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The parasitic dinoflagellates of the genus Hematodinium have been considered one of the most important emerging pathogens for a broad range of marine crustaceans around the world. In China, frequent outbreaks of Hematodinium infections have caused serious economic losses for local farmers since 2004. Wild crabs were recently indicated to play a vital role in the transmission and spreading of the Hematodinium disease in polyculture pond systems. Based on PCR amplification and histopathological examination, we demonstrated that H. perezi can naturally infect a wild crab species, Hemigrapsus takanoi, which were collected from the waterways located on the coast of Rizhao or Weifang, Shandong Peninsula, China. According to the sequence similarity analysis and phylogenetic analysis, the Hematodinium isolates were identified as H. perezi and belonged to genotype II. The prevalence of H. perezi ranged from 3.3% to 5.7% in H. takanoi originating from Rizhao (n = 165 wild crabs) and from 0.9% to 20.0% in that originating from Weifang (n = 1386 wild crabs), respectively. To our knowledge, H. takanoi is, for the first time, reported as a new host for Hematodinium. Given the wide distribution of H. takanoi on the coasts along the Shandong Peninsula and the relative high prevalence of infection we monitored in our study, we speculate that H. takanoi contributes to the introducing and spreading parasitic Hematodinium between ponds via waterways in a poly-culturing system. Findings in this study broaden the host range of this parasite and expand the scope of our surveillance for Hematodinium disease in China.

A novel pathogenic strain Vibrio 20190611023 was isolated from the hepatopancreas of moribund cultured Penaeus vannamei suffering from black gill disease. This strain was identified as V. brasiliensis based on the phylogenetic analyses of 16S rDNA gene and five other housekeeping genes (i.e., gapA, ftsZ, mreB, topA and gyrB). Some biochemical features of this strain were determined with an API 20NE system, and its haemolytic activity was determined using a sheep blood agar plate. The pathogenicity of this isolate 20190611023 was confirmed by the experimental challenge tests and histopathological examinations. P. vannamei were challenged via reverse gavage with different doses of bacterial suspensions. The calculated median lethal dose (LD50 ) was (3.16 ± 1.78) × 105 CFU/g (body weight). Moreover, antibiotic susceptibility tests were performed, the results of which showed that the strain 20190611023 was sensitive to chloramphenicol, compound sulphamethoxazole, ciprofloxacin, doxycycline and oxacillin, but resistant to erythromycin, kanamycin, gentamicin, cefoperazone, ceftriaxone, cefamezin and piperacillin. To our knowledge, this is the first report for demonstrating V. brasiliensis as a shrimp pathogen, which expands the host range of V. brasiliensis infection. The present study highlights that more attention should be paid to this novel pathogen in intensive shrimp aquaculture.

Acute hepatopancreatic necrosis disease (AHPND) is one of the most significant bacterial diseases in global shrimp culture, causing severe economic losses. In the present study, we carried out in vitro antimicrobial tests to investigate the disinfection efficacy of 14 common disinfectants toward different AHPND-causing Vibrio spp., including eight isolates of V. parahaemolyticus, four isolates of V. campbellii, and one isolate of V. owensii. Polyhexamethylene biguanidine hydrochloride (PHMB) was revealed to possess the strongest inhibitory activity. Through analyzing and evaluating the results of antimicrobial tests and acute toxicity test, we selected PHMB and hydrogen peroxide (H2O2) for further clinical protection test. Clinical manifestations indicated that both PHMB (2 mg/L and 4 mg/L) and H2O2 (12 mg/L) could effectively protect juvenile Penaeus vannamei from the infection of V. parahaemolyticus isolate Vp362 at 106 CFU/ml, and the survival rate was over 80%. When the bacterial concentration was reduced to 105 CFU/ml, 104 CFU/ml, and 103 CFU/ml, the survival rate after treated by 1 mg/L PHMB was 64.44%, 93.33%, and 100%, respectively. According to the results, PHMB and H2O2 showed a lower toxicity while a better protection activity, particularly against a lower concentration of the pathogens. Therefore, these two disinfectants are proved to be promising disinfectants that can be applied to prevent and control AHPND in shrimp culture. Moreover, the methods of this study also provided valuable information for the prevention of other important bacterial diseases and suggested a reliable means for screening potential drugs in aquaculture.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD50 ) was 9.75 ± 4.29 × 105 CFU/g (body weight) by challenging P. vannameivia reverse gavage. The diseased shrimp displayed enlarged hepatopancreatic tubules and sloughing of epithelial cells in tubular lumens. The strain 20190611022 was also characterized by the testing of API 20NE systems and antibiotic susceptibility. The results of disc diffusion test showed that strain 20190611022 was sensitive to chloramphenicol, compound sulfamethoxazole, cefoperazone, ceftriaxone, ceftazidime and cefuroxime. To our knowledge, this is the first report of isolation and characterization of Photobacterium damselae subsp. damselae from natural diseased P. vannamei. Our findings can serve as a basis for further studies of its pathogenicity and provide technological support for disease controlling in shrimp aquaculture.

User-friendly phenotypic antibiotic susceptibility testing (AST) methods are urgently needed in many fields including clinical medicine, epidemiological studies and drug research. Herein, we report a convenient and cost-effective phenotypic AST method based on online monitoring bacterial growth with a developed 8-channel contactless conductometric sensor (CCS). Using E. coli and V. parahaemolyticus as microorganism models, as well as enoxacin, florfenicol, ampicillin, kanamycin and sulfadiazine as antibiotic probes. The minimum inhibitory concentration (MIC) determination was validated in comparison with standard broth microdilution (BMD) assay. The total essential agreements between the CCS AST assays and the reference BMD AST assays are 68.8–92.3%. The CCS has an approximate price of $9,000 (USD). Requiring neither chemical nor biotic auxiliary materials for the assay makes the cost of each sample

To avoid the negative effects of antibiotics, using phage to prevent animal disease becomes a promising method in aquaculture. Here, a lytic phage provisionally named vB_VcaS_HC that can infect the pathogen (i.e., Vibrio campbellii 18) of prawn was isolated. The phage has an isometric head and a non-contractile tail. During phage infection, the induced host mortality in 5.5 h reached ca. 96%, with a latent period of 1.5 h and a burst size of 172 PFU/cell. It has an 81,566 bp circular dsDNA genome containing 121 open reading frames (ORFs), and ca. 71% of the ORFs are functionally unknown. Comparative genomic and phylogenetic analysis revealed that it is a novel phage belonging to Delepquintavirus, Siphoviridae, Caudovirales. In the phage genome, besides the ordinary genes related to structure assembly and DNA metabolism, there are 10 auxiliary metabolic genes. For the first time, the pyruvate phosphate dikinase (PPDK) gene was found in phages whose product is a key rate-limiting enzyme involving Embden-Meyerhof-Parnas (EMP) reaction. Interestingly, although the phage has a strong bactericidal activity and contains a potential lysogeny related gene, i.e., the recombinase (RecA) gene, we did not find the phage turned into a lysogenic state. Meanwhile, the phage genome does not contain any bacterial virulence gene or antimicrobial resistance gene. This study represents the first comprehensive characterization of a lytic V. campbellii phage and indicates that it is a promising candidate for the treatment of V. campbellii infections. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00271-w) contains supplementary material, which is available to authorized users.

Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming., Vibrio parahaemolyticus is an important foodborne pathogen and has recently gained particular notoriety because it causes acute hepatopancreatic necrosis disease (AHPND) in shrimp, which has caused significant economic loss in the shrimp industry. Here, we report a whole-genome analysis of 233 V. parahaemolyticus strains isolated from humans, diseased shrimp, and environmental samples collected between 2008 and 2017, providing unprecedented insight into the historical spread of AHPND. The results show that V. parahaemolyticus is genetically diverse and can be divided into 84 sequence types (STs). However, genomic analysis of three STs of V. parahaemolyticus identified seven transmission routes in Asia since 1996, which promoted the transfer of an AHPND-associated plasmid. Notably, the insertion sequence (ISVal1) from the plasmid subsequently mediated the genetic exchange among V. parahaemolyticus STs and resulted in the deletion of an 11-kb region regulating cell mobility and the production of capsular polysaccharides. Phenotype assays confirmed that this deletion enhanced biofilm formation, providing a novel mechanism for environmental adaptation. We conclude that the transmission mode of AHPND consists of two steps, the transmission of V. parahaemolyticus and the subsequent horizontal transfer of the AHPND-associated plasmid. This plasmid allows ISVal1 to mediate genetic exchange and improve pathogen fitness in shrimp ponds. Current shrimp farming practices promoted such genetic exchanges, which highlighted a risk of the emergence of new virulent populations, with potentially devastating consequences for both aquaculture and human health. This study addressed the basic questions regarding the transmission mechanism of AHPND and provided novel insights into shrimp and human disease management. IMPORTANCE Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming.

In Nov 2020, mass mortality of cultured Pacific white shrimp (Penaeus vannamei) occurred due to black spot disease (BSD) in some shrimp farms in China. To investigate the disease cause, a potential pathogenic fungus was isolated from melanized lesions of P. vannamei suffering from the BSD syndrome. The fungal isolate was then identified as Fusarium solani based on TEF1α and ITS-gene sequences. The isolate of fungus was used in infection studies. This study used Koch postulates confirmed that the studied agent was causing BSD symptoms in the white leg shrimp, which were significantly symptoms relieved after molting. Results showed that an identical fungus was re-isolated from black lesions after the artificial infection period. Virulence test revealed that F. solani could cause 88.66% mortality within 30 dpi at a dose of 1.02 × 106 CFU/mL, and the LC50 of F. solani at 30 dpi is 3.37 × 104 CFU/mL. The accumulative mortalities also displayed concentration dependence. The histopathological characterization displayed that responses of P. vannamei against F. solani are haemocytic infiltration, encapsulation, melanization, etc. in the black spot lesions. Furthermore, severe infection of F. solani in P. vannamei causes molting failure, and further contributes to the mortality of the diseased individuals. The findings of our study would be useful to provide a theoretical basis for early prevention and control of BSD.

As a newly found virus that seriously threatens the economic crustacean aquaculture, the susceptible host range of Decapod iridescent virus 1 (DIV1) plays a critical role in both epidemic prevention and international trade. The swimming crab, Portunus trituberculatus, is widely distributed in the coastal waters of Asia and has become an important economic species in China. In the present study, healthy swimming crabs Po. trituberculatus were artificially infected with DIV1 (isolate SHIV 20141215) through intramuscular injection and per os pathways. The infected individuals showed symptoms with decreased vitality, retarded reaction, anorexia, and mortalities of 100% and 50%, respectively. Nested PCR showed that all the moribund individuals and some surviving individuals were DIV1-positive. Histological examination revealed that eosinophilic inclusions and karyopyknosis existed in hemocytes of gills, hepatopancreas, ovaries, and muscles of diseased Po. trituberculatus, which was also verified by the positive signal of in situ DIG-labeling loop-mediated isothermal amplification (ISDL) at the same location of these tissues. Transmission electron microscopy (TEM) of ultrathin sections showed that typical DIV1 virions, the virogenic stroma, and the process of nucleocapsid formation existed in hemocytes. Quantitative detection results showed that gills and epidermis of the diseased Po. trituberculatus had the highest DIV1 load, while the gonads and hepatopancreas had the lowest DIV1 load. The above-mentioned results support that Po. trituberculatus is a newly confirmed susceptible host for infection with DIV1. Furthermore, the epidemiological survey showed that the DIV1 detection rates of farmed Po. trituberculatus, wild Helice tientsinensis, and wild Hemigrapsus penicillatus were relatively high, from 6.9% (2/29) to 42% (22/53). The findings warn that polyculture with shrimps and crabs has a nonnegligible risk of cross-species transmission of pathogens, and wild crabs are very likely to be vectors of DIV1 to an aquaculture system.

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post-infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 105 colony-forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD

Many Pseudoalteromonas strains can produce bioactive compounds with antimicrobial activities. This study focused on a probiotic candidate P.flavipulchra CDM8 to reveal its novel antibacterial mechanism and risks for antibiotic resistance dissemination. Strain CDM8 could form floating biofilm, displayed strikingly broad antibacterial activities against multiple Vibrio and Bacillus species, and decreased the competitor's concentration in their co-cultures in the microtiter plate tests. It could also form vesicle/pilus-like structures on the outer surface, which were indicated to participate in the bactericidal activity and represent a novel antibacterial mechanism of CDM8, according to the scanning electron microscopic observation. However, CDM8 displayed multi-antibiotic resistance, conferred by the multidrug resistance regions in hotspot 4 and variable region III of a novel SXT/R391-like integrative and conjugative element (ICEPflCDM8). Summing up, our results provided a better understanding of the bactericidal mechanism of P. flavipulchra and highlighted the role of SXT/R391-like ICEs in conferring multidrug resistance phenotype of probiotic P. flavipulchra candidates.

A double fluorescent staining method was used to differentiate life stages of Enterocytozoon hepatopenaei (EHP) with a chitin-binding dye, calcofluor white (CFW) and a nucleic acid dye, acridine orange (AO). The unreleased spores showed a significantly blue fluorescent chitin wall with weak orange-red RNA staining, while the plasmodia appeared grape cluster-like orange-red nucleic acid-containing particles without or with very weak blue fluorescence and sporophytes showed grape cluster-like orange-red particles with varying degree of blue fluorescence. The analysis of the red, blue and green intensity of different targets revealed that the ratio of relative concentration of CFW and AO (RRC/A) of plasmodia (0.36 ± 0.15) (n = 60), sporophytes (1.25 ± 0.41) (n = 157), and unreleased spores (3.06 ± 1.09) (n = 21) had extremely significant differences (P

White feces syndrome (WFS) is an emerging digestive system disease of intensively cultured penaeid shrimp in Asian countries. In this study, Penaeus vannamei suffering from WFS were collected from a pond with water salinity of 2 g/l. Histopathology and comparative analysis of dominant bacterial populations (DBPs) and their interactions through high-throughput sequencing bacterial 16S rRNA genes were conducted, using the digestive tracts of shrimp at different infection stages. Although Enterocytozoon hepatopenaei and acute hepatopancreatic necrosis disease-causing Vibrio (VAHPND) were detected using PCR in WFS-affected shrimp, the histopathology did not support their pathogenicity for WFS. Histopathology of WFS was characterized by collapsed epithelia and enlarged lumens in hepatopancreatic tubules. Vibrio predominated in the digestive tracts of diseased shrimp and served as a major hub to interact with the co-occurring DBPs in the networks. The purified opportunistic Vibrio isolates (V. sinaloensis and V. parahaemolyticus) could cause experimental shrimp to develop a similar histopathology to the naturally WFS-affected. By contrast, Acinetobacter and Candidatus Bacilloplasma dominated in the bacterial communities of hepatopancreas and gut from grossly healthy shrimp, respectively, and they were major hubs in the networks. Contributions of bacterial commensalism and amensalism were highlighted to shift the major hubs in the interaction networks and to change the DBPs of microbial communities from healthy to diseased. Taking together, these findings provide insights into the histopathology and microbiome dynamics in the shrimp digestive system during WFS infection, and provide a histopathology-based support for that Vibrio species are the major bacterial species responsible for WFS and the consequent mortality of infected shrimp, which are valuable for controlling WFS in shrimp farming ponds.

Acute hepatopancreatic necrosis disease (AHPND) is a recently discovered shrimp disease that has become a severe threat to global shrimp-farming industry. The causing agents of AHPND were identified as Vibrio parahaemolyticus and other vibrios harboring a plasmid encoding binary toxins PirAvp/PirBvp. However, the epidemiological involvement of environmental vibrios in AHPND is poorly understood. In this study, with an aim to reveal the possible transmission route of AHPND-causing V. parahaemolyticus, we sequenced and analyzed the genomes of four pairs of V. parahaemolyticus strains from four representative regions of shrimp farming in China, each including one strain isolated from diseased shrimp during an AHPND outbreak and one strain isolated from sediment before AHPND outbreaks. Our results showed that all the four shrimp-isolated and three of the sediment-isolated strains encode and secret PirAvp/PirBvp toxins and, therefore, are AHPND-causing strains. In silico multilocus sequence typing and high-resolution phylogenomic analysis based on single-nucleotide polymorphisms, as well as comparison of genomic loci in association with prophages and capsular polysaccharides (CPSs) consistently pointed to a close genetic relationship between the shrimp- and sediment-isolated strains obtained from the same region. In addition, our analyses revealed that the sequences associated with prophages, CPSs, and type VI secretion system-1 are highly divergent among strains from different regions, implying that these genes may play vital roles in environmental adaptation for AHPND-causing V. parahaemolyticus and thereby be potential targets for AHPND control. Summing up, this study provides the first direct evidence regarding the transmission route of AHPND-causing V. parahaemolyticus and underscores that V. parahaemolyticus in shrimp are most likely originated from local environment. The importance of environmental disinfection measures in shrimp farming was highlighted.

Vibrio parahaemolyticus is an important foodborne pathogen which commonly inhabits estuarine and marine environments and seafood. In the present study, 90 V. parahaemolyticus isolates from the main seafoods from three coastal provinces surrounding Bohai Sea and Yellow Sea, China were analyzed to elucidate their antimicrobial resistance, virulence and genetic relationship by multilocus sequence typing (MLST). The results showed that the virulence genes tdh and trh were detected in one isolate and five isolates respectively. Most of isolates showed resistance to ampicillin (86/90) and cephazolin (75/90). Some isolates were resistant to amikacin (27/90), cefuroxime sodium (18/90), tetracycline (16/90), sulphamethoxazole/trimethoprim (16/90) and streptomycin (13/90). Forty isolates (44.4%) possessed multiple antimicrobial resistance to at least three antimicrobials. The V. parahaemolyticus population was composed of 68 sequence types, of which 41 were novel to the pubMLST database, displaying a high level of genetic diversity. The phylogenetic relatedness of V. parahaemolyticus isolates was irrelevant to the collection sources. Moreover, there were no associations of antimicrobial resistance and trh positive virulence with genetic population of V. parahaemolyticus isolates. These results indicated that the diversity of antimicrobial-resistant or pathogenic V. parahaemolyticus isolates from coasts of Bohai Sea and Yellow Sea, China could pose a potential risk to human health.

Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirABvp. Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirABvp-bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirABvp-bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirABvp. Novel variations likely driven by ISVal1 in the genetic contexts of the pirABvp genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirABvp in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirABvp and also appeals for precautions to encounter the dissemination of the hazardous genes.

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification of E. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values (y) against log10 (E. tarda genomic DNA concentration) as x. The intra- and inter-assay coefficient of variation (CV) values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.

Edwardsiella tarda is one of the most important emerging pathogens in the global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQPCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values (y) against the common logarithmic copies (log10n c as x; n c is copy number) of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.

Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB - LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.

Edwardsiella tarda has become one of the most severe pathogens in aquaculture industries throughout the world; therefore, a specific and rapid identification method for this bacterium is urgently needed. In the present study, a novel loop-mediated isothermal amplification (LAMP) was developed by targeting the upstream region of the hlyb gene of E. tarda, which was then named as UH-LAMP. The Mg(2+) concentrations, the reaction temperature, and the reaction time of UH-LAMP were optimized to 10 mM, 65°C, and 45 min, respectively. The detection limit of the UH-LAMP was 100-times higher than that of conventional polymerase chain reaction (10 versus 1000 CFU/test). Furthermore, the new UH-LAMP assay showed no cross-reactivity to the E. ictaluri belonging to the other species in the genus Edwardsiella. The high specificity of the assay was also confirmed by testing the nine strains of E. tarda collected from different geographical locations and the other 20 bacteria species. The assay can be performed in a simple water bath or a heat block and the detection result can be visualized by adding a fluorescent reagent to the reaction mixture. Taken together, our preliminary results indicate that this UH-LAMP assay provided a rapid, sensitive, and species-specific diagnostic tool for E. tarda and can easily be applied for the diagnosis under clinical or onsite conditions.