Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure.

Background: The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods: Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results: We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions: Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.

First identification, molecular characterization, and pathogenicity assessment of Lactococcus garvieae isolated from cultured pompano in Taiwan.

Lactococcosis, caused by Lactococcus garvieae, is an acute hemorrhagic septicemia in fish recorded in marine and freshwater aquaculture during the summer months. In 2020-2021, several sea cage Pompano farms recorded sudden fish mortality events. Based on the results of phenotypic and biochemical tests, L. garvieae was predicted to be the cause. PCR with L. garvieae specific primers (pLG1 and pLG2) targeting the 16S rRNA region further confirmed the etiological agent as L. garvieae after amplifying an 1100 base pairs (bp) product. Furthermore, the 16S rRNA sequences of the two representative strains (AOD109-196-2B and AOD110-215-2B) shared 99.81% identity with L. garvieae (GenBank accession number: MT597707.1). The genetic profiles of the strains were classified using pulsed-field gel electrophoresis after digestion with SmaI and ApaI, which clustered our strains under the same pulsotype. Multiplex PCR targeting the capsule gene cluster and serotype-specific PCR collectively showed that the strains were non-capsulated; thus, they belonged to serotype I. An experimental infection was designed to fulfil Koch's postulates by infecting healthy Pompano with case-driven L. garvieae strains (AOD109-196-2B and AOD110-215-2B) with a cumulative mortality of 70%. Overall, L. garvieae infection in Pompano emphasizes the need for better monitoring and control procedures in aquaculture settings.

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C).

Background: Both bacterial and viral diseases are a major threat to farmed fish. As the antiviral immune mechanisms in lumpfish (Cyclopterus lumpus L.) are poorly understood, lumpfish leukocytes were stimulated with poly(I:C), a synthetic analog of double stranded RNA, which mimic viral infections, and RNA sequencing was performed. Methods: To address this gap, we stimulated lumpfish leukocytes with poly(I:C) for 6 and 24 hours and did RNA sequencing with three parallels per timepoint. Genome guided mapping was performed to define differentially expressed genes (DEGs). Results: Immune genes were identified, and transcriptome-wide analyses of early immune responses showed that 376 and 2372 transcripts were significantly differentially expressed 6 and 24 hours post exposure (hpe) to poly(I:C), respectively. The most enriched GO terms when time had been accounted for, were immune system processes (GO:0002376) and immune response (GO:0006955). Analysis of DEGs showed that among the most highly upregulated genes were TLRs and genes belonging to the RIG-I signaling pathway, including LGP2, STING and MX, as well as IRF3 and IL12A. RIG-I was not identified, but in silico analyses showed that genes encoding proteins involved in pathogen recognition, cell signaling, and cytokines of the TLR and RIG-I signaling pathway are mostly conserved in lumpfish when compared to mammals and other teleost species. Conclusions: Our analyses unravel the innate immune pathways playing a major role in antiviral defense in lumpfish. The information gathered can be used in comparative studies and lay the groundwork for future functional analyses of immune and pathogenicity mechanisms. Such knowledge is also necessary for the development of immunoprophylactic measures for lumpfish, which is extensively cultivated for use as cleaner fish in the aquaculture for removal of sea lice from Atlantic salmon (Salmo salar L.).

Nocardia seriolae cell wall lipids: An effective protective mechanism in resistance and virulence.

Piscine nocardiosis, caused by Nocardia seriolae, is a refractory granulomatous disease in South-East Asian aquaculture. This study investigates the virulence of nocardial lipids essential for pathogenesis among Actinomycetes. Petroleum ether (PE) was used to selectively delipidate two groups of N. seriolae, namely, live cell (LC) and killed cell (KC); resulting in delipidated live cell (DLC) and delipidated killed cell (DKC), respectively. Changes post-delipidation on genus characteristics, such as loss in acid-fast nature and resistance to lysozyme were observed. Transmission electron microscopy revealed notable changes in the lipid layer. Additionally, Lates calcarifer, Asian seabass intraperitoneally injected with LC and DLC had mortality rates of 90% and 50%, respectively, with the latter exhibiting a delay in mortality. Reverse-transcription quantitative PCR (RT-qPCR) analysis of host cytokines from the spleen and head kidney showed delipidation contributed to the induction of an immune response with increased transcriptional levels of interferon-γ (ifn-γ). Histopathological samples collected on day 7 post-inoculation displayed a varied granulomatous response between the treatment groups and scored for pathological changes. These findings affirm that the virulence of the lipids remains independent of the living state of the cell, significantly altering the immune and granulomatous responses in L. calcarifer to N. seriolae.

Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse-field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an evolving disease affecting many fish species and causing significant economic losses worldwide. Assessing pathogen relatedness and bacterial population structure is critical for determining the epidemiology of L. garvieae infections and in establishing effective pathogen management methods. The previously published morphological and genetic studies point to a clonal population structure, as seen in other fish bacteria. In the present study, the pulsed-field gel electrophoresis (PFGE) method was utilized to define a population of 41 Taiwanese isolates from outbreaks with comparisons to four well-characterized non-Taiwanese isolates previously published. Two restriction enzymes (ApaI and SmaI) were utilized individually for PFGE analysis (cut-off value = 90.0%), revealing genetic heterogeneity across L. garvieae isolates, with ApaI and SmaI yielding 12 and seven distinct PFGE band patterns, respectively. The phylogenic analysis using internal transcribed spacer region clustered all L. garvieae isolates in the same clad. Furthermore, the electron microscopic results confirmed the absence of capsular gene cluster (CGC) in previously characterized Taiwanese vaccine strain (S3) from grey mullet. Overall, our findings emphasize the importance of analysing the morphological and genetic diversity in L. garvieae being correlated for proper taxonomic classification in vaccine strain selection and epidemiological studies.

First report of acanthocephalan parasite, Longicollum pagrosomi Yamaguti, 1935 in cultured red snapper (Lutjanus erythropterus) in Taiwan.

For the first time, Longicollum pagrosomi Yamaguti, 1935, an acanthocephalan parasite, has been characterized with morphological, histopathological and molecular detail in farmed red snapper (Lutjanus erythropterus) in Taiwan. The diseased fish showed clinical signs of anorexia, emaciation and were gasping for air at the water's surface. Gross examination revealed extensive necrosis in the intestine and even penetration at the site of parasitic attachment, resulting in a large number of parasites being lodged in the peritoneal cavity, surrounded by black hyperplastic connective tissue. The parasites collected from the intestine were studied using optical microscopy, histopathology, scanning electron microscopy (SEM) and molecular phylogenetic analysis. They were creamy white in colour, and were separated into their proboscis, neck and metasoma (trunk) under optical microscopy. Histopathological examination revealed ovarian balls (floating ovaries) inside the ligament sac and eggs within the egg sorting apparatus. The SEM images revealed that the proboscis had 11-14 longitudinal rows with 9-12 recurrent, backward-facing, organized hooks. In the 18S and 28S phylogenetic tree, sequences of the specimens were identical to those of other Longicollum pagrosomi, in addition to the morphological features. Thus, we confirmed that the parasites belonged to Longicollum pagrosomi in this study.

First report on genetic characterization, cell-surface properties and pathogenicity of Lactococcus garvieae, emerging pathogen isolated from cage-cultured cobia (Rachycentron canadum).

The diseased cage-cultured cobia (Rachycentron canadum) displayed clinical signs, haemorrhagic eyes, dorsal darkness and gross pathological lesions, enlargement of spleen and liver. Haemorrhages were found in brain, heart and liver with cumulative mortality rates ranging from 20% to 50%. Extensive congestion in the heart, liver, spleen, kidney and brain was observed histopathologically. Epicarditis and meningitis were also revealed in diseased cobia. All isolates recovered from the organs (liver, spleen, head kidney, posterior kidney, brain and muscle) of cobia were found to be gram-positive, non-motile, ovoid cocci, short-chain-forming (diplococci) and α-haemolytic. The API 32 strep system together with the polymerase chain reaction assay for species-specific primers (pLG1 and pLG2) and the internal transcribed spacer (ITS) region (G1 and L1 primers) confirmed all four selected isolates as Lactococcus garvieae. Partial 16S rDNA nucleotide sequence (~1,100 bp) of one representative L. garvieae isolate AOD109191 (GenBank accession number, MW328528.1) shared 99.9% identities with the 16S rDNA nucleotide sequence of L. garvieae (GenBank accession numbers: MT604790.1). Transmission electron microscopy (TEM) evaluation of one representative L. garvieae isolate (AOD109191) and the results of multiplex PCR did not reveal the presence of the capsular gene cluster (CGC), thus categorizing the isolate as the KG+ phenotype. Capsule staining and TEM observations confirmed the presence of a hyaluronic acid-like capsule, a possible virulence factor in KG+ phenotype L. garvieae isolates. The pathogenic potential of the representative isolate (AOD109191) was assessed through intraperitoneal injection challenges in cobia. The gross lesions and histopathological changes found in experimentally infected cobia were similar to those seen in naturally infected fish. This is the first report that confirms L. garvieae-induced 'warm water lactococcsis' can cause outbreaks of diseases in cage-cultured cobia.

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan.

Chinese softshell turtles (Pelodiscus sinensis) (CST) are susceptible to infections by bacteria belonging to the Bacillus cereus group (Bcg). Bcg includes several closely related species, two of which, B. cereus and B. thuringiensis, are pathogens of aquatic animals or insects. In the present study, we collected 57 Bcg isolates obtained from diseased CST from 2016 to 2019 in Kaohsiung and Pingtung, the areas with the most CST farms in Taiwan. All isolates were divided into four genotypes with two restriction enzymes, SmaI and NotI, by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Representative isolates from each genotype were subjected to phylogenetic tree analysis using 16S rDNA and pyruvate carboxylase genes as phylogenetic markers, and these CST isolates appeared in different clades. PCR was performed targeting six selected virulence genes, four of which were detected in CST isolates, including cytotoxin K (1/57), hblC of the haemolysin BL complex (46/57), nheA of the non-haemolytic enterotoxin complex (52/57) and enterotoxin FM (57/57), whereas cereulide synthetase and cereulide peptide synthase-like genes were not detected in any isolates.

The moonlighting protein fructose 1,6-bisphosphate aldolase as a potential vaccine candidate against Photobacterium damselae subsp. piscicida in Asian sea bass (Lates calcarifer).

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.