Comparative pathogenicity and histopathological analysis of Edwardsiella anguillarum intraperitoneal infection in milkfish (Chanos chanos), Nile tilapia (Oreochromis niloticus) and Asian seabass (Lates calcarifer).

Edwardsiella anguillarum, a highly virulent species within the Edwardsiella genus, causes significant mortality in milkfish farms in Taiwan. This study aimed to investigate the comparison of milkfish susceptibility, a newly identified host species in Taiwanese aquaculture, with other species Nile tilapia (Oreochromis niloticus) and Asian seabass (Lates calcarifer), to E. anguillarum, elucidating its pathogenicity across both seawater and freshwater aquaculture environments. The results showed milkfish exhibited the highest mortality rate of 85% within 48 h of infection, whereas Nile tilapia exhibited a mortality rate of 70% between the second- and tenth-day post challenge, and seabass exhibited a mortality rate of 25% between the second- and sixth-day post challenge. Gross lesions observed in milkfish included splenomegaly and haemorrhage, whereas Nile tilapia exhibited signs of ascites, exophthalmia and brain haemorrhage. Seabass displayed spleen granulomas and haemorrhage at the injection site. Histopathological analysis revealed common features across all three species, including multifocal necrosis, bacterial presence in the necrotic areas, serositis and oedema. Asian seabass also exhibited chronic lesions in the form of splenic granulomas. This study highlights the high susceptibility of milkfish and Nile tilapia to E. anguillarum, emphasizing the urgent need for further investigation into targeted vaccine development for these fish species. These results not only deepen our understanding of the differing levels of pathogenicity among the three species but also offer valuable insights for improving disease prevention and management strategies in aquaculture, including those applied within polyculture systems and for the maintenance of aquaculture water environments.

Comparative genomics of Edwardsiella anguillarum and Edwardsiella piscicida isolated in Taiwan enables the identification of distinctive features and potential virulence factors using Oxford-Nanopore MinION® sequencing.

Edwardsiella tarda (ET) and Edwardsiella anguillarum (EA) are the most harmful bacterial fish pathogens in Taiwan. However, there is confusion regarding the genotypic identification of E. tarda and E. piscicida (EP). Therefore, we used a novel Nanopore MinION MK1C platform to sequence and compare the complete genomes of E. piscicida and E. anguillarum. The number of coding genes, rRNA, and tRNA recorded for E. anguillarum and E. piscicida were 8322, 25, and 98, and 5458, 25, and 98, respectively. Ribosomal multilocus sequence typing (rMLST) for E. piscicida indicated 35 rps. The shared clusters between E. anguillarum and E. piscicida indicated several unique clusters for the individual genomes. The phylogenetic tree analysis for all complete genomes indicated that E. anguillarum and E. piscicida were placed into two species-specific genotypes. Distribution of subsystems for annotated genomes found that genes related to virulence, defence, and disease for E. anguillarum were 103 and those for E. piscicida were 60 and pathogenic islands (PI) were 498 and 225, respectively. Vaccine candidates were identified in silico from the core genes using high antigenic, solubility, and secretion probabilities. Altogether, the genome data revealed distinctive features between E. anguillarum and E. piscicida, which suggest different pathogenicity and thus the need for separate preventive strategies.

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Molecular and immunological responses of the giant freshwater prawn, Macrobrachium rosenbergii, to the organophosphorus insecticide, trichlorfon.

Trichlorfon is an organophosphorus (OP) insecticide that is used as an agriculture pesticide to destroy insects, a human medicine to combat internal parasites, and an ectoparasiticide in the livestock and aquaculture industries, but which has caused aquatic toxicity in the prawn industry. The aim of this study was to investigate the effects of trichlorfon on molecular and enzymatic processes of the immunological response of the giant freshwater prawn, Macrobrachium rosenbergii, at 0, 0.2, and 0.4mgL(-1) with 0, 3, 6, 12, and 24h of exposure. The total hemocyte count (THC), respiratory bursts (RBs), phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity were examined to evaluate immunological responses and oxidative stress. Results showed that THCs of the prawn exposed to trichlorfon at both concentrations (0.2 and 0.4mgL(-1)) had increased after 12 and 24h; SOD and PO activities had significantly increased at 3h, whereas production of RBs had dramatically increased as oxidative stress at each sampling time after exposure to trichlorfon compared to the control. A potential biomarker of OPs, acetylcholinesterase (AChE) revealed a significant decrease after exposure for 6h, and showed a time-dependent tendency. Immune gene expressions, including prophenoloxidase (proPO), the lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglubulin (α2M), transglutaminase (TG), and copper, zinc (Cu,Zn)-SOD, of prawns exposed to trichlorfon at 0, 0.2, and 0.4mgL(-1) for 0, 6, and 24h were further evaluated. Expressions of all of the immune genes significantly decreased when prawns were exposed to 0.4mgL(-1) trichlorfon for 24h, and among them, an increase in SOD expression was seen after exposure to 0.4mgL(-1) for 6h. Prawns exposed to trichlorfon within 24h exhibited the decrease of circulating hemocytes, and also the induction of oxidative stress, which caused subsequent damage to DNA formation of immune genes. From these results, we concluded that immunological responses and immune gene expressions of prawn exposed to trichlorfon at 0.4mgL(-1) for 24h were perturbed, thus causing a deficiency in immunity and subsequent increased susceptibility to pathogen infections.