First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds

Duck enteritis virus (DEV) is an aetiological agent of duck plague (DP), which represents one of the most acute and lethal diseases of waterfowl (Anseriformes) including geese, ducks and swans [9]. The infection may spread between farmed and free-ranging birds. The estimated number of birds susceptible to DEV infection includes members of over 48 species [3, 9, 13]. The economic impact on waterfowl husbandry caused by DP is considerable, since the mortality, especially in young birds, can reach 100 % [10]. DEV, officially named anatid herpesvirus 1, belongs to the genus Mardivirus, family Herpesviridae [11]. Its genome comprises about 160 kbp of double-stranded linear DNA, organised as a unique long (UL) region, a unique short (US) region flanked by a unique short internal repeat (IR_S) region, and a unique short terminal repeat (TRS) region. The DEV genome encodes at least 67 genes that have homology to those of other members of the subfamily Alphaherpesvirinae. The fIR_St DEV infection was noted in 1923 in domestic ducks in the Netherlands [2]. Further occurrence of the disease caused by this virus has been observed frequently in China [7]. The clinical form of the disease may be not be observed due to the asymptomatic carrier state of the infection [5, 10, 28]. The only stage during which DEV identification is possible is associated with the intermittent virus shedding period, and therefore, firm and reliable detection of this virus remains an epizootically important issue. The classical methods of DEV identification, such as virus isolation in chicken (CEF) or duck embryo fibroblasts (DEF), electron microscopy, and serological tests including enzyme-linked immunosorbent assay (ELISA), are laborious and time-consuming [1, 12, 30]. A much better alternative for DEV detection is polymerase chain reaction (PCR) in different modifications [6, 21], whereas real-time PCR facilitates determination of the viral copy number, which is useful for assessing the distribution of the virus or the possible infection stage [22, 23, 34]. Recently, loopmediated isothermal amplification (LAMP) for DEV detection has been described [7]. The main advantage of LAMP is its simplicity and the fact that there is no absolute requirement to use expensive thermocyclers or real-time PCR systems [16]. The results may be read with the naked eye, since the LAMP products, produced in microgram quantities, cause a colour change after addition of SYBR Green or other intercalating dye. Similarly, the results can be confirmed by UV illumination. However, since the reaction mixtures contain fluorescent dyes, the reaction may be also recorded by real-time PCR, thus allowing additional observation of the product increase, or even rapid LAMP quantification. The present study describes the application of an upgraded real-time LAMP assay with additional loop primers for fast real-time identification of DEV in free-ranging water birds. The purpose of this study was to assess the importance of DEV infection in freeranging birds, because of the possibility of its transmission to domestic waterfowl. The occurrence of DEV in freeranging Polish water birds has not been investigated previously.

The DNA of DEV strain 1227, isolated from a wild duck in 2006, was used as positive control in our study for the development of a real-time LAMP method. Negative controls used to determine the specificity of the real-time LAMP assay included DNA from Derzsy’s disease virus (DDV) strain B38, DNA from goose hemorrhagic polyomavirus (GHPV) strain 2003 (obtained from the Department of Poultry Viral Diseases, National Veterinary Research Institute, Pulawy, Poland) and DNA from goose circovirus (GCV) P_1_03 (CEVA-Phylaxia, Ceva Sante Animale, Budapest, Hungary). One hundred thirty-two field isolates of DEV were collected during the years 2006-2013 from liver tissue from 38 dead wild ducks (Anas platyrhynchos), 90 mute swans (Cygnus olor), one graylag goose (Anser anser), one tundra bean goose (Anser fabalis) and one grey heron (Ardea cinirea). About 25-30 mg of liver tissue from the examined birds was homogenized in 1 mL of phosphate-buffered saline (PBS) supplemented with 1 % antibiotic-antimycotic (Gibco) and subjected to DNA extraction. Total DNA was extracted from liver homogenates according to the procedure recommended the manufacturer of the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Three sets of complementary primers were designed according to the complete sequence of the U_L30 gene of DEV (GenBank accession number EF643560) using Primer Explorer version 4 (NetLaboratory, Tokyo, Japan). Real-time LAMP was run on an ABI 7500 instrument (Applied Biosystems, Foster City, California United States) with operating software version 2.0.1. The assay volume was 15 μL and contained 7.5 μL of Isothermal Mastermix (OptiGene, Horsham, West Sussex, United Kingdom), 0.8-0.4 mM (1 μL) each inner primer FIP and BIP, 0.2-0.1 mM (0.25 μl) each outer primer F3 and B3, 0.4 to 0.2 mM each LF and LR primer, 1 μl of 1:10 000 concentrated ROX passive reference dye (EurX, Gdansk, Poland), 1 μl of PCR-grade water (EurX, Gdansk, Poland) and 2μl of DNA template. The incubation temperature was optimised in a water bath (57 – 67 °C) for times ranging from 15 to 90 min. The reaction was supplemented for additional melting curve analysis at temperatures ranging from 95 °C to 60°C. After this stage, 0.5 μl of 1:100 stock dilution of 10,000 X concentrated SYBR Green I solution in DMSO (Invitrogen) was added to each sample. The samples were observed with the naked eye and under UV illumination using a simple lamp designed for testing paper money to detect color change during LAMP. All of the reaction steps were recorded using an ABI 7500 machine (Applied Biosystems, Foster City, California, United States) with operating software version 2.0.1.

Three chosen DEV isolates, 1227, 327 and 776, diagnosed by real-time LAMP as positive, were isolated in chicken embryo fibroblasts (CEFs). These isolates were: CEFs were prepared from 11-day-old SPF chicken embryos (LTZ, Cuxhaven, Germany). The infected cells were incubated at 37.5 °C in 5 % CO₂ for 96 h. Three successive passages in CEFs were conducted in order to detect specific cytopathic effect (CPE) in cell monolayers.

The three pairs of primers that were designed were located within the unique long region (U_L30) of the DEV genome sequence encoding the viral DNA polymerase. The primer sequences and their locations are shown in Table 1. The optimal isothermal temperature was established at 60°C. However, the temperatures ranging from 58.3 °C to 64.6°C resulted in the same amplification efficiency (data not shown). A 30-min incubation time resulted in efficient amplification of LAMP products that were visible as green color, greenish fluorescence, and a signal detected by 7500 Applied Biosystems software. The parallel reaction in a water bath resulted in reliable detection starting at 30 min of incubation (Fig. 1A). The final reaction volume of the LAMP mixture was 15 μl, and it contained 7.5 μl of Isothermal Mastermix (OptiGene, Horsham, West Sussex, United Kingdom), 0.4 mM (1 ll) each inner primer FIP and BIP, 0.1 mM (0.25 μl) each outer primer F3 and B3, 0.2 mM (0.5 μl) each LF and LR primer, 1 μl of PCRgrade water (EurX, Gdansk, Poland), 1 μl of 1:10,000- diluted ROX passive reference dye (EurX, Gdansk, Poland) and 2 μl of DNA template (∼50 ng). Finally, 0.5 μl of SYBR Green® I dye (Invitrogen) (diluted 1:100) was added to the reaction mixture, resulting in an immediate green colour change in DEV-positive DNA samples, while in negative control samples, the reaction mixture turned orange. Under UV light, greenish fluorescence was observed in positive samples, while in the real-time PCR system, these samples produced clear fluorescent curves (Fig. 1B). 

Table 1 Sequences of LAMP primers used in this study. F3, forward outer primer; B3, backward outer primer; FIP, forward inner primer (F1c+F2); BIP, backward inner primer (B1c+B2); LF, forward loop primer; LB, backward loop primer; TTTT, thymine linker

 

Fig. 1 LAMP DEV (A) Incubation of reaction mixtures for different times under optimal reaction conditions. (I) Detection with the naked eye after addition of SYBR Green® I dye, (II) under UV light, (III) real-time LAMP plot. 1, 15 min; 2, 30 min; 3, 60 min; 4, 90 min. (B) Sensitivity of LAMP. Serial dilution of DNA extracted from strain 1127. 1, 100 pg; 2, 10 pg; 3, 1 pg; 4, 100 fg; 5, 10 fg; 6, 1 fg; 7, 100 ag; 8, 10 ag. (I) Detection with the naked eye after addition of SYBR Green I dye, (II) under UV light, (III) real-time LAMP plot

The detection limit of the real-time LAMP was 1 pg of strain 1227 DNA (Fig. 1B). No positive signal was observed in DNA samples extracted from control samples of DDV, GHPV or GCV. No visible green colour, fluorescence or fluorescent curves were detected with negative controls, indicating the reliability of the real-time LAMP results. In total, 132 different field samples were examined. In 96 samples (72.7 %), the observed C_T values ranged from 13.3 (sample 773 from a mute swan) to 51.6 (sample 682 from a wild duck). Melting temperature analysis of the LAMP products showed very similar melting temperatures (84.1-86.6 °C) for all of the products (data not shown). Real-time LAMP allowed the detection of DEV as early as 30 min. Parallel detection was possible in a waterbath for 30 min. Since there are three different ways to confirm the results, agarose gel separation is no longer needed. After three successive passages in CEFs, DEV strains caused an evident CPE, which was visible as cells forming foci and plaques with sharp boundaries and encompassed a clear area starting at 56 h of incubation. The plaques formed by isolate 1227, collected from a wild duck, were slightly larger than those produced by two other isolates: 327 and 776, originating from a mute swan and a grey heron, respectively. Virus isolation confirmed the results obtained by the rapid real-time LAMP technique.

During the past few years, LAMP has been found to be a rapid and very reliable diagnosis method for multiple emerging animal diseases including foot-and-mouth disease virus [32], West Nile virus [19], Crimean-Congo hemorrhagic fever virus [18], Chikungunya and dengue virus [14] and porcine circovirus 2 [24]. In the context of viral diseases of waterfowl, the technique was used for detection of avian influenza virus [20, 37], goose parvovirus [26, 36], Muscovy duck parvovirus [8], goose circovirus [29], Tembusu or Tembusu-related virus [27, 33], duck hepatitis virus 1 [25, 35], and duck plague virus – also called duck enteritis virus (DEV) [7].. In the past, the diagnosis of DP was mainly achieved by virus isolation in chicken (CEF) or duck embryo fibroblasts (DEF), and together with isolation of the virus in 1-day-old ducklings, these were the best ways to fulfill the requirements of viral diagnosis [9]. It the present study, DEV isolation in cell culture was conducted in order to confirm the results obtained by real-time LAMP. Apart from classical virus isolation methods, there has been an increase in the use of serological assays, including ELISA, for the last five years [1, 12, 30]. However, molecular biology methods, including PCR [6, 21] and qualitative and quantitative real-time PCR [22, 23, 31, 34] techniques have paved the way for the improvement in DP diagnosis, facilitating the monitoring of viral kinetics and replication. A huge step in DP diagnosis was the introduction of LAMP for in-farm diagnosis [7]. The authors of that study designed two sets of primers to detect a U_L6 gene fragment of DEV; however, in the present study, a more conserved region of the viral genome encoding the DNA polymerase was chosen. Moreover, the previously applied technique was developed with two pairs of primers, while the technique applied in this study used an additional third pair of primers forming ‘‘loops’’ to accelerate the reaction [16]. The present study showed that this improvement shortened the reaction time by half in comparison to the previously described LAMP. Moreover, the amplification was possible after 13-15 min, since the cycle treshold values (C_T) of samples 773 and 654 from mute swans reached 13.3 and 15.5, respectively. This may be caused by a higher viral load of DEV in these two samples in comparison to the standard 1227 strain from wild duck used for real-time LAMP optimisation. Previous papers on real-time LAMP described its advantages in the context of fast and semi-quantitative diagnosis of human influenza A infection (H7N9) by subtype-specific reverse transcription LAMP [17]. The authors measured turbidity within the reaction tubes using color changes of hydroxynaphtol blue dye. Similary, a LAMP assay in which an increase in fluorescence in a sample containing capripoxvirus DNA was measured on an Mx3005p PCR machine (Agilent Technologies, Santa Clara, USA) was recently described [15]. In the present study, similar results were achieved using an ABI 7500 system, which facilitated parallel observation of LAMP product formation. Having three ways to detect LAMP products (by the observed increase in the fluorescence signal or by visible color change by naked eye or under UV light), it was reasonable to omit gel electrophoresis. To ensure the specificity of the assay, a melting curve analysis was also included. An interesting aspect of our study was the identification of DEV in over 72.7 % of the free-ranging waterfowl investigated, highlighting a serious concern also for farmed geese and Muscovy duck flocks. Interestingly, DEV was also found in one grey heron, which might indicate its possible transmission to other water birds species. Previous reports of the occurrence of DEV in the United Kingdom showed that 46.4 percent of birds investigated from 1980 to 1989 were infected. These findings were confirmed by virus isolation [5]. Another report described an outbreak among Muscovy ducks in Illinois, USA, with clinical symptoms manifested as lethargy, diarrhea, dehydration, and death within 2-3 h of disease onset [4]. The material used in our study on DEV occurrence did not allow us to observe clinical symptoms or lesions, since it originated from dead waterfowl. At the beginning of the 1980s, a survey of DEV in North American migratory birds showed that DP was not established in a population of the examined birds. However, it is known that DEV is detectable only during the period when there is active shedding of the virus. This may explain the high prevalence of this virus in free-ranging Polish waterfowl. In our study, we developed a rapid and reliable real-time LAMP method that facilitated identification of DEV infection in free-ranging and domestic waterfowl. Our results show a very significant percentage of infected water birds, indicating that the risk of transmission of DEV to farmed geese and ducks in Poland remains very serious. This study requires further extension to DEV surveillance among domestic flocks of geese and ducks. 

Acknowledgments The authors acknowledge Prof. Zenon Minta and all co-workers of the Department of Poultry Diseases, National Veterinary Research Institute, for sharing the infectious material from wild ducks and swans used in this study. The study was supported by research and development project no. P/020, entitled ‘‘The free-living waterfowl as the reservoir and vector of chosen viral infections spreading’’. 

Conflict of interest The authors declare that they have no competing interests. 

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