Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii.

In this study, we established a murine cell line that expresses ginbuna crucian carp (ginbuna) CD4-2 and used it to develop an anti-CD4-2 monoclonal antibody (mAb). An established mAb, named D5, showed good reactivities to BALB/c 3T3 cells expressing CD4-2 and a lymphocyte population in the ginbuna leukocytes. Gene expression analysis showed that D5+ cells express CD4-2 and TCRß genes but not CD4-1 and IgM genes, meanwhile May Grunwald-Giemsa staining of sorted D5+ cells had the typical morphology of lymphocytes. Two-color immunofluorescence analysis with anti-CD4-1 mAb (6D1) and anti-CD4-2 mAb (D5) by flow cytometry revealed that the percentages of CD4-1 single positive (SP) and CD4-2 SP lymphocytes were comparatively higher than CD4-1/CD4-2 double positive (CD4 DP) lymphocytes in all tissues examined in ginbuna. The highest percentage of CD4-2 SP cells (∼40%) was found in the thymus, while the head-kidney exhibited the highest percentages of CD4-1 SP (∼30%) and CD4 DP (∼5%) cells. These findings indicated that ginbuna CD4+ lymphocyte population consists of two major subpopulations (CD4-1 SP and CD4-2 SP) and a minor subset (CD4 DP).

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/µg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

Intra vitam assays for detecting fish infected with cyprinid herpesvirus 3 (CyHV-3).

Koi carp is one of the most sensitive variants of common carp Cyprinus carpio to cyprinid herpesvirus 3, commonly known as koi herpesvirus (KHV). Given that this species is traded at high prices throughout the world, intra vitam assays for detecting KHV in targeted fish with a high detection efficiency are essential. In this study, 4 intra vitam assays were compared with regard to their efficiency of detecting KHV in koi carp on each day after viral exposure via experimental infection. The results indicated that PCR from the gills and scales sampled by biopsy using dissecting scissors and forceps, respectively, can detect KHV for apparently longer periods than the other assays. This study also suggests that a PCR detection assay for environmental samples could be developed as a convenient intra vitam assay to confirm the presence of virus in environments inhabited by virus-shedding fish.

Reactivation of cyprinid herpesvirus 2 (CyHV-2) in asymptomatic surviving goldfish Carassius auratus (L.) under immunosuppression.

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen of goldfish (Carassius auratus) and Prussian carp (Carassius auratus gibelio) causing herpesviral hematopoietic necrosis. Our previous study revealed that CyHV-2 can persistently infect the kidney and spleen of goldfish that recovered from a primary infection. In this study, we tried to identify the cells persistently infected with the virus in surviving fish and investigated virus reactivation in the survivors injected with immunosuppressants, namely dexamethasone (Dex) and cyclosporine A (CsA). Virus DNA was detected from the monocytes that were isolated from the trunk kidney of the asymptomatic survivors, suggesting that monocytes/macrophages are major cells that may be persistently infected with CyHV-2. A significant increase of virus DNA levels was detected in the group injected with Dex at 10 and 21 days post-injection (dpi). In the fish group injected with CsA, the virus DNA level was the same as that in the control group at 10 dpi but increased in some organs at 21 dpi. Compared with Dex-injected fish at 10 dpi, the group injected with both Dex and CsA showed a greater increase in virus DNA levels. The gene expression of phagocytosis-associated genes, major histocompatibility complex (MHC) class II and p47phox, and anti-virus antibody levels increased in the CsA group due to virus reactivation in the infected cells but not in the Dex and Dex & CsA groups, indicating that Dex effectively suppressed monocyte/macrophage function and antibody production. In addition, recombinant interferon γ (IFNγ) supplementation in the kidney leukocyte culture that was isolated from survivors showed a reduction of virus DNA. CsA may inhibit T-helper 1 (Th1) cells and consequently IFNγ production, causing a synergetic effect with Dex on virus reactivation. The results suggest that the activity of monocytes/macrophages stimulated by IFNγ can relate to virus latency and reactivation in asymptomatic virus carriers.

Immune responses induced by oil-adjuvanted inactivated vaccine against Flavobacterium psychrophilum in ayu Plecoglossus altivelis.

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery.

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20-33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV-2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non-permissive temperature for 4 days, initiated at three different time points post-infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post-infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR-positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV-2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.

Maturation-associated changes in the non-specific immune response against Flavobacterium psychrophilum in Ayu Plecoglossus altivelis.

In this study, we investigated maturation-associated changes in non-specific immune responses of ayu against Flavobacterium psychrophilum. The gonadosomatic index was minimum on 16 June, began to increase on 17 July, and reached the maximum value during August. The highest phagocytic rate (16.3%) was observed on 16 June, which decreased significantly to 5.6% on 26 August. The number of viable bacteria after the serum treatment was highest during August, suggesting that bactericidal activity of the serum decreased along with the sexual maturation. Gene expression levels of interleukin-8, and tumor necrosis factor-α in the spleen did not change significantly during this period, whereas the level of suppressor of cytokine signaling (SOCS)3 was significantly higher on 26 August than that on 16 July (p < 0.05). These results suggest that phagocytic activity of trunk kidney leukocytes and serum bactericidal activity against F. psychrophilum decreased with sexual maturation, and that SOCS3 may be related to the decrease in non-specific immune activity in ayu.

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi.

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7. Received June 16, 2016; accepted October 7, 2016.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

Genome characterization of a novel vibriophage VpKK5 (Siphoviridae) specific to fish pathogenic strain of Vibrio parahaemolyticus.

Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future.

Complete genome sequence analysis of two Pseudomonas plecoglossicida phages, potential therapeutic agents.

Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416-1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33-39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.

In vitro characteristics of cyprinid herpesvirus 2: effect of kidney extract supplementation on growth.

Herpesviral hematopoietic necrosis caused by goldfish hematopoietic necrosis virus (now identified as cyprinid herpesvirus 2, CyHV-2) has contributed to economic losses in goldfish Carassius auratus culture and is becoming a major obstacle in Prussian carp C. gibelio aquaculture in China. Several reports have described difficulties in culturing the virus, with the total loss of infectivity within several passages in cell culture. We succeeded in propagating CyHV-2 with a high infectious titer in a RyuF-2 cell line newly derived from the fin of the Ryukin goldfish variety using culture medium supplemented with 0.2% healthy goldfish kidney extract. The addition of kidney extract to the medium enabled rapid virus growth, resulting in the completion of cytopathic effect (CPE) within 4 to 6 d at 25°C. The extract also enabled reproducible virus culture with a titer of 105-6 TCID50 ml-1. The virus cultured using this protocol showed pathogenicity in goldfish after intraperitoneal injection. The virus grew in RyuF-2 cells at 15, 20, 25, 30, and 32°C but not at 34°C or higher. Higher incubation temperatures allowed earlier development of CPE, but culture at 30 and 32°C yielded a lower virus titer than that obtained at other temperatures because of heat inactivation of the propagated virus during cultivation. Cell lines derived from goldfish and ginbuna C. langsdorfii showed high susceptibility to the virus; cell lines from carp were susceptible to the virus using a medium containing goldfish kidney extract, but EPC, FHM, and BF-2 cell lines did not produce any CPE, even in the presence of the extract.

[The efficacy and safety of FOLFIRI or combined FOLFIRI and bevacizumab treatment as second-line chemotherapy for metastatic colorectal cancer patients aged 75 years and older].

The safety and efficacy of FOLFIRI as second-line chemotherapy for metastatic colorectal cancer patients ≥ 75 years was retrospectively evaluated. We analyzed 106 patients, who received FOLFIRI or a combination of FOLFIRI and bevacizumab following oxaliplatin-based first-line chemotherapy. The clinical characteristics and outcome in elderly patients ≥75 years(elderly[EP]group; n=18)were compared with those in patients aged<75 years(control group; n=88). The number of patients treated by a combination of FOLFIRI and bevacizumab in the EP group was lower than that in the control group (27.8% vs 55.7%; p=0.03). The comparison revealed no significant differences in response rate, progression-free survival, overall survival, and the frequency of overall adverse events after the start of second-line chemotherapy, although the frequency of anemia(Bgrade 3, p=0.07)and alopecia(grade 1/2, p=0.054)tended to be higher in the EP group than in the control group. Although this study retrospectively analyzed a limited number of patients, our results indicate that the safety and efficacy of FOLFIRI as second-line chemotherapy for metastatic colorectal cancer are almost equal in patients ≥ 75 years and those aged<75 years.

Evaluation of the Simple Suspension Method for Oral Anticancer Drugs.

The simple suspension method (SSM) involves administering tablets or capsules using a tube after disintegration and suspension in hot water without crushing or opening the capsule. Particularly, for anticancer drugs, it is an excellent method of administration that reduces the risk of exposure during dispensing. In contrast, information on SSM for individual drugs is insufficient. Anticancer drugs present a therapeutic challenge because their information is limited. We investigated whether SSM is possible with 36 anticancer drugs. Furthermore, we examined the pH of the suspension of these drugs, for which no information on SSM is available. We found that suspension was possible for 24 of the 36 drugs. Furthermore, the pH of the suspension was measured, which provided important information when considering dissolution solutions other than hot water. Little changes in the pH were observed before or after passing through the tube. The results of this study may improve medication adherence in patients with cancer experiencing dysphagia.

Potential risk of driving performance under combined conditions of taking second-generation antihistamines and attending calls using a hands-free function.

OBJECTIVE: Although second-generation antihistamines have reduced sedation-related side effects compared to first-generation antihistamines, sedation may still impair motor vehicle driving performance. Moreover, receiving/making phone calls using a hands-free function can negatively affect driving performance. Therefore, herein, driving performance was evaluated using a driving simulator to gain insights into the hazards of driving by combining second-generation antihistamines and a calling task, i.e., simulated calls using a hands-free function. METHODS: In this study, 20 subjects drove in a driving simulator in the absence or presence of a calling task while taking or not taking second-generation antihistamines. Driving performances for nonemergency and emergency events were determined, and a comparative analysis of intra-individual variability when taking and not taking second-generation antihistamines was conducted. RESULTS: First, when nonemergency and emergency were examined in the absence of a calling task, no significant difference in driving performance was observed between taking and not taking second-generation antihistamines. Next, when the nonemergency event was examined in the presence of a calling task, no significant difference in driving performance was observed between taking and not taking second-generation antihistamines. However, when the emergency event was examined in the presence of a calling task, a significant difference in driving performance was observed between taking and not taking second-generation antihistamines, thus resulting in reduced driving performance. CONCLUSIONS: The new system with added calling tasks allowed the extraction of the potential risks of driving performance of second-generation antihistamines that may have been previously overlooked. This study suggests that pharmacists and other healthcare professionals may need to instruct people taking any second-generation antihistamine to focus on driving and not on subtasks that require cognitive load such as talking while driving.

Comparative genomics reveals that a fish pathogenic bacterium Edwardsiella tarda has acquired the locus of enterocyte effacement (LEE) through horizontal gene transfer.

BACKGROUND: Edwardsiella tarda is an enterobacterium which causes edwardsiellosis, a fatal disease of cultured fishes such as red sea bream, eel, and flounder. Preventing the occurrence of E. tarda infection has thus been an important issue in aquaculture. E. tarda has been isolated from other animals and from many environments; however, the relationship between the genotype and evolutionary process of this pathogen is not fully understood. To clarify this relationship, we sequenced and compared the genomes of pathogenic and non-pathogenic E. tarda strains isolated from fish, human, and eel pond using next-generation sequencing technology. RESULTS: Eight strains of E. tarda were sequenced with high accuracy (>99.9%) with coverages from 50- to 400-fold. The obtained reads were mapped to a public reference genome. By comparing single nucleotide and insertion/deletion polymorphisms, we found that an attenuated strain of E. tarda had a loss-of-function mutation in a gene related to the type III secretion system (T3SS), suggesting that this gene is involved in the virulence of E. tarda. A comprehensive gene comparison indicated that fish pathogenic strains possessed a type VI secretion system (T6SS) and pilus assembly genes in addition to the T3SS. Moreover, we found that an E. tarda strain isolated from red sea bream harbored two pathogenicity islands of T3SS and T6SS, which were absent in other strains. In particular, this T3SS was homologous to the locus of enterocyte effacement (LEE) in enteropathogenic and enterohemorrhagic Escherichia coli. Evolutionary analysis suggested that this locus, here named Et-LEE (E. tarda LEE), was introgressed into the E. tarda genome through horizontal transfer. CONCLUSIONS: We found significant differences in the presence/absence of virulence-related genes among E. tarda strains, reflecting their evolutionary relationship. In particular, a single genotype previously proposed for fish-pathogenic strains may be further divided into two subgroups. Furthermore, the current study demonstrated, for the first time, that a fish pathogenic bacterium carried a LEE-like pathogenicity island which was previously reported only in zoonotic pathogenic enterobacteria. These findings will contribute to the exploration of strain-specific drug targets against E. tarda in aquafarms, while also shedding light on the evolution of pathogenesis in enterobacteria.

Growth of cyprinid herpesvirus 2 (CyHV-2) in cell culture and experimental infection of goldfish Carassius auratus.

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.