The role of marine pollution on the emergence of fish bacterial diseases.

Marine pollution and bacterial disease outbreaks are two closely related dilemmas that impact marine fish production from fisheries and mariculture. Oil, heavy metals, agrochemicals, sewage, medical wastes, plastics, algal blooms, atmospheric pollutants, mariculture-related pollutants, as well as thermal and noise pollution are the most threatening marine pollutants. The release of these pollutants into the marine aquatic environment leads to significant ecological degradation and a range of non-infectious disorders in fish. Marine pollutants trigger numerous fish bacterial diseases by increasing microbial multiplication in the aquatic environment and suppressing fish immune defense mechanisms. The greater part of these microorganisms is naturally occurring in the aquatic environment. Most disease outbreaks are caused by opportunistic bacterial agents that attack stressed fish. Some infections are more serious and occur in the absence of environmental stressors. Gram-negative bacteria are the most frequent causes of these epizootics, while gram-positive bacterial agents rank second on the critical pathogens list. Vibrio spp., Photobacterium damselae subsp. Piscicida, Tenacibaculum maritimum, Edwardsiella spp., Streptococcus spp., Renibacterium salmoninarum, Pseudomonas spp., Aeromonas spp., and Mycobacterium spp. Are the most dangerous pathogens that attack fish in polluted marine aquatic environments. Effective management strategies and stringent regulations are required to prevent or mitigate the impacts of marine pollutants on aquatic animal health. This review will increase stakeholder awareness about marine pollutants and their impacts on aquatic animal health. It will support competent authorities in developing effective management strategies to mitigate marine pollution, promote the sustainability of commercial marine fisheries, and protect aquatic animal health.

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.

Cyathocotylidae spp and motile aeromonads co-infections in farmed Nile tilapia (Oreochromis niloticus) causing mass mortality.

Motile aeromonads, and Cyathocotylidaespp.co-infections were identified in farmed Nile tilapia(Oreochromis niloticus) which suffering from mortalities. Moribund fish showed signs of septicemia, skin irritations, and respiratory distress. A total of 150 O. niloticus specimens showing signs of disease were collected from the affected earthmen ponds and examined. Bacteriological examination of fish samples revealed infections with motile aeromonads species. Phenotypic characteristics and phylogenetic analysis of gyrB gene sequences of aeromonads isolates identified them as Aeromonas hydrophila (12.6%), A.sobria (12.6%), and A. caviae (30.4%). Aeromonads strains harbored some virulence genes: Aer (78.62%); Hyl (60.86%); laf-A (52.17%); and Act (47.82%). The antibiogram of aeromonads showed high resistance against tetracycline (73.9%), and gentamycin (65.2%), while a high sensitivity was noticed to ciprofloxacin (82.6%),and trimethoprim/sulfamethoxazole (60.86%). Parasitological examination of fish revealed the presence of Cyathocotylidae spp. encysted metacercaria (EMC). High levels of interleukin 6 (IL-6) and cluster of differentiation 4 (CD4) were noticed in fish with parasitic and bacterial co-infection compared to those with a single infection or non-infected. Experimentally infected fish with Aeromonas spp. showed septicemic signs similar to that noticed in naturally infected tilapia with variable cumulative mortality. The study is one of the earlier reports identifying as Cyathocotylidae spp., and motile aeromonads co-infections, and their link with the exaggerated tilapia mortality which will be of value for incorporating these pathogens in the necessary management strategies to protect fish health.

Dactylogyrus extensus and Pseudomonas fluorescens dual infection in farmed common carp (Cyprinuscarpio).

Dactylogyrus extensus and Pseudomonas fluorescens are serious pathogens in Cyprinus carpio aquaculture causing severe impacts and substantial economic losses. During the early spring of 2021, abnormal mortalities were reported among farmed C. carpio. Moribund fish showed anorexia, respiratory distress, dermal ulcers, and septicemia. The water analysis revealed low dissolved oxygen (3.4 mg/L), and high un-ionized ammonia levels (0.65 mg/L). Seventy moribund C. carpio specimens were collected and subjected to parasitological and bacteriological examinations. The monogenetic trematode D. extensus was discovered in wet mounts from the gills of all the examined fish samples (100%). The identity of recovered parasites was confirmed by sequencing and alignment of the 28S rDNA gene. P. fluorescens was concurrently identified in the infested fish samples (58.5%) based on phenotypic characteristics using the API20 E. The identity of bacterial isolates was confirmed further by sequencing and alignment of 16S rRNA gene. The IL-1ß and MHCII were upregulated in infested fish in tandem with the severity of infections. P. fluorescens isolates displayed high resistance to most of the tested antibiotics. The study is one of the earlier reports on D. extensus and P. fluorescens co-infections in farmed C. carpio and highlights the need of effective control programs to protect fish health and minimize losses.

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.

Genetic characterization and antimicrobial profiling of bacterial isolates collected from Nile tilapia (Oreochromis niloticus) affected by summer mortality syndrome.

In recent years, Egyptian tilapia aquaculture has experienced mortality episodes during the summer months. The causative agents responsible for such mortalities have not been clearly identified. A total of 400 fish specimens were collected from affected tilapia farms within five Egyptian governorates. A total of 344 bacterial isolates were identified from the examined fish specimens. Bacterial isolates were grouped into seven genera based on API 20E results. The most prevalent pathogens were Aeromonas spp. (42%), Vibrio spp. (21%), and Streptococcus agalactiae (14.5%). Other emerging infections like, Plesiomonas shigelloides (10%), Staphyloccocus spp. (8%), Pseudomonas oryzihabitans, and Acinetobacter lwoffii (2.3%) were also detected. Sequence analysis of the 16S ribosomal RNA bacterial gene of some isolates, confirmed the phenotypic identification results. The analysis of antibiotic resistance genes revealed the presence of aac(6')-Ib-cr (35.7%), blaCTX gene (23.8%), qnrS (19%), ampC (16.7%), floR (14.3%), sul1, tetA, and van.C1 (2.4%) genes in some isolates. The antimicrobia resistance gene, qac was reported in 46% of screened isolates. Bacterial strains showed variable virulence genes profiles. Aeromonas spp. harboured (act, gcat, aerA, lip, fla, and ser) genes. All Vibrio spp. possessed the hlyA gene, while cylE, hylB, and lmb genes, were detected in S. agalactiae strains. Our findings point to the possible role of the identified bacterial pathogens in tilapia summer mortality syndrome and highlight the risk of the irresponsible use of antibiotics on antimicrobial resistance in aquaculture.

Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities.

Edwardsiellosis is a serious bacterial disease affecting Nile tilapia (Oreochromis niloticus), causing septicemia and mortalities. Edwardsiella tarda and Edwardsiella anguillarum were isolated from Nile tilapia summer mortality events in Egypt. Diseased fish showed hemorrhagic septicemia, skin erosions, and eye opacity. A total of 24 Edwardsiella spp. isolates were retrieved from the investigated fish specimens. Phenotypic and biochemical characteristics grouped isolates into typical Ed. tarda (n = 14 strains) and atypical Ed. tarda (n = 10 strains). The BLAST analysis of sodB gene sequencing confirmed the conventional identification of typical Ed. tarda strains (n = 14) and reidentified all the atypical strains (n = 10) as Ed. anguillarum. Isolates showed a combination of virulence factors, including biofilm formation (66.6%), hemolysis (100%), chondroitinase (50%), and proteolytic activity (20.8%). The major part of isolates showed high resistance to ampicillin, amoxicillin, gentamycin antibiotics and harbored tetA, blaCTX-M, and aadA1 resistance genes. Pathogenicity testing of isolates in O. niloticus confirmed their virulence. Challenged fish exhibited septicemic signs similar to naturally diseased fish. Infections in naturally infected tilapia triggered acute and chronic histopathological alterations. Degenerative and necrotic changes were noticed in hematopoietic organs. Granulomas were noticed in between the hepatic parenchyma. The data extracted from the study confirm that accurate identification of the causative agents of edwardsiellosis should be reliant on genetic-based approaches. Analysis of the bacterium virulence properties offers insights into establishing novel therapeutics for edwardsiellosis control. The findings refer to the need for antimicrobial sensitivity testing to minimize antimicrobial resistance and increase therapy efficacy.

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.

Coinfections of Aeromonas spp., Enterococcus faecalis, and Vibrio alginolyticus isolated from farmed Nile tilapia and African catfish in Egypt, with an emphasis on poor water quality.

The high mortality rate among Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus) polycultured in earthen ponds in Manzala, Egypt, was investigated. Mortality has been linked to poor water quality parameters accompanied with bacterial infections. Moribund farmed fishes exhibited general septicemic signs. Fish from both species (45 each) were sampled and analyzed bacteriologically. Vibrio alginolyticus (32.3%), Enterococcus faecalis (29.4%), Aeromonas caviae (23.5%), and A. veronii (14.7%) were isolated from moribund fishes using selective media and further identified by biochemical tests. 16S rRNA gene sequencing and phylogenetic analysis confirmed the identity of these isolates. Experimental infection of O. niloticus with different bacterial isolates resulted in clinical signs of hemorrhagic septicemia and mortality rates of 80%, 60%, 40%, and 30%, respectively, for E. faecalis, A. veronii, V. alginolyticus, and A. caviae. Water parameter analysis revealed marked divergence from typical values. In addition, different bacterial isolates (including Staphylococcus sciuri, S. aureus, E. faecalis, A. veronii, A. caviae, and V. alginolyticus) were identified and isolated from water samples. BLAST analysis of water bacterial isolates displayed a 100% similarity score with relevant fish isolates, indicating that the water was the likely source of infections. Histopathological examination revealed signs of bacterial infection in both fish species. In addition, common circulatory and degenerative changes with melanophore aggregation, and lymphocytic depletion in hematopoietic organs were recorded.

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.

Rapid identification of pathogenic streptococci isolated from moribund red tilapia (Oreochromis spp.).

Accurate and rapid identification of bacterial pathogens of fish is essential for the effective treatment and speedy control of infections. Massive mortalities in market-sized red tilapia (Oreochromis spp.) were noticed in mariculture concrete ponds in northern Egypt. Histopathological examination revealed marked congestion in the central vein of the liver with the presence of bacterial aggregates inside the lumen and in the vicinity of the central vein. A total of 12 isolates of streptococci were obtained from the moribund fish. This study documented the ability of the MicroSeq 500 16S bacterial sequencing method to accurately identify Streptococcus agalactiae and S. dysgalactiae mixed infections from moribund red tilapia that were difficult to be recognised by the commercial biochemical systems. The continuously decreasing cost of the sequencing technique should encourage its application in routine diagnostic procedures.