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Determining an optimal pool size for testing beef herds for Johne's disease in Australia
Anna Ly1, Navneet K Dhand1, Evan S G Sergeant2
1Faculty of Science, Sydney School of Veterinary Science, The University of Sydney, Camden, NSW, Australia.
Determining the optimal pool size for bovine Johne
Area of Science:
- Veterinary Medicine
- Molecular Diagnostics
- Bovine Health
Background:
- Bovine Johne's disease (JD) is a significant global cattle health concern.
- Current diagnostic methods for Mycobacterium avium subsp. paratuberculosis (Mptb) rely on lengthy culture techniques.
- High-throughput molecular testing offers a faster alternative, but optimal parameters for beef cattle are unknown.
Purpose of the Study:
- To identify the optimal pool size for direct fecal quantitative PCR (qPCR) testing of beef cattle for Mptb.
- To evaluate the impact of pool size on diagnostic sensitivity and specificity.
Main Methods:
- Individual fecal samples with known Mptb quantities were pooled with negative samples at sizes of 5, 10, 15, and 20.
- Mptb DNA was extracted using magnetic bead isolation.
- Quantitative PCR (qPCR) targeting the IS900 gene was performed on all pools.
Main Results:
- A pool size of 10 achieved the highest diagnostic sensitivity (73%) for Mptb detection.
- Diagnostic specificity was 100% across all tested pool sizes.
- Larger pool sizes (>10) increased PCR inhibition, manageable through DNA dilution.
Conclusions:
- A fecal pool size of 10 is optimal for direct qPCR testing of beef cattle for JD.
- This finding supports cost-effective JD surveillance strategies in beef herds.
- Optimized pooling strategies can enhance the efficiency of molecular diagnostics for Mptb.
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