Omkar Vijay Byadgi

Streptococcus iniae is one of the key etiological agents of Streptococcosis and is a significant pathogen affecting four‐finger Threadfin ( Eleutheronema tetradactylum ), leading to severe clinical manifestations, such as ascitic fluid accumulation in the abdominal cavity and intestinal haemorrhages. Understanding the interplay between fish infections, gut microbiota dysbiosis and immune responses is critical for effective management in aquaculture. This study investigates the impact of the acute infection caused by S. iniae on the gut microbiome composition and immune response of Threadfin fish. Following experimental infection, we observed significant differences in taxonomic richness and diversity between healthy and infected fish during the acute phase of the disease. The gut microbiota was primarily dominated by Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Notably, infection led to a marked decline in microbiota richness and diversity in the gut, accompanied by an increase in the relative abundance of pathogenic genera such as Streptococcus and Vibrio . Immune modulation, as evidenced by altered gene expression of selected immune parameters, was observed in tissue samples following clinical infection. Also, histological examinations revealed significant disruptions to intestinal villi architecture in infected fish. These findings suggest that S. iniae alters the gut microbiota composition, which may compromise immune function and elevate the risk of intestinal inflammation. This research underscores the importance of monitoring gut microbiota dynamics as a potential diagnostic tool for managing bacterial infections in aquaculture settings.

Nile tilapia ( Oreochromis niloticus ) farms face streptococcus agalactiae infections causing mass mortality following economic losses. To date, no commercial fish vaccine is available in India to protect against streptococcosis due to a lack of proper adjuvants and vaccine targets. Multiple studies justify that immunomodulators like β‐glucan and Alkoxy glycerol (AKG) have improved immunity in fish. β‐glucan and AKG are natural homopolymers and lipids, respectively, which have proven immunostimulant capabilities in fish and can be effectively used as a prominent adjuvant to improve vaccine efficacy. The present study aims to demonstrate the improved adjuvant efficacy of the formalin‐inactivated S. agalactiae vaccine. Nile tilapia fingerlings were immunised with formalin‐inactivated S. agalactiae vaccines mixed with β‐glucan and AKG. Each week postvaccination, the serum nonspecific responses were measured by lysozyme, myeloperoxidase, SOD and bactericidal activity. Following the challenge after 28 days postvaccination, the survival rate achieved was 32.14%, 57.14%, 53.57% and 71.43% in the respective experimental groups. The immune gene expressions of IgM, TCRβ and MHCII were evoked postvaccination. The specific serum IgM titre values were significantly upregulated in 14‐ and 28‐day postvaccinated experimental groups. The study highlights the enhanced activity of β‐glucan and AKG incorporated vaccine, which has the potency towards commercialisation for sustainable aquaculture.

Litopenaeus vannamei (Whiteleg shrimp) is a crucial crustacean species in global aquaculture, but infectious disease outbreaks, particularly from pathogens like Vibrio harveyi, threaten its sustainable growth. Understanding shrimp-pathogen interactions, particularly through Toll-like receptors (TLRs), is essential for improving disease resistance. This study identifies and characterizes TLR6 and TLR13 in Whiteleg shrimp through genome-wide analysis. Using bioinformatics tools, we analyzed their physiological properties, domain structures, multiple sequence alignments, phylogeny, and 3D models. Noticeably, TLR6 and TLR13 contain open reading frames (ORF) encoding 1349 and 1092 amino acids, respectively, with typical leucine-rich repeats, transmembrane, and toll-interleukin-1 receptor (TIR) domains. Multiple sequence alignment indicated the conservation of sequence in the leucine-rich repeat domain. Phylogenetic analysis showed that TLR6 and TLR13 are closely related to Penaeus japonicus and Penaeus monodon. Quantitative PCR (q-PCR) revealed TLR6 had higher expression in hemocytes and heart, while TLR13 was most expressed in the intestine. After Vibrio harveyi infection, TLR6 was upregulated in the gills, though TLR13 showed no significant change in the gills or hepatopancreas. These findings provide new insights into the immune function of whiteleg shrimp`s TLR6 and TLR13 against V. harveyi.

Giant freshwater prawn (Macrobrachium rosenbergii (MR)) is a significant aquafarm species commercially cultured in Taiwan. Intensive farming practices have led to the outbreak of Lactococcus garvieae (LG), which causes Lactococcosis in MR. Recently, LG has re‐emerged and the number of mortalities in prawn farms has increased in Taiwan. However, there is no preventative strategy described and a lack of knowledge on virulence factors and pathogenesis from LG in MR. The most virulent strain of L. garvieae from M. rosenbergii was screened in vivo among seven isolates selected for infectivity testing injecting 0.1 mL of 10⁸ CFU/mL bacterial concentration. Among the seven isolates screened, L. garvieae 109‐6 resulted in 100% mortality within 3 days post‐infection. Furthermore, 109‐6 L. garvieae LD₅₀ dosage from in MR was found to be 10⁶ CFU/mL. Subsequently, the most virulent strain 109‐6 was sequenced using MinIon Nanopore sequencing. Results indicated that the LG genome yielded a protein‐coding of 3857 with 59 tRNA and 16 rRNA and no plasmid. Interestingly, the distribution of subsystems in the annotated genome revealed genes related to virulence, defence, and disease among LG 50 genes. Altogether, the virulent strain and its genome data revealed distinctive features of LG, which hinted toward its pathogenicity and could facilitate for better preventive strategies.

Background: The high mortality rate and stunted growth of marine shrimp in the farm have raised concerns about the outbreak of co-infection involving Enterocytozoon hepatopenaei (EHP) and white spot syndrome virus (WSSV), which could lead to significant economic loss. In a mixed-culture farm consisting of Scylla serrata and Peneaus vannamei located in Yunlin Country, Taiwan, were reported mortality cases on culture animals. Methods: Based on clinical signs, histology and molecular analyses, the disease was diagnosed in this study. In the histopathology examination, infected organs, such as the carapace and gills, were stained with hematoxylin and eosin (H and E), then observed under microscope. For the molecular analysis, SalI DNA fragment (WSSV) and spore wall protein (SWP) gene (EHP) were targeted to detected the pathogen in animal host. Result: The cumulative mortality data for this co-infection indicates that S. serrata (1.5% mortality) exhibits higher susceptibility compared to P. vannamei ( greater than 99% mortality). Phylogenetic tree analysis reveals that the spore wall protein (SWP) gene of EHP and the SalI DNA fragment of WSSV strain extracted from S. serrata are genetically similar to those extracted from Peneaus spp., indicating the potential for cross-species infection. Histopathological examination of lesion tissue observed the presence of WSSV basophilic intranuclear inclusion bodies in the carapace tissue and gill fragments of S. serrata. The source of the disease outbreak is highly suspected to be the feed used, which consisted of raw shrimp and fish. This study provides the first documented case of natural EHP infection with co-infection of WSSV in S. serrata.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

The Chinese softshell turtle (CST; Pelodiscus sinensis) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen. Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin.

Red mark syndrome (RMS) is a widespread skin disorder of rainbow trout in freshwater aquaculture, believed to be caused by a Midichloria-like organism (MLO). Here, we aimed to study the pathologic mechanisms at the origin of RMS by analyzing field samples from a recent outbreak through gene expression, MLO PCR, quantitative PCR, and a histopathological scoring system proposed for RMS lesions. Statistical analyses included a One-Way Analysis of Variance (ANOVA) with a Dunnett’s multiple comparisons test to assess differences among gene expression groups and a nonparametric Spearman correlation between various categories of skin lesions and PCR results. In short, the results confirmed the presence of a high quantity of 16S gene copy numbers of Midichloria-like organisms in diseased skin tissues. However, the number of Midichloria-like organisms detected was not correlated to the degree of severity of skin disease. Midichloria-like organism DNA was found in the spleen and head kidney. The spleen showed pathologic changes mainly of hyperplastic type, reflecting its direct involvement during infection. The most severe skin lesions were characterized by a high level of inflammatory cytokines sustaining and modulating the severe inflammatory process. IL-1 β, IL-6, IL-10, MHC-II, and TCR were upregulated in severe skin lesions, while IL-10 was highly expressed in moderate to severe ones. In the moderate form, the response was driven to produce immunoglobulins, which appeared crucial in controlling the skin disease’s severity. Altogether our results illustrated a complex immune interaction between the host and Midichloria-like organism.