Coinfections by multiple pathogens, including viruses and bacteria, have severely impacted tilapia aquaculture globally. This study evaluated the impacts of dietary supplementation on red hybrid tilapia (Oreochromis spp.) coinfected with Tilapia lake virus (TiLV) and Aeromonas hydrophila. Fish were divided into three groups: a control group on a normal diet, and two experimental groups received diets supplemented with strategy A, an organic acid blend combined with a lyso-phospholipid-based digestive enhancer, and strategy B, an organic acid blend combined with natural immunostimulants and nutrients. Following exposure to both pathogens, the fish supplemented with strategies A and B showed lower cumulative mortality rates of 50.0% and 41.7%, respectively, compared to 76.3% in the control group. Notably, fish fed with strategy B-supplemented diet displayed a stronger immune response, with a lower expression of il-8, mx, and rsad2, and showed less pathological changes in the liver, spleen, and intestines, suggesting enhanced resistance to coinfection. In contrast, fish receiving strategy A did not exhibit significant changes in the immune-related gene expression or pathogen load, but demonstrate less pathological alterations, indicating intestinal protection. These findings highlight the potential of feed additives, particularly strategy B, to reduce the impact of virus-bacterial coinfections and improve outcomes in tilapia farming.

Tilapia lake virus (TiLV) presents a substantial threat to global tilapia production. Despite the development of numerous cell lines for TiLV isolation and propagation, none have been specifically derived from red hybrid tilapia (Oreochromis spp.). In this study, we successfully established a new cell line, RHTiB, from the red hybrid tilapia brain. RHTiB cells were cultured for 1.5 years through over 50 passages and demonstrated optimal growth at 25 °C in Leibovitz-15 medium supplemented with 10% fetal bovine serum at pH 7.4. Morphologically, RHTiB cells displayed a fibroblast-like appearance, and cytochrome oxidase I gene sequencing confirmed their origin from Oreochromis spp. Mycoplasma contamination testing yielded negative results. The revival rate of the cells post-cryopreservation was observed to be between 75 and 80% after 30 days. Chromosomal analysis at the 25th passage revealed a diploid count of 22 pairs (2n = 44). While no visible cytopathic effects were observed, both immunofluorescence microscopy and RT-qPCR analysis demonstrated successful TiLV propagation in the RHTiB cell line, with a maximum TiLV concentration of 10^{7.82 ± 0.22} viral copies/400 ng cDNA after 9 days of incubation. The establishment of this species-specific cell line represents a valuable advancement in the diagnostic and isolation tools for viral diseases potentially impacting red hybrid tilapia.

Tilapia lake virus (TiLV) poses a major threat to global tilapia aquaculture and contributes to significant economic losses due to the absence of effective vaccines and treatments. Given the high mortality rates and severe pathological effects of TiLV on tilapia, alternative strategies, such as immunoglobulin-based therapies, are being considered for disease control. In this study, we developed specific immunoglobulin Y (IgY) antibodies against TiLV and evaluated their neutralization activity. Laying hens were immunized via intramuscular injections of recombinant TiLV segment 4 protein, and IgY antibodies were extracted and purified from their egg yolks using polyethylene glycol precipitation. Western blot analysis confirmed the specificity of the IgY, which demonstrated no cross-reactivity with nontarget proteins. Neutralization assays revealed a dose-dependent reduction in TiLV infectivity, which declined from 5.01 × 10⁶ TCID₅₀/mL to 5.01 × 10⁴–1.26 × 10⁵ TCID₅₀/mL, with the highest efficacy observed at a 1:2 dilution. Despite the variability in neutralization infectivity among the different hens, IgY effectively inhibited TiLV-induced cytopathic effects. Immunofluorescence assays further confirmed a significant reduction in the TiLV antigen levels in IgY-treated RHTiB cells. Our findings highlight IgY as a promising strategy for TiLV control and suggest its potential application in the prevention of emerging viruses.

Background: Tilapia Lake virus (TiLV) poses a significant threat to global tilapia aquaculture, causing high mortality rates and severe economic losses. However, the molecular mechanisms underlying TiLV-host interactions remain largely unexplored. Methodology: We investigated the proteomic and phosphoproteomic changes in two piscine cell lines, E-11 and RHTiB cells, following TiLV inoculation at different time points. Differential protein expressions at 10-min and 24-h post infection were selected for constructing protein-protein interactions and analyzing enriched pathways related to the viral entry and replication. Results: Our findings revealed significant alterations in protein expression and phosphorylation states, highlighting distinct responses between the cell lines. In E-11 cells, TiLV infection suppressed proteins involved in the Janus kinase-signal transducer and activator of transcription and Fas-associated death domain protein-tumor necrosis factor receptor-associated factor pathways, leading to activation of nucleotide oligomerization domain signaling and apoptosis. In RHTiB cells, TiLV suppressed host cellular metabolism by reducing protein phosphatase activity to facilitate early viral entry, while later stages of infection showed increased activity of myosin heavy chain 9 and enhanced host immune responses via phosphorylation of ribosomal protein L17 and GTPase immunity-associated protein 7 (GIMAP7). Conclusion: Our study suggested that TiLV employs different strategies to manipulate host cellular pathways depending on the cell type. Further studies are essential to validate these findings and ultimately facilitate the development of effective antiviral strategies.

Background and Aim: Postpartum reproductive tract infections pose significant challenges to dairy farms, leading to economic losses due to reduced fertility associated with uterine inflammation. In veterinary practice, numerous research groups have explored the underlying causes of subfertility in cows, including surveying endemic viral infections related to endometritis in local areas. This study investigated bovine herpesvirus 4 (BoHV-4) infection in Thai dairy herds and assessed its impact on endometritis and subsequent reproductive outcomes. Materials and Methods: The present study analyzed BoHV-4 DNA in various samples, including milk, blood, and endometrial tissue, from 44 Holstein-Friesian cows 21–47 days postpartum across five dairy herds in Central Thailand. BoHV-4 glycoprotein B and thymidine kinase DNA sequences were detected using the polymerase chain reaction (PCR) and nested PCR, with sequence comparisons made to GenBank data for phylogenetic analysis. The endometritis status was diagnosed through vaginal mucus examination and endometrial cytology, with reproductive performance monitored up to the subsequent calving. Results: BoHV-4 DNA was identified in blood and endometrial tissues (15.91%) but not in milk samples. Phylogenetic analysis revealed that the local BoHV-4 strains are similar to those identified in Brazil and Japan. Notably, the presence of BoHV-4 was correlated with reduced postpartum reproductive performance, particularly extending the interval from calving to the first service. Conclusion: Our findings underscore the importance of integrating BoHV-4 genomic surveys and uterine health assessments to refine reproductive management strategies within the dairy industry. Keywords: bovine herpesvirus-4, dairy cattle, endometritis, phylogenetic analysis, reproductive performance, Thailand.