Abstract

Herpesvirus infections and diseases with similar clinical signs have been documented in free-ranging and domestic birds of prey in the United States, Europe, and Asia since the mid-1900s.^1,8,12 Clinically affected raptors typically exhibit a short duration of depression, lethargy and anorexia prior to death. Falcons in particular have been reported to be highly susceptible to herpesvirus infection.⁷

Within Europe, a recent survey of wild injured raptors in Germany determined that 35.5% of the birds evaluated showed antibodies against Falconid herpesvirus 1 (FHV-1).¹¹ Two subsequent retrospective studies in Germany, however, reported less than 4.0% seroprevalence of FHV-1 in free-living birds of prey.^10,14 The prevalence of FHV-1 within the United Kingdom has not been determined. Until recently, only three sporadic outbreaks of FHV-1 had been reported in the UK. Two cases were reported in 1982 in American and European kestrels and, in 1993, a third case was confirmed in a hybrid falcon.^6,9 Nine additional, unreported cases of FHV-1 occurred in various areas of the country since 1996, as verified by virus isolation.¹⁵ In August and September 2000, two new and unrelated cases were recorded in Southwest England.²

The presence of confirmed clinical cases of falcon herpesvirus infections in the UK, in conjunction with the recent seroprevalence results from Germany, prompted this cross-sectional epidemiologic study. The aims of the study were to quantify and compare the seroprevalence of FHV-1 antibodies in wild and captive raptors within the UK, to investigate the prevalence of owl herpesvirus (Strigid herpesvirus 1, StHV-1) in Strigidae tested, and to evaluate the data for a geographic, species-specific, or age-related predisposition for herpesvirus infection.

Blood was collected from wild, debilitated birds of prey upon presentation to rescue centers and veterinary clinics throughout the United Kingdom. The samples were collected opportunistically with no restrictions based on original presentation, species, or geographic locality. Serum was concurrently collected from a comparable group of captive raptors. All of the samples were collected over a 6-month period, from January to July 2001, and stored frozen (−20°C) until tested.

The serum samples were tested for FHV-1 antibodies using micro-neutralization techniques. Eight doubling dilutions of each test sample were prepared in 96-well microtiter plates (Nunc, Gibco, Paisley, Renfrewshire, UK) using 25 µl of serum and 25 µl of Eagles maintenance medium. Monospecific antiserum to FHV-1 (isolate CVL 23/92) was used as a positive control to detect specific antiviral antibodies.⁶ Specific-pathogen-free (SPF) serum was used as a negative control for each test. A volume of 25 µl of virus pre-titrated to 100 tcid₅₀ (mean tissue culture infective dose affecting 50% of the inoculated culture cells) was added to each dilution. The plates were incubated for 1 hour at 37°C, 5% CO₂. A volume of 25 µl of the serum/virus mixture was transferred to confluent cultures of CEL (chicken embryo liver) cells in 96-well plates. The CEL cultures were prepared from 15-day-old SPF embryos using routine methods.⁴ Pre-titrated virus was serially diluted 10-fold up to 10^-4 and inoculated onto the CEL cells in 25 µl volumes to check the titer of the virus. The plates were incubated for 1 hour at 37°C, 5% CO₂. Each well was overlaid with 200 µl maintenance medium, an adhesive plastic film was applied to each plate, and the plates were stored in a 5% CO₂, 37°C incubator. The plates were examined daily for four to seven days until the back-titration wells developed evidence of infection corresponding to 50% cytopathic effect (CPE) at a dilution of 1:100. Final neutralizing antibody titers were expressed as the highest dilution of serum causing complete neutralization of 100 tcid₅₀ of virus.

Samples collected from owls were also tested for antibody against StHV-1 using the same technique, as outlined above, with an isolate from a snowy owl (Nyctea scandiaca) (CVL 1082/94).⁵

One-hundred twenty-five hawk, eagle, falcon, and owl serum samples were collected from 11 different UK counties. A total of 22 raptor species belonging to three families were represented. Of the total samples tested, 44.8% were from captive raptors while 55.2% originated from wild birds of prey. The FHV-1 antibody seroprevalence results for the study group of captive and free-living Falconidae, Accipitridae, and Strigidae are shown in Table 1.

Table 1. Seroprevalence of FHV-1 antibodies
in Falconidae, Accipitridae and Strigidae
within the separate captive and wild study groups

Three of 56 (5.4%) captive birds were seropositive for FHV-1 antibody by serum neutralization. The positive samples originated from 5% (1/20) of the Accipitridae tested and 10.5% (2/19) of the Falconidae tested. The positive samples included a red kite (Milvus milvus) with a neutralizing titer of 1:2 and two peregrine falcons (Falco peregrinus) with titers of 1:2 and 1:8. None of the captive owls were found to have serum neutralization of FHV-1.

Neutralizing antibody to FHV-1 was detected in seven of 69 (10.1%) of the wild birds tested. Positive samples were obtained from 13% (3/23) of the Falconidae family, including three kestrels (F. tinnunculus) with antibody titers of 1:2, 1:16, and 1:32, and from 13.3% (4/30) of the Strigidae sample group, including four tawny owls (Strix aluco) with titers of 1:4, 1:8 (2) and 1:16. No positive samples were observed from the free-ranging hawks or eagles.

Serum samples from 47 owls were tested for antibody to StHV-1. Eight of the owls (8/47, 17%) were seropositive for StHV-1 neutralizing antibody, including five tawny owls, one little owl (Athene noctua), one barn owl (Tyto alba) and a Northern long-eared owl (Asio flammeus). The antibody titers ranged from 1:2 to 1:16, with all the seropositive samples originating from free-living owls (8/30, 26.7%). Of the eight wild owls testing positive for StHV-1, 50% also showed serum neutralization of FHV-1. These findings suggest that there are at least two different serotypes of herpesvirus infection in owls, with varied degrees of antigenic relatedness to FHV-1.

A summary of the details on the raptors testing positive for FHV-1 and StHV-1 neutralizing activity is presented in Table 2. No statistical significance was found between the captive and wild groups tested with respect to FHV-1 antibody prevalence, geographic distribution, distribution between raptor families, or comparison between age groups. A statistically significant correlation was detected between wild owls and the presence of StHV-1 antibodies within this study (Fisher exact test, p=0.0372, CI=0.108–0.425).

Table 2. Details of raptors showing antibody neutralization of FHV-1, StHV-1

^aOnly members of the Strigidae family were tested for StHV-1 neutralizing antibody
nd=not done

The results of this investigation are part of an ongoing study to evaluate the seroprevalence of herpesvirus infections in raptors within the UK. The preliminary findings demonstrate a relatively high prevalence of FHV-1 antibodies in the tested populations of captive and wild falcons. These results are consistent with previous reports of virus isolation and clinical disease from a predominance of falcons within the UK, USA, and UAE.^2,13 Members within the Falconidae family have been suggested to have an increased susceptibility to herpesvirus infection and disease. A low seroprevalence level was detected in both the captive and wild groups of Accipitridae in this study. In the tested population of Strigidae, wild owls were found to have a greater FHV-1 seroprevalence than captive owls. In early experimental herpesvirus transmission studies, a large proportion of the hawks and eagles as well as certain species of owls exposed to FHV-1 were either assumed to be refractory to infection or suffered from a nonfatal form of the disease with subsequent development of resistance.^1,12 It is likely that members of the Accipitridae and Strigidae families attain immunity to FHV-1 through infection with a less virulent herpesvirus serotype (i.e., StHV-1), resulting in a mild antigenic reaction with low production of antibodies.³

Acknowledgments

Several raptor rehabilitation centers, private aviaries, and individual veterinary surgeons were involved in the collection and submission of samples for this study. These include The Barn Owl Centre, Clockhouse Veterinary Hospital, Mr. John Chitty, Mr. Nigel Harcourt-Brown, Medlab, National Bird of Prey Centre, Portishead Veterinary Centre, The Raptor Foundation, and RSPCA facilities in Cheshire, Norfolk, and Westhatch. All laboratory testing was carried out with assistance from the staff at the Veterinary Laboratory Agency (Weybridge), UK. The funding for this work was provided by a grant from the Royal College of Veterinary Surgeons Trust. The research presented is part of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Wild Animal Health, University of London, 2001.

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