Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep

Sassu, Elena L.; Kangethe, Richard T.; Settypalli, Tirumala Bharani Kumar; Rufael, Tesfaye; Cattoli, Giovanni; Wijewardana, Viskam

doi:10.1016/j.vetimm.2020.110092

Cited by 7 publications

(13 citation statements)

References 30 publications

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“…The specificity of each primer pair was evaluated by melting curve analysis. All amplified PCR products showed distinct and unique Tms that were compatible with the amplification of ovine targets, as previously reported ( 20 ). The relative quantities of the target genes were normalized against the internal standard’s relative quantities (GAPDH).…”

Section: Methodssupporting

confidence: 84%

“…The resulting cDNA was stored at -20°C until further use. For quantitative analysis, a cDNA dilution of 1: 100 was used as a template for real-time PCR using specific primers presented in Supplementary Table 1 ( 20 ), using GAPDH as a housekeeping gene. Real-time PCR was performed in a total reaction volume of 10 μl containing 2 μl of diluted cDNA as template, 1 μl of forward and reverse primers ( Supplementary Table 1 ), 5 μl of iQ SYBR Green Supermix 2X (Bio-Rad, USA), and 1 µl of water.…”

Section: Methodsmentioning

confidence: 99%

“…Real-time PCR was performed in a CFX96 real-time PCR Detection System (Bio-Rad, Hercules, USA). The cycling conditions and quantification of targets were performed as previously described ( 20 ).…”

Section: Methodsmentioning

confidence: 99%

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Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

et al. 2021

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Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

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Section: Methodssupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

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Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

et al. 2021

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“…In PBMC culture supernatants, all cytokine concentrations increased over time in response to all treatments, including some spontaneous secretion in the unstimulated control sample (Figure 2A). Both PMA/ionomycin and LPS are known to induce production of bovine IL-10, TNF-α, and IFN-γ in PBMC (Jacobsen et al, 2007;Sassu et al, 2020). We observed the highest concentrations of all 3 cytokines in PBMC cultures in response to PMA/ionomycin stimulation.…”

Section: Development Of a Bead-based Multiplex Assay To Quantify Bovi...mentioning

confidence: 47%

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

et al. 2022

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“…To support participating laboratories further with ongoing activities, APH laboratories also developed tools that could be used in evaluating vaccine efficacy. Quantitative PCR panels that measure innate and adaptive immunity were developed for ruminants, pigs and chicken ( 70 ). Quantitative PCR panels are easy to adopt especially where collaborating partners have limited resources to carry out other assays.…”

Section: Technical Support To Member States Through Coordinated Resea...mentioning

confidence: 99%

Advances in Irradiated Livestock Vaccine Research and Production Addressing the Unmet Needs for Farmers and Veterinary Services in FAO/IAEA Member States

et al. 2022

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The Animal Production and Health section (APH) of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture at the International Atomic Energy Agency has over the last 58 years provided technical and scientific support to more than 100 countries through co-ordinated research activities and technical co-operation projects in peaceful uses of nuclear technologies. A key component of this support has been the development of irradiated vaccines targeting diseases that are endemic to participating countries. APH laboratories has over the last decade developed new techniques and has put in place a framework that allows researchers from participating member states to develop relevant vaccines targeting local diseases while using irradiation as a tool for improving livestock resources.

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Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep

Cited by 7 publications

References 30 publications

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

Advances in Irradiated Livestock Vaccine Research and Production Addressing the Unmet Needs for Farmers and Veterinary Services in FAO/IAEA Member States

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