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Journal of Clinical Microbiology, February 2004, p. 759-763, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.759-763.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comparison of Sensitivities of Virus Isolation, Antigen Detection, and Nucleic Acid Amplification for Detection of Equine Influenza Virus

Michelle Quinlivan,1 Ann Cullinane,1* Maura Nelly,1 Kees van Maanen,2 Jacco Heldens,3 and Sean Arkins4

Virology Unit, Irish Equine Centre, Johnstown, Naas, County Kildare,1 Department of Life Sciences, University of Limerick, Limerick, Ireland,4 Animal Health Service, 7400 AA Deventer,2 Intervet International BV, Department for Virological Research and Development, 5830 AA Boxmeer, The Netherlands3

Received 9 June 2003/ Returned for modification 19 August 2003/ Accepted 5 October 2003

Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 106 50% egg infective doses (EID50)/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID50 was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucleoprotein gene and a single set of primers from the matrix gene. RT-PCR using the matrix primers and virus isolation in embryonated eggs proved to be the most sensitive methods for the detection of virus. The Directigen Flu A test was the least sensitive method. The inclusion of 2% fetal calf serum in the viral transport medium inhibited the growth of virus from undiluted samples in MDCK cells but was essential for the maintenance of the virus titer in samples subjected to repeated freeze-thaw cycles.

* Corresponding author. Mailing address: Virology Unit, Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland. Phone: 353-45-866266. Fax: 353-45-866273. E-mail: acullinane{at}equine-centre.ie.

Journal of Clinical Microbiology, February 2004, p. 759-763, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.759-763.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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