Arch Virol (2015) 160:339–344 DOI 10.1007/s00705-014-2225-x

BRIEF REPORT

Beak and feather disease virus: correlation between viral load and clinical signs in wild Cape parrots (Poicepahlus robustus) in South Africa Guy L. Regnard • Rutledge S. Boyes • Rowan O. Martin • Inga I. Hitzeroth • Edward P. Rybicki

Received: 11 July 2014 / Accepted: 30 August 2014 / Published online: 7 September 2014 Ó Springer-Verlag Wien 2014

Abstract Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). This study assessed viral load using qPCR in a wild Cape parrot population affected by PBFD and compared it to overall physical condition based on clinical signs attributable to PBFD. A significant inverse correlation between viral load and overall physical condition was found, which confirmed that clinical signs may confidently be used to diagnose the relative severity of BFDV infections in wild populations. This is the first assessment of BFDV viral load in a wild psittacine population. Keywords Psittacine beak and feather disease  Beak and feather disease virus  BFDV  Viral load  Cape parrot Psittacine beak and feather disease (PBFD) is the most prevalent viral disease affecting captive and wild psittacine

Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2225-x) contains supplementary material, which is available to authorized users. G. L. Regnard  I. I. Hitzeroth (&)  E. P. Rybicki Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa e-mail: [email protected] R. S. Boyes  R. O. Martin DST/NRF Centre of Excellence, Percy FitzPatrick Institute of African Ornithology, University of Cape Town, Rondebosch 7701, South Africa E. P. Rybicki Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa

populations. The prevalence of the disease has been attributed largely to the international trade in psittacines [1]. The causative agent is beak and feather disease virus (BFDV), which belongs to the genus Circovirus of the family Circoviridae [2]. The virus has an icosahedral virion of approximately 20.5 nm [3], and a 2-kb circular ssDNA ambisense genome which encodes the replication-associated (Rep) and capsid (CP) proteins [4, 5]. Transmission of BFDV between birds can occur either through horizontal or vertical transmission. Horizontal transmission can occur via ingestion or inhalation of the virus, and ingestion of contaminants including faecal and feather material is seen as the primary route of transmission [6, 7]. Horizontal transmission is exacerbated by psittacines congregating into flocks and through contamination of nesting sites, which enables transmission between ecologically disconnected species [8–10]. Overall, transmission is dependent on the locus of persistence and the viral load during acute viraemia [11]. A chronic form of PBFD mainly affects adult psittacines and is characterised by beak and feather abnormalities and immunosuppression [12, 13]. In the chronic form, infection is generally followed by a gradual replacement of feathers with abnormal ones after each successive moult [14–16]. Feather abnormalities are accompanied by symmetric feather loss, and this in turn causes the skin to discolour if exposed to sunlight [12, 17]. Subclinical infections are also common, making detection and containment of the disease difficult [18]. These subclinical infections are difficult to detect from blood samples, as has previously been shown in clinically healthy but BFDV-positive budgerigars, where more often than not, the blood samples tested negative [19]. Hess et al. [20] suggested that there is a correlation between clinical signs and viraemia as assayed using PCR during a longitudinal study of captive budgerigars; however, the correlation between

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viral load and clinical signs has not been investigated for BFDV. Cape parrots (Poicepahlus robustus) are endemic to the Eastern Cape and adjacent regions in South Africa. With fewer than 1500 wild parrots remaining, it is listed as being critically endangered. Moreover, many birds in wild flocks are apparently affected by PBFD [21]; as it is impractical to test all of them for BFDV, a surrogate test is needed. In this study, we determined the viral load in BFDV-infected birds using qPCR DNA amplification from blood samples isolated from wild Cape parrots, to determine whether there was a useful correlation between viral load and clinical signs. This is the first reported assessment of BFDV viral load in Cape parrots, or in any wild psittacine population. A total of 49 Cape parrots from a wild population in the Eastern Cape Province of South Africa were trapped in mist nets (Department of Economic Development & Environmental Affairs (Eastern Cape) permit number: O8071A). Sex and biometric measurements were recorded. A weak correlation between viral load and overall condition in males and females was seen when condition was calculated based on the ratio of weight to wing length to the power of three (unpublished data), and therefore, an overall physical condition score based on the rounded average of six scores (1-5) for clinical signs attributable to PBFD was used (Table 1). Blood samples were taken from the brachial vein (University of Cape Town’s Animal Ethics Committee clearance number: 2010/V12/RB) and stored on FTA Classic Cards (Whatman, UK) at 4 °C. Blood was also collected from four captive Cape parrots in Cape Town of unknown origin with no history of BFDV infection and used as a PCR control group. No assessment of physical condition was performed on these captive birds. The quantitative real-time PCR experiment was performed following the MIQE guidelines as described by Bustin et al. [22]. Total DNA was extracted from nucleated blood using a DNeasyÒ Blood & Tissue Kit (QIAGEN, DE). Each qPCR reaction was performed in triplicate using a LuminoCT SYBR Green qPCR ReadyMix (Sigma-Aldrich, US) as per manufacturer’s instructions and primers (50 -CAGTTAAGGGCGCTTTTGTGGAG-30 and 50 -TTC GGGTCACAGTCCTCCTTG-30 ) specific to a 97-bp region of the BFDV rep gene (GenBank accession number GQ165756) together with primers specific to a 97-bp region of the P. robustus reference gene, TGF beta 2 (GenBank accession number EU660286) (50 -TCCCATCT GGCACTGTCTCTG-30 and 50 -ACAGAGCTTTCACCCT CATTTATGG-30 ). The Cape parrot TGF beta 2 has been used previously to describe phylogeny in psittacines [23]. This gene is present in both humans and mice as a singlecopy gene, and it is therefore our assumption that this is also true for psittacines [24]. A BLAT search of the recently sequenced budgerigar genome using Cape parrot

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G. L. Regnard et al. Table 1 Summary of the condition scores used to assess overall physical condition of Cape parrots based on possible symptoms of BFDV infection Clinical symptom

Overall physical condition scores: 1 (very poor) to 5 (very good)

Degraded beak

Lesions, cracks and black blotches are: (5) absent; (4) [15 mm apart or \10 % of beak area; (3) 10-15 mm apart or 10-25 %; (2) 5-10 mm apart or 25-50 %; (1) \5 mm apart or [50 %

Darkened cere

Colour of the cere is: (5) white; (4) light grey to white; (3) light grey and dry; (2) dark grey; (1) dark grey to black with lesions

Degraded feathers

Area degraded/dead feathers estimated on R wing: (5) absent; (4) \25 %; (3) 25-50 %; (2) 50-75 %; (1) 100 %

Black blotches

Area black blotches estimated on breast: (5) absent; (4) single blotch; (3) 25-50 %; (4) 50-75 %; (5) [75 %

Degraded down feathers

Area degraded or missing down feather estimated on breast: (5) absent; (4) \25 %; (3) 25-50 %; (2) 50-75 %; (1) 100 %

Excessive feather dust

Feather dust on breast is (5) absent; (4) visible on a few feathers; (3) accumulated on latex glove during handling; (2) dispersed into air when parrot moves; (1) visible on all feathers

TGF beta 2 produced only one alignment, which supports our assumption [25]. Cloned plasmid DNA containing the amplicons in serial dilution served as the standard curve for the determination of gene copy number for both rep and TGF beta 2. Thermocycling was performed using a RotorGene RG-6000 (QIAGEN, DE), and the parameters consisted of a 10-minute hold at 95 °C followed by cycling (40 repeats) between a 15-second hold for denaturation at 95 °C, a 15-second hold at 55 °C for annealing, and a 15-second hold 60 °C for elongation. Template specificity was confirmed from the reaction melt curve analysis. The Cq values from the NTCs fell below the lower limit. The qPCR efficiencies and r2 values for each reaction were 0.990 (standard deviation, 0.0418) and 0.997 (standard deviation, 0.00250) for the BFDV rep gene and 1.01 (standard deviation, 0.0577) and 0.996 (standard deviation, 0.00307) for the TGF beta 2 reference gene, respectively. For further information regarding MIQE, details can found as an online supplement. The viral load was determined as a ratio of BFDV rep gene copies to Cape parrot TGF beta 2 (Table 2). Studying virus infections in wild psittacine populations provides a unique opportunity to study the relationship between viral load and overall physical condition, as the population is homogenous and larger than those normally found in captivity, and the birds were at varying stages of disease progression. Most reports of viruses in captive birds have included small sample sizes and multiple

Beak and feather disease in Cape parrots in South Africa

341

Table 2 Summary of the data collected during sampling and qPCR data with technical variance presented as percentage relative standard deviation Sample code

Sex

Condition

FH02

M

5

FH13

M

4

FH11 FH08

M F

FH12 FH10

rep copy number

rep variance (%)

TGF beta 2 copy number

TGF beta 2 variance (%)

Viral load ratio*

10

57

5668

6.1

-2.8

8

42

2889

19

-2.6

4 4

4 20

61 12

847 4130

4.6 8.7

-2.4 -2.3

M

3

19

19

1257

9.9

-1.8

F

5

10

30

467

16

-1.7

FH09

M

5

6

17

252

14

-1.6

FH06

M

3

81

12

1189

4.1

-1.2

FH07

F

3

511

7.5

8567

6.0

-1.2

FH39

M

4

820

4.3

5448

6.6

-0.8

FH17

M

4

741

15

3012

7.1

-0.6

FH30

M

3

570

11

2080

8.9

-0.6

KWT02

F

3

278

10

1084

7.2

-0.6

KWT03

M

4

396

17

1426

9.7

-0.6

KWT05

F

3

162

22

510

9.3

-0.5

FH19

M

5

845

11

1647

6.9

-0.3

KWT09

F

1

465

37

940

7.0

-0.3

FH20 FH27

F F

3 3

242 277

8.0 13

395 447

11 22

-0.2 -0.2

FH32

F

4

563

5.0

884

5.1

-0.2

FH43

F

4

488

39

701

24

-0.2

FH34

M

4

340

33

426

29

-0.1

FH42

M

4

1447

10

1822

12

-0.1

KWT10

F

1

497

16

617

11

-0.1

FH18

F

4

186

9.6

167

16

0.0

FH41

M

3

1246

29

1287

2.5

0.0

FH26

M

3

804

12

320

18

0.4

FH40

M

4

4371

11

1779

2.0

0.4

KWT04

F

3

1426

7.1

499

6.7

0.5

FH03

F

2

2983

15

345

63

0.9

FH29

M

3

2966

4.8

295

23

1.0

FH33

M

2

894

16

100

12

1.0

FH01

M

2

205969

4.5

9994

20

1.3

FH23 FH37

F F

3 2

51181 62371

4.1 12

592 512

7.7 10

1.9 2.1

FH31

F

2

200542

6.0

646

12

2.5

FH04

F

1

1470280

28

2715

25

2.7

KWT07

M

1

975977

14

542

5.3

3.3

KWT01

M

1

2180971

12

834

12

3.4

FH28

M

2

6326690

5.0

1809

5.0

3.5

FH05

F

2

8040302

6.7

1842

9.5

3.6

FH38

M

1

4686372

13

1110

13

3.6

KWT06

M

1

6342852

21

1326

3.6

3.7

FH35

M

2

4618781

7.3

564

7.6

3.9

STUT01

M

1

2057767

23

232

5.0

3.9

FH21

F

3

9265699

24

738

14

4.1

FH16

M

3

4769109

37

201

70

4.4

123

342

G. L. Regnard et al.

Table 2 continued Sample code

Sex

Condition

rep copy number

rep variance (%)

TGF beta 2 copy number

TGF beta 2 variance (%)

Viral load ratio*

FH14

M

2

62704464

9.7

1541

11

FH24

F

2

40522823

5.3

880

5.1

4.7

SE04

-

-

2

214

1451

7.5

-3.0

SE03

-

-

2

99

1463

13

-2.8

SE01

-

-

4

25

1801

9.5

-2.7

SE02

-

-

3

33

1052

10

-2.5

4.6

*The viral load ratio is represented as the log10 of the ratio of BFDV rep copies to Cape parrot TGF beta 2

Fig. 1 (a) A box plot of viral load in the blood as determined by qPCR using the BFDV rep gene and the Cape parrot TGF beta 2 gene in relation to overall physical condition score for Cape parrots. Overall condition was scored as one through five based on observable physical condition, with one being poor condition (p \ 0.0001).

There is no statistical difference within groups ‘a’ and ‘b’, while groups ‘a’ and ‘b’ are significantly different from each other (b) A discontinuous plot of viral loads for males (d) and females (s) for each overall physical condition score

species, which complicates assessing overall physical condition [26, 27]. In our study, overall physical condition of wild Cape parrots was compared to BFDV viral load in the blood, measured by qPCR; previous studies of wild psittacine populations have relied on standard and nonquantitative PCR assays to detect the presence of BFDV [28–30]. The condition of the Cape parrots sampled varied, and many appeared to be malnourished and to display clinical signs of PBFD (Supplementary Fig. 1). There was a significant difference in viral load between the different physical condition groups (Kruskal-Wallis; df = 4 (N = 49), H = 25.563, p \ 0.0001) (Fig. 1a). Post hoc testing using multiple comparisons of mean ranks for all groups indicated that parrots with an overall condition score of 1 and 2 had viral loads that were significantly higher than birds in symptom score groups 4 and 5. Birds

with an overall condition score of below three had a mean viral load ratio of greater than 103, while those with a condition score of three and greater had a mean viral load ratio of less than one. Similar results have been reported in a controlled challenge experiment in long-billed corellas, in which a peak viraemia of around 106 copies/ll was detected from week two after challenge and continued past week six [31, 32]. This is similar to the maximum rep gene copy number found in this study (Table 2). This high viral load suggests that a proportion of Cape parrots in the present study had an active viral infection, corresponding to a greater severity of clinical signs. A similar finding for a similar virus has been reported in pigs infected with porcine circovirus type 2 [33]. Cape parrots with a viral load of less than one rep copy per reference gene, corresponding to 102 rep copies/ll, had a high overall condition score: this result was similar to the

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Beak and feather disease in Cape parrots in South Africa

vaccinated long-billed corellas study, where a value of around 102 copies/ll was seen. This could indicate that these Cape parrots are mounting a successful immune response against the virus. This is supported by a previous study, where birds with a high haemagglutination inhibition (HI) score had a negative blood sample PCR result [34]. Katoh et al. [27] showed an overall viral load range of between 10-2 and 105 copies per cell in clinical samples obtained from a range of captive psittacine species; however, no comparison with overall physical condition was made. The viral loads determined in the present Cape parrot study also closely mirror these findings. Interestingly, there have been no literature reports of a viral load ratio less than 10-3. In the present study, the four captive birds with no history of PBFD had rep gene copy numbers in this range. Similarly, healthy wild Cape parrots also had low rep gene copy numbers, and this indicates that this is possibly a useful cutoff value for assessing birds as being disease-free. Males and females showed a similar correlation between viral load and overall condition. Two females with an overall physical condition score of 1 but with a viral load ratio of less than 1 were found to be infected with a Pseudomonas sp. (Fig. 1b). This secondary infection may have contributed to the low overall physical condition score. This is the first reported assessment of BFDV viral load in a wild psittacine population. Our study confirms that the presence and degree of observed clinical signs of PBFD in Cape parrots correlate strongly with BFDV infection, and more specifically, with viral load. Therefore, clinical signs assessed as we describe may confidently be used to diagnose the presence and relative severity of BFDV infections in wild Cape parrots in the field. This will be very useful in ongoing Cape parrot population surveys, and will make it only periodically necessary to capture birds to track the incidence of BFDV in the wild population – which will significantly decrease their stress and capture- and handling-related injuries. Acknowledgments This work was supported by funding from the South African National Research Foundation (NRF) and National Geographic Society. The authors gratefully acknowledge David Mutepfa and David Nkosi for technical assistance, and the Poliomyelitis Research Foundation (PRF), the Harry Crossley Foundation as well as the NRF for student funding for Guy Regnard. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to the NRF.

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