Detection of lumpy skin disease virus in formalin-fixed paraffin-embedded tissues using immunohistochemistry and indirect immunofluorescence assay

Abstract

Abstract

Background: Lumpy skin disease (LSD) is a disease caused by the Lumpy skin disease virus (LSDV). This virus causes fever and the formation of skin nodules, mucosal lesions, and internal organ damage, resulting in significant losses in livestock. The diagnosis of LSD can be performed using PCR, ELISA, and histopathology. However, in some cases, additional techniques such as IHC and IFAT are necessary due to limitations of samples preserved in formalin. Therefore, the objective of this study was to produce a specific primary antibody for LSDV by expressing recombinant protein and expanding the capabilities and options for disease diagnosis.

Method: The p32 gene was amplified by PCR to increase the genetic material and cloned into a cloning vector, followed by subcloning into an expression cloning vector. The molecular weight of the recombinant protein P32 was confirmed through SDS-PAGE and western blotting. The p32 gene was sequenced and aligned to test its identity. Then, the antiserum obtained from immunized mice was tested against positive and negative samples using IHC and IFAT techniques.

Result: The experiments showed that the genetic material of LSDV, particularly the p32 gene, could be amplified successfully. Moreover, the recombinant P32 protein could be expressed in E. coli and the antiserum could be used in IHC and IFAT techniques. Additionally, the nucleotide sequence of p32 was closely related to the nucleotide sequence of the virus found in China.

Conclusion: The use of IHC and IFAT techniques expands the options for LSD diagnosis, allowing for the examination of samples preserved in formalin. This enhances the capabilities and expands the options of laboratory services, reducing the limitations of shortages or the need to import antibodies from foreign countries.