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August 2009 Issue

Overview of West Nile Virus (WNV)

West Nile fever is a disease caused by West Nile virus (WNV), a single-stranded RNA virus of the Flaviviridae family. Other viruses in this family cause Japanese encephalitis, yellow fever and Saint Louis encephalitis.

The name of the virus comes from the West Nile district of Uganda where the disease was first reported in 1937 in a woman with a high fever. In 1957, WNV was recognized as the cause of a severe meningitis and encephalitis outbreak in Israel. In 1999, the disease spread to the Western hemisphere, where it first appeared in the New York area and then entered Canada, Mexico and Latin America.

There are two virus lineages. Lineage 1 is found mostly in Europe, North Africa, Central Africa, Israel, India, Central America, North America, Argentina and Columbia. Lineage 2 is endemic in Central Africa, Southern Africa and Madagascar.

Clinical Signs

West Nile fever primarily affects birds, humans and horses. The disease is generally asymptomatic, but in about 20% of cases, flu-like symptoms such as fever, headache, anorexia and myalgia are reported. The disease can also cause encephalomyelitis in humans and horses.

In horses, the incubation period is 3–15 days. Infected horses usually show ataxia and can also show weakness, muscle fasciculation and cranial nerve deficits.

About 70% of horses do not present clinical signs, while 20% present mild signs. Neuro-invasive forms of infection are observed in about 1%–10% of infected horses; these forms include meningitis, encephalitis, paresis, motor-neuron infections and flaccid paralysis. Between 20% and 60% of horses with neuro-invasive infections die. There is no specific treatment for West Nile fever.

Disease Transmission

Wild birds are the reservoir of the West Nile virus, which is transmitted by mosquitoes. Mosquitoes become infected when they bite infected birds. They then spread the virus to other birds and other species, including humans and horses. Humans and horses are considered “dead-end” hosts because WNV does not replicate enough within them to reach a transmissible level.

Diagnosis of West Nile Fever Using IgM Antibody Detection

West Nile fever clinical signs raise the suspicion of WNV infection, but because cases of WNV infection may have no apparent signs, diagnosis requires both clinical assessment and laboratory tests.

West Nile fever can be diagnosed by detecting the virus using virology or polymerase chain reaction methods. But the most useful method is one that detects IgM antibodies to WNV.

IgM antibodies can be detected for up to three months after infection. Therefore, the presence of IgM antibodies reveals the presence of recent WNV infection, making this detection method useful for epidemiological studies, as well as for disease diagnosis. The OIE Terrestrial Manual calls the IgM-capture ELISA “particularly useful in detecting recent natural exposures and infections by West Nile virus.”¹

A positive result for an equine sample tested with an IgM-capture ELISA indicates that the horse had contact with West Nile virus within the last three months. Mosquitoes spreading West Nile virus were recently circulating in the area and may still be present in the environment.

Protective Measures

The key to protecting against West Nile virus is to control the mosquito population and reduce exposure to mosquitoes. Insect screens and repellents are useful. Other recommendations include: isolating horses from mosquitoes, using fans to keep mosquitoes away, and switching off lights at night.

IDEXX Pourquier* IgM WNV ELISA Detects Recent Exposure to West Nile Virus

The IDEXX Pourquier* IgM WNV ELISA is a two-step capture ELISA that accurately detects IgM antibodies against West Nile virus (WNV) in horse serum.

Because IgM antibodies can be detected for only three months after infection, positive test results indicate both recent WNV exposure and the presence of WNV circulating in the area.

Other WNV ELISAs detect IgG antibodies, which remain in the horse for several years after infection. The presence of IgG antibodies does not provide information about the timing of infection or the current presence of WNV in the environment.

Two-Step Capture ELISA Method

The following figure illustrates the two-step IgM capture method employed by the IDEXX Pourquier IgM WNV ELISA.

1. Each sample is placed in two wells of the microplate. If the sample contains IgM antibodies, they become fixed to the anti-equine IgM coating of the microplate wells.
2. The microplate is washed, then West Nile Recombinant Antigen (WNRA) is added to one well and Normal Cell Antigen (NCA) is added to the other well for each sample. If the sample contains WNV-specific IgM antibodies, the WNRA becomes fixed to the IgM in the wells.
3. The microplate is washed, then HRP conjugate against West Nile is added to all wells. The conjugate binds to any WNRA fixed to the IgM.
4. The microplate is washed, then TMB enzyme substrate is added to the wells. If the conjugate was bound to the WNRA (in step 3), the TMB enzyme forms a blue compound that becomes yellow after stop solution is added.
5. The microplate is read at 450 nm. The intensity of the color is proportional to the amount of antibody in the sample.

Immune Status Ratio (ISR) Provides a Clear Interpretation of Results

Results of the ELISA are expressed as an Immune Status Ratio (ISR), which is the ratio between the optical density of the WNRA well and the optical density of the NCA well for each sample.

Results are interpreted as follows:

• ISR ≤ 2.00 = Negative (the sample does not contain IgM antibodies against West Nile virus).
• ISR ≥ 3.00 = Positive (the sample does contain IgM antibodies against West Nile virus, indicating a recent exposure to the virus).
• ISR between 2.00 and 3.00 (2.00 < ISR < 3.00) = Doubtful (the sample requires a confirmatory test).

Accurate Timing of Infection Allows Appropriate Response

Because IgM are detectable from seven days to three months after WNV exposure (see chart in “Overview of West Nile Virus”), a positive result from an IgM-capture ELISA means the horse was exposed to West Nile Virus within the last three months.

Such a recent infection indicates that mosquitoes are spreading WNV in the area. Armed with this knowledge, you can take appropriate actions to protect animals and to eliminate mosquitoes.

Sensitivity and Specificity Studies

The IDEXX Pourquier IgM WNV ELISA demonstrated 99% sensitivity and 100% specificity in tested populations.

Although there is no reference serum for use with West Nile ELISAs, the IDEXX Pourquier IgM WNV ELISA was tested using a positive serum from the French National Reference Laboratory. The IDEXX kit detected this positive serum at a dilution of 1:102400.

Availability of IDEXX Pourquier IgM WNV ELISA

The IDEXX Pourquier IgM WNV ELISA is available outside of the U.S. with these reference IDs:

• P00730-2 : 2-plate kit
• P00730-5 : 5-plate kit

To order a kit or for further information, please contact your IDEXX representative or call Customer Service at +33 4 99 23 24 28.

Maintaining Your xChek* Database

IDEXX recommends that as your xChek* database grows in size, you perform two procedures that will help your database function with the speed and robustness you are accustomed to: archiving the database and compacting the database.

Archiving Your Database

The archiving process lets you remove data from a database and store it. Use archiving to remove old data that you do not access regularly and that is taking up valuable space in your database, but that you do not want to lose. Archived data will be removed from your existing database and stored in the archive.

How frequently you archive your database depends on your testing volume, as well as on your need to continually access old data. IDEXX recommends that you routinely back up your database for security reasons by using either the archive function or the export function.

1. Start the xChek software and log in.
2. Choose Database > Archive from the menu bar to open the Create Archive dialog box.
3. To specify a location for your archive database, enter a filename and click Save.
   A dialog box is displayed with the message, “Enter the date of the last plate set you wish to keep active”.
4. Enter a date and click OK. xChek will archive all data with a date older than the specified date.

Compacting Your Database

As the xChek database accumulates data and grows in size, it becomes less efficient. The compact function lets you clean up your database and remove any unnecessary clutter.

1. Start the xChek software and log in.
2. Choose Database > Compact Database from the menu bar.

Events Around the World

• Minneapolis, MN, USA—August 9–14, 2009
  10th International Colloquium on Paratuberculosis
  www.cvm.umn.edu/outreach/events/icp/home
• Budapest, Hungary—August 23–26, 2009
  8th International Congress of Veterinary Virology
  www.vmri.hu/esvv2009
• Copenhagen, Denmark—August 27–28, 2009
  First European Symposium on Porcine Health Management
  www.ecphm.org
• Surrey, England—September 2–4, 2009
  VLA International Conference—Animal Diseases 2009
  www.defra.gov.uk/vla/news/new_conf_vla2009.htm
• Giessen, Germany—Septemter 2–4, 2009
  DVG Meeting
  www.dvg.de
• Bern, Switzerland—Septemter 3, 2009
  Diagnostic Laboratories Meeting
  www.bvet.admin.ch
• Omaha, NE, USA—September 10–12, 2009
  American Association of Bovine Practioners (AABP)
  www.aabp.org/meeting/vendors/default.asp
• Kloster Banz, Germany—September 16–18, 2009
  AVID-Tagung "Bakteriologie" 2009
  www.avid.dvg.net
• Saint Paul, MN, USA—September 19–22, 2009
  Allen D. Leman Swine Conference
  www.cvm.umn.edu/outreach/events/adl
• Porto Carras, Greece—September 23–25, 2009
  Prion 2009
  www.prion2009.com
• Krakao, Poland—Septemter 28–29, 2009
  TSE Conference
  www.piwet.pulawy.pl.piwet7/index_b_eng.php?strona=conf1
• San Diego, CA, USA—October 7–14, 2009
  American Association of Veterinary Laboratory Diagnostics/U.S. Animal Health Association (AAVLD/USAHA)
  www.adilva.com
• Toulouse, France—October 14–16, 2009
  ADILVA
  www.aavld.org
• Greifswald, Germany—November 5–6, 2009
  Greifswald Congress
  
• Marrakesh, Morocco—November 8–12, 2009
  XVI Congress of the World Veterinary Poultry Association
  www.wvpa.net/fs_wvpa_congress.html
• Southport, England—November 26–28, 2009
  British Congress Veterinary Association (BCVA) Congress 2009
  www.bcva.org.uk
• Marseille, France—December 1–3, 2009
  European Buiatrics Forum
  www.buiatricsforum.com/anpartspons2009.html

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