Characterization of Photobacterium damselae subsp. damselae isolated from diseased Asian seabass (Lates calcarifer) and the preliminary development of a formalin-killed cell vaccine.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019-2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 µg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

Transcriptome profiling reveals the novel immunometabolism-related genes against WSSV infection from Fenneropenaeus merguiensis.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

A Hint of Primitive Mucosal Immunity in Shrimp through Marsupenaeus japonicus Gill C-Type Lectin.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is Marsupenaeus japonicus gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from M. japonicus displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and Vibrio counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.

Identification of an anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R) related to Vp_PirAB-like toxin resistance in Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by the pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxins. Formalin-killed cells of V. parahaemolyticus AHPND-causing strain D6 (FKC-VpD6) were used to select Vp_PirAB-like toxin-resistant Litopenaeus vannamei by oral administration. Stomach and hepatopancreas tissues of shrimps that survived for one week were subjected to RNA sequencing. Differentially expressed genes (DEGs) between surviving shrimp, AHPND-infected shrimp, and normal shrimp were identified. The expressions of 10 DEGs were validated by qPCR. Only one gene (a gene homologous to L. vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R)) was expressed significantly more strongly in the hepatopancreas of surviving shrimp than in the other groups. Significantly higher expression of LvALF AV-R was also observed in shrimp that survived two other trials of FKC-VpD6 selection. Recombinant ALF AV-R bound to LPS, PGN, Gram-negative bacteria, and some Gram-positive bacteria in ELISAs. ALF AV-R recombinant protein did not interact with native Vp_PirAB-like toxin in an ELISA or a Far-Western blot. For L. vannamei orally fed ALF AV-R protein for 3 days, the survival rate following challenge with VpD6-immersion was not significantly different from that of shrimp fed two control diets. These results suggest that LvALF AV-R expression was induced in the hepatopancreas of shrimp in response to the presence of Vp_PirAB-like toxin, although other factors might also be involved in the resistance mechanism.

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Development of DNA Markers for Acute Hepatopancreatic Necrosis Disease Tolerance in Litopenaeus vannamei through a Genome-Wide Association Study.

Shrimp aquaculture is facing a serious disease, acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio paraheamolyticus (VpAPHND). For sustainable shrimp aquaculture, massive losses of shrimp infected with VpAPHND must be prevented. Research and selection of shrimp tolerant to VpAPHND infection is a sustainable approach to reducing the risk of AHPND. This study focused on the identification and development of potential DNA markers associated with AHPND using DArT sequencing (DArTSeq) and a genome-wide association study. Three populations of post-larval Litopenaeus vannamei were immersed in VpAPHND to collect susceptible (D) and tolerant (S) samples. The 45 D and 48 S shrimp had their genotypes analyzed using DArTSeq. A total of 108,983 SNPs and 17,212 InDels were obtained from the DArTseq data, while the biallelic 516 SNPs and 2293 InDels were finally filtered with PIC < 0.1, MAF < 0.05, and a call rate ≥ 80%. The filtered variants were analyzed for their association with AHPND tolerance. Although there were no significantly associated SNPs and InDels above the Bonferroni correction threshold, candidate variants, four SNPs and 17 InDels corresponding to p < 0.01, were provided for further validation of the AHPND tolerance trait. The candidate SNPs are located on an exon of the zinc finger protein 239-like gene, an intron of an uncharacterized gene, and in intergenic regions. Most of the candidate InDels are in the intergenic regions, with fewer in the intronic and exonic regions. This study provides information on SNPs and InDels for white shrimp. These markers will support the variant database of shrimp and be useful in shrimp aquaculture for breeding selection.

Virulent properties and genomic diversity of Vibrio vulnificus isolated from environment, human, diseased fish.

The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.

Nucleus-forming vibriophage cocktail reduces shrimp mortality in the presence of pathogenic bacteria.

The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VPAHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VPAHPND strains and reduced shrimp mortality from VPAHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture.