Risk assessment of wild fish as environmental sources of red sea bream iridovirus (RSIV) outbreaks in aquaculture.

Red sea bream iridovirus (RSIV) causes substantial economic damage to aquaculture. In the present study, RSIV in wild fish near aquaculture installations was surveyed to evaluate the risk of wild fish being an infection source for RSIV outbreaks in cultured fish. In total, 1102 wild fish, consisting of 44 species, were captured from 2 aquaculture areas in western Japan using fishing, gill nets, and fishing baskets between 2019 and 2022. Eleven fish from 7 species were confirmed to harbor the RSIV genome using a probe-based real-time PCR assay. The mean viral load of the RSIV-positive wild fish was 101.1 ± 0.4 copies mg-1 DNA, which was significantly lower than that of seemingly healthy red sea bream Pagrus major in a net pen during an RSIV outbreak (103.3 ± 1.5 copies mg-1 DNA) that occurred in 2021. Sequencing analysis of a partial region of the major capsid protein gene demonstrated that the RSIV genome detected in the wild fish was identical to that of the diseased fish in a fish farm located in the same area in which the wild fish were captured. Based on the diagnostic records of RSIV in the sampled area, the RSIV-infected wild fish appeared during or after the RSIV outbreak in cultured fish, suggesting that RSIV detected in wild fish was derived from the RSIV outbreak in cultured fish. Therefore, wild fish populations near aquaculture installations may not be a significant risk factor for RSIV outbreaks in cultured fish.

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients.

OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.

Genome-wide association study of clinically defined gout identifies multiple risk loci and its association with clinical subtypes.

OBJECTIVE: Gout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only. METHODS: A GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls. RESULTS: Five gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10(-8)), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10(-12); OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10(-23); OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10(-9); OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case-control ORs for two distinct types of gout (r=0.96 [p=4.8×10(-4)] for urate clearance and r=0.96 [p=5.0×10(-4)] for urinary urate excretion). CONCLUSIONS: Our findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Common variants of a urate-associated gene LRP2 are not associated with gout susceptibility.

A recent genome-wide association study revealed that there is an association between serum uric acid (SUA) levels and rs2544390, a common variant in low-density lipoprotein-related protein 2 (LRP2/Megalin) gene. Two other variants of LRP2, rs2229268 and rs3755166, are also found to have associations with dyslipidemia and Alzheimer's disease, respectively, which also could have a relationship with SUA in human. Although no studies report that LRP2 transports urate, LRP2 is a multi-ligand receptor and expresses in many tissues including kidney, suggesting a direct and/or indirect relationship with gout. In the present study, we investigated the association between gout and these variants of LRP2 with 741 clinically diagnosed male gout patients and 1,302 controls. As a result, the three common LRP2 variants, rs2544390, rs2229268 and rs3755166, showed no association with gout (P = 0.76, 0.55, and 0.22, respectively). Our study is the first to reveal that an SUA-related gene LRP2 is not involved in gout susceptibility.

Assessing the transmission risk of red sea bream iridovirus (RSIV) in environmental water: insights from fish farms and experimental settings.

Aquatic animal viruses are considered to be transmitted via environmental water between fish farms. This study aimed to understand the actual transmission risk of red sea bream iridovirus (RSIV) through environmental water among fish farms. An environmental DNA (eDNA) method using iron-based flocculation coupled with large-pore filtration was used to monitor RSIV DNA copies in seawater from fish farms and from an experimental infection model. RSIV dispersion in seawater from a net pen where the disease outbreak occurred was visualized by the inverse distance weighting method using multiple-sampling data sets from a fish farm. The analysis demonstrated that the center of the net pen had a high viral load, and RSIV seemed to be quickly diluted by the tidal current. To evaluate the transmission risk of RSIV in environmental water, the red sea bream Pagrus major (approximately 10 g) was exposed to RSIV-contained seawater (103, 104, 105, 106, and 107 copies/L) for 3 days, which mimicked field exposure. A probit analysis of the challenge test indicated that the inferred infection rates of seawater containing 105.9 copies/L and 103.1 copies/L of RSIV were 50% and 0.0001%, respectively. In the surveillance for 3 years at 10 fixed points (n = 306), there were only seven samples in which the viral load exceeded 104 copies/L in seawater. These results suggest that the transmission of RSIV among fish farms via seawater is highly associated with the distance between the net pens, and the environmental water is not always an infection source for the transmission of RSIV between fish farms. IMPORTANCE Our surveillance of viral loads for red sea bream iridovirus (RSIV) by monitoring environmental DNA in fish farms suggested that the viral loads in the seawater were low, except for the net pens where RSIV outbreaks occurred. Furthermore, our experimental infection model indicated that the infection risk of RSIV-contained seawater with less than 103 copies/L was extremely low. The limited risk of environmental water for transmission of RSIV gives an insight that RSIV could be partly transmitted between fish farms due to the movement of equipment and/or humans from the fish farm where the disease outbreaks. Since our data suggest that seawater can function as a potential wall to reduce the transmission of RSIV, biosecurity management, such as disinfection of equipment associated with fish farms could be effective, even in the semi-open system aquaculture that the environmental water can be freely transferred, to reduce the risk of RSIV outbreaks.

Significant association of serum uric acid levels with SLC2A9 rs11722228 among a Japanese population.

Genome-wide association studies identified that SLC2A9 (GLUT9) gene polymorphisms were associated with serum uric acid (SUA) levels. Among the Japanese, a C/T polymorphism in intron 8 (rs11722228) was reported to be highly significant, though the function and strength of association were unknown. This study aimed to confirm the association, estimating the means of SUA according to the genotype, as well as OR of the genotype. Subjects were 5024 health checkup examinees (3413 males and 1611 females) aged 35 to 69 years with creatinine <2.0 mg/dL. Since SLC22A12 258X allele and ABCG2 126X allele are known to influence SUA levels strongly, the subjects with SLC22A12 258WW and ABCG2 126QQ (3082 males and 1453 females, in total 4535 subjects) were selected. The genotype frequency of SLC2A9 rs11722228 was 2184 for CC, 1947 for CT, and 404 for TT, being in Hardy-Weinberg equilibrium (p=0.312). Mean SUA was 6.10 mg/dL for CC, 6.25 mg/dL for CT, and 6.45 mg/dL for TT among males (p=1.5E-6), and 4.34 mg/dL, 4.59 mg/dL, and 4.87 mg/dL among females (p=4.6E-11), respectively. Males with SUA less than 5.0 mg/dL were 14.7% for CC, 10.6% for CT, and 7.8% for TT (p=2.3E-4), and females with SUA less than 4.0 mg/dL were 34.1%, 25.5%, and 15.4% (p=3.7E-6), respectively. This study was the first report to estimate the impact of SLC2A9 rs11722228 on SUA levels. Since the allele frequency of rs11722228 is similar among different ethnic groups, the impact remains to be examined in other ethnic groups.

Double-balloon endoscopy for treatment of long-term abdominal discomfort due to small bowel penetration by an eel bone.

BACKGROUND: Penetration of the GI tract by a fish bone was treated by laparotomy. DBE is a useful procedure for removal of a foreign body from the small bowel. CASE REPORT: A 33-year-old male presented with bothersome postprandial fullness. He was diagnosed previously with functional dyspepsia, and had been treated with medication for 8 months with no success. During antegrade DBE, which was performed to rule out of small intestinal abnormalities, an eel bone found stuck in the jejunum was grasped with forceps and pulled out. Following removal of the bone, the patient's postprandial fullness ameliorated dramatically. CONCLUSIONS: This is the first report of DBE-based diagnosis and treatment of a small bowel penetration by a fish bone.

Genetic polymorphism in collagen type 1 alpha 2 intron 33 can be used for the initial screening of East Asians quickly and easily by a heteroduplex analysis.

To save time and simplify the matching genetic information of bereaved family with the collected samples for identification of individuals in a mass-scale disaster and terrorism using molecular biological methods, we propose a method to initially screen samples according to different populations. We found a unique deletion (26822-3) in an allele that 62.0% of East Asian individuals had in intron 33 of type I collagen alpha 2 (COL1A2), while most Europeans and Africans did not have it. By making a heteroduplex complex, a visual discrimination was possible using 10% PAGE. A PCR and heteroduplex analysis could identify genetic polymorphism of COL1A2 intron 33 both clearly and accurately, even from decomposed samples or very small amounts of samples within a very short time period (approximately within 150min). As a result, genetic polymorphism of COL1A2 intron 33 is thus considered to be useful to quickly and easily screen East Asians from vast number of samples using a heteroduplex analysis. Consequently, we consider that an initial screening method for East Asians which use a heteroduplex analysis of genetic polymorphism of COL1A2 intron 33 can smoothly and effectively perform various identifications among East Asians when supplies are logistically poor after a mass-scale disaster or terrorism.

[Medico-legal autopsy case of an infant suffering anaphylactic shock during dental treatment. Potential hazards in the use of a rubber-dam-sheet for infants].

We report a medico-legal autopsy of a 4-year-old girl without any history of allergies, who died following anaphylactic shock due to local anesthesia with lidocaine during dental treatment. The shock symptoms were overlooked due to a rubber-dam-sheet covering her face. The autopsy revealed laryngeal edema, secretory fluid filling the trachea, severe pulmonary congestion and edema. An immunohistochemical investigation showed numerous mast cells releasing histamine in lungs, and the fluids in the pulmonary alveoli and veins also showed positive staining. In addition, the plasma concentration of histamine in the heart blood showed a high value. From these findings, the cause of death was determined to be respiratory failure due to anaphylactic shock induced by lidocaine. In this case, her death was considered to be due to medical malpractice; adequate life support had not been performed, because anaphylactic shock was overlooked until the rubber-dam-sheet removed. In addition, due to two previous autopsy case reports that described infantile fatalities involved with the misusage of a rubber-dam-sheet for the patients under local anesthesia, we therefore recommend immediate improvements in rubber-dam-sheets and/or better applications of them in pediatric dentistry.

Common variant of PDZ domain containing 1 (PDZK1) gene is associated with gout susceptibility: A replication study and meta-analysis in Japanese population.

PDZ domain containing 1 (PDZK1) is a scaffold protein that organizes a transportsome and regulates several transporters' functions including urate and drug transporters. Therefore, PDZK1 in renal proximal tubules may affect serum uric acid levels through PDZK1-binding renal urate transporters. Two previous studies in Japanese male population reported that a PDZK1 single nucleotide polymorphism (SNP), rs12129861, was not associated with gout. In the present study, we performed a further association analysis between gout and rs12129861 in a different large-scale Japanese male population and a meta-analysis with previous Japanese population studies. We genotyped rs12129861 in 1210 gout cases and 1224 controls of a Japanese male population by TaqMan assay. As a result, we showed that rs12129861 was significantly associated with gout susceptibility (P = 0.016, odds ratio [OR] = 0.80, 95% confidence interval [CI] 0.67-0.96). The result of the meta-analysis among Japanese populations also showed a significant association (P = 0.013, OR = 0.85, 95%CI 0.75-0.97). Our findings show the significant association between gout susceptibility and common variant of PDZK1 which reportedly regulates the functions of urate transporters in the urate transportsome.