Veterinary Microbiology 174 (2014) 229–232
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Evidence of possible vertical transmission of duck circovirus Zhiguo Li a,b,1, Xin Wang a,b,1, Ruihua Zhang a,b, Junhao Chen a,b,c, Linlin Xia a,b, Shaoli Lin a,b, Zhijing Xie a,b, Shijin Jiang a,b,* a
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agriculteral University, Taian, Shandong 271018, China b Agricultural University, Taian, Shandong, China c Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Taian, Shandong 271018, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 April 2014 Received in revised form 30 August 2014 Accepted 1 September 2014
To test the hypothesis that duck circovirus (DuCV) may be vertically transmitted from infected breeder ducks to their ducklings, we investigated 120 newly hatched ducklings, 30 dead duck embryos and 80 non-embryonated duck eggs with the duplex polymerase chain reaction (PCR). DuCV DNA was present in 15 newly hatched ducklings, 4 duck embryos and 3 non-embryonated eggs. Four ducklings from two flocks were co-infected by DuCV-1 and DuCV-2, three ducklings from three flocks were DuCV-1 single infection, and eight ducklings from six flocks were DuCV-2 single infection. One duck embryo and one non-embryonated egg were positive for both DuCV-1 and DuCV-2 DNAs, one embryo for DuCV-1 DNA, and two embryos and two non-embryonated eggs for DuCV-2 DNA. The findings provide evidence of possible vertical transmission of DuCV and simultaneous transmission of DuCV-1 and DuCV-2 from breeder ducks to ducklings. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Duck circovirus Vertical transmission Duplex PCR Co-infection
1. Introduction Duck circovirus (DuCV), one member of the genus Circovirus of the Circoviridae family, is a small, nonenveloped, 15–16 nm in diameter, icosahedral virus (Hattermann et al., 2003). The genome of DuCV is a single-stranded circular DNA of about 1.99 kb, which contains three major open reading frames (ORFs), ORF1, ORF2 and ORF3 (Hattermann et al., 2003; Xiang et al., 2012). Based on the analysis of capsid gene and genomic sequences, DuCV was classified into two genotypes, DuCV1 and DuCV-2 (Wang et al., 2011; Zhang et al., 2012; Zhang et al., 2013; Wen et al., 2014).
* Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong 271018, China. Tel.: +86 538 8245799; fax: +86 538 8245799. E-mail addresses: [email protected], [email protected] (S. Jiang). 1 Both the authors contributed equally to this work. http://dx.doi.org/10.1016/j.vetmic.2014.09.001 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
DuCV infection was characterized by feathering disorders, poor body condition and low body weight (Hattermann et al., 2003), and caused lymphocyte depletion, necrosis, and histiocytosis in the bursa of Fabricius (BF) of ducks (Soike et al., 2004). There were multiple local lesions in the spleen, thymus and BF of ducks infected with DuCV (Liu et al., 2010b). Surveys would suggest a high prevalence of the virus in flocks experiencing morbidity and mortality as well as those that appear clinically normal (Fringuelli et al., 2005; Chen et al., 2006; Banda et al., 2007; Jiang et al., 2008; Zhang et al., 2009; Liu et al., 2010a). Based on findings from other avian circoviruses, it is possible that DuCV can be transmitted both horizontally and vertically. It has been reported that there was a high prevalence of DuCV in symptom-free duck populations in China, and the DuCV-seropositive rate of long-term farmed breeder flocks was significantly higher than that of meat duck flocks, indicating that DuCV was transmitted horizontally (Liu et al., 2010a). However, the vertical transmission of the virus thus far remains unclear. The aim
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of the work was to investigate the possible transmission of DuCV from breeder ducks to newly hatched ducklings using the polymerase chain reaction (PCR). 2. Materials and methods On the basis of the whole genome sequence alignments of DuCV sequences retrieved from GenBank (http:// www.ncbi.nlm.nih.gov), four primers for DuCV were selected. DuCV-F (CGG GAA ATG ACG TAG TCG TCA TG, position 640–662) and DuCV-R (GGA(C) C(T)TG(A) AAC ATG AGA TGG GC, position 1653–1672) were designed to amplify a 1032 base pair (bp) fragment for DuCV-1 and DuCV-2. DuCV-1F (GTT CAC TCC G(T)GT TGT GTT GTC C(T)GG, position 1226–1249) was designed with DuCV-R to amplify a 446 bp fragment for DuCV-1 typing, and DuCV-2R (GAT AAT GCG ACC(T) GGC GAC G, position 1219–1239) was designed with DuCV-F to amplify a 599 bp specific fragment for DuCV-2 strains. Using the four primers, a duplex PCR assay was performed in a total volume of 25 ml, included 1 ml of DNA, 1 PCR buffer (50 mM KCl, 10 mM Tris–HCl [pH 8.3]), 1.5 mM MgCl2, 0.4 mM of each dNTP, 1 mM of the each primers and 1.0 U Taq DNA Polymerase (TaKaRa, Dalian, China). PCR parameters were as follows: a denaturation step at 95 8C for 5 min and then 35 cycles of a denaturation step at 95 8C for 45 s, an annealing and extension step of 68 8C for 90 s, with a final extension at 72 8C for 10 min. The PCR products were analyzed by electrophoresis in 0.8% agarose gel. DuCV-1 strain FJ0601 (EF370476) (Jiang et al., 2008), DuCV-2 strain WF0701 (EU022375) (Zhang et al., 2012), duck plague virus (DPV), egg drop syndrome virus (EDSV), duck hepatitis B virus (DHBV), Muscovy duck parvovrius (MDPV), Riemerella anatipestifer (RA), Salmonella and Escherichia coli (E. coli) were used to determine the specificity of the duplex PCR. Two plasmids, pDuCV-1 including the complete genomic DNA of DuCV-1 strain FJ0601 and the plasmid pDuCV-2 including the complete genomic DNA of DuCV-2 strain WF0701, were purified with a QIAprep Spin Miniprep kit (Qiagen, Chatsworth, CA, USA). Quantitation of the plasmid DNAs was performed spectrophotometrically at 260 nm, and a mixture of 1 ml of 108 to 100 copies of the plasmid DNAs was used to detect the sensitivity of the duplex PCR. Total of 120 1-day-old ducklings from 8 breeder Cherry Valley duck flocks (15 ducklings per flock) in eastern China were collected to investigate the presence of DuCV infection. The antibodies to DuCV in breeder duck sera were detected with the established iELISA method (Liu et al., 2010a). The positive rates of DuCV-specific antibody ranged from 40% to 64.7% among the 8 breeder duck flocks. The mortality of pre-hatching ducklings was 2–3%. Newly hatched ducklings were obtained within 10 min of hatch. The liver, spleen, kidney, thymus and BF samples from ducklings were collected. According to the manufacturer’s instructions, DuCV DNA was extracted by Dneasy tissue kit (Qiagen, Hilden, Germany). In addition, 30 duck embryos and 80 non-embryonated duck eggs from one of the three DuCV-1 and DuCV-2 coinfection flocks were also detected by the PCR. The
embryos died in the final stage of incubation, and the non-embryonated eggs were obtained within 1 h of laying. DNA derived from embryonic tissues was isolated using the Dneasy tissue kit (Qiagen, Hilden, Germany). DNA isolation from non-embryonated eggs was carried out following the established procedures (Rahaus et al., 2008). Briefly, the non-embryonated eggs were opened using a sterile scalpel, glair and yolk were separated and introduced into a phenol/chloroform extraction, and ethanol was used for precipitation of DNA. All the samples were detected by the pan DuCV PCR and the two type specific PCRs respectively. The expected DuCV-1-specific and DuCV-2-specific products amplified from clinical samples were sequenced on a commercial service (Shanghai Sangon Biological Engineering Technology & Service Co., Ltd, Shanghai, China).
3. Results The duplex PCR amplified two DNA fragments of 1032 bp and 446 bp from DuCV-1 DNA, two DNA fragments of 1032 bp and 599 bp from DuCV-2 DNA, and three DNA fragments of 1032 bp, 446 bp and 599 bp from DuCV-1 and DuCV-2 mixed DNAs (Fig. 1). Under the same conditions, no amplification occurred using nucleic acids from the 7 other duck pathogens (DPV, EDSV, DHBV, MDPV, RA, Salmonella and E. coli) and negative control (healthy duck) (Fig. 1). Using 108 to 100 copies of the plasmid DNAs of DuCV-1 and DuCV-2 as templates, the duplex PCR assay was able to detect 10 copies of DuCV-1 and DuCV-2 DNAs (Fig. 2). From 120 newly hatched ducklings from 8 anti-DuCV antibody positive breeder duck flocks, 3 (37.5%) flocks were identified as mixed infection of DuCV-1 and DuCV-2, and other 3 (37.5%) flocks were DuCV-2 single infection (Table 1). Among them, 3.33% (4/120) of duckling were detected as mixed infection of DuCV-1 and DuCV-2, 2.5% (3/120) of duckling were DuCV-1 single infection, and 6.67% (8/120) of duckling were DuCV-2 single infection. DuCV-1 was detected in seven BF samples from three duck flocks. DuCV-2 was found in nine liver samples from five duck flocks and four thymus samples from three duck flocks. All spleen and kidney samples were negative for DuCV-1 and DuCV-2.
Fig. 1. The specificity of the duplex PCR for simultaneous detection of the two genotypes of DuCVs. Lane M, DNA marker DL2000; lane 1, DuCV-1 strain FJ0601; lane 2, DuCV-2 strain WF0701; lane 3, DuCV strains FJ0601 and WF0701; lane 4, DPV; lane 5, EDSV; lane 6, DHBV; lane 7, MDPV; lane 8, RA; lane 9, Salmonella; lane 10, E. coli; lane 11, negative control (healthy duckling sample).
Z. Li et al. / Veterinary Microbiology 174 (2014) 229–232
Fig. 2. The sensitivity of the duplex PCR using plasmid DNA as template. The assay was performed using 108 copies DNAs (lane 1), 107 copies (lane 2), 106 copies (lane 3), 105 copies (lane 4), 104 copies (lane 5), 103 copies (lane 6), 102 copies (lane 7), 101 copies (lane 8), and 1 copy (lane 9). Lanes M, DNA marker.
To analyze the possibility of a vertical transmission of DuCV, we investigated the presence of viral DNA in embryonated and non-embryonated eggs. In duck embryos died in the final stage of incubation, 4 of 30 (13.33%) samples were found to be DuCV positive. As a result, one was positive for both DuCV-1 and DuCV-2 DNAs, one for DuCV-1 DNA, and two for DuCV-2 DNA. In the case of glair, 3 of 80 (3.75%) samples were found to be DuCV positive, one positive for both DuCV-1 and DuCV-2 DNAs, and two for DuCV-2 DNA. In the case of yolk, all samples were negative. It indicated that embryos can carry the viruses. There was 100% agreement between the results of the duplex PCR assay, the pan DuCV PCR and the two type specific PCRs. DuCV-1-specific sequences obtained from ducklings and duck embryos were initially aligned using DNAStar (DNAStar Inc., Madison WI, USA) and showed 87.1–92.4% nucleotide similarity with six published DuCV-1 strains (AY228555, NC007220, EF451157, EU022375, GU131340 and GU131342), but shared only 76.4–76.9% nucleotide similarity with six reference DuCV-2 strains (EF370476, AY394721, DQ166836, DQ166837, DQ166838 and EU499310). On the other hand, DuCV-2-specific sequences were exhibited high nucleotide similarity of 97.3–98.7% with the six DuCV-2 strains, whereas shared low nucleotide similarity of 78.0–79.6% with the six DuCV-1 strains. 4. Discussion Because of the absence of available cell culture for the isolation of DuCV, the PCR assays were often used for
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detecting DuCVs (Hattermann et al., 2003; Fringuelli et al., 2005; Jiang et al., 2008; Zhang et al., 2009; Wan et al., 2011; Wang et al., 2011). However, these methods cannot be used to distinguish mixed infection of DuCV-1 and DuCV-2. In this study, with one pair of universal primers and two typing primers, within a single reaction, two seminested PCR assays were established for diagnosis of DuCV1 and DuCV-2 respectively. The duplex PCR assay was shown to be high specificity and sensitivity for simultaneous detection of mixed infection of the two genotypes of DuCVs. Using the method, mixed infection of DuCV-1 and DuCV-2 was detected from newly hatched ducklings, duck embryos and non-embryonated eggs. For newly hatched ducklings, 3 of 8 (37.5%) flocks and 4 of 120 (3.33%) individuals were tested positive for mixed infection of DuCV-1 and DuCV-2. The mixed infection of DuCV-1 and DuCV-2 also presented in 1 of 30 (3.33%) duck embryos and 1 of 80 (1.25%) non-embryonated duck eggs. It has been reported that no DuCV specific amplicon was obtained from embryonated duck eggs, non-embryonated duck eggs, and newly hatched ducklings (Wan et al., 2011). In this study, the duplex PCR assay was performed on newly hatched ducklings from eight anti-DuCV antibody positive breeder duck flocks, and duck embryos and non-embryonated eggs from one DuCV-1 and DuCV-2 coinfection flock. DuCV-2 DNA was present in 10% (12/120) of newly hatched ducklings from 6 duck flocks, DuCV-1 DNA present in 5.83% (7/120) of ducklings from 3 of the 6 duck flocks (Table 1). Among 30 duck embryos and 80 non-embryonated eggs, 2 (6.67%) embryos and 1 (1.25%) non-embryonated egg were positive for DuCV-1 DNA, and 3 (10%) embryos and 3 (3.75%) non-embryonated eggs positive for DuCV-2 DNA. The result indicated that the infection of DuCV-2 was more common than that of DuCV1 in newly hatched ducklings, duck embryos and eggs. The different DuCV positive rates between our study and other reports might be due to the different sources of the sample origins and not the test methods. DuCV-1 and DuCV-2 coinfected duck embryos and newly hatched ducklings suggest that DuCVs may be vertically transmitted from breeder ducks via eggs to their progeny. Several avian circoviruses can spread vertically. Vertical transmission of chicken infectious anemia virus (CIAV) occurs when antibody-negative hens become infected or by semen from infected cocks (Hoop, 1993). In commercial flocks, vertical transmission of CIAV through the hatching egg is the most important means of dissemination (Chettle
Table 1 DuCV-1 and DuCV-2 in different meat duck flocks by duplex PCR. Flocks
1 2 3 4 5 6 7 8 Total
Age (days)
1 1 1 1 1 1 1 1
Positive rate (number positive/number tested) DuCV-1
DuCV-2
DuCV-1 and DuCV-2
0 (0/15) 6.67% (1/15) 0 (0/15) 6.67% (1/15) 0 (0/15) 0 (0/15) 6.67% (1/15) 0 (0/15) 2.5% (3/120)
0 (0/15) 6.67% (1/15) 0 (0/15) 0 (0/15) 6.67% (1/15) 13.33% (2/15) 20% (3/15) 6.67% (1/15) 6.67% (8/120)
0 (0/15) 0 (0/15) 0 (0/15) 13.33% (2/15) 0 (0/15) 0 (0/15) 13.33% (2/15) 0 (0/15) 3.33% (4/120)
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et al., 1989; Engstro¨m, 1999). In both psittacine and columbid birds with circovirus infection, psittacine beak and feather disease virus (BFDV) and pigeon circovirus (PiCV) have been suspected to be probable vertical transmission (Latimer et al., 1991; Pare´ et al., 1999). Recently, PiCV DNA was detected in embryos before hatching, and the ovary and testis of adult birds suggesting a vertical transmission (Duchatel et al., 2006). In addition, BFDV DNA was detected in both embryonated and nonembryonated budgerigar eggs, suggesting that the BFDV can be transmitted vertically (Rahaus et al., 2008). The data presented in this study correspond to these findings. In summary, this report presents the first evidence suggesting that DuCV may be vertically transmitted. Since horizontal transmission has already been demonstrated to occur (Liu et al., 2010a), it is likely that DuCV is transmitted via both horizontal and vertical routes. Furthermore, DuCV-1 and DuCV-2 can be vertically transmitted from breeder ducks to ducklings at the same time. Acknowledgments This study was funded by grants from Shandong Provincial Natural Science Foundation, China (ZR2013 CM009) and Shandong Modern Agricultural Technology & Industry System, China (SDAIT-13-011-15). References Banda, A., Galloway-Haskins, R.I., Sandhu, T.S., Schat, K.A., 2007. Genetic analysis of a duck circovirus detected in commercial Pekin ducks in New York. Avian Dis. 51, 90–95. Chen, C.L., Wang, P.X., Lee, M.S., Shien, J.H., Shien, H.K., Ou, S.J., Chen, C.H., Chang, P.C., 2006. Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus. Avian Dis. 50, 92–95. Chettle, N.J., Eddy, R.K., Wyeth, P.J., Lister, S.A., 1989. An outbreak of disease due to chicken anaemia agent in broiler chickens in England. Vet. Rec. 124, 211–215. Duchatel, J.P., Todd, D., Smyth, J.A., Bustin, J.C., Vindevogel, H., 2006. Observations on detection, excretion and transmission of pigeon circovirus in adult, young and embryonic pigeons. Avian Pathol. 35, 30–34. Engstro¨m, B.E., 1999. Prevalence of antibody to chicken anaemia virus (CAV) in Swedish chicken breeding flocks correlated to outbreaks of
blue wing disease (BWD) in their progeny. Acta Vet. Scand. 40, 97–107. Fringuelli, E., Scott, A.N., Beckett, A., McKillen, J., Smyth, J.A., Palya, V., Glavits, R., Ivanics, E., Mankertz, A., Franciosini, M.P., Todd, D., 2005. Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests. Avian Pathol. 34, 495–500. Hattermann, K., Schmmitt, C., Soike, D., Mankertz, A., 2003. Cloning and sequencing of duck circovirus (DuCV). Arch. Virol. 148, 2471–2480. Hoop, R.K., 1993. Transmission of chicken anaemia virus with semen. Vet. Rec. 133, 551–552. Jiang, S., Zhang, X., Liu, S., Wang, Y., Kong, Y., Wei, X., Sun, Y., Zhao, Q., 2008. PCR detection and sequence analysis of duck circovirus in sick Muscovy ducks. Virol. Sin. 23, 265–271. Latimer, K.S., Rakich, P.M., Niagro, F.D., Ritchie, B.W., Steffens, W.L., Campagnoli, R.P., Pesti, D.A., Lukert, P.D., 1991. An updated review of psittacine beak and feather disease. J. Assoc. Avian Vet. 5, 211–220. Liu, S.N., Zhang, X.X., Zou, J.F., Xie, Z.J., Zhu, Y.L., Zhao, Q., Zhou, E.M., Jiang, S.J., 2010a. Development of an indirect ELISA for the detection of duck circovirus infection in duck flocks. Vet. Microbiol. 145, 41–46. Liu, S.N., Yang, J.B., Zhang, X.X., Chen, Z., Kong, Y.B., Zhu, Y.L., Jiang, S.J., 2010b. Prokaryotic expression and antiserum preparation of replication protein of duck circovirus FJ0601 strain. Chin. J. Vet. Sci. 30,, 10– 12, 18 (in Chinese). Pare´, J.A., Brash, M.L., Hunter, D.B., Hampson, R.J., 1999. Observations on pigeon circovirus infection in Ontario. Can. Vet. J. 40, 659–662. Rahaus, M., Dwsloges, N., Probst, S., Loebbert, B., Lantermann, W., Wolff, M.H., 2008. Detection of beak and feather disease virus DNA in embryonated eggs of psittacine birds. Vet. Med. – Czech 53, 53–58. Soike, D., Albrecht, K., Hattermann, K., Schmitt, C., Mankertz, A., 2004. Novel circovirus in mulard ducks with developmental and feathering disorders. Vet. Rec. 154, 792–793. Wan, C., Huang, Y., Cheng, L., Fu, G., Shi, S.H., Chen, H., Peng, C., Lin, F., Lin, J., 2011. The development of a rapid SYBR green I-based quantitative PCR for detection of duck circovirus. Virol. J. 8, 465. Wang, D., Xie, X., Zhang, D., Ma, G., Wang, X., Zhang, D., 2011. Detection of duck circovirus in China: a proposal on genotype classification. Vet. Microbiol. 147, 410–415. Wen, H., Wu, Y., Yang, C., Zhang, X., Lian, C., Chen, H., Han, L., 2014. Comments on duck circovirus (DuCV) genotype definition. Gene 538, 207–208. Xiang, Q.W., Wang, X., Xie, Z.J., Sun, Y.N., Zhu, Y.L., Wang, S.J., Liu, H.J., Jiang, S.J., 2012. ORF3 of duck circovirus: a novel protein with apoptotic activity. Vet. Microbiol. 159, 251–256. Zhang, X., Jiang, S., Wu, J., Zhao, Q., Sun, Y., Kong, Y., Li, X., Yao, M., Chai, T., 2009. An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China. Vet. Microbiol. 133, 252–256. Zhang, X.X., Liu, S.N., Xie, Z.J., Kong, Y.B., Jiang, S.J., 2012. Complete genome sequence analysis of duck circovirus strains from Cherry Valley duck. Virol. Sin. 27, 154–164. 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.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Evidence of possible vertical transmission of duck circovirus Zhiguo Li a,b,1, Xin Wang a,b,1, Ruihua Zhang a,b, Junhao Chen a,b,c, Linlin Xia a,b, Shaoli Lin a,b, Zhijing Xie a,b, Shijin Jiang a,b,* a
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agriculteral University, Taian, Shandong 271018, China b Agricultural University, Taian, Shandong, China c Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Taian, Shandong 271018, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 April 2014 Received in revised form 30 August 2014 Accepted 1 September 2014
To test the hypothesis that duck circovirus (DuCV) may be vertically transmitted from infected breeder ducks to their ducklings, we investigated 120 newly hatched ducklings, 30 dead duck embryos and 80 non-embryonated duck eggs with the duplex polymerase chain reaction (PCR). DuCV DNA was present in 15 newly hatched ducklings, 4 duck embryos and 3 non-embryonated eggs. Four ducklings from two flocks were co-infected by DuCV-1 and DuCV-2, three ducklings from three flocks were DuCV-1 single infection, and eight ducklings from six flocks were DuCV-2 single infection. One duck embryo and one non-embryonated egg were positive for both DuCV-1 and DuCV-2 DNAs, one embryo for DuCV-1 DNA, and two embryos and two non-embryonated eggs for DuCV-2 DNA. The findings provide evidence of possible vertical transmission of DuCV and simultaneous transmission of DuCV-1 and DuCV-2 from breeder ducks to ducklings. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Duck circovirus Vertical transmission Duplex PCR Co-infection
1. Introduction Duck circovirus (DuCV), one member of the genus Circovirus of the Circoviridae family, is a small, nonenveloped, 15–16 nm in diameter, icosahedral virus (Hattermann et al., 2003). The genome of DuCV is a single-stranded circular DNA of about 1.99 kb, which contains three major open reading frames (ORFs), ORF1, ORF2 and ORF3 (Hattermann et al., 2003; Xiang et al., 2012). Based on the analysis of capsid gene and genomic sequences, DuCV was classified into two genotypes, DuCV1 and DuCV-2 (Wang et al., 2011; Zhang et al., 2012; Zhang et al., 2013; Wen et al., 2014).
* Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong 271018, China. Tel.: +86 538 8245799; fax: +86 538 8245799. E-mail addresses: [email protected], [email protected] (S. Jiang). 1 Both the authors contributed equally to this work. http://dx.doi.org/10.1016/j.vetmic.2014.09.001 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
DuCV infection was characterized by feathering disorders, poor body condition and low body weight (Hattermann et al., 2003), and caused lymphocyte depletion, necrosis, and histiocytosis in the bursa of Fabricius (BF) of ducks (Soike et al., 2004). There were multiple local lesions in the spleen, thymus and BF of ducks infected with DuCV (Liu et al., 2010b). Surveys would suggest a high prevalence of the virus in flocks experiencing morbidity and mortality as well as those that appear clinically normal (Fringuelli et al., 2005; Chen et al., 2006; Banda et al., 2007; Jiang et al., 2008; Zhang et al., 2009; Liu et al., 2010a). Based on findings from other avian circoviruses, it is possible that DuCV can be transmitted both horizontally and vertically. It has been reported that there was a high prevalence of DuCV in symptom-free duck populations in China, and the DuCV-seropositive rate of long-term farmed breeder flocks was significantly higher than that of meat duck flocks, indicating that DuCV was transmitted horizontally (Liu et al., 2010a). However, the vertical transmission of the virus thus far remains unclear. The aim
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of the work was to investigate the possible transmission of DuCV from breeder ducks to newly hatched ducklings using the polymerase chain reaction (PCR). 2. Materials and methods On the basis of the whole genome sequence alignments of DuCV sequences retrieved from GenBank (http:// www.ncbi.nlm.nih.gov), four primers for DuCV were selected. DuCV-F (CGG GAA ATG ACG TAG TCG TCA TG, position 640–662) and DuCV-R (GGA(C) C(T)TG(A) AAC ATG AGA TGG GC, position 1653–1672) were designed to amplify a 1032 base pair (bp) fragment for DuCV-1 and DuCV-2. DuCV-1F (GTT CAC TCC G(T)GT TGT GTT GTC C(T)GG, position 1226–1249) was designed with DuCV-R to amplify a 446 bp fragment for DuCV-1 typing, and DuCV-2R (GAT AAT GCG ACC(T) GGC GAC G, position 1219–1239) was designed with DuCV-F to amplify a 599 bp specific fragment for DuCV-2 strains. Using the four primers, a duplex PCR assay was performed in a total volume of 25 ml, included 1 ml of DNA, 1 PCR buffer (50 mM KCl, 10 mM Tris–HCl [pH 8.3]), 1.5 mM MgCl2, 0.4 mM of each dNTP, 1 mM of the each primers and 1.0 U Taq DNA Polymerase (TaKaRa, Dalian, China). PCR parameters were as follows: a denaturation step at 95 8C for 5 min and then 35 cycles of a denaturation step at 95 8C for 45 s, an annealing and extension step of 68 8C for 90 s, with a final extension at 72 8C for 10 min. The PCR products were analyzed by electrophoresis in 0.8% agarose gel. DuCV-1 strain FJ0601 (EF370476) (Jiang et al., 2008), DuCV-2 strain WF0701 (EU022375) (Zhang et al., 2012), duck plague virus (DPV), egg drop syndrome virus (EDSV), duck hepatitis B virus (DHBV), Muscovy duck parvovrius (MDPV), Riemerella anatipestifer (RA), Salmonella and Escherichia coli (E. coli) were used to determine the specificity of the duplex PCR. Two plasmids, pDuCV-1 including the complete genomic DNA of DuCV-1 strain FJ0601 and the plasmid pDuCV-2 including the complete genomic DNA of DuCV-2 strain WF0701, were purified with a QIAprep Spin Miniprep kit (Qiagen, Chatsworth, CA, USA). Quantitation of the plasmid DNAs was performed spectrophotometrically at 260 nm, and a mixture of 1 ml of 108 to 100 copies of the plasmid DNAs was used to detect the sensitivity of the duplex PCR. Total of 120 1-day-old ducklings from 8 breeder Cherry Valley duck flocks (15 ducklings per flock) in eastern China were collected to investigate the presence of DuCV infection. The antibodies to DuCV in breeder duck sera were detected with the established iELISA method (Liu et al., 2010a). The positive rates of DuCV-specific antibody ranged from 40% to 64.7% among the 8 breeder duck flocks. The mortality of pre-hatching ducklings was 2–3%. Newly hatched ducklings were obtained within 10 min of hatch. The liver, spleen, kidney, thymus and BF samples from ducklings were collected. According to the manufacturer’s instructions, DuCV DNA was extracted by Dneasy tissue kit (Qiagen, Hilden, Germany). In addition, 30 duck embryos and 80 non-embryonated duck eggs from one of the three DuCV-1 and DuCV-2 coinfection flocks were also detected by the PCR. The
embryos died in the final stage of incubation, and the non-embryonated eggs were obtained within 1 h of laying. DNA derived from embryonic tissues was isolated using the Dneasy tissue kit (Qiagen, Hilden, Germany). DNA isolation from non-embryonated eggs was carried out following the established procedures (Rahaus et al., 2008). Briefly, the non-embryonated eggs were opened using a sterile scalpel, glair and yolk were separated and introduced into a phenol/chloroform extraction, and ethanol was used for precipitation of DNA. All the samples were detected by the pan DuCV PCR and the two type specific PCRs respectively. The expected DuCV-1-specific and DuCV-2-specific products amplified from clinical samples were sequenced on a commercial service (Shanghai Sangon Biological Engineering Technology & Service Co., Ltd, Shanghai, China).
3. Results The duplex PCR amplified two DNA fragments of 1032 bp and 446 bp from DuCV-1 DNA, two DNA fragments of 1032 bp and 599 bp from DuCV-2 DNA, and three DNA fragments of 1032 bp, 446 bp and 599 bp from DuCV-1 and DuCV-2 mixed DNAs (Fig. 1). Under the same conditions, no amplification occurred using nucleic acids from the 7 other duck pathogens (DPV, EDSV, DHBV, MDPV, RA, Salmonella and E. coli) and negative control (healthy duck) (Fig. 1). Using 108 to 100 copies of the plasmid DNAs of DuCV-1 and DuCV-2 as templates, the duplex PCR assay was able to detect 10 copies of DuCV-1 and DuCV-2 DNAs (Fig. 2). From 120 newly hatched ducklings from 8 anti-DuCV antibody positive breeder duck flocks, 3 (37.5%) flocks were identified as mixed infection of DuCV-1 and DuCV-2, and other 3 (37.5%) flocks were DuCV-2 single infection (Table 1). Among them, 3.33% (4/120) of duckling were detected as mixed infection of DuCV-1 and DuCV-2, 2.5% (3/120) of duckling were DuCV-1 single infection, and 6.67% (8/120) of duckling were DuCV-2 single infection. DuCV-1 was detected in seven BF samples from three duck flocks. DuCV-2 was found in nine liver samples from five duck flocks and four thymus samples from three duck flocks. All spleen and kidney samples were negative for DuCV-1 and DuCV-2.
Fig. 1. The specificity of the duplex PCR for simultaneous detection of the two genotypes of DuCVs. Lane M, DNA marker DL2000; lane 1, DuCV-1 strain FJ0601; lane 2, DuCV-2 strain WF0701; lane 3, DuCV strains FJ0601 and WF0701; lane 4, DPV; lane 5, EDSV; lane 6, DHBV; lane 7, MDPV; lane 8, RA; lane 9, Salmonella; lane 10, E. coli; lane 11, negative control (healthy duckling sample).
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Fig. 2. The sensitivity of the duplex PCR using plasmid DNA as template. The assay was performed using 108 copies DNAs (lane 1), 107 copies (lane 2), 106 copies (lane 3), 105 copies (lane 4), 104 copies (lane 5), 103 copies (lane 6), 102 copies (lane 7), 101 copies (lane 8), and 1 copy (lane 9). Lanes M, DNA marker.
To analyze the possibility of a vertical transmission of DuCV, we investigated the presence of viral DNA in embryonated and non-embryonated eggs. In duck embryos died in the final stage of incubation, 4 of 30 (13.33%) samples were found to be DuCV positive. As a result, one was positive for both DuCV-1 and DuCV-2 DNAs, one for DuCV-1 DNA, and two for DuCV-2 DNA. In the case of glair, 3 of 80 (3.75%) samples were found to be DuCV positive, one positive for both DuCV-1 and DuCV-2 DNAs, and two for DuCV-2 DNA. In the case of yolk, all samples were negative. It indicated that embryos can carry the viruses. There was 100% agreement between the results of the duplex PCR assay, the pan DuCV PCR and the two type specific PCRs. DuCV-1-specific sequences obtained from ducklings and duck embryos were initially aligned using DNAStar (DNAStar Inc., Madison WI, USA) and showed 87.1–92.4% nucleotide similarity with six published DuCV-1 strains (AY228555, NC007220, EF451157, EU022375, GU131340 and GU131342), but shared only 76.4–76.9% nucleotide similarity with six reference DuCV-2 strains (EF370476, AY394721, DQ166836, DQ166837, DQ166838 and EU499310). On the other hand, DuCV-2-specific sequences were exhibited high nucleotide similarity of 97.3–98.7% with the six DuCV-2 strains, whereas shared low nucleotide similarity of 78.0–79.6% with the six DuCV-1 strains. 4. Discussion Because of the absence of available cell culture for the isolation of DuCV, the PCR assays were often used for
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detecting DuCVs (Hattermann et al., 2003; Fringuelli et al., 2005; Jiang et al., 2008; Zhang et al., 2009; Wan et al., 2011; Wang et al., 2011). However, these methods cannot be used to distinguish mixed infection of DuCV-1 and DuCV-2. In this study, with one pair of universal primers and two typing primers, within a single reaction, two seminested PCR assays were established for diagnosis of DuCV1 and DuCV-2 respectively. The duplex PCR assay was shown to be high specificity and sensitivity for simultaneous detection of mixed infection of the two genotypes of DuCVs. Using the method, mixed infection of DuCV-1 and DuCV-2 was detected from newly hatched ducklings, duck embryos and non-embryonated eggs. For newly hatched ducklings, 3 of 8 (37.5%) flocks and 4 of 120 (3.33%) individuals were tested positive for mixed infection of DuCV-1 and DuCV-2. The mixed infection of DuCV-1 and DuCV-2 also presented in 1 of 30 (3.33%) duck embryos and 1 of 80 (1.25%) non-embryonated duck eggs. It has been reported that no DuCV specific amplicon was obtained from embryonated duck eggs, non-embryonated duck eggs, and newly hatched ducklings (Wan et al., 2011). In this study, the duplex PCR assay was performed on newly hatched ducklings from eight anti-DuCV antibody positive breeder duck flocks, and duck embryos and non-embryonated eggs from one DuCV-1 and DuCV-2 coinfection flock. DuCV-2 DNA was present in 10% (12/120) of newly hatched ducklings from 6 duck flocks, DuCV-1 DNA present in 5.83% (7/120) of ducklings from 3 of the 6 duck flocks (Table 1). Among 30 duck embryos and 80 non-embryonated eggs, 2 (6.67%) embryos and 1 (1.25%) non-embryonated egg were positive for DuCV-1 DNA, and 3 (10%) embryos and 3 (3.75%) non-embryonated eggs positive for DuCV-2 DNA. The result indicated that the infection of DuCV-2 was more common than that of DuCV1 in newly hatched ducklings, duck embryos and eggs. The different DuCV positive rates between our study and other reports might be due to the different sources of the sample origins and not the test methods. DuCV-1 and DuCV-2 coinfected duck embryos and newly hatched ducklings suggest that DuCVs may be vertically transmitted from breeder ducks via eggs to their progeny. Several avian circoviruses can spread vertically. Vertical transmission of chicken infectious anemia virus (CIAV) occurs when antibody-negative hens become infected or by semen from infected cocks (Hoop, 1993). In commercial flocks, vertical transmission of CIAV through the hatching egg is the most important means of dissemination (Chettle
Table 1 DuCV-1 and DuCV-2 in different meat duck flocks by duplex PCR. Flocks
1 2 3 4 5 6 7 8 Total
Age (days)
1 1 1 1 1 1 1 1
Positive rate (number positive/number tested) DuCV-1
DuCV-2
DuCV-1 and DuCV-2
0 (0/15) 6.67% (1/15) 0 (0/15) 6.67% (1/15) 0 (0/15) 0 (0/15) 6.67% (1/15) 0 (0/15) 2.5% (3/120)
0 (0/15) 6.67% (1/15) 0 (0/15) 0 (0/15) 6.67% (1/15) 13.33% (2/15) 20% (3/15) 6.67% (1/15) 6.67% (8/120)
0 (0/15) 0 (0/15) 0 (0/15) 13.33% (2/15) 0 (0/15) 0 (0/15) 13.33% (2/15) 0 (0/15) 3.33% (4/120)
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et al., 1989; Engstro¨m, 1999). In both psittacine and columbid birds with circovirus infection, psittacine beak and feather disease virus (BFDV) and pigeon circovirus (PiCV) have been suspected to be probable vertical transmission (Latimer et al., 1991; Pare´ et al., 1999). Recently, PiCV DNA was detected in embryos before hatching, and the ovary and testis of adult birds suggesting a vertical transmission (Duchatel et al., 2006). In addition, BFDV DNA was detected in both embryonated and nonembryonated budgerigar eggs, suggesting that the BFDV can be transmitted vertically (Rahaus et al., 2008). The data presented in this study correspond to these findings. In summary, this report presents the first evidence suggesting that DuCV may be vertically transmitted. Since horizontal transmission has already been demonstrated to occur (Liu et al., 2010a), it is likely that DuCV is transmitted via both horizontal and vertical routes. Furthermore, DuCV-1 and DuCV-2 can be vertically transmitted from breeder ducks to ducklings at the same time. Acknowledgments This study was funded by grants from Shandong Provincial Natural Science Foundation, China (ZR2013 CM009) and Shandong Modern Agricultural Technology & Industry System, China (SDAIT-13-011-15). References Banda, A., Galloway-Haskins, R.I., Sandhu, T.S., Schat, K.A., 2007. Genetic analysis of a duck circovirus detected in commercial Pekin ducks in New York. Avian Dis. 51, 90–95. Chen, C.L., Wang, P.X., Lee, M.S., Shien, J.H., Shien, H.K., Ou, S.J., Chen, C.H., Chang, P.C., 2006. Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus. Avian Dis. 50, 92–95. Chettle, N.J., Eddy, R.K., Wyeth, P.J., Lister, S.A., 1989. An outbreak of disease due to chicken anaemia agent in broiler chickens in England. Vet. Rec. 124, 211–215. Duchatel, J.P., Todd, D., Smyth, J.A., Bustin, J.C., Vindevogel, H., 2006. Observations on detection, excretion and transmission of pigeon circovirus in adult, young and embryonic pigeons. Avian Pathol. 35, 30–34. Engstro¨m, B.E., 1999. Prevalence of antibody to chicken anaemia virus (CAV) in Swedish chicken breeding flocks correlated to outbreaks of
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