Infected Fish Research Articles

Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Enrofloxacin, Effective Treatment of Pseudomonas aeruginosa and Enterococcus faecalis Infection in Oreochromis niloticus

Enrofloxacin is a broad-spectrum synthetic antimicrobial drug widely used in veterinary medicine. The present study aimed to determine the effective enrofloxacin dose for treating Pseudomonas aeruginosa and Enterococcus faecalis infection in Oreochromis niloticus. P. aeruginosa and E. faecalis isolates were verified using selective differential media and biochemically using the Vitek 2 test. Bacterial isolates were virulent for O. niloticus with LD50 equal to 2.03 × 106 and 2.22 × 107 CFU fish−1 for P. aeruginosa and E. faecalis, respectively. Infected fish suffered from decreased feed intake followed by off-food, tail erosion, darkening of the external body surface, exophthalmia, ascites, and loss of escape reflex. Internally, congested hemorrhagic hepatopancreas with engorged distended gall bladder were dominant. The posterior kidney was congested with enlarged spleen, and empty elementary tract. Pathologically, severe degenerative changes were dominant in the hepatopancreas, posterior kidney, spleen, stomach, and gills of infected fish. Antimicrobial sensitivity test indicated the high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin with MIC estimated at 1 and 0.0625 µg/mL, respectively. Enrofloxacin effectively protected O. niloticus against E. faecalis and P. aeruginosa infection when used with medicated feed at doses of 10 and 20 mg kg−1 body weight.

Effects of Single and Co-infections of Proteus Mirabilis and Aeromonas Hydrophila on Baseline Hematological, Serological, and Histological Data in Cultured Clarias Gariepinus

Background and Aim Significant mortality and production disruptions in fish culture are brought on by diseases and parasites. The purpose of the current study was to collect baseline data on the effects of single and co-infections of Proteus mirabilis, and Aeromonas hydrophila in Clarias gariepinus. Materials and Methods One hundred and twenty sub-adults of C. gariepinus were divided into control, P. mirabilis, A. hydrophila, and co-infection groups (P. mirabilis X A. hydrophila). Standard methods were used to determine hematology, serology, and histology. Standard microbiology methods were used for microbial analysis. Results The single A. hydrophila-infected group had the highest mortality (60% versus 37%) in the co-infected and P. mirabilis groups. A marked decrease was observed in the RBC, hemoglobin, and Packed Cell Volume (PCV) of 2.9×1012/L, 32.8 g/L, and 33.5% in the co-infected fish, compared to the control with 3.6×1012/L, 35.8 g/L, and 41.0%, respectively. Alanine transaminase, alkaline phosphatase, and aspartate transaminase levels were significantly lower in the co-infected fish (13.8, 236.0, and 66.3, respectively) compared to the A. hydrophila-infected group. Creatinine and urea levels were, however, higher in the co-infected treatment. The kidneys and livers of the A. hydrophila and co-infected groups were more severely damaged than those of the P. mirabilis and control groups. Vacuolation and necrosis of hepatocytes led to the desquamation of tubular and glomerular epithelial cells in the livers and kidneys of infected fish. Fish infected with A. hydrophila had the highest bacterial load count. Conclusion It was concluded that an antagonistic association exists between A. hydrophila and P. mirabilis when they are co-infected.

Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

Prevalence of Edwardsiella ictaluri in cage cultured Pangasius spp in Pahang River and their risk factors

Striped catfish, Pangasianodon hypophthalmus is a native species to most Asian countries, including Malaysia which faced series of bacterial disease such as edwardsiellosis caused by E.ictaluri. Edwardsiella ictaluri is a Gram-negative intracellular bacterial pathogen which had caused mortalities in farmed or wild fish in lots of countries and accountable for large economic losses. This study was conducted to determine disease status of Pangasius spp due to Edwardsiella ictaluri and to determine the risk factors of E.ictaluri in striped catfish in Pahang River, Malaysia. Four sampling sites were chosen, two sites in Pekan (Kampung Belimbing and Kampung Tanjung Pulai) and two sites in Temerloh (Kampung Teluk Ira and Kampung Bintang). Thirty samples were collected from each farm for 6 months consecutively and four organ tissues were taken which were liver, spleen, kidney, and brain. External and internal clinical signs were recorded accordingly. DNA was extracted from all tissues and pursued for PCR to detect the presence of Edwardsiella ictaluri. Results show that the infected fish has gross internal clinical signs such as patchy liver, white nodular spleen, and congested kidney. The prevalence rate of E.ictaluri was highest at Kampung Tanjung Pulai in June with 16.67% compared to all farms. No infection of E.ictaluri shown at Kampung Belimbing during the whole sampling session. Spearman’s rho correlation showed that the prevalence of E.ictaluri has strong correlation with temperature in Kampung Tanjung Pulai (ρ > 0.7). Meanwhile those bacteria prevalence have strong relationship with ammonia, sulfide and total suspended solid (TSS) with ρ > 0.5. The susceptible size for this bacterium in striped catfish is between 1 to 50g and 150g to 200g. To our knowledge, this paper is the first report for Edwardsiella ictaluri in cage cultured P.hypophthalmus in Pahang River, Malaysia.

Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.

We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc. Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays. The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book. The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Prevalence, genetic diversity, and antimicrobial susceptibility of Vibrio spp. infected gilthead sea breams from coastal farms at Damietta, Egypt

BackgroundVibriosis is one of the most serious bacterial diseases and causes high morbidity and mortality among cultured sea breams. This study was undertaken to track the surveillance of Vibrio infection and its correlation to environmental factors. A total of 115 gilthead sea breams were collected seasonally from a private earthen pond fish farm in the Shatta area of Damietta, Egypt from September 2022 to July 2023. Physicochemical parameters of water were analyzed, and heavy metal levels were measured. The fish samples were subjected to clinical, bacteriological, Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting, and hematoxylin and Eosin histopathological staining.ResultsThe results revealed significant variations in the water quality parameters over different seasons, in addition to an increase in heavy metals. Naturally infected fish showed external signs and postmortem lesions that were relevant to bacterial infection. Two dominant Vibrio subspecies of bacteria were identified: V. alginolyticus (205 isolates) and V. fluvialis (87 isolates). PCR confirmed the presence of V. alginolyticus using the species-specific primer collagenase at 737 bp. The highest prevalence of V. alginolyticus was detected during the summer season (57.72%), and the lowest prevalence was observed in autumn (39.75%). The correlation analysis revealed a positive relationship between V. alginolyticus and water temperature (r = 0.69). On the other hand, V. fluvialis showed a high prevalence during the autumn season (25.30%) and the lowest prevalence during the summer season (10.56%), where it was negatively correlated with water temperatures (r =—0.03). ERIC fingerprinting showed genetic variation within the Vibrio isolates. Antimicrobial susceptibility testing revealed sensitivity to ciprofloxacin and doxycycline, and resistance to amoxicillin and erythromycin. The multiple antibiotic resistance (MAR) index values for V. alginolyticus and V. fluvialis ranged from 0.3 to 0.7, with a multi-drug resistance pattern to at least three antibiotics. Histopathological alterations in the affected tissues revealed marked hemorrhage, vascular congestion, and hemosiderosis infiltration.ConclusionThis study provides insights into the potential propagation of waterborne diseases and antibiotic resistance in the environment. Ensuring that the environment does not serve as a reservoir for virulent and contagious Vibrio species is a critical concern for regional aquaculture industries. Therefore, we recommend implementing environmental context-specific monitoring and surveillance tools for microbial resistance.

Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi (Lates calcarifer)

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections lead to an inflammatory response in the host that is characterized by changes in leukocyte profile and activity. We hypothesized that the profile of circulating peripheral blood leukocytes and markers of their activity, such as reactive oxygen species production, would change following an event of bacterial infection in fish. We therefore developed a flow cytometry-based methodology for analyzing blood leukocytes in barramundi (Lates calcarifer), then used it to compare healthy fish to those infected with a common pathogenic bacterium, Vibrio harveyi. The initial step of the analysis relied on the effective separation of erythrocytes and leukocytes, and two methods were tested: one based on hypotonic lysis of red blood cells, and the other involving stain-based differentiation using carboxyfluorescein succinimidyl ester (CFSE). Fluorescence-activated cell sorting analysis revealed significant differences in leukocyte parameters of infected and control fish using the stain-based approach vs. hypotonic lysis. Changes in blood leukocytes included an increase in the proportion of monocytes and decrease in that of lymphocytes in infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time in infected fish as compared to controls. Monitoring peripheral blood leukocytes provides a non-lethal analysis of inflammation that allows repeated sampling of individual fish over time. This is beneficial given the high variability between individual fish. The use of CFSE-staining for leukocyte isolation proved to be advantageous over a method based on lysis of erythrocytes.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Immunological and molecular evaluation of zoonotic metacercarial infection in freshwater fish: A cross-sectional analysis

Improperly cooked fish, carrying active metacercariae (MCs), can pose a significant risk for transmitting fish-borne zoonotic trematodes (FBZTs) to human consumers. This study aimed to enhance our understanding of FBZTs by conducting a comprehensive cross-sectional analysis involving various fish species, such as Nile tilapia (Oreochromis niloticus), African catfish (Clarias gariepinus), and red-belly tilapia (Tilapia zillii). These fish specimens were collected from distinct Egyptian governorates, specifically Giza, Kafr al-Shaykh, and Fayoum. The recovered flukes from experimentally infected domestic pigeons were identified as Prohemistomum vivax, Haplorchis pumilio, and Pygidiopsis genata based on morphological features. Furthermore, the identity of the retrieved adult flukes was confirmed using three species-specific primers for PCR amplification and sequencing analysis of the ITS rDNA region and have been deposited in GenBank with the following accession numbers: P. vivax (OR291421.1 and OR291422.1), P. genata (OP099561.1), and H. pumilio (OM439581.1-OP090510.1).Quantitative real-time PCR targeting the immunological genes Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-1 (IL-1Β) was employed to compare the cellular immune response between infected with EMCs and uninfected O. niloticus. The results indicated a significant increase in TNF- and IL-1Β levels in FBZTs-infected vs un-infected fishes. Importantly, the presence of adult flukes and EMCs led to substantial histological alterations in both experimentally infected pigeons and naturally infected fish tissues. These changes included the necrosis of fish muscle bundles and a pronounced inflammatory reaction with muscular necrosis in the digestive tracts of experimentally infected pigeons.

Intestinal microbial profiling and immune responses of hybrid snakehead (Channa maculata ♀ × Channa argus ♂) challenged with Nocardia seriolae strain

Nocardiosis in fish, caused by Nocardia seriolae (a kind of filamentous bacterium), is a severe disease in aquaculture, affecting over 40 species of marine and freshwater fish and causing great losses. To investigate the relationship between fish nocardiosis and the gut microbiota, we first artificially infected hybrid snakehead (female Channa maculata × male Channa argus) with N. seriolae (1.0 × 107 CFU/mL), then utilized techniques such as high-throughput sequencing of 16 S rRNA, histopathology, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to analyze the changes in microbial community structure, intestinal histopathological characteristics, and related immune genes expression in hybrid snakehead. Changes in gut microbial community structure and diversity were observed starting from day 2, followed by fluctuations on day 4, and significant alterations on day 6. Four major phyla (Actinomycetes, Proteobacteria, Firmicutes, and Bacteroidetes), particularly Actinomycetes, reflected the temporal variations in the gut microbiota structure. The composition of the gut microbiota also underwent significant changes, with beneficial microorganisms such as Lactobacillus decreasing and harmful microorganisms such as Aeromonas and Nocardia increasing. The pathological sections showed that after N. seriolae injection, the fish intestinal villi became distorted and widely spaced, with increasing damage over time, leading to severe shrinkage and atrophy by the sixth day. Additionally, immune-related genes, such as TLR2 and IRF2, exhibited higher mRNA expression levels (P < 0.01) in the intestinal tissues of the infected fish. Overall, this study provides preliminary insights into the dynamic changes in gut microbial community, immune response, and intestinal pathogenic mechanisms during N. seriolae infection in hybrid snakehead, at both the levels of gut microbial composition and mRNA expression. These findings are important for better understanding and managing Nocardia infection in fish.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

Threat of heavy metal pollutants and parasites to freshwater fish with special reference to their risk of cancer to humans in Egypt

Understanding how pollution and parasite infection work together to stress their hosts' health raises the possibility of a danger to aquatic life and humans. This study examined two freshwater fish species, Clarias gariepinus, and Oreochromis niloticus, at El-Abbasa Fish farm in Sharkia Governorate of Egypt. to determine their heavy metal content, parasite frequency, and potential health risks. Parasitological investigations revealed higher parasite prevalence in C. gariepinus, with trematodes being the most prevalent. Histopathological examinations displayed severe alterations in infected fish, including gill necrosis, intestinal damage, liver degeneration, and muscle necrosis. Moreover, heavy metal analysis. Heavy metals levels in water, Fe, Zn, Pb, Cu, As, and Cd levels were 0.95 ± 0.04, 0.75 ± 0.03, 0.14 ± 0.02, 0.09 ± 0.01, 0.07 ± 0.01, and 0.03 ± 0.01 mg/L, respectively. Sediment samples showed Cd as the lowest (2.38 ± 0.94 μg/g dry wt. basis) and Fe as the highest (241.01 ± 6.39 mg/kg dry wt. basis). The sequence of heavy metal concentrations in both water and sediment was Fe > Zn > Pb > Cu > As > Cd. Significant differences were found among metals in water and sediment (p < 0.05). Heavy metal concentrations in fish tissues followed the order liver > muscles > gills > intestine Iron was the most abundant, while cadmium was the lowest is the order in which bio-accumulation factors (BAF) between water and fish tissues followed a descending order: BAF-Liver < BAF-Gills < BAF-Intestine < BAF-Muscles. The sequence of heavy metal accumulation in tissues was BAF-Fe > BAF-Pb > BAF-Cd > BAF-As > BAF-Cu > BAF-Zn, indicating varying degrees of accumulation across different metals and tissues and bio-sedimentation (BSF) of metals in fish tissues are the order of heavy metal concentration within tissues was BSF-Cd > BSF-As > BSF-Fe > BSF-Zn > BSF-Cu > BSF-Pb. Additionally, data were recoded to reflect histological changes in several fish organs. The estimated daily intake of heavy metals (EDI-HMs) was far lower than the permissible tolerated daily intake (PTDI) limits, which were set by international authorities at 70 kg of body weight. As the hazard index value is below 1, the THQ and HI associated with the consumption muscle of fish demonstrate negligible non-carcinogenic harm to residents' health. Contrarily, As, and Cd pose a cancer threat for kids and adults who eat muscular fish, and it needs to be viewed as a warning sign from the perspective of data indices and human health.

Identification and Antibiotic Resistance Edwardsiella tarda from Clown Knifefish (Chitala chitala) in the Mekong Delta, Vietnam

This investigation is intended to isolate, identify, and assess the pathogenicity of Edwardsiella tarda, which originated from diseased clown knifefish. A total of 43 isolates were obtained from infected fish samples in Hau Giang and Dong Thap provinces of the Mekong Delta, Vietnam. Two isolates of DT37 and HG41 were identified as E. tarda by morphological, biochemical, and 16S rRNA gene sequencing. Experimental challenge studies revealed that isolate DT37 leads to 83.33% at a 108 CFU/ml concentration after 60 hours. Meanwhile, in isolate HG41, mortality reached 100% within 48 hours post-injection at the highest concentration of 108 CFU/ml. The challenged clown knifefish exhibited gross signs of abnormal swimming, skin ulcerations, and petechial hemorrhages in the body. Internally, ascites with hemoperitoneum, light-colored nodules on the liver, hemorrhagic kidneys, and splenomegaly were also recorded. The LD50 of two isolates, DT37 and HG41, was 4.89 × 105 and 4.07 × 105 CFU/ml, respectively. The antibiogram result showed that most of the isolates were highly susceptible to ampicillin (65%), enrofloxacin (85%), florfenicol (100%), flumequine (90%), cefotaxime (80%), and trimethoprim and sulfamethoxazole (70%). However, the bacterial isolates were highly resistant to doxycycline (75%) and streptomycin (100%).

Predictive Model for the Nematode (Hysterothylacium aduncum) in Horse Mackerel, Trachurus trachurus From the Black Sea

Hysterothylacium aduncum is a parasitic nematode that infects mainly marine fish. The information on the heterogeneity in the aggregation of macroparasites in fish species is still limited, particularly for the Raphidascarid nematode H. aduncum that infects exploitable marine fish species including horse mackerel (Trachurus trachurus). This study aimed to investigate the heterogeneity of the distribution of H. aduncum to understand macroparasite aggregation better in marine fish. Weibull tests comprising Maximum likelihood were used to analyze the frequency distribution of parasites within a fish host population over the three years period. The parasite, H. aduncum (third larval stage) prevalence was found to be 88% and the condition factor was 1.30∓0.029 in un-infected fish and 1.12∓0.022 in infected fish. The clustered pattern was observed in H. aduncum in horse mackerel. The relation between parasite load and the fish condition factor was found to be significant and conformity of length and weight was affected by parasite load. It is the first time that aggregation has been observed in H. aduncum distribution and made forecasts of nematode intensity in horse mackerel. The findings related to the large individual variations suggest that this pattern is significant and should be considered for host-parasite dynamics.

Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus

In the present study, Aeromonas hydrophila was isolated from Tilapia zillii and Mugil cephalus samples collected during different seasons from various Suez Canal areas in Egypt. The prevalence of A. hydrophila, virulence genes, and antibiotic resistance profile of the isolates to the commonly used antibiotics in aquaculture were investigated to identify multiple drug resistance (MDR) and extensive drug-resistant (XDR) strains. In addition, a pathogenicity test was conducted using A. hydrophila, which was isolated and selected based on the prevalence of virulence and resistance genes, and morbidity of natural infected fish. The results revealed that A. hydrophila was isolated from 38 of the 120 collected fish samples (31.6%) and confirmed phenotypically and biochemically. Several virulence genes were detected in retrieved A. hydrophila isolates, including aerolysin aerA (57.9%), ser (28.9%), alt (26.3%), ast (13.1%), act (7.9%), hlyA (7.9%), and nuc (18.4%). Detection of antibiotic-resistant genes revealed that all isolates were positive for blapse1 (100%), blaSHV (42.1%), tetA (60.5%), and sul1 (42.1%). 63.1% of recovered isolates were considered MDR, while 28.9% of recovered isolates were considered XDR. Some isolates harbor both virulence and MDR genes; the highest percentage carried 11, followed by isolates harboring 9 virulence and resistance genes. It could be concluded that the high prevalence of A. hydrophila in aquaculture species and their diverse antibiotic resistance and virulence genes suggest the high risk of Aeromonas infection and could have important implications for aquaculture and public health.

Tilapia lake virus infection disrupts the gut microbiota of red hybrid tilapia (Oreochromis spp.)

Viral infections have the potential to damage fish intestines and disrupt the composition and diversity of the bacterial populations. In this study, we investigated the gut microbiota profiles of 23 red hybrid tilapia (10 ± 3 g) infected with Tilapia lake virus (TiLV) via two different methods. For the intraperitoneal (IP)-injection, 50 μl of TiLV at a concentration of 105 TCID50/ml was administered into the fish's coelomic cavity. In the cohabitation challenge method, infection was achieved by injecting the virus into the coelomic cavity of the inducer fish and allow them to cohabited with cohabitation fish at a ratio of 1:3. The cohabitation infected fish had a cumulative mortality rate of 49%, while the IP-injection group had 81.5%, which was accompanied by clinical signs, severe histopathological changes in the intestines and high viral loads in the intestines over the infection period at 6, 12 and 21 days post challenge (dpc). Most importantly, an analysis of the bacterial populations based on the alpha and beta diversity patterns revealed distinct alterations in the intestines of the fish infected via IP injection and cohabitation and the control fish. Specifically, our findings highlighted a reduction in Cetobacterium (phylum Fusobacterium) and an increase in Brevinema (phylum Spirochaetes) in the infected fish at 6 and 12 dpc. Indeed, the elevation of Brevinema population is associated with a higher mortality rate within the IP-injection compared to the cohabitation group. Remarkably, the fish that survived TiLV infection at 21 dpc showed a higher abundance of Bacillus (phylum Firmicutes), which was in contrast to that at earlier time points (6 and 12 dpc). While Brevinema has been recognised as a potentially opportunistic pathogen in fish, different strains of Bacillus are commonly used as probiotics to promote fish health and enhance disease resistance. Our findings thus provide valuable insights into the distinct shifts occurring within fish gut microbiota during TiLV infection and suggest potential implications for improving gut health and resistance to viral infections in tilapia. Taken together, our study establishes that TiLV infection triggers significant changes in the diversity and composition of the intestinal microbiota in red hybrid tilapia.

Occurrence of herpesvirus in fish.

Herpesviruses are common agents in animals of the aquatic environment. They infect many species of fish but only lead to disease in one or two species. Nevertheless, infected fish without clinical symptoms can actively transfer infectious agents to disease-susceptible species. The aim of the study was to identify and prove the natural presence of different herpesviruses. Koi, Nile tilapia, grass carp, goldfish and crucian carp were infected with a herpesvirus isolate 99% identical to goldfish herpesvirus (GHV) or cyprinid herpesvirus 2 (CyHV-2) obtained from crucian carp. Before and after infection, samples were collected non-lethally at different time points from all five fish species to identify and evaluate the replication of viruses naturally infecting the fish as well as the CyHV-2 experimentally infecting them. Gill swabs and separated leukocytes were subjected to PCR and the results compared. These samples yielded DNA of koi herpesvirus (KHV, also referred to as CyHV-3), GHV and a new herpesvirus. While Asian-lineage CyHV-3 DNA was detected in samples from crucian carp and goldfish, CyHV-2 DNA was found in samples from koi and tilapia. A new, hitherto unknown herpesvirus was identified in samples from grass carp, and was confirmed by nested PCR and sequence analysis. The survival rates were 5% for grass carp, 30% for tilapia, 55% for crucian carp, 70% for koi and 100% for goldfish at 20 days post infection. Evolutionary analyses were conducted and five clusters were visible: CyHV-1 (carp pox virus), CyHV-2 with sequences from koi and tilapia, CyHV-3 with sequences from crucian carp and goldfish, probable CyHV-4 from sichel and a newly discovered herpesvirus - CyHV-5 - from grass carp. The results obtained with the molecular tools as well as from the animal experiment demonstrated the pluripotency of aquatic herpesviruses to infect different fish species with and without visible clinical signs or mortality.