Improved Stability and Activity of a Marine Peptide-N6NH2 against Edwardsiella tarda and Its Preliminary Application in Fish.

Edwardsiella tarda can cause fatal gastro-/extraintestinal diseases in fish and humans. Overuse of antibiotics has led to antibiotic resistance and contamination in the environment, which highlights the need to find new antimicrobial agents. In this study, the marine peptide-N6 was amidated at its C-terminus to generate N6NH2. The antibacterial activity of N6 and N6NH2 against E. tarda was evaluated in vitro and in vivo; their stability, toxicity and mode of action were also determined. Minimal inhibitory concentrations (MICs) of N6 and N6NH2 against E. tarda were 1.29-3.2 µM. Both N6 and N6NH2 killed bacteria by destroying the cell membrane of E. tarda and binding to lipopolysaccharide (LPS) and genomic DNA. In contrast with N6, N6NH2 improved the stability toward trypsin, reduced hemolysis (by 0.19% at a concentration of 256 µg/mL) and enhanced the ability to penetrate the bacterial outer and inner membrane. In the model of fish peritonitis caused by E. tarda, superior to norfloxacin, N6NH2 improved the survival rate of fish, reduced the bacterial load on the organs, alleviated the organ injury and regulated the immunity of the liver and kidney. These data suggest that the marine peptide N6NH2 may be a candidate for novel antimicrobial agents against E. tarda infections.

Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species.

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection.

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions.

Enteric Viral Surrogate Reduction by Chitosan.

Enteric viruses are a major problem in the food industry, especially as human noroviruses are the leading cause of nonbacterial gastroenteritis. Chitosan is known to be effective against some enteric viral surrogates, but more detailed studies are needed to determine the precise application variables. The main objective of this work was to determine the effect of increasing chitosan concentration (0.7-1.5% w/v) on the cultivable enteric viral surrogates, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and bacteriophages (MS2 and phiX174) at 37 °C. Two chitosans (53 and 222 kDa) were dissolved in water (53 kDa) or 1% acetic acid (222 KDa) at 0.7-1.5%, and were then mixed with each virus to obtain a titer of ~5 log plaque-forming units (PFU)/mL. These mixtures were incubated for 3 h at 37 °C. Controls included untreated viruses in phosphate-buffered saline and viruses were enumerated by plaque assays. The 53 kDa chitosan at the concentrations tested reduced FCV-F9, MNV-1, MS2, and phi X174 by 2.6-2.9, 0.1-0.4, 2.6-2.8, and 0.7-0.9 log PFU/mL, respectively, while reduction by 222 kDa chitosan was 2.2-2.4, 0.8-1.0, 2.6-5.2, and 0.5-0.8 log PFU/mL, respectively. The 222 kDa chitosan at 1 and 0.7% w/v in acetic acid (pH 4.5) caused the greatest reductions of MS2 by 5.2 logs and 2.6 logs, respectively. Overall, chitosan treatments showed the greatest reduction of MS2, followed by FCV-F9, phi X174, and MNV-1. These two chitosans may contribute to the reduction of enteric viruses at the concentrations tested but would require use of other hurdles to eliminate food borne viruses.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

Citrobacter-induced colitis in mice with murine acquired immunodeficiency syndrome.

Over the period of a year, colitis was observed in 44 mice raised in a conventional nonspecific pathogen-free colony, 41 of these having concomitant retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS). The lesions varied from bacterial colonization to hyperplasia of colonic mucosa to severe, often fatal, ulceration. Citrobacter rodentium was isolated from the colon and/or liver of 2 mice with colitis. When C57BL/6 mice with or without MAIDS were given graded doses of the bacterium, only those with MAIDS developed colitis, and C rodentium was reisolated from their livers. Thus, mice with MAIDS can develop severe disease following opportunistic infection with an environmental contaminant of the colony that is nonpathogenic for normal adult mice.

[Extended-spectrum beta-lactamase in Klebsiella pneumoniae and Escherichia coli isolates].

OBJECTIVE: To investigate the prevalence, drug resistance, gene typing, and epidemicity of extended-spectrum beta-lactamases (ESBL) Klebsiella pneumoniae and Escherichia coli isolates. METHODS: 559 strains of K. pneumoniae and 427 strains of E.coli were isolated form Huanshan Hospital from 1 January to 31 December 1999. The ESBL-producing strains were detected by double disc test and confirmed by minimal inhibitory concentration (MIC). The MIC of ESBL-producing strains was detected by agar dilution test. The beta-lactamase genes were detected by PCR. DNA fingerprinting was made by pulsed-field gel electrophoresis (PFGE). RESULTS: The incidence of ESBL-producing strains was 51% among the isolated K. pneumoniae (285/559) and 23.6% among the isolated E. coli (101/427), most of which were collected from the patients in the intensive care unit and neurosurgical ward. 63.5% of the ESBL-producing K. pneumoniae strains were collected from sputum specimens, and 64.3% of the ESBL-producing E. coli strains were collected from the urine specimens. Most ESBL-producing strains were resistant to most beta-lactam antibiotics, including the third-generation cephalosporins, and non- beta-lactam antimicrobial drugs, such as fluoroquinolones, aminoglycosides, tetracycline, and chloramphenicol. Most of the ESBL-producing strains were susceptible to imipenam, cefmetazole, and beta-lactam antibiotic/clavulanic acid. TEM type beta-lactamase was the main type among those EBSL-producing strains, followed by SHV type and CTX-M type. Some ESBL-producing E. coli and most ESBL-producing K. pneumoniae produced more than one type of beta-lactamase. CONCLUSION: ESBL-producing strains are common among hospital strains of E. coli and K. pneumoniae. Most of them are multidrug resistant. Prevalence and transmission of these strains exist in hospital.