Isolation of herpesvirus and Newcastle disease virus from White Storks (Ciconia ciconia) maintained at four rehabilitation centres in northern Germany during 1983 to 2001 and failure to detect antibodies against avian influenza A viruses of subtypes H5 and H7 in these birds

Abstract

Herpesvirus isolations from peripheral white blood cells of 253 White Storks (Ciconia ciconia) were obtained during a long-term study (1983 to 2001). The storks lived for a few months to 20 years at four rehabilitation centres. Isolates were obtained from 83 of 253 storks. This herpesvirus is indigenous for storks and unrelated to any other avian herpesvirus. Significantly more herpesvirus isolates were obtained during spring than in autumn samplings. The intervals between the first and last virus isolation ranged from 1 to 15 years. Herpesvirus isolates were simultaneously obtained from white blood cells and from pharyngeal swabs of four of 34 storks but not from cloacal swabs. Neutralizing antibodies to stork herpesvirus were detected in 178 of 191 examined blood plasma samples. Neutralizing antibodies against stork herpesvirus did not correlate with herpesvirus viraemia. The results further substantiate the persistence of herpesvirus in White Storks and underline the previously unrecorded long periods of virus and antibody presence. Virulent avian paramyxovirus type 1 (APMV-1; Newcastle disease virus) was isolated from white blood cells during 1992 and 1993 from four healthy migrating storks, and possessed virulence markers on the cleavage site of the H and F genes. These properties resemble the NE type of APMV-1. Haemagglutination inhibition antibodies against APMV-1 were detected in 16 of 191 blood plasma samples. Avian influenza A virus was not isolated and antibodies against subtypes H5 and H7 were not detected.

Introduction

The White Stork (Ciconia ciconia; Linnaeus, 1758) is known as a common and well-accepted anthropophilic bird in Europe. However, the total population has declined for various reasons since the 1950s but has become stable in recent decades at a lower level (Rossbach, 1983, 1984; Sies, 1989) due to artificial nest sites and various conservation measures (Löhmer & Schulz, 1989).

The large amounts of faecal droppings of storks may contain infectious agents that can threaten productivity of domestic poultry and the health of endemic avian species. White Storks are long-distance travellers between Europe and Africa. Depending on the climatic conditions, storks may rest during migration on farmed land and in close proximity to poultry facilities and can intermingle with free-living birds. Therefore, storks can represent an epidemiological link for the transmission of infectious agents that are present in African and European countries. The role of storks was demonstrated for the mosquito-borne West Nile virus (Malkinson et al., 2002a, b) and for Newcastle disease virus (Kaleta et al., 1983). As a risk-reduction strategy, governmentally authorized and supported rehabilitation centres or sanctuaries were established in several European countries with the intention of monitoring transmissible diseases and zoonoses, to treat sick or injured storks, and to return successfully cured birds to the wild.

The family Herpesviridae contains the stork herpesvirus as an unassigned virus to the family (Davison et al., 2009; Davison, 2010). The arrangement of the genes in the stork herpesvirus genome and additional properties of the virions are not yet available. However, all known properties allow the allocation of stork herpesvirus to this large family. The herpesviruses from Black and White Storks are serologically identical but unrelated to any other avian herpesvirus (Kaleta et al., 1980b).

This contribution presents data from a long-term study (1983 to 2001, i.e. 19 years) of captive storks with the central issue to study the prevalence of stork herpesvirus and to correlate the obtained results with survival in the presence of viraemia and neutralizing antibodies. We report also on the isolation of a virulent avian paramyxovirus type 1 (APMV-1) and the failure to detect antibodies against the haemagglutinin of avian influenza A of subtypes H5 and H7 in blood plasma samples.

Materials and Methods

Structure and functions of rehabilitation centres and sources of White Storks

At the times of investigation and sampling of blood plasma and swabs from the pharynx and cloaca during the years 1983 to 2001, all 253 White Storks lived for variable periods of time at four different rehabilitation centres (Centres V, L, S and B) in Northern Germany. These centres are equipped with houses for shelter and had large areas of pasture. Some storks stayed in the centres for complete recovery for only a few weeks while others stayed due to persistent disabilities for up to 20 years or more. The geographical coordinates of the four rehabilitation centres are: Centre V, 52° 92′ North and 9° 23′ East; Centre L, 52° 43′ North, 10° 43′ East; Centre S, 52° 87′ East and 10° 55′ East; and Centre B, 53° 18′ North and 8° 48′ East.

The four centres received storks that were unable to fly due to emaciation, undefined causes of sickness, various injuries or other disabilities. The storks were captured by private citizens, ornithologists, veterinarians and concerned persons. The centres were authorized and supported by governmental decision and operated privately to serve two main purposes. The primary aim was to restore physical health and fitness until the storks could be independent and were fit for release into the environment. Other storks were rehabilitated but without regaining the ability to fly and forage for themselves. Such birds remained in captivity for as long as 20 years. Recovered male storks with functional wings and only minor leg problems were able to mate, breed and produce healthy offspring with free-living storks migrating in autumn to wintering sites. Females with partial loss of legs but functional wings were regularly fertilized and produced healthy offspring. Occasionally free-living, completely healthy storks appeared at a centre, paired with captive storks and bred successfully. The second aim of the centres was to collect and analyse data on the prevalence of zoonoses and other stork-specific infectious agents. Thirdly, since almost all storks were ring-banded, additional data on routes and duration of migration of the total stork population were obtained.

Clinical examination

Every year during autumn and spring all resident storks at the centres were physically examined by one of us (NK) with the assistance of local personnel and veterinary students of the Foundation Veterinary School Hannover, and the Justus Liebig University, Giessen. The storks were examined for any bodily abnormalities and treated if necessary. Predominant health problems consisted of traumatic insults that caused luxations, fractures and partial loss of limbs (Wolf et al., 2001). Some storks survived contusions of the heads and fractured beaks but recovered. The plumage was examined for burning of feathers indicating direct contact with live electrical cables, and also for, other structural abnormalities and external parasites.

Pharyngeal and cloacal samples

To gain information on the site(s) of herpesvirus replication and excretion, pharyngeal and cloacal swabs were collected from 34 selected White Storks at three locations.

Cloacal excretions

Excreta were repeatedly collected from each of the 253 storks almost every year in spring and autumn (Table 1). In total, 948 samples were obtained from storks presented during sampling times, and tested for internal parasites. A few samples contained round worms but never cestodes (details not shown).

Table 1.  Number of White Storks examined at each centre and number of samples obtained from peripheral white blood cells, swabs from the pharynx and cloaca, and cloacal content.

		Types and total number of samples obtained from storks
Rehabilitation centre	Number of storks examined	Peripheral white blood cells: virus isolation^a	Peripheral blood plasma: antibody tests^b	Swabs from pharynx and cloaca: virus isolation^a	Cloacal content: bacteriology and parasitology^c
V	116	552	154	23	552
L	58	156	20	1	156
S	16	122	17	10	156
B	63	118	0	0	118
Total	253	948	191	34	948
^aVirus isolation in CEF cultures. Most but not all storks were repeatedly tested.
^bA total of 191 blood plasma samples from spring 1984 to 2000 were used in virus neutralization tests in CEF cultures for detection of neutralizing antibodies against stork herpesvirus, in haemagglutination inhibition tests for antibodies against APMV-1 and for avian influenza A virus of subtypes H5 and H7.
^cCultures for aerobic bacteria and microscopic examination for intestinal parasites.

Using standard procedures, cloacal contents were examined for aerobic bacteria, and especially Salmonella spp. (Arp, 1980; Mallison & Snoeyenbos, 1980; Rhoades et al., 1980). In response to regular examinations and appropriate treatments, pathogenic bacteria were rarely isolated. Only occasionally was Escherichia coli cultured, while Pasteurella spp., Erysipelothrix rhusiopathiae, Salmonella spp. and other pathogenic bacteria were never isolated (data not shown).

Blood plasma samples

A total of 948 blood plasma samples, approximately 2 ml each, were collected in sodium ethylenediamine tetraacetic acid, by puncture of the wing vein of 253 storks during the years 1983 to 2001 (Table 1). Most of the storks were bled repeatedly, and others only once, depending on the duration of their stay at the centres. All whole-blood samples were kept at 4 to 8°C until further processing.

Reference viruses

The stork herpesvirus isolate KS 213/79 was used for the detection of serum-neutralizing antibodies in microtitre plates. This virus was originally isolated from the liver, kidney and bone marrow of a dead Black Stork (Ciconia nigra; Linné, 1758) in chick embryo fibroblasts (Kaleta et al., 1980a) and passaged several times in cell culture. By cross-neutralization plaque reduction, this herpesvirus is unrelated to any of the other 14 avian herpesviruses (Kaleta et al., 1980b) but is serologically indistinguishable from other herpesvirus isolates of White Storks (Kaleta & Kummerfeld, 1986; Kaleta, 1990).

The Asplin F strain (1952) of paramyxovirus type 1 (APMV-1) (Newcastle disease virus) was used for detecting antibodies in blood plasma samples. This strain was supplied by Dr D. J. Alexander, Central Veterinary Agency, Weybridge, UK. Allantoic fluid was used as the test virus in haemagglutination inhibition tests.

For avian influenza A virus antibody detection in blood plasma samples, two strains were used: A/turkey/Ontario/7733/1966 (H5N9) kindly provided by Dr C. Scholtissek, Institute of Virology, University Giessen, Germany; and A/carduelis/Germany/1972 (H7N1) isolated in our laboratory (Kaleta & Hönicke, 2005). Both strains were propagated in embryonated chicken eggs, and allantoic fluids served as test viruses.

Chicken embryo fibroblast cultures

Ten-day-old specified pathogen free (SPF) chicken embryos (VALO, Cuxhaven, Germany) were used to prepare chicken embryo fibroblast (CEF) cultures according to standard procedures (Schat & Purchase, 1980). The cultures were grown and maintained in basal Eagle's medium with Earle's salt solution, supplemented with glutamine, gentamicin, nystatin and 2% or 5% foetal calf serum. All cultures were incubated at 37.5°C in a saturated humid atmosphere.

Virus isolation in CEF cultures from buffy coat cells and swabs

Fresh, uncoagulated peripheral blood samples were kept for 2 to 4 h at room temperature to allow all blood cells to settle. Buffy coat cells were carefully aspirated and placed on 1 ml Ficoll-Paque (Healthcare Bio-Science, Uppsala, Sweden) density gradient (specific gravity 1.077 g/ml), and were centrifuged at 1500×g for 10 min. The top layer, consisting of blood plasma, was collected and stored for antibody assays. The white blood cells (buffy coat cells) were transferred to a tube containing 8 ml phosphate-buffered saline (PBS) solution. To remove Ficoll-Paque, cells were thoroughly mixed in PBS and centrifuged at low speed (800×g) for 5 min. This washing procedure was repeated twice, then all cells were resuspended in 0.5 ml PBS; this suspension contained approximately 3.0×10⁶ to 5.0×10⁶ white blood cells per millilitre. Washed cells were used to inoculate duplicate confluent primary CEF cultures.

Cloacal swabs were immersed in cell culture medium (basal Eagle's medium with Earle's salts) soon after collection and kept frozen until processing. After a short ultrasonic treatment and low-speed centrifugation, aliquots were inoculated on confluent CEF cultures and further treated as described for the washed buffy coat cells.

All inoculated and mock-inoculated cultures were observed microscopically each day for 1 week for any cytopathic changes. Supernatant fluids and cells of cultures showing extended morphological changes were harvested and used for virus identification (see below). If only minor focal changes were noted in the monolayer, subcultures were prepared and again observed for 1 week. After 1 week of incubation, unchanged CEF cultures were subcultured or supernatant fluids were passaged in freshly prepared CEF cultures. Cultures without cellular changes after three consecutive passages were considered free of cytopathic virus.

Virus identification

Beginning at 2 to 3 days post inoculation, two different types of cytopathic change were visible microscopically (100×) in some of the inoculated CEF cultures. Commonly seen changes consisted of focal areas of rounded refractile cells comprising initially 5 to 10 mononucleated cells. During further incubation, these small foci enlarged and consisted of numerous rounded cells. These foci expanded further in size and neighbouring foci merged, which resulted in large areas of altered cells in the monolayer. While these changes are similar to those induced by known avian herpesviruses, additional tests were applied to identify the focus-forming isolates as avian herpesviruses. These tests included sensitivity to chloroform, co-cultivation with 5-iodine-5-desoxiuridine and neutralization of the infectivity by rabbit antisera against 14 avian herpesvirus isolates (Kaleta et al., 1980b). Restriction enzyme analysis of the stork herpesvirus allowed the isolates to be placed in a separate group of avian herpesviruses (Günther, 1995). In addition, negatively-stained supernatant fluids of cultures of the round-cell-type of cytopathic effect were examined by electron microscopy. The observed viral particles measuring 120 to 130 nm in diameter were surrounded by a rather small envelope and contained an icosahedral capsid of 162 hollow capsomeres (Kaleta et al., 1980a).

During 1992 and 1993, buffy coat cells of four storks yielded a second form of cytopathology in cell culture consisting of large, multinucleated syncytia that detached from the surface of the plates and floated in the medium. Supernatant fluids were inoculated into the allantoic cavity of SPF eggs pre-incubated for 10 to 11 days. Haemagglutination inhibition tests with monospecific polyclonal antisera against avian paramyxovirus types 1, 2 and 3 were used to identify the haemagglutination. In addition, subtyping of the isolate was performed with monoclonal antibodies and the H and F genes were sequenced (Lomniczy et al., 1998).

Polyclonal monospecific antisera to subtypes 5 and 7 served to identify avian influenza A virus. Selected samples of the haemagglutinating isolates were examined by electron microscopy after negative staining with 1% phosphotungstic acid to visualize the size, morphology and outer structures of virions, which corresponded well with avian paramyxoviruses.

Virus neutralization tests

The beta procedure (constant virus, two-fold diluted blood plasma) and the microtitre system in 96-well flat-bottom plates was used to test for neutralizing antibodies against stork herpesvirus isolate KS 213/79 (Kaleta et al., 1980b). The total of 948 blood plasma samples were aliquoted due to the available small volume for the detection of antibodies against West Nile virus (98 samples; Malkinson & Banet, 2002), for the detection of avihepadnavirus (566 samples; manuscript in preparation), and the remaining 191 blood plasma samples were used for the detection of antibodies against stork herpesvirus and antibodies against Newcastle disease virus and avian influenza A virus subtypes H5 and H7.

For the assay of stork herpesvirus antibodies, 191 blood plasma samples (Table 1) were heat-inactivated for 30 min at 56°C and prediluted 1:8 to circumvent the negative effects of ethylenediamine tetraacetic acid on CEF. A volume of 25 µl per blood plasma sample was further diluted in 96-well flat-bottom plates using a diluent tissue culture medium (basal Eagle's medium with Earle's salts) that was supplemented with 2% foetal calf serum. Each well contained 25 µl diluted blood plasma. To each of these wells, 25 µl test virus containing approximately 100 median tissue culture infective dose was added. Both components were mixed by tipping the plates. After incubation of the mixture of diluted blood plasma and test virus for 60 to 80 min at room temperature, 50 µl freshly prepared suspension of CEF were added. The negative control contained normal SPF chicken serum instead of stork blood plasma. As a positive control, rabbit anti-stork herpesvirus serum was used.

The titre of a given blood plasma sample was defined as the highest dilution that completely prevented the development of cellular changes due to the test virus. Titres of log₂<2 were interpreted as negative.

Haemagglutination inhibition tests for APMV-1 and avian influenza virus

A total of 191 blood plasma samples were used to determine antibody titres against APMV-1 and avian influenza A virus of the haemagglutinin subtypes H5 and H7 (Table 1). The procedure for the detection of antibodies against APMV-1 and for influenza A viruses of both haemagglutinin subtypes H5 and H7 followed the standard protocol formulated by the OIE Biological Standards Commission of the World Organization for Animal Health, Paris (formerly Office International des Epizooties [OIE], 2000). In brief, all heat-inactivated (56°C, 30 min) blood plasma samples were two-fold diluted in PBS in V-shaped 96-well plates, each suspension of the three test viruses was adjusted to four haemagglutinating units and 25 µl were added to the plasma dilutions. The reaction time for antibodies with test viruses was 60 min for all three test viruses. A 1% suspension in PBS of washed chicken erythrocytes was added to each well. The plates were kept at room temperature for 40 to 60 min for the detection of antibodies against APMV-1 and for the same period of time at 4 to 8°C for both influenza A viruses. Plates were read in an oblique position. SPF chicken sera served as a negative control, and hyperimmune serum produced in chickens following immunization with Newcastle disease virus, strain LaSota, served as a positive controls. The haemagglutinating inhibition (HI) titre of a given plasma is the highest dilution that completely inhibited haemagglutination. Titres > 4 are considered positive (OIE, 2000).

Statistical analysis

The chi-square test was applied to test for significant differences between groups of blood plasma samples collected at the four rehabilitation centres (Dixon, 1993). A value of P < 0.05 represented the level for significant differences.

Results

Physical examination

Based on detailed physical examination, the behaviour and general health of all the storks were considered appropriate under the conditions of indoor and outdoor management. The body weight was judged as normal due to extensive care and regular food and water intake. Storks with amputated legs but functional wings performed well and some were able to mate and breed successfully. Individual storks produced clutches in species-typical numbers of fertile eggs, incubated them and cared for their chicks appropriately. Outgrown young storks followed autumn free-living storks on their migratory routes to wintering sites in the south. Social interactions within groups of storks and with the bird technicians appeared relaxed following their adaptation to the new environment. Capture, physical restraint and collection of blood and samples were tolerated without major resistance.

Isolation and biological properties of White Stork herpesvirus

Using these criteria all isolates were identified as avian herpesviruses that originated from buffy coat cells of storks at all four rehabilitation centres. The cytopathology was very similar for all isolates. All isolates were sensitive to chloroform treatment, co-cultivation with 5-iodine-5-desoxiuridine reduced viral replication, and electron microscopy of selected isolates generated viral particles that were similar in diameter, shape, outer surface components and capsomer numbers. The frequencies of the stork isolates varied between the centres (Table 2). Most were obtained from storks living at Centre V (52 of 116 storks, 44.8% positive). Ten out of 63 (15.9%) storks at Centre B were virus-positive, and Centres L and S yielded 17 of 58 (29.3%) and 4 of 16 (25.0%) isolations, respectively.

Table 2.  Number of White Storks at four rehabilitation centres and rate of herpesvirus isolations from blood plasma.

	Number of herpesvirus isolations from White Storks
Rehabilitation centre	Number of tested storks	Herpesvirus isolated	No herpesvirus isolated	% rate isolation of number of tested storks
V	116	52	64	44.8
L	58	17	41	29.3
S	16	4	12	25.0
B	63	10	53	15.9
Total	253	83	170	32.8

Based on the total of 948 blood plasma samples, the frequency of recovered herpesviruses differed markedly between samples collected in spring or in autumn (Table 3); 73 isolates were collected in the spring at Centre V (30.5%), while the number of virus isolations in the autumn was 23 (8.9%). This difference was significant by chi-square test (P < 0.001). A significant difference (P < 0.001) was also calculated for blood samples from Centre L. During spring collection, 17 of 74 (23.0%) samples were virus-positive whereas only four of 82 (4.9%) samples were positive during autumn. Centre B yielded also a significantly higher isolation rate (P < 0.031) during spring collection compared with autumn (22.7% vs. 7.5%). Owing to the relatively small number of blood samples at Centre S, no significant difference could be calculated.

Table 3.  Frequency of stork herpesvirus isolations from peripheral blood leukocytes as affected by the season of sampling.

		Number of samples tested
Rehabilitation centre	Season of sampling	Positive	Negative	Total	% positive of total tested	χ^2	P value
V	Spring	73	197	270	30.5
	Autumn	23	259	282	8.9	34.227	<0.001
L	Spring	17	57	74	23.0
	Autumn	4	78	82	4.9	10.933	<0.001
S	Spring	4	55	59	6.8	Not applicable^a
	Autumn	5	58	63	7.9		0.060
B	Spring	5	17	22	22.7
	Autumn	7	89	96	7.3	4.668	0.031
Sub-total	Spring	99	326	425	23.3
	Autumn	39	484	523	7.5	47.282	<0.001
Total	Spring+	138	810	948	14.6	–	–
	Autumn
^aChi-squared test not applicable due to small numbers.

The duration of herpesvirus viraemia in individual White Storks apparently lasted for very long times in birds maintained at rehabilitation centres for more than 20 years (Table 4). Some storks that were initially virus-positive, although with virus-negative intervals, remained positive even after many years of rehabilitation. The number of samplings per stork increased the probability of virus isolations (Table 4). One to eight samplings per stork yielded an average of 28.8% virus isolations; 9 to 16 samplings yielded 64.3% isolations; 17 to 24 samplings were 85.7% positive; and storks tested 25 to 32 times were always virus-positive.

Table 4.  Frequencies of stork herpesvirus isolations as affected by the number of bleedings for viraemia per stork.

		Number of blood samplings from each stork and herpesvirus isolations
		1 to 8 samplings				9 to 16 samplings				17 to 24 samplings				25 to 32 samplings
Rehabilitation centre	Number of storks	n	+ve	−ve	% +ve	n	+ve	−ve	% +ve	n	+ve	−ve	% +ve	n	+ve	−ve	% +ve
V	116	98	38	60	38.8	10	7	3	70.0	5	5	0	100.0	3	3	0	100.0
L	58	58	17	41	29.3	0				0				0
S	16	11	1	10	9.1	3	2	1	66.7	2	1	1	50.0	0
B	63	62	10	52	16.1	1	0	1	0	0				0
Total	253	229	55	163	28.8	14	9	5	64.3	7	6	1	85.7	3	3	0	100.0
+ve, positive; −ve, negative.

Herpesvirus isolations from the pharynx and cloaca

An attempt was made to detect the route of herpesvirus excretion via the crop and cloaca in a limited number of storks (Table 1). For 34 storks examined, herpesvirus was isolated from pharyngeal swabs of four storks that were also viraemic on testing their white blood cells. Infectious virus was never obtained from the cloaca of these birds.

Neutralizing antibodies against White Stork herpesvirus

Neutralizing antibody titres against stork herpesvirus were detected in 178 of 191 tested blood plasma samples in the range of log₂=3 to log₂=11 (Table 5). An attempt was made to correlate the antibody titres with herpesvirus isolation from these samples. Obviously, the presence of antibodies does not correspond to the detection of infectious herpesvirus. The 15 samples with herpesvirus isolation either were negative for antibodies (nine samples) or the determined titres of 15 storks were scattered over a wide range from log₂=5 to log₂=10. A total 163 out of 167 samples without herpesvirus isolation did contain neutralizing antibodies in the range of log₂=3 to log₂=11.

Table 5.  Herpesvirus neutralizing antibodies in relation to isolations of stork herpesvirus.

	Neutralizing antibodies (log2) titres against stork herpesvirus in blood plasma samples with or without herpesvirus isolations
Herpesvirus isolation	≤2^a	3	4	5	6	7	8	9	10	11	Number of antibody-positive/number of tested samples
Yes	9	0	0	7	3	1	2	1	1	0	15/24
No	4	3	30	40	35	27	14	10	2	2	163/167
^aTitres of log2<2 are interpreted as non-specific.

Isolation of avian paramyxovirus type 1

APMV-1 was isolated from white blood cells for four of 35 healthy storks at Centres V and L only during 1992 and 1993. This virus induced large multinucleated syncytia in CEF cultures 2 to 3 days post inoculation, and caused embryonic death 2 to 3 days post inoculation. The allantoic fluids agglutinated chicken red blood cells. Haemagglutination inhibition tests using polyclonal antisera against APMV-1 and avian paramyxovirus types 2 and 3 indicated a close relationship to APMV-1.

Subtyping of two of these isolates with monoclonal antibodies directed against the haemagglutinin and fusion proteins confirmed the identity of the isolates as APMV-1 group. Restriction enzyme cleavage site analysis of the haemagglutinin (H) and fusion (F) protein genes enabled location of the stork APMV-1 into the recently established genotype VII. Lateral spread of APMV-1 between storks and the development of clinical signs of Newcastle disease were not present at clinical examinations in spring and autumn.

Antibodies against avian paramyxovirus type 1

A total of 191 blood plasma samples were tested by HI for APMV-1 antibodies. Only blood plasma samples of 16 storks that were collected in autumn 1992 and in spring 1993 yielded HI antibodies against APMV-1 that were positive in the range of log₂=4 to log₂>8 (Table 6). The seropositive birds lived at Centres V and L.

Table 6.  APMV-1 haemagglutination inhibition titres of plasma samples.

		Number of blood plasma samples with HI titre^a in log2
Rehabilitation centre	Number tested	≤3	4	5	6	7	≥8
V	154	139	0	1	2	5	7
L	20	19	1
S	17	17
Total	191	175	1	1	2	5	7
^aTitres of log2<3 are interpreted as non-specific (OIE, 2000).

Antibodies against avian influenza A virus of subtypes H5 and H7

All 191 blood plasma samples from storks at all four locations collected between 1983 and 2001 were free of detectable specific HI antibodies (log₂<1) against avian influenza A virus subtypes H5 and H7.

Discussion

We have examined 253 partially disabled, live storks at four rehabilitation centres and isolated a herpesvirus from 83 of these storks (Table 2). Of the 948 blood plasma samples, 191 had homologous neutralizing antibodies. In contrast to stork herpesvirus isolation, homologous neutralizing antibodies were detected in 178 of 191 tested blood plasma samples (Table 5). This result indicated that most of the storks were infected previous to capture, which resulted in antibody production and persistence for prolonged times whereas viraemia is of indeterminate duration.

Under certain poorly defined circumstances, stork herpesvirus may cause clinical signs and occasionally mortality (Kaleta et al., 1980a, b; Gómez-Villamandos et al., 1998). We have previously reported that natural infection by stork herpesvirus will result in long-lasting viraemia without associated morbidity and lethality (Kaleta & Kummerfeld, 1983, 1986). In this report, we again demonstrate that viraemia in naturally infected storks may last for at least 15 years. Such data on the long-term persistence of viraemia were never reported previously to our knowledge. We also demonstrate a persisting co-existence of herpesvirus viraemia in conjunction with circulating antibodies in peripheral blood (Tables 4 and 5). This indicates that neutralizing antibodies provide no protection against infection. Since the majority of birds without detectable viraemia had neutralizing antibodies (163 of 167 storks), it can be concluded that antibody testing is more appropriate than virus isolation for the detection of a previous herpesvirus infection.

An inoculum of semi-purified white blood cells, separated by density gradient centrifugation from 2 ml whole blood and containing approximately 3.0×10⁶ to 5.0×10⁶ buffy coat cells, was used to isolate herpesvirus. This high cell number contrasts to the small number of initial herpesvirus-induced foci. Subculturing the infected monolayers increased the focal count but the titrated virus content remained rather low (Kaleta & Kummerfeld, 1986). Owing to the low herpesvirus content in buffy coat cells, it was postulated that some samples were falsely considered virus free.

A significantly higher rate of virus isolations was found in blood plasma samples collected during spring than during the autumn. It is conjectured that a causal association exists with the initiation of the breeding season, which is known to reactivate latent viral infections including those caused by herpesviruses (Sharma, 1980; Kaleta & Docherty, 2007). However, definite proof for this assumption is currently lacking.

We also isolated an APMV-1, genotype VII (also termed the “NE type” of Newcastle disease virus) from white blood cell samples collected during 1992 and 1993 from four storks at two rehabilitation centres. The intracerebral pathogenicity test of the isolates was not determined; however, they were identified as APMV-1 by Dr D. J. Alexander (Central Veterinary Agency, Weybridge, UK), employing monoclonal antibodies directed against the HN and F proteins. In addition, Lomniczi et al. (1998) characterized two isolates as members of the novel virulent genotype VII that causes major losses in chicken flocks. Of interest were the reports that NE virus was present in ornamental and commercial chickens during 1992 to 1995 in northeastern parts of Europe (Collins et al., 1998; Lomniczi et al., 1998).

The introduction of this paramyxovirus into the stork population cannot be explained, but its presence provides circumstantial evidence for the storks’ epidemiological role in the lateral spread of APMV-1 and substantiates the necessity of continuous surveillance of White Storks and other wild birds for Newcastle disease viruses (Glünder et al., 1991). When they were blood sampled, the APMV-1-positive storks were free of clinical signs of Newcastle disease—which together with the presence of antibodies indicated that natural APMV-1 infection must have occurred earlier. We have reported previously that natural infection of White Storks by virulent Newcastle disease virus can result in severe signs and mortality (Kaleta & Kummerfeld, 1983). The detection of antibodies in clinically healthy storks provides evidence for survival of a natural infection by genotype VII viruses. Although vaccination of storks against Newcastle disease is possible and protective (Trinkaus et al., 1994), these storks had no history of previous vaccinations.

Using the HI test, the examination of 191 blood plasma samples for antibodies against avian influenza A viruses of the haemagglutinin subtypes H5 and H7 failed to yield evidence of infection. Since the HI test is of acceptable sensitivity and specificity, a previous infection by these viruses should have been detected (OIE, 2000). This negative result agrees with that of Müller et al. (2009), who failed to demonstrate the presence of antibodies against influenza A viruses in the surveillance of 88 live nestling White Storks during the years 2003 to 2008. In 2006, however, highly pathogenic avian influenza A virus of the subtype H5N1, clade 2.2, was isolated from two dead storks. Notwithstanding, the authors concluded that influenza A viruses in White Storks represent a low risk factor for domestic poultry and humans (Müller et al., 2009).

Acknowledgements

The authors appreciate the generous gift of the F strain of avian APMV-1 by Dr Dennis J. Alexander, Central Veterinary Agency, Weybridge, UK and for the subtyping of our APMV-1 isolates. The authors thank Dr Christoph Scholtissek, formerly Institute of Virology, University Giessen, for the influenza A virus of subtype H5. They also thank Dr Bela Lomniczi, Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary, for genotyping our APMV-1 as an additional member of the novel genotype VII. The authors also wish to thank Sandra David-Holl and Andrea Meiwes for excellent technical assistance during sample collections, virus isolations and antibody assays.