Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus).

ß-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-ßdefensin which express both the outer membrane protein of the bacterium and ß-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-ßdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-ßdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-ßdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-ßdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that ß-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Who should be screened for carbapenemase-producing Enterobacterales and when? A systematic review.

Carbapenemase-producing Enterobacterales (CPE) cases increases every year in Denmark and the proportion of CPE-positive cases with a travel history decreases. Several epidemiological links show transmission in Danish healthcare setting indicating infection prevention and control challenges and raising questions about the Danish CPE screening protocol. The aim of this review was to identify additional risk factors to those described in the Danish CPE-screening protocol in order to detect the Danish CPE-positive patients and thereby reduce the risk of transmission and outbreaks. A systematic literature search was conducted in PubMed, Embase and Cochrane Library during March 2022. A total of 1487 articles were screened, and 19 studies were included. Retrieved studies dealt with patients with laboratory-confirmed CPE (colonization and/or infection) and associated risk factors. Antimicrobial therapy, especially broad-spectrum antimicrobial agents, prior or current hospitalization of approximately one week in ICU and 20-28 days in other wards and travel history with or without hospitalization abroad were significant risk factors associated with CPE acquisition. Comorbidities and invasive procedures were identified as risk factors, but without identifying specific comorbidities or invasive procedures associated with risk for CPE-acquisition. This study suggests the need to develop an additional algorithm for CPE-screening in Denmark. In addition to risk-based screening on admission, screening of inpatients should be considered. The screening protocol might include screening of inpatients with comorbidities who are hospitalized >1 week in ICU or >3 weeks in other wards and who have previously received or currently are receiving antibiotic treatment. Further research is needed to develop a new CPE-screening algorithm.

Bacteriophage PVN06 protected catfish Pangasianodon hypophthalmus from Edwardsiella ictaluri infection.

Bacillary necrosis of pangasius (BNP) is a disease caused by Edwardsiella ictaluri bacteria in striped catfish Pangasianodon hypophthalmus that results in high mortality rates. To control this disease, bacteriophages have been considered as alternatives to antibiotics. In this study, we applied the lytic bacteriophage PVN06 in striped catfish fingerlings to prevent E. ictaluri infection. In an experimental trial, the phage was administered to fish by feeding phage-coated feed with doses of 7.17±0.09, 8.17±0.09 and 9.17±0.09 log PFU/g feed per day before bacterial infection. Fish were infected by bacteria once with concentrations ranging from 3.01 to 7.01 log CFU/ml tank water. A day after infection, phage treatment resumed at a rate of once per day until the end of the trial. The results of the trial show that bacterial infection caused typical symptoms of BNP in fish with the cumulative fish death rate of 36.7±2.9 to 75.0±5.0%, depending on the bacterial concentration used for infection. Phage treatment with 9.17±0.09 log PFU/g significantly reduced the mortality rate, while treatments with 8.17±0.09 and 7.17±0.09 log PFU/g did not. This phage dose resulted in a 61.7-fold reduction in the toxicity of the bacterial pathogen and the survival rate of 15-23.3% in fish. Our study has demonstrated that the bacteriophage PVN06 protected striped catfish from BNP.

Fructose potentiates the protective efficiency of live Edwardsiella tarda cell vaccine.

Vaccination is an effective measure to prevent infection by pathogens. Live vaccines have higher protective efficacy than inactivated vaccines. However, how live vaccines interact with the host from a metabolic perspective is unknown. The present study aimed to explore whether a live Edwardsiella tarda vaccine regulates host metabolism and whether this regulation is related to the protective efficacy of the vaccine. Therefore, a gas chromatography mass spectrometry (GC-MS)-based metabolomics approach was used to investigate the metabolomic profile of mice serum after vaccination with live E. tarda vaccine. Fructose was identified as a key biomarker that contributes to the immune protection induced by the live vaccine. Moreover, co-administration of exogenous fructose and the live vaccine synergistically promoted survival of mice and fish after bacterial challenge. These results indicate that metabolites, especially fructose, can potentiate the live E. tarda vaccine to increase its protective efficiency.

Quantification of time delay between screening and subsequent initiation of contact isolation for carriers of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales: A post hoc subgroup analysis of the R-GNOSIS WP5 Trial.

OBJECTIVES: The aim of this study was to quantify the time delay between screening and initiation of contact isolation for carriers of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-E). METHODS: This study was a secondary analysis of contact isolation periods in a cluster-randomized controlled trial that compared 2 strategies to control ESBL-E (trial no. ISRCTN57648070). Patients admitted to 20 non-ICU wards in Germany, the Netherlands, Spain, and Switzerland were screened for ESBL-E carriage on admission, weekly thereafter, and on discharge. Data collection included the day of sampling, the day the wards were notified of the result, and subsequent ESBL-E isolation days. RESULTS: Between January 2014 and August 2016, 19,122 patients, with a length of stay ≥2 days were included. At least 1 culture was collected for 16,091 patients (84%), with a median duration between the admission day and the day of first sample collection of 2 days (interquartile range [IQR], 1-3). Moreover, 854 (41%) of all 2,078 ESBL-E carriers remained without isolation during their hospital stay. In total, 6,040 ESBL-E days (32% of all ESBL-E days) accrued for patients who were not isolated. Of 2,078 ESBL-E-carriers, 1,478 ESBL-E carriers (71%) had no previous history of ESBL-E carriage. Also, 697 (34%) were placed in contact isolation with a delay of 4 days (IQR, 2-5), accounting for 2,723 nonisolation days (15% of ESBL-E days). CONCLUSIONS: Even with extensive surveillance screening, almost one-third of all ESBL-E days were nonisolation days. Limitations in routine culture-based ESBL-E detection impeded timely and exhaustive implementation of targeted contact isolation.

Increased evidence for no benefit of contact precautions in preventing extended-spectrum ß-lactamases-producing Enterobacteriaceae: Systematic scoping review.

INTRODUCTION: Extended-spectrum ß-lactamases-producing Enterobacteriaceae (ESBL-E) is a critical antimicrobial resistance pathogen, to which we need to pay the greatest attention. This study was aimed at uncovering the present evidence for the preventive effectiveness of contact precautions for patients colonized or infected with ESBL-E. METHODS: According to the Preferred Reporting Items for Systemic Reviews and Meta-analyses (PRISMA) Extension for Scoping Reviews, we searched MEDLINE for articles with relevant keywords from the beginning of 2010 to October 18, 2022. RESULTS: Of the 355 articles found, 9, including 8 observational studies and 1 randomized controlled trial, were selected. Safety of discontinuing contact precautions was evaluated mainly in acute-care and long-term care hospitals. Consistently, all authors concluded that contact precautions can be safely discontinued in patients colonized or infected with ESBL-E. CONCLUSION: The clinical impact of discontinuing contact precautions for patients with ESBL-E is minimal and can be safely withdrawn at acute, noncritical, adult care wards. Relevant data from pediatric and geriatric wards, as well as intensive care units, were insufficient and should be investigated in future research.

The Attenuation Mechanism and Live Vaccine Potential of a Low-Virulence Edwardsiella ictaluri Strain Obtained by Rifampicin Passaging Culture.

The rifampicin-resistant strain E9-302 of Edwardsiella ictaluri strain 669 (WT) was generated by continuous passage on BHI agar plates containing increasing concentrations of rifampicin. E9-302 was attenuated significantly by 119 times to zebrafish Danio rerio compared to WT in terms of the 50% lethal dose (LD50). Zebrafish vaccinated with E9-302 via intraperitoneal (IP) injection at a dose of 1 × 103 CFU/fish had relative percentage survival (RPS) rates of 85.7% when challenged with wild-type E. ictaluri via IP 14 days post-vaccination (dpv). After 14 days of primary vaccination with E9-302 via immersion (IM) at a dose of 4 × 107 CFU/ml, a booster IM vaccination with E9-302 at a dose of 2 × 107 CFU/ml exhibited 65.2% RPS against challenge with wild-type E. ictaluri via IP 7 days later. These results indicated that the rifampicin-resistant attenuated strain E9-302 had potential as a live vaccine against E. ictaluri infection. A previously unreported amino acid site change at position 142 of the RNA polymerase (RNAP) ß subunit encoded by the gene rpoB associated with rifampicin resistance was identified. Analysis of the whole-genome sequencing results revealed multiple missense mutations in the virulence-related genes esrB and sspH2 in E9-302 compared with WT, and a 189 bp mismatch in one gene, whose coding product was highly homologous to glycosyltransferase family 39 protein. This study preliminarily explored the molecular mechanism underlying the virulence attenuation of rifampicin-resistant strain E9-302 and provided a new target for the subsequent study of the pathogenic mechanism of E. ictaluri.

A matrix management of prevention and control for carbapenem-resistant Enterobacteriaceae in an urban compact medical union.

PURPOSE: To understand the epidemiological characteristics of nosocomial infection of carbapenem-resistant Enterobacteriaceae (CRE) in an urban medical union includes 10 medical hospitals with different number of beds in China. METHODS: Epidemiological data on age, department, and infection of CRE cases detected from January 2014 to December 2021 were collected via a real-time hospital-infection monitoring system or manually for subsequent characterization. A multi-departmental and multi-disciplinary matrix (MMM) management of CRE was established and implemented within a medical union. RESULTS: A total of 1327 cases of CRE infection were detected during the 8 years, of which 352 were due to nosocomial infection, with an infection morbidity of 0.046% and a resistance rate of 10.79%. The morbidity of CRE infection showed a trend of year-to-year fluctuation. The morbidity of CRE infection was significantly higher in winter and spring than that in summer and autumn, significantly higher in men than in women (χ2 â€‹= â€‹55.891, p â€‹< â€‹0.001), and 3 times higher in elderly patients ≥65 years old than in patients <65 years old (χ2 â€‹= â€‹117.517, p â€‹< â€‹0.001). The morbidity of CRE infection after intervention with MMM management decreased significantly from 0.071% to 0.042% (χ2 â€‹= â€‹15.628, p â€‹< â€‹0.001). CONCLUSIONS: CRE prevention and control practice should be adapted to seasonal variations, gender and age differences. The effective prevention and control of CRE nosocomial infections can be achieved by implementing MMM management within a medical association.

Audit and feedback as a tool to increase compliance with carbapenemase-producing Enterobacteriaceae (CPE) screening and decrease CPE transmission in the hospital.

OBJECTIVE: To increase compliance with carbapenemase-producing Enterobacteriaceae (CPE) screening through real-time audit and feedback in our hospital and decrease CPE transmissions. DESIGN: A before-and-after trial, using active enhanced surveillance of CPE carriers. SETTING: A 500-bed, secondary, university-affiliated hospital that serves a population of 450,000 in a northern district in Israel. METHODS: The study was conducted during 2016-2019 and included patients who were admitted to the hospital and fulfilled CPE screening criteria upon admission and during prolonged hospitalizations. On January 1, 2017, the infection control team implemented a new strategy of real-time feedback toward compliance with in-hospital screening guidelines. Other infection control measurements were performed without interventions. The primary outcome was compliance with appropriate CPE screening. Secondary outcomes included CPE acquisition and compliance with hand hygiene and contact precaution practices. Data were analyzed to calculate differences between compliance with CPE screening during the study period and to test the correlation between contact precautions and hand hygiene practices according to compliance with CPE screening. RESULTS: During the study period, 3,131 patients were eligible for CPE screening. We detected a statistically significant increase in compliance to CPE screening from 74% during 2017 to 92% in 2018 and 95% in 2019 (P < .0001 for both comparisons). We detected a decrease in CPE transmission from 12% in 2017 to 2% in 2019 (P < .0001). We did not find any correlation between other infection control interventions and CPE screening and acquisition. CONCLUSION: Audit and feedback can improve appropriate CPE screening and may reduce CPE transmission in the hospital.

The effect of re-directed patient flow in combination with targeted infection control measures on the spread of multi-drug-resistant Enterobacteriaceae in the German health-care system: a mathematical modelling approach.

OBJECTIVE: The introduction of multi-drug-resistant Enterobacteriaceae (MDR-E) by colonized patients transferred from high-prevalence countries has led to several large outbreaks of MDR-E in low-prevalence countries, with the risk of propagated spread to the community. The goal of this study was to derive a strategy to counteract the spread of MDR-E at the regional health-care network level. METHODS: We used a hybrid ordinary differential equation and network model built based on German health insurance data to evaluate whether the re-direction of patient flow in combination with targeted infection control measures can counteract the spread of MDR-E in the German health-care system. We applied pragmatic re-direction strategies focusing on a reduced choice of hospitals for subsequent stays after initial hospitalization but not manipulating direct transfers because these are most likely determined by medical needs. RESULTS: The re-direction strategies alone did not reduce the system-wide spread of MDR-E (system-wide prevalence of MDR-E is 18.7% vs. 25.7%/29.9%). In contrast, targeted hospital-based infection control measures restricted to institutions with the highest institutional basic reproduction numbers in the network were identified as an effective tool for reducing system-wide prevalence (system-wide prevalence of MDR-E is 18.7% vs. 9.3%). If these measures were applied to the top one-third of hospitals, the system-wide prevalence could be reduced by approximately 80% (system-wide prevalence of 18.7% vs. 3.5% for one-third of patients subjected to interventions). A combination of this hospital-based intervention and patient re-direction strategies could not improve the effectiveness of the hospital-based approach (system-wide prevalence of MDR-E is 9.3% vs. 14.2%/14.3%). CONCLUSIONS: The pragmatic patient re-direction strategies were not capable of restricting the spread of MDR-E in a simulation of the German health-care system; in contrast, hospital-based interventions focusing on institutions identified based on network transmission patterns seem to be a promising approach for sustainable reduction of the spread of MDR-E through the German population.

Reducing Carbapenem-Resistant Enterobacteriaceae Using the Targeted Solution Tool: A Quality Improvement Project.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) is a worldwide urgent health problem. Hand hygiene (HH) is an effective intervention to reduce the spread of CRE. LOCAL PROBLEM: In 2017, an increase in the rate of health care-associated (HA) CRE colonization was observed in a large multiorgan transplant center in Italy. This study aimed to reduce the HA-CRE colonization rates by improving HH compliance. METHODS: A pre-/post-intervention project was conducted from November 2017 through December 2020. INTERVENTIONS: The DMAIC (Define, Measure, Analyze, Improve, and Control) framework was used to implement the HH Targeted Solution Tool (TST). RESULTS: Hand hygiene compliance increased from 49% to 76.9% after the Improve phase ( P = .0001), and to 81.9% after the second Control phase ( P = .0001). The rate of HA-CRE decreased from 24.9% to 5.6% ( P = .0001). CONCLUSIONS: Using the DMAIC framework to implement the TST can result in significant improvements in HH compliance and HA-CRE colonization rates.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Phenotypic differences between Edwardsiella piscicida and Edwardsiella anguillarum isolates in Japan.

OBJECTIVE: Edwardsiella tarda has been regarded as the causative agent of edwardsiellosis in cultured marine and freshwater fish species in Japan. Our previous study genetically classified an E. tarda-like isolate from diseased Olive Flounder Paralichthys olivaceus as E. piscicida and that from diseased Red Seabream Pagrus major as E. anguillarum. This study aimed to understand the phenotypic differences between E. piscicida and E. anguillarum. METHODS: Fourteen E. piscicida and seven E. anguillarum isolates were used in this study. The colonies of each isolate were grown on brain-heart infusion agar plates and then subjected to DNA extraction. The extracted DNA was amplified using PCR. carbohydrate fermentation of the isolates was examined using API 50 CH test kits. Moreover, the growth of the two species was examined in defined media. Also, free amino acids in Olive Flounder and Red Seabream sera were detected and quantified via high-performance liquid chromatography-mass spectrometry. Statistical differences in the concentrations of free amino acids were analyzed using Welch's t-tests. RESULT: The API 50 CH test revealed that L-arabinose and D-mannitol were fermented by E. anguillarum isolates but not E. piscicida isolates. Furthermore, the growth of E. piscicida and E. anguillarum was reduced in the defined medium without methionine and iron sulfate. The growth of E. piscicida was reduced in the defined medium without phenylalanine, tyrosine, alanine, or nicotinic acid, whereas the growth of E. anguillarum was reduced in the defined medium without serine, cysteine, leucine, threonine, or isoleucine. Tyrosine and alanine were present in higher concentrations in the Olive Flounder serum, whereas threonine and isoleucine were present in higher concentrations in the Red Seabream serum, suggesting favorable growth conditions for E. piscicida and E. anguillarum. CONCLUSION: This study characterizes a minimal defined medium that can be used for developing vaccines against E. piscicida and E. anguillarum.

Molecular and functional characterization of teleost-specific Interleukin-17N in yellow catfish (Pelteobagrus fulvidraco).

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

Immune responses and protective immunity in Pangasius pangasius (Hamilton, 1822) as induced by outer membrane proteins of Edwardsiella tarda and aluminium hydroxide adjuvant complex.

Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to the control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

Innate immunity and protective effects of orally administered betaine against viral and bacterial diseases in the olive flounder Paralichthys olivaceus (Temminck & Schlegel).

Sustainable methods that increase farmed fish yield while controlling infections are required to prevent economic losses in aquaculture farms. In this study, we evaluated the effects of betaine-supplemented (0%, 0.1%, 0.5%, and 1.0%) feed on the growth and immunity of the olive flounder Paralichthys olivaceus. Feed conversion ratios, post-infection cumulative mortality rates and innate immune responses were monitored. Weight gain was significantly higher with 0.5% and 1.0% than with 0% and 0.1% betaine-supplemented feed. Lysozyme activity was highest with 1.0% betaine. Respiratory burst activity was highest with 0.5% and 1.0% betaine. Serum bactericidal activity against Edwardsiella tarda was highest with 1.0% betaine (40% increase in survival rates compared with those in the control). Furthermore, serum virucidal activity against the viral haemorrhagic septicaemia virus (VHSV) was higher with 1.0% betaine than with other concentrations. With 0.5% and 1.0% betaine, the survival rates against VHSV were higher than those in the control until day 11, after which they declined. Our study suggests that betaine is a promising agent for promoting the growth of and enhancing immunity against E. tarda in olive flounders. Our findings may further contribute to developing necessary alternatives to conventional antibiotics in fish farming.