Characterization of External Mucosal Microbiomes of Nile Tilapia and Grey Mullet Co-cultured in Semi-Intensive Pond Systems

The external mucosal surfaces of the fish harbor complex microbial communities, which may play pivotal roles in the physiological, metabolic, and immunological status of the host. Currently, little is known about the composition and role of these communities, whether they are species and/or tissue specific and whether they reflect their surrounding environment. Co-culture of fish, a common practice in semi-intensive aquaculture, where different fish species cohabit in the same contained environment, is an easily accessible and informative model toward understanding such interactions. This study provides the first in-depth characterization of gill and skin microbiomes in co-cultured Nile tilapia (Oreochromis niloticus) and grey mullet (Mugil capito) in semi-intensive pond systems in Egypt using 16S rRNA gene-based amplicon sequencing. Results showed that the microbiome composition of the external surfaces of both species and pond water was dominated by the following bacterial phyla: Proteobacteria, Fusobacteriota, Firmicutes, Planctomycetota, Verrucomicrobiota, Bacteroidota, and Actinobacteriota. However, water microbial communities had the highest abundance and richness and significantly diverged from the external microbiome of both species; thus, the external autochthonous communities are not a passive reflection of their allochthonous communities. The autochthonous bacterial communities of the skin were distinct from those of the gill in both species, indicating that the external microbiome is likely organ specific. However, gill autochthonous communities were clearly species specific, whereas skin communities showed higher commonalities between both species. Core microbiome analysis identified the presence of shared core taxa between both species and pond water in addition to organ-specific taxa within and between the core community of each species. These core taxa included possibly beneficial genera such as Uncultured Pirellulaceae, Exiguobacterium, and Cetobacterium and opportunistic potential pathogens such as Aeromonas, Plesiomonas, and Vibrio. This study provides the first in-depth mapping of bacterial communities in this semi-intensive system that in turn provides a foundation for further studies toward enhancing the health and welfare of these cultured fish and ensuring sustainability

Optimization of Low-Biomass Sample Collection and Quantitative PCR-Based Titration Impact 16S rRNA Microbiome Resolution

The major aquatic interface between host and environment in teleost finfish species is the gill. The diversity of this infraclass, high complexity of the organ, and its direct exposure to the surrounding environment make it an ideal candidate for furthering our understanding of the intertwined relationships between host and microbiome. Capturing the structure and diversity of bacterial communities from this low-biomass, inhibitor-rich tissue can, however, prove challenging. Lessons learned in doing so are directly applicable to similar sample types in other areas of microbiology. Through the development of a quantitative PCR assay for both host material and 16S rRNA genes, we tested and developed a robust method for low-biomass sample collection which minimized host DNA contamination. Quantification of 16S rRNA facilitated not only the screening of samples prior to costly library construction and sequencing but also the production of equicopy libraries based on 16S rRNA gene copies. A significant increase in diversity of bacteria captured was achieved, providing greater information on the true structure of the microbial community. Such findings offer important information for determining functional processes. Results were confirmed across fresh, brackish, and marine environs with four different fish species, with results showing broad homology between samples, demonstrating the robustness of the approach. Evidence presented is widely applicable to samples similar in composition, such as sputum or mucus, or those that are challenging due to the inherent inclusion of inhibitors. IMPORTANCE The interaction between the fish gill and surrounding bacteria-rich water provides an intriguing model for examining the interaction between the fish, free-floating bacteria, and the bacterial microbiome on the gill surface. Samples that are inherently low in bacteria, or that have components that inhibit the ability to produce libraries that identify the components of microbial communities, present significant challenges. Gill samples present both of these types of challenges. We developed methods for quantifying both the bacterial and host DNA material and established a sampling method which both reduced inhibitor content and maximized bacterial diversity. By quantifying and normalizing bacteria prior to library construction, we showed significant improvements with regards to the fidelity of the final data. Our results support wide-ranging applications for analyzing samples of similar composition, such as mucus and sputum, in other microbiological spheres

Exploring the impact of thermal delousing on gill health and microbiome dynamics in farmed Atlantic Salmon

The economic cost of salmon louse (Lepeophtheirus salmonis and Caligus rogercresseyi) infestations in the Atlantic salmon (Salmo salar) industry has been estimated to be around $900 million annually. This high cost has driven a concerted effort to develop, examine, understand, and implement various methods for louse control. Husbandry interventions utilising warm water exposure have been highly successful in complementing traditional chemotherapeutants, especially as the efficiency of the latter has reduced in recent years. In this study, we sought to examine the impact of thermal delousing on gill health in two commercial sites with different historical husbandry and treatment interventions prior to and post-thermal treatment. Methods to characterise gill health and the response to thermal treatment included a detailed examination of the gill for microparasites using both histology and qPCR and targeted immune gene expression analysis, most notably antigen-presenting cells (mhc ii), proinflammatory cytokines (il-1β and tnf-α) and inhibitory cytokines (tgf-β and il-10). Furthermore, we examined the bacterial communities present on the gill surface using 16S rRNA amplicon sequencing. Data obtained from these trials indicated a minimal impact on gill microparasite prevalence in response to the thermal treatment. The expression of immune markers exhibited a significant decrease across both sites after treatment. Intriguingly, marked differences in the gill bacterial communities in response to treatment between the two sites were clearly observed. This divergence could be attributed to the notable differences in husbandry history and health status of the fish at the two sites prior to the thermal treatment. Our data suggest that microbiome diversity is an informative indicator of fish gill health and could be used to define appropriate interventions when treating sea lice

Net cleaning impacts Atlantic salmon gill health through microbiome dysbiosis

Introduction: Net biofouling has a significant impact for the global salmon industry in the seawater grow-out stage in terms of its management. Current mitigation strategies occur primarily through the regular removal of biofouling using in situ cleaning. While in situ net cleaning is effective there is uncertainty as to whether the equipment or dispersed material has an impact upon the fish in the cages. Through direct contact with the environment, the significant surface area of the gill including its microbiome is directly exposed to the acute environmental changes generated by net cleaning. This study aimed to provide a detailed understanding of the impact of in situ net cleaning on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) gill health. Methods: Three field trials were conducted on commercial fish farms in western Norway. Fouling organisms on net pens and flushed particles during in situ cleaning were identified and screened for major fish pathogens. Hydrographic profile measurements were performed to measure the impact on water quality. Gill samples were examined for histopathological changes, immune gene expression, and the prevalence of major pathogens. 16S rRNA amplicon sequencing was employed to explore the impact of net cleaning on gill microbiome. Results and discussion: Data obtained from these trials identified a diversity of fouling species including hydroids, algae, skeleton shrimps, and filter feeders on net pens, a direct impact on measured water quality indicators, a moderate change in gill inflammatory and antigen presentation activity at the level of mRNA, and a large significant change in gill microbiome. Observed changes in gill microbial community involved a decrease in bacterial richness coupled to an increase in identified bacterial genera related to negative health consequences. Parallel analyses for pathogens load in biofouling organisms and flushed particles highlighted the presence of several fish bacteria and parasites. However, minor changes were detected in salmon gill pathogen diversity and loading. Our results suggest that biofouling organisms may act as transient reservoirs for some fish pathogens but not viruses and that gill microbial dysbiosis could be related to the host stress response during and post net cleaning

Collagen extract obtained from Nile tilapia (Oreochromis niloticus L.) skin accelerates wound healing in rat model via up regulating VEGF, bFGF, and α-SMA genes expression

Background Collagen is the most abundant structural protein in the mammalian connective tissue and represents approximately 30% of animal protein. The current study evaluated the potential capacity of collagen extract derived from Nile tilapia skin in improving the cutaneous wound healing in rats and investigated the underlying possible mechanisms. A rat model was used, and the experimental design included a control group (CG) and the tilapia collagen treated group (TCG). Full-thickness wounds were conducted on the back of all the rats under general anesthesia, then the tilapia collagen extract was applied topically on the wound area of TCG. Wound areas of the two experimental groups were measured on days 0, 3, 6, 9, 12, and 15 post-wounding. The stages of the wound granulation tissues were detected by histopathologic examination and the expression of vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-ß1) were investigated using immunohistochemistry. Moreover, relative gene expression analysis of transforming growth factor-beta (TGF-ß1), basic fibroblast growth factor (bFGF), and alpha-smooth muscle actin (α-SMA) were quantified by real-time qPCR. Results The histopathological assessment showed noticeable signs of skin healing in TCG compared to CG. Immunohistochemistry results revealed remarkable enhancement in the expression levels of VEGF and TGF-β1 in TCG. Furthermore, TCG exhibited marked upregulation in the VEGF, bFGF, and α-SMA genes expression. These findings suggested that the topical application of Nile tilapia collagen extract can promote the cutaneous wound healing process in rats, which could be attributed to its stimulating effect on recruiting and activating macrophages to produce chemotactic growth factors, fibroblast proliferation, and angiogenesis. Conclusions The collagen extract could, therefore, be a potential biomaterial for cutaneous wound healing therapeutics. Backgroun

Exploring the multimodal role of yucca schidigera extract in protection against chronic ammonia exposure targeting: Growth, metabolic, stress and inflammatory responses in nile tilapia (oreochromis niloticus l.)

Ammonia is a critical hazardous nitrogen metabolic product in aquaculture. Despite trials for its control, ammonia intoxication remains one of the most critical issues to overcome. In this study, we explored the modulatory effect and potential mechanism by which Yucca schidigera extract (YSE) can ameliorate ammonia intoxication-induced adverse effects on tilapia health and metabolism. A total number of 120 Nile tilapia were evenly assigned into four groups with three replicates each. The first group served as normal control group; the second group was exposed to ammonia alone from the beginning of the experiment and for four weeks. The third group was supplied with YSE in water at a dose of 8 mg/L and exposed to ammonia. The fourth group was supplied with YSE only in water at a dose of 8 mg/L. YSE supplementation succeeded in improving water quality by reducing pH and ammonia levels. Moreover, YSE supplementation markedly alleviated chronic ammonia-induced adverse impacts on fish growth by increasing the final body weight (FBW), specific growth rate (SGR), feed intake and protein efficiency ratio (PER) while reducing the feed conversion ratio (FCR) via improvements in food intake, elevation of hepatic insulin-like growth factor (ILGF-1) and suppression of myostatin (MSTN) expression levels with the restoration of lipid reserves and the activation of lipogenic potential in adipose tissue as demonstrated by changes in the circulating metabolite levels. In addition, the levels of hepato-renal injury biomarkers were restored, hepatic lipid peroxidation was inhibited and the levels of hepatic antioxidant biomarkers were enhanced. Therefore, the current study suggests that YSE supplementation exerted an ameliorative role against chronic ammonia-induced oxidative stress and toxic effects due to its free radical-scavenging potential, potent antioxidant activities and anti-inflammatory effects

Dietary clenbuterol modifies the expression of genes involved in the regulation of lipid metabolism and growth in the liver, skeletal muscle, and adipose tissue of Nile tilapia (Oreochromis niloticus)

The current study aimed to evaluate whether clenbuterol, a β2-adrenergic agonist, supplementation in Nile tilapia (Oreochromis niloticus) diets can influence growth and blood parameters. Besides, assessment of adipogenic genes as fatty acid synthase (FAS) and lipoprotein lipase (LPL) which is a key enzyme in the regulation of the flux of fatty acids in liver, muscle, and adipose tissue as well as muscle growth-regulating genes as myostatin (MYO) in muscle and insulin-like growth factor-1 (IGF-1) in liver. The fish were allocated into three equal groups; control group that fed basal diet only and the other two groups fed a basal diet containing clenbuterol at two doses 5 ppm and 10 ppm/kg diet for 30 consecutive days. Results revealed that clenbuterol supplementation significantly increased body weight, decreased liver, spleen and abdominal fat weights, and decreased total circulatory cholesterol and triacylglycerol levels. Moreover, clenbuterol inhibits lipogenesis by downregulation of FAS gene expression by dose and time-dependent manner in the liver while enhanced lipolysis in both the liver and in the adipose tissue. Moreover, lipolysis was reduced in muscle by dose 10 ppm on day 30. Furthermore, clenbuterol presented higher gene expression of MYO and IGF-1 in muscle and liver respectively by dose 5 ppm at day 15 on the other hand, these findings were reversed by day 30 compared with control. In conclusion, clenbuterol efficacy was apparent in a dose and time response pattern to boost growth and reduce fat deposition rates, indicating for the first time that clenbuterol has a profitable growth impact on Nile tilapia

Impact of husbandry practice on the gill microbiome of Salmo salar and Oreochromis niloticus

The major aquatic interface between host and environment in teleost finfish species is the gill. The diversity of this infraclass, high complexity of the organ and direct exposure to the surrounding environment make it an ideal candidate for furthering our understanding of the intwined relationships between host and microbiome and for monitoring health status in aquaculture practices. Capturing the structure and diversity of bacterial communities from this low biomass, ‘inhibitor-rich’ tissue can, however, prove challenging. Moreover, little is known about the gill microbiome of farmed fish with particular emphasis to the impact of farm husbandry practises, and its potential use as a health and welfare management tool. Through the development of qPCR assay for both host genomic DNA and bacterial 16S rRNA gene, and exploring the community composition using amplicon sequencing, a robust method for sample collection for gill microbiome analysis was developed. The impact of normalising 16S rRNA amplicon sequencing libraries to equal copy numbers of 16S rRNA, starting material concentration, and microbiome DNA enrichment on the recovered microbial diversity were explored. Results showed that the non-invasive approach of gill sampling (swabbing) significantly improved the bacterial 16S rRNA recovery, minimised host DNA, and thereby remarkably increased abundance and diversity of bacteria captured as opposed to the other sampling approaches (surfactant-washed gills and whole gill tissue). Quantifying 16S rRNA and normalising the input to equal copy numbers of 16S rRNA prior to costly library construction showed significant improvements with regards the fidelity of the final data. Utilising the optimised protocol for gill microbiome sampling and analysis, in-depth characterisation of gill and skin microbiomes in co-cultured Nile tilapia (Oreochromis niloticus) and grey mullet (Mugil capito) in semi-intensive pond systems was performed. In addition, the impact of farm practices on the Atlantic salmon (Salmo salar) gill health in terms of microbiome and the associated immune response were investigated. In semi-intensive pond systems, the external autochthonous communities of co-cultured tilapia and mullet were not a passive reflection of their allochthonous communities. The autochthonous bacterial communities of the skin were distinct from those of the gill in both species, indicating that the external microbiome is likely organ specific. Gill autochthonous communities were clearly species specific, whereas skin communities showed higher commonalities between both species. Finally, salmon farm practises including thermal treatment for sea lice, freshwater treatment for amoebic gill disease, and net cleaning had a significant impact on markers of gill immune response and gill microbial community composition. These observations highlight the promising use of gill microbiome as a health and welfare management tool

Biological fixation of comminuted subtrochanteric fractures with proximal femur locking compression plate

Introduction: Subtrochanteric fractures are challengeable fractures that are associated with high incidence of complications and various intramedullary and extramedullary implants, are available for their fixation.(1) Traditional extramedullary implants are associated with higher rate of implant failure and varus collapse while the intramedullary nails are biomechanically better but technically demanding and are associated with higher re-operation rates.(2)-(3) This study was done to evaluate the outcome following biological (indirect) fixation of comminuted subtrochanteric fractures with proximal femur locking plate (PF-LCP).  Methods: Thirty   patients with comminuted subtrochanteric fractures were operated upon with PF-LCP (Synthes) by using an indirect reduction technique. Russell Taylor types A and B fractures were included in the study. Operating time, blood loss and any technical difficulty with the implant were recorded. Patients were followed clinically and radiologically for union at fracture site and implantrelated complications. The Harris Hip Score was used to document hip function at final follow-up.  Results: The mean operating time was 90.5 min and total blood loss averaged 241.4 ml. Union was achieved in all cases with an average time of 15.87weeks. Complications included two cases of delayed union and two cases of infection.&nbsp

Assessment of functional capacity of patients with chronic heart failure by the use of echocardiography right atrial volume index

Introduction: Heart failure (HF) is a chronic syndrome that influences survival & quality of life. over the past 20 years, hospitalizations for heart failure have almost tripled, making heart failure the most common cause of hospitalization.  Objective: evaluation of relationship between functional capacity by duke activity status index(DASI) in chronic heart failure patients and the right atrial volume index. Materials and methods: study was conducted on 40 patients who were admitted in the hospital with heart failure.All patients were subjected to detailed history, physical examination, electrocardiography, laboratory investigations, 2D echocardiography & tissue Doppler imaging and assessment of functional capacity using DASI. Results: Out of 40 patients, 10 (25%) had DASI<10 and 30 (75%) patients had DASI>10, according to functional capacity between the two groups, group I Vo2 peak (ml/min) mean is 12.98±1.45 and DASI score ranged from (4.5-9.95) with a median 8.6, in group II Vo2 peak (ml/min) mean is 21.89±4 and DASI score ranged from (23.3- 36.7) with a median 28.7with p value <0.001. RAVI had a significant correlation between the two groups with p-value <0.001. Also, DASI have a strong correlation with RAVI (r=1.139 & p=0.029). Conclusions: RAVI predicts functional capacity in chronic stable heart failure patients.&nbsp