Evaluation of swab pooling and transport medium to improve the identification of avian influenza viruses in farmed ostriches in South Africa

Abstract

Avian influenza (AI) control in farmed ostriches relies on rapid and accurate diagnostic testing to detect the presence and spread of disease in avian populations, as flocks are in unavoidable contact with wild bird reservoirs, increasing the possibility of infection. Increasing swab pools from five to ten would significantly decrease the cost of testing by reducing the number of tests performed; however detection of highly pathogenic avian influenza (HPAI) at low virus titres by RT-qPCR and virus isolation is critical to ensure that the disease is detected and accurately characterised. The present study found that pooling a single HPAI positive ostrich swab with four or nine AI negative ostrich tracheal swabs did not significantly affect the detection of influenza A virus by RT-qPCR (p < 0.05) over the dilution range. RT-qPCR had a high sensitivity detecting up to 102.5 EID50/ ml (~ RT-qPCR Ct value = 35) virus titre compared to virus isolation, that detected only up 104.8 EID50/ml (~ RT-qPCR Ct value = 29) virus titre, confirming that weak positive swab pools with a low virus titre detected by RT-qPCR (Ct value ≥ 35) may not be detected by standard virus isolation techniques. Both virus transport media evaluated (10% v/v glycerol in brain heart infusion (BHI) broth supplemented with antibiotics (VTM) and 50% v/v glycerol PBS transport medium (pH 7.2)) facilitated the isolation of influenza A virus at ~ 104.5 EID50/ml (RT-qPCR Ct value ~ 27); however VTM improved the efficiency of the method, potentially reducing the reporting time. Sampling costs can be reduced by increasing swabs pools to ten and efficacy of virus isolation can be improved by using antibiotic supplemented VTM, without compromising the detection of influenza A virus by RT-qPCR. It is recommended that costs and efforts are focused at isolating virus from pooled swab samples, with a virus titre of ≥ 104 EID50/ ml (RT-qPCR Ct value ≤ 29), followed by full molecular sequencing and characterisation of the viral isolate. Samples with a low virus titre < 104 EID50/ ml (RT-qPCR Ct value > 29) should be characterised by direct sequencing of PCR amplicons or type-specific RT-qPCR assays.