Bovine respiratory disease (BRD) can involve multiple infectious agents, making it difficult to identify the pathogens contributing to an outbreak. Multiplex PCR testing offers a way to investigate several pathogens simultaneously, but the cost of specialized equipment and reagents can limit access to these diagnostics.
Researchers in Brazil have developed an alternative approach that could make viral BRD testing more accessible. Their endpoint multiplex PCR/RT-PCR assay uses conventional PCR technology rather than real-time PCR (qPCR), potentially reducing the equipment and reagent requirements for laboratories performing molecular diagnostics.
The Challenge of Diagnosing Bovine Respiratory Disease
BRD involves interactions among host, environmental and pathogen factors, including several viral and bacterial agents. Identifying the infectious contributors can require multiple laboratory tests, increasing diagnostic costs and the time needed to obtain results.
Multiplex assays address part of this challenge by detecting several pathogens in a single reaction. However, many existing molecular panels rely on qPCR technology, which requires specialized equipment and, depending on the assay, pathogen-specific probes.
An Alternative Approach to Multiplex PCR Testing
Endpoint PCR offers a different approach. Rather than measuring amplification as it occurs, the technique identifies amplified genetic material at the end of the reaction. Researchers separate the resulting DNA fragments using agarose gel electrophoresis. Because each target produces a fragment of a different size, the viral agents can be differentiated by examining the resulting bands.
The researchers designed their assay to detect five viral targets, covering seven viruses associated with BRD:
- Bovine herpesvirus 1 (BoAHV-1)
- Bovine viral diarrhea virus 1 (BVDV-1)
- Bovine viral diarrhea virus 2 (BVDV-2)
- HoBi-like pestivirus (HoBiPeV)
- Bovine parainfluenza virus 3 (BPIV-3)
- Bovine respiratory syncytial virus (BRSV)
- Bovine coronavirus (BCoV)
The three pestiviruses are detected through a shared target, allowing the assay to cover seven viruses using five sets of viral targets.
Assay Detects Major Viral Targets in Clinical Samples
Researchers evaluated the assay’s analytical performance before testing it against 289 respiratory specimens from cattle with clinical signs compatible with BRD. The sample set included 277 nasal swabs from Holstein dairy calves on 16 farms in São Paulo, Brazil, along with 10 lung tissue samples and two additional nasal swabs submitted to a diagnostic laboratory.
The multiplex assay produced the same results as the individual PCR/RT-PCR tests for all 289 samples. Researchers also assessed whether the assay would produce unintended amplification in the presence of three bacterial pathogens associated with BRD: Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. None produced nonspecific amplification.
In the clinical sample evaluation, 43 of 289 specimens (14.8%) tested positive for at least one viral target. BPIV-3 was most frequently detected, accounting for 44.2% of positives, followed by BCoV at 23.3%, BRSV at 16.3%, BoAHV-1 at 14.0% and bovine pestiviruses at 2.3%.
Balancing Diagnostic Accessibility and Practical Limitations
The assay could offer a more accessible way to test for multiple viral BRD pathogens without investing in a real-time PCR platform. The use of conventional PCR could make the protocol more affordable while maintaining adequate sensitivity, potentially giving laboratories with more limited resources another option for investigating respiratory disease cases.
The approach also comes with practical trade-offs. Unlike qPCR, endpoint PCR does not quantify viral load, which limits the information available from a positive result. The process also requires additional hands-on time for gel electrophoresis and interpretation. Keep in mind that the assay covers viral pathogens only, so bacterial testing may still be needed to build a more complete picture of the disease.
Expanding Access to Viral BRD Diagnostics
The assay offers a potential alternative for veterinary diagnostic laboratories seeking to expand molecular testing for BRD-associated viruses without relying on real-time PCR equipment. Its ability to detect multiple viral targets in a single reaction could help laboratories investigate viral contributors to respiratory disease with a more accessible testing approach.
The assay still has important limitations. It does not detect bacterial pathogens, quantify viral load or establish which agent is responsible for clinical disease. Veterinarians would need to interpret results alongside clinical findings and other diagnostic testing to understand the broader disease picture.
Further validation across different cattle populations and production systems will help determine how broadly the assay can be applied. For now, the research provides another approach to identifying viral agents associated with BRD and highlights the potential to expand access to molecular diagnostics in veterinary laboratories.
