FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / J Clin Microbiol / Comparison of sensitivities of virus isolation, antigen dete...
Journal of clinical microbiology2004; 42(2); 759-763; doi: 10.1128/JCM.42.2.759-763.2004

Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus.

Abstract: Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 10(6) 50% egg infective doses (EID(50))/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID(50) 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.
Get Full Text
Publication Date: 2004-02-10 PubMed ID: 14766849PubMed Central: PMC344455DOI: 10.1128/JCM.42.2.759-763.2004Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Comparative Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research aimed to assess the sensitivity of different methods in detecting equine influenza virus, including virus isolation, antigen detection, and nucleic acid amplification. Among the several methods used, virus isolation and nucleic acid amplification through the matrix primers displayed the highest sensitivity while the Directigen Flu A test was found to be the least sensitive.

Research Design and Methodology

  • This experiment was conducted on four seronegative foals of 6 to 7 months old, exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 10(6) 50% egg infective doses (EID(50))/ml.
  • The researchers collected nasopharyngeal swabs from the foals for 10 consistent days after this exposure.
  • The procedure of virus isolation was carried out in embryonated eggs and the EID(50) was determined for all the samples that were tested positive.
  • The 50% tissue culture infective dose was also determined using Madin-Darby Canine Kidney (MDCK) cells.
  • In addition to all these, the samples were also tested through an ‘in vitro enzyme immunoassay’ test, named Directigen Flu A, as well as by Reverse Transcription Polymerase Chain Reaction (RT-PCR) using nested primers from the nucleoprotein gene and a single set of primers from the matrix gene.

Findings

  • RT-PCR using the matrix primers and virus isolation in embryonated eggs turned out to be the most sensitive of all methods in detecting the virus. This signifies their effectiveness in identifying the presence of equine influenza virus as compared to the other methods.
  • The Directigen Flu A test was found out to be the least sensitive method, implying its reduced efficiency in detecting the virus presence.
  • The research data also indicated that the inclusion of 2% of the fetal calf serum in the viral transport medium could inhibit the growth of the virus in the undiluted samples in MDCK cells. However, this serum was found to be essential for maintaining the virus titer in samples which underwent several cycles of freezing and thawing.

Conclusion

  • The findings of this study provide valuable insights into a comparative study of different methods in detecting equine influenza virus. It emphasizes the effectiveness of rt-pcr method and virus isolation in embryonated eggs compared to the other methods.
  • The study could have significant implications for improving the virus detection methods in the future, subsequently contributing to the betterment of equine health through disease prevention and containment.

Cite This Article

APA
Quinlivan M, Cullinane A, Nelly M, Van Maanen K, Heldens J, Arkins S. (2004). Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus. J Clin Microbiol, 42(2), 759-763. https://doi.org/10.1128/JCM.42.2.759-763.2004

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 42
Issue: 2
Pages: 759-763

Researcher Affiliations

Quinlivan, Michelle
  • Virology Unit, Irish Equine Centre, Johnstown, Naas, County Kildare, Ireland.
Cullinane, Ann
    Nelly, Maura
      Van Maanen, Kees
        Heldens, Jacco
          Arkins, Sean

            MeSH Terms

            • Animals
            • Antigens, Viral / analysis
            • Base Sequence
            • DNA Primers
            • Female
            • Freezing
            • Horse Diseases / diagnosis
            • Horse Diseases / virology
            • Horses
            • Influenza A virus / genetics
            • Influenza A virus / immunology
            • Influenza A virus / isolation & purification
            • Orthomyxoviridae Infections / diagnosis
            • Orthomyxoviridae Infections / veterinary
            • Pregnancy
            • Reverse Transcriptase Polymerase Chain Reaction / methods
            • Sensitivity and Specificity
            • Specimen Handling / methods

            References

            This article includes 28 references
            1. Chambers TM, Shortridge KF, Li PH, Powell DG, Watkins KL. Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay.. Vet Rec 1994 Sep 17;135(12):275-9.
              pubmed: 7817505doi: 10.1136/vr.135.12.275google scholar: lookup
            2. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA. Antigenic and genetic evolution of equine H3N8 influenza A viruses.. J Gen Virol 1996 Apr;77 ( Pt 4):661-71.
              pubmed: 8627254doi: 10.1099/0022-1317-77-4-661google scholar: lookup
            3. Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwaan GF, Osterhaus AD. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene.. J Clin Microbiol 2000 Nov;38(11):4096-101.
              pmc: PMC87547pubmed: 11060074doi: 10.1128/jcm.38.11.4096-4101.2000google scholar: lookup
            4. Guo Y, Wang M, Kawaoka Y, Gorman O, Ito T, Saito T, Webster RG. Characterization of a new avian-like influenza A virus from horses in China.. Virology 1992 May;188(1):245-55.
              pubmed: 1314452doi: 10.1016/0042-6822(92)90754-dgoogle scholar: lookup
            5. Guthrie AJ, Stevens KB, Bosman PP. The circumstances surrounding the outbreak and spread of equine influenza in South Africa.. Rev Sci Tech 1999 Apr;18(1):179-85.
              pubmed: 10190213doi: 10.20506/rst.18.1.1155google scholar: lookup
            6. Johnston SL, Bloy H. Evaluation of a rapid enzyme immunoassay for detection of influenza A virus.. J Clin Microbiol 1993 Jan;31(1):142-3.
              pmc: PMC262637pubmed: 8417019doi: 10.1128/jcm.31.1.142-143.1993google scholar: lookup
            7. Karber G. 50% end point calculation. Arch. Exp. Pathol. Pharmakol. 162:480-483.
            8. Morley PS, Bogdan JR, Townsend HG, Haines DM. Evaluation of Directigen Flu A assay for detection of influenza antigen in nasal secretions of horses.. Equine Vet J 1995 Mar;27(2):131-4.
              pubmed: 7607146doi: 10.1111/j.2042-3306.1995.tb03049.xgoogle scholar: lookup
            9. Mumford JA, Hannant D, Jessett DM. Experimental infection of ponies with equine influenza (H3N8) viruses by intranasal inoculation or exposure to aerosols.. Equine Vet J 1990 Mar;22(2):93-8.
              pubmed: 2156688doi: 10.1111/j.2042-3306.1990.tb04217.xgoogle scholar: lookup
            10. Mumford J, Wood J. WHO/OIE meeting: consultation on newly emerging strains of equine influenza. 18-19 May 1992, Animal Health Trust, Newmarket, Suffolk, UK.. Vaccine 1993;11(11):1172-5.
              pubmed: 8249440doi: 10.1016/0264-410x(93)90092-cgoogle scholar: lookup
            11. Mumford JA, Jessett DM, Rollinson EA, Hannant D, Draper ME. Duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccines.. Vet Rec 1994 Feb 12;134(7):158-62.
              pubmed: 8160328doi: 10.1136/vr.134.7.158google scholar: lookup
            12. Mumford JA, Wilson H, Hannant D, Jessett DM. Antigenicity and immunogenicity of equine influenza vaccines containing a Carbomer adjuvant.. Epidemiol Infect 1994 Apr;112(2):421-37.
              pmc: PMC2271453pubmed: 8150017doi: 10.1017/s0950268800057848google scholar: lookup
            13. Mumford JA. Virus infections regarding the respiratory tract. Swiss-Vet 11-5:66-69.
            14. Nelly M. Characterisation of equine influenza virus isolates from the 1989 and 1992 influenza outbreaks in Ireland by nucleotide sequence determination of the haemagglutinin molecule. M.Sc. thesis. National University of Ireland, Dublin.
            15. Newton DW, Mellen CF, Baxter BD, Atmar RL, Menegus MA. Practical and sensitive screening strategy for detection of influenza virus.. J Clin Microbiol 2002 Nov;40(11):4353-6.
              pmc: PMC139677pubmed: 12409430doi: 10.1128/jcm.40.11.4353-4356.2002google scholar: lookup
            16. Office International des Epizooties (OIE). Equine influenza. Manual of standards for diagnostic tests and vaccines p. 546-557. OIE, Paris, France.
            17. Office International des Epizooties (OIE). Conclusions and recommendations from the consultation meeting of OIE and WHO experts on equine influenza, Newmarket, United Kingdom, Septemer 18-19, 1995. Office Int. Epiz. Bull. 108:482-484.
            18. Oxburgh L, Hagström A. A PCR based method for the identification of equine influenza virus from clinical samples.. Vet Microbiol 1999 Jun 30;67(3):161-74.
              pubmed: 10418871doi: 10.1016/s0378-1135(99)00041-3google scholar: lookup
            19. Rogers AL. A-equi-2 influenza in horses in the Republic of South Africa.. J S Afr Vet Assoc 1988 Jun;59(2):123-5.
              pubmed: 2839681
            20. Ryan-Poirier KA, Katz JM, Webster RG, Kawaoka Y. Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens.. J Clin Microbiol 1992 May;30(5):1072-5.
              pmc: PMC265226pubmed: 1583103doi: 10.1128/jcm.30.5.1072-1075.1992google scholar: lookup
            21. SOVINOVA O, TUMOVA B, POUSKA F, NEMEC J. Isolation of a virus causing respiratory disease in horses.. Acta Virol 1958 Jan-Mar;2(1):52-61.
              pubmed: 13533033
            22. Timoney PJ. Equine influenza.. Comp Immunol Microbiol Infect Dis 1996 Jun;19(3):205-11.
              pubmed: 8800546doi: 10.1016/0147-9571(96)00006-9google scholar: lookup
            23. van Maanen C, Cullinane A. Equine influenza virus infections: an update.. Vet Q 2002 Jun;24(2):79-94.
              pubmed: 12095083doi: 10.1080/01652176.2002.9695127google scholar: lookup
            24. van Maanen C, van Essen GJ, Minke J, Daly JM, Yates PJ. Diagnostic methods applied to analysis of an outbreak of equine influenza in a riding school in which vaccine failure occurred.. Vet Microbiol 2003 Jun 10;93(4):291-306.
              pubmed: 12713892doi: 10.1016/s0378-1135(03)00029-4google scholar: lookup
            25. WADDELL GH, TEIGLAND MB, SIGEL MM. A NEW INFLUENZA VIRUS ASSOCIATED WITH EQUINE RESPIRATORY DISEASE.. J Am Vet Med Assoc 1963 Sep 15;143:587-90.
              pubmed: 14077956
            26. Waner JL, Todd SJ, Shalaby H, Murphy P, Wall LV. Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus.. J Clin Microbiol 1991 Mar;29(3):479-82.
              pmc: PMC269804pubmed: 2037665doi: 10.1128/jcm.29.3.479-482.1991google scholar: lookup
            27. Webster RG. Are equine 1 influenza viruses still present in horses?. Equine Vet J 1993 Nov;25(6):537-8.
              pubmed: 8276003doi: 10.1111/j.2042-3306.1993.tb03009.xgoogle scholar: lookup
            28. Wernery R, Yates PJ, Wernery U, Mumford JA. An equine influenza outbreak in a polo club in Dubai, United Arab Emirates in 1995/96. Equine infectious diseases VIII p. 342-346. R&W Publications Ltd., Newmarket, United Kingdom.

            Citations

            This article has been cited 12 times.
            1. Kweon OJ, Lim YK, Kim HR, Choi Y, Kim MC, Choi SH, Chung JW, Lee MK. Evaluation of rapid SARS-CoV-2 antigen tests, AFIAS COVID-19 Ag and ichroma COVID-19 Ag, with serial nasopharyngeal specimens from COVID-19 patients. PLoS One 2021;16(4):e0249972.
              doi: 10.1371/journal.pone.0249972pubmed: 33831118google scholar: lookup
            2. Luo L, Han W, Du J, Yang X, Duan M, Xu C, Zeng Z, Chen W, Chen J. Chenodeoxycholic Acid from Bile Inhibits Influenza A Virus Replication via Blocking Nuclear Export of Viral Ribonucleoprotein Complexes. Molecules 2018 Dec 14;23(12).
              doi: 10.3390/molecules23123315pubmed: 30558117google scholar: lookup
            3. Mena J, Brito B, Moreira R, Tadich T, González I, Cruces J, Ortega R, van Bakel H, Rathnasinghe R, Pizarro-Lucero J, Medina R, Neira V. Reemergence of H3N8 Equine Influenza A virus in Chile, 2018. Transbound Emerg Dis 2018 Dec;65(6):1408-1415.
              doi: 10.1111/tbed.12984pubmed: 30054993google scholar: lookup
            4. Gora IM, Kwasnik M, Zmudzinski JF, Rozek W. Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus. Virol J 2017 Jun 21;14(1):120.
              doi: 10.1186/s12985-017-0788-3pubmed: 28637468google scholar: lookup
            5. Nzussouo NT, Kadjo HA, Coulibaly D, Ekaza E, Kouakou B, N'Golo DC, Tempia S, Davis R, Dosso M, Thompson M. Comparing Influenza Positivity Rates by Real-Time RT-PCR, Elisa and Viral Culture Methods in Côte D'Ivoire, West Africa, in 2009. Afr J Infect Dis 2013 Aug 9;7(2):31-35.
              doi: 10.4314/ajid.v7i2.3pubmed: 28451079google scholar: lookup
            6. Galvin P, Gildea S, Nelly M, Quinlivan M, Arkins S, Walsh C, Cullinane A. The evaluation of three diagnostic tests for the detection of equine influenza nucleoprotein in nasal swabs. Influenza Other Respir Viruses 2014 May;8(3):376-83.
              doi: 10.1111/irv.12235pubmed: 24512560google scholar: lookup
            7. Diaz-Mendez A, Viel L, Hewson J, Doig P, Carman S, Chambers T, Tiwari A, Dewey C. Surveillance of equine respiratory viruses in Ontario. Can J Vet Res 2010 Oct;74(4):271-8.
              pubmed: 21197227
            8. Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus. J Clin Microbiol 2009 Dec;47(12):3907-13.
              doi: 10.1128/JCM.00598-09pubmed: 19846644google scholar: lookup
            9. Fesenko EE, Kireyev DE, Gryadunov DA, Mikhailovich VM, Grebennikova TV, L'vov DK, Zasedatelev AS. Oligonucleotide microchip for subtyping of influenza A virus. Influenza Other Respir Viruses 2007 May;1(3):121-9.
              doi: 10.1111/j.1750-2659.2007.00018.xpubmed: 19453417google scholar: lookup
            10. Quinlivan M, Dempsey E, Ryan F, Arkins S, Cullinane A. Real-time reverse transcription PCR for detection and quantitative analysis of equine influenza virus. J Clin Microbiol 2005 Oct;43(10):5055-7.
              doi: 10.1128/JCM.43.10.5055-5057.2005pubmed: 16207961google scholar: lookup
            11. Jeong JM, Kang G, Kim JO, Lee JT, Park CI, Kim KH. Red Sea Bream Iridovirus Stability in Freeze-Thaw Cycles: Quantitative Assays of Infectious Particles. Animals (Basel) 2025 Jun 9;15(12).
              doi: 10.3390/ani15121699pubmed: 40564252google scholar: lookup
            12. Costa TP, Brown JD, Howerth EW, Stallknecht DE. Effect of a prior exposure to a low pathogenic avian influenza virus in the outcome of a heterosubtypic low pathogenic avian influenza infection in mallards (Anas platyrhynchos). Avian Dis 2010 Dec;54(4):1286-91.
              doi: 10.1637/9480-072210-Reg.1pubmed: 21313851google scholar: lookup
            Subscribe to our newsletter
            Join 99,383 horse owners like you who receive our email updates on horse health and nutrition.