Parvoviruses are etiological agents of multiple diseases of humans and animals. The study conducted at the beginning of the 80s. of the last century on chickens with symptoms of emaciation and enteropathy has shown the presence of viral particles in their gut. It has been shown that these viruses have a single stranded DNA (ssDNA) with features specific for autonomically replicating parvovirus. Further on these viruses were classified as chicken parvovirus (ChPV). In experimentally infected 1-d-old SPF chickens, the virus caused the occurrence of clinical symptoms resembling runting-stunting syndrome [1-3].

ChPV virions are non-enveloped particles about 20 nm in diameter with icosahedral symmetry assembled from 32 capsomers. The genome consists of 4-6 kilobase-pair DNA encoding three opened reading frames (ORFs): 5’ORF, 3’ORF and the third small ORF localised between junction of these two ORFs. 5’ORF encodes non-structural protein (NS) taking a part in viral replication while 3’ORF encodes structural proteins of capsid (VP1, VP2 and VP3). The function of the third ORF is not explained so far. ChPV is difficult to propagate in cell clutures and chicken embryos. Meanwhile, in young chickens, ChPV causes runting-stunting syndrome (RSS) frequently described as malabsorbtion syndrome (MS) and rarely as the pale bird syndrome or brittle bone disease. In fact, in young birds the clinical symptoms are noticeable as runting, diarrhoea with subsequent increase in morbidity and mortality with increased factor of fodder uptake [4-10].

During the last few years, an increase in RSS occurrence is observed in chicken broilers. In the USA, the parvoviral infection was found in 77% flocks of broilers and 78% of turkeys [9]. The results obtained by Palade et al., [7] suggest that all of isolated parvoviruses from poultry fall into two common groups of chicken (ChPV) and turkey (TuPV) parvovirus. Actually in Poland the most common problems in chickens are caused by enteropathy and emaciation. Therefore, the aim of this study was the development and application of real-time PCR method for the detection and determination of the prevalence of ChPV in chicken flocks.

The first part of the presented study was done for the development and validation of real-time PCR for the detection of ChPV. The sensitivity of new method allowed detecting ChPV DNA in 10¹ copy dilution of pNS114 plasmid and was specific only for ChPV. The fluorescent curves were detected only in the presence of ChPV and no signal was detected in negative control samples of other examined samples of GPV, MDV and DNA isolated from CEFs (Fig. 1). The presence of ChPV genetic material was detected in chickens from 25 flocks what presented about 17.6% of overall number of examined flocks. The virus was detected in the gut of layers and broiler chickens. The conducted analysis of melting temperature of real-time PCR products revealed that all of them were specific which was found as a common melting point at 86.6°C. The concentration of DNA of ChPV in the examined samples was expressed as 40- C_T value what was shown on Fig. (2). The highest concent of viral DNA was observed in the gut of broiler chickens (from C_T=25,5 up to C_T= 31,6). The age of birds was associated with the concentration of the virus and was the highest among 3-6 week-old broiler chickens and 14 to 21 week-old layers (Fig. 2). In case of four ChPV strains (48/08, 42/09, 8/11 and 18/11) (Table 1), the C_T value was close to 40. Therefore, these results were treated as doubtful. However, the coincidental contamination of reaction mixture was excluded by set of negative controls. The comparison of obtained sequence of PCR product specific for NS of 114/10 strain has shown 93% similarity to field ChPV strains isolated in Hungary (Fig. 3).

Fig. (1). Real-time PCR. Fluorescent curves of DNA samples extracted from field strains of ChPV. The amplification plot was taken by ABI 7500 software ver. 2.0.1(Applied Biosystems). ΔRn – normalised value of fluorescent signal in ChPV positive samples. 1-2/10 strain, 2-13/09 strain, 3-25-other positive samples.

Fig. (2). Concentration of ChPV DNA in samples extracted from chicken guts vs age of chickens. Standard error for CT values was marked. The significance was accepted level at p=0.05.

Fig. (3). Similarity of nucleotide sequence of NS gene of 114/10 Polish strain with GenBank accessible entires. (www.ncbi.nlm.nih.gov).

Primary occurrence of parvoviruses was found in chicken and turkey flocks in the United States of America [9], Hungary [7], and Poland [11]. Previously, it has been shown that both chicken and turkey parvoviruses share high similarity and homology of DNA sequence [9, 10]. Therefore, these viruses fall into common Parvovirus genus. Meanwhile, the other well-known parvovirus of geese (GPV) and Muscovy ducks (MDPV), which causes Derzsy’s disease, belongs to Dependovirus genus [12]. GPV and MDPV are distinct from poultry parvoviruses and have no common antigenic features. Due to the lack of fast diagnostic methods, which allow identifying poultry parvoviruses, Zsak et al., [9] developed PCR with application of hexamers, which consist of random three sets of primers complementary to sequence of poultry parvoviruses. In the presented study, we developed and conducted real-time PCR method as the more sensitive and less laborious alternative technique in comparison to previously described PCR. This advantagable technique was not previously used for the identification of chicken or turkey parvoviruses. The method was developed with use of primers designed by Zsak et al., [9], SYBR Green dye and optimisation of specific reaction conditions. Real-time PCR allowed detecting ChPV in chicken gut with the sensitivity equal to 10¹ copies of pNS114 plasmid. According to previous reports from Hungary and USA described by Palade et al., [7] and Zsak et al., [9], chicken parvoviruses have been isolated from the gut of birds with and without intestinal disorders. In our study, we have shown that infection with ChPV in frequently observed during the last weeks of broiler chicken life. Among these chickens no clinical symptoms were noted except emaciation and stunting. The highest concentration of ChPV expressed as C_T value was found in 2.5-week old broilers and 18-week old layers (Table 1). However, in layers older than 22 weeks from two flocks, the presence of parvovirus was negligible since the C_T value were close to 40 cycle. Infection among these birds were asymptomatic as well. Meanwhile, a considerable correlation was found between stunting of chickens and concentration of the virus in their gut because the C_T value reached 25.5 cycle.

Our results on the occurrence of parvoviral infection mostly in young chickens confirm previously published data [3, 7, 9]. This may indicate on vertical transmission of the infection. This hypothesis is supported by the occurrence of RSS symptoms in hatched nestlings infected by in ovo route [1]. Similarly, the study conducted by Domańska –Blicharz et al., [11] has shown the higher incidence of parvoviruses in poults from 3 to 7 weeks of age than in older birds. They revealed TuPV infection in 26% of turkey flocks. Additionally results described by Palade et al., [7] suggest all poultry parvoviruses isolated in Hungary fall into two groups of ChPV and TuPV

The occurrence of ChPV was detected in almost 18% of examined strains. Supporting the reports about raising incidence of TuPV and ChPV and their close similarity the conducted analysis of nucleotide sequence of NS gene fragment of 114/10 strain has shown 93% of homology with field isolated from Hungary. This may imply on spreading of ChPV in South and Eastern Europe. In summary, the conducted study indicates on the occurrence of ChPV in Polish chicken flocks but their exact role in enteropathies and asymptomatic infections is not understood. This will be investigated in the future.