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Abstract The intention of this study was to identify the bacterial pathogens infecting Oreochromis niloticus (Nile tilapia) and Clarias gariepinus (African catfish), and to establish the antibiotic susceptibility of fish bacteria in Uganda. A total of 288 fish samples from 40 fish farms (ponds, cages, and tanks) and 8 wild water sites were aseptically collected and bacteria isolated from the head kidney, liver, brain and spleen. The isolates were identified by their morphological characteristics, conventional biochemical tests and Analytical Profile Index test kits. Antibiotic susceptibility of selected bacteria was determined by the Kirby-Bauer disc diffusion method. The following well-known fish pathogens were identified at a farm prevalence of; Aeromonas hydrophila (43.8%), Aeromonas sobria (20.8%), Edwardsiella tarda (8.3%), Flavobacterium spp. (4.2%) and Streptococcus spp. (6.3%). Other bacteria with varying significance as fish pathogens were also identified including Plesiomonas shigelloides (25.0%), Chryseobacterium indoligenes (12.5%), Pseudomonas fluorescens (10.4%), Pseudomonas aeruginosa (4.2%), Pseudomonas stutzeri (2.1%), Vibrio cholerae (10.4%), Proteus spp. (6.3%), Citrobacter spp. (4.2%), Klebsiella spp. (4.2%) Serratia marcescens (4.2%), Burkholderia cepacia (2.1%), Comamonas testosteroni (8.3%) and Ralstonia picketti (2.1%). Aeromonas spp., Edwardsiella tarda and Streptococcus spp. were commonly isolated from diseased fish. Aeromonas spp. (n = 82) and Plesiomonas shigelloides (n = 73) were evaluated for antibiotic susceptibility. All isolates tested were susceptible to at-least ten (10) of the fourteen antibiotics evaluated. High levels of resistance were however expressed by all isolates to penicillin, oxacillin and ampicillin. This observed resistance is most probably intrinsic to those bacteria, suggesting minimal levels of acquired antibiotic resistance in fish bacteria from the study area. To our knowledge, this is the first study to establish the occurrence of several bacteria species infecting fish; and to determine antibiotic susceptibility of fish bacteria in Uganda. The current study provides baseline information for future reference and fish disease management in the country.

Over a decade since its discovery, piscine myocarditis virus (PMCV) remains a significant pathogen in Atlantic salmon aquaculture. Despite this significant impact, the genomic landscape, evolutionary dynamics, and virulence factors of PMCV are poorly understood. This study enhances the existing PMCV sequence dataset by adding 34 genome sequences and 202 new ORF3 sequences from clinical cardiomyopathy syndrome (CMS) cases in Norwegian aquaculture. Phylogenetic analyses, also including sequences from the Faroe Islands and Ireland revealed that PMCV sequences are highly conserved with distinct clustering by country of origin. Still, single CMS outbreaks display multiple PMCV variants, and although some clustering was seen by case origin, occasional grouping of sequences from different cases was also apparent. Temporal data from selected cases indicated increased sequence diversity in the population. We hypothesize that multiple bottlenecks and changing infection dynamics in the host population, with transfer to naïve individuals over time, represent a continuous selection pressure on the virus populations. No clear relation was found between PMCV variants and the severity of heart pathology. However, specific non-synonymous and synonymous mutations that might impact protein function and gene expression efficiency were identified. An additional factor that may impact PMCV replication is the presence of defective viral genomes, a novel finding for viruses of the order Ghabrivirales . This study provides new insights into PMCV genomic characteristics and evolutionary dynamics, highlighting the complex interplay of genetic diversity, virulence markers, and host-pathogen interactions, underscoring the epidemiological complexity of the virus. Keywords: piscine myocarditis virus; evolutionary dynamics; diversity; phylogeny; genomic sequencing; defective viral genomes., Competing Interests: M.L. was affiliated by the diagnostic service company PatoGen AS, which offers PMCV screening, when the analyses were performed. F.M. is affiliated by the aquaculture production company Mowi ASA., (© The Author(s) 2024. Published by Oxford University Press.)

Infectious Pancreatic Necrosis virus (IPNV) is one of the major threats to the animal welfare and economy of the rainbow trout farming industry. Previous research has demonstrated significant genetic variation for resistance against IPNV. The main objective of the study was to investigate the genetic architecture of resistance against IPNV in rainbow trout fry. To achieve this, 610 rainbow trout fry, from a full factorial mating between 5 sires and 5 dams, were bath challenged with the IPNV isolate (IPNV-AS) from Atlantic salmon reared at a commercial farm. The resistance against IPNV was accessed using three different phenotypes; binary survival (BS), total days survived (TDS) and virus load (VL) recorded on the fish throughout the 40-day challenge test. All fish were genotyped using a 57K Affymetrix SNP array. The IPNV-AS isolate resulted in an overall mortality of 62.1%. The heritability estimates for survival (BS h 2 = 0.21 ± 0.06, TDS h 2 = 0.25 ± 0.07) and VL traits (h 2 = 0.23 ± 0.08) were moderate and indicative of potential use of selection for increased resistance to IPNV in rainbow trout selective breeding programs. The unity estimated genetic correlation between the two survival traits (BS and TDS) indicates that the traits can be considered the same trait. In contrast, a moderate favourable negative genetic correlation was found between VL and the two survival traits (-0.61 ± 0.22 to -0.70 ± 0.19). The GWAS of the traits with many QTLs crossing the chromosome-wide Bonferroni corrected threshold indicates the polygenic nature of the studied traits. Most of the 10 possible identified genes were found to be linked with immunity or viral pathogenesis, which could be potentially responsible for the significant genetic variation in survival against the IPNV-AS. The QTL validation analysis revealed no significant difference in the mortalities and VL among the three genotypes of the detected QTL. The VL trait showed larger variation among the dead fry and with a concordant pattern with the two survival phenotypes, but with no significant difference in the proportion of IPNV VL positive samples in the dead and the survived fry. Overall, the results indicate the polygenic nature of the studied traits and support the use of genomic selection to improve resistance against IPNV in rainbow trout breeding companies., Competing Interests: Authors AB and TS are employed by Osland Genetics, AS, Norway. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ahmad, Aslam, Evensen, Gamil, Berge, Solberg, Schmitt and Gjerde.)

Streptococcosis caused by Streptococcus agalactiae 1a in Nile tilapia (Oreochromis niloticus) is a severe disease challenge for the global supply of tilapia. Currently, the extensive use of antibiotics is the primary curative tool used to minimize the impact of the disease. Vaccination is a prophylactic measure that has been shown to reduce antibiotic use in the aquaculture sector substantially. However, no commercially licensed vaccine against Streptococcus agalactiae 1a is currently available. This study aimed to investigate, through molecular and immunological methods, if Streptococcus agalactiae 1a isolates collected from North America (NAM), Central America (CAM), and Southeast Asia (SEA) were similarly suitable for the development of a potentially effective vaccine to serve the global tilapia farming industry. Our comparative data showed that the Streptococcus agalactiae 1a isolates from NAM, CAM and SEA had similar biochemical profiles, and genetic multi-locus sequence typing (MLST) analysis showed that the NAM and CAM isolates belonged to sequence type 7 (ST-7) and clonal complex 1, while isolates from SEA grouped into three sequence types (ST-1650, ST-500, and ST-7) and two distinct clonal complexes (CC1 and CC12). Isolates from NAM, CAM, and SEA displayed similar antigenic profiles determined by western blotting with polyclonal rabbit antisera, which was supported by in vivo cross-protection studies, showing that fish immunized with vaccines based on SEA and CAM isolates with different genetic MLST profiles were highly protected against cross-challenge using the same bacterial strains for challenge. Overall, the data obtained from our investigations provide strong indications that Streptococcus agalactiae 1a distributed in NAM, CAM, and SEA are serologically uniform pathogens, and vaccines based on isolates of Streptococcus agalactiae 1a from these regions may be suited for vaccination of tilapia worldwide., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon ( Oncorhynchus tshawytscha ) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log 10 ) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Xu, Gamil, Wang, Munang’andu and Evensen.)

Salmonid alphavirus (SAV) causes pancreas disease (PD), which negatively impacts farmed Atlantic salmon. In this study, fish were vaccinated with a DNA-PD vaccine (DNA-PD) and an oil-adjuvanted, inactivated whole virus PD vaccine (Oil-PD). Controls were two non-PD vaccinated groups. Fish were kept in one tank and challenged by cohabitation with SAV genotype 2 in seawater. Protection against infection and mortality was assessed for 84 days (Efficacy study). Nineteen days post challenge (dpc), subgroups of fish from all treatment groups were transferred to separate tanks and cohabited with naïve fish (Transmission study 1) or fish vaccinated with a homologous vaccine (Transmission study 2), to evaluate virus transmission for 26 days (47 dpc). Viremia, heart RT-qPCR and histopathological scoring of key organs affected by PD were used to measure infection levels. RT-droplet digital PCR quantified shedding of SAV into water for transmission studies. The Efficacy study showed that PD associated growth-loss was significantly lower and clearance of SAV2 RNA significantly higher in the PD-DNA group compared to the other groups. The PD-DNA group had milder lesions in the heart and muscle. Cumulative mortality post challenge was low and not different between groups, but the DNA-PD group had delayed time-to-death. In Transmission study 1, the lowest water levels of SAV RNA were measured in the tanks containing the DNA-PD group at 21 and 34 dpc. Despite this, and irrespective of the treatment group, SAV2 was effectively transmitted to the naïve fish during 26-day cohabitation. At 47 dpc, the SAV RNA concentrations in the water were lower in all tanks compared to 34 dpc. In Transmission study 2, none of the DNA-PD immunized cohabitants residing with DNA-PD-vaccinated, pre-challenged fish got infected. In contrast, Oil-PD immunized cohabitants residing with Oil-PD-vaccinated, pre-challenged fish, showed infection levels similar to the naïve cohabitants in Transmission study 1. The results demonstrate that the DNA-PD vaccine may curb the spread of SAV infection as the DNA-PD vaccinated, SAV2 exposed fish, did not spread the infection to cohabiting DNA-PD vaccinated fish. This signifies that herd immunity may be achieved by the DNA-PD vaccine, a valuable tool to control the PD epizootic in farmed Atlantic salmon., Competing Interests: Author RT was employed by company Elanco Animal Health. Author AR was employed by company VESO Aqualab. Author JCW was employed by company Experimental Pathology Laboratories Inc. Author JFR was employed by company Elanco Canada Ltd. Author ES provided the statistical analysis of all the study data the capacity of a private consultant. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Thorarinsson, Ramstad, Wolf, Sindre, Skjerve, Rimstad, Evensen and Rodriguez.)

This study aimed at identifying the presence of harmful cyanobacteria, detecting potential harmful algae toxins and their distribution in three seasons: December to February (hot dry season), March to May (rainy season), and June to November (cool dry season) of 2016. The samples were collected in five study sites in Tanzania: Tumbe, Chwaka, Paje, Bweleo in Zanzibar islands and Songosongo Island, mainland Tanzania, where skin irritation problems were observed in seaweed workers in an earlier study. The cyanobacteria from the Moorea genus were microscopically detected in the seawater, with highest concentrations in the months with the highest seawater temperature or hot dry season, than in the other two seasons. The concentration of Moorea species was significantly higher in Songosongo, Tanzania mainland than in Zanzibar Islands in all three seasons, corresponding to the higher level of nutrients of nutrients (PO 4 3- , NO 3 - and NH 4+ ) in the prior season. However, the concentrations were considered relatively low and thus not collected during an ongoing algal bloom. This is one of the first studies that detect Moorea sp. in Tanzanian seawater, and complementary studies including genome sequencing to characterize the species are warranted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Eco-friendly alternatives such as probiotics are needed to prevent economically relevant infectious diseases for a successful disease-free harvest in aquaculture. The use of antibiotics has been the favored practice, but its empirical and indiscriminate use has led to antibiotic resistance in the aquatic environment and residues in the food fish. With this rationale, a probiotic was isolated from tilapia, a commercially important cultured fish worldwide. The characteristics of the probiotic were checked against common bacterial pathogens affecting aquaculture. In vitro tests demonstrated the inhibitory effects of the isolated probiotic on the growth of Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and V. alginolyticus. The candidate probiotic, referred to as TLDK301120C24, was identified as Bacillus subtilis by a battery of biochemical tests and genotypic confirmation by 16S rDNA sequencing. The in vitro results revealed the ability of the probiotic to withstand the gut conditions that included pH range of 3-9, salt concentration of 0.5-6%, and bile salt concentration of up to 6%. The isolate could hydrolyze starch (12-14 mm clearance zone), protein (20-22 mm clearance zone), and cellulose (22-24 mm clearance zone). Further, the inhibitory ability of the probiotic against aquatic pathogens was determined in vivo using gnotobiotic zebrafish by employing a novel approach that involved tagging the probiotic with a red fluorescent protein and the pathogens with a green fluorescent protein, respectively. The colonizing ability of probiotics and its inhibitory effects against the pathogens were evaluated by fluorescence microscopy, PCR, and estimation of viable counts in LBA + Amp plates. Finally, the competitive inhibition and exclusion of fish pathogens A. hydrophila and E. tarda by B. subtilis was confirmed semi-quantitatively, through challenge experiments. This study shows the potential of B. subtilis as a probiotic and its excellent ability to inhibit major fish pathogens in vivo and in vitro. It also shows promise as a potent substitute for antibiotics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Biswajit Maiti reports financial support was provided by Department of Science and Technology (DST), Government of India., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Salmon louse, Lepeophtheirus salmonis, represents major challenge for salmon farming. Current treatments impose welfare issues and are costly, whereas prophylactic measures are unavailable. Two salmon louse heme peroxidases (LsPxtl-1 and LsPxtl-2) were tested for their importance for parasite development and as potential vaccine candidates. LsPxtl-1 possesses two heme peroxidase domains and is expressed in ovaries and gut, whereas LsPxtl-2 encodes one domain and contains N-terminal signal peptide and an Eph receptor ligand-binding domain. LsPxtl-1, but not LsPxtl-2, knockdown in nauplius II stage caused poor swimming and death, indicating its importance for parasite development. Immunizations using single DNA plasmid injection encoding the peroxidases or heterologous prime (DNA) and boost (recombinant LsPxtl-2 protein) gave non-significant reduction in lice numbers. Single injection gave low specific antibody levels compared with the prime-boost. The findings suggest LsPxtl-1 is important for parasite development but formulations and vaccination modalities used did not significantly reduce lice infestation., Competing Interests: The authors declare no competing interests apart from ØE being a co-inventor on the patent no. NO345611B1., (© 2023 The Author(s).)

The current study was carried out to investigate a wide variety of persistent organic pollutants (POPs) in wild and farmed tilapia (Oreochromis niloticus) in Lake Kariba, Zambia, and assess levels of POPs in relation to Environmental Quality Standards (EQSs). Concentrations of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyls (PBDEs), and perfluoroalkyl substances (PFASs) were determined in liver samples of tilapia. PFASs compounds PFOS, PFDA and PFNA were only detected in wild fish, with the highest median PFOS levels in site 1 (0.66 ng/g ww). Concentrations of POPs were in general highest in wild tilapia. The highest median ∑DDTs (93 and 81 ng/g lw) were found in wild tilapia from sites 1 and 2, respectively 165 km and 100 km west of the fish farms. Lower DDE/DDT ratios in sites 1 and 3 may indicate relatively recent exposure to DDT. The highest median of ∑ 17 PCBs (3.2 ng/g lw) and ∑ 10 PBDEs (8.1 ng/g lw) were found in wild tilapia from sites 1 and 2, respectively. The dominating PCB congeners were PCB-118, -138, -153 and -180 and for PBDEs, BDE-47, -154, and -209. In 78% of wild fish and 8% of farmed fish ∑ 6 PBDE concentrations were above EQS biota limits set by the EU. This warrants further studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus-host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses-infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)-and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

The present study evaluated the effects of increasing the dietary levels of EPA and DHA in Atlantic salmon ( Salmo salar ) reared in sea cages, in terms of growth performance, welfare, robustness and overall quality. Fish with an average starting weight of 275 g were fed one of four different diets containing 10, 13, 16 and 35 g/kg of EPA and DHA (designated as 1·0, 1·3, 1·6 and 3·5 % EPA and DHA) until they reached approximately 5 kg. The 3·5 % EPA and DHA diet showed a significantly beneficial effect on growth performance and fillet quality compared with all other diets, particularly the 1 % EPA and DHA diet. Fish fed the diet containing 3·5 % EPA and DHA showed 400-600 g higher final weights, improved internal organ health scores and external welfare indicators, better fillet quality in terms of higher visual colour score and lower occurrence of dark spots and higher EPA and DHA content in tissues at the end of the feeding trial. Moreover, fish fed the 3·5 % EPA and DHA diet showed lower mortality during a naturally occurring cardiomyopathy syndrome outbreak, although this did not reach statistical significance. Altogether, our findings emphasise the importance of dietary EPA and DHA to maintain good growth, robustness, welfare and fillet quality of Atlantic salmon reared in sea cages.

Aeromonas media is a Gram-negative bacterium ubiquitously found in aquatic environments. It is a foodborne pathogen associated with diarrhea in humans and skin ulceration in fish. In this study, we used whole genome sequencing to profile all antimicrobial resistance (AMR) and virulence genes found in A . media strain SD/21-15 isolated from marine sediments in Denmark. To gain a better understanding of virulence and AMR genes found in several A . media strains, we included 24 whole genomes retrieved from the public databanks whose isolates originate from different host species and environmental samples from Asia, Europe, and North America. We also compared the virulence genes of strain SD/21-15 with A . hydrophila , A . veronii , and A . salmonicida reference strains. We detected Msh pili, tap IV pili, and lateral flagella genes responsible for expression of motility and adherence proteins in all isolates. We also found hylA , hylIII , and TSH hemolysin genes in all isolates responsible for virulence in all isolates while the aerA gene was not detected in all A . media isolates but was present in A . hydrophila, A . veronii , and A . salmonicida reference strains. In addition, we detected LuxS and mshA-Q responsible for quorum sensing and biofilm formation as well as the ferric uptake regulator ( Fur ), heme and siderophore genes responsible for iron acquisition in all A . media isolates. As for the secretory systems, we found all genes that form the T2SS in all isolates while only the vgrG1 , vrgG3 , hcp , and ats genes that form parts of the T6SS were detected in some isolates. Presence of bla MOX-9 and bla OXA-427 β-lactamases as well as crp and mcr genes in all isolates is suggestive that these genes were intrinsically encoded in the genomes of all A . media isolates. Finally, the presence of various transposases, integrases, recombinases, virulence, and AMR genes in the plasmids examined in this study is suggestive that A . media has the potential to transfer virulence and AMR genes to other bacteria. Overall, we anticipate these data will pave way for further studies on virulence mechanisms and the role of A . media in the spread of AMR genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dubey, Ager-Wick, Peng, Evensen, Sørum and Munang’andu.)

Aeromonas species are Gram-negative bacteria that infect various living organisms and are ubiquitously found in different aquatic environments. In this study, we used whole genome sequencing (WGS) to identify and compare the antimicrobial resistance (AMR) genes, integrons, transposases and plasmids found in Aeromonas hydrophila , Aeromonas caviae and Aeromonas veronii isolated from Indian major carp ( Catla catla) , Indian carp ( Labeo rohita) , catfish ( Clarias batrachus ) and Nile tilapia ( Oreochromis niloticus ) sampled in India. To gain a wider comparison, we included 11 whole genome sequences of Aeromonas spp. from different host species in India deposited in the National Center for Biotechnology Information (NCBI). Our findings show that all 15 Aeromonas sequences examined had multiple AMR genes of which the Ambler classes B, C and D β-lactamase genes were the most dominant. The high similarity of AMR genes in the Aeromonas sequences obtained from different host species point to interspecies transmission of AMR genes. Our findings also show that all Aeromonas sequences examined encoded several multidrug efflux-pump proteins. As for genes linked to mobile genetic elements (MBE), only the class I integrase was detected from two fish isolates, while all transposases detected belonged to the insertion sequence (IS) family. Only seven of the 15 Aeromonas sequences examined had plasmids and none of the plasmids encoded AMR genes. In summary, our findings show that Aeromonas spp. isolated from different host species in India carry multiple AMR genes. Thus, we advocate that the control of AMR caused by Aeromonas spp. in India should be based on a One Health approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dubey, Ager-Wick, Kumar, Karunasagar, Karunasagar, Peng, Evensen, Sørum and Munang’andu.)

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID 50 /ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R 2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (10 0.301 TCID 50 /ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log 10 , 3.94 log 10 and 3.52 log 10 TCID 50 /ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon ( Salmo salar ). In seawater-farmed salmonids in the southern part of Norway SAV subtype 3 (SAV3) is dominating. PD continues to cause significant economic and fish health concerns in this region despite years of extensive use of oil-adjuvanted vaccines (OAVs) containing inactivated whole virus (IWV) antigens. In the current study, three commercially available PD vaccines were tested. Group A got a DNA vaccine (DNAV) injected intramuscularly (i.m.) plus an OAV without a PD component injected intraperitoneally (i.p.). Groups B and C got different OAV IWV vaccines injected i.p., respectively. The control group was i.p. injected with saline. Approximately 12 weeks after vaccination, the post smolt groups were challenged in seawater with SAV3 by cohabitation. Samples were collected pre-challenge, and at 19, 54 and 83 days post-challenge (dpc). There were no differences in growth or visible intraperitoneal side effects between the immunized groups prior to challenge. Fish in group A had significantly higher SAV3 neutralizing antibody titers than the other groups pre-challenge and significantly lower SAV3 viremia levels than the control group at 19 dpc. Fish in group A had significantly more weight gain than the other groups measured at 54 and 83 dpc. Prevalence and severity of heart necrosis at 19 dpc and loss of exocrine pancreas tissue at 54 and 83 dpc were significantly lower in groups A and B compared to group C and controls. The cumulative mortality in the control group during the challenge period was 10.5%. Group A experienced the lowest mortality (6.4%) albeit not statistically different from the controls. The results suggest that DNAV may reduce the clinical and economic impact of PD by improved protection against SAV3-induced changes in pancreas tissue and growth impairment., Competing Interests: Ragnar Thorarinsson is employed by Elanco Animal Health which funded this study and is the marketing authorization holder of the DNAV. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Elanco Animal Health Inc. Published by Elsevier Ltd.)

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda . In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri . There was no inhibition of three foodborne bacterial species, namely Salmonella enterica , Klebsiella pneumoniae , and Escherichia coli . In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration of 33 µg/mL exhibited low cytotoxicity, while concentrations ≤3.3 µg/mL had no cytotoxicity on CHSE-214 and RTG-2 cells. In vivo tests showed that zebrafish larvae treated with 33 µg/mL and 3.3 µg/mL GarKS prior to challenge had 53% and 48% survival, respectively, while concentrations ≤0.33 µg/mL were nonprotective. Altogether, these data show that GarKS has a broad inhibitory spectrum against Gram positive and negative bacteria and that it has potential applications as a therapeutic agent for a wide range of bacterial pathogens. Thus, future studies should include clinical trials to test the efficacy of GarKS against various bacterial pathogens in farmed fish.

Objectives: Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties., Results: API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture., (© 2021. The Author(s).)

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHμ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The pathogenesis of Lactococcus garvieae (L. garvieae) was assessed in Nile tilapia (Oreochromis niloticus) following administration by two different routes of infection (intraperitoneal versus immersion), using 180 fish divided into three groups. The first group of fish was injected intraperitoneally (IP) with 3 × 10 5 colony-forming units (cfu) of L. garvieae; the second group was infected by immersion (IMM) into water containing 9.6 × 10 5  cfu/ml L. garvieae, and in group 3 (Control), the fish were injected IP with sterile normal saline. Mortalities were recorded daily, and on 3, 5, 7, and 13 days post-infection (dpi), liver, kidney, spleen, brain and eyes were sampled. The level of infection between groups was assessed by number of mortalities that occurred, pathology/histopathology of internal organs, bacterial re-isolation and presence of bacteria in situ determined using immunohistochemistry. A significant difference (p < .0001) was observed between L. garvieae re-isolation from tilapia following administration by IP injection and IMM. Similarly, more clinical signs and mortalities (p < .001) were observed in the IP group compared to the IMM group where no mortalities were observed. These findings suggest that L. garvieae has a low invasive potential in Nile tilapia with intact skin/external barriers and highlights the importance of maintaining fish without cuts or abrasions under field conditions., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Aquaculture is the fastest food-producing sector in the world, accounting for one-third of global food production. As is the case with all intensive farming systems, increase in infectious diseases has adversely impacted the growth of marine fish farming worldwide. Viral diseases cause high economic losses in marine aquaculture. We provide an overview of the major challenges limiting the control and prevention of viral diseases in marine fish farming, as well as highlight potential solutions. The major challenges include increase in the number of emerging viral diseases, wild reservoirs, migratory species, anthropogenic activities, limitations in diagnostic tools and expertise, transportation of virus contaminated ballast water, and international trade. The proposed solutions to these problems include developing biosecurity policies at global and national levels, implementation of biosecurity measures, vaccine development, use of antiviral drugs and probiotics to combat viral infections, selective breeding of disease-resistant fish, use of improved diagnostic tools, disease surveillance, as well as promoting the use of good husbandry and management practices. A multifaceted approach combining several control strategies would provide more effective long-lasting solutions to reduction in viral infections in marine aquaculture than using a single disease control approach like vaccination alone.

In this study, fish farmers' management practices, occurrence, and knowledge of fish diseases in Nyeri County, Kenya, were evaluated. Fish farming management practices for small-scale farmers in Kenya have numerous challenges which have led to disease occurrence and reduced production. Moreover, the impact and association of these challenges to farmers' knowledge of fish diseases and their burden has not been fully studied. A semistructured questionnaire was used to capture farmers' biodata, fish species farmed, and farmers' management practices such as handling of nets, pond fertilization, and disposal of fish waste. Farmers' knowledge of fish diseases was based on their ability to identify independent and dependent variable indicators. Independent variables included clinical signs, decreased feeding, bulging eyes, floating on water, abdominal swelling, bulging eyes, abnormal skin color, reduced growth, and abnormal swimming with fish death as were the dependent variable. A total of 208 farmers were interviewed and included those of tilapia (134), mixed tilapia and catfish (40), catfish (22), rainbow trout, and five dams under cooperative management. Tilapia was the most kept fish species (66.8%) followed by polyculture of tilapia and catfish (20%) and rainbow trout (2%). Most respondents were male (78.5%) over 51 years of age (50%). Fifty percent of the respondents had secondary school education. There was a significant association between deaths and sharing of nets in Kieni East subcounty ( p =0.0049, chi-square), while on-farm fish waste disposing appeared to cause higher deaths compared to burning of the waste although not statistically significant ( p =0.13). Few respondents observed decreased feed uptake (<20%) and poor growth. Fifty-seven percent of farmers reported mortalities. Fish poor growth, floating in water, and management practices in subcounties had significant effect on fish deaths. The farmers had knowledge of signs of diseased fish, but there was paucity of knowing the specific causes of disease. Farmers need to be empowered on best aquaculture husbandry to avoid disease transmission and specific fish disease signs to enhance proper reporting of disease for subsequent mitigation measures., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Isaac R. Mulei et al.)

Pancreas disease (PD) caused by salmonid alphavirus subtype 3 (SAV3) is a serious disease with large economic impact on farmed Norwegian Atlantic salmon production despite years of use of oil-adjuvanted vaccines against PD (OAVs). In this study, two commercially available PD vaccines, a DNA vaccine (DNAV) and an OAV, were compared in an experimental setting. At approximately 1040° days (dd) at 12 °C post immunization, the fish were challenged with SAV3 by cohabitation 9 days after transfer to sea water. Sampling was done prior to challenge and at 19, 54, and 83 days post-challenge (dpc). When compared to the OAV and control (Saline) groups, the DNAV group had significantly higher SAV3 neutralizing antibody titers after the immunization period, significantly lower SAV3 viremia levels at 19 dpc, significantly reduced transmission of SAV3 to naïve fish in the latter part of the viremic phase, significantly higher weight gain post-challenge, and significantly reduced prevalence and/or severity of SAV-induced morphologic changes in target organs. The DNAV group had also significantly higher post-challenge survival compared to the Saline group, but not to the OAV group. The data suggest that use of DNAV may reduce the economic impact of PD by protecting against destruction of the pancreas tissue and subsequent growth impairment which is the most common and costly clinical outcome of this disease., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Here we have studied the impact of lice ( Lepeophtheirus salmonis ) infestation of donor fish on the ability of isolated peripheral blood monocytes (PMBCs) to control the replication of salmonid alphavirus (SAV) ex vivo. PMBC were collected by Percoll gradients at eight and nine weeks post copepodid infestation of Atlantic salmon post smolt. Uninfested fish were controls. PBMCs were then infected ex vivo with SAV (subtype 3), and samples were collected for analysis at two, four, and six days post virus infection. Virus titer in the supernatant was assayed in CHH-1 cells, and in addition, the relative expression of the virus structural protein E2 and selected host antiviral genes, IRF9, ISG15, Mx, and IFIT5, were assayed using real-time PCR. Significantly higher virus replication was detected in cells collected from lice-infested fish compared to controls. Higher virus titer coincided with an inability to upregulate the expression of different immune genes, IFIT5, IRF9, and Mx. These findings point towards compromised ability of PMBCs from lice-infested fish to control virus replication, and, to our knowledge, is the first report showing the direct effect of lice infestation on the interplay between viruses and immune cells. There is a possible impact on the dynamic spread of viral diseases in the aquatic environment.

Tilapia lake virus (TiLV) causes high mortality and high economic losses in tilapines. We describe an experimental challenge study focusing on early post challenge innate immune responses. Nile tilapia (Oreochromis niloticus) were infected with 10 5 TCID 50 /mL TiLV intraperitoneally, followed by virus quantification, histopathology and gene expression analysis in target (brain/liver) and lymphoid (spleen/headkidney) organs at 3, 7, 12, 17, and 34 days post challenge (dpc). Onset of mortality was from 21 dpc, and cumulative mortality was 38.5% by 34 dpc. Liver and kidney histopathology developed over the period 3-17 dpc, characterized by anisocytosis, anisokaryocytosis, and formation of multinucleated hepatocytes. Viral loads were highest at early time (3 dpc) in liver, spleen and kidney, declining towards 34 dpc. In brain, viral titer peaked 17 dpc. Innate sensors, TLRs 3/7 were inversely correlated with virus titer in brain and headkidney, and IFN-ß and Mx showed a similar pattern. All organs showed increased mRNA IgM expression over the course of infection. Overall, high virus titers downplay innate responses, and an increase is seen when viral titers decline. In silico modeling found that TiLV segments 4, 5 and 10 carry nucleolar localization signals. Anti-viral effects of TiLV facilitate production of virus at early stage of infection.

Aquaculture is one of the fastest-growing food-producing sectors in the world. However, its growth is hampered by various disease problems due to infectious microorganisms, including Gram-negative bacteria in finfish aquaculture. Disease control in aquaculture by use of antibiotics is not recommended as it leads to antibiotic residues in the final product, selection, and spread of antibiotic resistance in the environment. Therefore, focus is on disease prevention by vaccination. All Gram-negative bacteria possess surface-associated outer membrane proteins (OMPs), some of which have long been recognized as potential vaccine candidates. OMPs are essential for maintaining the integrity and selective permeability of the bacterial membrane and play a key role in adaptive responses of bacteria such as solute and ion uptake, iron acquisition, antimicrobial resistance, serum resistance, and bile salt resistance and some adhesins have virulence attributes. Antigenic diversity among bacterial strains even within the same bacterial species has constrained vaccine developments, but OMPs that are conserved across serotypes could be used as potential candidates in vaccine development, and several studies have demonstrated their efficacy and potential as vaccine candidates. In this review, we will look into the application of OMPs for the design of vaccines based on recombinant proteins, subunit vaccines, chimeric proteins, and DNA vaccines as new-generation vaccine candidates for major bacterial pathogens of fish for sustainable aquaculture., (Copyright © 2020 Maiti, Dubey, Munang'andu, Karunasagar, Karunasagar and Evensen.)

Immunoassays are among the frontline methods used for disease diagnosis and surveillance. Despite this, there are no immunoassays developed for the Chinese soft-shelled turtle ( Pelodiscus sinensis ), which has expanded into large scale commercial production in several Asian countries. One of the critical factors delaying the development of immunoassays is the lack of characterized soft-shelled turtle immunoglobulins. Herein, we used mass spectrometry together with the ProtQuest software to identify the soft-shelled turtle IgM heavy chain in serum, which again was used to produce a polyclonal anti-turtle-IgM in rabbits. Thereafter, the polyclonal anti-turtle-IgM was used as a secondary antibody in an indirect ELISA to evaluate antibody responses of soft-shelled turtles injected with the bovine serum albumin (BSA) model antigen. Our findings show that only turtle immunized with a water-in-oil BSA plus ISA 763A VG adjuvant (SEPPIC, France) emulsion had antibodies detected at 42 days post vaccination (dpv) while turtles injected with phosphate buffered saline (PBS) only as well as turtle injected with BSA dissolved in PBS had no significant antibody levels detected in serum throughout the study period. In summary, our findings show that rabbit polyclonal anti-turtle-IgM produced can be used in ELISA to measure serum antibody responses in immunized soft-shelled turtles. Future studies should explore its application in other immunoassays needed for the disease diagnosis and vaccine development for soft-shelled turtles.

The autovaccine was produced in-house using a bacterial isolate from a diseased fish from the target farm. Three groups of 150 fish each were injected with either 1) an oil-adjuvanted, inactivated whole cell autovaccine, 2) adjuvant only or 3) PBS (negative control). Approximately 660 degree days post vaccination, the fish were challenged with 9x105 cfu bacteria/fish by intraperitoneal injection and monitored for a further 28 days. Protection against infections was measured by lack of/reduced bacterial loads both by bacterial re-isolation and immunohistochemistry as well as absence of clinical signs/pathology. Significantly less L. garvieae (p<0.03) was re-isolated from either the adjuvant only or control groups compared to the vaccinated group. Furthermore, a significantly high amount (p<0.001) of anti-L. garvieae specific antibodies were observed in the vaccinated group compared to the adjuvant only or control groups at time of challenge. This coincided with protection against infection measured by absence/reduced L. garvieae re-isolation from internal organs, reduced clinical signs and lack of pathology in this group. In the adjuvant only and control groups, bacteria were re-isolated from the kidney, liver, spleen, brain and eyes during the first 14 days. The findings suggest that oil-based vaccines can protect tilapia against L. garvieae infection through an antibody mediated response., Competing Interests: The authors have declared that no competing interests exist.

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH 4 Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey ( Meleagris gallopavo ), Atlantic salmon ( Salmo salar L), and Nile tilapia ( Oreochromis niloticus ) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH 4 Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH 4 Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

Saprolegniosis is a worldwide fungal-like infection affecting freshwater fishes and their eggs. Reports show high mortalities and subsequent economic losses annually from Saprolegnia infections. Most therapeutants against Saprolegnia spp. infections are inefficient and some have negative impact on the environment. In this study, we have investigated the ability of boric acid (BA) to prevent Saprolegnia infection in Nile tilapia (Oreochromis niloticus). BA inhibited radial growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥0.6 g/L. Inhibitory effects were also observed in vivo when Nile tilapia were experimentally challenged with Saprolegnia spores and followed over 10 days post challenge and under continuous exposure to different BA concentrations. No signs of saprolegniosis were observed in fish treated with BA at concentrations of 0.4 g/L and above. Comet assay revealed that BA has low toxicity in tilapia continuously exposed to concentrations of 0.2-0.6 g/L for 96 h. Additionally, no significant histomorphological changes were observed in BA-treated fish compared to non-treated controls. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) enzyme levels indicated reduction in systemic tissue damage associated with Saprolegnia infection. This study demonstrates the potential of BA as a prophylactic measure against Saprolegnia infection in tilapia, and we recommend additional studies on environmental impact.

Long-term immunity is of great importance for protection against pathogens and has been extensively studied in mammals. Successive heterologous infections can affect the maintenance of immune memory, inducing attrition of T memory cells and diminishing B cell mediated protection. In fish, the basis of immune memory and the mechanisms of immunization to heterologous pathogens remain poorly understood. We sequentially immunized isogenic rainbow trout with two immunologically distinct viruses, VHSV and IPNV, either with one virus only or in combination, and analyzed the antibody responses and repertoires. Neutralizing antibodies and ELISPOT did not reveal an effect of heterologous immunization. Using a consensus read sequencing approach that incorporates unique barcodes to each cDNA molecule, we focused on the diversity expressed by selected responding VH/C combinations. We identified both public and private responses against VHSV and/or IPNV in all groups of fish. In fish immunized with two viruses, we registered no significant reduction in the persistence of the response toward the primary immunization. Similarly, the response to the second immunization was not affected by a prior vaccination to the other virus. Our data suggest that heterologous immunization does not enforce attrition of pre-existing antibody producing cells, which may impair the protection afforded by multiple successive vaccinations. These observations are potentially important to improve vaccination strategies practiced in aquaculture., (Copyright © 2019 Navelsaker, Magadan, Jouneau, Quillet, Olesen, Munang'andu, Boudinot and Evensen.)

Piscine myocarditis virus (PCMV) is a double-stranded RNA virus structurally similar to the Totiviridae family. PCMV is the causative agent of cardiomyopathy syndrome (CMS), a severe cardiac disease that affects farmed Atlantic salmon (Salmo salar). A recent study characterized the host immune response in infected salmon through a transcriptome immune profiling, which confirmed a high regulation of immune and anti-viral genes throughout infection with PCMV. Previously we developed a novel model based on repeated non-lethal blood sampling, enabling the individual monitoring of salmonids during an infection. In the present work, we used this model to describe the host immune response in the blood cells of Atlantic salmon after intramuscular infection with PCMV-containing tissue homogenate over a 77-day period. At the final stage heart samples were also collected to verify the PCMV load, the pathological impact of infection and to compare the transcript profiles to blood. The expression level of a range of key immune genes was determined in the blood and heart samples by real-time PCR. Results indicated selected immune genes (mx, cd8α and γip) were up-regulated in the heart tissue of infected animals at the terminal time point, in comparison to the non-infected fish. When analyzing the blood samples over the course of infection, a significant n up-regulation of mx gene was also observed. The time and number of peaks in the kinetics of expression was different between individuals. The PCMV load and CMS pathology was verified by real-time PCR and histopathology, respectively. No pathogen and no pathology could be detected during the course of the experiment except at the terminal stage (viral load by qPCR and pathology by histology). This study emphasizes the value of non-lethal monitoring for evaluating the health status of fish at early stages of infection and in the absence of clinical signs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tilapia is the second most farmed fish species after carp in the world. However, the production has come under threat due to emerging diseases such as tilapia lake virus (TiLV) that causes massive mortalities with high economic losses. It is largely unknown whether different tilapia strains are equally susceptible to TiLV infection. In the present study we compared the susceptibility of gray ( Oreochromis niloticus x O. aureus ) and red tilapia ( Oreochromis spp.) to experimental TiLV infection. Virus was injected intraperitoneally at a concentration of 10 4 TCID 50 /mL. Our findings show that gray tilapia had a lower mortality, 86.44%, but statistically not significantly different ( p = 0.068) from red tilapia (100%). The duration of the mortality period from onset to cessation was similar for the two species, starting at 2-3 days post challenge (dpc) with a median at 10-11 dpi and ending on 20-22 dpi. In addition, there was no difference between species in mean viral loads in brain, liver and headkidney from fish collected soon after death. As for host response, expression levels of IL-1β and TNFα were equally high in brain and headkidney samples while levels in liver samples were low for both red and gray tilapia, which coincides with lower viral loads in liver compared to brain and headkidney for both species. We find that red and gray tilapia were equally susceptible to TiLV infection with similar post challenge mortality levels, equal virus concentration in target organs and similar proinflammatory cytokine responses in target and lymphoid organs at time of death. Nonetheless, we advocate that the search for less susceptible tilapia strains should continue with the view to reduce losses from TiLV infection in aquaculture.

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API-20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates., (© 2019 John Wiley & Sons Ltd.)

Type I interferons (IFNs) have been shown to play an important role in shaping adaptive immune responses in addition to their antiviral properties in immune cells. To gain insight into the impact of IFN-I-induced pathways involved in early adaptive immune responses, i.e., antigen-presenting pathways, in an Atlantic salmon-derived ( Salmo salar L.) macrophage cell line (TO-cells), we used a comparative de novo transcriptome analysis where cells were treated with IFN-I or kept untreated and concurrently infected with salmonid alphavirus subtype 3 (SAV3). We found that concurrent treatment of TO-cells with IFN-I and SAV3 infection (SAV3/IFN + ) significantly enriched the major histocompatibility complex class I (MHC-I) pathway unlike the non-IFN-I treated TO-cells (SAV3/IFN - ) that had lower expression levels of MHC-I pathway-related genes. Genes such as the proteasomal activator ( PA28 ) and β-2 microglobulin ( β2M ) were only differentially expressed in the SAV3/IFN + cells and not in the SAV3/IFN - cells. MHC-I pathway genes like heat shock protein 90 ( Hsp90 ), transporter of antigen associated proteins ( TAPs ) and tapasin had higher expression levels in the SAV3/IFN + cells than in the SAV3/IFN - cells. There were no MHC-II pathway-related genes upregulated in SAV3/IFN + -treated cells, and cathepsin S linked to the degradation of endosomal antigens in the MHC-II pathway was downregulated in the SAV3/IFN - cells. Overall, our findings show that concurrent IFN-I treatment of TO-cells and SAV3 infection enriched gene expression linked to the MHC-I antigen presentation pathway. Data presented indicate a role of type I IFNs in strengthening antigen processing and presentation that may facilitate activation particularly of CD8+ T-cell responses following SAV3 infection, while SAV3 infection alone downplayed MHC-II pathways.

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry., (© 2019 John Wiley & Sons Ltd.)

The present study was conducted to explore the occurrence of Flavobacteriaceae in wild Nile Tilapia Oreochromis niloticus (n = 108) collected from Lake Victoria and farmed Nile Tilapia (n = 187) collected from 12 ponds in the Morogoro region of Tanzania. The size of the ponds surveyed ranged from 130 to 150 m 2 . Pond parameters and fish morphometric data were recorded during sampling. In total, 67 Flavobacterium-like isolates (n = 44 from farmed fish; n = 23 from wild fish) were identified on the basis of colony morphology and biochemical tests. Sequences from the 16S ribosomal RNA (rRNA) gene revealed that all 67 isolates belonged to the genera Flavobacterium and Chryseobacterium. Based on 16S rRNA nucleotide identity, 26 isolates showed high similarity with C. indologenes (99-100% identity), 16 showed similarity to C. joostei (98-99.9%), and 17 were similar to diverse species of Chryseobacterium (97-99%). Three isolates were similar to F. aquatile and three were similar to F. indicum, with 99-100% nucleotide identity in both cases, and two isolates were similar to F. oryzae (99-100% identity). The findings obtained in this study provide a baseline for future studies and contribute to an understanding of the threats presented by the aquatic Flavobacteriaceae reservoir toward the development of healthy fish farming in Tanzania. Such knowledge is vital for the development of a sustainable aquaculture industry in Tanzania that will contribute to increased food security., (© 2018 American Fisheries Society.)

Vaccination is one of the most effective disease control strategies that has contributed to the significant reduction of disease outbreaks and antibiotics usage in salmonid aquaculture. To date, licensing of fish vaccines is to a limited extent based on in vitro correlates of protection, as done for many mammalian vaccines. This is because the immunological mechanisms of vaccine protection have not been clearly elucidated for most fish vaccines. Herein, we provide an overview of the different steps required to establish correlates of protective immunity required to serve as benchmarks in optimizing vaccine production in aquaculture. We highlight the importance of optimizing challenge models needed to generate consistent results used during vaccine development as a basis for establishing immune correlates of protection. Data generated this far shows that antibodies are potentially the most reliable correlates of protective immunity for fish vaccines. Our findings also show that antigen dose can be optimized to serve as a correlate of protection for fish vaccines. Further, there is need to establish signatures of T-cell protective immunity when antibodies fail to serve as proxies of immune protection, particularly for vaccines against intracellular pathogens. We can anticipate that documentation of efficacy for future vaccines in aquaculture, particularly batch testing will be based on in vitro correlates of protective immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)