• In 2001 it was noted that "Surveillance for WN virus disease in equines should be conducted, because they are potential sentinels of WN virus epizootic activity, and equine health is an important economic issue. Although equines did not appear to be good sentinels of increased human risk for WN virus infection in 1999-2000, more experience is clearly needed. Veterinarians and veterinary service societies/agencies are essential partners in any surveillance activities involving equines with WN virus disease. Any utility of equines as sentinels of increased human risk may soon be reduced if equine WN virus vaccines become available." (D67)
• Many equines in North America are now vaccinated and occurrence of clinical disease in these animals has decreased, from the peak of more than 15,000 reported cases in the USA in 2002 to under 500 reported cases in 2007; however, they may still play a role in surveillance. (D341, D343, N7.54.w2, N7.56.w1) [2008]

The goals of equine surveillance are to:

• Assess the public and equine health impact of WNV disease and to monitor national trends;
• Demonstrate the need for public and animal health intervention programs and to allocate resources; and 
• Identify factors for high-risk population groups or geographic areas to target for interventions. 

(D67, D147, N7.54.w2)

Not all horses which become infected develop clinical disease, but those becoming ill generally show neurological signs which, while not pathognomonic for WNV infection, do encourage a consideration of this disease in areas where it is known to occur or may be an emerging disease. Consideration of WNV infection as a differential diagnosis for a horse with neurological signs is likely to be increased if veterinarians are aware of this disease, its likely presence in an area, and the clinical signs which it causes.

• For arboviruses such as WN virus, EEE virus and WEE virus which clinically affect horses, reports by local veterinarians of equine encephalomyelitis provide a warning of increased arbovirus activity in an area. Surveillance of equine cases may be a practical and sensitive tool for the recognition of a potential public health problem with such diseases. (D68).
• Active equine surveillance involves regularly contacting large animal/equine veterinarians in an area to encourage them to report clinically suspect equine cases and to submit blood samples and necropsy samples, as appropriate, for laboratory confirmation of disease. (D68)
• As with other surveillance methods, results of laboratory testing are useful only if adequate records, including a case history and a vaccination history, accompany each sample. (D68)
• Totals of equines with WNV infection reported to CDC and USDA-APHIS were 25 in 1999, 60 in 2000; 738 in 2001; 15,257 in 2002, decreasing to 5,181 in 2003; 1,406 in 2004; 1,088 in 2005; 1,086 in 2006; and 507 in 2007. (D341.IIB.w3 - full text included, D343 - full text included, D345 - full text included, N7.56.w1, W30.May08.w3, W30.May08.w5, W184.May08.w25)

Timing and level of surveillance:

• Northeastern and Midwestern USA: Active ecological surveillance should begin in early spring and continue through the fall (autumn) until mosquito activity ceases because of cold weather. (D147)
  • During 2001-2002, onset of human illness in the northeastern states occurred from early July to mid-November; in the same years, cases in birds occurred as early as the first week of April and as late as the second week of December. (D147)
• Southern USA: Active ecological surveillance should be conducted year round in these areas. (D67, D147)
  • N.B. in the Southern states, WN virus circulates throughout the year, with activity detected from January to December. In 2001-2004, onset of human illness was reported from as early as April to as late as mid-December; equine and avian infections were reported in all months of the year in these geographical areas. (D147, P39.3.w1, J84.10.w5, J84.11.w4)
• Western USA: Ecological surveillance should be encouraged beginning in early spring and continuing through the fall until mosquito activity ceases due to cold weather. (D147)
  • In 2002, WNV activity was first reported among humans and animals in Rocky Mountain states and among animals in Pacific coast states. These events occurred relatively late in the year (mid-August). (D147)
• Western Hemisphere outside the USA: Surveillance in other countries of the Western Hemisphere should be encouraged. (D67)
  • In Canada, in 2002 a WNV epidemic occurred in Ontario and Quebec provinces and an equine/avian epizootic occurred extending from the maritime provinces to Saskatchewan. By 2005, WV virus had been detected in Alberta, Manitoba, New Brunswick, Nova Scotia, Ontario, Quebec and Saskatchewan. (B526.21.w21, D147)
    • WNV infection in equines in Canada is an immediately notifiable disease (veterinary laboratories are required to notify suspicion or diagnosis of WNV in horses and other domestic animals to CFIA, but equine surveillance is no longer a part of WNV early detection activities. (W43.May08.w2)
  • Development of surveillance systems capable of detecting WNV activity should be encouraged in the Caribbean and Central and South America. WNV surveillance should be integrated with dengue surveillance in these areas, and with yellow fever surveillance in areas where urban or peri-urban transmission of this virus occurs. (D147)
    • Equine surveillance has been recommended as part of surveillance for WNV in the Caribbean and in South America (Brazil). (J408.40.w1, W705.May08.w1)
    • By 2005, WV virus had been detected in equines and/or birds in Mexico, the Caribbean (the Bahamas, the Cayman Islands, Cuba, the Dominican Republic, Guadeloupe, Jamaica, Puerto Rico, Trinidad), Central America (Belize, Guatemala, El Salvador), and South America (Colombia); in 2006 it was detected in Venezuela and caused fatal illness in equines in Argentina. (B526.21.w21, J84.12.w3, J84.13.w1, J270.45.w1, J491.19.w1)
• Southern Africa: transmission of WNV increases in the early months of the year, following heavy rainfall in spring and summer. (J84.11.w4)
  • Testing of equines has been used to confirm the presence of WNV in several countries of sub-Saharan Africa. (J84.12.w5, J87.35.w1)

Minimal components of an equine surveillance programme for WN virus include:

• Prompt reporting of all equine neurological cases with testing of affected individuals for WN virus, other arboviruses (as appropriate for the geographical area) and rabies.
• Prompt investigation of clusters of equine neurological disease.

(D147)

NOTE: Data on veterinary cases (mainly equines) in the USA to 2007 is available summarised in map form. See: Map0001 - Spread of West Nile Virus in the USA (2000-2007) - Veterinary maps

• Guidelines for Arbovirus Surveillance Programs in the United States

• Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention, and Control

• Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control Third Revision

• New York State West Nile Virus Response Plan - Guidance Document

• Health Canada - Municipal Mosquito Control Guidelines

• West Nile Virus In Equids in the Northeastern United States in 2000

• Equine 2005 Part II- Changes in the U.S. Equine Industry,1998-2005

• Western equine encephalomyelitis (WEE);
• Eastern equine encephalomyelitis (EEE);
• Venezuelan equine encephalomyelitis (VEE);
• Equine herpesvirus 1 (EHV-1) infection (equine herpesvirus myeoloencephalopathy, also called EHM);
• Rabies;
• Borna disease;
• Japanese encephalitis (JE) virus infection;
  • (in eastern, southern and southeastern Asia, Papua New Guinea and the Torres Strait of northern Australia) (J242.S10.w1)
• Equine protozoal myeloencephalopathy (EPM);
• Bacterial meningoencephalomyelitis;
• Verminous meningoencephalomyelitis;
• Leukoencephalomacia (fumonisin B1 toxicosis);
• Encephalopathy associated with:
  • Hepatic disease,
  • Renal disease,
  • Intestinal disease resulting is severe electrolyte abnormalities and dehydration;
• Wobbler syndrome (myelopathy due to cervical stenosis);
• Hypocalcaemia;
• Botulism;
• Space occupying masses in brain or in spinal canal such as neoplasia or abscesses;
• Trauma;
• Other toxicities.

Note: 

• It is unlikely that more than one horse in a single premises will develop neurological signs due to causes such as trauma or a space-occupying lesion in the CNS at one time.
• Clustering of cases in an area where WN virus is known to be active would increase the level of suspicion. 

(J4.218.w2, J89.16.w1, J89.22.w1, J214.267.w2, J242.S10.w1, J484.35.w1, B249.11.w1, P51.48.w1, P51.49.w3, V.w117)

• Assessment of Individual Cases by Veterinary and Medical Personnel
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Editorial Overview of Disease Characteristics for specified SPECIES-TAXA

Specimens required:

• Serum and cerebrospinal fluid (CSF) for antibody testing
  • N.B. Due to the widespread use of WNV vaccines in equids, a full vaccination history should be provided with all equine specimens submitted for antibody testing. (D147)
  • Seroconversion (negative to positive), or a four-fold or greater rise in titre in paired serum samples taken 10-14 days apart and tested with the PRNT; note that neutralising antibodies detected by the PRNT may persist for several months after infection. IgM-capture ELISA may be used to detect recent infection but there is a greater risk of cross reaction with other flaviviruses. (J215.24.w1)
    • IgM in serum, detected by MAC-ELISA, is reliable for identification of recently-infected horses. The presence of IgM in serum probably indicates recent infection and appears to be minimally affected by vaccination against WNV (IgM is present from about 6-7 day after infection and detectable for less than three months - e.g. for about 6-8 weeks); its presence in CSF is highly suggestive of recent infection, since IgM cannot cross the blood-brain barrier. (J4.228.w1, J89.22.w1, J275.20.w1, J484.35.w1)
    • IgG may remain detectable in serum for as long as 15 months post infection; it is also induced by vaccination. (J89.22.w1, J484.35.w1)
    • IgG ELISA may cross-react with other flaviviruses, but a four-fold or greater increase in IgG titre on paired samples collected 14 days apart, without a history of recent vaccination against WNV, indicates recent infection. (J89.22.w1, J484.35.w1)
    • PRNT is more specific than IgG ELISA. (J89.22.w1)
    • PRNT requires live WN virus and a higher level Biosafety Facility, therefore is not used by many laboratories; the MAC-ELISA is used more commonly. (V.w117)
    • An epitope-blocking ELISA can detect WNV specific antibodies. (J93.41.w1, J484.35.w1)
• Whole blood or CSF may be taken for virus isolation or detection of WN Virus RNA by RT-PCR. (J212.16.w1, J484.35.w1)
  • Note: viraemia is generally of low level and short duration in equines, therefore a negative finding does not rule out WNV Infection (diagnostic sensitivity of this method is low). (J212.16.w1, J484.35.w1)
  • On occasion (three of 140 clinically affected horses in one study), viral RNA may be detected in blood while no IgM is detected. (J212.16.w1)
  • When grown in cell culture, cytopathic effect (CPE) may not be evident; a monoclonal antibody indirect immunofluorescence test is needed for confirming and identifying the virus. (J484.35.w1)
  • This method is not generally used for diagnosis of field cases, due to the low likelihood of detection of virus and the Biosafety Level 3 facilities required. (V.w117)
• Following death or euthanasia, tissues, particularly brain and spinal cord, should be collected at necropsy for gross pathological examination, histopathology, RT-PCR, virus isolation and immunohistochemistry. (D67, D147) Note: Those performing necropsy examination should take appropriate precautions as several zoonotic agents are differentials for equine neurological disease, including rabies.
  • Brainstem, cerebellum, cerebrum and spinal cord should be kept for virus isolation; tissues should be fixed also and examined histologically. (J215.24.w1)
  • In equines, brainstem and medulla appear to be preferable samples for detection of WNV RNA by PCR techniques. Brain may be positive by PCR when spinal cord is negative. Viral RNA concentration in medulla is generally at least as high as that in cerebrum. (J212.16.w1)
  • Unfixed (fresh, or frozen at -80 °C) brain tissue is preferable, but viral RNA may also be detected in formalin-fixed tissues. Fixing may be preferable to allowing sample degradation due to autolysis. (J212.16.w1)
  • Virus can be detected in CNS tissues by immunohistochemistry. (J26.38.w1, J484.35.w1)
• NOTE: Details of samples required, shipping and handling conditions should be obtained prior to sending specimens to a laboratory for testing. Details of requirements for samples sent to CDC are provided in: West Nile Virus - Detection and Identification Techniques (Viral Reports) - Sample Collection & Shipping

Samples recommended by APHIS/USDA for diagnosis in cases of suspected WNV infection in equines include:

• From a live animal:
  • Serum;
  • Cerebrospinal fluid (less important than serum).
• At necropsy (post mortem examination):
  • Fresh brain tissue for rabies testing;
  • Brain tissue, fresh and formalin fixed;
  • Cerebrospinal fluid (cervical or lumbosacral);
  • Spinal cord segments (cervical, thoracic and lumbar, divide each sample in half), fresh and formalin fixed.

  (W30.Nov01.WNV4)

The following Surveillance Case Definition for West Nile Virus Infection in Equines is taken directly from the CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control Third Revision (D147) Appendix B: [Text copied directly]

Laboratory criteria for diagnosis Compatible clinical signs[1] plus one or more of the following: Isolation of West Nile (WN) virus from or demonstration of specific viral antigen or genomic sequences in tissue, blood, cerebrospinal fluid (CSF) or other body fluid;[2] or Detection of IgM antibody against WN virus by IgM-capture ELISA in serum (at 1:400 or greater dilution) or cerebrospinal fluid (CSF) (at dilution 1:2 or greater dilution); or An associated 4-fold or greater change in IgG-capture ELISA or plaque-reduction neutralization test (PRNT) antibody titer to WN virus in appropriately timed,[3] paired serum specimens from an equid that is unvaccinated against WN virus; or  Positive immunohistochemistry (IHC) for WN virus antigen in tissue. Case classification Probable: compatible clinical signs occurring during a period when arboviral transmission is likely, and with the following supportive serology: 1) a single or stable (less than or equal to two-fold change) but elevated titer of WN virus-specific IgM-capture ELISA or neutralizing serum antibodies without knowledge of prior WN virus vaccination. Confirmed: compatible clinical signs with laboratory-confirmed evidence of WN virus infection. Notes: [1] Clinical signs are associated with central and/or peripheral nervous system dysfunction. Most horses exhibit secondary CNS-derived neurological manifestations such as ataxia (including stumbling, staggering, wobbly gait, or incoordination) or at least two of the following: circling, hind limb weakness, inability to stand, multiple limb paralysis, muscle fasciculation, proprioceptive deficits, altered mental status, blindness, lip droop/paralysis, teeth grinding. (Ostlund et al, Equine West Nile Encephalitis, United States, Emerging Infectious Diseases, Vol 7, No 4. Jul – Aug 2001 [J84.7.w12]) Fever is not a consistent finding. [2] Preferred diagnostic tissues from equids are brain or spinal cord; isolation of WN virus or detection of WN viral nucleic acid sequences in equine blood or CSF are infrequent. (Bunning et al, Experimental Infection of Horses with West Nile virus, Vol 8, No. 4. April 2002 [J84.8.w4]) [3] The first serum should be drawn as soon as possible after onset of clinical signs and the second drawn at least 14 days post-onset. _______________________________________________ Assumptions on which case definitions are based: IgM-capture ELISA testing may give nonspecific results; cross-reactions to closely related flaviviruses (e.g., St. Louis encephalitis virus) may occur. Because closely related arboviruses exhibit serologic cross-reactivity, positive results of serologic tests using antigens from a single arbovirus can be misleading. In some circumstances (e.g., in areas where two or more closely related arboviruses occur, or in imported arboviral disease cases), it may be epidemiologically important to attempt to pinpoint the infecting virus by conduction cross-neutralization tests using an appropriate battery of closely related viruses. Vaccination refers to one or more doses of the current USDA-licensed inactivated WN virus vaccine. [Wildpro Editor's note, May 2008: Three vaccines have been licensed by USDA, are also licensed in Canada, and are commercially available. See: Vaccination for West Nile Virus] IgM antibody in equine serum is relatively short-lived; a positive IgM-capture ELISA means infection with WN virus or a closely related flavivirus has occurred, probably within the last three months. (personal communication Eileen N. Ostlund, USDA) Neutralizing antibody, as detected by PRNT, may not be present in equine serum until two weeks or more after exposure to WN virus; it is possible that clinical signs may be present in an equine before a serum PRNT is positive. Neutralizing antibody detected in serum by PRNT indicates past infection with WN virus or vaccination with WN virus vaccine; equines exposed to WN virus in prior years may test positive by PRNT.

For further information on different serological tests and virus identification see:

• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Detection and Identification Techniques (Viral Reports)

• Clinical Approach to Cases for Veterinary and Medical Personnel
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Detailed Clinical Signs (Disease Reports)
  • West Nile Virus Disease - Editorial Overview of Disease Characteristics for specified SPECIES-TAXA
• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Detection and Identification Techniques (Viral Reports)

• Equines are highly conspicuous, numerous, easily identified to individual or herd level and widely distributed in some areas; this may make them useful sentinels particularly in some rural areas where detection of dead birds may be less likely to occur. (D67, D147, J112.27.w2)
• When this disease occurs clinically in equines (which are not fully vaccinated) it has a high case fatality rate ranging from 29 up to 40% or even higher (See: Mortality Rate (Percentage of the Species Population that die) (Disease Reports), making recognition of the disease likely. 
• Veterinary networks and public veterinary services are available in many countries. (J112.27.w2)
• Equines can be sampled quite easily, and tested using available serological tools such as the IgM antibody-capture ELISA. (J112.27.w2)
• Some equines are routinely bled and tested for other pathogens. (D67)
• Detection of WNV infection in equines may be combined with detection of other diseases in these animals. (J112.27.w2)
• Equines have a high intensity of exposure to mosquitoes and are therefore relatively likely to become infected with WN virus (more so than humans). (D147)
• Sick horses may be the first indication of WN virus activity in rural areas. (D67)
  • In some geographical areas equines have been one of the earliest sentinels for WN virus activity. (D147)
  • In 2001, in 39/359 counties (10.8%) the earliest detection of WN virus activity was by cases in horses. (P39.3.w7)
  • In western areas [of the USA] with numerous, efficient bridge vectors, equine illness may be an early indicator of WN virus activity. (P39.4.w4)
• Detecting equine cases will assist in defining areas of WN virus activity. (J240.71.w1, P39.4.w4, J488.6.w1, N7.54.w2)
  • This may assist in development of disease control strategies. (J13.69.w1)

• Regional Assessment of West Nile Virus
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Time Course-Persistence of Disease in a Susceptible Population (Disease Reports)

• In some geographical areas cases in equines may be seen only simultaneously with or just before human cases, so that they would not act as an early sentinel. (D147)
  • Data from 1999 and 2000 in the USA showed that surveillance for veterinary (primarily equine) cases of disease due to WNV infection was a relatively insensitive method of WN virus surveillance, as such cases were likely to occur only when disease activity was high. (J112.27.w2, P39.2.w1)
• Necropsies are expensive and logistically difficult. (D67)
  • Personal protective equipment recommended to be worn while conducting a necropsy on suspected WNV infection horses are considered to make the process of necropsy more difficult. (P48.4.w13)
  • Gross pathological findings at necropsy of fatal cases are non-specific; no pathognomonic lesions have been described. (D147)
• Horses are not present or abundant in many areas of the U.S., such as metropolitan areas with dense human populations; the proximity of equines to human populations varies. (D67, D147)
• In areas where the disease has not previously been recognised or is only newly recognised, there may be a delay in disease being detected and reported. (J67.71.w2)
• In North America, the incidence of WNV-associated clinical disease in equines has decreased as use of vaccines became widespread, decreasing the usefulness of equines as sentinels. 
  • An increasing number of equids in the USA have been vaccinated against WN virus infection (63.8% of equine operations vaccinated horses against WNV in 2005; 85% of establishments which vaccinated against any disease, vaccinated against WNV). (D340 - full text provided, D341 - full text provided)
  • Horses vaccinated with the licensed vaccines do not develop detectable levels of IgM antibodies (although they do develop neutralising antibodies); therefore detection of IgM antibodies in a horse, even if it has been vaccinated, are very likely to be a result of natural infection. (J485.122.w1, J490.5.w1, P39.3.w2, P51.49.w2)
  • [Wildpro Editor's note, May 2008: Three vaccines have been licensed by USDA, are also licensed in Canada, and are commercially available. See: Vaccination for West Nile Virus]
  • Note: Despite vaccination, equine cases still occurring in the USA continue to correlate geographically and temporally with human cases. (N7.54.w2)
• Costs of collection and testing of equine clinical specimens are usually borne by the owner therefore there are economic reasons why equine specimens may not be submitted and tested for arboviral infections. (D147)
  • In 1999-2001, when WNV Infection was considered an emerging infectious disease in the USA, the National Veterinary Services Laboratory performed WNV diagnostic testing at no charge; once the disease was categorised as endemic in the USA, testing was performed by multiple different veterinary diagnostic laboratories, and charged for. (V.w117)

• Regional Assessment of West Nile Virus
• Clinical Approach to Cases for Veterinary and Medical Personnel
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Time Course-Persistence of Disease in a Susceptible Population (Disease Reports)
• Vaccination for West Nile Virus
  • West Nile Virus Disease - Vaccine Developments (Viral Reports)
  • West Nile Virus Disease - Vaccination Regimes (Disease Reports)

• In New York State in the USA in 1999 and 2000, cases in horses were first recognised after the first cases had occurred in humans. (J84.7.w27)
• In Israel in 2000 during the outbreak of WNV infection, cases in equines occurred after the first cases in humans. (J73.57.w1, J84.7.w16)
• In 2001 in the USA, in 39/359 counties (10.8%) the earliest detection of WN virus activity was by equine surveillance. (P39.3.w7)
• In the northeastern USA in 2001, spatial analysis of case-control and other data indicated that exposure of equids to WN virus was geographically clustered. (D345 - Full text included)
• In Florida, USA, in 2002, infections in equines, as well as sentinel chickens and dead birds, were considered to contribute to early warning. (P48.1.w4)
• In the USA in 2002, equine WNV disease cases provided the first indication of WN virus activity in 16% of the counties (95 of 589) in which human disease was reported. (D147)
• In urban areas of Texas, USA, in 2002, equine cases pre-dated nearby human cases by an average of 12 days, but in rural areas of Texas dates of onset in horses did not significantly differ from dates of onset in humans in the same areas. (J238.129.w1)
• Analysis of the distribution of equine cases of WNV infection in Texas in 2002 identified high-risk areas which were spatially related to high-risk areas for human infection in Texas in 2003. (J488.6.w1)
• In Saskatchewan, Canada, 2003, in most areas clusters of cases in horses occurred after clusters of human cases in the same area. (J67.76.w1)
• Cases of WNV disease in equines in the USA have generally been scattered; there have been few clusters of cases. (D147)
• In 2007, it was noted that although equine cases of clinical WNV infection had declined (1,086 cases in equines in 2006), the cases which did occur continued to correlate geographically and temporally with human cases "suggesting that equine surveillance can continue to help indicate areas of increased risk for human WNV disease." (N7.56.w1)
• Data from equine cases in France, along with land cover and landscape structure data, have been used to indicate areas of "high" and "low" WN virus circulation in southern France. (J279.8.w1)
• Gross pathological findings at necropsy of fatal cases have been non-specific; no pathognomonic lesions have been described. (D147)

NOTE: Data on veterinary cases (mainly equines) in the USA to 2007 is available summarised in map form. See: Map0001 - Spread of West Nile Virus in the USA (2000-2007) - Veterinary maps

• Regional Assessment of West Nile Virus
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Time Course-Persistence of Disease in a Susceptible Population (Disease Reports)