Chitosan nanoparticles and green synthesized silver nanoparticles as novel alternatives to antibiotics for preventing A.hydrophila subsp. hydrophila infection in Nile tilapia, Oreochromis niloticus.

Recently, nanoparticles have attracted attention as a preventive tool for certain infectious diseases affecting fish in aquaculture. Furthermore, freshwater fishes are frequently vulnerable to summer mass morality caused by Aeromonas bacteria. In this regard, we focused on the evaluation of the in vitro and in vivo antimicrobial activity of chitosan (CNPs) and silver (AgNPs) nanoparticles against Aeromonas hydrophila subsp. hydrophila. CNPs and AgNPs were prepared at a mean particle size of 9.03 and 12.8 nm and a charge equalled+36.4 and -19.3 mV for CNPs and AgNPs, respectively. A. hydrophila subsp. hydrophila, Aeromonas caviae, and Aeromonas punctata were retrieved and identified by traditional and molecular techniques. The sensitivity of the obtained bacteria to eight different antibiotic discs was also tested. The antibiotic sensitivity studies revealed the presence of multidrug-resistant (MDR) Aeromonas species (spp.). The bacterium that showed the highest multidrug resistance against the tested antibiotic discs was Aeromonas hydrophila subsp. hydrophila. Therefore, CNPs and AgNPs were in vitro tested against the isolated bacterium and exhibited inhibition zones of 15 and 25 mm, respectively. TEM images also showed that CNPs and AgNPs had an antagonistic action against the same bacterium causing loss of architecture and bacterial death.

The antibacterial activity and immunomodulatory effect of naturally synthesized chitosan and silver nanoparticles against Pseudomonas fluorescence infection in Nile tilapia (Oreochromis niloticus): An in vivo study.

A sixty-day feeding trial was conducted to assess the effects of silver nanoparticles (AgNPs) and chitosan nanoparticles (CNPs) on the growth and immunity of Nile tilapia (Oreochromis niloticus), compared with the control group. CNPs and AgNPs were green synthesized and added to a control diet (30% crude protein) at levels of 2.0 g CNPs/kg diet and 1.0 mg AgNPs/kg diet. One hundred and eighty fish (101 ± 3.98 g) were randomly distributed into nine fiberglass tanks (200 cm × 200 cm x 100 cm, twenty fish each) to represent three equal groups (60 fish per group). After one and two months of the feeding trial, parameters of water quality, growth indices, hematology, and liver and kidney biomarkers were evaluated. At the end of the experiment, 10 fish from each group were challenged experimentally via the intraperitoneal injection with Pseudomonas fluorescence and fish mortality was observed for further ten days. Then, specimens from the liver, kidney, spleen, and anterior intestine were examined to assess the histopathological alterations. Incorporating a 2.0 g CNPs/kg diet was a promising growth enhancer; however, a 1.0 mg AgNPs/kg diet had no effects on tilapia performance. Furthermore, AgNPs appeared to reduce water pollution, leading to water filtration via decreasing both total dissolved solids (TDS) and electrical conductivity (EC). A significant role of AgNPs in improving tilapia's erythrogram (RBCs number and Hb concentration) was evident. Compared with the control group, both groups of CNPs and AgNPs improved non-specific immune parameters and showed defense effects against P. fluorescence. The fish mortality after P. fluorescence infection in CNPs and AgNPs-fed fish groups revealed significant decreases (P < 0.05) of 10% and 25%; respectively, while the control group exhibited a mortality rate of 40%. The current investigation evoked that using dietary CNPs (2.0 g/kg feed) as an antibacterial agent against P. fluorescence infection in Nile tilapia culture was better than dietary AgNPs (1.0 mg/kg diet) which, induced cells inflammation causing tissues necrosis.

Investigating the etiologies behind emergent mass mortalities of farmed Liza carinata juveniles from coastal farms at Damietta, Egypt.

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.

Mass kills in hatchery-reared European seabass (Dicentrarchus labrax) triggered by concomitant infections of Amyloodinium ocellatum and Vibrio alginolyticus.

Amyloodiniosis and vibriosis are serious diseases in European seabass (Dicentrarchus labrax) hatcheries with noticeable high mortality. This study was conducted on tank-cultured D. labrax frys at a private marine hatchery near Mariout Lake (Alexandria, Egypt). Frys showed a high mortality rate (70%), lethargy, darkening, asphyxia, ascites, and velvety skin appearance. Both infectious agents were presumptively identified in all investigated frys. The identities of the two recovered agents were confirmed by molecular assay and phylogenetic analysis. On the tissue level, histopathological examination of skin, splenic, and renal tissue indicated severe alterations due to the direct impacts of both infections. On the cellular level, scanning electron micrographs showed both protozoal and bacterial pathogens on/in gill epithelial cells in solitary and colonial forms. Vibrio alginolyticus showed variable results for tested antibiotics, with a higher sensitivity to florfenicol. A successful control strategy was strictly adopted to overcome infections and stop mortalities. Copper sulphate and hydrogen peroxide were efficiently applied to tank water to overcome A. ocellatum infections. Further, florfenicol was effectively used to overcome systemic V. alginolyticus infections. The efficacy of treatments was confirmed by the absence of infectious agents in randomly collected fish samples. To the best of the authors' knowledge, this study is one of the earliest Egyptian studies that dealt with the dilemma of mass kills associated with external parasitic/systemic bacterial infections among hatchery-reared European seabass.

Effect of poultry offal silage with or without betaine supplementation on growth performance, intestinal morphometry, spleen histomorphology of Nile tilapia (Oreochromis niloticus) fingerlings.

Fishmeal (FM) is the main protein source in fish feed. However, it is quite expensive due to its limited resources. Therefore, finding a dietary alternative to the FM to sustain fish production is crucial, and the current study was performed to assess the impact of poultry offal silage (POS) with or without betaine supplementation; as an effective and cheaper alternative to FM; on feed efficiency, growth performance, spleen morphology and intestinal morphometry of Nile tilapia (Oreochromis niloticus) fingerlings. Four dietary treatments were formulated: (1) FM based diet, (2) FM-B; FM diet +0.7% betaine, (3) POS diet and (4) POS-B; POS diet +0.7% betaine. Each dietary treatment consisted of three replicates (n = 10/replicate), and the experiment was continued for 16 weeks. By the end of the experiment, spleen and intestine specimens were collected from 15 fish (n = 5/replicate) for histopathological assessment. The results were statistically analysed using GLM procedures of SAS 9.4. Feed efficiency increased in both POS-B and FM-B groups (p = 0.01), while body weight and body weight gain showed only weak tendencies towards an increase (p = 0.10 and 0.12, respectively). The villi length was the highest in POS-B fed group (p < 0.01). In addition, melanomacrophage centres of the spleen increased in both betaine-supplemented groups (p < 0.01). From our findings, we conclude that betaine supplementation with poultry offal silage improved production performance and immune status of Nile tilapia fish.

Streptococcus, Centrocestus formosanus and Myxobolus tilapiae concurrent infections in farmed Nile tilapia (Oreochromis niloticus).

Stress triggered concurrent microbial/parasitic infections are prevalent in earthen pond based farmed Nile tilapia Oreochromis niloticus. In the current study, a total of thirty five O. niloticus were collected from a commercial fish farm with a history of severe mortalities at Port Said, Egypt. Nile tilapia samples were subjected to bacteriological, parasitological and pathological examinations. Twenty one Enterococcus fecalis and 15 Streptococcus agalactiae isolates were presumptively identified utilizing the semi-automated API 20 Strept test kit. The identities of the retrieved bacteria were confirmed by the sequencing of 16 S rRNA gene. Moribund O. niloticus were found to be heavily infected by one or both of Centrocestus formosanus encysted metacercariae (EMC) and/or Myxobolus tilapiae spores presenting a unique form of synergistic and/or symbiotic relationship. The identities of both parasites were confirmed through morphological and molecular characterization. Variable circulatory, degenerative, necrotic and proliferative changes were also noticed in hematopoietic organs. Interestingly, multiple myxobolus spores and EMC were noticed in some histological sections. It was obvious that the current concurrent bacterial and parasitic infections are triggered by the deleterious effects of some stressing environmental conditions. The unfavorable climatic conditions (high temperature and high relative humidity) recorded at the surge of mortalities are probable predisposing stress factors.

Efficiency of monovalent and polyvalent Vibrio alginolyticus and Vibrio Parahaemolyticus vaccines on the immune response and protection in gilthead sea bream, Sparus aurata (L.) against vibriosis.

This experimental studies investigated the protective efficiencies and the potential immune mechanisms of vibrio monovalent and polyvalent autogenous formalin-inactivated whole-cell bacterins (FKC) in Gilthead sea bream (Sparus aurata) cultured in Egypt. Two months post-vaccination, the relative percentage survival (RPS) was estimated after challenge with the vaccine's homologues pathogenic strains. The survival values were 100% and 83.3% in groups immunized with monovalent V. alginolyticus or V. parahaemolyticus FKC bacterins, respectively. On the other hand, survival values were 91.75% and 75% in fish groups subjected to polyvalent (V. parahaemolyticus O11: K40 & V. alginolyticus) and (V. parahaemolyticus O3: K6 & V. alginolyticus) FKC bacterins, respectively. Overall, the tested vaccine preparations were significantly increased (P < 0.05) the agglutination antibody titer, phagocytic activity, respiratory burst activity, when compared to the non-immunized control group. The current results conclude that, autogenous Vibrio vaccines provoked a promising protection against vibriosis in Gilthead sea bream cultured in Egypt, it was superior in monovalent FKC V. alginolyticus vaccine and polyvalent FKC of V. parahaemolyticus O11: K40 with V. alginolyticus vaccine that could be useful means of prevention and control of vibriosis.

Assessment of the immune-modulatory and antimicrobial effects of dietary chitosan on Nile tilapia (Oreochrmis niloticus) with special emphasis to its bio-remediating impacts.

Fish, pathogen and environment are three counterparts who are sharing the same circle of life. To keep fish up to their optimal health, environment should be competently improved and pathogen count/virulence should be seized. Using of bioactive immunostimulants to achieve these objectives is the hypothesis under assessment. Thus, the present study was performed to evaluate the use of shrimp shells derived chitosan as an immunostimulant as well as preventive regime against Aeromonas hydrophila infection of Nile tilapia and to assess its antibacterial/aquatic bio-remediating effects. Results achieved by feeding 1% chitosan as preventive/therapeutic regimes have revealed a remarkably enhanced several innate immunological parameters (e.g., Phagocytic activity/index, NBT, Lysozyme activity and ACH50), increased resistance against A. hydrophila and strikingly improved water quality compared to the 0.5 and 2% chitosan containing diets. Conclusively, experimental results suggest the commercial usage of chitosan as an efficient immunostimulant and bio-remediating agent in aquaculture.

Epidemiological investigation of Renibacterium salmoninarum in three Oncorhynchus spp. in Michigan from 2001 to 2010.

Bacterial kidney disease (BKD) has caused mortalities and chronic infections in wild and farm-raised salmonids throughout the world. In the Laurentian Great Lakes of North America, BKD was associated with several large-scale mortality events of Oncorhynchus spp. throughout the 1980s and 1990s. In response to these mortality events, the state of Michigan implemented several enhanced biosecurity measures to limit the occurrence of BKD in state-operated hatcheries and gamete-collection weirs. The objectives of this study were to assess if infection levels (prevalence and intensity) of Renibacterium salmoninarum, the causative agent of BKD, have changed in broodstock and pre-stocking fingerlings of three feral Oncorhynchus spp. (Chinook salmon (O. tshawytscha), coho salmon (O. kisutch), and steelhead (O. mykiss)) over a decade, following the implementation of the enhanced biosecurity measures. Between 2001 and 2010, a total of 3,530 broodstock salmonids collected from lakes Huron and Michigan tributaries during spawning runs and 4,294 propagated pre-stocking salmonid fingerlings collected from three state of Michigan fish hatcheries were tested for the presence of R. salmoninarum antigens using the enzyme-linked immunosorbent assay. Substantial declines in the overall prevalence of the bacterium were detected in each of the examined broodstocks. Most propagated pre-stocking fingerlings also exhibited substantial declines in R. salmoninarum prevalence. Prevalence was typically higher in Chinook salmon from Lake Michigan than from Lake Huron; prevalence was also generally higher in the Hinchenbrooke strain of coho salmon than in the Michigan-adapted strain. For most strains and stocks examined, intensity of R. salmoninarum infection was found to have declined. Although there were declines in the potential for shedding the bacteria for both male and female Chinook and coho salmon, overall shedding rates were generally low (<15%) except for Hinchenbrooke coho salmon strain, which had shedding prevalences in excess of 50% at the beginning of the study. This study provides evidence that enhanced biosecurity measures at culture facilities and collection sites are capable of severely curtailing disease infection in wild populations even at the scale of Lake Michigan fisheries.

Spread of the emerging viral hemorrhagic septicemia virus strain, genotype IVb, in Michigan, USA.

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.