Yersinia ruckeri Infection and Enteric Redmouth Disease among Endangered Chinese Sturgeons, China, 2022.

During October 2022, enteric redmouth disease (ERM) affected Chinese sturgeons at a farm in Hubei, China, causing mass mortality. Affected fish exhibited characteristic red mouth and intestinal inflammation. Investigation led to isolation of a prominent bacterial strain, zhx1, from the internal organs and intestines of affected fish. Artificial infection experiments confirmed the role of zhx1 as the pathogen responsible for the deaths. The primary pathologic manifestations consisted of degeneration, necrosis, and inflammatory reactions, resulting in multiple organ dysfunction and death. Whole-genome sequencing of the bacteria identified zhx1 as Yersinia ruckeri, which possesses 135 drug-resistance genes and 443 virulence factor-related genes. Drug-susceptibility testing of zhx1 demonstrated high sensitivity to chloramphenicol and florfenicol but varying degrees of resistance to 18 other antimicrobial drugs. Identifying the pathogenic bacteria associated with ERM in Chinese sturgeons establishes a theoretical foundation for the effective prevention and control of this disease.

Genetic and morphological characterization of a new genotype of nervous necrosis virus circulating among Nile tilapia in the south of Egypt.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis in freshwater and marine fishes. In this study, NNV circulating among wild and farmed Nile tilapia (Oreochromis niloticus) was genetically and morphologically characterized using reverse transcription polymerase chain reaction (RT-PCR), sequencing analysis, and transmission electron microscopy (TEM). Brain, eye, and other organ (spleen, kidney, heart, and liver) specimens were collected from 87 wild (66) and farmed (21) Nile tilapia fish during their adult or juvenile stage at different localities in Qena and Sohag governorates in southern Egypt. Among them, 57/87 fish showed suspected NNV clinical signs, and 30/87 were healthy. The results revealed that NNV was detected in 66 out of 87 fish (58.62% in the wild and 17.24% in farmed Nile tilapia by RT-PCR), and the prevalence was higher among diseased (55.17%) than in healthy (20.69%) fish. NNV was detected in the brain, eye, and other organs. Using TEM, virion size variations based on the infected organs were observed. Nucleotide sequence similarity indicated that NNVs had a divergence of 75% from other fish nodaviruses sequenced in Egypt and worldwide. Phylogenetic analysis distinguished them from other NNV genotypes, revealing the emergence of a new NNV genotype in southern Egypt. In conclusion, NNV is circulating among diseased and healthy Nile tilapia, and a new NNV genotype has emerged in southern Egypt.

Reaching the steady state: 30 years of Anguillicola crassus infection of European eel, Anguilla anguilla L., in Northern Germany.

A 30 years long data series on the infection dynamics of European eel (Anguilla anguilla L.) with the non-native invasive nematode Anguillicola crassus Kuwahara, Niimi & Hagaki, 1974 is presented. Parasite burden was evaluated for 30 years in inland and coastal waters in Mecklenburg-Western Pomerania from 1991 to 2020. The total prevalence, mean intensity and damage status of the swim bladders were very high during the first decade (1991­2000), and significantly decreased in both marine and freshwater eel populations in the following decades (2001­2010, 2011­2020). The parasite intensity of eels in coastal waters was significantly lower compared with the freshwater systems (61.3% vs 79.5% in the first decade), indicating the vulnerability of the parasites to brackish water conditions and the fact that the life cycle of A. crassus cannot be completed under high saline conditions. Eel caught in the western part of the Baltic Sea (west of Darss sill) had the lowest mean infection (51.8% in first decade) compared to the eastern part with 63.8%. Thus, besides different infection patterns caused by the environmental conditions, a temporal trend towards a reduced parasite intensity and a more balanced parasite­host relationship developed in the 30 years of interaction after the first invasion. Possible reasons and mechanisms for the observed trends in parasite­host interactions are discussed.

A multidisciplinary study on Clinostomum infections in Nile tilapia: micro-morphology, oxidative stress, immunology, and histopathology.

Yellow grub disease, caused by Clinostomum metacercaria, is an endemic zoonotic infection in freshwater fish, responsible for Halzoun syndrome transmitted through the consumption of raw infected fish. This study aimed to conduct a multidisciplinary investigation integrating detailed morphology, oxidative stress, immunology, and histopathology alteration to advance our understanding of Clinostomum infection. In this annual study, 400 Nile tilapia (Oreochromis niloticus) were collected from the Nile River at Al Bahr Al Aazam, Giza Governorate to assess Clinostomum infection prevalence. Of the examined fish, 160 individuals (40.0%) harboured larval Clinostomum infections. Clinostomum metacercariae were observed in various anatomical locations, with 135 fish (33.8%) in buccal cavities, 21 fish (5.25%) in gill chambers, and 4 fish (1.0%) on the skin. Infection intensity ranged from 2 to 12 cysts per fish, averaging 5 cysts, notably with skin infections characterized by a single cyst in each fish. Macroscopic encysted metacercariae were collected from buccal cavities, gills, and skin. Micro-morphology revealed distinct features in C. complanatum, including an elliptical oral sucker with collar-like rings and large sensory papilla-like structures, contrasting with the absence of these features in C. phalacrocoracis. Oxidative stress, assessed through malondialdehyde (MDA) and nitric oxide levels, revealed an elevation in MDA to 35.13 ± 6 nmol/g and nitric oxide to 25.80 ± 3.12 µmol/g in infected fish. In infected fish, MHC-I gene expression increased approximately 13-fold, MHC-II peaked at 19-fold, and IL-1ß significantly upregulated by 17-fold, compared to control levels. Histopathology detailed associated lesions, such as cyst encapsulation and eosinophilic infiltration. Clinstomiasis and its impacts on fish hosts offer crucial insights to control this emerging fish-borne zoonotic disease, threatening wildlife, aquaculture, and human health.

Two proteocephalid cestodes in the fish Malapterurus electricus and Heterobranchus bidorsalis from Lake Nasser, Egypt: a morphological, molecular, and histopathological study.

Despite the importance of the electric catfish (Malapterurus electricus) and the African giant catfish (Heterobranchus bidorsalis) in the foodweb of Lake Nasser, Egypt, little is known about their diseases and parasitic fauna. This work describes, for the first time, cestodiasis in M. electricus and H. bidorsalis. Corallobothrium solidum and Proteocephalus sp. were identified morphologically and molecularly from M. electricus and H. bidorsalis, respectively. Using PCR, sequencing, and phylogenetic analysis, the two cestodes shared rRNA gene sequence similarities yet were unique and the two new sequences for the proteocephalid genera were submitted to the GenBank database. The prevalence of infection was 75% and 40% for the two fish species, respectively. Infections significantly increased in the summer and spring and were higher in female fish than in male fish. The intestine was the preferred site of the two adult cestodes. However, in the case of C. solidum some larval cestodes were found outside the intestine in between the skin and abdominal musculature, attached to the mesentery, and within intestinal tunica muscularis. Desquamation of the intestinal epithelium and inflammation at the site of infection in addition to congestion of the intestinal wall of the tapeworm infected fish were evident, indicating that C. solidum and Proteocephalus sp. impacted the infected fish. The larval stages of C. solidum attempted to penetrate the intestine and sometimes they were encircled within fibrous layers infiltrated with inflammatory cells. The infected fish's musculature was free of cestode infections. Preventive measures should be implemented to prevent the spread of infections.

Cryptosporidium spp. and Eimeria spp. (Apicomplexa: Eimeriorina) of freshwater Cyprinid fish species in the Kura River basin in Azerbaijan territory.

This study aims to determine the prevalence of Cryptosporidium and Eimeria spp. oocysts in fish specimens in the river Kura. It was conducted during the 2021-2022 at two sites: Mingachevir reservoir in central Azerbaijan and in Neftchala district where the river finally enters the Caspian Sea through a delta of the Kura River estuary. The diagnosis of oocysts was performed microscopically. Fine smears from the intestine epithelial layers stained by Ziehl-Neelsen for Cryptosporidium oocysts. To identify Eimeria oocysts, each fish's faecal material and intestinal scrapings were examined directly under a light microscope in wet samples on glass slides with a coverslip. Results revealed a prevalence of Cryptosporidium and Eimeria species infections in fish hosts from both territories Rutilus caspicus, Alburnus filippi, Abramis brama orientalis and Carassius gibelio. Of 170 investigated fish specimens, 8.8% (15/170) were infected with Cryptosporidium species oocysts. Eimeria species oocysts were identified in 20.6% (35/170). The presence of Cryptosporidium and Eimeria infections in fish specimens are natural infections. However, their presence in fish species may be attributed to the age of the fish species and water pollution. This is the first report regarding the prevalence of Cryptosporidium oocysts in fish species in Azerbaijan.

Association of ectoparasite Lepeophtheirus salmonis counts on farmed Atlantic salmon and wild sea trout in Scotland.

Parasitic sea lice (Copepoda: Caligidae) colonising marine salmonid (Salmoniformes: Salmonidae) aquaculture production facilities have been implicated as a possible pressure on wild salmon and sea trout populations. This investigation uses monitoring data from the mainland west coast and Western Isles of Scotland to estimate the association of the abundance of adult female Lepeophtheirus salmonis (Krøyer) colonising farmed Atlantic salmon Salmo salar L. with the occurrence of juvenile and mobile L. salmonis on wild sea trout, anadromous S. trutta L. The associations were evaluated using generalised linear mixed models incorporating farmed adult female salmon louse abundances which are temporally lagged relative to dependent wild trout values. The pattern of lags, which is consistent with time for L. salmonis development between egg and infective stage, was evaluated using model deviances. A significant positive association is identified between adult female L. salmonis abundance on farms and juvenile L. salmonis on wild trout. This association is consistent with a causal relationship in which increases in the number of L. salmonis copepodids originating from lice colonising farmed Atlantic salmon cause an increase of L. salmonis abundance on wild sea trout.

Risk assessment of wild fish as environmental sources of red sea bream iridovirus (RSIV) outbreaks in aquaculture.

Red sea bream iridovirus (RSIV) causes substantial economic damage to aquaculture. In the present study, RSIV in wild fish near aquaculture installations was surveyed to evaluate the risk of wild fish being an infection source for RSIV outbreaks in cultured fish. In total, 1102 wild fish, consisting of 44 species, were captured from 2 aquaculture areas in western Japan using fishing, gill nets, and fishing baskets between 2019 and 2022. Eleven fish from 7 species were confirmed to harbor the RSIV genome using a probe-based real-time PCR assay. The mean viral load of the RSIV-positive wild fish was 101.1 ± 0.4 copies mg-1 DNA, which was significantly lower than that of seemingly healthy red sea bream Pagrus major in a net pen during an RSIV outbreak (103.3 ± 1.5 copies mg-1 DNA) that occurred in 2021. Sequencing analysis of a partial region of the major capsid protein gene demonstrated that the RSIV genome detected in the wild fish was identical to that of the diseased fish in a fish farm located in the same area in which the wild fish were captured. Based on the diagnostic records of RSIV in the sampled area, the RSIV-infected wild fish appeared during or after the RSIV outbreak in cultured fish, suggesting that RSIV detected in wild fish was derived from the RSIV outbreak in cultured fish. Therefore, wild fish populations near aquaculture installations may not be a significant risk factor for RSIV outbreaks in cultured fish.

First molecular confirmation of cyprinid herpesvirus 1 (CyHV1) in diseased carp in Serbia.

Mass mortality of farmed 1 yr old common carp Cyprinus carpio occurred at a carp farm in April 2022. In addition to high mortality, diseased fish exhibited papillomatous growths on the skin and fins, characteristic of carp pox. To investigate a possible viral cause, tissue samples were collected and nucleic acid was extracted using standard procedures. In a pooled sample from the gills and kidneys, carp edema virus (CEV) was detected by real-time PCR. In a skin tissue sample with papillomatous growths, cyprinid herpesvirus 1 (CyHV1) was detected by a conventional PCR targeting a conserved region of the DNA polymerase of cyprinid herpesviruses. PCR products were visualized through agarose gel electrophoresis, and the presence of CyHV1 DNA was confirmed by Sanger sequencing. This represents the first molecular confirmation of CyHV1 in common carp in Serbia.

Epidemiological cut-off values for Yersina ruckeri disc diffusion data generated by a standardised method.

In order to establish the meaning of data generated in antimicrobial agent susceptibility tests, it is necessary to develop internationally harmonised interpretive criteria. Currently, such criteria have not been developed for data generated in studies of the susceptibility of the fish pathogen Yersinia ruckeri. This work generated the data that would be required to set epidemiological cut-off values for the susceptibility data of this species that had been generated using a standardised disc diffusion method that specified the use of Mueller Hinton agar and incubation at 22°C for 24-28 h. Using this method, sets of inhibition zones data for 4 antimicrobial agents were generated by 3 independent laboratories. The data from these laboratories were aggregated and analysed using the statistically based normalised resistance interpretation. For ampicillin, florfenicol, oxytetracycline and trimethoprim-sulfamethoxazole the cut-off values calculated by this analysis were ≥16, ≥23, ≥24 and ≥30 mm, respectively. Evidence is presented demonstrating that the data for these 4 agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for Y. ruckeri.

First record of Pseudoterranova decipiens in sprat (Sprattus sprattus) from the Baltic Sea.

A dispersion of Anisakidae nematodes (particularly Contracaecum osculatum) among marine organisms in the Baltic Sea has been reported over the last decade. This is in line with an increase in the number of grey seal that act as final host for Contracaecum osculatum and Pseudoterranova sp., and are thus indispensable for the completion of their life cycles. Most attention has been paid to zoonotic nematode species, like Pseudoterranova sp., which have been noted in commercially important fish in the area. Little is known about the spread and transmission of Pseudoterranova sp. in the Baltic Sea. The aim of this study was to investigate whether sprat may play a role as a transport host for this Anisakidae. Samples were collected in three areas of the southern Baltic Sea (south and east of Bornholm, Slupsk Farrow and the Gulf of Gdansk) during a research cruise in August 2019. A visual inspection of the viscera of 556 sprats was conducted. Parasites were identified using anatomomorphological and molecular methods. Nematodes were recorded only in sprat caught southeast of Bornholm (prevalence 2.7%; intensity of infection 1-4; abundance 0.05). Molecular identification revealed the presence of Pseudoterranova decipiens. This is the first report of P. decipiens in sprat from the Baltic Sea. Sprat is likely a transmitter of P. decipiens in the Baltic Sea food web.

Diversity of fungus-like stramenopilous organisms (Oomycota) in Lithuanian freshwater aquaculture: Morphological and molecular analysis, risk to fish health.

The present work is the first comprehensive study of fungus-like stramenopilous organisms (Oomycota) diversity in Lithuanian fish farms aimed at proper identification of saprolegniasis pathogens, which is important for water quality control, monitoring infection levels and choosing more effective treatments for this disease in aquaculture. Pathogenic to fish, Saprolegnia and other potentially pathogenic water moulds were isolated from adult fish, their eggs, fry and from water samples. All detected isolates were examined morphologically and confirmed by sequence-based molecular methods. A total of eight species belonging to the genera Saprolegnia, Achlya, Newbya and Pythium were identified. Four species (S. parasitica, S. ferax, S. australis and S. diclina) were found to be the main causative agents of saprolegniasis in Lithuania. S. parasitica and S. ferax dominated both in hatcheries and open fishponds, accounting for 66.2% of all isolates. S. parasitica was isolated from all farmed salmonid fish species as well as from the skin of Cyprinus carpio, Carassius carassius and Perca fluviatilis. S. australis was isolated from water and once from the skin of Oncorhynchus mykiss, and S. diclina was detected only once on the skin of Salmo salar fish. In addition, Achlya ambisexualis, Saprolegnia anisospora and Newbia oligocantha isolated during this study are noted as a possible source of saprolegniasis. The results of this study are relevant for assessing the risk of potential outbreaks of saprolegniasis or other saprolegnia-like infection in Lithuanian freshwater aquaculture.

Multiple Aeromonas strains isolated from Atlantic salmon (Salmo salar) displaying red skin disease signs in Scandinavian rivers.

Since 2014, Atlantic salmon (Salmo salar L.) displaying clinical signs of red skin disease (RSD), including haemorrhagic and ulcerative skin lesions, have been repeatedly observed in Swedish rivers. Although the disease has since been reported in other countries, including Norway, Denmark, Ireland and the UK, no pathogen has so far been conclusively associated with RSD. In this study, the presence of 17 fish pathogens was investigated through qPCR in 18 returning Atlantic salmon with clinical signs of the disease in rivers in Sweden and Norway between 2019 and 2021. Several potential pathogens were repeatedly detected, including a protozoan (Ichthyobodo spp.), an oomycete (Saprolegnia spp.) and several bacteria (Yersinia ruckeri, Candidatus Branchiomonas cysticola, Aeromonas spp.). Cultivation on different media from ulcers and internal organs revealed high concentrations of rod-shaped bacteria typical of Aeromonadaceae. Multilocus phylogenetic analysis of different clones and single gene phylogenies of sequences obtained from the fish revealed concurrent isolation of several bacterial strains belonging to the species A. bestiarum, A. piscicola and A. sobria. While these bacterial infections may be secondary, these findings are significant for future studies on RSD and should guide the investigation of future outbreaks. However, the involvement of Aeromonas spp. as putative primary etiological agents of the disease cannot be ruled out and needs to be assessed by challenge experiments.

Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway.

Viral diseases are a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway, often leading to reduced fish welfare and increased mortality. Disease outbreaks in salmon farms may lead to spread of viruses to the surrounding environment. There is a public concern that viral diseases may negatively affect the wild salmon populations. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus-1 (PRV-1) are common viral diseases in salmon farms in western Norway. In the current study, we investigated the occurrence of SAV and PRV-1 infections in 651 migrating salmon post-smolt collected from three fjord systems (Sognefjorden, Osterfjorden and Hardangerfjorden) located in western Norway in 2013 and 2014 by real-time RT-PCR. Of the collected post-smolts, 303 were of wild origin and 348 were hatchery-released. SAV was not detected in any of the tested post-smolt, but PRV-1 was detected in 4.6% of them. The Ct values of PRV-1 positive fish were usually high (mean 32.0; range: 20.1-36.8). PRV-1 prevalence in post-smolts from the three fjords was 6.1% in Sognefjorden followed by 4.8% in Osterfjorden and 2.3% in Hardangerfjorden. The prevalence PRV-1 was significantly higher in wild (6.9%) compared to hatchery-released post-smolt (2.6%). The occurrence of PRV-1 infection in the fish was lowest in the Hardangerfjorden which has the highest fish farming intensity. Our results suggest that SAV infection are uncommon in migrating smolt while PRV-1 infection can be detected at low level. These findings suggest that migrating smolts were at low risk from SAV or PRV-1 released from salmon farms located in their migration routes in 2013 and 2014.

Mixed Eimeria and Cryptosporidium infection and its effects on pathology and clinical outcomes in juvenile Asian seabass (Lates calcarifer) cultured in Thailand.

Coccidiosis is an important disease in juvenile fish because of severe intestinal injury during infection. We first reported the mixed infection of intestinal coccidia and its association with health status and pathological findings in juvenile Asian seabass (Lates calcarifer) cultured in Thailand. Two groups of Asian seabass, 60-day fish and 90-day fish, were sampled to investigate prevalence and coccidian infection intensity using morphological characterization and PCR. Phylogenetic analysis of 18S rRNA gene amplified from the intestines revealed Eimeria sp. and Cryptosporidium sp. infection. The prevalence of Eimeria sp. and Cryptosporidium sp. in sampled fish was 100%. Clinical outcomes assessed, using health assessment index (HAI) scoring and semi-quantitative grading of intestinal lesions and inflammation, demonstrated that all fish developed variety of pathology and clinical illness; however, infection intensity in 60-day fish was significantly higher (p < .05) than 90-day fish. The HAI score of 60-day fish was poorer than 90-day fish, which correlated to a high infection intensity (r = .397), analysed by Pearson correlation coefficient. Overproduction of intestinal oxidants contributing to mucosal injury was examined by nitrotyrosine expression. The high production of reactive nitrogen species indicated severe inflammatory response, and intestinal injuries occurred mainly in the 60-day fish.

Patterns of hyperostosis during the ontogenic development of Atlantic spadefish (Broussonet, 1782) in Brazilian coast.

Marine teleost species of commercial interest are often reported with hyperostosis, an osteological condition that results in bone thickening. Various specimens of Atlantic Spadefish Chaetodipterus faber (n = 86) obtained from artisanal fishermen in Rio de Janeiro, Brazil, were radiographed and assessed to detect the occurrence of hyperostosis across four different size classes. Of the examined individuals, 58.62% displayed signs of hyperostosis, which manifested in eight skeletal regions, notably in the supraoccipital crest, cleithrum and supraneural areas. In the vertebral column, hyperostosis was more frequently observed in haemal spines than in neural spines, predominantly between the sixth and eighth caudal vertebrae. The smallest size class (<200 mm total length) showed a low frequency of hyperostosis at 7.89%. This frequency escalated for larger classes, reaching 94.12% in individuals measuring 200-300 mm in total length and was observed in all individuals exceeding 300 mm. Hyperostosis exhibited an ontogenetic development pattern, where both the occurrence frequencies and the sizes of the affected bones expanded in proportion to the fish size. This is the first description of the hyperostosis pattern of development for the species, an important commercial resource.

Nocardiosis and other common diseases of cultured golden pompano (Trachinotus blochii) in Taiwan.

Golden pompano (Trachinotus blochii) is a carnivorous teleost cultured in the Asia-Pacific region. Fish culture in high densities and numbers results in disease outbreaks, causing huge economic losses. Here, we collected cultured golden pompanos from 2021 to 2022 and identified the pathogens isolated from the diseased fish. Out of a total of 64 clinical cases observed in both sea cages and fish ponds, it was found that Nocardia seriolae was the predominant pathogen (26%), followed by Lactococcus garvieae (13%). Trichodina spp. was the most prevalent parasite in sea cages and earthen ponds (21%), while Neobenedenia spp. was the primary parasitic pathogen (16%) in sea cages. Given these findings, further investigations were conducted, including antibiotic susceptibility and pathogenicity tests specific to N. seriolae in golden pompanos. Antibiotic susceptibility tests of N. seriolae revealed that all strains were susceptible to doxycycline, oxytetracycline, florfenicol and erythromycin but resistant to amoxicillin and ampicillin. Additionally, a pathogenicity assessment was carried out by administering an intraperitoneal injection of 0.1 mL containing 107 CFU of N. seriolae per fish. The mortality rates observed varied between 40% and 90%, with the P2 strain exhibiting the highest level of virulence, resulting in a cumulative mortality of 90%. Therefore, disease outbreaks in fish can be minimized by developing effective treatments and prevention methods.

Most of the escaped farmed salmon entering a river during a 5-year period were infected with one or more viruses.

Disease interactions between farmed and wild populations have been poorly documented for most aquaculture species, in part due to the complexities to study this. Here, we tested 567 farmed Atlantic salmon escapees, captured in a Norwegian river during 2014-2018, for five viral infections that are prevalent in global salmonid aquaculture. Over 90% of the escapees were infected with one or more viruses. Overall prevalences were: 75.7% for piscine orthoreovirus (PRV-1), 43.6% for salmonid alphavirus (SAV), 31.2% for piscine myocarditis virus (PMCV), 1.2% for infectious pancreatic necrosis virus (IPNV) and 0.4% for salmon anaemia virus (ISAV). A significantly higher prevalence of PMCV infection was observed in immature compared to mature individuals. The prevalence of both SAV and PMCV infections was higher in fish determined by fatty acid profiling to be 'recent' as opposed to 'early' escapees that had been in the wild for a longer period of time. This is the first study to establish a time-series of viral infection status of escapees entering a river with a native salmon population. Our results demonstrate that farmed escapees represent a continuous source of infectious agents which could potentially be transmitted to wild fish populations.

Natural product honokiol exhibits antiviral effects against Micropterus salmoides rhabdovirus (MSRV) both in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is a formidable pathogen, presenting a grave menace to juvenile largemouth bass. This viral infection frequently leads to epidemic outbreaks, resulting in substantial economic losses within the aquaculture industry. Unfortunately, at present, there are no commercially available vaccines or pharmaceutical treatments to combat this threat. In order to address the urgent need for therapeutic strategy to resist MSRV infection, the antiviral activity of natural product honokiol against MSRV was explored in this study. Firstly, cellular morphology was directly observed in an inverted microscope when treated with honokiol after MSRV infection. The results clarified that honokiol significantly lessened cytopathic effect (CPE) induced by MSRV and protected the integrity of GCO cells. Furthermore, the viral nucleic acid expression (G gene) was detected by reverse transcription real-time quantitative PCR (RT-qPCR) and the results indicated that honokiol significantly decreased the viral loads of MSRV in a concentration-dependent manner, and honokiol showed a high antiviral activity with IC50 of 2.92 µM. Besides, honokiol significantly decreased the viral titre and suppressed apoptosis caused by MSRV. Mechanistically, honokiol primarily inhibited the initial replication of MSRV and discharge of progeny virus to exert anti-MSRV activity. More importantly, in vivo experiments suggested that honokiol (40 mg/kg) expressed a fine antiviral activity against MSRV when administrated with intraperitoneal injection, which led to a notable 40% improvement in the survival rate among infected largemouth bass. In addition, it also resulted in significant reduction in the viral nucleic acid expression within liver, spleen and kidney at 2, 4 and 6 days following infection. What is more, 100 mg/kg honokiol with oral administration also showed certain antiviral efficacy in MSRV-infected largemouth bass via improving the survival rate by 10.0%, and decreasing significantly the viral nucleic acid expression in liver, spleen and kidney of largemouth bass on day 2. In summary, natural product honokiol is a good candidate to resist MSRV infection and has promising application prospects in aquaculture.

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.