Adel Shaheen

Construction and evaluation of an Edwardsiella ictaluri fhuC mutant

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in channel catfish. Iron is an essential micronutrient needed for bacterial virulence, and to acquire iron, many Gram-negative bacteria secrete ferric iron chelating siderophores. The ferric hydroxamate uptake (Fhu) system consists of four genes (fhuC, fhuD, fhuB, and fhuA), and is involved in the uptake of hydroxamate type siderophores across bacterial membranes. However, the Fhu system and its importance in E. ictaluri virulence have been uninvestigated. Here, we present construction and evaluation of an E. ictaluri ΔfhuC mutant. The E. ictaluri fhuC gene was deleted in-frame by allelic exchange, and the mutants growth in media and virulence in catfish were determined. Our results indicated that deletion of the E. ictaluri fhuC gene did not affect the growth of E. ictaluri largely in both iron-replete and iron-depleted media. Addition of ferric iron sources into the iron-depleted medium improved the growth of both E. ictaluri ΔfhuC and wild type (WT). Catfish mortalities indicated that E. ictaluri ΔfhuC mutant was attenuated 2.05-fold compared with the parent strain. The catfish immunized with the E. ictaluri ΔfhuC mutant showed a high relative percent survival rate (97.50%) after re-challenge with the WT E. ictaluri strain. Taken together, our data indicates that the fhuC gene contributes to E. ictaluri virulence.

Expression of Hsp70, Igf1, and Three Oxidative Stress Biomarkers in Response to Handling and Salt Treatment at Different Water Temperatures in Yellow Perch, Perca flavescens

Stress is a major factor that causes diseases and mortality in the aquaculture industry. The goal was to analyze the expression of stress-related biomarkers in response to different stressors in yellow perch, which is an important aquaculture candidate in North America and highly sensitive to handling in captivity. Three fish groups were established, each having four replicates, and subjected to water temperatures of 14, 20, and 26°C and acute handling stress was performed followed by a salt treatment for 144h at a salinity of 5 ppt. Serum and hepatic mRNA levels of heat shock protein (hsp70), insulin-like growth factor 1 (Igf1), glutathione peroxidase (Gpx), superoxide dismutase 1 (Sod1), and glutathione reductase (Gsr) were quantified at seven times interval over 144 h using ELISA and RT-qPCR. Handling stress caused a significant down-regulation in Hsp70, Gpx, Sod1, and Gsr at a water temperature of 20°C compared to 14 and 26°C. Igf1 was significantly upregulated at 20°C and down-regulated at 14 and 26°C. Salt treatment had a transient reverse effect on the targeted biomarkers in all groups at 72 h, then caused an upregulation after 144 h, compared to the control groups. The data showed a negative strong regulatory linear relationship between igf1 with hsp70 and anti-oxidative gene expressions. These findings could provide valuable new insights into the stress responses that affect fish health and could be used to monitor the stress.

A novel viral responsive protein (MjVRP) from Marsupenaeus japonicus haemocytes is involved in white spot syndrome virus infection

ABSTRACT A viral responsive protein (MjVRP) was characterized from Marsupenaeus japonicus haemocytes. In amino acid homology and phylogenetic tree analyses, MjVRP clustered in the same group with the viral responsive protein of Penaeus monodon (PmVRP15), showing 34% identity. MjVRP transcripts were mainly expressed in haemocytes and the lymphoid organ. Western blotting likewise showed that MjVRP was strongly expressed in haemocytes and the lymphoid organ. Immunostaining detected MjVRP within the cytosol next to the perinuclear region in some haemocytes. Experimental challenge with white spot syndrome virus (WSSV) significantly up‐regulated the mRNA level of MjVRP in the M. japonicus haemocytes at 6 and 48 h. Flow cytometry and indirect immunofluorescence assays revealed that the ratio of MjVRP+ haemocytes significantly increased 24 and 48 h post‐WSSV infection. These results suggest that MjVRP+ haemocytes have a supporting role in the pathogenesis of WSSV. HighlightsA viral responsive protein of M. japonicus was cloned and characterized.MjVRP is mainly expressed in haemocytes and located close to the nucleus of semi‐granular and granular cells.MjVRP mRNA expression level is increased after WSSV challenge.The number of positive haemocytes was increased after WSSV challenge.

Saprolegniosis in Nile Tilapia: Identification, Molecular Characterization, and Phylogenetic Analysis of Two Novel Pathogenic Saprolegnia Strains

Saprolegniosis is a fungal infection that leads to huge economic losses in tilapia aquaculture. Saprolegnia spp. are usually implicated as the etiological agents, but their identification is sometimes troublesome and confusing. In this study, two Saprolegnia strains (ManS22 and ManS33) were isolated from Nile Tilapia Oreochromis niloticus suffering from saprolegniosis. Both isolates were characterized morphologically and from internal transcribed spacer (ITS) sequence data. Additionally, both strains were tested for pathogenicity, and they were highly pathogenic and caused cumulative mortalities of 88.9% and 95.6%, respectively. Initially, the two strains were identified, by morphology of sexual and asexual stages, as members of the genus Saprolegnia. For more definitive identification and characterization, the ITS region of the ribosomal RNA genes was amplified and sequenced, and sequences were compared with other known sequences in GenBank. A phylogenetic tree constructed using the neighbor-joining method revealed that the two strains fell into two clusters within the species Saprolegnia parasitica. Cluster 1 included the ManS33 strain and cluster 2 the ManS22 strain. Cluster 1 grouped the ManS33 strain with other S. parasitica stains and shared 97-99% sequence similarity. Cluster 2 contained only the ManS22 strain and shared 93-94% similarity to several reference sequences of S. parasitica strains. Therefore, our findings suggest that ManS22 represents a newly described strain of S. parasitica. Received April 19, 2016; accepted October 27, 2016.