Originally prepared for Proceedings of the Association of Avian Veterinarians, 2004.

Abstract

West Nile virus (WNV) has caused morbidity and mortality in more than 150 bird species in the United States since 1999. The threat to avian collections and conservation programs led to the investigation for an effective vaccine to protect birds. A recombinant E. coli DNA-plasmid preparation that contained WNV genes coding for specific antigenic proteins was mixed with proprietary aluminum hydroxide adjuvant to produce the experimental vaccine. The vaccine was administered IM to 20 permanently disabled red-tailed hawks (Buteo jamaicensis) twice 3 weeks apart. Five hawks served as sham-vaccinated controls. Four weeks after completion of the vaccine series, hawks were challenged with a Louisiana strain of WNV. Blood samples were collected throughout the study, initially to evaluate serologic status by plaque reduction neutralization test for WNV antibodies after vaccination, and later to evaluate the degree of viremia after WNV antigen challenge. A significant difference in level of viremia post-challenge between vaccinates and non-vaccinates was found.

Introduction

West Nile virus (WNV) became one of the first arthropod-associated viral diseases ever described when it was isolated from the blood of a febrile woman in Uganda in 1937.⁵ Historically, this member of the Flavivirus genus in the Flaviviridae family has caused occasional disease in humans and horses in Africa and Asia, with sporadic outbreaks in Europe.⁷ West Nile virus is considered one of the most widely distributed of all flaviviruses.³ In Africa, up to 70% of humans in endemic areas are seropositive, suggesting that the majority of WNV infections are mild or subclinical.¹

West Nile virus was introduced into the United States in 1999 and became a significant cause of morbidity and mortality among wild birds (especially crows), horses, and humans. A member of the flavivirus group, WNV is a vector-borne disease transmitted primarily by Culex spp. mosquitos.⁴ By fall of 2003, it had become endemic in all states but those west of the Rocky Mountains, with epidemics occurring at the front of westward movement in each of the years 2000, 2001, 2002, and 2003.

Owing to the more than 150 bird species in which WNV has been documented since its introduction, and since many of these are components of collections and conservation programs, protection in the form of effective vaccination was deemed desirable.⁶ Introduced in 2000, a killed vaccine licensed for use in horses received limited testing in birds⁸ and became widely used by avian veterinarians. A recombinant DNA-plasmid vaccine was reported effective in protecting crows against challenge with WNV.⁹ The DNA-plasmid vaccine was further tested in California condors (Gymnogyps californianus).⁶ The E. coli DNA-plasmid contains WNV genes pCBWN that express the prM and E glycoproteins of WNV which elicit an immune response.²

Methods

In mid-September 2003, we began a vaccine trial with this recombinant DNA-plasmid West Nile vaccine obtained from a manufacturer of stable DNA plasmids (Aldevron, Inc., Fargo, ND, USA) under a research agreement with the Centers for Disease Control and Prevention (CDC), Department of Health and Human Services, USA. The DNA concentration was 500 µg/500 µl in PBS, and it was mixed with proprietary aluminum hydroxide adjuvant (Biomune Co., Lenexa, KS, USA).

A group of 20 permanently disabled red-tailed hawks (Buteo jamaicensis) was obtained from The Raptor Center and various rehabilitators throughout the USA in accordance with provisions of permits issued by the United States Fish and Wildlife Service. All subsequent procedures were conducted in accordance with an approved IACUC protocol. Hawks were given physical examinations. Blood samples were collected from the hawks for CBC and serology. Blood samples were tested for antibodies to WNV by the plaque reduction neutralization test (PRNT), and only antibody-negative birds were used for further study. Fifteen birds were selected for immunization at random and placed in a treatment group room; five birds were used as sham-vaccinated controls and placed in a separate room.

Hawks were housed indoors in controlled light and ventilation rooms constructed of cinder block walls and concrete floors all sealed with epoxy resin finishes. Temperature was maintained constant at 20°C, and photoperiod was regulated at 12L:12D. Hawks were fed ad lib coturnix quail (Coturnix japonica) supplied at the rate of approximately ¾ of a quail per hawk per day. Water for drinking and bathing was provided in shallow floor pans. Rope-covered perches of suitable size and height for non-flying red-tailed hawks were placed in various locations on the floor. All rooms were cleaned once daily with the hawks left in situ.

Hawks received two 1-ml doses of the vaccine with a 3-week interval between doses. Inoculations were delivered by IM injection in the pectoral musculature with a 25-ga needle. Blood samples were drawn before the first vaccination, at the time of the second vaccination, and 3 weeks after the second vaccination. Blood samples were spun by centrifuge, and plasma was drawn off for antibody testing by PRNT.

Following the third phlebotomy, the hawks were loaded into fiberglass animal holding kennels and driven by van to Baton Rouge, Louisiana for challenge with live WNV at the School of Veterinary Medicine, Louisiana State University. Upon arrival, hawks were transferred to suitable animal holding space, similar to that described previously, and allowed 1 week of acclimation prior to additional experimentation.

For challenge, birds were inoculated SC in the inguinal web with 0.1 ml of a suspension containing 10⁵ PFU of the Louisiana strain of WNV. This virus strain had been passaged once in Vero cells after isolation from the kidney of a blue jay (Cyanocitta cristata), which had died in Louisiana during the summer of 2001. Blood samples were collected on day 1, 2, 4, 6, 8, 10, 12, and 14 to assess the degree of viremia. Swabs were taken from the pharynx and cloaca during this time to assess viral shed patterns. Birds were humanely euthanatized upon completion of the study. Due to the development of neurologic signs, one bird was euthanatized on day 10 post-challenge and was subjected to full necropsy. One bird that exhibited no viremia post-inoculation was reinoculated 6 weeks after the first inoculation and was sampled for viremia as previously.

Geometric mean values for viremia were compared using a one-tailed t-test. A p value <0.05 was taken as an indication of significant difference between vaccinates and non-vaccinates.

Results and Discussion

A significant difference in level of viremia post-challenge between vaccinates and non-vaccinates was found. In the vaccinated hawk that was challenged twice with live WNV, viremia results were negative at all samplings. These findings render the possibility of developing an avian-specific vaccine for WNV more probable in the near future. Details of differences between groups along with other findings from this experiment will be presented and ultimately published.

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