Circovirus infection in Croatian population of Eurasian griffon vultures (Gyps fulvus)

Abstract

Circoviruses cause diseases in a wide range of hosts, including avian species. One of the most studied circoviruses is beak and feather disease virus (BFDV), which has spread worldwide due to global trade of birds and characteristics of the virus that allow it to persist in the environment. The Eurasian griffon vulture (Gyps fulvus) is one of the largest birds in the world and is among several vulture species that are considered endangered today, and currently the only remaining vulture species in Croatia. Several juvenile Eurasian griffon vultures undergoing rehabilitation at the Beli Visitor Centre and Rescue Centre for Griffon Vultures on the island of Cres, Croatia, had feather lesions similar to those caused by BFDV in psittacine birds. Signs included partially retained feather sheaths, discoloration and circumferential constrictions of the feather shafts, gnawed feather tips, keratin and egg deposits near the feather shafts, as well as feather breakage and spontaneous feather loss. Feather samples were collected for molecular detection of viral pathogens and macroscopic detection of ectoparasites; blood samples were taken for complete blood count (CBC), biochemical analysis and oxidative stress analysis. The molecular analysis confirmed a circovirus infection, which was supported by the blood analyses indicating a viral infection. The collected ectoparasites were identified as chewing lice - Colpocephalum turbinatum and Falcolipeurus quadripustulatus, which have previously been detected in Eurasian griffon vultures. To our knowledge, this is the first report of circovirus infection in Eurasian griffon vultures. Considering that circoviruses are highly contagious, very resistant and easily transmissible, it is extremely important to perform continuous monitoring in order to improve the breeding and survival conditions of Eurasian griffon vulture populations worldwide.

Introduction

Circoviruses are small, non-enveloped viruses with icosahedral virions and a single-stranded circular DNA genome^1,2. They are members of the Circoviridae family, within which there are 101 established species classified into two genera - Circovirus and Cyclovirus^2,3. They have been reported in a wide range of hosts, causing varying clinical signs but mostly feather lesions in avian species⁴. One of the most studied circoviruses is beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD), which was first described in 1907 in a population of red-rumped grass parrots (Psephotus haematonotus) in Australia⁵. Due to the global trade of wild-caught Australian birds, the disease has spread worldwide and nowadays affects many wild and captive avian species^6,7. It has been reported in many non-psittacine birds such as finches, columbids, corvids and raptors, but usually as a sporadic spillover infection^4,7–9.

The BFDV infection can be acute or chronic, and usually manifests as feather dystrophy and immunosuppression^7,10,11. The physicochemical properties of the virus enable its persistence in the environment and its ability to survive high temperatures, desiccation and a wide range of chemicals^12,13. However, high doses are required to establish an infection, and the age of the bird, stage of feather development and bird immune status have a major influence on the incubation period and outcome of the infection, with the youngest birds being much more susceptible to developing the disease⁷. Transmission occurs mostly horizontally through ingestion or inhalation of contaminated faeces and feather dust, as well as through ingestion of regurgitated feed from infected birds. Vertical transmission has been postulated but has yet to be clarified. In wild birds, the transmission most likely occurs through oral or intracloacal ingestion of the virus⁷. Additionally, some ectoparasites, such as hippoboscid flies and mites, were reported as potential passive vectors of BFDV¹³.

Vultures play a very important role in ecosystems by feeding on dead animals and preventing the spread of pathogens¹⁴. The survival of an increasing number of different bird populations around the world is threatened primarily by anthropogenic activities leading to climate change, as well as environmental and habitat changes and degradation affecting individual bird species¹⁵. The Eurasian griffon vulture (Gyps fulvus) is one of the largest birds in the world and is among several vulture species that are considered endangered today¹⁶. More than 85% of the European griffon vulture population is located in Spain, where it has significantly increased over the past few decades and is now the largest griffon vulture population globally. In contrast, the griffon vulture population in the Balkans has experienced a significant decline over the past century and is therefore considered regionally endangered^15,17. In the territory of the Republic of Croatia, the griffon vulture is the only remaining vulture species. The Kvarner population, located in the Kvarner region in Croatia, currently consists of around 150 pairs and is one of the few viable populations in Southeast Europe [M. Modrić, personal communication, July 14, 2025]. The griffon vulture is protected by the international Bonn, Bern, and Washington Conventions, as well as the European Union Birds Directive, and in Croatia, it is a strictly protected species under the Nature Protection Act¹⁸. The applied conservation measures increased the population numbers and led to griffon vultures being classified as a Least Concern category according to the Global IUCN 2024 Red List¹⁸. The main threats to the Kvarner population are lack of available food in the habitat, disturbance of nesting sites and accidents such as electrocution and poisoning [¹⁹, M. Modrić, personal communication, 2025]. In addition to the continuous work of the local and national organisations on the conservation of the griffon vulture population in Croatia, the LIFE SUPport project was established to mitigate the above-mentioned threats and improve breeding and survival conditions.

Vultures have a specialized gastrointestinal tract and gut microbiome that enables them to feed on decaying carcasses. As a result, they contribute to the removal of the potential hazardous sources of infection and regulate the transmission of pathogens in nature^20–22. On the other hand, circoviruses also have physicochemical features and defence mechanisms which enable them to confound the host’s immune system in order to replicate and persist in the environment^13,23. Although a great variety of circoviruses have been reported in vertebrates and insects, they have often not been associated with any clinical signs or pathogenic lesions²⁴. Here, we present a comprehensive case of circovirus infection in a Croatian Eurasian griffon vulture population that manifested clinically as feather lesions consistent with psittacine beak and feather disease.

Materials and methods

All animal procedures were approved by the Committee for Ethics in Veterinary Medicine of Faculty of Veterinary Medicine University of Zagreb (approval no. 640-01/23 − 02/06; 251-61-01/139-23-23) and were performed in accordance with the ARRIVE guidelines and relevant regulations.

Case history

Several Eurasian griffon vultures (Gyps fulvus) that are in a rehabilitation process at the Beli Visitor Centre and Rescue Centre for Griffon Vultures on the island of Cres, Croatia, were rescued from the wild. All birds were rescued as juveniles during 2022–2023 after falling into the sea during their first flight attempts from the nest, which is a common reason for rescue, as the Croatian population nests on cliffs just above the sea level. The aviary at the Rescue Centre was built from metal mesh with an indoor shelter area, where all admitted birds were placed with a few other birds of different ages. Three birds had chronic, extensive feather lesions with no other clinical signs. The affected birds lost almost all primary and many secondary feathers, while the tail feathers remained intact. The detected changes included feather breakage at one or two thirds of the quill, partially retained feather sheaths, discoloration and circumferential constrictions of the feather shaft, gnawed tips of the feathers, keratin and ectoparasite egg deposits on the individual feathers adjacent to the feather shaft, and spontaneous feather loss without breakage (Figs. 1, 2 and 3). The same problem occurred approximately five years ago in two juvenile birds that spent two years in the Rescue Centre, but after two moults, the feathers were completely normal again. In this case, the affected birds were shedding the developing feathers and the new feathers showed abnormal development so that they could no longer fly. The differential diagnoses included folliculitis of fungal/bacterial aetiology, ectoparasites, viral infection, hemoparasites, endocrinologic and metabolic disorders.

Fig. 1.

Security camera footage from the aviary in the Rescue Centre shows griffon vultures with feather lesions.

Fig. 2.

Feather samples with retained feather sheath (A), gnawed feather barbs (A, B) and egg deposits (B).

Fig. 3.

Feather with shaft discolorations, retained sheath and dystrophic quill with keratin deposits (A, B); hyperkeratotic feather shaft with egg deposits and completely gnawed barbs (C).

Blood samples were collected from the metatarsal vein in three birds with extensive clinical signs (CV7, CR4, CZ4) for a complete blood count, biochemical analysis, oxidative stress analysis, molecular testing for circovirus (BFDV) and avian polyomavirus (APV). Blood EDTA samples for viral testing were sent to an external veterinary diagnostic laboratory. The samples were negative for both viruses. Blood samples for other analyses were taken in Li-heparin tubes and analysed at the Department of Pathophysiology, Faculty of Veterinary Medicine, University of Zagreb, Croatia. Despite negative results, the analyses of viruses were performed again at the Department of Poultry Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Croatia, from the feather samples collected from the same birds. The feathers were also examined for ectoparasites, and live specimens were sent to the Department of Parasitology and Invasive Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Croatia, for identification.

Complete blood count and biochemical analysis

Tubes with anticoagulant (lithium heparin) were sent for biochemical and hematological tests. Biochemical analysis was done on an automated biochemical analyser Cobas c111 (Roche Diagnostics, Switzerland) using blood plasma. Parameters used in analysis included glucose (mmol/L), total protein (g/L), albumins (g/L), globulins (g/L), triglycerides (mmol/L), cholesterol (mmol/L), calcium (mmol/L), total bilirubin (µmol/L), blood urea nitrogen (mmol/L), creatinine (µmol/L), aspartate aminotransferase - AST (U/L), alanine aminotransferase - ALT (U/L), alkaline phosphatase - AP (U/L), creatine kinase - CK (U/L), lactate dehydrogenase LDH (U/L) and uric acid (mg/dL). Hematological tests (complete blood count) were performed manually using Natt-Herrick’s solution and Neubauer-improved counting chamber, while blood smears were stained with Wright-Giemsa stain for reptiles and birds at the Department of Pathophysiology, Faculty of Veterinary Medicine, University of Zagreb, Croatia. We compared our results with previously published hematological and biochemical values for vultures^14,25.

Analysis of antioxidant enzyme activity

The activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined. Enzyme activities were determined using whole blood samples. The catalytic activity of SOD was determined using the commercial RANSOD antioxidant kit (Randox Laboratories, Crumlin, UK) according to the manufacturer’s instructions while GPx activity was determined using the commercial RANSEL kit (Randox Laboratories), following the manufacturer’s instructions²⁶. Sample absorbances were measured using a Tecan Infinite 200 microplate reader (Tecan Group Ltd., Switzerland).

DNA isolation for molecular analyses

Feather samples were collected from the affected birds in the Rescue Centre on two occasions and sent to the Department of Poultry Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Croatia, for repeated molecular testing for circovirus and APV. In total, 24 samples were analysed. For the first sampling, several feathers per bird with clinical signs were collected and DNA was individually isolated. At the same time, several feathers from the aviary were pooled and analysed altogether. For the second sampling, in the pooled feather sample from the aviary, each feather was analysed individually. Total DNA was isolated from the feathers’ calami. The calami were aseptically mechanically homogenized, after which the fragments were used for isolation using the NucleoSpin DNA RapidLyse kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions.

PCR

The samples were tested for circovirus and APV. Circovirus was tested using both BFDV-specific primers and broad-spectrum primers (Table 1). The reaction mixtures for initial detection were composed of 7.5 µl GoTaq G2 Hot Start Green Master Mix (Promega, Madison, WI, USA), 0.6 µl of each 10 pmol primers, 2 µl of DNA and 4.3 µl of Nulease-free water (Promega, Madison, WI, USA) (total volume = 15 µl). The cycling parameters were set as previously described by Ypelaar et al., Roy et al. and Halami et al.^11,27,28.

Table 1.

Primers used for molecular analyses.

Pathogen	Region/gene	PCR product size	Primer	Primer sequence	Reference
Circovirus (BFDV)	ORF-1	717 bp	PBFD-F	5’-AACCCTACAGACGGCGAG-3’	27
			PBFD-R	5’-GTCACAGTCCTCCTTGTACC-3’
Circovirus (nested broad-spectrum)	Rep	-	Cv-a	5’-AGAGGTGGGTCTTCACNHTBAAYAA-3’	11
			Cv-as	5’-AAGGCAGCCACCCRTARAARTCRTC-3’
		350 bp	Cn-s	5’-AGCAAGGAACCCCTCAYYTBCARGG-3’
			Cn-as	5’-ACGATGACTTCNGTCTTSMARTCACG-3’
Polyomavirus (APV)	VP1	550 bp	F	5’-CTTATGTGGGAGGCTGTCAGTGTT-3’	28
			R	5’-TACTGAAATAGCGTGGTAGGCCTC-3’

Gel electrophoresis, DNA purification and sequencing

The PCR products were visualised using 1.5% agarose gel (Nippon Genetics Europe GmbH, Düren, Germany) and stained with Midori Green Advance (Nippon Genetics Europe GmbH, Düren, Germany). The expected amplicon sizes are presented in Table 1.

For sequencing, 50 µl reaction mixtures were prepared in order to obtain a higher concentration of DNA. The reaction mixture for isolation and purification of DNA for sequencing contained 25 µl of GoTaq G2 Hot Start Green Master Mix, 2 µl of each 10 pmol primer, 5 µl of DNA and 16 µl of Nuclease-free water.

Purification of the amplicons from gel slices was done using ReliaPrep DNA Clean-up Concentration System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After checking the concentration and purity using BioDrop (BioDrop Ltd., Cambridge, UK), the samples were submitted for Sanger (dideoxy) sequencing (Macrogen Inc., Amsterdam, The Netherlands) using the inner primers set (Cn) for the nested broad-spectrum PCR (Table 1).

Phylogenetic analysis

The raw sequences were edited using BioEdit Sequence Alignment Editor v7.2.5²⁹. After editing, they were aligned, cut to equal lengths and analysed using Molecular Evolutionary Genetics Analysis software (MEGA12) v12.0.11³⁰. The data were analysed using the Maximum Likelihood method and Kimura 2-parameter model with gamma distributed rates among sites³¹. Basic local alignment search tool (BLAST) of the National Center of Biotechnology Information (NCBI, USA) was used to search for genetically similar sequences based on the data available in the GenBank. In total, 50 publicly available sequences were used for the construction of the phylogenetic tree, and the gull circovirus sequence (MN164711) was used as an outgroup. The accession numbers of other sequences used in the analysis are shown on the phylogenetic tree (Fig. 4).

Fig. 4.

Phylogenetic tree based on the Rep coding gene sequences of BFDV.

Identification of ectoparasites

The collected lice were prepared for identification following³² with some modifications: specimens were preserved in 70% ethanol, mounted in lactophenol for at least 24 h, and identified based on the identification keys^33,34 using an Olympus BH 40 microscope and a Touptek camera with QuickPHOTO Micro 2.3 software.

Results and discussion

All analysed samples were negative for APV, but 12 out of 24 samples were positive for circovirus. Interestingly, the samples were positive for both BFDV and broad-spectrum circovirus. Although several positive samples were prepared and submitted for sequencing, only one sample provided concentrated and pure DNA sufficient for phylogenetic analysis. The sequenced sample (BFDV_CV7_Gyps fulvus/Croatia/2024) showed the highest similarity (98.6%) with publicly available sequences from Saudi Arabia and Egypt (Fig. 4). Interestingly, Sub-Saharan Africa, the Eastern Mediterranean region and the Middle East are common migration sites for juvenile Eurasian griffon vultures according to the telemetry data. Although the affected birds did not migrate to these regions since they were rescued as juveniles, they had contact with birds that migrate there and with birds from other populations that showed similar clinical signs, suggesting that the older birds may have been infected during their migrations or that the infected birds from other populations spread the virus within the Rescue Centre [M. Modrić, personal communication, July 14, 2025]. Previous studies by Sarker et al.^6,35 showed a high number of circovirus genome variants in the infected birds, which is a result of a high mutation rate and presents a replication strategy that allows flexible host-switching. The species threshold for members of the Circoviridae family is 80% genome-wide pairwise identity, while sequences that share less than 55% identity with classified circovirids are likely members of unclassified cressdnaviricots or a different family^2,24. Although we did not perform whole-genome sequencing in this case, the similarity level according to BLAST confirms identification as BFDV. The phylogenetic analysis included sequences of BFDV from two Moluccan eclectus parrots diagnosed with PBFD in Croatia. Interestingly, one of them showed a high similarity with the sequence from the vulture. As the breeder from which the above-mentioned parrots originate imports birds from various countries [Ž. Gottstein, personal communication, 2024], the high similarity could be a result of bird introduction from different origins without previous quarantine and screening for pathogens such as BFDV.

The following key findings from CBC correspond to the three BFDV-positive birds. Birds CZ4 and CR4 were anemic with macrocytic-hypochromasia and normocytic-normochromasia, respectively. Leukocyte changes were observed in all three birds (Table 2). Changes in biochemical findings were observed in all three birds, with hypoalbuminemia and decreased creatinine concentration being the most significant changes (Table 2), while other biochemical parameters were similar to findings of Polo et al.²⁵. SOD activity was low in bird CZ4 while in birds CV7 and CR4 high (Table 2). Due to low sample volume, activity of GPx in CZ4 was not determined, while in other two birds (CV7 and CR4) it was moderately elevated. The laboratory findings in bird CV7 could indicate infection, possibly viral, due to leucocytosis, moderate heterophilia, severe lymphopenia and monocytosis, while eosinophilia and basophilia in CV7 and CR4 might be a result of ectoparasite infestation, although they are not a regular feature of parasitic infestation in birds. The rest of the complete blood count were similar to the parameters described by Polo et al.²⁵. These changes could be due to the different diet, season, stress and physical activity in captive conditions.

Table 2.

Results of the hematological, biochemical and antioxidant enzyme analyses.

Measurement	CZ4	CV7	CR4	Reference values25
RBC (106/µL)	0.9	2.3	2.12	2.19–2.87
HCT (%)	47	47	38	40.0–51.8.0.8
Hgb (g/dL)	6.9	13.5	12.3	12.4–18.2
MCV (fL)	522.2	204.3	180	150.9–189.3.9.3
MCH (pg)	76.7	58.7	58	46.8–66.6
MCHC (g/dL)	14.7	28.7	32.4	29.7–36.6
WBC 103/µL (%)	20	26	19.8	5.0–24.0
Heterophils 103/µL (%)	8.8 (44)	18.9 (73)	11.7 (59)	1.95–16.56 (51–72)
Lymphocytes 103/µL (%)	9.2 (46)	1.6 (6)	6.7 (34)	1.92–9.0.92.0 (24–49)
Monocytes 103/µL (%)	0.4 (2)	1.8 (7)	0	0–0.2.2 (0–1)
Eosinophils 103/µL (%)	1 (5)	3.4 (13)	0	0–3.2.2 (0–4)
Basophils 103/µL (%)	0.4 (2)	0.3 (1)	1.4 (7)	0.00
Glucose (mmol/L)	13.9	N/A*	17.4	14.2–22.2
Total protein (g/L)	50	45	33	30–51
Albumin (g/L)	13	11	10	13–22.3.3
Globulin (g/L)	37	34	23	N/A
Triglycerides (mmol/L)	0.5	N/A	0.7	0.56–1.9
Cholesterol (mmol/L)	4.1	N/A	5.1	3.1–7.1
Ca (mmol/L)	2.8	N/A	2.4	2.4–4.7
Total bilirubin (µmol/L)	4.4	2.4	1.7	N/A
BUN (mmol/L)	1.6	N/A	1.1	0.53–2.82
Creatinine (µmol/L)	25	N/A	25	30.9–86.7
AST (U/L)	280	N/A	163	93.5–156.8.5.8
ALT (U/L)	46	11	14	3.9–19.8
ALP (U/L)	95	37	69	30–354.4.4
CK (U/L)	111	N/A	236	113–774
LDH (U/L)	209	N/A	303	30.9–86.7
Uric acid (mg/dL)	4.77	2.93	8.45	2.81–11.4
SOD (U/mL)	62.3	287.14	298.7	N/A
GPx (U/mL)	N/A*	7.2	9.11	N/A

*Parameter value was not determined because of the low sample volume.

There are several possible reasons why the initial blood tests were negative for circovirus. As the affected birds showed no clinical signs other than feather dystrophy, the infection might have caused a mild viremia at the time of first sampling, as supported by the CBC results, which did not indicate significant immunosuppression or liver damage. However, all three birds had increased AST activity, which, in combination with lymphopenia, indicates a viral infection. The second possible explanation could be the use of different sets of primers that have a narrow spectrum. Since the positive test results in this study were later achieved with both specific and broad-spectrum primers, the first explanation was considered more likely.

Two chewing lice species were identified through microscopic examination of the collected ectoparasites - Colpocephalum turbinatum (Amblycera: Menoponidae) and Falcolipeurus quadripustulatus (Ischnocera: Philipteridae) Fig. 5, although the eggs had dried out on most feather samples. Both lice species have previously been detected in griffon vultures and various birds of prey in other countries³⁶, but not in Croatia. As vultures have a high close contact behaviour between birds, the transmission of parasites is often very high^36,37. As the vultures’ gastrointestinal tract and gut microbiome provide specialized defence mechanisms that allow them to feed on potentially contaminated carcasses without becoming infected, it is difficult to conclude the route of infection that occurred in these birds. Based on previous reports of mechanical transmission of the infection by mites and flies^4,13 and the fact that these birds were infested with chewing lice, there is a possibility that the infection could spread via ectoparasites, but further scientific research is needed for confirmation.

Fig. 5.

Chewing lice collected from the feather samples - Colpocephalum turbinatum (A) and Falcolipeurus quadripustulatus (B).

To our knowledge, there is only one previous report on the circovirus detection in vultures, in the Egyptian vulture (Neophron percnopterus) populations in Greece and Bulgaria in 2012–2013³⁸. In said report, avian circovirus was detected by qPCR in seven out of twelve blood samples examined, but the birds showed no clinical signs or lesions indicating the infection might have been subclinical³⁸. Similar feather lesions were reported in Eurasian griffon vultures in Italy, in which case pathohistological and immunohistochemical tests confirmed polyomavirus infection³⁹ that was previously described in wild birds only in a common buzzard and a kestrel in Germany⁴⁰.

Considering the properties of circovirus that enable its resistance and virulence, it is difficult to eradicate it, especially in the aviaries or on the feeding sites where many birds come into contact. In this case, the staff at the Rescue Centre was advised on how to improve the biosecurity measures to try to prevent viral spread. The aviaries were thoroughly cleaned and disinfected; birds showing feather lesions were sampled and tested for circovirus, and those testing positive were euthanised. Some of the birds may be asymptomatic and spread the infection within the population, which can lead to decreased flock immunity and increased susceptibility to other diseases. Because of that, all new birds will be tested during their quarantine period at the Rescue Centre in the future. For endangered species such as Eurasian griffon vultures in Croatia, it is extremely important to monitor the existing population and try to improve their living conditions to enable their successful rehabilitation and release into the wild.

Author contributions

L.L. Conceptualization, methodology, investigation, writing, prepared all figures. S.F. Investigation, methodology, formal analysis, writing, review and editing. M.L. Investigation, resources, editing. I.K. Methodology, formal analysis. F.M. Investigation, formal analysis, writing. Ž.G. Investigation, methodology, writing and editing, supervision. All authors reviewed the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The sequence generated and analysed during the current study is available in the GenBank repository, under the accession number PV780460.

Declarations

Competing interests

The authors declare no competing interests.