Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses

Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) cause substantial loss in the production of goslings and Muscovy ducklings. The infection with both parvoviruses spreads rapidly worldwide and causes high morbidity and mortality [1–3]. GPV and MDPV belong to Dependovirus genus of Parvovirdae family and cause Derzy's disease in geese or 3-w disease in Muscovy ducklings [3–6]. The emergence of the disease was observed in middle 60 s of 20^th century in European countries. The disease was noted among goslings and ducklings characterised by anorexia, wheezing, and locomotor dysfunction [1, 2]. The specific lesions observed in the affected geese and ducks include myopathy of skeletal muscle, hepatitis, myocarditis, sciatic neuritis and polioencephalomyelitis [4]. Other commonly observed lesions include atrophy of lymphoid organs (bursa of Fabricius, spleen and thymus). The disease affects mainly young goslings and Muscovy ducklings between 2 and 4 weeks old. However, infections in older birds also occur and are often asymptomatic except ascites or loss in featheriness.

The capsids of GPV and MDPV are non-enveloped, 20-22 nm in diameter and assembled from 32 capsomers. Their genome is single-stranded DNA about 5106 nt (GPV) and 5132 nt (MDPV) [7–10]. The classical detection of GPV and MDPV by virus isolation in gosling or duckling embryos, cell cultures and serological assays like ELISA and seroneutralisation test (SN) is dependent on the availability of SPF gosling embryos and standard positive control sera [2]. These limitations caused an increase in application of polymerase chain reaction (PCR) based techniques which allowed for the identification of GPV or MDPV [11–13]. Recent reports by Ji et al. [14] and Jang et al. [15] presented application of loop-mediated isothermal amplification assay (LAMP) which remarkably simplified the detection of both viruses. In spite of the diagnostic power of these methods they remain qualitative and are incapable to quantitate DNA viral copy which is important from both pathogenic and epidemiological point of view. Quantification of GPV by real-time PCR in experimentally infected goslings was previously described by Bi et al. [16] and Yang et al. [15]. However, these reports did not mention detection and quantification of MDPV. Moreover, the described research was conducted as an experimental approach on goslings and did not include detection and quantification of GPV or MDPV from field cases of Derzsy's disease in goslings or 3-w disease in Muscovy ducklings. The main goal of the presented study was to develop the real-time PCR for parallel detection and quantification of GPV and MDPV. The advantage of a single quantitative real-time PCR is the unification of GPV and MDPV detection and quantification. The second goal was to apply the developed real-time PCR method in order to assume correlation between the age of the infected birds, the observed clinical symptoms and viral DNA copy number. The presented data may be beneficial for better understanding of the pathogenesis of both parvoviral infections and their relationship with the observed clinical symptoms or lesions.

In spite of the prophylaxis against Derzsy's disease of geese and 3-w disease in Muscovy ducks infctions still pose a threat for the production of waterfowl. The comparison of both aetiological agents (GPV and MDPV) by southern hybridization has shown close relation between them in the nucleotide and aminoacid sequence of their genomes [8, 10, 17]. However, both parvoviruses have different host and antigenic features [1, 10]. An important tool to control both diseases is their fast and firm diagnosis. The classical virus isolation methods require at least 2 weeks and are dependent on the laying season of goslings and ducklings [2, 3]. Serological assays provide important data regarding anti-GPV or MDPV antibody level in goslings and Muscovy ducklings but they are also dependent on SPF goose embryos and prone to the influence of maternal antibodies in reproduction flocks [2]. The polymerase chain reaction (PCR) was the first molecular method used for waterfowl parvoviruses detection [12, 13]. Another fast LAMP molecular method for the detection of MDPV was introduced by Ji et al. [14] and Jang et al. [15]. The described LAMP-based methods are the milestone in GPV and MDPV diagnosis. On the other hand PCR and LAMP methods do not allow for the quantification of GPV and MDPV. So far, there was no molecular method developed for parallel detection and quantification of both waterfowl parvoviruses. Real-time PCR is an alternative for the classical PCR and is comparable in speed and labour with the LAMP technique. The method also provides quantitative data which may be used in the in-depth analysis of the pathogenesis or epidemiological study what is non-achievable by PCR or LAMP. It was previously confirmed that real-time PCR is reliable, simple and reproducible [16, 18, 19]. In the presented study the developed real-time PCR allowed for the quantification of GPV and MDPV copy number therefore it can be used for the monitoring of the viral load and determination of the possible stage of both diseases and their correlation with the age and the observed clinical symptoms or lesions in birds. Baigent et al. [20] used the quantitative real-time PCR in order to find the correlation of the CVI988 attenuated strain of Marek's disease virus load and its distribution in visceral organs of chickens. The presented study has shown significant correlation with the infected birds age and the observed clinical symptoms or lesions. In young geese and Muscovy ducks aging up to 2-w no clinical symptoms were observed and also the level of DNA copy number was the lowest and ranged from 0.45 to 3.32 log₁₀ copies/g. The specific symptoms and lesions of the disease observed as ascites, loss of featheriness, pericarditis, enteritis and liver enlargement were observed in birds aging from 3 to 5-w what was confirmed with quantitative real-time PCR data since the number of DNA copy in these birds reached from 1.34 up to 7.55 log₁₀/g. This indicated the most intensive replication of GPV and MDPV in the liver and heart of these birds. A slightly lower copy number - about 6.59 log₁₀ copies/g was also detected in 9-w old ducks suggesting a decrease in the rate of viral replication. On the other hand the observed low DNA copy number in young 1-2-w old goslings and ducklings may indicate early infection with GPV or MDPV and transovarial transmission. Meanwhile, the previously described reports conducted on the experimentally infected goslings revealed the peak in GPV replication between 48 and 72 h post inoculation (PI) [19]. Bi et al. [16] found that fluorescent real-time PCR may be used for the detection and quantification of GPV in goslings as soon as 48 h PI. Similarly Limn et al. [11] detected GPV in liver, heart, brain, heart, pacreas and rectum of Muscovy ducklings as early as 2-d PI. The conducted studies on the infected goslings and ducklings from field farms have shown the field isolate of the virus was detected in 1-w old goslings while the highest viral load was observed in 4-5-w old birds. Yang et al. [19] and Limn et al. [11] described liver, spleen, thymus, Harder's glands and BF to be a good reservoir of GPV and MDPV. In the presented study due to standard laboratory diagnosis procedure the liver and heart of the examined birds were used. The DNA copy number in the examined liver and heart samples were comparable with the results obtained by Yang et al. [19]. Interestingly in the presented study the DNA copy number in young goslings and ducklings aging from 3-5 weeks were consistent with the observed clinical symptoms and pathological changes. Also the results described by Glávitis et al. (4) supported the data obtained in our study. The conducted analysis of correlation between the age of the examined birds and DNA copy number of GPV and MDPV gene revealed a significant lineage between them since the calculated R_S values for liver and heart samples reached 0.8294 and 0.8176 which was above the critical level for the Spearman's rank correlation test (Figure 4). The obtained data are also consistent with the observed clinical symptoms and lesions in the affected birds. Moreover, these results were supported by the data obtained from the correlation test for the age of birds and the observed clinical symptoms or lesions (Figure 1).

Concluding the conducted study the presented quantitative real-time PCR method may be applied as a routine procedure for veterinary laboratories for parallel detection and quantification of GPV and MDPV and may be beneficial for updating the actual data regarding pathogenesis, epidemiology and dynamics of GPV and MDPV replication during the infection in goslings and Muscovy ducklings.