Efficacy of recombinant enolase as a candidate vaccine against Haemaphysalis longicornis tick infestation in mice.

Tick infestation causes a significant threat to human and animal health, requiring effective immunological control methods. This study aimed to investigate the potential of recombinant Haemaphysalis longicornis enolase protein for tick vaccine development. The exact mechanism of the recently identified enolase protein from the H. longicornis Jeju strain remains poorly understood. Enolase plays a crucial role in glycolysis, the metabolic process that converts glucose into energy, and is essential for the motility, adhesion, invasion, growth, and differentiation of ticks. In this study, mice were immunized with recombinant enolase, and polyclonal antibodies were generated. Western blot analysis confirmed the specific recognition of enolase by the antiserum. The effects of immunization on tick feeding and attachment were assessed. Adult ticks attached to the recombinant enolase-immunized mice demonstrated longer attachment time, increased blood-sucking abilities, and lower engorgement weight than the controls. The nymphs and larvae had a reduced attachment rate and low engorgement rate compared to the controls. Mice immunized with recombinant enolase expressed in Escherichia coli displayed 90% efficacy in preventing tick infestation. The glycolytic nature of enolase and its involvement in crucial physiological processes makes it an attractive target for disrupting tick survival and disease transmission. Polyclonal antibodies recognize enolase and significantly reduce attachment rates, tick feeding, and engorgement. Our findings indicate that recombinant enolase may be a valuable vaccine candidate for H. longicornis infection in experimental murine model.

Effects of histamine and antihistamine on the hard tick Haemaphysalis longicornis during blood sucking.

At the time of host attachment, ticks are very sensitive to histamine, but during rapid blood sucking they paradoxically require histamine. Using a rabbit model, we studied the effects of histamine and antihistamine during attachment and fast-feeding in different life stages of Haemaphysalis longicorns. We examined how they responded to histamine and antihistamine by analyzing the detachment rate, histology of feeding lesions, and post-feeding behavior. A significant difference (P<0.01) was found in the detachment rate between experimental and control treatments throughout the observation period. Ticks exhibited a higher detachment rate (30.1%) at 12 h after histamine application during attachment time and on antihistamine-treated skin (25.4%) at 96 h during fast-feeding. After feeding on histamine-treated rabbits, the fully engorged body weights of larvae and nymphs were 0.7±0.36 mg and 3.5±0.65 mg, respectively. An average increase in body weight of 0.6±0.05 mg and 3.2±0.30 mg was observed for larvae and nymphs compared to the respective control weights. Nymphs and adults engorged after antihistamine treatment had an average body weight of 1.3±0.54 mg and 54±0.81 mg, respectively. An average decrease in body weight was observed in antihistamine-treated H. longicornis compared with control nymphs (3.3±0.42 mg) and adults (174±1.78 mg). Skin biopsies were collected after treatment, and differential histopathological characteristics were found between the treatment and control groups. Tick-infested skin collected from rabbits in the antihistamine-treated group lacked erythrocytes in the feeding pool, indicating that antihistamine impaired tick fast-feeding stage.

Protective Effects of Phosphatidylcholine against Hepatic and Renal Cell Injury from Advanced Glycation End Products.

Background and Objectives: Receptors of the advanced glycation products (RAGE) are activated to promote cell death and contributes to chronic diseases such as diabetes and inflammation. Advanced glycation end products (AGEs), which interact with RAGE are complex compounds synthesized during diabetes development and are presumed to play a significant role in pathogenesis of diabetes. Phosphatidylcholine (PC), a polyunsaturated fatty acid found in egg yolk, mustard, and soybean, is thought to exert anti-inflammatory activity. We investigated the effects of PC on AGEs-induced hepatic and renal cell injury. Materials and Methods: In this study, we evaluated cytokine and NF-κB/MAPK signal pathway activity in AGEs induced human liver (HepG2) cells and human kidney (HK2) cells with and without PC treatment. Results: PC reduced RAGE expression and attenuated levels of inflammatory cytokines and NF-kB/MAPK signaling. Moreover, cells treated with PC exhibited a significant reduction in cytotoxicity, oxidative stress, and inflammatory factor levels. Conclusions: These findings suggest that PC could be an effective functional material for hepatic and renal injury involving with oxidative stress caused by AGEs during diabetic conditions.

Pathogenicity of severe fever with thrombocytopenia syndrome virus in mice regulated in type I interferon signaling: Severe fever with thrombocytopenia and type I interferon.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease, which causes high fever, thrombocytopenia, and death in humans and animals in East Asian countries. The pathogenicity of SFTS virus (SFTSV) remains unclear. We intraperitoneally infected three groups of mice: wild-type (WT), mice treated with blocking anti-type I interferon (IFN)-α receptor antibody (IFNAR Ab), and IFNAR knockout (IFNAR-/-) mice, with four doses of SFTSV (KH1, 5 × 105 to 5 × 102 FAID50). The WT mice survived all SFTSV infective doses. The IFNAR Ab mice died within 7 days post-infection (dpi) with all doses of SFTSV except that the mice were infected with 5 × 102 FAID50 SFTSV. The IFNAR-/- mice died after infection with all doses of SFTSV within four dpi. No SFTSV infection caused hyperthermia in any mice, whereas all the dead mice showed hypothermia and weight loss. In the WT mice, SFTSV RNA was detected in the eyes, oral swabs, urine, and feces at 5 dpi. Similar patterns were observed in the IFNAR Ab and IFNAR-/- mice after 3 dpi, but not in feces. The IFNAR Ab mice showed viral shedding until 7 dpi. The SFTSV RNA loads were higher in organs of the IFNAR-/- mice compared to the other groups. Histopathologically, coagulation necrosis and mononuclear inflammatory cell infiltration in the liver and white pulp atrophy in the spleen were seen as the main lesions in the IFN signaling lacking mice. Immunohistochemically, SFTSV antigens were mainly detected in the marginal zone of the white pulp of the spleen in all groups of mice, but more viral antigens were observed in the spleen of the IFNAR-/- mice. Collectively, the IFN signaling-deficient mice were highly susceptible to SFTSV and more viral burden could be demonstrated in various excreta and organs of the mice when IFN signaling was inhibited.

AK-1, a Sirt2 inhibitor, alleviates carbon tetrachloride-induced hepatotoxicity in vivo and in vitro.

Background/Aim: Acute liver injury (ALI) is a life-threatening clinical syndrome that is usually caused by toxic chemicals, drugs, or pathogen infections. Sirtuin2 (Sirt2), an NAD+-dependent deacetylase, appears to play detrimental roles in liver injury. Here, we evaluated the therapeutic application targeting Sirt2 in carbon tetrachloride (CCl4)-induced ALI, by using AK-1 (a Sirt2 inhibitor).Methods: For in vivo experiments, a single injection of CCl4 was used to induce ALI. One hour later, mice were intraperitoneally injected with AK-1 and were sacrificed 24 h after CCl4 administration. For in vitro experiments, primary mouse hepatocytes were used to determine the effects of AK-1 on oxidative stress and hepatocellular death induced by CCl4.Results: AK-1 alleviated CCl4-induced ALI as confirmed by histopathologic analysis, and decreased levels of serum biochemicals and inflammatory cytokines. Although it barely affected the expression of hepatic cytochrome P450 enzymes, AK-1 attenuated CCl4-induced oxidative stress and its related cell death. Mechanistically, Sirt2 inhibition significantly increased the nuclear protein level of nuclear factor erythroid 2-related factor 2 (Nrf2), and meanwhile decreased phosphorylation of c-Jun N-terminal kinases (JNK), in normal and injured livers. Similar results were observed in vitro. AK-1 significantly attenuated CCl4-induced cytotoxicity and oxidative stress by up-regulating the activity of Nrf2, and down-regulating JNK signaling in hepatocytes.Conclusions: Our results suggest that AK-1 treatment attenuated oxidative stress and cell death in the ALI model, at least partially, via activating Nrf2 and inhibiting JNK signaling, and that Sirt2 inhibition might be a potential approach to cure ALI.

Molecular cloning, expression and impact of ribosomal protein S-27 silencing in Haemaphysalis longicornis (Acari: Ixodidae).

Ticks, obligatory blood-feeding arthropods, are a major pathogen vector in humans and animals worldwide. Anti-tick vaccines are an exciting alternative to chemical acaricides for controlling these disease-transmitting vectors. However, identification of protective antigens for anti-tick vaccine development is challenging. Different ribosomal proteins play multifunctional roles in tick survival and feeding. Here, we first report the cloning and molecular characterization of ribosomal protein S27 (RPS-27) from the hard tick Haemaphysalis longicornis. We identified a complete open reading frame (ORF) of RPS-27: a 255-bp (base pair) cDNA encoding a mature protein of 84 amino-acid residues with a 9.4-kDa predicted molecular mass. Amino-acid sequence analysis revealed that RPS-27 was highly conserved among different tick and vertebrate animals with identity ranges of 97-98% and 60-85%, respectively. Phylogenetic tree analysis showed that RPS-27 from different tick species clustered together. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the RPS-27 mRNA transcript was expressed in all life stages. At the tissue level, it was more highly expressed in the salivary gland than in the midgut for both the fed and unfed conditions, which indicates a role for RPS-27 in tick feeding. In vitro analysis showed that recombinant RPS-27 (10-RPS-27) was successfully expressed in a pGEMEX-2 vector with an estimated 45-kDa molecular mass. The functional importance of RPS-27 was determined by gene silencing through RNA interference (RNAi). RPS-27 silencing showed a significant (P < 0.05) reduction of feeding abilityand engorgement weight after the blood meal in both nymph and adult female ticks and also significantly (P < 0.05) reduced molting rate in nymph. In addition, RPS-27 silencing in eggs led to abnormalities in shape and hatching. Taken together, our results suggest that RPS-27 is an important molecule that plays multiple roles in the tick life cycle including in both feeding and reproduction. Therefore, RPS-27 is an exciting target for future tick control strategies.

Molecular cloning and transcriptional and functional analysis of glycogen synthase kinase-3ß in Haemaphysalis longicornis (Acari, Ixodidae).

Glycogen synthase kinase 3 (GSK-3), which belongs to the serine/threonine kinase family, regulates glycogen metabolism, Wnt signaling, hormonal regulation, and embryonic development in many eukaryotes. Here, we cloned a complete open reading frame (ORF) of glycogen synthase kinase 3ß (GSK-3ß) from Haemaphysalis longicornis and characterized its transcriptional and functional status. The ORF of GSK-3ß possesses 1242 nucleotides encoding a mature protein of 413 amino acid residues. GSK-3ß nucleotide and protein sequences are highly conserved among different vertebrate and invertebrate animals, with identity between 47.8-100% and 63.2-88.7%, respectively. Sequence comparison showed one signature domain between the residues of 51 and 335 amino acids, which was identified as a protein kinase (serine/threonine). RT-PCR showed GSK-3ß mRNA present in all developmental stages of H. longicornis. Interestingly, a higher transcript level was observed in nymph and 7-day-old eggs compared with others by real-time PCR, indicating a role of GSK-3ß in the early stages of life. The functional status of GSK-3ß was characterized by RNA interference (RNAi) and caused significant (p < 0.05) reduction in feeding and reproduction, as well as an abnormality in eggs and hatching. Taken together, our results suggest that GSK-3ß may be an important candidate for a multiple antigen vaccine for controlling the tick population.

Impact of Subolesin and Cystatin Knockdown by RNA Interference in Adult Female Haemaphysalis longicornis (Acari: Ixodidae) on Blood Engorgement and Reproduction.

Currently, multi-antigenic vaccine use is the method of choice for the strategic control of ticks. Therefore, determining the efficacy of combined antigens is a promising avenue of research in the development of anti-tick vaccines. The antigen responsible for blood intake and reproduction has proven suitable as a vaccine antigen. It has been shown to silence Haemaphysalis longicornis salivary cystatin (HlSC-1) and subolesin by RNA interference. Adult unfed female ticks were injected with double-stranded RNA of (A) subolesin, (B) cystatin, (C) subolesin plus cystatin, and (D) injection buffer, then fed alongside normal unfed males up to spontaneous drop-down. The percentage of knockdowns was determined by real-time polymerase chain reaction. Sixty-three percent and 53% knockdown rates were observed in subolesin and cystatin double-stranded RNA-injected ticks respectively, while 32 and 26% knockdown rates of subolesin and cystatin transcript were observed in subolesin plus cystatin double-stranded RNA-injected ticks. Subolesin and/or cystatin knockdown causes a significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Most importantly, combined silencing did not act synergistically, but caused a similarly significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Therefore, the elucidation of multiple antigens may be helpful in the future of vaccines.

Expression Patterns of Host Inflammatory Cytokine Genes during Infestation with Haemaphysalis longicornis, a Zoonotic Vector, in Blood Sucking Periods.

Tick saliva is critically important for continuous attachment to the host, blood feeding for days, and transmission of tick-borne pathogens. To characterize the patterns of inflammatory cytokine gene expression during its attachment and blood sucking time, peripheral blood samples of rabbits infested with Haemaphysalis longicornis ticks were collected at different intervals. Blood histamine concentration was evaluated as well as gene encoding IFN-γ, TNF-α, IL-2, IL-6, IL-4, and IL-10 were compared with non-infested rabbits. Blood histamine concentration of tick-infested rabbits during fast feeding time was significantly higher than that of non-infested rabbits. In both nymph and adult tick infested rabbits, expression of TNF-α and IFN-γ genes were decreased significantly (P<0.05), while expression of IL-4, IL-6, and IL-10 were increased 1.3 to 7 folds in adult infested rabbits with the exception of IL-6 that was significantly (P<0.05) decreased in nymph infested rabbits. IL-2 was not expressed in either nymph or adult infestation. H. longicornis saliva is capable of modulate host responses through a complex correlation with histamine and Th1, Th2 mediated cytokines that suppress the inflammatory responses directed toward inflammatory mediators introduced into the host during tick feeding.

Convallaria keiskei as a novel therapeutic alternative for salivary gland cancer treatment by targeting myeloid cell leukemia-1.

BACKGROUND: Various chemotherapeutic agents have been used largely for the treatment of salivary gland cancer. However, results are disappointing, and these agents can cause some serious side effects. Therefore, recent studies have focused on the possible roles of natural products to overcome these limitations. METHODS: Salivary gland cancer cells treated with or without Convallaria keiskei (MECK) for 24 hours. Apoptotic changes were evaluated by live/dead assay, immunoblotting, and expression levels of caspase-3 and B-cell lymphoma-2 family member. RESULTS: MECK significantly inhibited salivary gland cancer growth. At the molecular level, MECK dramatically reduced myeloid cell leukemia-1 (Mcl-1) in a translation-dependent manner and thereby induced apoptosis through Bax/Bid. Furthermore, we found that Mcl-1 could be a potential therapeutic target of MECK-induced apoptosis and its stability is regulated by extracellular signal-regulated kinases 1/2 (ERK1/2) signaling CONCLUSION: MECK can be used as a safe and efficient therapeutic alternative for the treatment of salivary gland cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: E761-E770, 2016.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

Proteomic screening of antigenic proteins from the hard tick, Haemaphysalis longicornis (Acari: Ixodidae).

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.

Clostridium tertium bacteremia in a patient with glyphosate ingestion.

BACKGROUND: Clostridium tertium is distributed in the soil and in animal and human gastrointestinal tracts. C. tertium has been isolated from patients with blood diseases, immune disorders, and abdominal surgeries. Glyphosate is toxic, causing cause eye and skin irritation, gastrointestinal pain, and vomiting. Ingestion of herbicides modifies the gastrointestinal environment, which stresses the living organisms. However, there has been little attention to cases of bacteremia in patients recovering from suicide attempt by ingesting herbicide. CASE REPORT: Clostridium tertium was identified in a 44-year-old female who attempted suicide by glyphosate (a herbicide) ingestion. The 16S rRNA sequences from all colonies were 99% identical with that of C. tertium (AB618789) found on a BLAST search of the NCBI database. The bacterium was cultured on TSA under aerobic and anaerobic conditions. Antimicrobial susceptibility tests performed under both aerobic and anaerobic conditions showed that the bacterium was susceptible to penicillin, a combination of ß-lactamase inhibitor and piperacillin or amoxicillin, and first- and second- generation cephalosporins. However, it was resistant to third- and fourth-generation cephalosporins. CONCLUSIONS: Glyphosate herbicide might be a predisposing factor responsible for the pathogenesis of C. tertium. The results highlight the need for careful diagnosis and selection of antibiotics in the treatment of this organism.

Suppression of Eimeria tenella sporulation by disinfectants.

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.

Effects of different sizes of glass beads on the release of sporocysts from Eimeria tenella oocysts.

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.

Detection of four important Eimeria species by multiplex PCR in a single assay.

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.

Inhibition of specificity protein 1 by dibenzylideneacetone, a curcumin analogue, induces apoptosis in mucoepidermoid carcinomas and tumor xenografts through Bim and truncated Bid.

OBJECTIVES: Dibenzylideneacetone (DBA), a curcumin analogue that has anti-cancer activity in a variety of tumor cells. In this study, we investigated the apoptotic effects of DBA and its molecular mechanism in human mucoepidermoid carcinoma (MEC) cell lines and tumor xenografts. MATERIAL AND METHODS: The apoptotic effects and related molecular mechanisms of DBA on MEC cell lines were evaluated using cell viability assay, DAPI staining, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and Dual-luciferase Reporter Assay. The anti-tumor activity using in vivo were determined by Nude mouse xenograft assay and histopathological examination. RESULTS: DBA decreased cell viability and induced apoptosis in MEC cells. These events were accompanied by inhibition of specificity protein 1 (Sp1). DBA did not induce major changes in Sp1 mRNA and promoter activity. Furthermore, inhibition of protein synthesis by cycloheximide demonstrated that DBA decreased Sp1 protein stability, but DBA did not attenuate phosphorylation of eIF4E. DBA also increased Bim and truncated Bid (t-Bid) via Sp1. Finally, DBA exhibited significant anti-tumor activity in athymic nude mice xenografts bearing MC-3 cells by regulating Sp1, Bim and t-Bid without any systemic toxicity. CONCLUSION: These results elucidate a crucial apoptotic mechanism of DBA and suggest that DBA may be a potent anticancer drug candidate for MEC.

Severe sepsis due to Aeromonas aquariorum in a patient with liver cirrhosis.

Thus far, Aeromonas aquariorum infection has been unrecorded in Korea. Herein, we report a fatal case of A. aquariorum infection in a 77-year-old male patient with liver cirrhosis. The bacterium isolated from a blood culture was initially mistaken as Aeromonas hydrophila using the Vitek2 identification system. In spite of intravenous ceftriaxone therapy, the patient was exacerbated by multiple organ dysfunction. By 4 days after admission, there was no hope for treatment or remission of symptoms and the patient was discharged. In the detailed microbiological investigations, the bacterium was identified as A. aquariorum harboring the act and alt genes, which encode cytotoxic and cytotonic enterotoxins.

Molecular characterization of Aeromonas species isolated from farmed eels (Anguilla japonica).

Seventy Aeromonas strains were identified by phylogenetic analysis using housekeeping genes (gyrB and rpoD) in order to investigate etiological agents for aeromoniasis in farmed eels (Anguilla japonica). The phylogenetic analysis showed that Aeromonas aquariorum (n=22, 31.4%) was the predominant species among the investigated eel strains, followed by Aeromonas caviae (n=16, 22.9%), A. veronii (n=13, 18.6%), A. hydrophila (n=12, 17.1%), A. jandaei (n=4, 5.7%), A. media (n=2, 2.9%), and A. trota (n=1, 1.4%). The potential virulence of the present strains was estimated by performing PCR assays using the following seven virulence genes: cytotoxic enterotoxin (act), two cytotonic enterotoxins (alt and ast), glycerophospholipid:cholesterol acyltransferase (gcaT), DNase (exu), lipase (lip), and flagellin (fla). The detection rates of act, alt, ast, gcaT, exu, lip, and fla among all 70 strains were 91.4%, 55.7%, 27.1%, 97.1%, 95.7%, 100%, and 98.6%, respectively. In genotyping of enterotoxin genes, act(+)/alt(+)/ast(+), act(+)/alt(+)/ast(-), and act(+)/alt(-)/ast(-) genotypes were prevalent in A. hydrophila (8/12 strains), A. aquariorum (13/22 strains), and A. caviae (14/16 strains), respectively, suggesting a high heterogeneity among Aeromonas species. In this study, A. aquariorum, which has been an unrecorded species in Korea, can be an etiological agent for aeromoniasis of eel.