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A High Prevalence of Beak and Feather Disease In Captive Parrots in China-Complete

A High Prevalence of Beak and Feather Disease In Captive Parrots in China-Complete

分类:
疾病防治
来源:美联众合动物医院转诊中心
发布时间:
2018-12-11 10:56:53


A High Prevalence of Beak and Feather Disease
In Captive Parrots in China.
Tang Guoliang
Mei Lian Zhong He Veterinary Hospital Referral Center, No 168 Beiyuan street, Chaoyang District, 100101, Beijing, China
Abstract:
Psittacine Beak and Feather Disease (PBFD) is a major life-threatening disease in parrots. In this study, 157 blood samples of captive parrots in China were collected, and screened for PBFD virus using nested PCR. The total positive rate was 61.1% (96/157). Phenotype analysis revealed that the clinical signs of virus carriers vary among different species. In African grey parrots and cockatoos usually have unique and obvious signs, whereas Macaws may be asymptomatic.
Key words:
Psittacine Beak and Feather Disease, Nested PCR, Prevalence, Leukopenia
Background:
Psittacine Beak and Feather Disease (PBFD) was first described in Australian cockatoos in 1970s [1]. The  disease, caused by a virus, commonly causes clinical  disease in young parrots. The disease occurs in a number of psittacine species including Sulphur-crested Cockatoos, lovebirds, budgerigars, Galahs, cockatiels, African grey parrots, Eclectus parrots, Amazon parrots and macaws. [2,3]. The etiologic agent of PBFD is Beak and feather disease virus (BFDV) [4], which belongs to the family of Circoviridae [5]. It is a single-stranded circular DNA virus with a diameter of 14-17 nm [6].
BFDV is widely spread. The infected individuals can release the virus to the environment through feather powder and feces after months of infection. Birds may be infected by inhaling, drinking water or eating food contaminated with the virus [1,7,8]. Thus, the virus can be easily transmitted to other individuals through contaminated clothing, bird toys, transport boxes, cages and other items. Females can also transmit the virus to their offspring through eggs [9]. Therefore, the morbidity and virus-carrying rates are very high, and it is extremely difficult to eliminate the virus.
The main target of the virus is active cells. When feathers become infected either apoptosis or abnormal hyperplasia of epithelial cells can occur [10], which in turn can lead to abnormal shedding or growth of feathers [11,12]. Changes that can be noted include  retained feather sheaths, blood feathers; and short, curly, deformed feathers, sometimes with a large number of stress bars[1,4,13,14]. The feather abnormalitiesoften worsen upon moulting. Beak abnormalities may also occur, although the changes can vary among species, and not all individuals exhibit abnormal beak [6]. The virus also suppresses the immune system. Most deaths caused by PBFD in clinical practice are due to the secondary infections caused by immunosuppression [6,15]. PBFD has three major clinical presentations, i.e. peracute, acute and chronic. [16]. Cases with peracute and acute phenotypes are most seen in fledging and juvenile birds. Some cases with these forms die suddenly without showing any pre-existing clinical signs. The others present with the main manifestations of leukocyte reduction, anemia, liver necrosis, pneumonia and enteritis caused by immunosuppression. [6,17,18]. Chronic forms are mainly manifested in adult individuals, often characterized by lack of feathers and deformities, and secondary infection. In general, the mortality of PBFD is very high. Current treatments mainly comprise supportive care and treatments to help the affected birds develop immunity against viral infection and secondary infections.
The prevalence of PBFD and infection rate of BFDV have been investigated in captive parrots in many countries and regions. For example, the positive rate of virus is 41.2% in Taiwan [19], 31.3% in Japan, and 39.2% in Germany [20]. However, thus far, no studies have been performed on PBFD prevalence in mainland China. In this study, I collected blood samples from clinical parrot cases to detect BFDV, investigated the infection rate and clinical phenotypes.
Materials and Methods
Samples
All the samples used in this study were left-over blood from clinical examinations, and the pet owners approved the usage of the samples. Dependent on the condition of the bird and the requests by the pet owner, the blood collection was carried out under isoflurane inhalation anesthesia. Blood samples were drawn from the ulnar vein or right jugular vein. About 0.1ml blood was extracted and placed rapidly in the 1.5ml centrifuge tube containing EDTA anticoagulants. After CBC, the rest of the blood was storaged at -20℃.
CBC
PCV and TP were read by capillary centrifugation. The blood smears were Wright-Gimsa stained, following which leucocyte counts and differentiation were performed using a microscope. The blood cells were stained by Natt-Herrick’s stain, and a blood cell counting board was adopted for RBC and WBC counting.
PCR test
DNA was extracted from blood according to manufacturer’s instruction (HIPURE Tissue DNA Kit, Magen). Genome of BFDV was amplified by nested PCR according to previously described methods [22]. PCR products were detected by agarose electrophoresis. Purified PCR amplicons were sequenced by Sanger Sequencing.
Results:
Sample and Phenotype Analysis
This study collected 157 blood samples during 2016-2017 from 26 species of parrots, including 49 African grey parrots, 29 Cockatoos, 24 Macaws, 10 Amazon parrots, eight Eclectus parrots, eight Conures, seven Caiques, six Cockatiels, four Quaker parakeets, three Lovebirds, two Ring-necked parakeets, two Senegal parrots, one Budgerigar, one Lory and one Pacific parrotlet. The ages of the birds ranged from 3 weeks to 11 years.
Through clinical examination and consultation, 43 individuals showed no detectable clinical  signs. The others (114/157) presented with different clinical manifestations. Sixty-nine individuals (43.9%, 69/157) had gastrointestinal tract  signs, including diarrhea, vomit/regurgitation, crop stasis; 19 (12.1%, 19/157) showed respiratory signs, including upper/lower respiratory infections; 29 (18.5%, 29/157) presented with skin, feather, beak and/or claw deformities; and 5 (3.2%, 5/157) individuals showed signs affecting multiple organ systems.
Molecular Detection of BFDV 
Representative DNA bands of PCR amplicons in agarose gel electrophoresis are shown in Figure 1. For negative samples, the first round of PCR showed non-specific amplification bands, and there was no positive band in second round of PCR (Figure 1A). For positive samples, some samples showed a clear 718bp band in first round of PCR (Figure 1B), whereas the others had no target band (Figure 1C). Both the samples showed a clear 359bp target band in second round of PCR. The difference in first round of PCR in positive samples indicated the former might have had higher titers of BFDV. Thus we designated the former as super-positive and the latter as positive.
The second round of PCR products from positive samples were subjected to Sanger Sequencing. The sequences were aligned using BLAST. Alignment proved that sequence of our samples were highly similar to the BDFV genome (Figure 1D). Thus the molecular detection method is applicable and reliable.
A High Prevalence of Beak and Feather Disease In Captive Parrots in China-Complete 
Figure 1 (A-C) Nested PCR detected BDFV in blood samples. (A-C) Representative PCR amplification results are shown in A (Negative), B (Super-Positive) and C (Positive). Left lane: 100 bp Plus ladder, Middle lane: first round of PCR; Right lane: second round of PCR.D: BLAST sequence alignment shows the similarity between PCR product and BDFV genome.
According to the molecular detection, an overall BFDV positive rate was 61.1% (96/157), including 47 positive cases and 49 strong positive cases. The top three species with highest affection rates were African grey parrots (63.3%, 31/49), Cockatoos (65.5%, 19/29), and Macaws (73.1%, 19/26). 
Clinical signs.
There were 24 positive cases that had no clinical signs. Among the 96 PBFD positive cases, nine peracute cases were around 3 months old. All of them died in very short time after blood extraction and examination. All of them were subjected to necropsy, and all of those cases showed liver injury. Forty acute cases were all juvenile around 1 year old. 53 chronic cases are more than 1 year old or Macaws. The result is consistent with previous data [16,17,18].
Species PBFD With clinical signs No
clinical signs
Gastrointestinal tract Respiratory system Skin/feather/beak/claws Leukopenia
African Grey + 45.2% 
(14/31)
12.9% 
(4/31)
22.6% 
(7/31)
71.0% (22/31) 25.8% 
(8/31)
- 33.3% 
(6/18)
16.7% 
(3/18)
33.3% 
(6/18)
0 16.7% 
(3/18)
Cockatoos + 52.6% 
(10/19)
0 36.8% 
(7/19)
52.6% (10/19) 15.8% 
(3/19)
- 40% 
(4/10)
10% 
(1/10)
0 10%
(1/10)
50% 
(5/10)
Macaws + 68.4% 
(13/19)
5.3% 
(1/19)
5.3% 
(1/19)
0 26.3% 
(5/19)
- 28.6% 
(2/7)
14.3% 
(1/7)
0 0 57.1% 
(4/7)
 
Table 1. Clinical signs analysis of top three species groups
I analyzed detailed phenotypes in three major species to explore potential phenotype characters for differential diagnosis. BFDV could cause immunosuppression. As illustrated in Table 1, most African grey parrots (22/31) and cockatoos (10/19) that tested positive for BFDV presented with leukopenia (WBC less than 5000 / uL). Among the positive cases with leukopenia, more than half of the African grey parrots (68.2%, 15/22) and Cockatoos (70%, 7/10)had severely low WBC (less than 1000 / uL). All of these leukopenia samples were detected as strongly positive in nested PCR. In negative cases, none of the African grey parrots, and only one Cockatoo, showed leukopenia. The significant difference between positive and negative cases in these two species indicates that leukopenia may be used as a clinical sign for PBFD. Using leukopenia as an indicator, the sensitivity and specificity was 100% and 70.97% respectively for African grey parrots, and the sensitivity and specificity was 90% and 52.6% respectively for Cockatoos. In addition, skin/feather/beak/claw abnormalities could be used as another indicator for cockatoos. The sensitivity and specificity was 100% and 36.8% respectively. However, there was no significant clinical manifestation in the Macaw positive cases. This may be because Macaws are very resistant to BFDV virus induced disease. The majority of these immunocompetent birds, when exposed to the virus, develop sensitized T & B lymphocytes, develop BFDV specific antibody, and successfully eliminate the viral infection. As a result, these birds generally retest negative for the virus within 90 days post exposure.
Discussion:
Because PBFD positive cases have a variety of clinical signs and manifestations, there often appears to be no specific clinical indicator for clinicians to diagnose the disease. In this study, based on molecular virus detection and phenotype analysis, we found leukopenia to be an important indicator for PBFD infection in African grey parrots and Cockatoos, whereas presense of skin/feather/beak/claws abnormalities could be another indicator for Cockatoos. However, the sample size in this study was relatively small, therefore these indicators should be tested in a greater number of samples and different cohorts. A number of positive cases tested in this study had been tested with negative results in other agencies using nested-PCR. All of the previous tests used feather samples. The false negative results indicated the low level titer of BFDV in the follicles. This finding was consistent with a previous study by Tomasek et al. [22], suggesting that blood samples are a better sample for molecular testing.
The overall positive rate of BFDV was 61.1% (96/157), which is higher than the previous results in Taiwan (41.2%, 68/165) [19]. It is also higher than the overall prevalence reported in other countries (31.3% in Japan, 39.2% in Germany) [20]. This finding may be related to the sample collection bias, since all of the selected samples are clinical samples, some of birds that already showed clinical signs. Nevertheless this study still suggests there is a high prevalence of PBFD in pet birds in mainland, China.
In this study, I could only identified the positive and strong positive cases qualitatively by nested PCR. Real-time PCR will be adopted in the future study to determine and quantify the viral load, which could help to identify environmental exposure to the virus compared to actual progressive infection in birds. In addition, some birds, especially Macaws and other New World Parrots, are very resistant to BFDV. They can develop sensitive immune response to eliminate the virus. Thus, tracking the viral infection status in virus-resistant birds is also a very interesting subject to study. Collecting multiple samples from a bird at different time points to monitor the viral titer will enable us to determine and predict prognosis.
In conclusion, it is recommended that the breeding and trade of birds in mainland China should be under strict population purification, epidemic prevention and control.
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