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.
•Using TiLV-Ab, a sensitive iELISA for the direct detection of TiLV antigen in fish tissue and mucus samples was developed.•The estimated PPV and NPV of the test were 76.19% and 65.62%, respectively, and the accuracy was estimated as 73.28%..•The highest TiLV positive rate is observed in the mucus samples compared to tissues (kidney, liver, and brain)..•Extensive examination of infection can be done even from apparently healthy fishes using non-invasive collection of mucus.
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 β-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 β-glucan at 100μg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with β-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 β-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-β-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 β-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.
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•Expression profiles of trained immunity markers in common carp post immune training with β-glucan.•Elevated expression profiles of trained immunity markers and proinflammatory cytokines were observed post-training.•Increase in metabolic activity and production of metabolites revalidates the phenomenon of the metabolic shift in trained immune cells.
The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this ...study, the expression patterns of inflammasome genes (
NLRC3
,
ASC
, and
CAS-1
), antiviral genes (
IFNγ
and
MX
), and immune genes (
IL-1β
and
IL-18
) were analysed in
Oreochromis niloticus
liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 10
6
TCID
50
of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS,
NLRC3
levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance,
NLRC3
and
ASC
did not show any response to PGN stimulation, and the expression of
IFNγ
was downregulated at 25 and 50 µg of PGN per ml.
CAS-1
and
IL-18
expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1β showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent.
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.
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•Comparative lesion scoring is conducted to determine the pathogenesis of clinical/subclinical TiLV infection.•Subclinical infection showed histopathological changes with no external gross signs.•Prolonged infection can develop protective immunity but survivors may act as a carrier.•The study improved the understanding of the pathology of inapparent TiLV infection.
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 β-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 β-glucan. A survival rate of 60% was observed in β-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.
•Innate immune training using β-glucan can elevate cytokine transcript levels.•Expression of trained immunity markers, mTOR and HIF1α elevated post immune training with β-glucan in tilapia.•Higher lactate and LDH production post β-glucan induction in the supernatant of tilapia head kidney macrophage culture.•Survival rate of 60% in Streptococcus agalactiae-challenged immune trained tilapia.
Emerging and existing diseases are the major havoc to the shrimp aquaculture industry. Outbreaks of viral epidemics severely hinder the sustainable farming system with significant economic losses ...worldwide. The Pacific white shrimp, Penaeus vannamei farming was seriously affected by infectious myonecrosis caused by a double-stranded RNA virus, infectious myonecrosis virus (IMNV). Early and rapid diagnostics is the priority on considering the efficient management and prevention measures. So, the present study utilized the accurate, rapid and specific detection capabilities of real-time PCR on SYBR Green platform to diagnose and quantify the viral load from the infected tissues by designing an efficient PCR primer set. The developed PCR could detect the virus at 98% efficiency with 10 viral copy number as the limit of detection. The standard curve analysis and amplification arithmetics have shown that it can even detect even less than 10 copy numbers of virus in a sample. The standard curve of the assay has shown R2 value of 0.98 and slope of −3.3834 without any significant variations in inter- and intra- assays. The validated PCR primer pairs and developed SYBR green-based real-time PCR is highly specific, equally sensitive and comparatively economic than the existing TaqMan probe-based PCR for detection of IMNV.
•Validated a new set of PCR primers for SYBR green-based qPCR for the detection and quantification of IMNV infection in India•The developed PCR could detect the virus at 98% efficiency with 10 viral copy number as the limit of detection.•The standard curve of assay was showing an R2 value of 0.98 and a slope of −3.3834 without any statistical variations.•The assay can be implemented for the accurate diagnosis and viral load quantification of the IMNV from infected shrimps.
Abstract
Tilapia parvovirus (TiPV) has been associated with heavy mortalities in tilapia as a single infection or in co‐infection with Tilapia lake virus (TiLV). In this study, TiPV was detected in ...farmed Nile tilapia,
Oreochromis niloticus
, from two geographical regions of India, Maharashtra and Uttar Pradesh. TiPV‐specific polymerase chain reaction (PCR) reported earlier was used in the screening. Tilapia collected from Maharashtra showed characteristic clinical signs, and TiPV was detected along with TiLV and/or
Aeromonas
spp. However, fish from Uttar Pradesh were apparently healthy and only TiPV could be detected in these samples. A high prevalence of TiPV was recorded from both the geographical locations, Maharashtra and Uttar Pradesh (59.6% and 95.0% respectively). The virus could be detected in tissues such as the spleen, liver, kidney, brain and mucus. The spleen appeared to be the best tissue for detecting TiPV in apparently healthy tilapia. The presence of TiPV was further confirmed through sequencing the PCR products, isolation of the virus in the cell line and electron microscopy. Sequences of the NS1 gene of the two TiPV isolates showed similarity to the earlier reported TiPV isolates. The virus could be successfully propagated in
O. niloticus
Liver (OnL) cell line, and cytopathic effect was observed as early as 3 days post‐infection. Furthermore, the presence of non‐enveloped icosahedral to round virus particles measuring about 26–35 nm could be demonstrated in the cytoplasm and nucleus of infected OnL cells in transmission electron microscopy. With this confirmation of the presence of the virus, India is the third country to report TiPV after China and Thailand. The detection of TiPV in co‐infection cases with TiLV and in apparently healthy Nile tilapia suggests its wide distribution and potential synergistic effect in co‐infection cases. Therefore, this emerging virus needs holistic attention to understand its virulence, host‐specificity and epidemiological risk factors.
Recombination activating genes (RAGs) mediates the process of rearrangement and somatic recombination (V(D)J) to generate different antibody repertoire. Studies on the expression pattern of adaptive ...immune genes during ontogenic development are crucial for the formulation of fish immunization strategy. In the present study, Nile tilapia was taken to explore the relative expression profile of RAG genes during their developmental stages. The developmental stages of Nile tilapia, i.e., unfertilized egg, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 days post-hatch (dph) and kidney, blood, gill, liver and spleen tissues from adult fish were collected and the cDNA synthesis was carried out. Gene specific primers for RAG-1 and RAG-2 of Nile tilapia were designed and their annealing temperature (Tm) was optimized by gradient PCR. Consequently, PCR was performed to confirm the specific amplification of RAG-1 and RAG-2 genes. Quantitative real-time PCR (qRT-PCR) gene expression of RAG-1 and RAG-2 were noticed in all the developmental stages; however, a significant increase was observed after 12 dph and peaked at 24 dph, followed by a gradual decrease until 30 dph. Tissue-specific gene expression profiling revealed that the highest expression of RAG-1 and RAG-2 was observed in the kidney, followed by spleen, gill, liver and blood. The findings of the study explored the suitable timing of lymphoid maturation that could be technically used for the adoption of strategies to improve disease resistance of fish larvae for mitigating larval mortality.
•The expression of RAG-1 and RAG-2 starts from the twelve days post hatch.•Kidney shows the abundance of RAG-1 mRNA.•The expression of RAG-1 is higher than RAG-2 across the tissues.
