatic microsporidia has led to a serious decline in shrimp production and severe economic losses. With the increasing demand for pathogen detection in shrimp farms, rapid DNA extraction technology has become more sophisticated. In this study, a rapid and crude method of extracting genomic DNA from shrimp muscle and hepatopancreas using Chelex-100 was established. ResultsDNA was successfully extracted from muscle and hepatopancreatic tissues using both the Chelex-100 method and commercial kits. The internal reference genes of shrimp were successfully amplified via PCR and real-time PCR using the obtained DNA samples. Moreover, a field assay was successfully conducted using real-time PCR and real-time enzymatic recombinase amplification (real-time ERA), indicating that the quality of the DNA extracted using Chelex-100 is sufficient for use in conjunction with nucleic acid amplification to detect pathogens in shrimps. ConclusionsChelex-100 is an efficient method for extracting DNA from shrimp muscle or hepatopancreas tissues, with a short extraction time, high extraction efficiency, and simple operation, making it appropriate for use in the detection of pathogens in shrimp.How to cite: Yang H, Zhou Q, Hu J, et al. A Chelex-100-based rapid DNA extraction method and its application in the detection of shrimp pathogens. Electron J Biotechnol 2024;70. https://doi.org/10.1016/j.ejbt.2024.04.004.

Passive surveillance for shrimp pathogens in Penaeus vannamei submitted from 3 regions of Latin America

Multiple PCR analyses were conducted to broaden the search spectrum for shrimp pathogens, revealing some pathogens without histological lesions. This underscores the importance of selecting appropriate primers for obtaining reliable results. Notably, there was significant variability among primers, emphasizing the need for seasonal and regional assessment to optimize primer selection. It's crucial to understand that a positive PCR test result alone doesn't confirm the presence of a viable pathogen or a disease state. Therefore, histological analysis was employed to confirm the presence of characteristic lesions for each pathogen. Overall, most shrimp exhibited coinfections, including WzSV8, DHPV, chronic midgut inflammation, and tubule distension/epithelial atrophy consistent with AHPND. Additionally, non-EHP-microsporidia, striated muscle necrosis in the abdomen, and gregarines in the hindgut caecum were observed. The presence of spheroids in the lymphoid organ might indicate host responses to often unidentified causes.

Multi-omics characterization and identification of small non-coding RNAs in Spiroplasma eriocheiris

Spiroplasma eriocheiris is the major pathogen in the aquaculture shrimp and crab tremor disease; its infection causes heavy losses in aquaculture. Previous investigations concentrated on how the host responds to S. eriocheiris infection, with little known on the regulation of virulence and pathogenesis by the bacteria themselves. A body of increasing evidence shows that bacterial small noncoding RNAs (sRNAs) play a crucial part in regulating bacterial virulence and pathogenesis; however, whether there are some sRNAs in S. eriocheiris and how those sRNAs regulate virulence and pathogenesis are still unclear. This study analyzed multi-omics data integration to identify the potential sRNAs and their mediated regulatory network in S. eriocheiris. First, through an integration analysis of the public genomic and transcriptomic data, 54 potential sRNAs were identified in S. eriocheiris with a 50–250 nt length distribution and the base composition mainly AT. Next, sRNA profiling of the in vitro logarithmic and decline phase of S. eriocheiris, as well as the early and onset stage of infection in Eriocheir sinensis, was performed, showing that 4 sRNAs (SR01, SR05, SR08, and S04) were both down-regulated in the logarithmic and the onset stage of infection. Subsequently, integrated with sRNAs target gene prediction and target gene expression analysis, a regulatory network composed of four sRNAs and 96 target genes was constructed. Last, an RNA pull-down experiment was performed on the SR01, which exhibited the highest abundance and the highest number of target genes, resulting in 37 highly credible target genes with multiple genes related to bacterial virulence, such as GTPases and virulence factor coding genes. Collectively, this study identified the sRNAs in S. eriocheiris and revealed their mediated regulatory network for regulating virulence and pathogenesis, thus paving a new avenue for disease prevention and control in aquaculture.

Determining patterns of tissue tropism for IHHNV, GAV and YHV-7 infection in giant black tiger shrimp (Penaeus monodon) using real-time RT-qPCR

Disease management in shrimp aquaculture is heavily dependent on accurate and rapid pathogen detection and diagnosis. Understanding the presence and distribution of pathogens within various tissues of the shrimp host is critical to ensure detection efforts are targeted for optimal sensitivity and reliability. Modern advancements in molecular technologies, including nucleic acid extraction and RT-qPCR, have yielded significant improvements in pathogen detection capabilities. Despite these advancements and their widespread adoption within both research and industry applications, evidence to inform their optimised use, such as revisions to tissue-specific viral detection to support selection of appropriate target tissues, has not been established. To address these gaps, this study aimed to establish contemporary evidence of viral tissue tropism for three major shrimp pathogens. TaqMan qPCR analysis was used to determine tissue-specific loading of viral pathogens in naturally infected Penaeus monodon pleopod, gill, hepatopancreas, lymphoid organ, abdominal muscle tissue, hindgut and ventral nerve cord. The viral targets analysed included Penaeus stylirostris penstyldensovirus 1 (PstDV1), formerly named infectious hypodermal haematopoietic necrosis virus (IHHNV) and, two genotypes of the yellow head virus complex, including genotype 2 known as gill associated virus (YHV-2/ GAV), and genotype 7 (YHV-7). QPCR analyses demonstrated the highest level of genetic IHHNV detection from gill tissue, followed sequentially by hindgut, pleopod, hepatopancreas, lymphoid organ, ventral nerve cord and abdominal muscle. For the yellow head viruses (GAV and YHV-7), no significant differences in genetic viral detection were demonstrated, although a non-significant advantage was observed for lymphoid organ and hindgut tissue. Beyond establishing contemporary evidence of viral distribution to improve understanding of host-viral interactions, these findings offer supporting evidence for revisions to appropriate target tissue recommendations, within the World Organisation for Animal Health (WOAH, founded as OIE) Manual of Diagnostic Tests for Aquatic Animals, for optimised modern shrimp pathogen detection.

Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens.

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.

Compound isolation through bioassay-guided fractionation of Tectona grandis leaf extract against Vibrio pathogens in shrimp.

Tectona grandis Linn, commonly known as teak, is traditionally used to treat a range of diseases, including the common cold, headaches, bronchitis, scabies, diabetes, inflammation, and others. The present study was conducted with the purpose of isolating and identifying the active compounds in T. grandis leaf against a panel of Vibrio spp., which may induce vibriosis in shrimp, using bioassay-guided purification. The antimicrobial activity was assessed using the microdilution method, followed by the brine shrimp lethality assay to determine toxicity. Following an initial screening with a number of different solvents, it was established that the acetone extract was the most effective. The acetone extract was then exposed to silica gel chromatography followed by reversed-phase HPLC and further UHPLC-orbitrap-ion trap mass spectrometry to identify the active compounds. Three compounds called 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin were identified with substantial anti-microbial action against V. parahaemolyticus, V. alginolyticus, V. harveyi, V. anguillarum, and V. vulnificus. The IC50 values of the three compounds viz. 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin varied between 2 and 28, 7 and 38, and 7 and 56 μg/mL, respectively, which are as good as the standard antibiotics such as amoxicillin and others. The in vivo toxicity test revealed that the compounds were non-toxic to shrimp. The results of the study suggest that T. grandis leaf can be used as a source of bioactive compounds to treat Vibrio species in shrimp farming.

Exploring the Antimicrobial Potential of Phyllanthus emblica L. (Amla) using Molecular Docking Studies against Shrimp Pathogens

Vibriosis is a major problem in shrimp farm. Farmers indiscriminately use hormones, antibiotics, disinfectants and other chemicals in fish feed and culture water to protect their crops. E. coli and Aeromonas sp were highly predominant isolates. Herbs act as agents in aquaculture to control or reduce pathogen infections. The results of phytochemicals screening of fruit extract of Phyllanthus emblica showed the presence of various phytchemicals. Phyllanthus emblica extract shows anti-bacterial properties against biofilm producing shrimp pathogens. Among the 10 bacterial genera, E. coli and P.aeruginosa were highly suppressed. The extracts exhibited the bacteria growth inhibitory activity in a dose-dependent manner. The compound sitosterol has potent antibiofilm activity was showed by molecular docking, which revealed a significant binding energy and interaction (-8.4 Kcal/mol) between it and key biofilm-forming protein. Diving analysis was done using the chemical compounds found in GCMS analysis.

Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

Estimating the rejection risk of Indonesian shrimp exports from Salmonella spp. contamination using the food safety objective approach

Contamination by non-typhoidal Salmonella spp. in aquacultured shrimp poses a risk for human health. In Indonesia, the presence of the pathogen in shrimp raises a major concern for international trade because shrimp is one of the important exported fishery commodities. The aim of this study was to use food safety objective approach in combination with a quantitative microbiological risk assessment to determine the contamination of Salmonella spp. in raw materials, to estimate the risk of export rejection and to identify the contributing factors of export rejections. The prevalence and concentration of Salmonella spp. in shrimp based on real-time polymerase chain reaction of 7.02% and 0.72–1.46 log CFU/gr (mean: 1.00 ± 0.28 log CFU/g), respectively, were used to develop an exposure model. The exposure model was used to estimate the level of Salmonella spp. in frozen shrimp at the border of an export destination country, which was ca. −6.02 to −2.07 (mean −3.73 ± 0.73) log CFU/g. The value was compared to the food safety objective value determined from the required microbiological criterion of Salmonella spp. of n = 30, c = 0, and m = negative/25 g. As a result, the risk estimate for rejection was 27 rejections per 100,000 exports. The Salmonella spp. contamination level in raw materials and freezing technology were the primary factors contributing to the rejections. Moreover, maintaining the quality control of raw materials purchased or controlled quick freezing may be the mitigation formulae of export rejection of frozen shrimp due to Salmonella spp.

Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea

White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen with a broad host range. Among the hosts, though mud crab, Scylla olivacea is reported to be more susceptible to WSSV than S. serrata and S. paramamosain, a detailed study on the pathogenicity and genome stability of the virus after multiple passages has yet to be reported. Firstly, to test the pathogenicity of the virus, WSSV was intramuscularly injected into healthy shrimp, Penaeus vannamei. Experimentally infected P. vannamei showed the first mortality at 36 h post-injection (hpi), followed by 100 % cumulative mortality in 7 days post-injection (dpi). However, S. olivacea injected with the WSSV inoculum derived from infected shrimp showed the first mortality at 48 hpi and a cumulative mortality of 70 % at the end of the ten days experiment. Subsequently, WSSV was sequentially passaged five times in Scylla olivacea to find out any change in the virulence of the virus in each passage. S. olivacea groups injected with 1st, second, third and fourth passages derived from the crab recorded the first mortality between 48 and 56 hpi and the cumulative mortality of 60 to 70 % at the end of the ten days experiment. Injection of WSSV inoculum in P. vannamei derived from multiple passages in S. olivaceae revealed the retention of the pathogenicity of the virus. Shrimp groups injected with WSSV derived from different passages showed first mortality between 24 and 36 hpi and cumulative mortality of 100 % between 6 and 7 dpi. The average viral load in the shrimp groups injected with WSSV inoculum derived from shrimp was 3.6 × 108, whereas in shrimp injected with the inoculum derived from 1st, third and fifth passages from crab showed 4.0 × 108, 4.7 × 108 and 4.3 × 108 copies per 100 ng DNA. Histological examination of the gill and stomach tissue of shrimp injected with inoculum prepared from shrimp as well as the inoculum derived from 1st, third and fifth passages in S. olivacea revealed characteristic pathological manifestations of the WSSV infection in gill and stomach tissues such as hypertrophied nuclei, Cowdry A-type inclusions as well as massive basophilic intranuclear inclusions. Further, to study the genome stability, the primers targeting highly variable regions of the WSSV genome (ORF94, ORF125, ORF75, variable region (VR) 14/15 and VR 23/24) were used to amplify WSSV derived from different passages and the amplified PCR products were sequenced. The sequence analysis revealed the WSSV genome stability after multiple passages in mud crab, S. olivacea.

Establishment of a SYBR Green I-based real-time PCR for the detection of decapod iridescent virus 1

Decapod iridescent virus 1 (DIV1) is an emerging pathogen that mainly threatens decapod crustaceans, causing high mortalities and leading to huge economic losses. In this study, a pair of specific primers were designed for the major capsid protein (MCP) gene of DIV1, and a SYBR Green I-based real-time PCR method was developed. The method displayed good linearity (R2 = 1.000) and good repeatability in detecting standards of DIV1 MCP fragments ranging from 6.2 × 101 to 6.2 × 108 DNA copies/μl. Specificity analysis revealed that the real-time PCR was specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. Sensitivity analysis revealed that the real-time PCR could efficiently detect DIV1 DNA as low as 62 copies/μl within 35 cycles. In summary, the established real-time PCR provides an efficient, sensitive, and reliable detection method for DIV1.

Specific gut bacterial taxa inhabited in healthy shrimp (Penaeus vannamei) confer protection against Vibrio parahaemolyticus challenge

Host gut bacterial community has strong resistance to the invasion of pathogens. However, the specific gut taxa that could confer the resistance against pathogens in shrimp are largely unknown. Here, we demonstrated that the shrimp in the same tank showed different resistance after pathogenic Vibrio parahaemolyticus challenge. Subsequently, the gut bacterial communities of healthy, diseased, and moribund shrimp were analyzed. The results indicated that the gut bacterial community in the healthy shrimp still kept a high stability after challenge, while they were gradually disordered with the severity of disease in the diseased, and moribund shrimp. Certain specific taxa belonging to Rhodobacteraceae (e.g., Ruegeria, Paracoccus, Sulfitobacter, and Phaeobacter), Flavobacteriaceae (e.g., Tenacibaculum) and Demequinaceae (e.g., Demequina) were mainly depleted in the moribund shrimp, compared to that in healthy, and diseased shrimp. Seven corresponding strains of above specific taxa including five Rhodobacteraceae, one Tenacibaculum and one Demequina were isolated from the shrimp gut. Then, four different synthetic communities (SynComs) were constructed by using these isolates: SynComA, all five strains from Rhodobacteraceae; SynComB, SynComA + T. lutimaris HBG23; SynComC, SynComA + D. globuliformis TCG4; and SynComD, SynComA + T. lutimaris HBG23 + D. globuliformis TCG4. The addition of these SynComs could not only improve the fitness of shrimp, but also protect them against subsequent V. parahaemolyticus challenge, especially the addition of SynComD. Our study revealed that specific gut bacteria inhabited in healthy shrimp could improve shrimp resistance against pathogenic microbes.