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Diagnosis
The following pages provide an overview of methods for laboratory diagnosis of equine influenza. Further details of the methods including the practicalities of performing the assays can be found in the Office International des Epizooties Manual of Standards for Diagnostic Tests and Vaccines (2000) and in the references given.
Virus isolation
Virus detection
Serological diagnosis
It is important to obtain samples for the isolation or detection of equine influenza virus as soon as possible after the onset of clinical signs. The samples that may be taken include nasopharyngeal swabs or nasal and tracheal washings (the latter taken by endoscopy).
Isolation of infectious virus in may be carried out in embryonated hens’ eggs or cell cultures (Madin-Darby canine kidney, MDCK, cells). Although eggs are generally accepted as the permissive host for equine influenza and isolation in MDCK cells may select variant viruses that are not representative of the predominant virus in clinical specimens, in recent years some viruses have been isolated in MDCK cells and not eggs, therefore it is advisable, where possible, to use both substrates in parallel. Up to 5 passages may be necessary to isolate viruses therefore virus isolation can be a lengthy process. It is, however, important to isolate viruses for further characterization as part of the international surveillance programme, and to yield isolates that may be included in updated vaccines.
In situations where laboratory facilities for virus isolation are unavailable, influenza virus antigen in nasal secretions may be detected directly by an antigen-capture enzyme-linked immunosorbent assay (ELISA) employing a monoclonal antibody against the nucleoprotein. An ELISA specific for equine influenza has been developed at the Animal Health Trust (Cook et al., 1988; Livesay et al., 1993). However, most of the rapid diagnostic kits that are commercially available for the rapid diagnosis of human influenza A viruses are designed to detect highly conserved internal proteins and will therefore also detect equine influenza viruses. They share the advantages that results are available in 15 to 45 minutes and they are technically less complex than most laboratory assays. However, they are generally less sensitive than other methods and are too expensive for screening of large numbers of isolates. Further details of the different rapid diagnostic assays available are given below. These assays offer the additional advantage that they allow virus isolation efforts to be focused on samples known to be positive for equine influenza.
Rapid diagnostic kits
Directigen Flu A (Becton Dickinson)
An enzyme immunoassay (EIA) test for influenza A viral antigen (nucleoprotein).
+ Results are available in 15 minutes.
+ Suitable for rapid diagnosis of equine influenza (Chambers et al., 1994).ZstatFlu (ZymeTx)
+ Detects equine influenza (T Chambers, personal communication).
- Specificity for equine influenza not validated.Flu OIA (Biostar)
An Optical ImmunoAssay (OIA) test for influenza A and B viral antigen (nucleoprotein).
+ Results are available in 15 minutes.
- Cross-reacts with equine herpesvirus (EHV)-2, therefore not suitable for diagnosis of equine influenza due to lack of specificity (T Chambers, University of Kentucky, personal communication).QuickVue Influenza (Quidel)
Another enzyme immunoassay (EIA)-based test for influenza A viral antigen.
+ Results are available in 10 minutes.
- Specificity for equine influenza not validated.NOW Flu A (Binax)
Assay based on what Binax term immunochromatographic (ICT) technology.
+ Results are available in 15 minutes.
+ Requires only one step.
- Specificity for equine influenza not validated.
Other virus detection methodsOther methods are in use for the detection of equine influenza virus which, although usually more rapid than virus isolation, are not as rapid as the rapid assay kits described above and generally require more technical expertise. These include applying the polymerase chain reaction to detect genetic material (Donofrio et al., 1994; Oxburgh & Hagstrom, 1999; Ozaki et al., 2001) and indirect immunofluorescence (Anestad & Maagaard). The polymerase chain reaction is a highly sensitive technique and can provide material for genetic characterisation of viruses.
References for virus detection
ANESTAD G & MAAGAARD O (1990) Rapid diagnosis of equine influenza. Vet Rec 126, 550-551.
CHAMBERS TM, SHORTRIDGE KF, LI PH, POWELL DG & WATKINS KL (1994) Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay. Veterinary Record 135, 275-279.
COOK RF, SINCLAIR R & MUMFORD JA (1988) Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses. Journal of Virological Methods 20, 1-20.
DONOFRIO JC, COONROD JD & CHAMBERS TM (1994) Diagnosis of equine influenza by the polymerase chain reaction. J Vet Diagn Invest 6, 39-43.
LIVESAY J, O’NEILL T, HANNANT D, YADAV MP & MUMFORD JA (1993) The outbreak of equine influenza (H3N8) in the United Kingdom in 1998; diagnostic use of an antigen capture ELISA. Veterinary Record 133, 515-519.
OXBURGH L & HAGSTROM A (1999) A PCR based method for the identification of equine influenza virus from clinical samples. Veterinary Microbiology 67, 161-174.
OZAKI H, SUGITA S & KIDA H (2001) A rapid and highly sensitive method for diagnosis of equine influenza by antigen detection using immuno-PCR. Jpn J Vet Res 48, 187-195.Two simple methods exist to detect equine influenza-specific antibodies, haemagglutination inhibition (HI) and single radial haemolysis (SRH). The complement fixation (CF) test can also be applied, but is not standardised or in general use.
The HI assay measures the ability of antibody present in a serum sample to inhibit agglutination of red blood cells (usually chicken) by a standardised dose of virus. The HI titre is expressed as the reciprocal of the highest dilution of serum that will still inhibit at least half the agglutination in a microtitre well. In the SRH assay, viral antigens are coupled to fixed sheep red blood cells that are suspended in agarose containing guinea-pig complement. Wells are punched in the agarose and filled with test sera. Influenza antibodies in the test sera (if present) and complement lyse the antigen-coated red blood cells, resulting in a clear, haemolytic zone around the well. The area of this zone of haemolysis (measured in mm) is directly proportional to the amount of antibody present in the test serum {Figure}.
Diagnosis of equine influenza can usually only be accomplished by tests on paired sera; the first sample should be taken as soon as possible after the onset of clinical signs, and the second around 2 weeks later. Both of the paired serum samples should be tested together at the same time, to minimise test variability. Sera are heat inactivated, and for the HI assay are pre-treated to remove non-specific inhibitors of agglutination. For diagnosis of H3N8 viruses by HI, treatment of the antigens used with Tween 80/ether enhances the haemagglutination titre and hence the sensitivity of the assay (John & Fulgitini, 1966). The SRH assay may be more sensitive for infections in vaccinated horses, and vaccine-induced SRH antibody is proportional both to the amount of haemagglutinin present in the vaccine and the degree of protection afforded against challenge with a homologous or closely-related virus strain. The HI assay can be used in the characterisation of new isolates by comparing the reactivity of different viruses against specific antisera. The species in which antisera are raised will influence the outcome, with ferrets providing the most strain-specific antibody (Mumford, 1992).
The European Pharmacopoeia Biological Reference Preparations for equine influenza (A/eq/Newmarket/1/93 (H3N8), A/eq/Newmarket/2/93 (H3N8) and A/eq/Newmarket/77 (H7N7)) have been accepted as OIE international reference standards for antibody assays for the diagnosis of equine influenza.
References for serological diagnosis
JOHN TJ & FULGITINI VA (1966) Parainfluenza 2 virus: increase in haemagglutinin titre on treatment with Tween 80 and ether. Proc. Soc. Exp. Med. 121, 109-111.
MORLEY PS, BOGDAN JR, TOWNSEND HGG & HAINES DM (1995) The effect of changing single radial haemolysis assay method when quantifying influenza A antibodies in serum. Veterinary Microbiology 44, 101-110.
MUMFORD JA (1992) Progress in the control of equine influenza. In: Equine Infectious Diseases VI. Eds. W Plowright, PD Rossdale & JF Wade. R&W Publications, UK. 207-217.
PLATEAU E & CRUCIERE C (1983) Study on radial haemolysis method for the detection of anti influenza antibodies in equine sera: reliability and expression of the results. Zentralbl. Veterinarmed. [b] 30, 512-520.
SCHILD GC, PEREIRA MS & CHAKRAVERTY P (1975) Single radial haemolysis: a new method for the assay of antibody to influenza haemagglutinin. Bull. WHO 52, 43-50.
