By Sarah Levine, VMD
In Israel in the year 2000, due to a 2-day history of reluctance to work, a 10 year old, Dutch warmblood gelding was presented to his veterinarian. The horse had been imported from Europe in 1996 to be used for show jumping.¬†¬† Initial clinical exam was normal except for mild fever. The hematology and serum chemistry results were within normal range with just a mildly elevated bilirubin. Decreased appetite and ataxia in the hindlimbs developed within 24 hours and the horse was then referred to the Koret School of Veterinary Medicine for further workup and treatment.
Drs. A. Steinman, G. A. Sutton and S. Hadar from the Koret School of Veterinary Medicine managed the case at the veterinary school. The horse initially was tachycardic, tachypnoeic, hyperexcitable and was grinding its teeth. It had proprioceptive deficits in all four legs while standing, walking freely and circling.¬† Appetite remained poor and he showed mild signs of colic within the first 48 hours. WNV was diagnosed by Drs. C. Banet, H. Yadin and A. Brill from the Kimron Veterinary Institute.
By the third day the horse‚Äôs neurologic condition worsened and it developed inspiratory dyspnea.¬† Endoscopy of the upper airway showed a narrowed glottis with a failure to abduct the arytenoids. A tracheostomy was performed which greatly improved the horse‚Äôs respiratory situation. Steroids (dexamethasone, 0.1mg/kg IV) and antibiotics (sulphamethoxazole trimethoprim, 30mg/kg PO q 12 hours) were given in an attempt to improve the deteriorating neurologic condition.
On the fourth day mannitol¬† (1gm/kg IV) was given. The horse progressed to recumbancy and was assisted to stand for short periods of time with a sling. The horse died on the fourth day.
Tissues were taken from the brain and spinal cord for virus isolation, however after four days no West Nile Virus was found. Histologic findings were consistent with a non-suppurative encephalomyelitis. Using a reverse transcriptase PCR (RT-PCR) West Nile Virus was isolated and identified.
Encephalomyelitis caused by the West Nile Virus (WNV) was once reported only in Africa, the Middle East, Southern Europe and Asia. Now it is a disease that is being reported throughout the world. Reported cases of human and equine outbreaks of West Nile Viral encephalomyelitis are definitely increasing.¬† Cases of WNV in birds and horses prove to be sentinels for human WNV outbreaks. Therefore diagnosis of the disease is extremely important.
West Nile Virus (WNV) is an arbovirus (arthropod-borne virus) from the family flavivirus. WNV causes severe meningioencephalomyelitis in equids, humans and birds(1).
WNV is primarily maintained in nature by transmission cycles between mosquitoes and birds. Migrating birds are the carriers of WNV and are responsible for the vast spread of disease. Mosquitoes are the vectors that spread viral encephalomyelitis for which mammals serve as dead end hosts. There is a vast range of avian and mosquito species that can be carriers of WNV and spread disease(2). In temperate regions (i.e. North America, Europe) most outbreaks occur in late summer and early fall when the insect numbers are high and the temperatures are warm. In tropical and subtropical regions there is a continuous circulation between birds and mosquitoes.
Clinical signs are variable and include fever, depression, lethargy, ataxia, hind limb weakness, muscle tremors, recumbancy, seizures, coma, paresis of tongue, dysphagia, facial paralysis, recumbancy, hyperaesthesia and less commonly photophobia. Treatment: There is no specific treatment and therefore all that can be offered is supportive care that includes anti-inflammatory drugs. Recovery may occur over weeks to months. Surviving horses either fully recover with little residual problems or some neurologic abnormalities may persist(3,5). The mortality rate ranges from 20-40% in symptomatic horses(5).
Differential diagnoses that must be considered are: alphaviral encephalitides (WEE, EEE, VEE), rabies, equine herpesvirus-1, equine protozoal myelitis, as well as noninfectious etiologies like spinal cord lesions, hypocalcemia and tremorigenic toxicities(1,2,5).
Post mortem examination of brain tissue/spinal cord via immunohistochemistry, viral isolation or viral nucleic acids assay should be performed with sufficient biosafety levels due to the potential for human infection.
When performing serological testing, no such cautions are necessary since there is no contact with infected tissues. Anti-WNV IgM antibodies can be detected 5-60 days post-infection using the IgM capture ELISA (MAC-ELISA) test. Positive results indicate recent exposure to WNV. These antibodies are short-lived and usually only last a few weeks.
WNV IgG ELISA tests measure the longer-lived IgG antibodies. Both vaccination and natural infection cause detectable serum IgG titers within 2-3 weeks. Fully immunized horses are expected to mount a detectable neutralizing antibody response. A 4-fold rise in IgG titers from paired sera samples taken 14 days apart reflects infection(4,5). A single positive IgG result may reflect virus exposure from the prior year.¬† The IgG ELISA test is often used for serosurveillance monitoring(2). Antibodies can last for up to 15 months post-exposure. Neither the WNV IgM antibody test nor the WNV IgG antibody test cross-reacts with similar tests for alphaviruses (i.e., WEE, EEE, VEE).
Control predominantly consists of vector management and vaccination. Knowledge of bird species that serve as carriers and which mosquitoes feed on them is crucial to plan control programs(2). An effort must be made to remove all standing water to prevent mosquito breeding grounds. Any known mosquito nesting grounds should be destroyed or sprayed with pesticides. Horses should be stabled at night and insecticides should be used around and within the stables(2,5). The overwhelming majority of equine cases of WNV are seen in horses that are unvaccinated or incompletely vaccinated(5). Therefore, care must be taken to fully vaccinate horses prior to mosquito season and to follow local vaccine protocols. In some affected areas vaccination is recommended once a year and others biannually.
West Nile Virus is an example of a zoonosis that has leaped geographic barriers and can cause severe disease in humans and
1)¬†¬†¬†¬†¬† Davidson, A. H., et al. (2005). Immunologic responses to West Nile Virus in vaccinated and clinically affected horses.¬†JAVMA,¬†226 (2): 240-45.
2)¬†¬†¬†¬†¬† Dauphin, G., et al. (2004). West Nile: Worldwide current situation in animals and humans.¬†Comp. Immunol. Microbiol. Infect. Dis.,¬†27 (5): 343-55.
3)¬†¬†¬†¬†¬† Ostlund, E. N., et al. (2002). West Nile Virus: Epidemiology, pathogenesis, immunologic response.Atlantic Coast Veterinary Internal Medicine Proceedings.
4)¬†¬†¬†¬†¬† Castillo-Olivares, J., et al. (2004). West Nile Virus infection in horses.¬†Vet. Res.,¬†35 (4): 467-83.
5)¬†¬†¬†¬†¬† Ostlund, E. (2004). Equine encephalitis arboviruses: EEE, WEE, VEE, WNV.¬†Proceedings Western Veterinary Conference.
6)¬†¬†¬†¬†¬† Case Study‚ÄĒSteinman, A., et al. (2002). Clinical signs of West Nile virus encephalomyelitis in horses during the outbreak in Israel in 2000.¬†The Veterinary Record,¬†July 13: 47-49.
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Technical Tip:¬† A Proper Use of the Controls
The ImmunoComb¬ģ¬†Equine West Nile Virus (WNV) Antibody Test Kit contains in addition to the standard content (i.e., a developing plate, a Comb, Capillary tubes, CombScale, Tweezers) positive and negative controls.
The positive and negative controls are included in the kit to assist the user to interpret the results. The positive control (C+) is calibrated to VN 1:20. Specimens with identical or higher color intensity than the positive control are considered positive. Namely, results are consistent with protective levels of antibodies to WNV.
The negative control (C-) should give a value of less than S1 (see the CombScale). A greater value indicates an invalid test.
You can place the controls in any empty well of row A of the developing plate. However, it is recommended to place both controls next to the wells in which specimens are inserted. For example, if you perform three tests (for three horses) and you place the specimens in wells A1, A2 & A3, then it is suggested to place the positive and negative controls in rows A4 & A5, respectively.
Each time you run the test, make sure to use both controls. Therefore, it may be economically worthwhile to perform several tests at once.
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