Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.

CD4+ T lymphocyte responses to viruses and virus-relevant stimuli in teleost fish.

Fish diseases caused by viruses are a major threat to aquaculture. Development of disease protection strategies for sustainable fish aquaculture requires a better understanding of the immune mechanisms involved in antiviral defence. The innate and adaptive arms of the vertebrate immune system collaborate to mount an effective defence against viral pathogens. The T lymphocyte components of the adaptive immune system, comprising two major classes (helper T, Th or CD4+ and cytotoxic T lymphocytes, CTLs or CD8+ T cells), are responsible for cell-mediated immune responses. In particular, CD4+ T cells and their different subsets orchestrate the actions of various other immune cells during immune responses, making CD4+ T cells central drivers of responses to pathogens and vaccines. CD4+ T cells are also present in teleost fish. Here we review the literature that reported the use of antibodies against CD4 in a few teleost fish species and transcription profiling of Th cell-relevant genes in the context of viral infections and virus-relevant immunomodulation. Studies reveal massive CD4+ T cell proliferation and expression of key cytokines, transcription factors, and effector molecules that evoke mammalian Th cell responses. We also discuss gaps in the current understanding and evaluation of teleost CD4+ T cell responses and how development and application of novel tools and approaches to interrogate such responses could bridge these gaps. A greater understanding of fish Th cell responses will further illuminate the evolution of vertebrate adaptive immunity, inform strategies to address viral infections in aquaculture, and could further foster fish as model organisms.

Feeding Behavior Comparison of Bean Bugs, Riptortus pedestris and Halyomorpha halys on Different Soybean Cultivars.

Riptortus pedestris (Fabricius) and Halyomorpha halys (Stål) are the major pests that feed on soybean pods, seeds, and fruits. Higher populations and damage occur during the soybean maturity stages (podding to harvest). To compare the feeding behavior of R. pedestris and H. halys, we used the six most cultivated cultivars (Daepung-2ho, Daechan, Pungsannamul, Daewon, Seonpung, and Seoritae) in Korea using the electropenetrography (EPG) technique. Both R. pedestris and H. halys, the NP (non-penetration), a non-probing waveform, was the shortest in the Pungsannamul (298 and 268 min) and the longest in the Daepung-2ho (334 and 339 min), respectively. The feeding waveforms Pb (phloem feeding: E1-Salivation and E2-Sap feeding) and G (xylem feeding) were the longest in Pungsannamul and the shortest in Daepung-2ho. In addition, as a result of investigating the damage rate by planting six cultivars of beans in the field, as expected, the proportions of damage types B and C were highest in Pungsannamul and lowest in Daepung-2ho. These results reveal that both bug species ingest xylem sap from leaflets and stems using a salivary sheath strategy to acquire water and nutrients from soybean pods/seeds with cell-rupture tactics. This study provides perceptive information to understand the feeding behavior, field occurrence, and damage patterns of R. pedestris and H. halys, which may have key implications for the management of hemipteran pests by determining the specificity and susceptibility of host plants.

Serotype-identifying ions in Listeria monocytogenes using matrix-associated laser desorption ionization-time of flight mass spectrometry.

Listeria monocytogenes is a foodborne pathogen that can cause a potentially life-threatening infection, and almost all cases of human listeriosis are caused by L. monocytogenes isolates in serotypes 1/2a, 1/2b, 1/2c, and 4b. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In the current study, we examined the potential of MALDI-TOF MS for rapid identification of the foodborne pathogen L. monocytogenes and to identify high-risk serotypes. To achieve this, MALDI-TOF MS was applied to 50 L monocytogenes strains. All strains were identified as L. monocytogenes species based on pattern matching against reference spectra for the species. Importantly, 83 specific mass ions were consistently and uniquely found in high-risk L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b. These 83 mass ions were also unique to specific combinations of these serotypes, which enabled specific identification of these four serotypes using MALDI Biotyper analysis. Hence, this method shows potential for using MALDI-TOF MS for the rapid identification of L. monocytogenes species and to discriminate high-risk L. monocytogenes serotypes through specific serotype-specific biomarker ions.

Antimicrobial Resistance, Pathogenic, and Molecular Characterization of Escherichia coli from Diarrheal Patients in South Korea.

Diarrheal diseases due to foodborne Escherichia coli are the leading cause of illness in humans. Here, we performed pathogenic typing, molecular typing, and antimicrobial susceptibility tests on seventy-five isolates of E. coli isolated from stool samples of patients suffering from foodborne diseases in Busan, South Korea. All the isolates were identified as E. coli by both biochemical analysis (API 20E system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The bacteria displayed entero-pathogenic E. coli (EPEC) (47.0%), entero-aggregative E. coli (EAEC) (33.3%), entero-toxigenic E. coli (ETEC) (6.6%), ETEC and EPEC (6.6%), EPEC and EAEC (4%), and ETEC and EAEC (2.7%) characteristics. The E. coli isolates were highly resistant to nalidixic acid (44.0%), tetracycline (41.3%), ampicillin (40%), ticarcillin (38.7%), and trimethoprim/sulfamethoxazole (34.7%); however, they were highly susceptible to imipenem (98.6%), cefotetan (98.6%), cefepime (94.6%), and chloramphenicol (94.6%). Although 52 strains (69.3%) showed resistance against at least 1 of the 16 antibiotics tested, 23 strains (30.7%) were susceptible to all the antibiotics. Nine different serotypes (O166, O8, O20, O25, O119, O159, O28ac, O127a, and O18), five genotypes (I to V, random-amplified polymorphic DNA), and four phenotypes (A to D, MALDI-TOF MS) were identified, showing the high level of heterogeneity between the E. coli isolates recovered from diarrheal patients in South Korea.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay To Detect Hemolysin Gene of Vibrio vulnificus in Oysters.

ABSTRACT: Vibrio vulnificus inhabits estuarine waters around the world and can cause severe infections in people who eat contaminated raw or undercooked oysters. Although current detection methods are sensitive and specific, there are continuous demands for the development of rapid and accurate methods without a trained operator and equipment in the field conditions. Herein, we developed a simple and rapid method by detecting the hemolysin (vvh) gene of V. vulnificus by using recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD). The RPA-LFD could detect 100 fg of DNA (P < 0.05) and 20 CFU of V. vulnificus per reaction within 30 min (P < 0.01) and showed the result with incubation temperature ranges from 30 to 45°C (P < 0.001). The test was specific only to V. vulnificus and was not responsive to 10 other closely related Vibrio species and 18 foodborne pathogenic bacteria. Compared with PCR, quantitative PCR, and colony hybridization assays by using naturally contaminated oyster samples, our RPA-LFD showed the same detection ability as quantitative PCR assay. Therefore, the current RPA-LFD would be a valuable tool to detect V. vulnificus in oysters, especially in field conditions.

Molecular Typing, Antibiotic Resistance and Enterotoxin Gene Profiles of Staphylococcus aureus Isolated from Humans in South Korea.

The emergence of antimicrobial-resistant Staphylococcus aureus has become a grave concern worldwide. In this study, 95 strains of S. aureus isolated from stool samples were collected from Busan, South Korea to characterize their antimicrobial susceptibility, enterotoxin genes, and molecular typing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and random amplification of polymorphic DNA (RAPD) assay. Only two strains showed no drug resistance, whereas resistance to three or more antibiotics was observed in 87.4% of strains. Ampicillin resistance was the most common at 90% and all strains were susceptible to vancomycin. The distribution of enterotoxin genes encoded in isolates was sea (32.6%), sec (11.6%), seg (19%), sea & sec (2.1%), and sec & seg (34.7%). Molecular typing using both MALDI-TOF MS and RAPD indicated that S. aureus exhibited diverse clonal lineages and no correlations were observed among the profiling of enterotoxin, MALDI-TOF MS, and RAPD. This investigation provides useful information on foodborne pathogenic S. aureus that has a significant public health impact in South Korea.

Membrane vesicles from antibiotic-resistant Staphylococcus aureus transfer antibiotic-resistance to antibiotic-susceptible Escherichia coli.

AIM: Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi-drug resistant bacteria (MDR) by delivering antibiotic-resistant substances to antibiotic-susceptible bacteria. Here, we aim to show that MVs from Gram-positive bacteria are capable of transferring ß-lactam antibiotic-resistant substances to antibiotic-sensitive Gram-negative bacteria. MATERIALS AND METHODS: MVs were collected from a methicillin-resistant strain of Staphylococcus aureus (MRSA) and vesicle-mediated fusion with antimicrobial-sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial-resistant E. coli (RC85-T). RESULTS: The RC85-T exhibited a higher resistance to ß-lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85-T and the parent strain were nearly equal, the ß-lactamase activity of the MVs from RC85-T was 12-times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85-T were able to protect ß-lactam-susceptible E. coli from ß-lactam antibiotic-induced growth inhibition in a dose-dependent manner. CONCLUSION: MVs play a role in transferring substances from Gram-positive to Gram-negative bacteria, shown by the release of MVs from RC85-T that were able to protect ß-lactam-susceptible bacteria from ß-lactam antibiotics. SIGNIFICANCE AND IMPACT OF STUDY: MVs are involved in the emergence of antibiotic-resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug-resistant bacteria could be reduced.

Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress.

Extracellular vesicles (EVs) containing specific cargo molecules from the cell of origin are naturally secreted from bacteria. EVs play significant roles in protecting the bacterium, which can contribute to their survival in the presence of antibiotics. Herein, we isolated EVs from methicillin-resistant Staphylococcus aureus (MRSA) in an environment with or without stressor by adding ampicillin at a lower concentration than the minimum inhibitory concentration (MIC). We investigated whether EVs from MRSA under stress condition or normal condition could defend susceptible bacteria in the presence of several ß-lactam antibiotics, and directly degrade the antibiotics. A comparative proteomic approach was carried out in both types of EVs to investigate ß-lactam resistant determinants. The secretion of EVs from MRSA under antibiotic stressed conditions was increased by 22.4-fold compared with that of EVs without stress. Proteins related to the degradation of ß-lactam antibiotics were abundant in EVs released from the stressed condition. Taken together, the present data reveal that EVs from MRSA play a crucial role in the survival of ß-lactam susceptible bacteria by acting as the first line of defense against ß-lactam antibiotics, and antibiotic stress leads to release EVs with high defense activity.

The Importance of Porins and ß-Lactamase in Outer Membrane Vesicles on the Hydrolysis of ß-Lactam Antibiotics.

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of ß-lactam antibiotics. ß-lactamases are enzymes that are able to inactivate the antibacterial properties of ß-lactam antibiotics. Interestingly, porins and ß-lactamase are found in outer membrane vesicles (OMVs) of ß-lactam-resistant Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the ß-lactam antibiotic. In this study, OMVs isolated from ß-lactam-resistant E. coli and from mutants, lacking porin or ß-lactamase, were evaluated to establish if the porins or ß-lactamase in OMVs were involved in the degradation of ß-lactam antibiotics. OMVs isolated from E. coli deficient in ß-lactamase did not show any degradation ability against ß-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of ß-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by ß-lactamase.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Outer membrane vesicles from ß-lactam-resistant Escherichia coli enable the survival of ß-lactam-susceptible E. coli in the presence of ß-lactam antibiotics.

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from ß-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental ß-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried ß-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from ß-lactam-induced death and/or directly degrade ß-lactam antibiotics. Several proteins that can be involved in degrading ß-lactam antibiotics were more abundant in OMVs from ß-lactam-resistant E. coli, and thus OMVs from ß-lactam resistant E. coli could directly and dose-dependently degrade ß-lactam antibiotics and fully rescue ß-lactam-susceptible E. coli and other bacterial species from ß-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from ß-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against ß-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.

Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus).

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry based identification of Edwardsiella ictaluri isolated from Vietnamese striped catfish (Pangasius hypothalamus).

Edwardsiella (E.) ictaluri is a major bacterial pathogen that affects commercially farmed striped catfish (Pangasius hypothalamus) in Vietnam. In a previous study, 19 strains of E. ictaluri collected from striped catfish were biochemically identified with an API-20E system. Here, the same 19 strains were used to assess the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; applied using a MALDI Biotyper) to conduct rapid, easy and accurate identification of E. ictaluri. MALDI-TOF MS could directly detect the specific peptide patterns of cultured E. ictaluri colonies with high (＞ 2.0, indicating species-level identification) scores. MALDI Biotyper 3.0 software revealed that all of the strains examined in this study possessed highly similar peptide peak patterns. In addition, electrophoresis (SDS-PAGE) and subsequent immuno-blotting using a specific chicken antibody (IgY) against E. ictaluri revealed that the isolates had highly similar protein profiles and antigenic banding profiles. The results of this study suggest that E. ictaluri isolated from striped catfish in Vietnam have homologous protein compositions. This is important, because it indicates that MALDI-TOF MS analysis could potentially outperform the conventional methods of identifying E. ictaluri.

Development of an immunochromatography assay kit for rapid detection of ranavirus.

Ranaviruses are large, double-stranded DNA viruses of the family Iridoviridae and are known to be primary pathogens in frogs, fish and other amphibians. These viruses have been shown to be highly adaptable and have the ability to cross species barriers, making them a potent threat to global biodiversity. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of these viruses. To address this, monoclonal antibodies (MAbs) were developed against ranavirus strain FV-3 (standard frog virus 3) to detect the major capsid protein and FV-3gorf19R related hypothetical protein in both the FV-3 and KRV-1 (Korean ranavirus) strains. The antibodies were then applied on a colloidal gold-immunochromatographic assay (GICA) as a kit for the detection of ranaviruses. The kit was able to detect low concentrations of the virus (10(1)TCID50/ml) and showed analytical specificity when tested against other viral pathogens, including those belonging to the same family. It was possible to detect ranavirus in experimentally infected frogs within 30 min using the kit. The kit described here is expected to be a valuable and informative tool for on-site detection of ranavirus in frog.

Combination treatment against scuticociliatosis by reducing the inhibitor effect of mucus in olive flounder, Paralichthys olivaceus.

The olive flounder, Paralichthys olivaceus, is an economically important food fish in Japan and Korea. Scuticociliatosis is a major parasitic disease, and fatal infection with scuticociliates, or mixed infections with scuticociliates and other pathogenic agents (e.g., Vibrio spp.) cause severe mortalities in farmed olive flounders. To date, however, effective chemotherapeutic treatment of scuticociliatosis has only been reported at the in vitro level. In this study, we employed combination treatment, using benzalkonium chloride (to remove excess mucus from the body surface) and bronopol (to kill the parasites), to overcome the protective effect of mucus by some medicine to the scuticociliates. In the presence of the mucus mixture, the higher dose of bronopol (156 ppm) yielded morphologies and motilities similar to those of ciliates treated with the lower dose of bronopol (80 ppm) in the absence of mucus. We also investigated the in vivo effects of this treatment in field trials involving a total of 15,025 naturally infected flounders. We observed that short-term bath treatments with benzalkonium chloride (50 ppm) followed by bronopol (500 ppm) were effective, assessed by the relative percentage mortality (RPS) value. Thus, this study provides a notable therapeutic strategy by removing the mucus to treat scuticociliatosis in olive flounders at the aquaculture field level.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus).

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

Comparative genomic characterization of three Streptococcus parauberis strains in fish pathogen, as assessed by wide-genome analyses.

Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus) in northeast Asia, can be distinctly divided into two groups (type I and type II) by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109) were determined and compared with the previously determined genome of a Korean strain (KCTC 11537). The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS) for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Phylogenomic network and comparative genomics reveal a diverged member of the ΦKZ-related group, marine vibrio phage ΦJM-2012.

Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ΦJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ΦJM-2012 was connected to only five members of the Pseudomonas ΦKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ΦKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ΦJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ΦJM-2012 genome showed that segments of the conserved N-terminal parts of ΦKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ΦKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.

Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis.

Heat shock proteins (HSPs) have been observed in cells exposed to a variety of stresses, including infectious pathogens. This study used a label-free, quantitative proteomic approach and transcriptional gene expression analysis to investigate infection-related HSP proteins and their encoding genes in whole kidneys from olive flounder (Paralichthys olivaceus). During Streptococcus parauberis infection in the flounder, the genes encoding Hsp10, Hsp40A4, Hsp40B6, Hsp40B11, Hsp60, Hsp70, glucose regulated protein 78 (Grp78), Hsp90α, Hsp90ß and Grp94 were induced, and the protein levels of Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 were differentially regulated over time. Subsequent results also revealed that Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 appear to be the dominant and critical HSPs in olive flounder during bacterial infection. This is the first estimation of the differential involvement of HSPs in the immune response of olive flounder exposed to bacterial infection.