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This study aimed to identify the mortality present in private fish farm Amyloodinium ocellatum and Cryptocaryon irritans were isolated from this outbreak affecting Liza carinata fingerlings at an earthen-based aquaculture facility in Damietta , Egypt. A total of 140 moribunds, L. carinata, were collected from the fish ponds during the mortality events. Physico-chemical analysis of water was analyzed. The skin, fins, gills, and eyes of each fish specimen were scraped gently onto slides in areas over 2 cm area. All smears were examined separately under the light microscope. Molecular identification of the parasites using analysis of ITS rDNA regions flanking both 18S and 28S rDNA genes of Amyloodinium protozoa and C. irritans . Identities of the detected parasites were confirmed by gene sequence and phylogenetic analysis. The majority of the examined fish (90%) were infected, 66.42% had a mixed infection, and 23.57% had a single infection either with A. ocellatum (10.71%) or C. irritans (12.85%).The mean intensity of A. ocellatum was 16.5 ± 2.03 in the skin and 13.18 ± 1.90 in the gills of infected fish, while that of C. irritans was 4.75 ± 1.05 in gills and 7.43 ± 1.45 in the skin, respectively. To control the emergent mortalities, affected ponds were treated using copper sulfate pentahydrate, hydrogen peroxides solutions, and amprolium hydrochloride powder in feed. Fish across the treated ponds were gradually improved with low morbidity and mortalityrates during the treatment period. The clinical disease was almost diminished at the end of the second week of treatment. Coinciding with the clinical improvement of the treated juveniles, microscopical examination of skin/gill scraps exhibited a marked decline in the number of protozoan parasites at the end of the second week of treatment.

In 2003, viral hemorrhagic septicemia virus (VHSV) emerged in the Laurentian Great Lakes causing serious losses in a number of ecologically and recreationally important fish species. Within six years, despite concerted managerial preventive measures, the virus spread into the five Great Lakes and to a number of inland waterbodies. In response to this emerging threat, cooperative efforts between the Michigan Department of Natural Resources (MI DNR), the Michigan State University Aquatic Animal Health Laboratory (MSU-AAHL), and the United States Department of Agriculture-Animal and Plant Health Inspection Services (USDA-APHIS) were focused on performing a series of general and VHSV-targeted surveillances to determine the extent of virus trafficking in the State of Michigan. Herein we describe six years (2005–2010) of testing, covering hundreds of sites throughout Michigan’s Upper and Lower Peninsulas. A total of 96,228 fish representing 73 species were checked for lesions suggestive of VHSV and their internal organs tested for the presence of VHSV using susceptible cell lines. Of the 1,823 cases tested, 30 cases from 19 fish species tested positive for VHSV by tissue culture and were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Gene sequence analyses of all VHSV isolates retrieved in Michigan demonstrated that they belong to the emerging sublineage “b” of the North American VHSV genotype IV. These findings underscore the complexity of VHSV ecology in the Great Lakes basin and the critical need for rigorous legislation and regulatory guidelines in order to reduce the virus spread within and outside of the Laurentian Great Lakes watershed.

Mycoplasma is associated with fatal diseases in human, animals, and fish. Mycoplasma of aquatic origin has not been thoroughly studied. The current study has shed the light on isolation and characterization of unique piscine Mycoplasma isolates retrieved from diverse geographical locations across the Egyptian state. Mycoplasma spp. was isolated using specific culture media. Mycoplasma was identified using the morphochemical tests and then confirmed molecularly by PCR of the 16S rRNA gene. The results indicated that the incidence of Mycoplasma from Cyprinus carpio, Oreochromis niloticus, Aulopiformes synodontida, and Clarias gariepinus were 33.33%, 16.36%, 8.108%, and 6.45%, respectively, while Mugil cephalus were negative for Mycoplasma isolation. Mycoplasma was detected only from gills and swim bladder of affected fish. Biochemically, the isolated Mycoplasmas were grouped into two clusters. Mycoplasma cluster 1 (35 isolates) and Mycolplasma cluster 2 (7 isolates). Mycoplasma cluster 1 was positive for tetrazolium reduction while Mycoplasma cluster 2 was negative. The phylogenetic tree of partial sequences of 16S rRNA showed that both clusters were grouped in one branch and separated from other Mycoplasma spp., suggesting that both clusters are belonging to one species. Interestingly, all fish Mycoplasma isolates were PCR negative for both Mycoplasma mobile and Mycoplasma monodon using specific species primers. This result confirmed that these two clusters belonged to unspecified Mycoplasma species, for which the temporary names Mycoplasma cluster 1 and Mycoplasma cluster 2 were designated. Pathogenicity trials of both Mycoplasmas clusters revealed that all inoculated Nile tilapias were susceptible to the unspecified Mycoplasmas.

In this study, three farmed Gilthead seabream exhibited tumor-like mass above the left pectoral fin. The mass measured 9 mm in length, 8 mm in width. Morphometrically, the mass was fleshy, raised, circumscribed, and rosy colored with grayish-white center. The affected fish showed poor growth with 280 g average body weight after 30 months rearing period. Internally, moribund fish exhibited pale kidney/liver, mottled spleen with remarkable visceral adhesions. The presumptive bacteriological and conventional biochemical assays revealed that fish were clinically infected with Photobacterium damselae subspecies piscicida. The histopathological picture of the tumor-like mass concurred with the standard cellular criteria of fibropapilloma. In brief, the epidermal papilloma was denoted by the remarkable hyperplasia of epidermis together with eosinophilic intracytoplasmic inclusion bodies and prominent mitosis in the hyperplastic cells. Ultimately, the proliferated dermal fibroblasts appeared whirling and elongated with spindle nuclei. Such pathological alterations have confirmed a typical form of dermal fibroma upon positively reacted with Masson’s trichrome stain. In the spleen, characteristic granulomatous reactions were observed especially in the area of melanomacrophage centers where the granuloma showed different developmental stages. The occurrence of skin tumors in farmed marine fish is an alarm for the critical impacts of the erratic usage of pesticides on marine aquaculture presenting a staggering prototype for the growing aquatic environmental pollution.

Egypt is the largest producer of farmed mullets worldwide. The mullet seeds are collected from natural waters at the north and northern east of Egypt. Thus, the occurrence of parasitic diseases is relatively high even after being stocked in aquaculture facilities. The current study was conducted on a fish farm located at the coastal area of the Suez Canal region, Egypt. Farmed flathead grey mullet (Mugil cephalus L.) and thin-lipped grey mullet (Liza ramada) showed pinkish-white granular cysts on their body surfaces that measured approximately 5 mm (4–6 mm) in width and 7 mm (5–9 mm) in length and contained a milky coloured exudate. The proximal portion of the fins and apical part of the scales were the only sites of infection. These cysts are randomly scattered throughout the fish body surface. No mortalities were detected among moribund mullets, but these cysts make the fish unsightly, causing commercial rejection. The overall prevalence of infection was 40%. Giemsa-stained wet smears initially indicated that the spores were of Myxobolus species. Subsequently, the phylogenetic analysis of the 18S rRNA gene confirmed the identification of spores as being those of Myxobolus episquamalis. Furthermore, amprolium-salinomycin mixture, which has an established track record for treating myxozoan infections in multiple fish species, was successfully tested in a large-scale field trial. Moribund mullets displayed significant reduction in myxosporean infection following treatment with amprolium-salinomycin mixture. To the best of our knowledge, this is the first report describing the detection of M. episquamalis in farmed flathead grey mullet (Mugil cephalus L.) and thin-lipped grey mullet (Liza ramada) collected from fish farms in the Suez Canal, Egypt.

The aim of this study is to identify the hidden etiologies of the black chromo-shifting transient phenomenon affecting Nile tilapia, Oreochromis niloticus , farmed in RAS-controlled ponds together with the assessment of the immunological reaction against the chronic irritating effects of the invasive agent. A total of 100 Nile Tilapia were collected from a private farm at Kafrelsheikh Province, located on the northern side of the Egyptian Nile Delta. The clinical history of the affected fish farm showed an unknown chromo-shifting phenomenon where tilapias were exhibiting severe black skin coloration, which gradually disappeared after removal from the tank’s water. A comprehensive gross examination of the collected fish; including parasitological examination of skin and gill scraps, was performed. Blood biochemical testing was performed on the infested blackish O. niloticus and control non-infested fish. The current study showed that the monogenean parasite, Gyrodactylus cichlidarum , was the abundant parasite detected in the infested fish leading to abnormal black discoloration of skin and disruption of the immune system represented by significant increase of cortisol levels, lysozyme activity and different liver enzymes compared to the control. Treatment trials have been applied with moderate degrees of success, where the monogenean count was sharply decreased, and the normal skin color was remarkably restored, at the end of day 14 post-treatment. A triple treatment plan was initiated through 7 days’ application of 0.7 g/m 3 copper sulfates pentahydrate preceded by 1.5 ml/m 3 hydrogen peroxide 40% solution for the same period. One day after the end of the initial treatment, a maintenance dosage of 0.095 ml/m 3 of glutaraldehyde (15%)/quaternary ammonium compounds (QACs) mixture was administered for 3 days. As a supportive/immune-stimulant regimen, a weekly dosage of vitamin C (0.45 g g/m 3 ) and Saccharomyces cerevisiae (0.45 g/m 3 ) was added into the tank’s water to improve the general fish health.

Bacterial Kidney Disease (BKD) caused by a Gram-positive bacterium, Renibacterium salmoninarum (R. salmoninarum) is a systemic disease that threatens the expansion of both cultured and wild salmonines worldwide. Historical records show that BKD affected Michigan's brook trout as early as 1955 (Allison 1958) and continued its spread to most of other salmonid species such as coho salmon (Oncorhynchus kisutch ) spawners in the late sixties (MacLean and Yoder 1970) followed by a potent epizootic among the chinook salmon (Oncorhynchus tshawytscha ) populations in Lake Michigan in the late 1980's (Holey et al. 1998). Despite the magnitude of the setback to fisheries conservation efforts in Michigan, relatively little research has been done on R. salmoninarum , primarily due to its slow-growing nature, which takes up to 12 weeks to obtain growth upon primary isolation. The tissue processing protocol and optimized culture technique developed in this study, has facilitated the isolation of large number of R. salmoninarum and achieved a remarkably shorter incubation time for primary isolation. A sort of disagreement in results among the three used diagnostic assays (nPCR, Culture, Q-ELISA) was recorded during BKD testing of the feral and captive salmonid stocks for BKD, which may reflect the different phases of R. salmoninarum infection at the time of sampling. Further, the testing results demonstrated the presence of six patterns, with each of the patterns representing a probable stage along the course of R. salmoninarum infection. Data also demonstrated that male spawner salmon play the same role played by female in transmission of R. salmoninarum by shedding of the bacteria and its soluble antigens in milt. Moreover, results revealed that Hinchenbrook coho strain is more susceptible to R. salmoninarum than the Michigan adapted coho salmon strain. Data also, supported the previous reports, which indicated that brook trout are highly susceptible to BKD. Also, data showed that prevalence of R. salmoninarum in the hatchery-raised brook trout is the same as wild strains residing in the water source and that Iron River brook trout are more vulnerable to R. salmoninarum infection than Assinica strain. Last, external parasites could played a possible role in initiation, speeding up the R. salmoninarum infection and BKD occurrence and mortalities is not related to changes in seasons. Finally, findings indicated that the adult parasitic sea lamprey is a new host range for R. salmoninarum where the bacteria were isolated from the kidneys of a number of sea lampreys from Lake Ontario in 2003 and 2004.

A new series of benzoxazole derivatives were designed and synthesised to have the main essential pharmacophoric features of VEGFR-2 inhibitors. Cytotoxic activities were evaluated for all derivatives against two human cancer cell lines, MCF-7 and HepG2. Also, the effect of the most cytotoxic derivatives on VEGFR-2 protein concentration was assessed by ELISA. Compounds 14o, 14l, and 14b showed the highest activities with VEGFR-2 protein concentrations of 586.3, 636.2, and 705.7 pg/ml, respectively. Additionally, the anti-angiogenic property of compound 14b against human umbilical vascular endothelial cell (HUVEC) was performed using a wound healing migration assay. Compound 14b reduced proliferation and migratory potential of HUVEC cells. Furthermore, compound 14b was subjected to further biological investigations including cell cycle and apoptosis analyses. Compound 14b arrested the HepG2 cell growth at the Pre-G1 phase and induced apoptosis by 16.52%, compared to 0.67% in the control (HepG2) cells. The effect of apoptosis was buttressed by a 4.8-fold increase in caspase-3 level compared to the control cells. Besides, different in silico docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

Among a group of 310 natural antiviral natural metabolites, our team identified three compounds as the most potent natural inhibitors against the SARS-CoV-2 main protease (PDB ID: 5R84), Mpro. The identified compounds are sattazolin and caprolactin A and B. A validated multistage in silico study was conducted using several techniques. First, the molecular structures of the selected metabolites were compared with that of GWS, the co-crystallized ligand of Mpro, in a structural similarity study. The aim of this study was to determine the thirty most similar metabolites (10%) that may bind to the Mpro similar to GWS. Then, molecular docking against Mpro and pharmacophore studies led to the choice of five metabolites that exhibited good binding modes against the Mpro and good fit values against the generated pharmacophore model. Among them, three metabolites were chosen according to ADMET studies. The most promising Mpro inhibitor was determined by toxicity and DFT studies to be caprolactin A (292). Finally, molecular dynamics (MD) simulation studies were performed for caprolactin A to confirm the obtained results and understand the thermodynamic characteristics of the binding. It is hoped that the accomplished results could represent a positive step in the battle against COVID-19 through further in vitro and in vivo studies on the selected compounds.