Medicinal plants are increasingly used in aquaculture owing to their beneficial impacts on the health status of farmed fish. The current study was conducted to investigate the effect of nutmeg (Myristica fragrans) extract on growth, immunity, antioxidant parameters, and resistance of common carp (Cyprinus carpio) against Aeromonas hydrophila. In addition, in vitro antibacterial activity of the skin mucus of fish fed on nutmeg extract was evaluated against three major fish pathogenic bacteria through the standard disk diffusion method. Fish (17.27 ± 0.11 g) were divided into four groups and fed on experimental diets containing different levels of nutmeg extract, including zero (control), 0.5% (M1), 1% (M2), and 2% (M3) per kg diet. Results showed that nutmeg significantly enhanced growth parameters after a four-week feeding trial. Feed conversion ratio was remarkably reduced with the lowest value reported for the M3 group, whereas weight gain was notably increased in M2 and M3. No significant effect was found on the hematological profile, including mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular volume, and hematocrit, while the highest levels of red blood cells and white blood cells were found in the M3 group. Stress biomarkers, including glucose and cortisol, were the lowest in the M3 group. Serum and skin mucus immunological and antioxidant parameters were significantly higher in M3, followed by M2, where the highest resistance was also observed. In addition, skin mucus samples effectively inhibited Streptococcus iniae, Yersinia ruckeri, and Aeromonas hydrophila. Overall, the present results suggest that dietary nutmeg (20 g/kg diet) could be used as a growth promotor and immunostimulant in common carp.

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7–3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno.

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 β, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.