Induction of trained immunity using ß-glucan and its protective responses in Nile tilapia, Oreochromis niloticus.

Emerging and re-emerging diseases in fish cause drastic economic losses in the aquaculture sector. To combat the impact of disease outbreaks and prevent the emergence of infections in culture systems, understanding the advanced strategies for protecting fish against infections is inevitable in fish health research. Therefore, the present study aimed to evaluate the induction of trained immunity and its protective efficacy against Streptococcus agalactiae in tilapia. For this, Nile tilapia and the Tilapia head kidney macrophage primary culture were primed using ß-glucan @200 µg/10 g body weight and 10 µg/mL respectively. Expression profiles of the markers of trained immunity and production of metabolites were monitored at different time points, post-priming and training, which depicted enhanced responsiveness. Higher lactate and lactate dehydrogenase (LDH) production in vitro suggests heightened glycolysis induced by priming of the cells using ß-glucan. A survival rate of 60% was observed in ß-glucan trained fish post challenge with virulent S. agalactiae at an LD50 of 2.6 × 107 cfu/ml, providing valuable insights into promising strategies of trained immunity for combating infections in fish.

Isolation, in vitro, and in vivo pathogenicity test of Tilapia lake virus (TiLV) and development of a prognostic semi-quantitative lesion scoring system for differentiating clinical/subclinical infection in farmed tilapia (Oreochromis niloticus L.).

Tilapia lake virus ('TiLV-MH-2022') was recently recovered from the naturally infected farmed tilapia. Reverse transcription-polymerase chain reaction (RT-PCR) using segment 1 specific primers, followed by Sanger sequencing, confirmed the infection. The pairwise sequence homology of segment 1 showed its close relationship with the previous isolates. The virus was successfully detected from the mucus, which emphasised the possibility of non-invasive screening of tilapia on a large scale. The virus inoculum prepared from the infected tissues was tested for in vivo and in vitro pathogenicity. Around 100-140 nm-sized electron-dense virus particles were observed in the infected OnlL cells. Based on the onset of symptoms and lesions, all RT-PCR-positive fish were categorised into two groups, 'clinical' and 'subclinical'. A lesion-scoring technique was developed for assessing the pathogenicity of the virus isolate. The external and internal gross lesions and histopathological alterations in the critical organs of the fish, such as the brain, kidney, gills, and liver, were assessed on a scale of 0 (no gross lesion) to 5 (most severe lesions). Overall lesion score was significantly high in the clinical and subclinical groups for gross and histopathology, respectively. This study is the first such attempt to standardise a semi-quantitative lesion scoring technique for TiLV infection, which establishes a clinical relevance and prognostic ability to distinguish between the apparent and inapparent infection.

Development of an indirect ELISA test for the detection of Tilapia lake virus (TiLV) in fish tissue and mucus samples.

A serological test for screening of TiLV in Oreochromis niloticus would be useful for the epidemiological investigations. Using polyclonal antisera against TiLV (TiLV-Ab), an indirect enzyme-linked immune sorbent assay (iELISA) was developed for the detection of TiLV antigen in fish tissue and mucus. After a cutoff value was established and antigen and antibody concentrations were optimized, the iELISA's sensitivity and specificity were assessed. We found the ideal dilutions of TiLV-Ab as 1: 4000 and secondary antibody as 1:65,000. High analytical sensitivity and moderate specificity were displayed by the developed iELISA. The Positive and Negative Likelihood Ratio (LR+, LR-) were 1.75 and 0.29, respectively. The estimated Positive and Negative Predictive Values (PPV and NPV) of the test were 76.19% and 65.62%, respectively. The accuracy of the developed iELISA was estimated as 73.28%. An immunological survey was performed using the developed iELISA with samples from the field and 155/195 fishes tested positive, indicating a 79.48% TiLV antigen positives. Among the pooled organs and mucus tested, the highest positive rate of 92.3% (36/39) is observed in mucus compared to other tissues, and least positive rate is found in liver of 46% (18/39). The newly designed iELISA proved sensitive and may be helpful for extensive examinations of TiLV infections and monitoring disease status even from apparently healthy samples using a non-invasive technology by collecting mucus as sample for iELISA.

Βeta-glucan stimulation induces trained immunity markers in common carp, Cyprinus carpio.

Trained immunity refers to the memory acquired by innate immune cells, leading to cross-protection and non-specific responses to subsequent infection, thereby improving host survival. Trained immunity induction is a combined effect of immune signaling, metabolic changes, and epigenetic modifications. The present study evaluated the induction of markers of the phenomenon of trained immunity in common carp, which is trained using ß-glucan. The mammalian target of rapamycin (mtor) and hypoxia-inducible factor (hif1α), the metabolic basis of trained immunity; the histone deacetylase (hdac7), one of the markers of epigenetic modifications, metabolic activity of activated cells and expression profiles of proinflammatory cytokines viz. il6a, tnfαa2, and ifnγ were targeted in the study and analyzed in vivo. Besides in vivo analysis, in vitro analysis of mtorc2, hif1α, hdac7, and ifnγ were analyzed. In vitro analyses were performed on head kidney macrophages isolated and maintained in L-15 media and double trained with ß-glucan at 100µg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with ß-glucan at 20 mg/kg body weight for training followed by a resting phase for 6 days and were restimulated with the same dose. Head kidney was collected from the fish post-induction as well as post-restimulation. The expression profile of mtorc2, hdac7, and hif1α were found elevated post-stimulation of ß-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-ß-glucan stimulation was confirmed by the high lactate and LDH production detected in the cell culture supernatant. Overall, the study revealed the expression profile of the trained immunity markers and the increased metabolic activity in cells induced with ß-glucan, which further validates that the action of trained immunity is indispensable in fish on encounter with a potential ligand. The study supports the existing reports on trained immunity in teleost fish with evidence at the genomic level. However, further studies are required to understand the responses and actions of trained immune cells during infection in detail.