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Home / Equine Research Bank / Front Genet / Development of a TaqMan® Allelic Discrimination qPCR Assay ...
Frontiers in genetics2022; 13; 871875; doi: 10.3389/fgene.2022.871875

Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions.

Abstract: Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive tract. Two alleles (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16  S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.
Copyright © 2022 Thieulent, Carossino, Balasuriya, Graves, Bailey, Eberth, Canisso, Andrews, Keowen and Go.
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Publication Date: 2022-04-13 PubMed ID: 35495124PubMed Central: PMC9043104DOI: 10.3389/fgene.2022.871875Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study showcases the development of a rapid detection method, the TaqMan allelic discrimination qPCR, to identify horses at high risk of becoming long-term carriers of the Equine Arteritis Virus (EAV). A consistent and timely research method for detecting allelic variants associated with EAV carrier state was deemed much needed.

Overview of the Research

  • The research is centered around Equine Arteritis Virus (EAV), a virus causing a respiratory, systemic, and reproductive disease in horses known as Equine viral arteritis (EVA). The EAV can persist within infected stallions, which can then shed the virus and thus spreading it further in the horse population.
  • The study focuses on a certain host cell factor — the equine C-X-C motif chemokine ligand 16 (CXCL16), which has been previously identified as influential in long-term persistent infection (LTPI) progression.
  • Two alleles ( and ) were found in the equine population and are linked to the likeliness of a CD3 T cell subpopulation in the blood to resist EAV infection.

Research Methods and Findings

  • To identify the high-risk horses, researchers developed a TaqMan allelic discrimination qPCR assay — a method to examine the specific gene related to the LTPI progression in stallions.
  • They tested 160 horses of four different breeds and sequenced the horses to determine their genetic composition. By using Sanger sequencing, they identified that all horses with the resisting phenotype were homozygous for the allele, whereas those susceptible carried at least one allele.
  • The findings from the TaqMan qPCR assay were consistent with results from Sanger sequencing and flow cytometric analysis.

Conclusions and Recommendations

  • The researchers concluded that the new TaqMan allelic discrimination genotyping qPCR assay can potentially become a useful screening method for identifying the high-risk genotype in colts before puberty.
  • They further recommended that any colts carrying the susceptible genotype ( or ) should be vaccinated against EAV after 6 months of age to prevent the establishment of future LTPI carriers that could result in the natural infection spreading further among the equine population.

Cite This Article

APA
Thieulent CJ, Carossino M, Balasuriya UBR, Graves K, Bailey E, Eberth J, Canisso IF, Andrews FM, Keowen ML, Go YY. (2022). Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions. Front Genet, 13, 871875. https://doi.org/10.3389/fgene.2022.871875

Publication

Frontiers in genetics
ISSN: 1664-8021
NlmUniqueID: 101560621
Country: Switzerland
Language: English
Volume: 13
Pages: 871875

Researcher Affiliations

Thieulent, Come J
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Carossino, Mariano
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Balasuriya, Udeni B R
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Graves, Kathryn
  • Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, United States.
Bailey, Ernest
  • Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, United States.
Eberth, John
  • Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, United States.
Canisso, Igor F
  • Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, United States.
Andrews, Frank M
  • Equine Health Studies Program, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Keowen, Michael L
  • Equine Health Studies Program, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Go, Yun Young
  • Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine, City University of Hong Kong, Kowloon, Hong Kong SAR, China.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Citations

This article has been cited 1 times.
  1. Thieulent CJ, Sarkar S, Carossino M, Bhowmik M, Zhu H, Balasuriya UBR. Cell Surface Vimentin Is an Attachment Factor That Facilitates Equine Arteritis Virus Infection In Vitro. Viruses 2026 Jan 15;18(1).
    doi: 10.3390/v18010113pubmed: 41600875google scholar: lookup
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