Development of simple, rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification.

A quantitative rapid detection method based on loop-mediated isothermal amplification has been developed for red-spotted grouper nervous necrosis virus (RGNNV). The nested polymerase chain reaction (PCR) assay is the mainstream inspection of the brooder in the hatchery. In this study, a real-time loop-mediated isothermal amplification (LAMP) method has been applied for RGNNV detection, known as a high-speed gene amplification procedure. Of the three temperatures (60 °C, 63 °C and 65 °C) attempted, it has been found that 63 °C is giving higher amplification from 11th minute onwards. Sensitivity analysis performed in comparison with real-time polymerase chain reaction, reverse transcriptase PCR and nested RT-PCR using various concentrations of template revealed that real-time LAMP method is efficient in terms of cost and time consumption. Specificity analysis revealed that the method developed is specific to RGNNV, whereas it has sequence cross-match with tiger puffer NNV giving advantage in detecting both the viruses. This method could be much efficient in analysing RGNNV in combination with TPNNV.

The SOCS and STAT from JAK/STAT signaling pathway of kuruma shrimp Marsupenaeus japonicus: molecular cloning, characterization and expression analysis.

Signal transducer and activators of transcription (STAT) gene, suppressors of cytokine signaling (SOCS) has been isolated from kuruma shrimp, Marsupenaeus japonicus and characterized. The kuruma shrimp STAT (MjSTAT) cDNA was composed of 2901 bp consisting of 801 amino acid residues which includes a protein interaction domain, all alpha domain, DNA binding domain and SH2 domain. Homology analysis of MjSTAT showed 94.1% and 34.0% identities with Penaeus monodon STAT (PmSTAT) and Drosophila melanogaster STAT92E (DmSTAT), respectively. The kuruma shrimp SOCS (MjSOCS) cDNA was composed of 1675 bp consisting of 342 amino acid residues including a SH2 domain and C-terminal SOCS domain. Homology analysis of MjSOCS showed 52.6% and 21.3% identities with Chinese mitten crab (Eriocheir sinensis) SOCS2 and fruit fly (D. melanogaster) SOCS44A, respectively. The MjSTAT and MjSOCS genes are constitutively expressed in the muscle, stomach, brain and gill of kuruma shrimp. In lymphoid organ cells, an enhanced expression of both MjSTAT and MjSOCS genes are observed following stimulation with peptidoglycan and polycytidylic acid. These observations suggest that MjSTAT and MjSOCS might play a major role in the innate immune defense of kuruma shrimp. The discovery of JAK/STAT signaling pathway in shrimp will allow a complete and concrete understanding of shrimp cytokine signaling.

Deciphering the DNA repair protein, Rad23 from kuruma shrimp Marsupenaeus japonicus: full-length cDNA cloning and characterization.

AIMS: Lesions of DNA are removed by nucleotide excision repair (NER) process in the living systems. NER process-related host factors are believed to aid recovery steps during viral integration. Here, we report identification and characterization of a DNA repair molecule Rad23 from kuruma shrimp Marsupenaeus japonicus. METHODS AND RESULTS: The full-length cDNA of M. japonicus Rad23 gene (MjRad23) has 1149 bp coding for a putative protein of 382 amino acids with a 5' untranslated region (UTR) of 92 bp and 3' UTR region of 1116 bp. Quantitative expression analysis revealed MjRad23 is constitutively expressed in all the organs of healthy shrimp, whereas with high level in muscle tissue. Although MjRad23 expression is observed in every haemolymph samplings to post-white spot syndrome virus infection, high expression is recorded at 2 h post infection (h.p.i.). MjRad23 consists of putative functional domains including one ubiquitin domain (UBQ), two ubiquitin-associated domains (UBA) and one heat-shock chaperonin-binding motif (STI1). Multiple alignment of MjRad23 with Rad23 of other species showed highly significant identity ranging from 37 to 53%; however, high homology is observed with Rad23 of Bombyx mori (BmRad23). UBQ domain region alignment revealed maximum of 66% homology with Rad23 of Apis melifera (AmRad23). MjRad23 clustered with invertebrate sector along with insect species in evolution analysis. Three-dimensional structural analyses demonstrated the highest identity between MjRad23 and human Rad23A (hHR23A). CONCLUSIONS: The present work revealed the presence of MjRad23 gene, which is essential in DNA repair process. Further studies are required to clarify the involvement of MjRad23 in NER process. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on identification and characterization of DNA repair protein in crustaceans, which will lead to further investigation to explore the molecular mechanisms behind the NER process.

Molecular cloning and transcriptional analysis of a newly identified anti-lipopolysaccharide factor gene in kuruma shrimp, Marsupenaeus japonicus.

AIM: In the present study, we have cloned a new family of anti-lipopolysaccharide factor (ALF) from haemocytes of kuruma shrimp Marsupenaeus japonicus (MjALF2) using RACE method. METHODS AND RESULTS: Transcriptional analysis of MjALF2 gene in the organs of healthy shrimp revealed prominent expression in gills and muscle. In vitro LPS stimulation in the lymphoid organ cells resulted in significant increase in expression at 48, 8 and 12 h poststimulation, compared to the nonstimulated cells. In vivo injection of V. penaeicida does not show any high expression in time course assay. Phylogenetic analysis showed MjALF2 is placed in the group closer to P. monodon isoform 1 and 2 than to MjALF1. The full-length MjALF2 gene consists of 558 bp with a 363 -bp open reading frame, encoding 121 amino acids. The deduced peptide contains a putative signal peptide of 22 amino acids with molecular mass of about 13.8 kDa molecular mass. The deduced amino acid sequence of MjALF2 showed 83.3 and 56.7% identity with ALF sequences of P. monodon. CONCLUSIONS: The present work revealed the presence of MjALF2 gene, which is highly expressed in gills and muscle of healthy kuruma shrimp. Further studies are required to clarify the involvement of MjALF2 in immune responses for using as a therapeutic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial peptides are promising potential therapeutic agents for disease control in aquaculture. Understanding the relation of MjALF2 with innate immunity mechanism will lead to develop better health management strategies for long-term sustainability of the shrimp industry.

Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus.

AIMS: White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) for quantitative detection of WSSV. MATERIALS AND METHODS: A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63 degrees C. Detection and quantification can be achieved by real-time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time (T(t)) using plasmid standards with high correlation coefficient (R(2) = 0.988). CONCLUSIONS: Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng microl(-1) to 35 ag microl(-1)) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross-reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV-infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV. SIGNIFICANCE AND IMPACT OF THE STUDY: WSSV real-time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.

A simple non-enzymatic method for the preparation of white spot syndrome virus (WSSV) DNA from the haemolymph of Marsupenaeus japonicus using FTA matrix cards.

White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates.

Histopathological Features of Mycobacterium chelonae Infection in Two Farmed Japanese Pufferfish (Takifugu rubripes).

Granulomatous lesions were observed in the swim bladder, kidney, spleen and gills of two farmed Japanese pufferfish (Takifugu rubripes) infected with Mycobacterium chelonae. Three types of lesions were noted: unencapsulated clusters of epithelioid cells without central necrosis (type 1), encapsulated granulomas without central necrosis (type 2) and encapsulated granulomas with central necrosis (type 3). Type 3 lesions occurred most frequently in the swim bladder, while type 1 and type 2 lesions occurred frequently in the kidney and spleen, and the gills exhibited mostly type 1 lesions. This suggests that the lesions in the swim bladder were more fully developed than those occurring elsewhere and that the swim bladder may be more susceptible to infection with M. chelonae. This is the first report describing the histopathological features of M. chelonae infection in Tetraodontidae.

Variable-speech-rate audiometry for hearing aid evaluation.

A new hearing aid evaluation method using variable-speech-rate audiometry (VSRA) was developed. VSRA was newly created based on the Japanese speech audiometry authorized by the Japan Audiological Society. The ordinary speech audiometry can not reveal a temporal factor in word discrimination ability of the hearing impaired. Since, with VSRA, we can compare several performance-intensity curves obtained from different speech-rate speech audiometries, the impact on the auditory system of each patient by the fast or slow speech rate could be easily determined. Taking the temporal factor of the auditory systems into consideration by using VSRA, hearing aid evaluation was performed for a master hearing aid with three types of signal processing and fitting for 36 hearing impaired subjects. Then hearing aid evaluation was performed using VSRA for a newly developed portable multi-function digital hearing aid with two types of signal processing and analog hearing aids which had been used by hearing-impaired patients. As a result, VSRA was useful for hearing aid evaluation, in particular, for cases when ordinary normal speech rate audiometry does not provide a significant difference in word discrimination scores. In addition, using VSRA revealed that amplitude compression is more effective for improvement of word discrimination than linear amplification.