Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR

Area of Science:

• Veterinary immunology and diagnostics
• Molecular biology and genetic epidemiology

Background:

• Bovine leukemia virus (BLV) causes chronic, untreatable livestock diseases, impacting productivity and requiring control strategies.
• Controlling BLV transmission involves removing superspreaders and selectively breeding resistant cattle.
• Accurate measurement of BLV proviral load (PVL) and host genetic markers is crucial for effective management.

Purpose of the Study:

• To develop a simple, rapid method for measuring BLV PVL and identifying susceptible/resistant cattle.
• To assess the impact of specific bovine major histocompatibility complex (BoLA) alleles on BLV PVL.

Main Methods:

• Development of a fourplex droplet digital PCR assay (IPATS-BLV).
• Targeting BLV pol gene, susceptible BoLA-DRB3*016:01 allele, resistant BoLA-DRB3*009:02 allele, and RPP30 housekeeping gene.
• Precise measurement of BLV-infected cell percentage and allele typing, discriminating homozygous and heterozygous carriers.

Main Results:

• IPATS-BLV accurately quantified BLV PVL and determined BoLA allele types.
• Cattle carrying the resistant BoLA-DRB3*009:02 allele suppressed BLV PVL, even with a susceptible allele.
• Cattle with the susceptible BoLA-DRB3*016:01 allele exhibited significantly higher, but variable, PVLs.

Conclusions:

• IPATS-BLV is a simple, rapid, single-well assay for combined BLV PVL and host biomarker diagnosis.
• The assay aids in identifying BLV-resistant cattle and strengthens risk-based herd management.
• This platform has potential for diagnosing other infectious diseases by combining pathogen load and host genetic markers.