Jiangyong Wang

Detection of red-spotted grouper nervous necrosis virus by loop-mediated isothermal amplification.

Red-spotted grouper nervous necrosis virus (RGNNV) causes high mortality in marine fish larvae cultured in China. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on loop-mediated isothermal amplification (LAMP) was developed. A set of four primers, two outer and two inner, was designed from RGNNV genome RNA. The LAMP reaction mix was optimized. The method was specific as no cross-reaction was observed between white spot syndrome virus, koi herpesvirus, infectious spleen and kidney necrosis virus, mud crab reovirus, and grass carp hemorrhage virus. The sensitivity of LAMP was 100-fold higher than the nested PCR in detecting the presence of RGNNV. RGNNV was detected in the brain of Trachinotus ovatus that showed typical symptoms of NNV infection, with the standardized LAMP procedure.

Studies on the isolation of Photobacterium damselae subsp. piscicida from diseased golden pompano (Trachinotus ovatus Linnaeus) and antibacterial agents sensitivity

An epizootic occurred among cultured golden pompano (Trachinotus ovatus); it involved mass mortality on fish farms in Linshui in Hainan province, China, in 2008. Diseased fish exhibited no obvious clinical signs, but pathological studies showed that nodules were scattered over the spleen and kidney. To investigate the nature of the pathogen, we studied the surviving fish, and a Gram-negative bacterium (designated strain TOS1) was isolated from the spleens of golden pompano. Pathogenicity assays revealed that TOS1 was virulent for golden pompano when they were challenged by intraperitoneal injection, and the lethal dose (LD(50)) was 1.1 × 10(6)colony forming units (CFU)g(-1). 16S rDNA gene sequence of TOS1 demonstrated high similarity (99%) to that of Photobacterium damselae subsp. piscicida. Phylogenetic analysis also showed a clear association of strain TOS1 with P. damselae subsp. piscicida, and this agreed with the results of morphological, physiological and biochemical identification. The results also showed that TOS1 was sensitive to norfloxacin, gentamicin, sulfamethoxazole/trimethoprim, neomycin, streptomycin and tetracycline, and was very sensitive to Psidium guajava and Atractylodes lancea (minimum bactericidal concentration, MBC=10(-6)g/mL). This paper describes a systematic study of P. damselae subsp. piscicida isolated from diseased golden pompano in China, including its sensitivity to different antibiotics and Chinese herbal extracts, which will contribute to the diagnosis and prevention of the associated disease.

Identification of a cobia (Rachycentron canadum) CC chemokine gene and its involvement in the inflammatory response.

The chemokines regulate immune cell migration under inflammatory and physiological conditions. We investigated a CC chemokine gene (RcCC1) from cobia (Rachycentron canadum). The full-length RcCC1 cDNA is comprised 673 nucleotides and encodes a four-cysteine arrangement 99-amino-acid protein typical of known CC chemokines. The genomic DNA of RcCC1 consists of three exons and two introns. Phylogenetic analysis showed that RcCC1 was closest to the MIP group of CC chemokines. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed RcCC1 was constitutively expressed in all tissues examined, with relative strong expression in gill, blood, kidney, spleen, and head kidney. The RcCC1 transcripts in the head kidney, spleen, and liver were quickly up-regulated after stimulation with formalin-inactivated Vibrio carchariae (bacterial vaccine) or polyriboinosinic polyribocytidylic acid (poly I:C). These results indicate RcCC1 not only plays a role in homeostasis, but also may be involved in inflammatory responses to bacterial and viral infection.

Quantitative PCR detection for abalone shriveling syndrome-associated virus.

Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-μL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years.

Real-time isothermal detection of Abalone herpes-like virus and red-spotted grouper nervous necrosis virus using recombinase polymerase amplification

Abstract Abalone herpes-like virus (AbHV) and Red-spotted grouper nervous necrosis virus (RGNNV) are two serious viruses that infect animal populations in aquaculture. Both viruses cause diseases associated with high mortality rates, resulting in dramatic economic losses in the aquaculture industry. There are currently no effective treatments for either of these two viral diseases. Thus, early, rapid, and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Traditional methods of diagnosis, such as virus culture, enzyme-linked immunoassay, and polymerase chain reaction (PCR), are either time consuming or require sophisticated temperature control devices. In this study, one sets of specific primers and probes were designed for the real-time quantitative recombinase polymerase amplification (qRPA) detection of AbHV and RGNNV separately. The sensitivity and specificity of detection were evaluated by comparison with detection by conventional PCR and quantitative PCR. The optimal reaction temperature and time for virus detection is 37°C for 20min. The detection limit is 100 copies per reaction, making this approach faster and more sensitive than qPCR in this study. In a field application, the detection percentage of qRPA was higher than that of qPCR for both AbHV and NNV. Additionally, good correlation was found between qRPA and qPCR detection (R2 >0.8). The methods presented here can be used as alternatives to qPCR for quick and quantitative detection of pathogens infecting aquaculture species.

Nested PCR detection of abalone shriveling syndrome-associated virus in China.

Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong).

Real-time quantitative isothermal detection of Ostreid herpesvirus-1 DNA in Scapharca subcrenata using recombinase polymerase amplification

Ostreid herpesvirus-1 (OsHV-1) is a well-known pathogen associated with high mortality rates in hatchery-reared larvae and juveniles of different bivalve species worldwide. Early, rapid and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Recombinase polymerase amplification (RPA) is a novel isothermal amplification method, which can amplify detectable amount of DNA at 37 °C-39 °C within 20 min. In the present study, two sets of specific primers and probes were designed for the real-time quantitative RPA (qRPA) detection of OsHV-1 DNA. The sensitivity and specificity of detection were evaluated by comparison with quantitative polymerase chain reaction (qPCR). The detection limit for qRPA assays was shown to be 5 copies DNA/reaction for the primer set ORF95, which was lower than the 100 copies required for the qPCR test. The optimal reaction temperature and time were 37 °C for 20 min, making this approach faster than qPCR. This is the first study to apply qPCR and qRPA methods to detect OsHV-1 in Scapharca subcrenata. The percentage of viral load sample detected by the two methods was 22% and the correlation of the two virus quantitative results was 0.8. Therefore, qRPA assays is sensitive, fast, and high-temperature independent relative to qPCR and is suitable for critical clinical diagnostics use and rapid field analysis in resource-limited settings.

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling.

Heterotrophic Bacterial Abundance and Diversity in the Farming Environment and Guts of the Oyster Crassostrea hongkongensis

ABSTRACT Currently, the knowledge of ecology and function of bacteria harbored by molluscs is very limited. In this study, quantity and richness of culturable heterotrophic bacteria (CHB) in seawater of the farming environment and guts of the oyster Crassostrea hongkongensis were determined monthly, with classical, biochemical, and molecular methods. About 800 randomly selected bacterial strains were purified and identified.Quantities of CHB and vibrios, ranging from2.3×103 to 2.2×105 CFU/ml and 1.6×102 to 3.0×103 CFU/ml, respectively, were present in the farming seawater, whereas those in the oyster C. hongkongensis ranged from 5.3×104 to 5.4×106 CFU/g and 6.0×103 to 1.4×105 CFU/g, respectively. Vibrio spp., Pseudoalteromonas spp., Bacillus spp., and Shewanella spp. appeared almost every month. Richness of heterotrophic microbial communities of the seawater and the oysters was very similar and peaked in August. Variation in the diversity (Shannon index) of CHB in the seawater and in the oysters was similar, a significant positive correlation was showed between them (r = 0.894), and their diversities were both peaked in August. Statistical analysis showed that salinity hasmore important impact on quantity of CHB in seawater. Similarly, temperature hadmore important impact on that in the oysters. Important threshold data were analyzed to further explain the relationships.

Mudworm Polydora lingshuiensis sp. n is a new species that inhabits both shell burrows and mudtubes.

A new polydorin species, Polydora lingshuiensis sp. n., which is found not only in burrows of pearl oyster shells (shell-boring type) but also in mudtubes on the surface of pearl oyster cages (tube-dwelling type), is described with the use of light microscopy, scanning electron microscopy, and molecular phylogeny. Morphological and molecular distinctions between P. lingshuiensis and other related species reveal that P. lingshuiensis is a valid new species. The reproduction characteristic that the eggs of P. lingshuiensis are gathered together in one hollow cylinder is another piece of evidence confirming that it is indeed a valid new species. Sequence comparisons based on nuclear 18S rDNA, 28S rDNA, and mitochondrial 16S rDNA show that strains of the shell-boring type possess as high as 99.9% to 100% sequence identity relative to those of the tube-dwelling type. This finding evidently indicates that these species types are conspecific. We also find that a comparison of mitochondrial 16S rDNA sequences can provide a higher resolution of polydorin species than those of the nuclear 18S rDNA because the former has a higher interspecific/intraspecific difference ratio. Phylogenetic analyses based on 18S rDNA sequences indicate that all P. lingshuiensis samples group together to forming a sister clade to Polydora uncinata and thus fall within Polydora aura/P. uncinata clade.