Development of silver nano-based indirect ELISA and Dot-ELISA methods for serological diagnosis of a bacterial fish pathogen Aeromonas veronii.

Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach using nano­silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 µg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 µg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis.

Livoneca redmanii (Isopoda, Cymothoidae) in meagre Argyrosomus regius: parasitological and molecular diagnosis and proposed control measure.

Isopodiosis in cultured meagre Argyrosomus regius was investigated at 3 farms in the northern lakes of Egypt throughout 2018, based upon prevalence rate, parasitological examination, and molecular identification by PCR targeting the large ribosomal subunit 16S of the rRNA gene. Further, the susceptibility of A. regius to isopod infection was experimentally evaluated under hyposalination of 25, 15, and 8 ppt for 1 wk. The isolated isopod stages were morphologically identified as Livoneca redmanii Leach, 1818 with prevalence rates of 77.05 and 77.9% in Al-Madiyyah and Sidi Krir, respectively. The highest prevalence and salinity of 78.85% and 30-34 ppt were reported in El Matareya. DNA sequencing and molecular analysis confirmed the identification of L. redmanii. A. regius experimentally infected with L. redmanii under a hyposalination protocol at 15 and 8 ppt showed marked reduction in mortality (20 and 50%, respectively), and infection prevalence (40 and 63.33%, respectively). The serum lysozyme concentration and nitric oxide of treated fish at 15 and 8 ppt were significantly increased compared to those held at 25 and 33 ppt, whereas serum osmolality levels were higher at 25 and 33 ppt. To our knowledge, this is the first molecular characterization of L. redmanii in cultured A. regius in Egypt.

Amyloodiniosis in cultured Dicentrarchus labrax: parasitological and molecular diagnosis, and an improved treatment protocol.

Amyloodinium ocellatum, the causative agent of amyloodiniosis (marine velvet, velvet disease), affects marine and brackish fish in various warm and temperate habitats. We recorded disease outbreaks with high morbidity and mortality rates in marine-cultured European seabass Dicentrarchus labrax fry at 2 locations in northwest Egypt. The sudden outbreak, high morbidity and mortality rates, and skin lesions with a velvety appearance in affected fish all indicated A. ocellatum infection. This was further confirmed by microscopic findings of the parasitic stage (trophonts) in skin and gill smears. While ecological factors including water temperature and salinity were all amenable to parasite establishment and propagation, mortality rates differed between the 2 farms, with rates of mortality well correlated with prevalence and intensity of A. ocellatum infections. Characterization by PCR targeting rDNA gene fragments and subsequent DNA sequencing and phylogenetic analysis further confirmed the molecular identity of the A. ocellatum isolate, which was genetically similar to isolates from other geographical locations. Finally, an improved treatment method using dual hyposalination and copper sulfate exposure to increase the efficiency and decrease the toxicity of copper sulfate was tested. The gradual reduction in water salinity coupled with copper sulfate treatment was more efficient at controlling the disease than only applying copper sulfate. To our knowledge, this is the first parasitological and molecular characterization of A. ocellatum in marine cultures in Egypt. The high molecular identity and close phylogenetic relationship further confirmed the monophyletic nature of A. ocellatum isolates.

Epizootics of Pseudomonas anguilliseptica among cultured seabream (Sparus aurata) populations: Control and treatment strategies.

The present study was investigating the clinical pictures, prevalence, as well as the ecological conditions associated with Pseudomonas anguilliseptica outbreaks in four cultured seabream, Sparus aurata farms at different localities in Egypt during winter of 2016. The phenotypic and genotypic patterns of Pseudomonas isolates were investigated. The existence of intraspecific heterogeneity among different isolates was analyzed using Restriction Fragment Length Polymorphism (RFLP) technique. Attempts on disease control using antibiogram or dietary supplement were also considered. To achieve these goals, various commercial antibiotic discs were analyzed against Ps. anguilliseptica isolates using the disc diffusion method. Additionally, the impact of one-month dietary incorporation with 3% garlic extract or 0.5% potassium diformate on S. aurata viability and response for prolonged bathing treatment with florfenicol was evaluated following challenge with the virulent strain of Ps. anguilliseptica. Most of the naturally infected fish displayed spiral-swimming behavior with no obvious external lesions. The prevalence of infections in the four investigated farms (F1, F2, F3, and F4) were 44.9, 69.04, 67.72, and 83.4%, respectively. Water analysis revealed a significant variation in total hardness, pH, dissolved oxygen (D.O), ammonia and salinity among different localities. All isolates were rather uniform in most of the biochemical characteristics and were identical on the basis of RFLP analysis. The analyses of PAF-PAR gene pointed out specific amplification bands of 439 bp length. The antibiogram revealed a potential activity of florfenicol, ciprofloxacin, nitrofurantoin, and oxytetracycline against all isolates. Experimentally challenged fish fed on garlic extract or potassium diformate presented lower mortality and better therapeutic response to florfenicol than those fed on a normal basal diet. In conclusion, Ps. anguilliseptica is a prevalent pathogen among cultured seabream where dietary inclusion of 3% garlic extract or 0.5% potassium diformate seemed to improve seabream health status and subsequently, increase the efficacy of the treatment with the selective antibiotic.