Evaluation of Formalin-Inactivated Vaccine Efficacy against Red Seabream Iridovirus (RSIV) in Laboratory and Field Conditions.

Red seabream iridovirus (RSIV) is a major cause of marine fish mortality in Korea, with no effective vaccine available since its first occurrence in the 1990s. This study evaluated the efficacy of a formalin-killed vaccine against RSIV in rock bream under laboratory and field conditions. For the field trial, a total of 103,200 rock bream from two commercial marine cage-cultured farms in Southern Korea were vaccinated. Farm A vaccinated 31,100 fish in July 2020 and monitored them for 18 weeks, while farm B vaccinated 30,700 fish in August 2020 and monitored them for 12 weeks. At farm A, where there was no RSIV infection, the vaccine efficacy was assessed in the lab, showing a relative percentage of survival (RPS) ranging from 40% to 80%. At farm B, where natural RSIV infections occurred, cumulative mortality rates were 36.43% in the vaccinated group and 80.32% in the control group, resulting in an RPS of 54.67%. The RSIV-infectious status and neutralizing antibody titers in serum mirrored the cumulative mortality results. This study demonstrates that the formalin-killed vaccine effectively prevents RSIV in cage-cultured rock bream under both laboratory and field conditions.

Molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis isolated from skin abscesses of native Korean goats (Capra hircus coreanae).

AIMS: This study aimed to investigate the molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis from skin abscesses of Korean native black goats (KNBG, Capra hircus coreanae) in South Korea. METHODS AND RESULTS: A total of 83 isolates were recovered from skin abscesses of KNBG. Of these isolates, 74 isolates were identified as C. pseudotuberculosis by phospholipase D (PLD) gene-based PCR assay. Each of the isolates possessed all 18 virulence genes (FagA, FagB, FagC, FagD, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE). The genetic diversity of C. pseudotuberculosis isolates was assessed by the phylogenetic analysis using the concatenated sequences (3073 bp) of five housekeeping genes (fusA, dnaK, infB, groeL1 and leuA) for investigating their genetic diversity. In the results, the isolates belonged to three groups: group 1 (67 isolates), group 2 (one isolate) and group 3 (six isolates) within biovar ovis. However, the groups exhibited low genetic diversity (0.20%-0.41%). In the antimicrobial susceptibility test, most isolates were susceptible to tetracycline, vancomycin, chloramphenicol, ciprofloxacin, erythromycin, enrofloxacin, cefoxitin, ampicillin, gentamycin, cephalothin and doxycycline, whereas they were not susceptible to cefotaxime, trimethoprim and streptomycin. CONCLUSION: This results suggest the involvement of relatively few clones of C. pseudotuberculosis in Korea. Further, present isolates can threaten public health due to potentially virulent strains with all 18 virulence genes and non-susceptible strains to clinically important antibiotics (CIA) and highly important antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to investigate the genetic diversity and potential pathogenicity of C. pseudotuberculosis biovar ovis isolates from skin abscesses of KBNG in South Korea, and could provide useful information in controlling its infections.

An indirect competitive assay-based aptasensor for detection of oxytetracycline in milk.

Oxytetracycline (OTC) is a common antibacterial agent used for the control of animal diseases. OTC abuse can seriously affect human health; therefore, we developed a biosensor using single-stranded DNA (ssDNA) aptamers for the detection of OTC. The binding probe aptamers for OTC were selected by a Systematic Evolution of Ligands by the exponential enrichment (SELEX) process and identified by the enzyme-linked aptamer assay (ELAA). Among the selected 5 aptamers, aptamer OTC3 showed the strongest affinity (Kd=4.7 nM) and highest specificity for OTC compared to structurally similar antibiotics, tetracycline and chlortetracycline. OTC was detected using indirect competitive ELAA. The limit of detection and quantitation with aptamer OTC3 were 12.3 and 49.8 µg/L, respectively, in milk and showed recovery rates of more than 90% in OTC-spiked milk. This biosensor method with high sensitivity and specificity based on indirect competitive ELAA can be applied to OTC detection in food products on-site because of the simplicity of detection.

Characterization of Kunitz-type protease inhibitor purified from hemolymph of Galleria mellonella larvae.

We characterized a Kunitz-type protease inhibitor (Gm KTPI) obtained from the hemolymph of Galleria mellonella larvae immunized with Escherichia coli. The structural analysis of the cloned cDNA showed that it consists of 56 residues derived from the precursor of 75 amino acids. The peptide was constitutively produced in the fat bodies, but not in the midgut nor the integument of larvae. In our analysis of stage-dependent expression, its transcript was detected within the midgut, the fat bodies and the integument of the prepupae, which undergo tissue remodeling. The inhibition assays showed that Gm KTPI was capable of inhibiting only the trypsin-like activity of the larval midgut extracts. Furthermore, it was determined that Gm KTPI induced the activation of extracellular signal-regulated kinase (ERK) in the fat bodies and integument cells, and this kinase is known to perform a central role in cell proliferation signaling. Its effect on ERK activation was also verified in a control experiment using a human endothelial cell culture. Collectively, it was suggested that Gm KTPI might be responsible for the protection of other tissues against proteolytic attack by trypsin-like protease(s) from larval midgut during metamorphosis, and might play a role in the proliferation of cells in the fat body and integument.

An insect multiligand recognition protein functions as an opsonin for the phagocytosis of microorganisms.

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and beta-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b.

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.

cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4.

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthiaaurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5'-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Cionaintestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (-) bacteria, while evidencing no hemolytic activity toward human erythrocytes.

Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella.

Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III.

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella.

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.

Biological activities of synthetic analogs of halocidin, an antimicrobial peptide from the tunicate Halocynthia aurantium.

Halocidin is a heterodimer antimicrobial peptide previously isolated from the tunicate Halocynthia aurantium. Based on the larger monomer (18Hc) of halocidin, nine halocidin congeners, including a series of 6 peptides truncated successively from the carboxyl-terminal end of 18Hc and 3 analogs (18HcKK, K19Hc, and K19HcKK), which have lysine residues in place of two internal histidines or have a lysine added to the amino terminus of the 18Hc molecule, were prepared. Each peptide was also converted into a homodimeric version. The antimicrobial activities of halocidin congeners truncated from the C terminus were dramatically decreased, suggesting that the full length of 18Hc is required for maintaining its maximum antimicrobial activity. Dimer forms of halocidin congeners exhibited stronger antimicrobial activities than the monomer of the corresponding peptide. Four dimer peptides (di-18Hc, di-18HcKK, di-K19Hc, and di-K19HcKK) were analyzed for antimicrobial activities against 10 clinically isolated antibiotic-resistant bacteria in elevated concentrations of NaCl or MgCl(2). Of the peptides studied here, di-K19Hc retained invariably strong activity against all bacteria in diverse conditions and also showed much reduced hemolytic activity against human erythrocytes.

A mark-release-recapture experiment with Anopheles sinensis in the northern part of Gyeonggi-do, Korea.

In order to study the range of flight and feeding activity of Anopheles sinensis, the dispersal experiment was conducted in Paju city, located in the northern part of Gyeonggi-do, Republic of Korea, during the period of 7th to 28th September 1998. Unfed females An. sinensis were collected in cowshed and released after being marked with fluorescent dye at 23:00 hours on the same day. Released female mosquitoes were recaptured everyday during 21 days using light traps, which were set at 10 sites in the cowsheds located 1, 3, 6, 9 and 12 km north-northwest and north-northeast and at 3 sites located 1, 6 and 9 km toward south-west from the release point. In addition, to study the longest flight distance in one night, we set the light traps at 16 and 20 km toward north-northeast from the release site. All the collected mosquitoes were placed on filter papers and observed on UV transilluminator after treatment with one drop of 100% ethanol. Out of 12,773 females of An. sinensis released, 194 marked females mosquitoes were recaptured, giving 1.52% recapture rate. Of 194, 72 mosquitoes (37.1%) were recaptured in light traps from three places set at 1 km from the release point, 57 mosquitoes (29.4%) from two places at 1-3 km, 41 mosquitoes (21.1%) from three places at 3-6 km, 20 mosquitoes (10.3%) from three places at 6-9 km, and 4 mosquitoes (2.1%) from two places at 9-12 km. Since 170 female mosquitoes (87.6%) out of 194 marked mosquitoes were captured within 6 km from the release point, this flight radius represents the main activity area. An sinensis was found to be able to fly at least 12 km during one night.