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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Evaluation of Bacillus spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (Litopenaeus vannamei) Farms.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75-95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80-95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly.

Insights Into a Chitin Synthase of Kuruma Shrimp Penaeus japonicus and Its Role in Peritrophic Membrane and Cuticle Formation.

Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans.

Design of a Chimeric Multi-Epitope Vaccine (CMEV) against Both Leishmania martiniquensis and Leishmania orientalis Parasites Using Immunoinformatic Approaches.

Leishmaniasis is a parasitic disease caused by protozoan flagellates of the genus Leishmania. Recently, Leishmania martiniquensis and Leishmania orientalis, emerging species of Leishmania, were isolated from patients in Thailand. Development of the vaccine is demanded; however, genetic differences between the two species make it difficult to design a vaccine that is effective for both species. In this study, we applied immuno-informatic approaches to design a chimeric multi-epitope vaccine (CMEV) against both L. martiniquensis and L. orientalis. We identified seven helper T lymphocyte (HTL) epitopes, sixteen cytotoxic T lymphocyte (CTL) epitopes, and eleven B-cell epitopes from sixteen conserved antigenic proteins found in both species. All these epitopes were joined together, and to further enhance immunogenicity, protein and peptides adjuvant were also added at the N-terminal of the molecule by using specific linkers. The candidate CMEV was subsequently analyzed from the perspectives of the antigenicity, allergenicity, and physiochemical properties. The interaction of the designed multi-epitope vaccine and immune receptor (TLR4) of the host were evaluated based on molecular dockings of the predicted 3D structures. Finally, in silico cloning was performed to construct the expression vaccine vector. Docking analysis showed that the vaccine/TLR4 complex took a stable form. Based on the predicted immunogenicity, physicochemical, and structural properties in silico, the vaccine candidate was expected to be appropriately expressed in bacterial expression systems and show the potential to induce a host immune response. This study proposes the experimental validation of the efficacy of the candidate vaccine construct against the two Leishmania.

Analysis of microbiota in the stomach and midgut of two penaeid shrimps during probiotic feeding.

In mammals, the intestine harbors numerous bacteria that play an important role in health. Intestinal microbiota have also been thought to be an important factor in the health of shrimp. However, the barrier systems of the digestive tracts of shrimp seem to be different from those of mammals. In this study, we analyzed the bacterial composition in the stomach and midgut of two species of shrimp during administration of a probiotic, Bacillus amyloliquefaciens strain TOA5001 by analysis of 16S rRNA genes with Illumina sequencing technology. Whiteleg shrimp Litopenaeus vannamei were observed under laboratory conditions and kuruma shrimp Marsupenaeus japonicus were observed in an aquaculture farm. The diversities of bacteria in the stomachs of both shrimps were significantly higher than those in the midgut. Also, the microbiota changed during probiotic feeding. Feeding whiteleg shrimp the probiotic after being challenged with an acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus increased their survival compared to the control group, which suggested that the probiotic prevented AHPND. These results appear to show that a probiotic can affect the microbiota throughout digestive tract of penaeid shrimps and that probiotic can have a role in preventing disease.

[MAKING OF THE DISEASE SEVERITY PREDICTION INDEX FOR ACUTE PYELONEPHRITIS ASSOCIATED WITH URINARY OBSTRUCTION DUE TO URETERAL CALCULI].

(Purpose) Both administration of antibiotics and drainage of urine are necessary for the treatment of acute pyelonephritis associated with urinary obstruction by the ureteral calculi. Though most patients get better after the treatment, some patients deteriorate accompanying with low blood pressure, and need the intensive care. Such patients sometimes visit small hospital, even at night with a few medical staffs. It is sometimes difficult to predict the patient's outcome. The disease severity prediction index for the patients was investigated. (Object and method) We examined 134 patients, who visited our hospital from 2001 to 2013, retrospectively. Ureteral stenting or nephrostomy was undergone within 24 hours in principle. If the blood pressure became under 90 mmHg, or lowered more than 40 mmHg than usual, the case was defined as serious. Blood data and physical findings were compared between serious and non-serious cases. The factors affecting the seriousness were found. Multiple logistic analysis was done to make a disease severity prediction index. (Result) 42 cases were judged as serious and 92 cases as non-serious. Six factors consisting of heart rate, serum creatinine, platelets, ages, PS and CRP affected the consequence significantly (p<0.05), however, white blood cells did not.Multiple logistic analysis was done, four factors consisting of serum creatinine, platelets, PS and CRP affected the consequence significantly (p<0.05), and the standardizing coefficients of each points were found to be 2, 2, 1, 1, respectively.The disease severity prediction index was proposed. If the index was 4 or more, the sensitivity and specificity were found to be 73.8% and 82.6%, respectively. (Conclusion) This index is useful and reliable for the prediction of the outcome of the disease.

[Clinical study on renal trauma].

We analyzed 26 cases of renal trauma, which occurred during the last 7 years and 6 months. Computed tomography was performed in all cases. Four cases were of type Ib, 13 cases of type II, 3 cases of type IIIa, 5 cases of type IIIb and 1 case of type IVa, according to the classification of renal injury by the Japanese association for the surgery of trauma. Conservative treatment was done in 21 cases, selective transcatheter arterial embolization (TAE) in 4 cases, and surgical treatment in 1 case. Conservative treatment was effective for type I and II renal trauma. In the cases of type IIIa and IIIb renal trauma, open surgery could be avoided and the affected kidney preserved by early TAE.