Pathogenesis of duck circovirus genotype 1 in experimentally infected Pekin ducks

Pathogenesis of duck circovirus genotype 1 in experimentally infected Pekin ducks ABSTRACT Ducks infected with duck circovirus (DuCV) exhibit feathering disorder, growth retardation, and low body weight. The virus can induce immunosuppression and increase rates of infection caused by other pathogens. The purpose of the present study was to investigate the pathogenesis of DuCV in experimentally infected Pekin ducks. At postmortem examination, gross lesions were observed in the immune organs including bursa of Fabricius (BF), thymus, and spleen. Hemorrhage, lymphocytic depletion, necrosis, and degeneration were observed in the bursal tissues by histological examination. The TUNEL assay was performed with bursal tissue. There was a significant difference of the apoptosis rate between the negative and DuCV-infected ducks. The earliest time point for detection of DuCV DNA in sera, cloacal swabs, and organs was 1 wk post-infection (WPI). Viral shedding was persistent and detectable at the end of the experiment (10 WPI). The findings provide evidence that horizontal transmission and persistent infection are the characteristics of DuCV. The organ with the highest mean viral load was the spleen, followed by BF, cecal tonsil, lung, thymus, liver, and kidney. We successfully established an experimental DuCV genotype 1 (DuCV-1) infection in Pekin ducks and demonstrated the pathogenicity and persistence of DuCV-1. In conclusion, DuCV-1 caused extensive damage to the immune organs that may have resulted in immunosuppression. Pathobiological characteristics of DuCV-1 include systemic infection, persistent infection, and horizontal transmission. These features allow DuCV-1 to circulate more easily in farms and increase the susceptibility of ducks to other diseases. INTRODUCTION The genus Circovirus is characterized by small, nonenveloped, spherical, and icosahedral viruses with a circular single-stranded DNA genome (Allan and Ellis, 2000; Todd, 2000). Circoviruses have two major open reading frames (ORF), ORF V1 and ORF C1, which have an opposite orientation. The ORF V1 (ORF1/rep) encodes a putative replication protein, whereas ORF C1 (ORF2/cap) encodes capsid protein (Todd et al., 2001; Hattermann et al., 2003). Another major conserved ORF (ORF3) encodes ORF3 protein, which has apoptotic activity (Liu et al., 2005, 2007; Xiang et al., 2012). Circovirus has been isolated from a wide range of vertebrates, such as mammals (pig, dog, bat, chimpanzee, mink, human), fish (barbel, catfish), and birds (cockatoo, budgerigar, pigeon, goose, duck). Porcine circovirus (PCV) (Hamel et al., 1998), psittacine beak and feather disease virus (Ritchie et al., 1989), pigeon circovirus (PiCV) (Woods et al., 1993; Mankertz et al., 2000), goose circovirus (GoCV) (Soike et al., 1999), and duck circovirus (DuCV) (Hattermann et al., 2003) are included in the genus Circovirus. Circovirus infections are commonly associated with damage of immune organs and immunosuppression (Todd, 2000; Todd et al., 2001). For example, infection with PCV2 causes post-weaning multisystemic wasting syndrome (Hamel et al., 1998), and psittacine beak and feather disease is characterized by a chronic, progressive, and symmetrical feather dystrophy and occasional beak deformity (Pass and Perry, 1984). Duck circovirus has been reported in many countries and the infectious rate is quite high. The prevalence was 46.2% in Germany (Hattermann et al., 2003), 84.2% in Hungary (Fringuelli et al., 2005), 38.2% in Taiwan (Chen et al., 2006), 10 to 81.6% in China (Liu et al., 2009; Wang et al., 2011), and 21.9% in South Korea (Cha et al., 2013). The virus has two genotypes: DuCV genotype 1 (DuCV-1) and DuCV genotype 2 (DuCV-2) (Zhang et al., 2013). Worldwide, there have been more reports of DuCV-1 in Germany, Hungary, the United States, China, South Korea, and Poland compared to DuCV-2 in Taiwan and China. Ducks infected with DuCV have poor growth, marked feather dystrophy, loss of feathers, and hemorrhagic feather shafts (Soike et al., 2004; Chen et al., 2006). In addition, surveys suggest that ducks of a wide age range are susceptible to DuCV (Fringuelli et al., 2005; Wan et al., 2011b) and that DuCV infections are often subclinical (Wan et al., 2011b; Cha et al., 2014). Duck circovirus may induce immunosuppression and predispose ducks to other infectious diseases. Coinfection with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, and duck hepatitis virus has been reported (Fringuelli et al., 2005; Zhang et al., 2009). Gross lesions identified in infected ducks include serositis, cloudy or cheese-like masses within the air sacs, and arthritis (Chen et al., 2006; Banda et al., 2007). Histopathologic examination of the bursa of Fabricius (BF) demonstrated lymphocyte depletion, necrosis, and histiocytosis (Soike et al., 2004). The widespread prevalence of DuCV is an issue of global concern, especially for farmed ducks, because DuCV infection has been reported mostly from farms. Despite the widespread prevalence of DuCV, the pathogenesis is poorly understood. This is due to the absence of cell culture systems for viral propagation, and most studies describe naturally occurring DuCV- cases. Additionally, the pathogenesis of DuCV-1 is less well known than DuCV-2. The only report of experimental infection with DuCV was performed to construct an infectious DuCV-2 DNA clone and compare the clone with parental virus (Li et al., 2015). To our knowledge, there are no reports describing the pathogenesis of DuCV-1 following experimental infection. The purpose of the present study was to investigate the pathogenesis of DuCV-1 in experimentally infected Pekin ducks. MATERIALS AND METHODS Virus The virus used in this study originated from D11-JW-008 (DuCV-1; GenBank No. JQ740363.1). The D11-JW-008 was isolated from 19-day-old Pekin ducks of commercial farm raising meat ducks in Gyeonggi Province, South Korea in 2011. The clinical signs were torticollis, lameness, hyperthermia, and hyperpnea (Cha et al., 2013). The ducks were diagnosed with DuCV infection, colibacillosis, and salmonellosis (data not shown). Spleen and BF homogenates from the affected ducks were clarified by centrifugation. The homogenates were propagated in the Pekin ducks. First passage was done to determine the infectivity of D11-JW-008 and obtain additional DuCV-infected samples. The same procedure was repeated to generate sufficient suspensions with high viral loads by using the passaged inoculum. Spleen and BF were homogenized as 20% suspensions in sterile phosphate-buffered saline (PBS) containing 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) and the suspensions were centrifuged. Viral load of the samples from second passage was 1.71 × 106 copies/mg by real-time quantitative polymerase chain reaction (qPCR), and no other pathogens were detected. The virus from second passage was stored at –70°C until further use. Birds Eighty-three 1-day-old Pekin ducklings were obtained from a commercial farm. The ducklings were confirmed to be free from DuCV and other pathogens. The birds were reared in a positive pressure isolator in an environmentally controlled facility, with food and water provided ad libitum. All birds were weighed at pre-infection, and no birds had clinical signs of illness. At 24 d of age, the birds were randomly divided into 2 groups: a negative control group (n = 29) and a DuCV-infected group (n = 54). All experimental and animal management procedures were undertaken in accordance with the requirements of the Animal Care and Ethics Committee of Chonbuk National University (CBNU2017–0074). The animal facility at Chonbuk National University is fully accredited by the National Association of Laboratory Animal Care. Experimental Design At 24 d of age, the 54 ducks in the DuCV group were infected with a 1-mL suspension containing a virus load equal to 3.4 × 109 copies. The 1-mL suspension was divided in half, 0.5 mL given orally and 0.5 mL given intramuscularly. The 29 ducks in the negative group were inoculated with sterile PBS. Clinical signs were monitored throughout the experimental period. To trace the change of body weight and viral load in sera and cloacal swabs, identification bands were placed on 7 ducks from the negative group and 14 ducks from the DuCV group. The marked ducks were weighed at 1, 2, 4, 6, and 8 weeks post-infection (WPI). Sera and cloacal swabs were obtained at 3 days post-infection (DPI), and at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 WPI. Cloacal swabs were collected into 1-mL PBS tubes and shaken for 1 h. The samples were then centrifuged at low speed and the supernatant stored at –70°C to be later used for extraction of total nucleic acids. To examine the pathological lesions and identification of virus in organs, ducks from each group were sacrificed at 3 DPI and at 1, 2, 3, 4, 6, 8, and 10 WPI. Seven inoculated ducks were killed at each time point, except at 3 DPI, where 5 ducks were killed. Four un-inoculated ducks were killed at each time point, except at 3 DPI, and 8 and 10 WPI, where 3 ducks were killed. All ducks were euthanized by cervical dislocation. Body weight and weights of BF and spleen were individually determined. A portion of the BF was collected and fixed in 10% formaldehyde solution for histopathological examination. Organs were collected for PCR analysis. The BF, spleen, and thymus were individually homogenized and liver, lung, cecal tonsil (CT), kidney, and oviduct were integrally homogenized as 20% suspensions in sterile PBS containing 1% antibiotic-antimycotic, using a Precellys Evolution homogenizer (Bertin Corp, Rockville, MD). The suspensions were centrifuged and the supernatants were stored at –70°C. Evaluation of Clinical Signs and Pathological Lesions During the experimental period, the clinical signs of the ducks as well as any external and internal gross lesions were recorded. On necropsy, gross lesions were observed and scored in the BF, thymus, and spleen. To examine histological changes, fixed bursal tissues were used. The fixed samples were dehydrated and embedded in paraffin. Four-micrometer sections were cut and stained with hematoxylin and eosin. In Situ Apoptosis Detection (TUNEL Assay) In situ apoptosis detection was performed using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The TUNEL assay was performed on sections of each BF from all ducks in both groups sacrificed at 4 and 8 WPI. Apoptotic cells were stained with ApopTag Peroxidase In Situ Apoptosis Detection Kits (EMD Millipore Corporation, Billerica, MA), according to the manufacturer's instructions. The apoptosis rate and prevalence of apoptotic cells versus all cells were calculated using Image Pro-Plus 7.0 software (Media Cybernetics, San Diego, CA) in 5 randomly selected fields at ×100 magnification (×10 objective) in each section. PCR, Real-Time Quantitative PCR, and Identification of Virus in Organs To detect DuCV DNA, PCR was performed on sera, cloacal swabs, and organ homogenates. Total viral DNA was extracted using the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher Scientific, Waltham, MA) and processed on a KingFisher Duo purification system (Thermo Fisher Scientific) following the manufacturer's instructions. The DNA was used to confirm the positive rate of DuCV in PCR assays. The common gene was detected using common DuCV detection primers (408 bp; DuCVaF 5΄-MGA GCT GCC GCC CTT GAG-3΄ and DuCVaR 5΄-TCC CGA GTA ACC GTC CCA CCA C-3΄; Banda et al., 2007). The PCR thermal cycles were 1 cycle of 94°C for 3 min, 35 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 20 s, extension at 72°C for 2 min, and 1 cycle of final extension at 72°C for 7 min. To quantify DuCV DNA in the samples, we used the qPCR method that was established in this laboratory (data not published). A primer set (forward, 5΄-GCA CGC TCG ACA ATT GCA AGT T-3΄; reverse, 5΄-TCA CCA ACT CKM AGG TAT GTC GA-3΄) was designed to amplify a fragment of 217 bp. The final PCR reaction mixture (20 μL) contained 10 μL Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA), 1 μL each primer (each 10 pmol/μL), 2 μL DNA template, and 6 μL distilled water. The thermal profile consisted of 95°C for 3 min, 35 cycles of 95°C for 15 s, and 60°C for 20 s with 2-step. The qPCR was performed using Mx3000P (Stratagene, La Jolla, CA). The results of the qPCR were expressed as the logarithm of the copies of the DuCV genome per milligram (log copies/mg) or microliter (log copies/μL) of organ, serum, and cloacal swab. Statistical Analysis The data of weight percent gain, bursa to body weight (b/B) ratio, spleen to body weight (s/B) ratio, and apoptosis rate were analyzed by Student t-test using SPSS 23 software (SPSS, Chicago, IL). P values of < 0.05 were considered statistically significant. RESULTS Clinical Signs All ducks in both groups remained clinically normal throughout the experimental period. The weight percent gain was comparatively lower in the DuCV group than in the negative group (except at 4 WPI), although differences were not significant (Table 1). Table 1. Comparison of weight gain and gross lesions. Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 1Weight percent gain (%) = [(final body weight—pre-infection body weight)/pre-infection body weight] × 100. Mean ± SD; ND, Not done. 2Bursa/body weight (b/B) and spleen/body weight (s/B) ratio = (organ (g)/body weight ratio (g)) × 1000. Mean ± SD 3DPI, days post-infection; WPI, weeks post-infection. 4–, not shown; +, mild; + +, moderate; +++, severe for gross lesions (number of ducks observed gross lesions/no. of ducks sacrificed). View Large Table 1. Comparison of weight gain and gross lesions. Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 1Weight percent gain (%) = [(final body weight—pre-infection body weight)/pre-infection body weight] × 100. Mean ± SD; ND, Not done. 2Bursa/body weight (b/B) and spleen/body weight (s/B) ratio = (organ (g)/body weight ratio (g)) × 1000. Mean ± SD 3DPI, days post-infection; WPI, weeks post-infection. 4–, not shown; +, mild; + +, moderate; +++, severe for gross lesions (number of ducks observed gross lesions/no. of ducks sacrificed). View Large Gross Lesions and Organ-to-Body Weight Ratio (b/B and s/B Ratio) At postmortem examination, gross lesions were observed in the BF, thymus, and spleen of the DuCV group (Table 1). The BF showed swelling and hemorrhage throughout the experimental period. Mild hemorrhage in BF was observed at 1 WPI. As the time post-infection increased, the severity and number of hemorrhages increased and were highest and most common (71.4%) at 10 WPI. Gross lesions of the thymus, hemorrhage, or atrophy were detected at 1 to 3 WPI. A few spleens were enlarged and appeared mottled in color varying white to red except at 1, 3, and 4 WPI. The b/B and s/B ratio were recorded at the postmortem (Table 1). The b/B ratio was higher in the DuCV group than in the negative group from 3 WPI and the s/B ratio in the DuCV group was higher at 3 DPI, 2 WPI, 8 WPI, and 10 WPI. No significant differences were detected between groups. Histology Similar to the gross findings, the bursal tissues of ducks in the DuCV group showed mild hemorrhage from 1 WPI. Lymphocytic depletion, necrosis, and degeneration were also observed. The severity and proportion of the lesions increased with the time post-infection. In addition, collapse and disappearance of follicle architecture was occasionally identified in the BF (Figure 1). No abnormalities were detected in ducks of the negative group (Figure 1a). Figure 1. View largeDownload slide Histopathological features of bursal tissue at 8 WPI. (a) Normal in the negative group, (b) depletion of lymphocytes and degeneration (arrow), (c) severe hemorrhage (arrow), and (d) necrosis (arrow) in the DuCV group. (a) and (b) ×200 magnification. (c) and (d) ×100 magnification. Hematoxylin and eosin stain. DuCV = duck circovirus; WPI = weeks post-infection. Figure 1. View largeDownload slide Histopathological features of bursal tissue at 8 WPI. (a) Normal in the negative group, (b) depletion of lymphocytes and degeneration (arrow), (c) severe hemorrhage (arrow), and (d) necrosis (arrow) in the DuCV group. (a) and (b) ×200 magnification. (c) and (d) ×100 magnification. Hematoxylin and eosin stain. DuCV = duck circovirus; WPI = weeks post-infection. Apoptosis Rate Apoptosis in the bursal tissues from all ducks sacrificed at 4 and 8 WPI was detected by the TUNEL assay. In both groups, there were more apoptotic cells in the medulla of follicles and follicle-associated epithelium than in the cortex of follicles. However, compared with the negative group, relatively more apoptotic cells and higher signal strength were detected in the DuCV group (Figure 2). The bursal tissues in the DuCV group showed more than 1.5 times higher apoptosis rate. When compared to the negative ducks, the apoptosis rate was significantly higher in the infected ducks at 4 WPI (*P<0.05) and 8 WPI (**P<0.01) (Figure 2d). Figure 2. View largeDownload slide In situ apoptosis of the bursal tissue detected by the TUNEL method. High magnification (×400) of the bursal tissue from the DuCV group at (a) 4 WPI, (b) 8 WPI, and (c) negative group. Proportion of apoptotic cells per total cells (d; *P < 0.05, **P < 0.01). DuCV = duck circovirus; WPI = weeks post-infection. Figure 2. View largeDownload slide In situ apoptosis of the bursal tissue detected by the TUNEL method. High magnification (×400) of the bursal tissue from the DuCV group at (a) 4 WPI, (b) 8 WPI, and (c) negative group. Proportion of apoptotic cells per total cells (d; *P < 0.05, **P < 0.01). DuCV = duck circovirus; WPI = weeks post-infection. PCR Analysis and Quantification Duck circovirus DNA was detected in sera, cloacal swabs, and organs from the DuCV group but it was not detected in the samples and organs obtained from ducks of the negative group. Ducks in the DuCV group showed viremia at 1 and 2 WPI, and the positive rates were 50 and 28.6%, respectively (Table 2). The viral load was 3.44 × 103 copies/μL at 1 WPI, and decreased to 3.79 × 102 copies/μL at 2 WPI. Viral DNA was detected in cloacal swabs from 1 to 10 WPI. The highest positive rate was 92.6% from 3 to 7 WPI, and then decreased to 42.8% (Table 2). The qPCR results showed that the viral loads were similar at each period with more than 104 copies/μL. The highest viral load in various organs identified was 1.74 × 107 copies/mg at 3 WPI in the spleen (Table 3). From 1 to 10 WPI, the mean viral loads were approximately 106 copies/mg in the spleen and BF; 105 copies/mg in the CT, lung, and thymus; and 104 copies/mg in the liver and kidney. The positive rate was high in the immune organs, such as BF, spleen, and thymus, from 1 to 10 WPI. The positive rate of the thymus was 57.1% at 1 WPI; increased to 100% at 3 WPI; and decreased to 85.7% at 8 WPI and 71.4% at 10 WPI. In contrast, the positive rate of the BF peaked at 2 WPI and persistent to 10 WPI. Viral DNA was not detected from oviduct (Table 3). Table 2. Positive rate and quantification of DuCV DNA in serum and cloacal swabs from the DuCV-infected ducks. Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ 1DPI, days post-infection; WPI, weeks post-infection. 2Positive rate (%) (Number of positive ducks/no. of live ducks identified by band). 3–, not detected; +, < 103 copies/μL; ++, 103 to 106 copies/μL; +++, > 106 copies/μL. View Large Table 2. Positive rate and quantification of DuCV DNA in serum and cloacal swabs from the DuCV-infected ducks. Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ 1DPI, days post-infection; WPI, weeks post-infection. 2Positive rate (%) (Number of positive ducks/no. of live ducks identified by band). 3–, not detected; +, < 103 copies/μL; ++, 103 to 106 copies/μL; +++, > 106 copies/μL. View Large Table 3. Quantification of DuCV DNA and viral positive rate in DuCV-infected ducks. Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) 1DPI, days post-infection; WPI, weeks post-infection. 2–, no detected; +, < 103 copies/mg; ++, 103 - 106 copies/mg; +++, > 106 copies/mg; NA, not applicable. 3Positive rate (%) (number of positive ducks/no of sacrificed ducks). View Large Table 3. Quantification of DuCV DNA and viral positive rate in DuCV-infected ducks. Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) 1DPI, days post-infection; WPI, weeks post-infection. 2–, no detected; +, < 103 copies/mg; ++, 103 - 106 copies/mg; +++, > 106 copies/mg; NA, not applicable. 3Positive rate (%) (number of positive ducks/no of sacrificed ducks). View Large DISCUSSION We successfully established an experimental DuCV-1 infection in Pekin ducks and demonstrated the pathogenicity and persistence of DuCV-1 following experimental infection. On postmortem examination, gross lesions were observed in the immune organs including BF, thymus, and spleen (Table 1). In inoculated geese, Guo et al. (2011) reported hemorrhage in the thymus and BF, and splenomegaly in their GoCV experimental model. However, the size of thymus described by Guo et al. (2011) was in contrast with ours. In our experiment, there were also histopathologic lesions in the BF (Figure 1). Our findings are similar to results of experimental infection with other avian circoviruses. Lymphocytic depletion and necrosis in the BF were observed in the PiCV and GoCV experimental infection, respectively (Schmidt et al., 2008; Guo et al., 2011). To detect apoptosis activated by ORF3, the TUNEL assay was performed with bursal tissue (Figure 2). There was a significant difference of the apoptosis rate between the negative and DuCV group. Guo et al. (2011) also reported that apoptotic cells in age-matched GoCV-inoculated geese increased significantly compared with uninoculated geese. These findings suggest that DuCV damages immune organs, especially the BF, resulting in immunosuppression. The level of immunosuppression remains unclear and should be studied. The earliest time point for detection of DuCV DNA in sera, cloacal swabs, and organs was 1 WPI (Tables 2 and 3). These data indicate that DuCV infection may induce a systemic infection after the virus spreads through the blood circulation system. Viremia was also detected at 2 WPI (Table 2). Similarly, viremia was previously detected by experimental infection with DuCV-2 and other avian circoviruses (Schmidt et al., 2008; Guo et al., 2011; Li et al., 2015). Li et al. (2015) reported that the duration of viremia was from 15 to 28 d post-challenge in ducks inoculated with DuCV-2. Our results suggest that ducks infected with DuCV-1 may show a similar period of viremia without a secondary viremia, even though there are differences related to experimental design, virus strains, and genotypes of DuCV. Given the lack of experimental studies of DuCV infection, more experimental research is required to prove the pathogenic differences related to various strains and genotypes in DuCV. Ducks began to shed the virus at 1 WPI, and viral shedding was persistent and still detectable at the end of the experiment (10 WPI) (Table 2). During that time, the positive rate was more than 42.8% and the viral loads from cloacal swabs exceeded 104 copies/μL. In a preliminary experiment, viral shedding was observed to 16 WPI (data not shown). The findings provide evidence that horizontal transmission and persistent infection are characteristics of the DuCV-1. This could increase the risk of DuCV-1 re-infection in duck farms. In the present investigation, organ viral loads peaked at 2 and 3 WPI (Table 3). The organ with the highest mean viral load was the spleen, followed by BF, CT, lung, thymus, liver, and kidney. However, Li et al. (2015) reported that the organ with the highest mean viral load was BF, followed by liver and kidney, which showed higher viral loads and positive rates than the spleen. This indicates that there are differences in organ tropism related to various strains and genotypes of DuCV. In our experiment, the number of copies of the DuCV DNA was high in the secondary immune organs, the CT and spleen. The viral load of Harderian gland, also a secondary immune organ, is reported to be similar to the viral load of the thymus (Li et al., 2015). These results indicate that DuCV has tropism for all immune organs. Viral DNA was detected in most of the examined organs until the end of the experiment (10 WPI), and in a preliminary experiment the spleen from a duck infected with DuCV was PCR-positive at 27 WPI (data not shown). In the absence of an in vitro cell culture method for the isolation of DuCV, PCR is often used for diagnosis (Banda et al., 2007; Fu et al., 2011; Wan et al., 2011a). Selection of optimal samples is needed for detection of DuCV DNA by PCR. In this study, cloacal swabs showed higher positive rate and viral loads than the sera (Table 2). The BF was the organ of the highest positive rate, and the spleen contained the highest viral loads (Table 3). These results indicate that both are major organs for diagnosis of DuCV from a carcass. Since the BF begins to regress at about 12 wk of age (Gille and Salomon, 1999), and ducks of varying ages are susceptible to DuCV (Wan et al., 2011b; Cha et al., 2013), the spleen may a better specimen than the BF for diagnosis. We suggest that cloacal swabs are better than sera for samples to detect DuCV-1 in live ducks, and that the spleen is the principal organ for the diagnosis of DuCV-1 in carcasses. Furthermore, our data showed that for the detection of viremia in early-stage infections, testing a serum sample is more useful than the other samples. Despite successful experimental DuCV-1 infection, clinical signs were not observed in DuCV group. These findings differ from previous reports of naturally infected cases (Soike et al., 2004; Chen et al., 2006; Banda et al., 2007). Li et al. (2015) reported that 10-d ducklings inoculated with DuCV-2 showed clinical symptoms that included wasting, feathering disorder, and low average daily weight gain. It is conceivable that development of clinical signs is involved in the time of challenge with DuCV as well as various strains and genotypes of DuCV. In conclusion, the results of this study demonstrate that DuCV-1 can induce immunosuppression by causing extensive damage to immune organs. Pathobiological characteristics of DuCV-1 include systemic infection, persistent infection, and horizontal transmission. Considering these findings and the demonstrated environmental stability of Circovirus (Allan et al., 1994; Raidal and Cross, 1994; Yilmaz and Kaleta, 2004), prolonged exposure to DuCV-1 may result in re-infection. We suggest that these features allow DuCV-1 to more easily circulate in farms and increase the susceptibility of ducks to other diseases. Despite the threat of coinfection by DuCV-1, the severity and virulence of coinfection have not been proven. Further experimental studies are needed to investigate the risk of coinfection and level of economic losses to duck farms. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Supplementary Figure S1. Gross lesions of the bursa of Fabricius (BF) and thymus in the negative and DuCV groups. The BF at 4 WPI (a, b) and 10 WPI (c, d) in the negative (a, c) and DuCV (b, d) groups. The thymus at 2 WPI in the negative (e) and DuCV (f) groups. Bar = 3 cm. DuCV = duck circovirus; WPI = weeks post-infection. ACKNOWLEDGMENTS This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (316039–3, 317009–3, 716002–7) and Research of Animal and Plant Quarantine Agency, South Korea. REFERENCES Allan G.M. , Ellis J.A. . 2000 . Porcine circoviruses: a review . J. Vet. Diagn. Invest. 12 : 3 – 14 . Google Scholar CrossRef Search ADS PubMed Allan G.M. , Phenix K.V. , Todd D. , McNulty M.S. . 1994 . Some biological and physico-chemical properties of porcine circovirus . Zentralbl. Veterinarmed. B. 41 : 17 – 26 . Google Scholar PubMed Banda A. , Galloway-Haskins R.I. , Sandhu T.S. , Schat K.A. . 2007 . 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An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China . Vet. Microbiol. 133 : 252 – 256 . Google Scholar CrossRef Search ADS PubMed Zhang Z. , Jia R. , Lu Y. , Wang M. , Zhu D. , Chen S. , Yin Z. , Chen X. , Cheng A. . 2013 . Identification, genotyping, and molecular evolution analysis of duck circovirus . Gene . 529 : 288 – 295 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Pathogenesis of duck circovirus genotype 1 in experimentally infected Pekin ducks

Poultry Science , Volume 97 (9) – Sep 1, 2018

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey177
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Abstract

ABSTRACT Ducks infected with duck circovirus (DuCV) exhibit feathering disorder, growth retardation, and low body weight. The virus can induce immunosuppression and increase rates of infection caused by other pathogens. The purpose of the present study was to investigate the pathogenesis of DuCV in experimentally infected Pekin ducks. At postmortem examination, gross lesions were observed in the immune organs including bursa of Fabricius (BF), thymus, and spleen. Hemorrhage, lymphocytic depletion, necrosis, and degeneration were observed in the bursal tissues by histological examination. The TUNEL assay was performed with bursal tissue. There was a significant difference of the apoptosis rate between the negative and DuCV-infected ducks. The earliest time point for detection of DuCV DNA in sera, cloacal swabs, and organs was 1 wk post-infection (WPI). Viral shedding was persistent and detectable at the end of the experiment (10 WPI). The findings provide evidence that horizontal transmission and persistent infection are the characteristics of DuCV. The organ with the highest mean viral load was the spleen, followed by BF, cecal tonsil, lung, thymus, liver, and kidney. We successfully established an experimental DuCV genotype 1 (DuCV-1) infection in Pekin ducks and demonstrated the pathogenicity and persistence of DuCV-1. In conclusion, DuCV-1 caused extensive damage to the immune organs that may have resulted in immunosuppression. Pathobiological characteristics of DuCV-1 include systemic infection, persistent infection, and horizontal transmission. These features allow DuCV-1 to circulate more easily in farms and increase the susceptibility of ducks to other diseases. INTRODUCTION The genus Circovirus is characterized by small, nonenveloped, spherical, and icosahedral viruses with a circular single-stranded DNA genome (Allan and Ellis, 2000; Todd, 2000). Circoviruses have two major open reading frames (ORF), ORF V1 and ORF C1, which have an opposite orientation. The ORF V1 (ORF1/rep) encodes a putative replication protein, whereas ORF C1 (ORF2/cap) encodes capsid protein (Todd et al., 2001; Hattermann et al., 2003). Another major conserved ORF (ORF3) encodes ORF3 protein, which has apoptotic activity (Liu et al., 2005, 2007; Xiang et al., 2012). Circovirus has been isolated from a wide range of vertebrates, such as mammals (pig, dog, bat, chimpanzee, mink, human), fish (barbel, catfish), and birds (cockatoo, budgerigar, pigeon, goose, duck). Porcine circovirus (PCV) (Hamel et al., 1998), psittacine beak and feather disease virus (Ritchie et al., 1989), pigeon circovirus (PiCV) (Woods et al., 1993; Mankertz et al., 2000), goose circovirus (GoCV) (Soike et al., 1999), and duck circovirus (DuCV) (Hattermann et al., 2003) are included in the genus Circovirus. Circovirus infections are commonly associated with damage of immune organs and immunosuppression (Todd, 2000; Todd et al., 2001). For example, infection with PCV2 causes post-weaning multisystemic wasting syndrome (Hamel et al., 1998), and psittacine beak and feather disease is characterized by a chronic, progressive, and symmetrical feather dystrophy and occasional beak deformity (Pass and Perry, 1984). Duck circovirus has been reported in many countries and the infectious rate is quite high. The prevalence was 46.2% in Germany (Hattermann et al., 2003), 84.2% in Hungary (Fringuelli et al., 2005), 38.2% in Taiwan (Chen et al., 2006), 10 to 81.6% in China (Liu et al., 2009; Wang et al., 2011), and 21.9% in South Korea (Cha et al., 2013). The virus has two genotypes: DuCV genotype 1 (DuCV-1) and DuCV genotype 2 (DuCV-2) (Zhang et al., 2013). Worldwide, there have been more reports of DuCV-1 in Germany, Hungary, the United States, China, South Korea, and Poland compared to DuCV-2 in Taiwan and China. Ducks infected with DuCV have poor growth, marked feather dystrophy, loss of feathers, and hemorrhagic feather shafts (Soike et al., 2004; Chen et al., 2006). In addition, surveys suggest that ducks of a wide age range are susceptible to DuCV (Fringuelli et al., 2005; Wan et al., 2011b) and that DuCV infections are often subclinical (Wan et al., 2011b; Cha et al., 2014). Duck circovirus may induce immunosuppression and predispose ducks to other infectious diseases. Coinfection with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, and duck hepatitis virus has been reported (Fringuelli et al., 2005; Zhang et al., 2009). Gross lesions identified in infected ducks include serositis, cloudy or cheese-like masses within the air sacs, and arthritis (Chen et al., 2006; Banda et al., 2007). Histopathologic examination of the bursa of Fabricius (BF) demonstrated lymphocyte depletion, necrosis, and histiocytosis (Soike et al., 2004). The widespread prevalence of DuCV is an issue of global concern, especially for farmed ducks, because DuCV infection has been reported mostly from farms. Despite the widespread prevalence of DuCV, the pathogenesis is poorly understood. This is due to the absence of cell culture systems for viral propagation, and most studies describe naturally occurring DuCV- cases. Additionally, the pathogenesis of DuCV-1 is less well known than DuCV-2. The only report of experimental infection with DuCV was performed to construct an infectious DuCV-2 DNA clone and compare the clone with parental virus (Li et al., 2015). To our knowledge, there are no reports describing the pathogenesis of DuCV-1 following experimental infection. The purpose of the present study was to investigate the pathogenesis of DuCV-1 in experimentally infected Pekin ducks. MATERIALS AND METHODS Virus The virus used in this study originated from D11-JW-008 (DuCV-1; GenBank No. JQ740363.1). The D11-JW-008 was isolated from 19-day-old Pekin ducks of commercial farm raising meat ducks in Gyeonggi Province, South Korea in 2011. The clinical signs were torticollis, lameness, hyperthermia, and hyperpnea (Cha et al., 2013). The ducks were diagnosed with DuCV infection, colibacillosis, and salmonellosis (data not shown). Spleen and BF homogenates from the affected ducks were clarified by centrifugation. The homogenates were propagated in the Pekin ducks. First passage was done to determine the infectivity of D11-JW-008 and obtain additional DuCV-infected samples. The same procedure was repeated to generate sufficient suspensions with high viral loads by using the passaged inoculum. Spleen and BF were homogenized as 20% suspensions in sterile phosphate-buffered saline (PBS) containing 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) and the suspensions were centrifuged. Viral load of the samples from second passage was 1.71 × 106 copies/mg by real-time quantitative polymerase chain reaction (qPCR), and no other pathogens were detected. The virus from second passage was stored at –70°C until further use. Birds Eighty-three 1-day-old Pekin ducklings were obtained from a commercial farm. The ducklings were confirmed to be free from DuCV and other pathogens. The birds were reared in a positive pressure isolator in an environmentally controlled facility, with food and water provided ad libitum. All birds were weighed at pre-infection, and no birds had clinical signs of illness. At 24 d of age, the birds were randomly divided into 2 groups: a negative control group (n = 29) and a DuCV-infected group (n = 54). All experimental and animal management procedures were undertaken in accordance with the requirements of the Animal Care and Ethics Committee of Chonbuk National University (CBNU2017–0074). The animal facility at Chonbuk National University is fully accredited by the National Association of Laboratory Animal Care. Experimental Design At 24 d of age, the 54 ducks in the DuCV group were infected with a 1-mL suspension containing a virus load equal to 3.4 × 109 copies. The 1-mL suspension was divided in half, 0.5 mL given orally and 0.5 mL given intramuscularly. The 29 ducks in the negative group were inoculated with sterile PBS. Clinical signs were monitored throughout the experimental period. To trace the change of body weight and viral load in sera and cloacal swabs, identification bands were placed on 7 ducks from the negative group and 14 ducks from the DuCV group. The marked ducks were weighed at 1, 2, 4, 6, and 8 weeks post-infection (WPI). Sera and cloacal swabs were obtained at 3 days post-infection (DPI), and at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 WPI. Cloacal swabs were collected into 1-mL PBS tubes and shaken for 1 h. The samples were then centrifuged at low speed and the supernatant stored at –70°C to be later used for extraction of total nucleic acids. To examine the pathological lesions and identification of virus in organs, ducks from each group were sacrificed at 3 DPI and at 1, 2, 3, 4, 6, 8, and 10 WPI. Seven inoculated ducks were killed at each time point, except at 3 DPI, where 5 ducks were killed. Four un-inoculated ducks were killed at each time point, except at 3 DPI, and 8 and 10 WPI, where 3 ducks were killed. All ducks were euthanized by cervical dislocation. Body weight and weights of BF and spleen were individually determined. A portion of the BF was collected and fixed in 10% formaldehyde solution for histopathological examination. Organs were collected for PCR analysis. The BF, spleen, and thymus were individually homogenized and liver, lung, cecal tonsil (CT), kidney, and oviduct were integrally homogenized as 20% suspensions in sterile PBS containing 1% antibiotic-antimycotic, using a Precellys Evolution homogenizer (Bertin Corp, Rockville, MD). The suspensions were centrifuged and the supernatants were stored at –70°C. Evaluation of Clinical Signs and Pathological Lesions During the experimental period, the clinical signs of the ducks as well as any external and internal gross lesions were recorded. On necropsy, gross lesions were observed and scored in the BF, thymus, and spleen. To examine histological changes, fixed bursal tissues were used. The fixed samples were dehydrated and embedded in paraffin. Four-micrometer sections were cut and stained with hematoxylin and eosin. In Situ Apoptosis Detection (TUNEL Assay) In situ apoptosis detection was performed using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The TUNEL assay was performed on sections of each BF from all ducks in both groups sacrificed at 4 and 8 WPI. Apoptotic cells were stained with ApopTag Peroxidase In Situ Apoptosis Detection Kits (EMD Millipore Corporation, Billerica, MA), according to the manufacturer's instructions. The apoptosis rate and prevalence of apoptotic cells versus all cells were calculated using Image Pro-Plus 7.0 software (Media Cybernetics, San Diego, CA) in 5 randomly selected fields at ×100 magnification (×10 objective) in each section. PCR, Real-Time Quantitative PCR, and Identification of Virus in Organs To detect DuCV DNA, PCR was performed on sera, cloacal swabs, and organ homogenates. Total viral DNA was extracted using the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher Scientific, Waltham, MA) and processed on a KingFisher Duo purification system (Thermo Fisher Scientific) following the manufacturer's instructions. The DNA was used to confirm the positive rate of DuCV in PCR assays. The common gene was detected using common DuCV detection primers (408 bp; DuCVaF 5΄-MGA GCT GCC GCC CTT GAG-3΄ and DuCVaR 5΄-TCC CGA GTA ACC GTC CCA CCA C-3΄; Banda et al., 2007). The PCR thermal cycles were 1 cycle of 94°C for 3 min, 35 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 20 s, extension at 72°C for 2 min, and 1 cycle of final extension at 72°C for 7 min. To quantify DuCV DNA in the samples, we used the qPCR method that was established in this laboratory (data not published). A primer set (forward, 5΄-GCA CGC TCG ACA ATT GCA AGT T-3΄; reverse, 5΄-TCA CCA ACT CKM AGG TAT GTC GA-3΄) was designed to amplify a fragment of 217 bp. The final PCR reaction mixture (20 μL) contained 10 μL Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA), 1 μL each primer (each 10 pmol/μL), 2 μL DNA template, and 6 μL distilled water. The thermal profile consisted of 95°C for 3 min, 35 cycles of 95°C for 15 s, and 60°C for 20 s with 2-step. The qPCR was performed using Mx3000P (Stratagene, La Jolla, CA). The results of the qPCR were expressed as the logarithm of the copies of the DuCV genome per milligram (log copies/mg) or microliter (log copies/μL) of organ, serum, and cloacal swab. Statistical Analysis The data of weight percent gain, bursa to body weight (b/B) ratio, spleen to body weight (s/B) ratio, and apoptosis rate were analyzed by Student t-test using SPSS 23 software (SPSS, Chicago, IL). P values of < 0.05 were considered statistically significant. RESULTS Clinical Signs All ducks in both groups remained clinically normal throughout the experimental period. The weight percent gain was comparatively lower in the DuCV group than in the negative group (except at 4 WPI), although differences were not significant (Table 1). Table 1. Comparison of weight gain and gross lesions. Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 1Weight percent gain (%) = [(final body weight—pre-infection body weight)/pre-infection body weight] × 100. Mean ± SD; ND, Not done. 2Bursa/body weight (b/B) and spleen/body weight (s/B) ratio = (organ (g)/body weight ratio (g)) × 1000. Mean ± SD 3DPI, days post-infection; WPI, weeks post-infection. 4–, not shown; +, mild; + +, moderate; +++, severe for gross lesions (number of ducks observed gross lesions/no. of ducks sacrificed). View Large Table 1. Comparison of weight gain and gross lesions. Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 Weight percent gain1 Bursa of Fabricius Thymus Spleen b/B ratio2 s/B ratio2 Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV Neg DuCV 3 DPI3 ND ND –(0/3)4 –(0/5) –(0/3) –(0/5) –(0/3) +(1/5) 1.47 ± 0.37 1.32 ± 0.32 1.13 ± 0. 21 1.15 ± 0.45 1 WPI 223.9 ± 35.6 219.7 ± 23.7 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) –(0/7) 1.13 ± 0.36 1.00 ± 0.32 0.85 ± 0.18 0.78 ± 0.18 2 WPI 329.1 ± 38.2 316.9 ± 35.1 –(0/4) +(1/7) –(0/4) ++ (3/7) –(0/4) ++ (2/7) 0.95 ± 0.18 0.95 ± 0.22 0.84 ± 0.29 1.16 ± 0.48 3 WPI ND ND –(0/4) –(0/7) –(0/4) +++ (1/7) –(0/4) –(0/7) 0.86 ± 0.29 1.02 ± 0.34 1.10 ± 0.33 0.90 ± 0.24 4 WPI 397.2 ± 60.7 397.7 ± 56.5 –(0/4) ++ (4/7) –(0/4) –(0/7) –(0/4) –(0/7) 0.72 ± 0.12 0.81 ± 0.16 0.84 ± 0.17 0.83 ± 0.10 6 WPI 444.1 ± 65.6 405.0 ± 66.9 –(0/4) ++ (3/7) –(0/4) –(0/7) –(0/4) +(2/7) 0.58 ± 0.08 0.73 ± 0.21 0.86 ± 0.16 0.81 ± 0.50 8 WPI 434.3 ± 78.7 402.3 ± 55.3 –(0/3) +++ (4/7) –(0/3) –(0/7) –(0/3) ++(1/7) 0.46 ± 0.18 0.83 ± 0.48 0.78 ± 0.12 1.00 ± 0.30 10 WPI ND ND –(0/3) +++ (5/7) –(0/3) –(0/7) –(0/3) +(1/7) 0.20 ± 0.12 0.45 ± 0.22 0.78 ± 0.41 0.95 ± 0.31 1Weight percent gain (%) = [(final body weight—pre-infection body weight)/pre-infection body weight] × 100. Mean ± SD; ND, Not done. 2Bursa/body weight (b/B) and spleen/body weight (s/B) ratio = (organ (g)/body weight ratio (g)) × 1000. Mean ± SD 3DPI, days post-infection; WPI, weeks post-infection. 4–, not shown; +, mild; + +, moderate; +++, severe for gross lesions (number of ducks observed gross lesions/no. of ducks sacrificed). View Large Gross Lesions and Organ-to-Body Weight Ratio (b/B and s/B Ratio) At postmortem examination, gross lesions were observed in the BF, thymus, and spleen of the DuCV group (Table 1). The BF showed swelling and hemorrhage throughout the experimental period. Mild hemorrhage in BF was observed at 1 WPI. As the time post-infection increased, the severity and number of hemorrhages increased and were highest and most common (71.4%) at 10 WPI. Gross lesions of the thymus, hemorrhage, or atrophy were detected at 1 to 3 WPI. A few spleens were enlarged and appeared mottled in color varying white to red except at 1, 3, and 4 WPI. The b/B and s/B ratio were recorded at the postmortem (Table 1). The b/B ratio was higher in the DuCV group than in the negative group from 3 WPI and the s/B ratio in the DuCV group was higher at 3 DPI, 2 WPI, 8 WPI, and 10 WPI. No significant differences were detected between groups. Histology Similar to the gross findings, the bursal tissues of ducks in the DuCV group showed mild hemorrhage from 1 WPI. Lymphocytic depletion, necrosis, and degeneration were also observed. The severity and proportion of the lesions increased with the time post-infection. In addition, collapse and disappearance of follicle architecture was occasionally identified in the BF (Figure 1). No abnormalities were detected in ducks of the negative group (Figure 1a). Figure 1. View largeDownload slide Histopathological features of bursal tissue at 8 WPI. (a) Normal in the negative group, (b) depletion of lymphocytes and degeneration (arrow), (c) severe hemorrhage (arrow), and (d) necrosis (arrow) in the DuCV group. (a) and (b) ×200 magnification. (c) and (d) ×100 magnification. Hematoxylin and eosin stain. DuCV = duck circovirus; WPI = weeks post-infection. Figure 1. View largeDownload slide Histopathological features of bursal tissue at 8 WPI. (a) Normal in the negative group, (b) depletion of lymphocytes and degeneration (arrow), (c) severe hemorrhage (arrow), and (d) necrosis (arrow) in the DuCV group. (a) and (b) ×200 magnification. (c) and (d) ×100 magnification. Hematoxylin and eosin stain. DuCV = duck circovirus; WPI = weeks post-infection. Apoptosis Rate Apoptosis in the bursal tissues from all ducks sacrificed at 4 and 8 WPI was detected by the TUNEL assay. In both groups, there were more apoptotic cells in the medulla of follicles and follicle-associated epithelium than in the cortex of follicles. However, compared with the negative group, relatively more apoptotic cells and higher signal strength were detected in the DuCV group (Figure 2). The bursal tissues in the DuCV group showed more than 1.5 times higher apoptosis rate. When compared to the negative ducks, the apoptosis rate was significantly higher in the infected ducks at 4 WPI (*P<0.05) and 8 WPI (**P<0.01) (Figure 2d). Figure 2. View largeDownload slide In situ apoptosis of the bursal tissue detected by the TUNEL method. High magnification (×400) of the bursal tissue from the DuCV group at (a) 4 WPI, (b) 8 WPI, and (c) negative group. Proportion of apoptotic cells per total cells (d; *P < 0.05, **P < 0.01). DuCV = duck circovirus; WPI = weeks post-infection. Figure 2. View largeDownload slide In situ apoptosis of the bursal tissue detected by the TUNEL method. High magnification (×400) of the bursal tissue from the DuCV group at (a) 4 WPI, (b) 8 WPI, and (c) negative group. Proportion of apoptotic cells per total cells (d; *P < 0.05, **P < 0.01). DuCV = duck circovirus; WPI = weeks post-infection. PCR Analysis and Quantification Duck circovirus DNA was detected in sera, cloacal swabs, and organs from the DuCV group but it was not detected in the samples and organs obtained from ducks of the negative group. Ducks in the DuCV group showed viremia at 1 and 2 WPI, and the positive rates were 50 and 28.6%, respectively (Table 2). The viral load was 3.44 × 103 copies/μL at 1 WPI, and decreased to 3.79 × 102 copies/μL at 2 WPI. Viral DNA was detected in cloacal swabs from 1 to 10 WPI. The highest positive rate was 92.6% from 3 to 7 WPI, and then decreased to 42.8% (Table 2). The qPCR results showed that the viral loads were similar at each period with more than 104 copies/μL. The highest viral load in various organs identified was 1.74 × 107 copies/mg at 3 WPI in the spleen (Table 3). From 1 to 10 WPI, the mean viral loads were approximately 106 copies/mg in the spleen and BF; 105 copies/mg in the CT, lung, and thymus; and 104 copies/mg in the liver and kidney. The positive rate was high in the immune organs, such as BF, spleen, and thymus, from 1 to 10 WPI. The positive rate of the thymus was 57.1% at 1 WPI; increased to 100% at 3 WPI; and decreased to 85.7% at 8 WPI and 71.4% at 10 WPI. In contrast, the positive rate of the BF peaked at 2 WPI and persistent to 10 WPI. Viral DNA was not detected from oviduct (Table 3). Table 2. Positive rate and quantification of DuCV DNA in serum and cloacal swabs from the DuCV-infected ducks. Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ 1DPI, days post-infection; WPI, weeks post-infection. 2Positive rate (%) (Number of positive ducks/no. of live ducks identified by band). 3–, not detected; +, < 103 copies/μL; ++, 103 to 106 copies/μL; +++, > 106 copies/μL. View Large Table 2. Positive rate and quantification of DuCV DNA in serum and cloacal swabs from the DuCV-infected ducks. Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ Serum Cloacal swab Positive rate Viral load Positive rate Viral load 3 DPI1 0 (0/14)2 –3 0 (0/14) – 1 WPI 50 (7/14) ++ 35.7 (5/14) ++ 2 WPI 28.6 (4/14) + 85.7 (12/14) ++ 3 WPI 0 (0/14) – 92.6 (13/14) ++ 4 WPI 0 (0/14) – 92.6 (13/14) ++ 5 WPI 0 (0/14) – 92.6 (13/14) ++ 6 WPI 0 (0/14) – 92.6 (13/14) ++ 7 WPI 0 (0/14) – 92.6 (13/14) ++ 8 WPI 0 (0/14) – 85.7 (12/14) ++ 9 WPI 0 (0/7) – 42.8 (3/7) ++ 10 WPI 0 (0/7) – 42.8 (3/7) ++ 1DPI, days post-infection; WPI, weeks post-infection. 2Positive rate (%) (Number of positive ducks/no. of live ducks identified by band). 3–, not detected; +, < 103 copies/μL; ++, 103 to 106 copies/μL; +++, > 106 copies/μL. View Large Table 3. Quantification of DuCV DNA and viral positive rate in DuCV-infected ducks. Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) 1DPI, days post-infection; WPI, weeks post-infection. 2–, no detected; +, < 103 copies/mg; ++, 103 - 106 copies/mg; +++, > 106 copies/mg; NA, not applicable. 3Positive rate (%) (number of positive ducks/no of sacrificed ducks). View Large Table 3. Quantification of DuCV DNA and viral positive rate in DuCV-infected ducks. Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) Viral load Positive rate Spleen Bursa of Fabricius Cecal tonsil Lung Thymus Liver Kidney Oviduct Bursa of Fabricius Spleen Thymus 3 DPI1 –2 – – – – – – NA 0 (0/5)3 0 (0/5) 0 (0/5) 1 WPI ++ ++ + ++ ++ ++ – NA 71.4 (5/7) 57.1 (4/7) 57.1 (4/7) 2 WPI +++ +++ +++ +++ +++ ++ ++ NA 100 (7/7) 100 (7/7) 85.7 (6/7) 3 WPI +++ ++ ++ +++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 4 WPI +++ +++ ++ ++ ++ ++ ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 6 WPI +++ ++ ++ ++ ++ + ++ NA 100 (7/7) 100 (7/7) 100 (7/7) 8 WPI +++ +++ ++ ++ ++ ++ ++ – 100 (7/7) 100 (7/7) 85.7 (6/7) 10 WPI +++ +++ ++ ++ ++ + + – 100 (7/7) 85.7 (6/7) 71.4 (5/7) 1DPI, days post-infection; WPI, weeks post-infection. 2–, no detected; +, < 103 copies/mg; ++, 103 - 106 copies/mg; +++, > 106 copies/mg; NA, not applicable. 3Positive rate (%) (number of positive ducks/no of sacrificed ducks). View Large DISCUSSION We successfully established an experimental DuCV-1 infection in Pekin ducks and demonstrated the pathogenicity and persistence of DuCV-1 following experimental infection. On postmortem examination, gross lesions were observed in the immune organs including BF, thymus, and spleen (Table 1). In inoculated geese, Guo et al. (2011) reported hemorrhage in the thymus and BF, and splenomegaly in their GoCV experimental model. However, the size of thymus described by Guo et al. (2011) was in contrast with ours. In our experiment, there were also histopathologic lesions in the BF (Figure 1). Our findings are similar to results of experimental infection with other avian circoviruses. Lymphocytic depletion and necrosis in the BF were observed in the PiCV and GoCV experimental infection, respectively (Schmidt et al., 2008; Guo et al., 2011). To detect apoptosis activated by ORF3, the TUNEL assay was performed with bursal tissue (Figure 2). There was a significant difference of the apoptosis rate between the negative and DuCV group. Guo et al. (2011) also reported that apoptotic cells in age-matched GoCV-inoculated geese increased significantly compared with uninoculated geese. These findings suggest that DuCV damages immune organs, especially the BF, resulting in immunosuppression. The level of immunosuppression remains unclear and should be studied. The earliest time point for detection of DuCV DNA in sera, cloacal swabs, and organs was 1 WPI (Tables 2 and 3). These data indicate that DuCV infection may induce a systemic infection after the virus spreads through the blood circulation system. Viremia was also detected at 2 WPI (Table 2). Similarly, viremia was previously detected by experimental infection with DuCV-2 and other avian circoviruses (Schmidt et al., 2008; Guo et al., 2011; Li et al., 2015). Li et al. (2015) reported that the duration of viremia was from 15 to 28 d post-challenge in ducks inoculated with DuCV-2. Our results suggest that ducks infected with DuCV-1 may show a similar period of viremia without a secondary viremia, even though there are differences related to experimental design, virus strains, and genotypes of DuCV. Given the lack of experimental studies of DuCV infection, more experimental research is required to prove the pathogenic differences related to various strains and genotypes in DuCV. Ducks began to shed the virus at 1 WPI, and viral shedding was persistent and still detectable at the end of the experiment (10 WPI) (Table 2). During that time, the positive rate was more than 42.8% and the viral loads from cloacal swabs exceeded 104 copies/μL. In a preliminary experiment, viral shedding was observed to 16 WPI (data not shown). The findings provide evidence that horizontal transmission and persistent infection are characteristics of the DuCV-1. This could increase the risk of DuCV-1 re-infection in duck farms. In the present investigation, organ viral loads peaked at 2 and 3 WPI (Table 3). The organ with the highest mean viral load was the spleen, followed by BF, CT, lung, thymus, liver, and kidney. However, Li et al. (2015) reported that the organ with the highest mean viral load was BF, followed by liver and kidney, which showed higher viral loads and positive rates than the spleen. This indicates that there are differences in organ tropism related to various strains and genotypes of DuCV. In our experiment, the number of copies of the DuCV DNA was high in the secondary immune organs, the CT and spleen. The viral load of Harderian gland, also a secondary immune organ, is reported to be similar to the viral load of the thymus (Li et al., 2015). These results indicate that DuCV has tropism for all immune organs. Viral DNA was detected in most of the examined organs until the end of the experiment (10 WPI), and in a preliminary experiment the spleen from a duck infected with DuCV was PCR-positive at 27 WPI (data not shown). In the absence of an in vitro cell culture method for the isolation of DuCV, PCR is often used for diagnosis (Banda et al., 2007; Fu et al., 2011; Wan et al., 2011a). Selection of optimal samples is needed for detection of DuCV DNA by PCR. In this study, cloacal swabs showed higher positive rate and viral loads than the sera (Table 2). The BF was the organ of the highest positive rate, and the spleen contained the highest viral loads (Table 3). These results indicate that both are major organs for diagnosis of DuCV from a carcass. Since the BF begins to regress at about 12 wk of age (Gille and Salomon, 1999), and ducks of varying ages are susceptible to DuCV (Wan et al., 2011b; Cha et al., 2013), the spleen may a better specimen than the BF for diagnosis. We suggest that cloacal swabs are better than sera for samples to detect DuCV-1 in live ducks, and that the spleen is the principal organ for the diagnosis of DuCV-1 in carcasses. Furthermore, our data showed that for the detection of viremia in early-stage infections, testing a serum sample is more useful than the other samples. Despite successful experimental DuCV-1 infection, clinical signs were not observed in DuCV group. These findings differ from previous reports of naturally infected cases (Soike et al., 2004; Chen et al., 2006; Banda et al., 2007). Li et al. (2015) reported that 10-d ducklings inoculated with DuCV-2 showed clinical symptoms that included wasting, feathering disorder, and low average daily weight gain. It is conceivable that development of clinical signs is involved in the time of challenge with DuCV as well as various strains and genotypes of DuCV. In conclusion, the results of this study demonstrate that DuCV-1 can induce immunosuppression by causing extensive damage to immune organs. Pathobiological characteristics of DuCV-1 include systemic infection, persistent infection, and horizontal transmission. Considering these findings and the demonstrated environmental stability of Circovirus (Allan et al., 1994; Raidal and Cross, 1994; Yilmaz and Kaleta, 2004), prolonged exposure to DuCV-1 may result in re-infection. We suggest that these features allow DuCV-1 to more easily circulate in farms and increase the susceptibility of ducks to other diseases. Despite the threat of coinfection by DuCV-1, the severity and virulence of coinfection have not been proven. Further experimental studies are needed to investigate the risk of coinfection and level of economic losses to duck farms. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Supplementary Figure S1. Gross lesions of the bursa of Fabricius (BF) and thymus in the negative and DuCV groups. The BF at 4 WPI (a, b) and 10 WPI (c, d) in the negative (a, c) and DuCV (b, d) groups. The thymus at 2 WPI in the negative (e) and DuCV (f) groups. Bar = 3 cm. DuCV = duck circovirus; WPI = weeks post-infection. ACKNOWLEDGMENTS This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (316039–3, 317009–3, 716002–7) and Research of Animal and Plant Quarantine Agency, South Korea. REFERENCES Allan G.M. , Ellis J.A. . 2000 . Porcine circoviruses: a review . J. Vet. Diagn. Invest. 12 : 3 – 14 . Google Scholar CrossRef Search ADS PubMed Allan G.M. , Phenix K.V. , Todd D. , McNulty M.S. . 1994 . Some biological and physico-chemical properties of porcine circovirus . Zentralbl. Veterinarmed. B. 41 : 17 – 26 . Google Scholar PubMed Banda A. , Galloway-Haskins R.I. , Sandhu T.S. , Schat K.A. . 2007 . 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Poultry ScienceOxford University Press

Published: Sep 1, 2018

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